Thèses sur le sujet « Antibiotici - Resistenza »

Oggetto di questa tesi è la traduzione dall'inglese all'italiano di due articoli di rassegna scientifica sul tema della resistenza agli antibiotici apparsinel 2015 sulla rivista statunitense P&T. I testi costituiscono una preziosa fonte di informazioni per il panorama scientifico internazionale e offrono importanti spunti di riflessione e di iniziativa per tutte le altre aree del mondo. La rilevanza dell’argomento trattato, infatti, si estende oltre i confini del singolo Paese e coinvolge tutte le parti interessate dal fenomeno, dal professionista sanitario al cittadino. Il presente lavoro si propone di produrre un testo completo e organico per il pubblico italiano di esperti del settore che intende approfondire l’argomento da una prospettiva multidiscplinare, ma non esclude la possibililtà di raggiungere un pubblico di persone “non addette ai lavori” che possiedono sufficienti interesse personale e strumenti conoscitivi per comprendere, almeno in maniera sommaria, le informazioni riportate negli articoli, grazie alla possibilità di consultarli liberamente online. La traduzione proposta potrebbe, inoltre, contribuire ad arricchire la letteratura scientifica disponibile in lingua italiana che, talvolta, passa in secondo piano per via della predominanza dell’inglese all’interno della produzione scientifica internazionale. Il primo capitolo fornisce alcune informazioni preliminari sull'argomento; il secondo offre una panoramica teorica sulle lingue speciali e, in particolare, sulla lingua della medicina; il terzo capitolo contiene l'analisi dei testi di partenza, mentre il quarto presenta le risorse che sono state utilizzate per la traduzione; nel quinto capitolo sono esposti il TP e il TA in italiano, mentre il sesto capitolo è dedicato a un'analisi dettagliata delle principali difficoltà riscontrate in fase traduttiva e le strategie adottate per risolverle.

Antibiotico-resistenza a Pseudomonas aeruginosa: studio molecolare ed epidemiologico in un ospedale marchigiano. Negli ultimi anni P.aeruginosa è diventato uno dei microrganismi più resistenti ai farmaci. Nonostante l'introduzione di nuovi antibiotici come Ceftolozane/tazobactam (C/T), una nuova combinazione di inibitori della cefalosporina/β-lattamasi con una potente attività contro gli isolati di Pseudomonas aeruginosa, sono stati riportati diversi isolati di P. aeruginosa resistenti.
Antibiotic resistance in Pseudomonas aeruginosa: molecular and epidemiological study in a hospital in the Marche region Background: In the last years P.aeruginosa has became one of most drug resistant microorganism. Despite introduction of new antibiotics such as Ceftolozane/tazobactam (C/T), a novel cephalosporin/β-lactamase inhibitor combination with a potent activity against Pseudomonas aeruginosa isolates, several resistant P. aeruginosa isolates have been reported. From November 2016 to April 2019 we performed both a retrospective study on C/T prescriptions and activity both a survey on clinical strains of P. aeruginosa isolated at “Ospedali Riuniti” of Ancona, Italy, characterising the resistant isolates. Materials/methods: From November 2016 to April 2019 we have collected data about C/T activity and efficacy against all multidrug resistant gram negative isolated at Ospedali Riuniti of Ancona. Particularly we have studied activity of C/T against P.aeruginosa, and microbiological and genetic charateristics of this microorganism. MICs to C/T were determined with gradient test for all P. aeruginosa recovered at the clinical laboratory of “Ospedali Riuniti” from October 2018 to March 2019. Resistant strains were characterized and typed by SpeI-PFGE. We have determined also AmpC production, we have performed DNA extraction and PCR exam. NGS with an Illumina Miseq platform was performed on representative strains to identify the mechanisms of C/T resistance. Results: Over 34 pt, who have received C/T as therapy against multidrug resistant gram negative infections, 53% had CCI >3, 21% underwent to surgery in the previous three months, 32% had pneumonia as acute comorbidities, 18%has died, 26% have experienced a therapeutic failure. CCI>3, pneumonia, P.aeruginosa infection and a previous corticosteroid therapy were a negative prognostic factors. P.aeruginosa resulted resistant to carbapenem, cephalosporin, piperacillina/tazobactam and fluorochinolons, but not to colistin. Over 317 isolated and screened isolates, 15 were resistant to C/T (MIC > 8 mg/L; 4.73%). PFGE showed that 8/15 were strictly related. NGS revealed 6 different STs. The resistance mechanisms to C/T included the metallo β-lactamase (MBL)-econding genes blaVIM-2 in 8 isolates belonging to ST111, and blaIMP in 2 isolates (blaIMP-19 in ST175 and blaIMP-13 in ST621). Additionally, blaPER-1 β-lactamase gene was detected in 2 isolates (ST235) and the blaGES β-lactamase gene in 1 isolate (ST175). Notably, in 2 strains (ST70 and ST3354) no acquired β-lactamase genes involved in C/T resistance has been detected but they showed alterations in ampC. Modifications in these genes and in ampC promoter (ampR) were also detected in all resistant strains except in ST175 isolates (possessing a wild type ampC and ampR). Conclusions: C/T has confirmed its high activity and efficacy against multidrug gram negative infections. There was a low rate of resistance to C/T, but several resistance mechanisms were identified, among which production of MBLs was the most common. Moreover, we found a possible mini-outbreak of blaVIM positive strains. Despite what has been pointed out, we must recognize that this study is limited by the low number of enrolled patients, by the retrospectivity and by being monocentric, but it can be considered an initial approach for further prospective future studies, involving other hospitals in the Region, to better to define both the therapeutic efficacy of C / T and the epidemiology of P. aeruginosa

Le tossinfezioni alimentari causate dal consumo di alimenti di origine animale sono un fenomeno piuttosto diffuso tra i quali la carne di pollo è una delle principali fonti, in quanto presenta diverse contaminazioni con potenziale zoonotico. Infatti, il pollame può presentare diverse malattie infettive di origine batterica che sono in grado di provocare patologie anche a carattere setticemico. È il caso delle infezioni da Escherichia coli: un microrganismo Gram-negativo normalmente presente nell’intestino degli animali e dell’uomo. In tali casi il trattamento farmacologico risulta indispensabile ma purtroppo questo approccio è frequentemente associato alla crescente capacità di acquisizione, da parte del microrganismo, di determinati genetici di resistenza antibiotica portando conseguentemente al fallimento delle terapie. Questo fenomeno in continuo aumento provoca una crescente preoccupazione delle autorità sanitarie in tutto il mondo tanto da consigliare il divieto di utilizzo degli antibiotici negli allevamenti animali. Nel presente studio, isolati di E. coli raccolti da due aziende di broiler allevati senza l’impiego di antibiotici sono stati analizzati al fine di comparare la suscettibilità antimicrobica a quattordici antibiotici (fenotipo di resistenza) con il genotipo di resistenza ottenuto mediante sequenziamento dell’intero genoma, che ha permesso di identificare i determinanti genetici di antibiotico-resistenza e virulenza. Lo studio ha messo in evidenza come il WGS sia in grado di predire il fenotipo di resistenza ma non quello di suscettibilità. L’analisi del viruloma ha indicato la presenza di geni interessanti per la valutazione della patogenicità degli isolati. La caratterizzazione genetica ha messo in luce una certa diversità tra gli isolati intra ed inter-allevamento tranne per alcuni campioni. Infine, l’analisi MLST ha confermato precedenti evidenze scientifiche riguardo ad alcuni ST-tipo indicati come resistenti ad antibiotici beta-lattamici.

2013-2014
Since the 1940s, the ever-increasing use of antibiotics for human, veterinary and agricultural purposes, contributes to their continuous release into the environment due to incomplete metabolism or due to disposal of unused antibiotics. The concern for the release of antibiotics into the environment isrelated to the development of antibiotic resistance genes (ARGs) and bacteria (ARB), which reduce the therapeutic potential against human and animal pathogens. Urban wastewater treatment plant (UWWTP) effluents, hospital discharges, livestock farms represent today the major contamination sources of surface water from antibiotics and ARB. The consequence is that antibiotics, exerting selective pressure, may facilitate the selection of ARB or the acquisition of resistance genes by horizontal transfer. The aim of this work was to investigate the spread of ARB in the environment, particularly in water system, as well as to minimize the related risk through the investigation of effective wastewater disinfection methods. Accordingly, experimental activity was addressed to (i) the monitoring of ARB in river, (ii) modelling ARB fate in river and (iii) minimize ARB release in river through effective wastewater disinfection. [edited by author]
XIII n.s.

Determinanti, contesti, ed elementi genetici associati alla resistenza ai macrolidi e ad altri antibiotici in streptococchi di gruppo viridans. Nell’uomo gli streptococchi di gruppo viridans (VGS) sono parte del microbiota delle vie aeree superiori, del tratto gastrointestinale e del tratto genitale femminile. I VGS, considerati a basso potenziale di patogenicità in soggetti sani, possono tuttavia rendersi responsabili, in particolari tipologie di paziente, di gravi malattie invasive. Obiettivi di questo lavoro sono stati: analizzare la diffusione delle resistenze ad eritromicina, cloramfenicolo e tetraciclina, e chiarire i meccanismi, i determinanti di resistenza e i relativi contesti/elementi genetici in una collezione di VGS recentemente isolati da tamponi faringei da laboratori del centro Italia. Infatti le attuali conoscenze sul resistoma streptococcico riguardano principalmente le specie più patogene e risultano alquanto limitate sui VGS. Dall'indagine svolta, sono stati rilevati: 98/263 VGS sensibili a tutti gli antibiotici testati, 148 isolati eritromicino-resistenti (56.3%), di cui 37 appartenenti al fenotipo cMLS e 111 appartenenti al fenotipo M; 72 isolati (27.4%) tetracyclino-resistenti e 7 (2.7%) cloramfenicolo-resistenti. L'analisi dei determinanti di resistenza ha evidenziato che tutti gli isolati cMLS portavano il gene erm(B), da solo (n=28) o associato al gene d'efflusso mef(E) (n=9). Non sono stati rilevati altri geni erm. Quasi tutti i ceppi di fenotipo M avevano mef(E) (n=107), 2 avevano mef(A) e 2 ceppi rispettivamente mef(I) e mef(G). La resistenza alla tetraciclina è stata evidenziata in 72 isolati VGS, di cui 61 Ery-R e 11 eritromicino-sensibili (Ery-S). Tra tutti i determinanti tet saggiati, tet(M) era di gran lunga il più comune ed era individuato in 43 ceppi Ery-R e in 5 isolati Ery-S. Un isolato aveva il gene tet(O), mentre in 23 isolati non era possibile identificare il determinante di resistenza alla tetraciclina. tet(M) è stato rilevato anche in 2 ceppi di VGS erm(B)-positivi e tetraciclino-sensibili (Tet-S). La resistenza al cloramfenicolo è stata evidenziata in 7 isolati VGS: 6 Ery-R e di fenotipo M possedevano il gene catQ, mentre un isolato Ery-S aveva il determinante catpC194. Per quanto concerne lo studio dei contesti/elementi genetici relativi ai determinanti di resistenza, accanto ad elementi genetici già descritti negli streptococchi (mega, Φ10394.4, Tn2009, Tn2010, elemento IQ, Tn917, Tn3872, Tn6002, Tn916, Tn5801, frammento contenente tet(O) di ICE2096-RD.2, e ICESp23FST81), sono state evidenziate nuove varianti di elementi già noti. Questi risultati mettono nuova luce sulla distribuzione delle antibiotico resistenze, dei determinanti di resistenza e dei relativi contesti/elementi genetici nei VGS, per i quali ad oggi sono disponibili poche informazioni. L'alta frequenza e la varietà degli elementi genetici riscontrati, rafforzano l’ipotesi che i VGS possano rappresentare un importante serbatoio di geni di resistenza per gli streptococchi più patogeni.
Genetic determinants and elements associated with antibiotic resistance in viridans group streptococci. In humans, viridans group streptococci (VGS) are normal inhabitants of the upper respiratory tract, gastrointestinal tract and female genital tract. Though generally considered to have low pathogenic potential in immunocompetent individuals, VGS can nonetheless cause invasive disease. Current knowledge of the resistome of streptococci from the upper respiratory tract is fairly poor as regards VGS compared with the major pathogens. The present study addresses erythromycin, tetracycline and chloramphenicol resistance in VGS. The relevant genetic determinants, environments and elements were investigated in a collection of 263 VGS, identified at the species level, that recently have been isolated from throat swabs in central Italy. Although this type of information is available for major b-haemolytic streptococci and pneumococci, this is not true of VGS. Of the 263 VGS isolates, 148 (56.3%) were resistant to erythromycin, 72 (27.4%) to tetracycline and 7 (2.7%) to chloramphenicol. Of the 148 erythromycin-resistant VGS, 37 (25.0%) belonged to the cMLS and 111 (75.0%) to the M macrolide resistance phenotype (the iMLS phenotype was not detected). All cMLS isolates bore the target-site modification gene erm(B), either alone (n=28) or together with the efflux gene mef(E) (n=9). Other erm genes reported in other streptococcal species, were not detected. Of the M phenotype isolates, the vast majority (n=107) harboured mef(E), two carried mef(A) and one each carried mef(I) and mef(G). Tetracycline resistance was recorded in 72 VGS, including 61 erythromycin-resistant and 11 erythromycin-susceptible isolates. Of the tet determinants assayed, tet(M) was by far the most common, detected in 43 erythromycin-resistant (23 cMLS and 20 M) and 5 erythromycin-susceptible isolates. One isolate carried tet(O), but the tetracycline resistance determinant could not be identified in 23 isolates. tet(M) was also sought in erm(B)-positive tetracycline-susceptible VGS and was detected in two of them. Chloramphenicol resistance was recorded in seven VGS, including six erythromycin-resistant isolates belonging to the M phenotype and carrying the catQ gene, and one erythromycin-susceptible isolate carrying the catpC194 determinant. Moreover a number of variants of known genetic contexts and elements carrying determinants of resistance to these antibiotics were detected, including the mega element, ɸ10394.4, Tn2009, Tn2010, the IQ element, Tn917, Tn3872, Tn6002, Tn916, Tn5801, a tet(O) fragment from ICE2096-RD.2 and ICESp23FST81. These findings shed new light on the distribution of antibiotic resistance mechanisms and determinants and their genetic environments in VGS, for which very few such data are currently available. The high frequency and broad variety of such elements supports the notion that VGS may be important reservoirs of resistance genes for the more pathogenic streptococci.

Negli ultimi decenni l'utilizzo degli antibiotici a scopo terapeutico o come promotori della crescita nell'allevamento animale ha portato alla comparsa e alla diffusione di microrganismi resistenti. In questo contesto, la presenza di Lattobacilli (LAB) antibiotico resistenti non rappresentano di per sé un rischio clinico. Tuttavia la possibilità che essi ma possono essere veicolo di geni codificanti l'antibiotico-resistenza verso batteri patogeni presenti negli alimenti o nel tratto gastro-intestinale umano (inclusi enterococchi, streptococchi e listeria), costituisce un possibile rischio per la salute umana che deve essere attentamente valutato. Obiettivo di questo lavoro è stato quello di valutare attraverso metodi di indagine fenotipica con le tecniche delle microdiluizioni in brodo, Etest e disc-diffusion, i livelli di antibiotico resistenza per le specie S. thermophilus e L. plantarum verso gli antibiotici tetraciclina, eritromicina, clindamicina, streptomicina, gentamicina, ampicillina. Ceppi atipici appartenenti alla specie S. thermophilus sono stati sottoposti ad analisi genetiche con lo scopo di caratterizzare e localizzare i geni responsabili della resistenza. E' stato inoltre testato il possibile trasferimento orizzontale dei geni di antibiotico resistenza nativi da S. thermophilus verso i batteri Gram-positivi E. faecalis e Listeria monocytogenes. In alcuni ceppi di S. thermophilus resistenti si sono infine osservati e studiati particolari caratteri fenotipici ( fitness ) correlati alla presenza delle determinanti genetiche di antibiotico resistenza nell'ospite batterico.
In the last decades, the use of antibiotics in human therapy or in animal husbandry as growth promoters has induced the development and the diffusion in antibiotic resistant micro-organisms. In this context antibiotic resistant Lactic Acid Bacteria (LAB) do not represent a clinical risk in themselves. However, the possibility that S. thermophilus cultures might transfer antibiotic resistance genes to pathogenic species either present in food or in the gastrointestinal tract (including enterococci, streptococci and listeria) represents a potential clinical risk that needs to be carefully evaluated. The aim of this study was to evaluate by means of phenotypic methods (microdilution, E-test, disc-diffusion) the levels of antibiotic resistance for S. thermophilus and L. plantarum species against the antibiotic tetracycline, erythromycin, clyndamicin, streptomycin, gentamycin and ampicillin. The atypical resistant S. thermophilus strains were subjected to genetic analyses in order to characterise and to localise the antibiotic resistance determinants. Furthermore the ability of the resistant S. thermophilus strains in transferring the antibiotic resistant determinant was assessed in mating experiments using as recipients the Gram-positive bacteria E. faecalis and Listeria monocytogenes. In same resistant S. thermophilus strains, special bacterial fitness related with the presence of the antibiotic resistance determinants in the bacterial hosts were observed and studied.

Negli ultimi decenni l'utilizzo degli antibiotici a scopo terapeutico o come promotori della crescita nell'allevamento animale ha portato alla comparsa e alla diffusione di microrganismi resistenti. In questo contesto, la presenza di Lattobacilli (LAB) antibiotico resistenti non rappresentano di per sé un rischio clinico. Tuttavia la possibilità che essi ma possono essere veicolo di geni codificanti l'antibiotico-resistenza verso batteri patogeni presenti negli alimenti o nel tratto gastro-intestinale umano (inclusi enterococchi, streptococchi e listeria), costituisce un possibile rischio per la salute umana che deve essere attentamente valutato. Obiettivo di questo lavoro è stato quello di valutare attraverso metodi di indagine fenotipica con le tecniche delle microdiluizioni in brodo, Etest e disc-diffusion, i livelli di antibiotico resistenza per le specie S. thermophilus e L. plantarum verso gli antibiotici tetraciclina, eritromicina, clindamicina, streptomicina, gentamicina, ampicillina. Ceppi atipici appartenenti alla specie S. thermophilus sono stati sottoposti ad analisi genetiche con lo scopo di caratterizzare e localizzare i geni responsabili della resistenza. E' stato inoltre testato il possibile trasferimento orizzontale dei geni di antibiotico resistenza nativi da S. thermophilus verso i batteri Gram-positivi E. faecalis e Listeria monocytogenes. In alcuni ceppi di S. thermophilus resistenti si sono infine osservati e studiati particolari caratteri fenotipici ( fitness ) correlati alla presenza delle determinanti genetiche di antibiotico resistenza nell'ospite batterico.
In the last decades, the use of antibiotics in human therapy or in animal husbandry as growth promoters has induced the development and the diffusion in antibiotic resistant micro-organisms. In this context antibiotic resistant Lactic Acid Bacteria (LAB) do not represent a clinical risk in themselves. However, the possibility that S. thermophilus cultures might transfer antibiotic resistance genes to pathogenic species either present in food or in the gastrointestinal tract (including enterococci, streptococci and listeria) represents a potential clinical risk that needs to be carefully evaluated. The aim of this study was to evaluate by means of phenotypic methods (microdilution, E-test, disc-diffusion) the levels of antibiotic resistance for S. thermophilus and L. plantarum species against the antibiotic tetracycline, erythromycin, clyndamicin, streptomycin, gentamycin and ampicillin. The atypical resistant S. thermophilus strains were subjected to genetic analyses in order to characterise and to localise the antibiotic resistance determinants. Furthermore the ability of the resistant S. thermophilus strains in transferring the antibiotic resistant determinant was assessed in mating experiments using as recipients the Gram-positive bacteria E. faecalis and Listeria monocytogenes. In same resistant S. thermophilus strains, special bacterial fitness related with the presence of the antibiotic resistance determinants in the bacterial hosts were observed and studied.

L’attenzione degli organismi di controllo nei confronti della problematica dell’antibiotico resistenza sta diventando sempre più pressante e l’individuazione di ceppi lattici che hanno mostrato resistenze e che potrebbero costituire una riserva di geni trasmissibili ad eventuali patogeni lungo la catena alimentare è diventato uno dei temi caldi della ricerca mondiale. Infatti, il consumo di batteri vivi attraverso gli alimenti fermentati (e non) può essere un potente veicolo di disseminazione di resistenza agli antibiotici, attraverso il passaggio di elementi genetici mobili tra specie che vengono a trovarsi in un medesimo habitat (compreso l’intestino umano). La possibilità di acquisire nuove resistenze è stata dimostrata anche per lattobacilli utilizzati per le fermentazioni alimentari ma pochi lavori sono stati condotti su Lactobacillus sakei, estensivamente utilizzato come starter dall’industria dei salumi e caratterizzato da un’ampia variabilità genetica che si riflette in una grande variabilità fenotipica. Poiché la conoscenza dell’antibiogramma è un aspetto cruciale indicato da EFSA per le colture starter, in questo elaborato è stato preso in considerazione il profilo di antibiotico resistenza di L. sakei, attraverso i dati riportati in letteratura e tramite specifiche analisi condotte su ceppi di collezione o isolati da fermentazioni spontanee. I dati sottolineano un’ampia variabilità fenotipica mettendo in luce differenti capacità dei ceppi studiati di reagire alla presenza di questi antimicrobici e mostrando alcuni casi di resistenza, ad esempio al cloramfenicolo e alla tetraciclina. Al contrario, uno dei ceppi si è mostrato sensibile alla vancomicina, considerata invece una resistenza intrinseca dei Lactobacillus. L’analisi critica della letteratura e dei dati acquisiti mostra come sia indispensabile un approfondimento di questa tematica, data l’importanza industriale di questa specie.

I lactobacilli sono considerati dei microorganismi non-patogeni. Molti di loro appartengono al gruppo batterico GRAS e/o sono nell’elenco QPS. Dal momento che i lactobacilli intenzionalmente aggiunti agli alimenti possono agire come reservoir di geni di resistenza, la valutazione del rischio deve essere continuamente aggiornata. Lo scopo di questa tesi era la valutazione di alcuni metodi usati per testare e caratterizzare le specie del genere Lactobacillus per quanto riguarda la sicurezza e la potenziale attività probiotica. Nella prima parte due metodi di micro diluzione, il metodo ISO e CLSI, soni stati comparati testando la resistenza agli antibiotici di 54 ceppi L. plantarum. Sulla base di risultati ottenuti il metodo ISO era più adatto per valutare la resistenza di questa specie. Il test del limite di sensibilità della PCR per 8 paia di primers specifici per il rilevamento dei lactobacilli e bifido batteri da feci ha confermato i loro diversi livelli di efficacia. La seconda parte della tesi descrive un progetto di ricerca mirato sulla identificazione di nuovi ceppi probiotici fra diversi ceppi di Lactobacillus paracasei e Lactobacillus rhamnosus identificando dei geni o loci responsabili della interazione con l’ospite, immunomodulazione, e l’inibizione della crescita dei patogeni. Le analisi fenotipiche dei 40 ceppi hanno confemato una grande variabilità fra di loro, che può servire per associare delle caratteristiche fenotipiche a quelle genotipiche. Tra i ceppi dello stesso progetto è stato individuato un ceppo di L. mucosae. Dal momento che questa è una specie relativamente nuova, le sue caratteristiche sono state analizzate comparandole con altri 3 ceppi appartenenti alla stessa specie. In questo modo sono state confermate alcune informazioni su L. mucosae, ma soprattutto sono stati forniti dei dati nuovi sulle proprietà di questa specie.
The species of the Lactobacillus genus are generally believed to be microorganisms with no pathogenic potential. Many of them have granted GRAS and QPS status. Non-pathogenic bacteria as lactobacilli-intentionally added or accidentally present in food-are under evaluation, as they could act as reservoir of resistant genes. This thesis was aimed to evaluate some methods used for testing and to characterize some Lactobacillus species, as regards their safety and potential probiotic activity. The first part of the research focused on the comparison of two broth microdilution methods: ISO and CLSI, in order to assess the resistance of 54 L. plantarum strains to antimicrobial agents. The results suggest better performances of the phenotypic assay developed by ISO, at least for strains belonging to L. plantarum species.Then the assessment of the PCR detection limit for 8 sets of primers for the detection of lactobacilli and bifidobacteria from infant faeces confirmed different levels of effectiveness for the primers. Next part of the thesis was the research project aimed at identifying genes or genetic loci of different strains of two Lactobacillus species (i.e. Lactobacillus paracasei and Lactobacillus rhamnosus) involved in the interaction with the host, immune-modulation of host cells and pathogen growth inhibition in order to find new probiotic strains. The phenotypic analysis of 40 selected strains demonstrated large variability between strains of these species, which could serve to the association of phenotypic differences to genome specificities. A strain of Lactobacillus mucosae was found within the framework of the same project. As it is a relatively new species, it was chosen to further investigate its properties, comparing it with three other L. mucosae strains. This study led to confirm some information but first and foremost it has provided new data on the examined species.

I lactobacilli sono considerati dei microorganismi non-patogeni. Molti di loro appartengono al gruppo batterico GRAS e/o sono nell’elenco QPS. Dal momento che i lactobacilli intenzionalmente aggiunti agli alimenti possono agire come reservoir di geni di resistenza, la valutazione del rischio deve essere continuamente aggiornata. Lo scopo di questa tesi era la valutazione di alcuni metodi usati per testare e caratterizzare le specie del genere Lactobacillus per quanto riguarda la sicurezza e la potenziale attività probiotica. Nella prima parte due metodi di micro diluzione, il metodo ISO e CLSI, soni stati comparati testando la resistenza agli antibiotici di 54 ceppi L. plantarum. Sulla base di risultati ottenuti il metodo ISO era più adatto per valutare la resistenza di questa specie. Il test del limite di sensibilità della PCR per 8 paia di primers specifici per il rilevamento dei lactobacilli e bifido batteri da feci ha confermato i loro diversi livelli di efficacia. La seconda parte della tesi descrive un progetto di ricerca mirato sulla identificazione di nuovi ceppi probiotici fra diversi ceppi di Lactobacillus paracasei e Lactobacillus rhamnosus identificando dei geni o loci responsabili della interazione con l’ospite, immunomodulazione, e l’inibizione della crescita dei patogeni. Le analisi fenotipiche dei 40 ceppi hanno confemato una grande variabilità fra di loro, che può servire per associare delle caratteristiche fenotipiche a quelle genotipiche. Tra i ceppi dello stesso progetto è stato individuato un ceppo di L. mucosae. Dal momento che questa è una specie relativamente nuova, le sue caratteristiche sono state analizzate comparandole con altri 3 ceppi appartenenti alla stessa specie. In questo modo sono state confermate alcune informazioni su L. mucosae, ma soprattutto sono stati forniti dei dati nuovi sulle proprietà di questa specie.
The species of the Lactobacillus genus are generally believed to be microorganisms with no pathogenic potential. Many of them have granted GRAS and QPS status. Non-pathogenic bacteria as lactobacilli-intentionally added or accidentally present in food-are under evaluation, as they could act as reservoir of resistant genes. This thesis was aimed to evaluate some methods used for testing and to characterize some Lactobacillus species, as regards their safety and potential probiotic activity. The first part of the research focused on the comparison of two broth microdilution methods: ISO and CLSI, in order to assess the resistance of 54 L. plantarum strains to antimicrobial agents. The results suggest better performances of the phenotypic assay developed by ISO, at least for strains belonging to L. plantarum species.Then the assessment of the PCR detection limit for 8 sets of primers for the detection of lactobacilli and bifidobacteria from infant faeces confirmed different levels of effectiveness for the primers. Next part of the thesis was the research project aimed at identifying genes or genetic loci of different strains of two Lactobacillus species (i.e. Lactobacillus paracasei and Lactobacillus rhamnosus) involved in the interaction with the host, immune-modulation of host cells and pathogen growth inhibition in order to find new probiotic strains. The phenotypic analysis of 40 selected strains demonstrated large variability between strains of these species, which could serve to the association of phenotypic differences to genome specificities. A strain of Lactobacillus mucosae was found within the framework of the same project. As it is a relatively new species, it was chosen to further investigate its properties, comparing it with three other L. mucosae strains. This study led to confirm some information but first and foremost it has provided new data on the examined species.

L’insorgenza e la diffusione dell’antibiotico resistenza sta diventando un problema a livello mondiale. Molti sono gli ambienti in cui può avvenire tale diffusione, ma una delle principali vie di trasmissione passa attraverso la catena alimentare. Infatti, l’utilizzo di sostanze antimicrobiche è largamente diffuso negli allevamenti di animali ad uso alimentare e in agricoltura. In particolare, negli allevamenti gli antibiotici non solo vengono usati per trattare eventuali patologie, ma anche come profilassi e come promotori di crescita. Di conseguenza, questo uso a volte sconsiderato ha portato all’insorgenza di batteri resistenti a tali sostanze. Un ruolo fondamentale nella trasmissione e diffusione di tali resistenze a livello alimentare è svolto da batteri non patogeni che sono parte del naturale microbiota degli alimenti. Questi microorganismi infatti, pur non essendo essi stessi nocivi per l’uomo, possono fungere da reservoir di antibiotico resistenze per eventuali batteri patogeni. I batteri che generalmente svolgono questo ruolo sono i batteri lattici. Per questo motivo molto importante è stato identificare e studiare l’antibiotico resistenza anche di tali microorganismi. Negli ultimi anni, tuttavia, c’è stato un crescente interesse per un’altra classe di microorganismi, chiamata Haloarchaea o alobatteri o archaea alofili, poiché la loro presenza è stata rilevata in alimenti particolarmente salati. Dal momento che in letteratura ci sono pochi lavori che studiano i profili di antibiotico resistenza di tali microorganismi e, comunque, tali profili non sono stati studiati su un numero significativo di microorganismi appartenenti alla stessa specie, il presente lavoro di tesi è volto a definire il profilo di antibiotico resistenza del capostipite degli archaea alofili, che è l’Halobacterium salinarum, verificare se ci sono ceppi che presentano antibiotico resistenze e controllare se tali resistenze possono essere trasferite a batteri patogeni.
Antimicrobial resistance is now widely acknowledged as a major global public health challenge. There are many environments through which the transmission and diffusion of antibiotic resistance could happen, but one of the main routes of transmission is the food chain. As a matter of fact, antibiotic use is widely spread in animal husbandry and in agriculture. In particular, in animal husbandry antimicrobials have been used both for therapeutic reasons and as growth promoters. As a consequence, a selective pressure on pathogenic and commensal bacteria of animal origin has been exerted during the time, leading to the onset of microorganisms resistant to such compounds. A pivotal role in the spread in the food chain of antibiotic resistance has been played by non-pathogenic bacteria present in food. These microorganisms are not harmful for humans, but they could represent a reservoir of antibiotic resistance for foodborne pathogenic bacteria. Usually lactic acid bacteria play this role, since they are present in all fermented food. For this reason, the antibiotic resistance profile of lactic acid bacteria has been assessed. In recent years, another class of microorganisms called halophilic archaea have raised an increasing scientific interest, since they have been found in the human intestinal mucosa as well as in foods such as salted codfish and fermented Asiatic seafood. As a few papers have studied the antibiotic resistance profiles of halophilic archaea, and the only present do not consider a statistically significant number of microorganisms belonging to the same species, the aim of the present work is to define the antibiotic resistance profile of the major exponent of halophilic archaea, named Halobacterium salinarum, and consequently to verify if some strains present antibiotic resistances and if they can transfer these resistances to bacteria present in the food chain.

L’insorgenza e la diffusione dell’antibiotico resistenza sta diventando un problema a livello mondiale. Molti sono gli ambienti in cui può avvenire tale diffusione, ma una delle principali vie di trasmissione passa attraverso la catena alimentare. Infatti, l’utilizzo di sostanze antimicrobiche è largamente diffuso negli allevamenti di animali ad uso alimentare e in agricoltura. In particolare, negli allevamenti gli antibiotici non solo vengono usati per trattare eventuali patologie, ma anche come profilassi e come promotori di crescita. Di conseguenza, questo uso a volte sconsiderato ha portato all’insorgenza di batteri resistenti a tali sostanze. Un ruolo fondamentale nella trasmissione e diffusione di tali resistenze a livello alimentare è svolto da batteri non patogeni che sono parte del naturale microbiota degli alimenti. Questi microorganismi infatti, pur non essendo essi stessi nocivi per l’uomo, possono fungere da reservoir di antibiotico resistenze per eventuali batteri patogeni. I batteri che generalmente svolgono questo ruolo sono i batteri lattici. Per questo motivo molto importante è stato identificare e studiare l’antibiotico resistenza anche di tali microorganismi. Negli ultimi anni, tuttavia, c’è stato un crescente interesse per un’altra classe di microorganismi, chiamata Haloarchaea o alobatteri o archaea alofili, poiché la loro presenza è stata rilevata in alimenti particolarmente salati. Dal momento che in letteratura ci sono pochi lavori che studiano i profili di antibiotico resistenza di tali microorganismi e, comunque, tali profili non sono stati studiati su un numero significativo di microorganismi appartenenti alla stessa specie, il presente lavoro di tesi è volto a definire il profilo di antibiotico resistenza del capostipite degli archaea alofili, che è l’Halobacterium salinarum, verificare se ci sono ceppi che presentano antibiotico resistenze e controllare se tali resistenze possono essere trasferite a batteri patogeni.
Antimicrobial resistance is now widely acknowledged as a major global public health challenge. There are many environments through which the transmission and diffusion of antibiotic resistance could happen, but one of the main routes of transmission is the food chain. As a matter of fact, antibiotic use is widely spread in animal husbandry and in agriculture. In particular, in animal husbandry antimicrobials have been used both for therapeutic reasons and as growth promoters. As a consequence, a selective pressure on pathogenic and commensal bacteria of animal origin has been exerted during the time, leading to the onset of microorganisms resistant to such compounds. A pivotal role in the spread in the food chain of antibiotic resistance has been played by non-pathogenic bacteria present in food. These microorganisms are not harmful for humans, but they could represent a reservoir of antibiotic resistance for foodborne pathogenic bacteria. Usually lactic acid bacteria play this role, since they are present in all fermented food. For this reason, the antibiotic resistance profile of lactic acid bacteria has been assessed. In recent years, another class of microorganisms called halophilic archaea have raised an increasing scientific interest, since they have been found in the human intestinal mucosa as well as in foods such as salted codfish and fermented Asiatic seafood. As a few papers have studied the antibiotic resistance profiles of halophilic archaea, and the only present do not consider a statistically significant number of microorganisms belonging to the same species, the aim of the present work is to define the antibiotic resistance profile of the major exponent of halophilic archaea, named Halobacterium salinarum, and consequently to verify if some strains present antibiotic resistances and if they can transfer these resistances to bacteria present in the food chain.

Obiettivo di questo lavoro è stato valutare la diffusione di AR in differenti ceppi di S. thermophilus isolati tra il 1947 e il 2004 e provenienti da differenti ambienti, in modo da avere un chiaro andamento del fenomeno; questo è stato possibile analizzando un numero significativo di ceppi isolati in un periodo di tempo che va da prima dell'utilizzo degli antibiotici fino ai giorni nostri. L'espressione fenotipica è stata valutata con tre differenti metodi (microdiluizioni in brodo, E-test e Disk Diffusion), in accordo con gli standard NCCLS, per la determinazione delle MICs (Minimum Inhibitory Concentration, ovvero Concentrazioni Minime Inibenti). Per la valutazione genetica è stata impiegata la tecnica dei microarrays a DNA utilizzando oligonucleotidi da 50 e 60-mer, per un totale di 300, appartenenti a 10 diverse classi di antibiotici. La conferma dei risultati è stata ottenuta mediante PCR e sequenziamento. In 9 ceppi di S. thermophilus è stato possibile mettere in evidenza la presenza di almeno uno dei geni tetS ed ermB responsabili della resistenza agli antibiotici Tetraciclina e Eritromicina rispettivamente.
The aim of the present work was to assess the AR diffusion in a total of 70 different strains of Streptococcus thermophilus, collected between 1950 and 2004 and from different environments; in this way we had the possibility to obtain a clear overview of the response of these bacteria to a large variety of antibiotics, having been able to analyze a significant number of different strains, originated from different areas and distributed over a wide time period, since before the use of antibiotics up to the present day. The phenotypic expression has been evaluated by using three different methods: microdilution, E-test and disk diffusion. The genetic analysis was performed using 50 and 60-mer oligonucleotides DNA based micro array for the identification of AR genes; the AR genes represented by the oligonucleotides on the micro array belong to: Aminoglycoside, Extended Spectrum ?-lactamase (ESBL), Chloramphenicol, Macrolide Lincosamides and Streptogramin (MLS) group, Sulfonamide, Tetracycline, Trimethoprim and Vancomycin. tetS and ermB genes were found and sequenced in 4 out of the total of the S. thermophilus investigated. Furthermore we have wanted to establish the genetic location of above-mentioned genes and assess their transfer intra and inter species adopting the conjugation technique in plate.

Obiettivo di questo lavoro è stato valutare la diffusione di AR in differenti ceppi di S. thermophilus isolati tra il 1947 e il 2004 e provenienti da differenti ambienti, in modo da avere un chiaro andamento del fenomeno; questo è stato possibile analizzando un numero significativo di ceppi isolati in un periodo di tempo che va da prima dell'utilizzo degli antibiotici fino ai giorni nostri. L'espressione fenotipica è stata valutata con tre differenti metodi (microdiluizioni in brodo, E-test e Disk Diffusion), in accordo con gli standard NCCLS, per la determinazione delle MICs (Minimum Inhibitory Concentration, ovvero Concentrazioni Minime Inibenti). Per la valutazione genetica è stata impiegata la tecnica dei microarrays a DNA utilizzando oligonucleotidi da 50 e 60-mer, per un totale di 300, appartenenti a 10 diverse classi di antibiotici. La conferma dei risultati è stata ottenuta mediante PCR e sequenziamento. In 9 ceppi di S. thermophilus è stato possibile mettere in evidenza la presenza di almeno uno dei geni tetS ed ermB responsabili della resistenza agli antibiotici Tetraciclina e Eritromicina rispettivamente.
The aim of the present work was to assess the AR diffusion in a total of 70 different strains of Streptococcus thermophilus, collected between 1950 and 2004 and from different environments; in this way we had the possibility to obtain a clear overview of the response of these bacteria to a large variety of antibiotics, having been able to analyze a significant number of different strains, originated from different areas and distributed over a wide time period, since before the use of antibiotics up to the present day. The phenotypic expression has been evaluated by using three different methods: microdilution, E-test and disk diffusion. The genetic analysis was performed using 50 and 60-mer oligonucleotides DNA based micro array for the identification of AR genes; the AR genes represented by the oligonucleotides on the micro array belong to: Aminoglycoside, Extended Spectrum ?-lactamase (ESBL), Chloramphenicol, Macrolide Lincosamides and Streptogramin (MLS) group, Sulfonamide, Tetracycline, Trimethoprim and Vancomycin. tetS and ermB genes were found and sequenced in 4 out of the total of the S. thermophilus investigated. Furthermore we have wanted to establish the genetic location of above-mentioned genes and assess their transfer intra and inter species adopting the conjugation technique in plate.

Enterococcus faecium è un componente fondamentale del microbiota di diversi alimenti fermentati quali formaggi e salumi e viene spesso isolato in alto numero in alimenti pronti al consumo. É inoltre largamente utilizzato come probiotico sia per l’uomo che per gli animali. Allo stesso tempo, però, questa specie batterica rappresenta una delle cause principali di infezioni nosocomiali quali endocarditi ed infezioni al tratto urinario. Studi recenti hanno dimostato che la specie E. faecium è costituita da due sub-popolazioni principali: la prima è denominate hospital associated (HA) clade “A” ed include la maggior parte dei ceppi responsabili di infezioni umane; la seconda è chiamata community associated (CA) clade “B”, e contiene principalmente ceppi commensali dell’uomo. Analisi più approfondite hanno rivelato un ulteriore suddivisione all’interno del clade A, nel sub-clade A1 (che raggruppa la maggioranza dei ceppi clinici) e nel sub-clade A2, associato agli animali e più sporadicamente ad infezioni umane. Nel 2012, EFSA ha redatto una linea guida per la valutazione della sicurezza di E. faecium usato come probiotico per gli animali, concludendo che i cepi appartenenti all’hospital-associated clade non devono essere utilizzati in nutrizione animale. Comunque, la distinzione tra le due sub-popolazioni è stata fatta utilizzando dati ottenuti prevalentemente da isolati umani e animali e solo un numero limitato di ceppi isolati dagli alimenti è stato considerato. Obiettivo di questa tesi di dottorato è stato quello di valutare la sicurezza di E. faecium negli alimenti fermentati, considerando ceppi isolati da formaggi artigianali e prodotti carnei e utilizzando sia tecniche di genomica che analisi fisiologiche. Nessuno dei ceppi alimentari studiati è risultato parte del clade A1, ma un ceppo isolato da un salame stagionato pronto al consumo ha rivelato diversi tratti tipici dei ceppi A1, tra cui particolari IS, transposase e geni di resistenza agli antibiotici. Questi risultati, così come altri dati, sottolineano la necessità di approfondire le conoscenze circa il ruolo dei ceppi di E. faecium isolati da alimenti come fattore di rischio per la salute umana.
Enterococcus faecium is commonly found in high numbers in ready to eat foods, being a member of the bacterial communities of a variety of fermented foods, including cheese and sausages, and is widely used as human and animal probiotic. However, this bacterial species is a leading cause of nosocomial infection, mainly endocarditis and urinary tract infections. Recent studies have demonstrated that E. faecium species consists of two very distinct clades: the hospital associated (HA) clade “A”, which includes most of the strains responsible for human infections, and the community associated (CA) clade “B”, that contains primarily human commensal isolates. Deeper analysis revealed a further split within clade A into sub-clade A1 (which groups the vast majority of clinical isolates), and sub-clade A2, associated with animals and sporadic human infections. In 2012, the European Food Safety Authority has issued a guideline for the safety assessment of E. faecium used as animal probiotics, concluding the strains belonging to the hospital-associated clade should not be used in animal nutrition. However, the differentiation of the two clades has been performed using data mainly deriving from human and animal isolates, and only a limited number of strains from the food chain were considered. Aim of this doctoral thesis was to assess the safety of E. faecium in fermented food, considering strains isolated from artisanal cheese and meat products, and using both whole genome-based techniques and physiological studies. None of the food isolates studied in this work belong to the epidemic clade A1, however a strain isolated from a ready to eat salami revealed several A1-specific traits, such as specific IS, transposases and antibiotic resistance genes. These results, as well as other data, underline the emergency of deeper understanding the role of E. faecium isolated from fermented foods as risk factor for human health.

Enterococcus faecium è un componente fondamentale del microbiota di diversi alimenti fermentati quali formaggi e salumi e viene spesso isolato in alto numero in alimenti pronti al consumo. É inoltre largamente utilizzato come probiotico sia per l’uomo che per gli animali. Allo stesso tempo, però, questa specie batterica rappresenta una delle cause principali di infezioni nosocomiali quali endocarditi ed infezioni al tratto urinario. Studi recenti hanno dimostato che la specie E. faecium è costituita da due sub-popolazioni principali: la prima è denominate hospital associated (HA) clade “A” ed include la maggior parte dei ceppi responsabili di infezioni umane; la seconda è chiamata community associated (CA) clade “B”, e contiene principalmente ceppi commensali dell’uomo. Analisi più approfondite hanno rivelato un ulteriore suddivisione all’interno del clade A, nel sub-clade A1 (che raggruppa la maggioranza dei ceppi clinici) e nel sub-clade A2, associato agli animali e più sporadicamente ad infezioni umane. Nel 2012, EFSA ha redatto una linea guida per la valutazione della sicurezza di E. faecium usato come probiotico per gli animali, concludendo che i cepi appartenenti all’hospital-associated clade non devono essere utilizzati in nutrizione animale. Comunque, la distinzione tra le due sub-popolazioni è stata fatta utilizzando dati ottenuti prevalentemente da isolati umani e animali e solo un numero limitato di ceppi isolati dagli alimenti è stato considerato. Obiettivo di questa tesi di dottorato è stato quello di valutare la sicurezza di E. faecium negli alimenti fermentati, considerando ceppi isolati da formaggi artigianali e prodotti carnei e utilizzando sia tecniche di genomica che analisi fisiologiche. Nessuno dei ceppi alimentari studiati è risultato parte del clade A1, ma un ceppo isolato da un salame stagionato pronto al consumo ha rivelato diversi tratti tipici dei ceppi A1, tra cui particolari IS, transposase e geni di resistenza agli antibiotici. Questi risultati, così come altri dati, sottolineano la necessità di approfondire le conoscenze circa il ruolo dei ceppi di E. faecium isolati da alimenti come fattore di rischio per la salute umana.
Enterococcus faecium is commonly found in high numbers in ready to eat foods, being a member of the bacterial communities of a variety of fermented foods, including cheese and sausages, and is widely used as human and animal probiotic. However, this bacterial species is a leading cause of nosocomial infection, mainly endocarditis and urinary tract infections. Recent studies have demonstrated that E. faecium species consists of two very distinct clades: the hospital associated (HA) clade “A”, which includes most of the strains responsible for human infections, and the community associated (CA) clade “B”, that contains primarily human commensal isolates. Deeper analysis revealed a further split within clade A into sub-clade A1 (which groups the vast majority of clinical isolates), and sub-clade A2, associated with animals and sporadic human infections. In 2012, the European Food Safety Authority has issued a guideline for the safety assessment of E. faecium used as animal probiotics, concluding the strains belonging to the hospital-associated clade should not be used in animal nutrition. However, the differentiation of the two clades has been performed using data mainly deriving from human and animal isolates, and only a limited number of strains from the food chain were considered. Aim of this doctoral thesis was to assess the safety of E. faecium in fermented food, considering strains isolated from artisanal cheese and meat products, and using both whole genome-based techniques and physiological studies. None of the food isolates studied in this work belong to the epidemic clade A1, however a strain isolated from a ready to eat salami revealed several A1-specific traits, such as specific IS, transposases and antibiotic resistance genes. These results, as well as other data, underline the emergency of deeper understanding the role of E. faecium isolated from fermented foods as risk factor for human health.

La scoperta e il successivo uso di antibiotici hanno reso resistenti molte specie batteriche sia di origine animale sia umana. I geni di resistenza agli antibiotici possono essere trasferiti tramite la catena alimentare, a partire dagli animali e alimenti, fino al tratto gastrointestinale degli esseri umani. Il presente studio descrive la proprietà coniugativa di alcuni nuovi plasmidi, in particolare di uno identificato in un ceppo di Lactococcus lactis spp. lactis, isolato dall'intestino di pesce, e di altri plasmidi individuati in ceppi di Lactobacillus brevis, Lb. plantarum e Lb. reuteri, isolati da salame. La trasferibilità dei plasmidi che portano i geni di resistenza per l’eritromicina o tetraciclina è stata valutata con metodi di elettroporazione e coniugazione in vitro. Nello specifico è riportato il trasferimento di tali plasmidi a specie batteriche patogene per l’uomo come Listeria monocytogenes e Staphylococcus spp. e a un agente responsabile di Lactococcosi nei pesci come Lc. garvieae. Dopo lo studio sulle proprietà coniugative si è proceduto alla caratterizzazione di questi elementi extracromosomici con esperimenti di comobilizzazione e stabilità. I dati ottenuti suggeriscono come i LAB possano essere un serbatoio di diffusione dei geni per l’antibiotico resistenza, con gravi rischi per l’allevamento di prodotti ittici e salute umana.
The discovery and subsequent widespread use of antibiotics have rendered many bacterial species of human and animal origin resistant to some antibiotics. Antibiotic resistance gene may be transferred via food chain, from animals into fermented and other food or in the human gastrointestinal tract. The transferability of some plasmids that harbor the tetracycline or erythromycin resistance genes to animal and human pathogens was assessed using electrotrasformation and conjugation. The present study describes the proprieties of some new plasmids, originally isolated from fish intestinal Lactococcus lactis ssp. lactis and from fermented sausage Lactobacillus brevis, Lb. plantarum and Lb. reuteri. In particular, here I report the potentially of transferable antibiotic resistance determinants to human pathogenic bacterial like Listeria monocytogenes and Staphylococcus spp. and to an etiologic agent of Lactococcus infection like Lc. garvieae. The possibility of transferring natural Lactococcus and Lactobacillus plasmids into pathogenic bacterial strains involved the characterization of these elements, like comobilization and plasmid stability. These data suggest that lactic acid bacteria (LAB) might be reservoir organism for acquired resistance genes that can be spread both to fish and human pathogens, posing a risk to aquaculture and human health.

La scoperta e il successivo uso di antibiotici hanno reso resistenti molte specie batteriche sia di origine animale sia umana. I geni di resistenza agli antibiotici possono essere trasferiti tramite la catena alimentare, a partire dagli animali e alimenti, fino al tratto gastrointestinale degli esseri umani. Il presente studio descrive la proprietà coniugativa di alcuni nuovi plasmidi, in particolare di uno identificato in un ceppo di Lactococcus lactis spp. lactis, isolato dall'intestino di pesce, e di altri plasmidi individuati in ceppi di Lactobacillus brevis, Lb. plantarum e Lb. reuteri, isolati da salame. La trasferibilità dei plasmidi che portano i geni di resistenza per l’eritromicina o tetraciclina è stata valutata con metodi di elettroporazione e coniugazione in vitro. Nello specifico è riportato il trasferimento di tali plasmidi a specie batteriche patogene per l’uomo come Listeria monocytogenes e Staphylococcus spp. e a un agente responsabile di Lactococcosi nei pesci come Lc. garvieae. Dopo lo studio sulle proprietà coniugative si è proceduto alla caratterizzazione di questi elementi extracromosomici con esperimenti di comobilizzazione e stabilità. I dati ottenuti suggeriscono come i LAB possano essere un serbatoio di diffusione dei geni per l’antibiotico resistenza, con gravi rischi per l’allevamento di prodotti ittici e salute umana.
The discovery and subsequent widespread use of antibiotics have rendered many bacterial species of human and animal origin resistant to some antibiotics. Antibiotic resistance gene may be transferred via food chain, from animals into fermented and other food or in the human gastrointestinal tract. The transferability of some plasmids that harbor the tetracycline or erythromycin resistance genes to animal and human pathogens was assessed using electrotrasformation and conjugation. The present study describes the proprieties of some new plasmids, originally isolated from fish intestinal Lactococcus lactis ssp. lactis and from fermented sausage Lactobacillus brevis, Lb. plantarum and Lb. reuteri. In particular, here I report the potentially of transferable antibiotic resistance determinants to human pathogenic bacterial like Listeria monocytogenes and Staphylococcus spp. and to an etiologic agent of Lactococcus infection like Lc. garvieae. The possibility of transferring natural Lactococcus and Lactobacillus plasmids into pathogenic bacterial strains involved the characterization of these elements, like comobilization and plasmid stability. These data suggest that lactic acid bacteria (LAB) might be reservoir organism for acquired resistance genes that can be spread both to fish and human pathogens, posing a risk to aquaculture and human health.

Lo studio ha analizzato la sensibilità ai chinoloni e al cloramfenicolo di 368 isolati clinici di Streptococcus agalactiae raccolti nel periodo 2010-2016 da tre ospedali della Regione Marche. La resistenza ai chinoloni è risultata pari al 2,99% (11 ceppi), mentre quella al cloramfenicolo è stata dell’1,6% (6 isolati). In 10 isolati la resistenza ad alto livello ai chinoloni era conferita da mutazioni delle regioni QRDR in entrambi i bersagli enzimatici GyrA (Ser81Leu) e ParC (Ser79Phe). Un unico isolato, mostrante la singola mutazione in ParC, era caratterizzato da una resistenza a basso livello. 4 degli 11 ceppi resistenti ai chinoloni erano resistenti anche al cloramfenicolo. La caratterizzazione genetica e gli esperimenti di trasferibilità hanno portato alla dimostrazione di un nuovo elemento genetico mobile (~110 kb) denominato ICESag236, che veicola i determinanti catQ, erm(TR) e mef(I), che conferiscono resistenza al cloramfenicolo e ai macrolidi. ICESag236 è un nuovo elemento genetico mosaico derivante dalla ricombinazione molecolare di ICESpn529IQ e ICESagTR7, identificati rispettivamente in Streptococcus pneumoniae e in S. agalactiae. I risultati ottenuti in questo studio confermano la grande flessibilità genomica di S. agalactiae. Inoltre, evidenziano come in questa specie la diffusione dell’antibiotico-resistenza può dipendere sia dalla circolazione di specifici cloni (resistenza ai chinoloni), sia dall’evoluzione di particolari elementi genetici (resistenza al cloramfenicolo).
In this study 368 clinical isolates of Streptococcus agalactiae, collected in 2010–2016 from three hospitals of central Italy, were screened for quinolone and chloramphenicol resistance. The rate of quinolone resistance was 2,99% (11 strains), while chloramphenicol resistance rate was 1,6% (6 isolates). In 10 isolates the high-level quinolone resistance was conferred by mutations of the QRDR regions in both enzymatic targets GyrA (Ser81Leu) and ParC (Ser79Phe). An isolate, showing the single mutation in ParC, was characterized by a low-level resistance. Interestingly, 4 of the 11 quinolone-resistant strains were also resistant to chloramphenicol. Transferability assays and sequencing experiments led to the characterization of a new mobile genetic element (~110 kb) designated ICESag236, harbouring catQ, erm(TR) and mef(I) determinants, which confer resistance to chloramphenicol and macrolides. ICESag236 is a new mosaic genetic element resulting from recombination of two integrative and conjugative elements (ICEs) originally described in different streptococcal species: S. agalactiae ICESagTR7, and Streptococcus pneumoniae ICESpn529IQ. The results obtained in this study confirm the great genomic flexibility of S. agalactiae. Moreover, we show how in this species the diffusion of the antibiotic-resistance may depend both on the spread of specific clones (e.g. for resistance to quinolones), and on the evolution of peculiar genetic elements (e.g. for resistance to chloramphenicol).

Nella lotta alle infezioni batteriche causate da Gram-positivi, gli oxazolidinoni (linezolid e tedizolid) rappresentano l’ultima classe di antibiotici ad essere stati sviluppati per l’utilizzo in clinica. La resistenza a questi farmaci è dovuta a mutazioni nel ribosoma (23S rRNA, proteine L3 e L4) e a geni acquisiti [cfr, cfr(B) e optrA]. In questo studio sono stati caratterizzati i meccanismi di resistenza agli oxazolidinoni in ceppi clinici di Staphylococcus spp. ed Enterococcus spp. È stato identificato un clone di Staphylococcus epidermidis, endemico negli Ospedali Riuniti di Ancona da 12 anni, che è resistente al linezolid a causa d’una mutazione del 23S rRNA. Sono stati caratterizzati due ceppi di S. epidermidis linezolid-resistenti provenienti dall’ospedale Careggi (Firenze): la resistenza era mediata da mutazioni sul 23S rRNA, sulle proteine L3 e dal gene cfr, localizzato su due nuovi plasmidi coniugativi multi-resistenti correlati. È stato caratterizzato un isolato di Staphylococcus aureus linezolid-resistente proveniente da Firenze, primo S. aureus cfr-positivo isolato in Italia: il cfr era localizzato nel cromosoma all’interno di un plasmide enterococcico linearizzato (pRE25-like), in una nuova struttura a mosaico portante anche i geni di resistenza erm(B) e fexB. È stato effettuato uno screening su enterococchi con MIC del linezolid ≥4 mg/L alla ricerca di determinanti di resistenza agli oxazolidinoni. Sono stati identificati due ceppi di Enterococcus faecium con ridotta sensibilità al linezolid, che portavano i geni cfr e optrA sullo stesso elemento, un plasmide pRE25-like. Infine è stato caratterizzato un ceppo di E. faecium pienamente resistente al linezolid a causa di una mutazione sul 23S rRNA. Pur se la percentuale di resistenza rimane molto bassa, la sorveglianza e l’uso consapevole degli oxazolidinoni sono necessari per preservare l’efficacia di questi antibiotici.
In the fight against bacterial infections due to Gram-positive bacteria, oxazolidinones (linezolid and tedizolid) represent the latest class of antibiotics developed for clinical use. Oxazolidinone resistance is caused by mutations in the ribosome (23S rRNA, L3 and L4 proteins) and acquired genes [cfr, cfr(B) and optrA]. In this study oxazolidinone resistance mechanisms have been characterised in clinical strains of Staphylococcus spp. and Enterococcus spp. A clone of Staphylococcus epidermidis, resistant to linezolid due to mutations in 23S rRNA, has been recognised as being endemic in the Ancona Regional Hospital for 12 years. Two linezolid-resistant S. epidermidis isolates from Careggi Hospital (Florence) have been characterised: resistance was mediated by 23S rRNA mutations, L3 protein mutations, and the cfr gene, located on two new related multi-resistance plasmids. A linezolid-resistant Staphylococcus aureus strain from Florence, the first cfr-positive isolate in Italy, has been studied: cfr was located on the chromosome, within a linearized plasmid of enterococcal origin (pRE25-like), in a mosaic structure carrying also resistance genes erm(B) and fexB. A screening was conducted on enterococci showing a linezolid MIC ≥4 mg/L in order to look for oxazolidinones resistance determinants. Two Enteroccocus faecium with reduced linezolid susceptibility were identified which carried both cfr and optrA on the same genetic element, a pRE25-like plasmid. Finally, an E. faecium isolate fully resistant to linezolid has been characterised: resistance was mediated by mutations in rRNA 23S. Even if the incidence of linezolid resistance remains very low, surveillance and conscious use of oxazolidinones are essential to preserve their effectiveness.

I takt med att allt mer antibiotika används och att världen blir allt mer globaliserad ökar och sprids antibiotikaresistensen. Djurhållningen i världen kantas av stressgivande miljöer som för små utrymmen och för många djur per yta. Det får djuren att drabbas av infektioner som vi botar med antibiotika. Antibiotika används även inom djurhållning i tillväxtfrämjande syfte och för att förebygga sjukdom och minska stress. Denna fel- och överbehandling av antibiotika i kombination med att vi använder samma sorts antibiotika inom human sjukvård som inom djurhållning gör att våra livsmedelsproducerande djur utgör en smittorisk för resistenta bakterier som hotar att nå oss via bland annat livsmedelskedjan. I och med att djuren medicineras via tillägg i foder och vatten och att upp till 90% av antibiotikan följer med fekalierna ut, sprids resistensen i naturen då stor del av fekalierna distribueras på jordbruksåkrar i fertiliserande syfte. Det ökar på spridningsrisken samt utgör ytterligare en risk för oss när vi äter grödorna. Från akvakulturer hamnar ungefär 80% av antibiotikan i det omgivande vattnet och i sedimentet och kan därifrån spridas till havets mikrober, vidare till fisk- och skaldjurspatogener och sedan till terrestra bakterier. Åtgärder till dessa problem innefattar att minska spridningen och förhindra uppkomsten av resistenta bakterier. Man bör forska fram fler antibiotika exklusivt för en sektor, i första hand vaccinera och när man måste använda antibiotika bör det vara en smalspektrumsvariant. Man måste också förbättra den globala djurhållningsstandarden, så att risken för spridning minskar vid resor och handel. Det krävs också ett ökat kunskapsläge och ett gemensamt internationellt samarbete för minskad och mer restriktiv antibiotikaanvändning.
As more antibiotics are being used in the world, and as the world gets more globalized, antibiotic resistance is a problem that is growing and spreading. Animal husbandry all over the world provides animals with stressful environments such as too small spaces and too many animals per area. The stress makes the animals suffer from infections that we cure with antibiotics. Antibiotics are also used in animal husbandry as a growth promoter and to prevent illness and decrease stress. This mis- and overuse of antibiotics and the fact that we are using the same type of antibiotics for human health care as well as for animal husbandry, makes our livestock a threat - we can get infected with antibiotic resistant bacteria through the food chain. As a result of us medicating the animals by putting antibiotics in their feed and water (where up to 90% of the antibiotics ends up in the faeces), the resistance is spread in nature, since the faeces often are used as fertilizers in agriculture. This increases the risk of spreading and is another threat for us when we eat the crops from the fields. From aquacultures about 80% of the antibiotics ends up in the nearby water and sediment and can spread through the microbes of the ocean, via fish and shellfish pathogens to terrestrial bacteria. Measuring steps includes decreasing the spread and preventing the rise of resistant bacteria. More research is needed to find new antibiotics, that should be used exclusively for one sector. We should also vaccinate more and when antibiotics are needed, use narrow spectrum antibiotics. Another step is to improve the global animal husbandry standards, so the risk for spreading decreases when travelling and importing/exporting. More education and international teamwork for reduced and more strict antibiotic usage is also needed.

La specie Staphylococcus haemolyticus tra gli stafilococchi coagulasi-negativi (CoNS) rappresenta uno dei più frequenti agenti d’infezione in ambito ospedaliero. La maggior parte degli isolati clinici sono meticillino-resistenti e per molti anni i glicopeptidi sono stati utilizzati nel trattamento delle infezioni stafilococciche nosocomiali. Recentemente è stato introdotto nella pratica clinica la daptomicina in seguito all’aumento di ceppi di Staphylococcus aureus e di stafilococchi CoNS con sensibililità ridotta ai glicopeptidi e la comparsa di ceppi di S. aureus VanA. Ceppi resistenti alla daptomicina sono ancora rari, e sembrano emergere in seguito ad esposizione prolungata alla vancomicina ma il meccanismo di resistenza è ancora da chiarire. Lo scopo di questo lavoro è stato quello di valutare l’attività della daptomicina nei confronti di coppie isogeniche di S. haemolyticus (teicoplanino-sensibile/resistente) e di derivati di laboratorio resistenti all’antibiotico. I mutanti ottenuti in seguito a selezione con la daptomicina, acquisivano la resistenza ad alto livello anche alla teicoplanina, mentre diminuiva la resistenza ai beta-lattamici, soprattutto nel derivato ottenuto dal ceppo meticillino-resistente. Questo effetto altalena non sembrava associato alla perdita dei geni mecA e blaZ e neanche alla loro mancata espressione. Si ipotizza che modificazioni nella struttura e/o nel turnover della parete possano influenzare la sensibilità ad antibiotici che agiscono sulla parete (glicopeptidi e beta-lattamici) e ostacolare anche l’azione di peptidi cationici come la daptomicina. La riduzione dell’attività autolitica e mutazioni nel gene vicK, coinvolto nella regolazione della sintesi della parete batterica, osservate nel ceppo mutante rispetto al parentale daptomicino-sensibile sembrano avvalorare tale ipotesi. Tuttavia altri sistemi di regolazione dovrebbero essere indagati poiché in S. haemolyticus la resistenza alla daptomicina, così come quella alla teicoplanina, sembra dipendere da più fattori responsabili della produzione di una superficie cellulare modificata.
Among coagulase-negative staphylococci (CoNS), Staphylococcus haemolyticus is one of the most common agents of hospital-acquired infection. Most clinical isolates are methicillin-resistant, and for many years glycopeptides have been the treatment of choice for these nosocomial infections. Daptomycin has recently been introduced into clinical practice due to the increase of Staphylococcus aureus and CoNS strains with reduced susceptibility to glycopeptides, and to the appearence of S. aureus VanA isolates. Daptomycin-resistant strains are still rare and they are recovered mostly from patients exposed to prolonged vancomycin treatment. The mechanism of daptomycin resistance is still unclear. The purpose of this study was to assess the activity of daptomycin against isogenic pairs (teicoplanin-susceptible and resistant) and antibiotic-resistant laboratory derivatives of S. haemolyticus. The mutants selected on daptomycin-containing agar exhibited high-level resistance to teicoplanin too. Moreover the derivative obtained from the methicillin-resistant parental strain showed a highly increased susceptibility to beta-lactam antibiotics. This “seesaw effect” seems not to be associated with the loss or deletion of mecA and blaZ genes, or with a variation of their expression. It was hypothesized that changes in the structure and/or the turnover of the bacterial wall and surface charge may influence the susceptibility to cell wall active antibiotics (glycopeptides and beta-lactams) and to cationic peptides such as daptomycin. This hypothesis was supported by the reduction in autolytic activity and by mutations (in the mutant compared to the parent) in the vicK gene involved in bacterial cell wall biosynthesis. Further studies are nevertheless needed since daptomycin resistance, as well as glycopeptide resistance, could arise in S. haemolyticus from multiple factors leading to the production of an altered cell surface.

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

Salmonele su najčešći prouzrokovači.crevnihinfekcija kod ljudi i životinja Široko surasprostranjene u prirodi a mogu kolonizovatirazličite domaćine Poebno su značajne oneživotinje čije se meso koristi za ishranu ljudi, akoje su i glavni izvor infekcije ljudi. Inficiraneživotinje ne ispoljavaju simptome i terapija sesprovodi nalazom salmonela prilikom rutinskekontrole zdravstvenog stanja. Posebno, kod ljudi iživotinja salmoneloze su značajne i zbogkliconoštva koje može trajati veoma dugo zavisnood starosti inficiranog organizma. Čest su uzrokalimentarnih toksiinfekcija kod ljudi. Duž čitavoglanca ishrane moguća je i sekundarnakontaminacija salmonelama. Pored rutinskihmikrobioloških analiza u otkrivanju izvora iputeva širenja infekcije koriste se i molekularnemetode koje daju precizne podatke o klonalnomporeklu bakterija izolovanih iz obolelih ljudi,namirnica i životinja. Međunarodnom trgovinomhrane isti tipovi bakterija mogu se pojaviti nageografski udaljenim lokacijama. Molekularnakarakterizacija Salmonella je značajna zbogodređivanja raznolikosti sojeva. Potrebno je da seizolati tipiziraju ne samo do nivoa vrste i serotipanego i preciznije. Tipizacija je bitna za utvrđivanjeepidemiološke povezanosti izolata, agenotipizacija podrazumeva direktnu analizuDNK. Geni rezistencije koji se od saprofita mogupreneti na patogene vrste imaju važnu ulogu upojavljivanju rezistentnih i multirezistentnihsojeva. Međusobni odnos gena rezistencije iupotrebe antimikrobnih sredstava određujejednačinu rezistencije na antimikrobna sredstva.Rezistencija predstavlja problem iako jesalmoneloza samolimitirajuća infekcija zbog čegase terapija antibioticima primenjuje samo koddece, starijih ljudi i u slučajevima sistemskihinfekcija. Navedena problematika je aktuelna i uCrnoj Gori jer ne postoji stalan monitoringprimene antimikrobnih sredstava kod domaćihživotinja. Iz navedenih razloga cilj ove doktorskedisertacije bilo je utvrđivanje prisustva Salmonellavrsta na farmama živine sa tri lokaliteta u CrnojGori, izolacija i identifikacija primenom standardnihmikrobioloških metoda, serotipizacija pomoćuaglutinacije na predmetnom staklu (slideagglutination) uz primenu anti-O i anti-H seruma.(Staten Serum Institute, Danska). Primenomstandardnih mikrobioloških metoda utvrđeno jeprisustvo serovarijeteta Salmonella Enteritidis iSalmonella Tiphymurium. SerovarijetetiSalmonella .Gallinarum biotip Gallinarum iSalmonella Gallinarum biotip Pullorum nisu bileserološki tipizirane. Identifikacija navedenihserovarijeteta je potvrđena metodom multipleksPCR detekcijom amplifikovanih DNK fragmenatau agaroznom gelu.. Nakon digestije 50 izolataSalmonella enterica podvrste enterica odabranihprema lokalitetima i zastupljenosti pomoću SpeIrestrikcionog enzma i njihovom analizomprimenom PFGE utvrđeno je 5 različitih SpeIpulsotipova. Kod 10% ispitivanih sojevaustanovljena je rezistencija na tetraciklin istreptomicin. Svi ispitivani serovarijetetisalmonela bili su osetljivi na amoksicilin saklavulanskom kiselinom, enrofloksacin,ciprofloksacin, sulfametoksazol-trimetoprim,cefuroksim, ceftriakson i norfloksacin. VrednostiMIK (minimalnih inhibitornih koncentracija)određenih antibiotika za odabrane sojeveserovarijeteta Salmonella Enteritidis primenomaparata VITEK 2 iznosile su za: piperacilin ≤ 4 –64 μg/ml (S – R) , cefuroksim 4 – 32 μg/ml (S -R), cefuroksim aksetil 4 –32 μg/ml (S– R),cefiksim ≤ 0,25 – 2 μg/ml (S - I), ceftriakson ≤ 1 –2 μg/ml (S - I) i minociklin ≤ 1 – 4 μg/ml (S),tetraciklin ≤ 1 μg/ml (S), tigeciklin ≤ 0,5 – 1 μg/ml(S), hloramfenikol ≤ 2 – 8 μg/ml (S), kolistin ≤0,5–1 μg/ml (S) i sulfametoksazol/trimetoprim ≤0,5 μg/ml (S).
Salmonella is the most common cause of alimentarytoxic infections among humans. They have beenadapted to a number of warm-blooded animals. Theinfected animals do not exhibit symptoms and thetreatment performs by finding of salmonella in routinehealth check. The secondary contamination bysalmonella ois possible in during entire food chain.Apart from the routine microbiological analysis indetection of sources and pathways of spreading theinfection, there are also used the molecular methodsthat provide accurate information about the clonalorigin of bacteria isolated from diseased humans, foodand animals. During international trade of food, thesame types of bacteria can occur in geographicallyremote locations. Molecular characterization ofSalmonella is important in determination of diversity ofstrains. It is necessary isolates to be typified not only tothe level of species and serotypes but also moreprecisely. Typification is essential to determine theepidemiological connection of isolates. Genotypingincludes a direct analysis of DNA. The resistance genesthat can be transferred from saprophytes to pathogenicmicroorganisms play an important role in theemergence of resistant and multiresistant strains. Theabove mentoned is also a current issue in our countrybecause there is a constant monitoring of using ofantimicrobials drugs to farm animals. For these reasons,the aim of this dissertation is to examine the serovars ofSalmonella in Montenegro, their molecularcharacterization using biomolecular methods based onisolation of DNA and subsequent amplification ofserovar-specific genes.(multiplex PCR method andPFGE), and testing sensitivity, or resistance toantimicrobial drugs used in clinic vet practice.

I examensarbetet Det post-antibiotiska köket har samhällsfrågan om antibiotikaresistens undersökts ur ett designperspektiv. Detta för att uppmärksamma och sprida kunskap om problemet med den ökande resistensen och den nutida konsumtionen av läkemedlet. Designmetoden som använts är spekulativ design och syftet har varit att tillverka fem objekt som skall påverka och motivera en publik till att förändra den aktuella användningen av mirakelmedicinen. Genom grundliga efterforskningar om problematiken och om en framtid utan antibiotika har ett scenario formulerats. Detta scenario utspelar sig 30 år framåt i tiden, år 2049, i en värld där det inte längre finns fungerande antibiotika. Hur utförs en vardagsaktivitet, som att laga spagetti och köttfärssås, när ett litet sår kan leda till en dödlig infektion? Baserat på efterforskningar, workshops och samtal kunde köksobjekten fastställas; en kniv som minskar risken för stick- och skärskador, skyddande handskar, halkfria skor, en ansiktsmask och bakteriekryddor. Objekt som kan bli en del av det post-antibiotiska köket och vardagslivet om vi inte ändrar vårt nutida beteende.
In the degree work The Post-Antibiotic Kitchen the societal issue concerning antibiotic resistance has been explored through design. The purpose of the project has been to bring attention and spread knowledge about the growing resistance and current consumption of antibiotics. Speculative design has been the used design method and the goal has been to create five objects that will influence and motivate an audience to change the present usage of antibiotics. Through thorough research on the issue and on a future without antibiotics, a scenario has been formulated. The scenario takes place 30 years in the future, in 2049, in a world where there are no longer any functioning antibiotics. How will a day to day activity, such as cooking a meal, be performed when a small cut could lead to a deadly infection? Based on research, workshops and conversations five objects were created; a knife to prevent cut- and stab injuries, protective gloves, non-slip shoes, a face-guard and bacteria spices. Five objects that could be a part of the post-antibiotic kitchen and the daily life unless we change our current behavior today.

Posten kompletterad 20190813 med uppdaterad version av uppsatsen.

L’emergenza globale dell’antibiotico-resistenza, unitamente al ridotto sviluppo di nuove molecole antibiotiche ha ravvivato l'interesse nei confronti delle sostanze naturali come agenti antimicrobici adiuvanti e/o alternativi agli antibiotici. Oltre ad un’azione battericida, tali sostanze possono sia agire in sinergia con gli antibiotici che esercitare un’azione diretta contro la virulenza batterica. In questo studio è stata valutata l’attività antimicrobica e anti-virulenza di differenti oli essenziali e principi attivi vegetali nei confronti di alcuni importanti patogeni antibiotico-resistenti quali Streptococcus pyogenes, Streptococcus suis, Listeria monocytogenes e Mycobacterium abscessus. I risultati dello studio dimostrano un’attività antimicrobica degli oli essenziali di Timo e Origano e dei loro principali costituenti, carvacrolo e timolo, nei confronti di ceppi clinici, antibiotico-resistenti di S. pyogenes e S. suis; in particolare, in S. pyogenes, il carvacrolo mostra un’azione sinergica con eritromicina. La capsaicina, principio attivo di piante appartenenti al genere Capsicum, oltre ad avere un’azione battericida, riduce l’invasione cellulare e l’attività emolitica di ceppi invasivi di S. pyogenes. L’olio essenziale di Cannabis sativa, estratto da due varietà monoiche francesi di canapa industriale, pur non mostrando alcun effetto battericida nei confronti di ceppi clinici di L. monocytogenes, è comunque capace di ridurre la capacità di formare biofilm, la motilità e l’invasione cellulare da parte del patogeno. La curcumina, fitocomposto di Curcuma longa, mostra invece un effetto sinergico con diversi antibiotici ai quali M. abscessus risulta resistente. L’utilizzo di una strategia antimicrobica basata sulla sinergia tra sostanze naturali e antibiotici e/o che abbia come bersaglio la virulenza potrebbe essere dunque molto interessante sia per la bassa pressione selettiva che per la conseguente scarsa propensione allo sviluppo di resistenze.
The global burden of antimicrobial resistance, together with the reduced development of new antibiotic molecules, has revived the interest in plant products as adjuvants/alternative to antibiotics. Actually, beside a bactericidal action, some products from plants can also act in synergy with antibiotics and/or have an anti-virulence action. In this study, the activities of different essential oils and other plant compounds were evaluated against antibiotic-resistant bacterial pathogens Streptococcus pyogenes (pharynx), Streptococcus suis (blood, CSF), Listeria monocytogenes (blood, CSF), and Mycobacterium abscessus (lung). Oregano and Thyme essential oils, and their main constituents carvacrol and thymol, demonstrated a bactericidal activity against S. pyogenes and tetracycline-resistant S. suis isolates; carvacrol also showed a synergistic action with erythromycin against erythromycin-resistant S. pyogenes. Capsaicin, the spicy component of plants belonging to the genus Capsicum, was bactericidal against S. pyogenes and also was able to inhibit, at sub-inhibitory concentrations, cell invasion and haemolytic activity, i.e. important virulence traits. Curcumin, a bioactive phenolic compound of Curcuma longa, showed a synergistic effect with several antibiotics (amikacin, clarithromycin, ciprofloxacin and linezolid) to which M. abscessus was highly resistant. Although not showing a bactericidal activity, the essential oil of Cannabis sativa from two French varieties of monoecious hemp reduced the virulence of L. monocytogenes by inhibiting biofilm formation, motility, and cell invasion. The study demonstrates that an antimicrobial strategy based on plant products showing synergy with antibiotics and/or targeting bacterial virulence may represent a new approach to fight antibiotic resistance, also considering the low selective pressure and thus the low propensity to the development of resistance.

Orientador: Aline Aparecida Pizzirani-Kleiner
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
Made available in DSpace on 2018-07-14T21:12:35Z (GMT). No. of bitstreams: 1 Legaspe_MaraFonsecaChiarelli_M.pdf: 1661323 bytes, checksum: 10c00ac805ea800b6e675e368a997a62 (MD5) Previous issue date: 1988
Resumo: Um total de 88 amostras de s. aureus, das quais 44 isoladas da narina e pele de estudantes e outras 44 isoladas do ambiente hospitalar, foram testadas frente à estreptomicina, eritromicina, rifampicina, cloranfenicol, canamicina, netilmicina, oxacilina, cefalotina, amicacina, tetraciclina e penicilina; foram as amostras discriminadas em resistentes, intermediárias e sensíveis de acordo com o método de difusão com discos de Kirby-Bauer. Os resultados obtidos mostraram níveis de resistência relativamente altos, principalmente com relação à penicilinnas amostras de estudantes. Dentre os isolados, de S. aureus, verificou-se uma grande incidência de resistência múltipla, podendo-se presumir a presença de plasmídios; para isso, as amostras M5 e M9 foram tratadas pelo brometo de etídio com a finalidade de se observar a possível origem plasmidial dos genes de resistência
Abstract: A total of 88 samples of S. aureus, 44 of which isolated from the nostril and skin of students, and the remaining 44 samples isolated from a hospital site, were tested with kanamycin, streptomycin, erythromycin, rifampicin, chloran phenicol, netilmicin, oxacillin, cephalotine, amikacin, tetracy cline, and penicillin G, according to the difusion method with discs by Kirby-Bauer, the samples were identified as resistant, intermediary and susceptible. The data showed resistance levels relatively high, specially those related to penicillin in the student samples. Among the isolated S. aureus, a great incidence of multiple resistance was verified, leading us to presume, Lhe presence of plasmids; the samples M5 and M9were treated with ethidium bromide for this with the purpose of observing the possible plasmidial origin of resistance genes
Mestrado
Farmacologia
Mestre em Odontologia

Aquesta tesi proporciona dades sobre la prevalença de la resistència antibiòtica en Mycoplasma genitalium i suggereix la implementació de noves estratègies terapèutiques per fer front a les infeccions causades per aquest patogen de transmissió sexual. A través d’una anàlisi exhaustiva de la infecció en el nostre entorn, aquest estudi ofereix reflexions globals al voltant de la infecció per M. genitalium i els seus mecanismes de resistència antimicrobiana. El capítol 1 actualitza i resumeix l’evidència clínica i epidemiològica en relació a la infecció , destacant alhora alguns aspectes bàsics de la fisiologia i la patogènesi del bacteri. El capítol 2 “Antibiotic resistance: where are we now?” proporciona estimacions sobre la resistència a macròlids i fluoroquinolones en M. genitalium a Barcelona, Espanya, a través d’un estudi de cohorts realitzat entre l’any 2016 i el 2017. A més, el capítol revisa i descriu l’evolució regional i europea de la resistència antibiòtica en M. genitalium durant l’última dècada. D’altra banda, el capítol 3 “Mycoplasma genitalium: should we screen and how?” i el capítol 4 “Transmission dynamics in Mycoplasma genitalium” se centren en les infeccions asimptomàtiques, aprofundint en la prevalença de M. genitalium, així com en les resistències antibiòtiques en la població asimptomàtica, tot revelant la dinàmica de transmissió de la infecció. Finalment, el capítol 5 resumeix les principals conclusions de la tesi, culminant amb la proposta d’un nou algoritme terapèutic basat en els resultats i evidències obtinguts al llarg d’aquest treball.
Esta tesis proporciona las primeras estimaciones en relación a la resistencia antibiótica en Mycoplasma genitalium y sugiere la implementación de nuevas estrategias terapéuticas para hacer frente a las infecciones causadas por este patógeno de transmisión sexual. A través de un análisis exhaustivo de la infección en nuestro entorno, este estudio ofrece reflexiones globales en torno a la infección por M. genitalium y sus mecanismos de resistencia antimicrobiana. El capítulo 1 actualiza y resume la evidencia clínica y epidemiológica en relación a la infección, destacando también algunos aspectos básicos de la fisiología y patogénesis de la bacteria. El capítulo 2 “Antibiotic resistance: where are we now?” proporciona estimaciones sobre la resistencia a macrolidos y fluoroquinolonas en M. genitalium en Barcelona, España, a través de un estudio de cohortes realizado entre 2016 y 2017. Además, el capítulo revisa y describe la evolución regional y Europea de la resistencia antibiótica en M. genitalium durante la última década. Por otro lado, el capítulo 3 “Mycoplasma genitalium: should we screen and how?” y el capítulo 4 “Transmission dynamics in Mycoplasma genitalium” se centran en las infecciones asintomáticas, profundizando en la prevalencia de M. genitalium y las resistencias antibióticas en población asintomática, y revelando la dinámica de transmisión de la infección. Finalmente, el capítulo 5 resume las principales conclusiones de la tesis, culminando con la propuesta de un novedoso algoritmo terapéutico basado en los resultados y evidencias obtenidos a lo largo de este trabajo.
This thesis provides the first antibiotic resistance estimates in Mycoplasma genitalium in Spain and suggests the implementation of novel treatment strategies against infections caused by this sexually transmitted pathogen. This study offers, through a comprehensive analysis of the infection in our settings, global insights regarding M. genitalium infection and its antimicrobial resistance mechanisms. Chapter 1 updates and summarizes the clinical and epidemiological evidence regarding the infection, highlighting also some basic aspects of the physiology and pathogenesis of the bacterium. Chapter 2 "Antibiotic resistance: where are we now?" provides estimates regarding macrolide and fluoroquinolone resistance in M. genitalium in Barcelona, Spain, through a cohort study performed between 2016 and 2017. Additionally, the chapter reviews and describes the regional and European evolution of antibiotic resistance in M. genitalium in the last decade. On the other hand, chapter 3 "Mycoplasma genitalium: should we screen and how?" and chapter 4 "Transmission dynamics in Mycoplasma genitalium" are focused on asymptomatic infections, addressing the prevalence of M. genitalium and antimicrobial resistance among asymptomatic individuals, and revealing the transmission dynamics of the infection. Finally, chapter 5 summarizes the main conclusions of this thesis work, culminating with the proposal of a novel treatment algorithm based on the results and the evidence obtained along this manuscript.
Universitat Autònoma de Barcelona. Programa de Doctorat en Microbiologia

La mastitis bovina es la enfermedad de mayor impacto económico para la industria lechera. Siendo el Staphylococcus aureus uno de los principales agentes patógenos involucrados, algunas de estas cepas son resistentes a los antimicrobianos β-lactámicos haciendo más difícil el control y tratamiento de esta enfermedad ocasionando grandes pérdidas económicas para los productores. La Organización Mundial de la Salud ha declarado la resistencia a los antibióticos como una de las mayores amenazas a la salud humana, la seguridad alimentaria y el desarrollo. El objetivo de esta investigación fue evaluar el uso de aceites esenciales para la prevención y control de la mastitis bovina en Cundinamarca – Colombia mediante el desarrollo de tres fases: 1.Una fase preliminar para la obtención, caracterización de principales componentes y evaluación de la citotoxicidad de los aceites esenciales de Thymus vulgaris (L), Lippia origanoides (Mill) y Lippia citriodora (L'He'r.); 2. Una fase in vitro que evaluó la actividad antimicrobiana, la sinergia de la mezcla de estos aceites esenciales y determinó la concentración mínima bactericida del género Staphylococcus spp (N=80) y S. aureus Oxacilina- resistente (n=15) mediante la técnica de disco de Kirby-Bauer (Bernal & Guzman, 1984) y la técnica de micro dilución en caldo (CLSI, 1999). Y una tercera fase in vivo que contempló tres etapas: diagnóstico de los hatos lecheros, formulación de un sellador de barrera para bovinos a base de aceites esenciales y evaluación a través de dos estudios: un estudio preventivo (N=14 pezones) y un estudio control de mastitis (N=33 pezones) realizado en un total de 12 animales comparado frente a sellador a base de una solución yodófora comercial en el grupo de control. Para el análisis estadístico se calcularon los tamaño de la muestra con GRANMO 7.12. paquete estadístico IBM SPSS y se concluyó la diferencia significativa a un p<0.05. Los principales compuestos caracterizados son limoneno, neral y geranial, y el timol, responsables de la capacidad antimicrobiana. La citotoxicidad de estos aceites, determinada con el bioensayo de toxicidad de Artemia (Meyer, y otros, 1982) está en un rango CL50 10-19 µg/ml, comparando con la CL50 y dosis de otros agentes microbianos como Triclosan e Itroconazol, son poco tóxicos y seguros. En la evaluación de la actividad antimicrobiana, se concluye que existen diferencias significativas en la sensibilidad de las cepas de Staphylococcus spp. aisladas frente a los aceites esenciales de Lippia citriodora (AEC) y Thymus vulgaris (AET) mostrando una actividad antimicrobiana (p < 0.05), así como una sensibilidad de estas cepas frente al Cloranfenicol y una resistencia a la Clindamicina (p < 0.05). El Staphylococcus aureus presenta mayor resistencia frente a los antibióticos convencionales sin embargo mantiene la sensibilidad frene a los aceites esenciales de Lippia citriodora y Thymus vulgaris. Igualmente, se concluye que existen efecto sinérgico de la mezcla de aceites esenciales (75% AEC /25% AET) que potencia su actividad antimicrobiana optimizando su eficiencia terapéutica (p < 0.05). La concentración mínima bactericida (CBM) de la mezcla (75% AEC /25% AET) fue determinada en 2.5% efectiva para el 92.5 % de las muestras (n=80). En los estudios In vivo que evaluaron la prevención y control de la mastitis bovina se concluye con un nivel de confianza del 95%, que no existen diferencias significativas entre el grupo sellador a base de aceites esenciales y el sellador a base de la solución yodófora. Es decir, alcanza a ser igualmente efectivo que el producto comercial. Por lo anterior, se concluye que los aceites esenciales evaluados son capaces de inhibir el crecimiento de los microorganismos predominantes de la mastitis en bovinos y son una posible estrategia para su prevención y control.
Bovine mastitis is the disease with the most significant economic impact for the dairy industry. Being the Staphylococcus aureus one of the primary pathogens involved, some of these strains are resistant to β-lactam antimicrobials making it more difficult to control and treat this disease, causing high economic losses for producers. The World Health Organization has declared antibiotic resistance as one of the greatest threats to human health, food security, and development. The objective of this research was to evaluate the use of essential oils for the prevention and control of bovine mastitis in Cundinamarca - Colombia through the development of three phases: 1. A preliminary phase for obtaining, characterization of principal components and evaluation of cytotoxicity of the essential oils of Thymus vulgaris (L), Lippia origanoides (Mill) and Lippia citriodora (L'He'r.); 2. An in vitro phase that evaluated the antimicrobial activity, the synergy of the mixture of these essential oils and determined the minimum bactericidal concentration, compared to isolated strains of clinical cases of mastitis in the region, of the genus Staphylococcus spp (N = 80) and S. aureus Oxacillin-resistant (n = 15) through the Kirby-Bauer disc technique (Bernal & Guzman, 1984) and the broth micro dilution technique (CLSI, 1999). And a third phase in vivo that contemplated three stages: diagnosis of dairy herds, formulation of a barrier sealant for bovines based on essential oils and evaluation through two studies: a preventive study (N = 14 nipples) and a study control (N = 33 nipples) performed on a total of 12 animals with control groups treated with sealant based on a commercial iodophor solution. For the statistical analysis, the sample size was calculated with GRANMO 7.12, and the IBM SPSS statistical package, and the significant difference was concluded at p <0.05. The main compounds characterized according to the analytical method for the characterization of essential oils, gas chromatography, and mass spectrometry, are limonene, neral, and geranial, and thymol, responsible for antimicrobial capacity. The cytotoxicity of these oils, determined with the Artemia toxicity bioassay (Meyer, et al., 1982) is in a 10-19 µg / ml LC50 range, compared with the LC50 and doses of other microbial agents such as Triclosan and Itraconazole, are less toxic and safe. On the other hand, in the evaluation of antimicrobial activity, it is concluded that there are significant differences in the sensitivity of Staphylococcus spp. isolated against the essential oils of Lippia citriodora (AEC) and Thymus vulgaris (AET) showing an antimicrobial activity (p <0.05), as well as a sensitivity of these strains against Chloramphenicol and resistance to Clindamycin (p <0.05). Staphylococcus aureus exhibits greater resistance against conventional antibiotics, great it maintains sensitivity to the essential oils of Lippia citriodora and Thymus vulgaris. Likewise, it is concluded that there is a synergistic effect of the mixture of essential oils (75% AEC / 25% AET) that enhances its antimicrobial activity optimizing its therapeutic efficiency (p <0.05). The minimum bactericidal concentration (CBM) of the mixture (75% AEC / 25% AET) was determined in 2.5% effective for 92.5% of the samples (n = 80). In the In vivo studies that evaluated the prevention and control of bovine mastitis, it is concluded with a 95% confidence level, that there are no significant differences between the sealant group based on essential oils and the sealer based on the iodophor solution. That is, it becomes equally effective as the commercial product. Therefore, it is concluded that the essential oils evaluated are capable of inhibiting the growth of the more relevant microorganisms of mastitis in cattle and are a possible strategy for their prevention and control.

Smittsamma sjukdomar kostar det svenska samhället enorma summor varje år. Behandlingen av smittade patienter har tidigare uppskattats till 5-10 miljarder svenska kronor årligen. Vidare estimeras de förebyggande åtgärderna kosta samhället runt en miljard svenska kronor. Detta betyder att det finns en ekonomisk drivkraft för att reducera antalet smittade patienter inom vården, speciellt de fall som är orsakade av resistenta bakterier. Samtidigt pågår det en debatt om resistenta bakterier och antibiotikaförbrukningen i både forskning och media. Resistenta bakterier kan bli ett hot mot vår framtid om vi inte minskar antibiotikaförbrukningen och vidtar åtgärder för att förhindra smittspridning. Om antalet personer som blir smittade av resistenta bakterier kan reduceras minskar även antibiotikaförbrukningen som i sin tur leder till att färre bakterier utvecklar ett resistensmönster för antibiotika. Detta betyder att det är viktigt att studera och effektivisera hanteringen av smittsamma patienter.   För att reducera antalet smittade patienter måste förebyggande åtgärder vidtas och smittkällan måste kartläggas vid ett upptäckt fall av en smitta. Svensk sjukvård arbetar idag aktivt med smitthantering. Detta begrepp omfattar upptäckt, kontroll och spårning av smitta. Uppdragsgivaren till detta examensarbete, Cambio Healthcare Systems, saknar en fullständig bild över hur smitthanteringen egentligen går till på ett sjukhus. Deras målsättning är att utveckla ett IT-system som kan underlätta smitthanteringsprocessen. Denna studie syftar till att kartlägga informationsflödet vid smitthanteringsprocessen och identifiera de inblandade aktörernas ansvarsområden och skyldigheter enligt regelverket. Vidare syftar arbetet till att presentera åtgärdsförslag som kan minska de identifierade riskerna och effektivisera smitthanteringsprocessen av resistenta bakterier.   För att kartlägga smitthanteringsprocessen genomfördes en fallstudie av ett svenskt universitetssjukhus under våren 2013. Aktörer som studerades var det mikrobiologiska laboratoriet, vårdhygien, smittskyddsenheten, smittskyddsinstitutet samt läkare och sjuksköterskor vid två avdelningar på sjukhuset. Datainsamlingen består av intervjuer, observationer och dokument.   Resultatet av fallstudien visade att smitthanteringsprocessen är ett komplext system med ett omfattande informationsflöde. Huvudaktörerna är vårdhygien, sjukvårdspersonalen och det mikrobiologiska laboratoriet. De är viktiga eftersom deras praktiska handlande är avgörande för att smitthanteringen genomförs. Smittskyddsenheten är inblandad till viss del, men tillhör inte huvudaktörerna. Studien visade även att smittskyddsinstitutet inte hade någon framträdande roll i smitthanteringsprocessen av resistenta bakterier på sjukhuset.   Ansvarsfördelningen är till viss del styrd av smittskyddslagen och enligt denna lag har den behandlande läkaren en central roll i processen. I verkligheten är läkarens roll mindre framträdande vid smittspårningen, vanligtvis delegerar läkaren uppgifter till sjuksköterskor eller till vårdhygien. Mycket av kommunikationen mellan aktörerna är muntlig och detta innebär att ett flertal risker kan uppstå. Vid identifieringen av risker för hela processen konstaterades det att de flesta risker kan uppstå på grund av den mänskliga faktorn, ofta i kombination med användandet av ett otillräckligt datasystem. Åtgärdsförslagen för att effektivisera processen fokuserar därför på att minimera de identifierade riskerna med hjälp av framtida IT-system.   Slutsatsen av studien är att det finns ett stort behov av IT-lösningar för att effektivisera smitthanteringsprocessen av resistenta bakterier. Min rekommendation är att Cambio Healthcare Systems AB bör fokusera på att utveckla ett system för att digitalisera arkiveringen av beläggningslistorna och spåra patientflöden tillbaka i tiden i Cosmic då detta är ett starkt önskemål från kunderna. En annan viktig åtgärd är att utveckla smittspecifika checklistor som visas på datorn i samband med att läkaren får ett positivt provsvar. Slutligen rekommenderar jag Cambio Healthcare Systems AB att utveckla ett smittlarm som kan integreras med deras befintliga whiteboardtavla som nyligen lanserades.
Infectious diseases are a major cost item for the Swedish society. The treatment of infected patients has previously been estimated to 5-10 billion SEK annually and preventive actions cost the Swedish society around one billion SEK every year. Therefore, there are strong economic incentives to reduce the number of infected patients in care, particularly cases caused by resistant bacteria. There is an ongoing debate in both media and research about bacterial resistance and antibiotic consumption. Resistant bacteria can be a threat to our future if we do not reduce the consumption of antibiotics and take measure against infection spreading. If it is possible to reduce the number of resistant bacteria infected patients in the future it enables a decline in antibiotic consumption. This in turn leads to a decreased quantity of bacteria that is able to develop a resistance pattern to antibiotic. Thus, it is highly motivated to study and streamline the process of infection control.   Preventive measures must be taken and the source of the infection must be identified in order to reduce the number of infected patients. The Swedish health care sector is currently working actively with infection control. The concept of infection control encloses the detection, the control and the tracing of the infection. The requestor of this master thesis, Cambio Healthcare Systems AB, does not have a complete picture of the process of the infection control. Their goal is to develop an IT system to facilitate the process of infection control. This study aims to map the information flow of the process and to identify the involved actors’ field of responsibility and obligations according to the law. Further, this thesis aims to present action proposals that can reduce the identified risks and streamline the infection control of resistant bacteria.   A case study of a Swedish university hospital was performed in the spring of 2013 in order to map the process of infection control. The investigated actors were the microbiological laboratory, the local health protection unit (Vårdhygien), the unit of infection control at a regional level (Smittskyddsenheten), the Swedish Institute of Infectious Disease Control (Smittskyddsinstitutet) and physicians and nurses at two hospital departments. The data collection consists of interviews, observations and documents.   The result of this case study shows that the process of infection control is a complex system with an extensive flow of information. The main actors are the local health protection unit, the microbiological laboratory and the medical staff. Their practical actions are essential for the process of infection control. The unit of infection control at a regional level is involved to some extent, but does not belong to the main actors. Furthermore, the study showed that the Swedish Institute of Infectious Disease Control does not have a prominent role in the process at the hospital.   The division of responsibilities is to some extent controlled by the law. According to the law, the physician in charge has a central role in the process of infection control. However, the physician’s role in reality is less prominent. Usually, the physician delegates the tasks to the other actors such as nurses or to the local health protection unit. The communication between the actors is mainly oral and this can cause risks. Most of the identified risks occurred due to human error, often in combination with use of an insufficient IT-system. Therefore, the proposed actions to streamline the process focus on minimizing the identified risks with help of future IT solutions.   The conclusion of this study is that there is a strong demand for IT solutions to streamline the process of infection control of resistant bacteria. My recommendation is that Cambio Healthcare Systems AB should focus on developing a system to digitalize the archiving of the occupancy lists, which also enables tracing the flow of patients back in time. This is a request from several health care professionals. Another important proposed action is to develop a checklist that is specific for every infection disease. Simultaneously as the physician receives the positive test results, this checklist will appear on the physician’s screen. Finally, I recommend Cambio Healthcare Systems AB to develop an alarm to infection diseases that can be integrated with their existing whiteboards that were recently introduced to the market.

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Staphylococcus aureus juntamente com outras espécies de estafilococos coagulase-negativa são importantes patógenos responsáveis por infecções nosocomiais associadas ao uso de dispositivos implantáveis. O fator mais importante na patogênese de infecções estafilocócicas associadas a estes dispositivos é a habilidade do patógeno de formar biofilme, que confere proteção contra o sistema imunológico do hospedeiro e da ação de antimicrobianos, sendo o Polissacarídeo de Adesão Intercelular (PIA) codificado pelo operon icaADBC o principal componente do biofilme estafilocócico. Esse estudo objetivou estudar a estrutura do biofilme de diferentes espécies de Staphylococcus, avaliar a antibioticoterapia utilizada para tratamento dessas infecções em células livres e em biofilme e alternativas para prevenção da formação de biofilme. Foram estudadas 200 amostras de Staphylococcus spp. sendo 50 da espécie S. aureus e 150 do grupo dos estafilococos coagulase-negativa (ECN), incluindo 50 amostras de S. epidermidis, 20 S. haemolyticus, 20 S. warneri, 20 S. hominis, 20 S. lugdunensis e 20 amostras de S. saprophyticus isoladas de pacientes do Hospital das Clínicas (HC) da Faculdade de Medicina de Botucatu (FMB). As amostras foram submetidas à pesquisa dos genes icaADBC pela reação em cadeia da polimerase (PCR) e à expressão pela técnica de Transcriptase Reversa-PCR (RT-PCR). A produção de biofilme foi verificada através dos métodos fenotípicos de aderência ao tubo de borossilicato e na placa de poliestireno. A determinação da concentração inibitória mínima em células planctônicas e em biofilme foi testada para as drogas oxacilina, vancomicina, eritromicina, gentamicina, linezolida e sulfametoxazol-trimetropim pelo método de microdiluição em caldo. O teste do peptídeo (RIP) na prevenção da formação de biofilme foi realizado com cultura de bactérias em TSB glicose 2% com pontas de cateteres e ...
Staphylococcus aureus, together with other coagulase-negative staphylococci (CoNS), is an important pathogen that causes nosocomial infections associated with the use of implantable devices. The most important factor in the pathogenesis of staphylococcal infections associated with these devices is the ability of the pathogen to form a biofilm, which protects bacteria against the host immune system and against the action of antimicrobial drugs. The main component of staphylococcal biofilms is polysaccharide intercellular adhesin (PIA), which is encoded by the icaADBC operon. The objectives of this study were to investigate the structure of biofilms of different Staphylococcus species, to evaluate the effect of antibiotics used to treat these infections on planktonic and biofilm cels, and to identify alternatives for the prevention of biofilm formation. A total of 200 Staphylococcus spp., including 50 S. aureus and 150 CoNS strains (50 S. epidermidis, 20 S. haemolyticus, 20 S. warneri, 20 S. hominis, 20 S. lugdunensis and 20 S. saprophyticus), isolated from patients seen at the University Hospital of the Botucatu School of Medicine (HC-FMB), were studied. The presence of the icaADBC genes was investigated by the polymerase chain reaction (PCR) and their expression was determined by reverse transcriptase-PCR (RT-PCR). Biofilm formation was evaluated using the phenotypic method of adherence to borosilicate tubes and polystyrene plates. The minimum inhibitory concentration (MIC) of oxacillin, vancomycin, erythromycin, gentamicin, linezolid and sulfamethoxazole-trimethoprim for planktonic and biofilm cells was determined by the broth microdilution method. The effect of RNA-inhibiting peptide (RIP) on the prevention of biofilm formation was tested using bacterial cultures grown in TSB-2% glucose containing catheter tips and visualization by scanning electron microscopy and on polystyrene plates. The icaA gene was detected by PCR in 97 (48.5%) ...
FAPESP: 11/07285-5

Introdution: Infections are one of the most important causes of morbility and mortality in the intensive care units all around the world. The indiscriminate use, often not appropriate, of the antibiotics, led to the development of the multidrug resistant species. In the last years there was a large spread of multidrug resistant Klebsiella Pneumoniae Carbapenemase-producing (KPC), that, due to the small therapeutic option and therefore its rapid diffusion, it is becoming a serious health problem. Aim of the study: First aim of the study has been to understand if the KPC infection could be able to aggravate the outcome of the patients admitted in intensive care units. Second aim was to understand if in the intensive car units there are correct strategies of prevention, if it exists a peculiar type of patient more sensitive to the infection, and to understand the real efficacy of the antibiotic therapy used. Materials and Methods: A prospective observational study that has included all the patients admitted to intensive care units, with at list one biological positive sample for KPC, from January 2013 to October 2015. General features of patients as well as data about comorbidity, trend of inflammation indexes, antibiotic therapy and mortality rate was recorded. Results: Of the 109 patient analyzed, 64% of cases came from surgical units. The simple positive in a swab of KPC does not cause increase in inflammatory markers, and the same we observed after infection confirmed by blood colture. Seventeen percent of the patients have 3 or more comorbidities and, of which, 60% died with a relative risk of death of 4,6. Twenty five percent of the patients had infection of KPC confirmed by blood culture, of which 54,5% died with a relative risk of death of 4,1. The association of three or more comorbidities and infection for KPC clinically documented increase the relative risk of death until 13,6. Conclusions: Among patients afferent to the intensive care unit, the most sensitive to the infection of KPC seem the patients from surgery unit. The only colonization by KPC does not seem to be able to worse the outcome, while the clinically documented infection confirmed by blood culture appears to increase the relative risk of death, even more if associated with a number of comorbidities greater than or equal to three.
Presupposti dello studio: Le infezioni sono una delle principali cause di morbilità e mortalità nei reparti di terapia intensiva di tutto il mondo. L’abuso e l’uso spesso inappropriato degli antibiotici ha portato nel corso degli anni allo sviluppo di specie antibiotico-resistenti. Negli ultimi anni c’è stata grande diffusione della Klebsiella Pneumoniae produttrice di carbapenemasi (KPC) multiresistente agli antibiotici che, proprio per le ridotte opzioni terapeutiche e la rapida capacità di colonizzazione ed infezione, desta sempre maggiore preoccupazione. Scopo dello studio: Obiettivo primario del nostro studio è stato capire se l’infezione da KPC sia effettivamente in grado da sola di peggiorare l'outcome dei nostri pazienti fino a determinarne il decesso. Obiettivi secondari: valutare se nelle terapie intensive in esame vengono messe in atto le strategie di prevenzione raccomandate, indagare se esiste una tipologia di pazienti più suscettibili all'infezione, capire se la terapia antibiotica messa in atto sia realmente efficace. Materiali e metodi Studio osservazione prospettico che ha incluso tutti i pazienti che all'ingresso presso le unità di terapia intensiva prese in esame o durante il ricovero nelle stesse abbiano avuto almeno un campione biologico positivo per KPC, nel periodo di tempo da Gennaio 2013 a Ottobre 2015. Di questi pazienti sono state registrate variabili individuali, esami ematochimici, indicatori di flogosi e condotta terapeutica. E’ stato inoltre calcolato il rischio relativo di mortalità in base alla presenza di 3 o più comorbidità, in base all'infezione confermata all'emocoltura da KPC e il rischio relativo di morte delle due precedenti variabili aggregate. Risultati Dei 109 casi analizzati, il 64% dei pazienti erano provenienti da reparti chirurgici. In seguito al riscontro di positività per KPC al tampone, non si evidenzia un aumento significativo dei globuli bianchi né degli indici di flogosi nei nostri pazienti. Anche dopo lo sviluppo di una emocultura positiva per KPC, non è stato osservato un aumento significativo dei globuli bianchi né degli indici di flogosi. Quindici (17%) Pazienti presentavano 3 o più comorbidità, e di questi il 9 (60%) sono deceduti, con un rischio relativo di decesso di 4.6. Ventidue (25%) Pazienti hanno sviluppato un'emocoltura positiva per KPC, e di questi 12 (54.5%) sono deceduti, con un rischio relativo di decesso di 4.1. Nel caso di pazienti con comorbidità maggiori o uguali a 3 e emocoltura positiva il rischio relativo di decesso è 13.6. Conclusione/discussione Dei Pazienti che afferiscono alle terapie intensive, quelli più suscettibili di infezione da KPC sembrano essere quelli provenienti dai reparti chirurgici. La colonizzazione da sola non sembra in grado di peggiorare l’outcome mentre l’infezione clinicamente documentata da emocoltura positiva appare aumentare il rischio di decesso, tato più se associata a un numero di comorbidità maggiore o uguale a tre.

L’evoluzione delle specie batteriche è un processo guidato da un rapido adattamento alle mutevoli, e spesso avverse, condizioni ambientali. Lo scambio di materiale genetico attraverso meccanismi di trasferimento genico orizzontale, unito alla plasticità intrinseca del genoma batterico, gioca un ruolo fondamentale nell’evoluzione di microorganismi con potenziate capacità di adattamento ad habitat ostili. In questo processo, un ruolo centrale è ricoperto dall’arsenale di agenti antimicrobici (antibiotici, antivirali etc.) utilizzati in vari settori (contesto clinico, agricolo etc.) per trattare le malattie infettive. L’abuso e l’uso improprio di questi farmaci guida l’evoluzione e selezione di microorganismi in grado di sopravvivere al trattamento con agenti antimicrobici precedentemente risultati efficaci per debellare l’infezione da essi provocata. Questo fenomeno si definisce Resistenza Antimicrobica (AMR). Il dato più allarmante riguarda la rapida diffusione della resistenza antibiotica che, giorno dopo giorno, erode l’efficacia degli antibiotici attualmente disponibili e minaccia la capacità di trattare efficacemente infezioni potenzialmente letali. Questo scenario porta alla luce la necessità urgente di sviluppare terapie innovative capaci di sostituire o affiancare l’uso degli antibiotici. In questo contesto, la Biologia Sintetica può dare un contributo significativo. Ad esempio, per trattare un’infezione batterica localizzata, un biologo sintetico può riprogrammare razionalmente un microrganismo affinchè rilasci in situ una molecola battericida alternativa ad un antibiotico che agisce selettivamente sulla popolazione di patogeni da debellare. Questo programma genetico può essere codificato all’interno di un circuito sintetico sfruttando una collezione di parti biologiche e l’elevata versatilità della tecnologia CRISPR. Quest’ultima può essere sfruttata per progettare degli agenti antimicrobici selettivi in grado di riconoscere una sequenza distintiva nel genoma del batterio bersaglio. Disegnando opportunamente la sequenza di una guida a RNA è infatti possibile pilotare il taglio della nucleasi Cas9 sui geni di resistenza antibiotica: la degradazione del DNA bersaglio si risolve poi nella morte cellulare o nel ripristino della sensibilità antibiotica. Sebbene questo approccio sia già stato esplorato con promettenti risultati da diversi gruppi di ricerca, almeno due questioni fondamentali rimangono ancora aperte: il rischio di generare nuove varianti dei geni di resistenza nelle cellule che sfuggono alla morte tramite la riparazione del DNA danneggiato, e la necessità di sviluppare una piattaforma in grado di veicolare efficacemente la circuiteria CRISPR nei batteri resistenti. Entrambe queste sfide sono state affrontate nel progetto di ricerca presentato in questa tesi. In primo luogo, per aggirare i rischi connessi al taglio del DNA nelle cellule target, è stata sviluppata una circuiteria sintetica basata sulla tecnologia CRISPRi, la quale sfrutta la capacità della proteina dCas9 di silenziare l’espressione di un gene target senza tuttavia comprometterne la sequenza nucleotidica. L’efficienza di repressione della circuiteria CRISPRi è stata caratterizzata in due casi di studi: inibizione trascrizionale di resistenze antibiotiche modello e ad alto impatto clinico. Successivamente, una piattaforma di trasferimento genico basata sulla coniugazione batterica è stata sviluppata per veicolare il circuito CRISPRi nei batteri resistenti. Infine, è stato implementato un nuovo modello matematico per simulare l’effetto di una terapia che impiega il sistema CRISPRi, mettendo a confronto diversi scenari riguardanti il meccanismo di inibizione e il trasferimento della circuiteria, al fine di predire in silico la strategia terapeutica più efficace da utilizzare in vivo.
Bacterial evolution is driven by rapid adaptation to changing environments where adverse conditions must be faced. The horizontal exchange of genetic information, along with the inherent bacterial genome plasticity, are key players in the evolution of microbial populations with increased tolerance towards challenging conditions, which also include the selective pressure exerted by physical or chemical agents. A central role in microbial adaptation is exerted by the arsenal of antimicrobial agents (antibiotics, antivirals, anti- fungals etc.) used in different settings (from clinical to agriculture sector) to threat or prevent infectious diseases. The abuse and misuse of these medicines drive the evolution and selection of microbes able to survive exposure to an antimicrobial agent that was originally effective to kill the cell or arrest its growth. This phenomenon is defined as Antimicrobial Resistance (AMR). Of a great concern is especially the spread of antibiotic resistance which, day by day, erodes the efficacy of available antibiotics and compromises our ability to cure life-threatening infections caused by multidrug-resistant (MDR) pathogens. This scenario poses an urgent need for new strategies to counteract AMR. With this regard, Synthetic Biology may significantly contribute to the development of non-traditional therapies able to supplant or accompanying antibiotics use. In particular, by rewriting the genetic program of a cell, synthetic biologists aim at designing sophisticated living systems able to carry out a defined task in a reliable and predictable manner. For instance, to treat a localized AMR-associated infection, a microorganism can be rationally programmed to act as a vehicle for the in situ delivery of an antimicrobial agent different from an antibiotic and able to selectively kill resistant bacteria. This genetic program can be encoded in a synthetic circuit by leveraging a collection of biological regulatory parts and the strong programmable nature of a genetic tool named CRISPR technology. The latter can be exploited to design sequence-specific antimicrobials as a guide RNA sequence can be ad hoc designed to drive the cleavage of Cas9 nuclease towards target genes encoding for resistance determinants. In target cells, this event results in bacterial death or re-sensitization to antibiotic therapy. Although this approach has already been explored by several research groups with promising results, at least two major hurdles still have to be faced: the risk of generating new variants of resistance genes in escaper cells that have survived CRISPR targeting by repairing the DNA damage, and the need to develop a robust delivery strategy to mobilize in vivo the synthetic circuit in target bacteria. Both challenges were addressed with the research work presented in this thesis. First, to avoid the threatening consequences of Cas9 cleavage, a synthetic circuitry based on CRISPRi technology was developed as it relies on the ability of dCas9 protein to inhibit the expression of target genes without damaging the relative nucleotide sequence. This is expected to exert re-sensitization of a target pathogen population. In particular, the CRISPRi circuitry was characterized in terms of repression efficiency and multi-targeting capability in two case studies: transcriptional inhibition of model- and clinically-relevant resistance genes. Second, a delivery platform based on bacterial conjugation was exploited to mobilize the CRISPRi circuitry in target resistant bacteria. Finally, a mathematical model was implemented with the purpose to simulate the effect of a CRISPRi-based therapy on AMR pathogens and to compare different biological scenarios including the targeting and the delivery mechanisms, and eventually gaining insight into the best therapeutic strategies for in vivo use.

Orientador: Thales Rocha de Mattos Filho
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Deter a contaminação nos consultórios dentários tem sido uma tarefa muito difícil. Na maioria das vezes os microrganismos tem vencido as medidas de segurança adotadas, expondo profissionais e pacientes à situações de risco. Em escolas de Odontologia, esse risco é ainda maior, pois há dificuldades na implementação de procedimentos de controle de infecção. Para identificar os microrganismos do ambiente clínico de uma Faculdade de Odontologia e avaliar a resistência antimicrobiana desses, foi desenvolvida esta pesquisa. Para isso utilizou se 60 placas de petri (15 cm de diâmetro), contendo ágar-soja-triptecaseína, dispostas em diversos locais da clínica (box, corredor, sala de esterilização e plantão de urgência) e em três situações: antes, durante e após a atividade. As placas foram abertas (120 segundos) e então fechadas e levadas à estufa (pressão de CO2 a 10% à 37ºC), durante 48 horas, e então transferidas para uma estufa em aerobiose (37ºC), por mais 24 horas. As unidades formadoras de colônias que cresceram foram contadas e fotografadas, e para a identificação utilizou-se a técnica de coloração de Gram. A resistência foi avaliada em placas de petri contendo ágar Muller Hinton, acrescido de 1,5°/0 de sangue de carneiro estéril previamente inoculadas com 100 µL dos microrganismos, onde foram inseridos discos de papel de 6,35mm contendo antibióticos, e os halos de inibição, que se desenvolveram após incubação, foram medidos. A análise estatística (KrusKall Wallis, 5%) mostrou diferença significante durante as diversas situações clínicas. Observou-se ainda que, de todos os microrganismos, 26% foram resistentes à penicilina G 10UI, 18% à ampicilina 10g, 19% à amoxicilina 10µg, 6% à amoxicilina 10µg + ácido clavulânico 20µg, 7% ao cefadroxil 30µg, 27% à eritromicina 15µg, 25% à daritromicina 15µg, 27% à azitromicina 15µg, 14% à clindamicina 2µg e 3% ao cloranfenicol 30µg. Condui-se que: 1) Durante atividade clínica há um maior crescimento de microrganismos, prevalecendo cocos, bacilos e fungos respectivamente; 2) A resistência encontrada foi maior para os antibióticos do 9rupo dos macrolídeos (26,4%), seguida pelo grupo das penicilinas (21,1%), sendo que a amoxicilina associada ao ácido clavulânico e o doranfenicol apresentaram as menores resistências, 6,47% e 2,8%, respectivamente
Abstract: It has been a very hard task to stop contamination in dental clínies. Most of the times, microorganisms have beaten the preventive measures used exposing health practitioners and patients to risk situations. ln Dental Schools this risk becomes even higher, for three are difficulties implementing procedures of infection control. This research was carried out in order to identify the microorganisms in the dental clínica I environment of a Faculty of Dentistry, and also to evaluate their resistance. For this purpose, 60 petri dishes (15 cm of diameter) with tripticasein soy agar were placed on different places in the clinic environment (between the dental clínies, on the corridors, sterilization room and emergency room) in three different situations: before, during and after the clinical work. The dishes were opened during 120 seconds, and then closed and incubated (at 10% CO2 pression) at 37°C, during 48 hours, and after moved to a stove at 37°C for 24 hours. The colony formation units (du) that grew on the dishes were counted and photographed. The Gram coloring technique for identification was applied. The resistance was evaluated on petri dishes with Muller Hinton agar plus 1.5% sheep blood sterile, previously inoculated with 100µg of microorganisms, there paper disks of 6.35 mm with antibiotics were placed. After incubation, the halos, which developed, were measured. The statistical analysis (KrusKaIl-Wallis 5%) showed significant difference during the various clinical situations. It was observed that 26% of the microorganisms were resistant to penicillin G [10Ul], 18% to ampicillin [10µg], 6% to amoxycillin [10µg] associated with clavulanic acid [20µg], 7% to cefadroxil [30µg], 27% to erythromycin [15µg], 27% to azithromycin [15µg], 250% to clarithromycin [15µg], 14% to clyndamicin [2µg] and 3% to cloranphenicol [30µg]. Therefore, it was concluded that: a) During clinical work there is a higher growth of microorganisms, being the most predominant cocci, bacillus and fungus, respectively; b) The higher resistance found was to the antibiotics from the macrolides group (26%), followed by the penicillin group (21%), having amoxycillin associated with clavulanic acid and doranphenicol, showed the lowest resistance
Mestrado
Mestre em Odontologia

Detta arbete har utförts som en litteraturstudie med artiklar funna dels i PubMed, dels på två farmaceutiska företags hemsidor. Syftet med arbetet var att undersöka antimikrobiella peptider som nya potentiella alternativ till traditionella antibiotika mot meticillinresistent Staphylococcus aureus (MRSA), med fokus på substanserna NZ2114, Agplectasin, Brilacidin/PMX-30063, PXL-150, Lytixar/LTX-109 och NAI-107. Antimikrobiella peptider är små katjoniska molekyler som ingår i det naturliga immunförsvaret hos i princip alla livsformer. De är kända för att ha en hög effekt och ett brett antibiotiskt spektrum, och har därför blivit föremål för omfattande forskning med förhoppning om att kunna omvandla dessa peptider till läkemedel. Samtliga i arbetet granskade substanser uppvisar god effekt mot S aureus, såväl meticillinkänslig som –resistent. De visar på goda möjligheter att utvinna och utveckla verksamma och effektiva molekyler, såväl från naturliga peptider som syntetiserade. Då peptiderna är en utmaning att omvandla till läkemedel med hänsyn till produktionskostnader och stabilitet kan det dröja innan ett läkemedel för systemiska infektioner finns på marknaden. För lokalt bruk, till exempel för sårinfektioner och nasal eradikering, ser framtiden mer lovande ut. Kanske kommer antimikrobiella peptider aldrig att bli förstahandsbehandling, men det borde vara möjligt att utnyttja dem till förebyggande och/eller kompletterande behandling.

A tuberculose (TB) é uma das 10 principais causas de morte no mundo, e o surgimento de estirpes de Mycobacterium tuberculosis multirresistente (MDR) e extensivamente resistente a medicamentos (XDR) é um grande problema de saúde pública. O uso de antibióticos que normalmente não são incluídos no tratamento da tuberculose tem sido considerado, e estudos recentes destacaram o uso de β-lactâmicos, como os carbapenemos, no tratamento da MDR-TB. Os carbapenemos são uma subclasse dos β-lactâmicos, que têm como alvo a biossíntese do peptidoglicano e que são particularmente resistentes à inativação pela β-lactamase BlaC, produzida por M. tuberculosis. As micobactérias têm um envelope celular característico, constituído por uma camada de ácidos micólicos de cadeia longa, um polissacárido de arabinogalactano altamente ramificado e uma malha de peptidoglicano reticulada e modificada. Essa barreira contribui para a virulência, persistência e resistência intrínseca das micobactérias a vários antibióticos e modula a resposta imune do hospedeiro. O objetivo desta tese foi estudar como a exposição à isoniazida e ao etambutol - antibióticos que inibem a síntese de ácidos micólicos e arabinogalactano - leva ao aumento da acessibilidade do peptidoglicano aos antibióticos que inibem a sua biossíntese, os β-lactâmicos. Para este fim, foram determinadas concentrações mínimas inibitórias e bactericidas (MIC e MBC) de β-lactâmicos (amoxicilina, cefotaxima, meropenem e imipenem), isoniazida e etambutol para quatro espécies de micobactérias (Mycobacterium smegmatis, Mycobacterium fortuitum, Mycobacterium bovis e Mycobacterium tuberculosis). Todas as espécies mostraram-se suscetíveis a pelo menos um dos β-lactâmicos testados, com melhor eficácia registada para o meropenem, e o clavulanato – um inibidor de β-lactamases – foi essencial para o aumento da atividade dos β-lactâmicos. Adicionalmente, verificámos se a exposição de micobactérias à isoniazida ou ao etambutol em duas sub-MIC (½ MIC e ¼ MIC) e subsequente e/ou simultânea exposição a β-lactâmicos poderia melhorar a sua eficácia. Notavelmente, o etambutol teve um efeito potencializador da atividade dos β-lactâmicos, com as MICs da amoxicilina e do meropenem significativamente mais baixas quando combinados com etambutol e clavulanato, alterando frequentemente a classificação das bactérias de resistente para suscetível. A combinação com isoniazida também foi benéfica, mas as condições devem ser otimizadas, especialmente para as micobactérias de crescimento lento. Este resultado sugere que o tratamento com sub-MICs de isoniazida e etambutol interfere na biossíntese correta dos componentes do envelope celular externo, deixando o peptidoglicano mais acessível para a atividade dos β-lactâmicos. Para quantificar a exposição do peptidoglicano micobacteriano após tratamento com isoniazida ou etambutol, foram realizados ensaios de co-precipitação com recetores de Drosophila que reconhecem o peptidoglicano, proteínas de reconhecimento de peptidoglicano (PGRPs). A microscopia de fluorescência corroborou os resultados dos ensaios sinergísticos de antibióticos, mostrando que o peptidoglicano micobacteriano apenas é reconhecido pelas PGRPs após a exposição a sub-MICs de isoniazida e etambutol. Os resultados apresentados são preliminares e os ensaios estão a ser otimizados para serem realizados com isolados clínicos de M. tuberculosis, incluindo estirpes MDR e XDR-TB, no futuro próximo. Este trabalho ajudará a estabelecer uma conexão entre mecanismos desconhecidos de resistência a medicamentos anti-TB e possíveis vulnerabilidades no envelope celular de micobactérias, que poderão ser exploradas para fins terapêuticos.
Tuberculosis (TB) is one of the top 10 causes of death worldwide, and the emergence of multi (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis strains is a major public health concern. The potential use of antibiotics that are not usually included in tuberculosis treatment is currently being considered and recent studies have highlighted the potential use of β-lactams such as carbapenems to treat MDR-TB. Carbapenems are a subclass of β-lactam antibiotics, which target peptidoglycan biosynthesis, that are particularly resistant to inactivation by the BlaC a β-lactamase, produced by Mycobacterium tuberculosis. Mycobacteria have a characteristic cell envelope, consisting of a long chain mycolic acids layer, a highly branched arabinogalactan polysaccharide and a very cross linked and modified meshwork of peptidoglycan. This barrier contributes to the virulence, persistence and intrinsic resistance of mycobacteria to several drugs, and modulates host-pathogen immune response. The aim of this thesis was to study how exposure to isoniazid and ethambutol – antibiotics that inhibit the synthesis of mycolic acids and arabinogalactan – lead to increased accessibility of peptidoglycan to antibiotics that target its biosynthesis, the β-lactams. To address this, minimum inhibitory and bactericidal concentrations (MIC and MBC) of β-lactams (amoxicillin, cefotaxime, meropenem and imipenem), isoniazid and ethambutol were determined for four different mycobacteria species (Mycobacterium smegmatis, Mycobacterium fortuitum, Mycobacterium bovis and Mycobacterium tuberculosis). All four species were susceptible to at least one of the β-lactams tested, with better efficacy registered for meropenem, and clavulanate – a β-lactamase inhibitor – was essential for enhancement of β-lactams activity. Additionally, we tested if exposure of mycobacteria to isoniazid or ethambutol in two sub-MIC (½ MIC and ¼ MIC) and subsequent and/or simultaneous exposure to β-lactams could improve its efficacy. It was notable that ethambutol had an enhancing effect over the activity of β-lactams, with amoxicillin and meropenem MIC being significantly lower when combined with ethambutol and clavulanate, frequently changing the bacteria classification from resistant to susceptible. Isoniazid was also advantageous, but further work must be done, especially for slow growing mycobacteria. These data suggests that treatment with sub-MICs of isoniazid and ethambutol halters the proper biosynthesis of outer cell envelope components, leaving peptidoglycan more accessible for β-lactams activity. In order to confirm the exposure of the mycobacterial peptidoglycan after treatment with isoniazid or ethambutol, co-precipitation assays were done with Drosophila receptors that specifically recognize peptidoglycan, peptidoglycan recognition proteins (PGRPs). Fluorescence microscopy corroborated the results from antibiotics synergistic assays, showing that mycobacterial peptidoglycan was only recognized by PGRPs after exposure to sub-MIC of isoniazid and ethambutol. The results presented here are preliminary and assays are being optimized to be tested in with clinical isolates of M. tuberculosis, including MDR and XDR-TB strains, in the near future. This work will help to establish a connection between unknown mechanisms of resistance to anti-TB drugs and potential vulnerabilities in the cell envelope of mycobacteria, which could be further exploited for therapeutic purposes.

Normally present in water, soil and waste water, Acinetobacter baumannii has become an important nosocomial pathogen, as causal agent of pneumonias, septicemias and urinary tract infections, among other complications in compromised patients from hospital’s intensive care units. One of its last acquired abilities is the resistance to colistin (polymixin E), the last therapeutic option for its infections. In this thesis, descriptive and quantitative differential expression proteomics is used in the study of acquired colistin resistance. As result of this research, 1,097 proteins belonging to the Acinetobacter genus have been identified by combined application of bidimensional gel electrophoresis (2DE), differential gel electrophoresis (DIGE), and peptide labeling with stable isobaric isotopes tags (iTRAQ). Analyses have been performed on the global expressed proteome of a reference, colistin-sensible strain (A. baumannii ATCC 19606) and, for comparative purposes, on a derived strain on which colistin resistance has been induced in vitro. The resistant phenotype shows reduced fitness, with significant differences in expression found in outer membrane proteins, membrane active transporters, diverse metabolic enzymes (fatty acids, citrate, phenylacetate, piruvate and nitrogen), proteins involved in stress response and biofilm formation, as well as in protein synthesis and folding pathways. The work has allowed to assess the strengths and weaknesses of the different techniques currently used in this type of proteomic analysis.
Acinetobacter baumannii, normalmente aislado en suelos y aguas (corrientes o residuales), se ha convertido en importante patógeno nosocomial, siendo agente causal de, entre otras complicaciones, neumonías, septicemias e infecciones del tracto urinario de pacientes comprometidos en unidades hospitalarias de cuidados intensivos. La más reciente de sus capacidades adquiridas es la resistencia a colistina (polimixina E), antibiótico peptídico considerado la última opción terapéutica en contextos clínicos. Esta tesis doctoral emplea la proteómica descriptiva y de expresión diferencial cuantitativa para investigar la resistencia adquirida por A. baumannii a dicho antibiótico. Los resultados han supuesto la identificación de 1.097 proteínas de Acinetobacter mediante el empleo combinado de electroforesis bidimensional convencional (2DE), 2DE diferencial (DIGE) y marcaje peptídico mediante isótopos isobáricos estables (iTRAQ). Los análisis se han realizado en el proteoma expresado por una cepa de referencia sensible a colistina (A. baumannii ATCC 19606), así como en una cepa derivada de ésta en la que se ha inducido, a efectos comparativos, resistencia a colistina in vitro. El fenotipo resistente manifestó reducida adaptabilidad biológica, encontrándose las principales diferencias en la estructura de la membrana externa, en la expresión de transportadores activos de membrana, en diversos enzimas metabólicos (ácidos grasos, citrato, fenilacetato, piruvato, nitrógeno) y de respuesta a condiciones de estrés, así como en la expresión de proteínas participantes en la formación de biopelículas y en el proceso de síntesis y plegamiento de proteínas. Además, el trabajo ha permitido evaluar los puntos fuertes y débiles de las técnicas empleadas actualmente en este tipo de análisis proteómicos.

En aquesta tesi es va estudiar la resistència de la microbiota oral a 3 de les classes d’antibiòtics més utilitzades en la pràctica dental: les tetraciclines, els macròlids i els β-lactàmics. En primer lloc, es va analitzar la resistència a tetraciclina de la microbiota subgingival d’individus sense periodontitis, per tal de conèixer quin era l’abast de la resistència a un antibiòtic de molt baixa exposició entre la població actual. A aquest treball el va seguir una rèplica en la que es va estudiar la microbiota subgingival d’individus amb periodontitis. En ambdós tipus de mostres es va fer un screening de gens de resistència a tetraciclina i a altres gens que podien indicar la presència d’elements genètics mòbils. Ambdós estudis van mostrar alts nivells de microorganismes resistents a tetraciclina a les mostres obtingudes, i van ser comparades entre elles al tercer estudi, on es van observar diferències entre la microbiota obtinguda i la prevalencia dels gens de resistència en les dues poblacions. La transferència genètica horitzontal a l’ambient oral va ser investigada al quart estudi, en el qual es va demostrar que el gen de resistencia a tetraciclina tet(B), el qual no s’havia descrit abans en grampositius, s’havia integrat al genoma de dos aïllats d’Streptococcus oralis. La resistència a macròlids va ser analitzada al cinqué estudi, en el que es va investigar la susceptibilitat de bacteris del gènere Prevotella a eritromicina i azitromicina, sent el segon un macròlid l’ús del qual ha anat creixent degut a la seva eficacia demostrada i als seus interessants efectes beneficiosos. Per conèixer si la resistència a macròlids en aïllats de Prevotella estava asociada a la presència de gens de resistència a macròlids, es va fer un screening per a aquests gens, així com un anàlisi estadístic que va confirmar l’increment de resistència a macròlids en presència del gen erm(F). Per últim, al sisé estudi es va analitzar la resistència a β-lactàmics, la classe d’antibiòtics més utilitzada en la pràctica dental, en mostres obtingudes de pacients amb periodontitis. La resistència a amoxicilina i cefotaxima, els dos β-lactàmics estudiats, va ser trobada a la majoria de les mostres analitzades juntament amb gens que codifiquen per a β-lactamases i β-lactamases d’espectre estès.
En esta tesis se estudió la resistencia de la microbiota oral a 3 de las clases de antibióticos más utilizadas en la práctica dental: las tetraciclinas, los macrólidos y los β-lactámicos. En primer lugar, se analizó la resistencia a tetraciclina de la microbiota subgingival de individuos sin periodontitis para conocer cual era el alcance de la resistencia a un antibiótico de muy baja exposición actualmente. A este estudio le siguió una réplica en la que se trabajó con la microbiota subgingival de individuos con periodontitis. En ambos tipos de muestras se hizo un screening de genes de resistencia a tetraciclina y a otros genes que podían indicar la presencia de elementos genéticos móviles. Ambos estudios mostraron altos niveles de microorganismos resistentes a tetraciclina en las muestras obtenidas, y fueron comparados entre sí en el tercer estudio, donde se observaron diferencias en la microbiota obtenida y la prevalencia de los genes de resistencia en una y otra población. La transferencia genética horizontal en el ambiente oral fue estudiada en el cuarto estudio, en el que se demostró que el gen de resistencia a tetraciclina tet(B), nunca antes descrito en grampositivas, se había integrado en el genoma de dos aislados de la especie Streptococcus oralis. La resistencia a macrólidos fue analizada en el quinto estudio, en el que se investigó la susceptibilidad de bacterias del género Prevotella a eritromicina y azitromicina, siendo el segundo un macrólido cuyo uso ha ido creciendo debido a su eficacia demostrada y sus efectos beneficiosos. Para conocer si la resistencia a macrólidos en aislados de Prevotella estaba asociada a la presencia de genes de resistencia a macrólidos, se hizo un screening para dichos genes, así como un análisis estadístico que confirmó el incremento de resistencia a macrólidos en presencia del gen erm(F). Por último, en el sexto estudio se analizó la resistencia a β-lactámicos, la clase de antibióticos más utilizada en la práctica dental, en muestras obtenidas de pacientes con periodontitis. La resistencia a amoxicilina y a cefotaxima, los dos β-lactámicos estudiados, fue encontrada en la mayoría de las muestras analizadas junto con genes que codifican para β-lactamasas y β-lactamasas de espectro extendido.
The oral environment is widely colonised by bacteria, which grow in a multispecies biofilm structure that confers many benefitial properties to the microorganisms, including better resistance to mechanical and chemical stress and a greater comunication and genetic exchange among them. The undisturbed growth of the oral biofilm can lead to certain pathologies such as caries, gingivitis or periodontitis. Antibiotics can be necessary as adjuvants in the treatment of these pathologies. However, antimicrobials treatments could fail in the presence of genetic determinants that confer resistance to the antimicrobial given. Resistance to antibiotics is a growing problem of modern medicine. The use of antibiotics is widespread and they are essential for the treatment of many infectious diseases. International organizations, such as the World Health Organization and the European Centre for Disease Prevention and Control, have underlined the importance of surveillance of antibiotic resistance and their causes as an essential mesure to avoid its propagation and to design strategies to fight them. In this thesis, we studied the resistance of the oral microbiota to the 3 main classes of antibiotics used in the dental practice: tetracyclines, macrolides and β-lactams. In the first place, we analised tetracycline resistance in the subgingival microbiota of healthy individuals without periodontitis in order to know which was the extent of the resistance to an antibiotic with a low level of exposition among the current population. A replica to this study followed, in which subgingival microbiota of patients with periodontitis was used. Screening of tetracycline resistance genes and other genes, that could indicate the presence of mobile genetic elements, was performed in both types of samples. Both studies showed high numbers of microorganisms resistant to tetracycline, and both were compared between them in a third study, where differences were observed in the microbiota obtained and the prevalence of the resistance genes. Horizontal genetic transfer in the oral environment was investigated in the fourth study, in which the integration of the tetracycline resistance gene tet(B), that has never before been described in grampositive bacteria, in the genome of two Streptococcus oralis isolates was proven. Resistance to macrolides was analised in the fifth study, in which the susceptibility of bacteria of the Prevotella genus against erythromycin and azythromycin, being the latter a macrolide which use has been growing due to its proven effectiveness and its interesting beneficial effects, was also tested. In order to know if resistance to macrolides in Prevotella isolates could be related to the presence of macrolide resistance genes, an screening for such genes was performed, together with a statistical analysis which confirmed the increase of macrolide resistance in the presence of the gen erm(F). Finally, resistance to β-lactams, the most used class of antibiotics in the dental practice, was analised in the sixth study in samples obtained from patients with periodontitis. Resistance to amoxicilin and cefotaxime, the two β-lactams studied, was found in most of the analised samples together with genes that code for β-lactamases and Extended Spectrum β-lactamases.

Orientador: Ronilson Agnaldo Moreno
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: A infecção por Helicobacter pylori é uma das mais comuns em todo o mundo estando relacionada ao câncer gástrico, gastrite crônica, úlceras, adenocarcinomas e linfomas gástricos. O metronidazol foi muito usado como componente de terapias contra H. pylori. No entanto metronidazol é um composto mutagênico e a resistência a esse antimicrobiano é muito comum. Isso estimulou o interesse em antimicrobianos que tivessem resistência incomum para H. pylori. No Brasil, onde o índice de resistência para metronidazol foi determinado em 42%, a furazolidona passou a ser usada nas terapias de erradicação. A furazolidona e o metronidazol, são compostos classificados como nitro-heterocíclicos ou nitroaromáticos e possuem mecanismos de ação similares. O mecanismo de resistência da H. pylori ao metronidazol foi relacionado com alterações nos genes NAD(P)H nitroredutase oxigênio insensível (rdxA) e NAD(P)H flavina oxidorre- dutase (fixA) produtores de nitroredutases que reduzem o metronidazol gerando produtos intermediários os quais tem ação tóxica. Devido a algumas linhagens apresentarem resistência simultânea para metronidazol e furazolidona foi sugerido que os mecanismos de resistência para essas drogas poderiam ser os mesmos. Entretanto linhagens.construídas em laboratório com os genes rdxA e frxA inativados não apresentaram resistência a furazolidona. Nesse estudo foi utilizado o método de transformação natural, feitos com os produtos de PCR dos genes rdxA e frxA de oito amostras brasileiras resistentes simultaneamente aos dois antimicrobianos para verificar o envolvimento dos genes rdxA e fdxA com a resistência ao metronidazol e a furazolidona. Das oito amostras utilizadas foram obtidos foram obtidos seis transformantes de diferentes produtos de PCR do gene rdxA, resistentes ao metronidazol. Não foram obtidos transformantes com o gene fixA resistentes ao metronidazol, nem transformantes resistentes a furazolidona com nenhum dos dois genes. Os resultados .obtidos nesse trabalho indicam que à resistência ao metronidazol esta relacionada ao gene rdxA , mas não ao frxA, e sugere que o gene rdxA influencia a concentração inibitória mínimade furazolidona, mas não confere resistência
Abstract: Helicobacter Pylori infection is one of the most common infections worldwide and recognized as the major cause of peptic ulcer disease, risk factor for gastric adenocarcinoma and primary gastric Iymphoma. Metronidazole has often used in combination therapies against H. pylori. However, metronidazole is highly mutagenic and resistance to this drug is very common.These stimulated the use the altematives drugs that resistance was uncommon in H. pylori. In Brazil, where the levei of resistance for metronidazole was detected in 42%, the nitrofuran furazolidone passed to be one of the components therapies. Metronidazole and furazolidone are classified as nitroeterocyclic and nitroaromatic compounds, hence the modes of drug action are similar and nitroreduction is required for activation these. The mechanism of metronidazole resistance among H. pylori strains has been reIated to alterations in gene products having metronidazole nitroreductase activity, like the oxygen insensitiveNAD(p)H nitroredutase (RdxA), and NAD(p)H flavin oxidoredutase (FrxA). Due some strains present simultaneous resistance for furazolidone and metronidazole suggests that the resistance mechanismfor these drugs may be the same. However, frxA and rdxA knockout mutations constructed in laboratory reference strains are not resistant to furazolidone, which indicates that mutations in others genes were also need for resistance. We used natural transformation with PCR products of eigth brazilian clinical isolates, simultaneous resistant to this these two drugs, for verifify the involvement of the genes rdxA and ftxA with resistance to metronidazole and furazolidone drugs. Our results showed that metronidazole resistance are associated with rdxA gene, but not fixA, and suggest the involvement of rdxA gene in increased leveI of furazolidone minimum inhibitoryconcentration , but not coffer resistance
Mestrado
Mestre em Farmacologia

[eng] Chronic respiratory infection (CRI) by Pseudomonas aeruginosa is the main cause of morbidity and mortality in cystic fibrosis (CF). During the progression from early infection to chronic non-eradicable colonization P. aeruginosa undergoes a complex evolutionary adaptation and diversification process which eventually leads to a mixed and persistent infecting population in which multidrug resistant variants frequently rise compromising the selection of appropriate antibiotic therapies. In this work the interplay between three key microbiological aspects of these infections was investigated: the occurrence of transmissible and persistent strains, the emergence of variants with enhanced mutation rates (mutators) and the evolution of resistance to antibiotics. Clonal epidemiology, antibiotic susceptibility profiles, contribution of P. aeruginosa classical resistance mechanisms and the role of mutator variants were investigated in two large collections of CF P. aeruginosa isolates from the Balearic Islands and Spain. As well, whole genome sequencing (WGS) was used to decipher the phylogeny, interpatient dissemination, within-host evolution, WGS mutator genotypes (mutome) and resistome of widespread P. aeruginosa clonal complex 274 (CC274), in isolates from two highly-distant countries, Australia and Spain, covering an 18-year period. Finally, due to the Chronic respiratory infection (CRI) by Pseudomonas aeruginosa is the main cause of morbidity and mortality in cystic fibrosis (CF). During the progression from early infection to chronic non-eradicable colonization P. aeruginosa undergoes a complex evolutionary adaptation and diversification process which eventually leads to a mixed and persistent infecting population in which multidrug resistant variants frequently rise compromising the selection of appropriate antibiotic therapies. In this work the interplay between three key microbiological aspects of these infections was investigated: the occurrence of transmissible and persistent strains, the emergence of variants with enhanced mutation rates (mutators) and the evolution of resistance to antibiotics. Clonal epidemiology, antibiotic susceptibility profiles, contribution of P. aeruginosa classical resistance mechanisms and the role of mutator variants were investigated in two large collections of CF P. aeruginosa isolates from the Balearic Islands and Spain. As well, whole genome sequencing (WGS) was used to decipher the phylogeny, interpatient dissemination, within-host evolution, WGS mutator genotypes (mutome) and resistome of widespread P. aeruginosa clonal complex 274 (CC274), in isolates from two highly-distant countries, Australia and Spain, covering an 18-year period. Finally, due to the relevance of aminoglycosides in the management of CF-CRI, the dynamics of P. aeruginosa resistance development to aminoglycosides was also studied in vitro by WGS approaches. Despite discrepancies between molecular genotyping methods, a high degree of genetic diversity was documented among CF isolates from the Balearic Islands and Spain with scarce representation of CF epidemic strains. However, for the first time in Spain, we documented a superinfection with the multidrug resistant Liverpool Epidemic Strain (LES) in a chronically colonized patient. As well, P. aeruginosa CC274, previously detected in several CF individuals from Austria, Australia and France, was detected in 5 unrelated chronically colonized patients from the Balearic Islands and, therefore, this clone-type should be added to the growing list of CF epidemic clones. Subsequent analysis of the whole genomes sequences of P. aeruginosa isolates from the CC274 P. aeruginosa collection provides evidence of interpatient dissemination of mutator sublineages and denotes their potential for unexpected short-term sequence type (ST) evolution and antibiotic resistance spread, illustrating the complexity of P. aeruginosa population biology in CF. As well, epidemiological studies demonstrated the coexistence of two divergent lineages but without evident geographical barrier. Antibiotic resistance significantly accumulated overtime accompanied by hypersusceptibility to certain antibiotics such as aztreonam, which can be explained in terms of collateral susceptibility. Correlation between phenotypes and WGS genotypes of clonal isolates from the CC274 collection allowed us to define the mutational resistome of CF P. aeruginosa which extends beyond the classical mutational resistance mechanisms. Among the new chromosomic resistance determinants encountered, mutations within the penicillin-binding-protein 3 (PBP3), shaping up beta-lactam resistance, are noteworthy as well as mutations within the fusA1 gene, coding for elongation factor G, which along with MexXY overexpresion contribute to high-level aminoglycoside resistance. Strikingly, we encountered that MexXY overexpression is dispensable for in vitro resistance development to aminoglycosides which suggests an evolutionary advantage of its overexpression in the CF respiratory tract. Altogether this work demonstrates that clonal epidemiology and antibiotic resistance evolution in the CF setting results from the complex interplay among mutation-driven resistance mechanisms, within host diversification and interpatient transmission of epidemic strains.
[spa] La infección respiratoria crónica por P. aeruginosa es la principal causa de morbilidad y mortalidad en pacientes con fibrosis quística (FQ). Durante la progresión desde la infección temprana a la colonización crónica, P. aeruginosa experimenta un complejo proceso adaptativo y de diversificación que resulta en una población heterogénea y persistente en la que la aparición de resistencias a los antibióticos comprometen la selección de terapias apropiadas. En este trabajo se investigó la interacción entre tres aspectos microbiológicos clave de estas infecciones: la presencia de cepas transmisibles y persistentes, la aparición de variantes con tasas de mutación incrementadas y la evolución de la resistencia a los antibióticos. La epidemiología clonal, los perfiles de sensibilidad antibiótica, la contribución de los mecanismos clásicos de resistencia de P. aeruginosa y el papel de las variantes hipermutadoras se estudiaron en dos grandes colecciones de aislados procedentes de pacientes con fibrosis quística de las Islas Baleares y España. Asimismo, mediante secuenciación de genoma completo, se determinó la filogenia, diseminación interpaciente, evolución intrapaciente, genotipo hipermutador y resistoma de una colección de aislados clonales pertenecientes al complejo clonal 274 (CC274), proviniendo dichos aislados de dos países muy distantes, Australia y España, y cubriendo un período de 18 años. Finalmente, dada la relevancia de los aminoglucósidos en el manejo de estos pacientes, se estudió la dinámica del desarrollo de resistencia a aminoglucósidos in vitro mediante secuenciación de genoma completo. A pesar de encontrarse discrepancias entre los métodos de genotipado molecular, se documentó un alto grado de diversidad genética en las colecciones de las Islas Baleares y España, siendo escasa la representación de cepas epidémicas. No obstante, por primera vez en España, se documentó un caso de sobreinfección con el clon epidémico multirresistente de Liverpool. Además, en 5 pacientes de Baleares, crónicamente colonizados y sin aparente relación epidemiológica, se detectó el CC274. Puesto que este complejo clonal también ha sido detectado en pacientes de países como Austria, Australia y Francia, éste debería incluirse en la creciente lista de cepas epidémicas. El análisis posterior de las secuencias de genoma completo de los aislados del CC274 evidenció la diseminación interpaciente de un sublinaje hipermutador, denotando además el potencial de estas variantes para la inesperada evolución a corto plazo del secuenciotipo y la rápida diseminación de resistencias. Además, los estudios epidemiológicos demostraron la coexistencia de dos linajes divergentes, no evidenciándose barrera geográfica. Asimismo se documentó una tendencia generalizada a la acumulación de resistencias a los antibióticos en el tiempo, acompañada de hipersensibilidad a ciertos antibióticos como aztreonam, lo cual se puede explicar en términos de sensibilidad colateral. La correlación entre los fenotipos y genotipos determinados mediante secuenciación del genoma completo de los aislados pertenecientes al CC274 nos permitió definir el resistoma mutacional de P. aeruginosa en la FQ, el cual se extiende más allá de los mecanismos mutacionales clásicos. Entre los nuevos determinantes de resistencia cromosómica encontrados caben destacar tanto las mutaciones en la proteína fijadora de penicilina PBP3, que confieren resistencia a betalactámicos, como las mutaciones en fusA1, que codifica para el factor de elongación G, y que junto con la hiperexpresión de MexXY contribuyen a la resistencia de alto nivel a aminoglucósidos. Paradójicamente, encontramos que la hiperexpresión de MexXY es prescindible para el desarrollo de resistencia in vitro a aminoglucósidos, lo que sugiere que dicha hiperexpresión confiere una ventaja evolutiva in vivo. En conjunto, este trabajo demuestra que, en la FQ, la epidemiología clonal y la evolución de la resistencia a los antibióticos son el resultado de una compleja interacción entre los mecanismos de resistencia mutacionales, la diversificación de la población infectante y la transmisión interpaciente de cepas epidémicas.
[cat] La infecció respiratòria crònica per P. aeruginosa és la principal causa de morbiditat i mortalitat en els pacients amb fibrosi quística (FQ). Durant la progressió des de la infecció primerenca a la colonització crònica, P. aeruginosa experimenta un complexe procés adaptatiu i de diversificació que resulta en una població heterogènia i persistent en la qual l'aparició de variants resistents a múltiples antibiòtics comprometen la selecció de teràpies antibiòtiques apropiades. En aquest treball es va investigar la interacció entre tres aspectes microbiològics clau: la presència de soques transmissibles i persistents, l'aparició de variants amb taxes de mutació incrementades i l'evolució de la resistència als antibiòtics. L'epidemiologia clonal, els perfils de sensibilitat antibiòtica, la contribució dels mecanismes clàssics de resistència i el paper de les variants hipermutadores es van estudiar en dos grans col·leccions d'aïllats procedents de pacients amb FQ de les Illes Balears i Espanya. Així mateix, mitjançant seqüenciació del genoma complet, es va determinar la filogènia, disseminació interpacient, evolució intrapacient, genotip hipermutador i resistoma d'una col·lecció d'aïllats pertanyents al complexe clonal 274 (CC274), provenint de dos països molt distants, Austràlia i Espanya, i cobrint un període de 18 anys. Finalment, donada la rellevància dels aminoglicòsids en el maneig d’aquests pacients, es va estudiar la dinàmica del desenvolupament de resistència a aminoglicòsids in vitro mitjançant seqüenciació de genoma complet. Tot i trobar discrepàncies entre els mètodes de genotipat molecular, es va documentar un alt grau de diversitat genètica en les col·leccions de les Illes Balears i Espanya, sent escassa la representació de soques epidèmiques. No obstant això, per primera vegada a Espanya, es va documentar un cas de sobreinfecció amb el clon epidèmic multiresistent de Liverpool. A més, en 5 pacients de les Illes Balears, crònicament colonitzats i sense aparent relació epidemiològica, es va detectar el CC274. Ja que aquest complexe clonal també ha estat detectat en països com Àustria, Austràlia i França, aquest clon hauria d'incloure a la creixent llista de soques epidèmiques. L'anàlisi posterior de les seqüències de genoma complet dels aïllats pertanyents al CC274, va evidenciar la disseminació interpaciente d'un subllinatge hipermutador, denotant a més el potencial d'aquestes variants per a la inesperada evolució a curt termini del sequenciotip i per a la ràpida disseminació de la resistència antibiòtica. A més, els estudis epidemiològics van demostrar la coexistència de dos llinatges divergents, no existint barrera geogràfica. Així mateix es va evidenciar una tendència generalitzada a l'acumulació de resistències en el temps, acompanyada d'hipersensibilitat a certs antibiòtics com l’aztreonam, la qual cosa es pot explicar en termes de sensibilitat col·lateral. La correlació entre els fenotips i genotips determinats mitjançant seqüenciació del genoma complet dels aïllats pertanyents al CC274 ens va permetre definir el resistoma mutacional de P. aeruginosa en la FQ, el qual s'estén més enllà dels mecanismes de resistència mutacionals clàssics. Entre els nous determinants de resistència cromosòmica trobats cal destacar tant les mutacions en la proteïna fixadora de penicil·lina PBP3, que confereixen resistència a betalactàmics, així com les mutacions en fusA1, que codifica per al factor d'elongació G, i que juntament amb la hiperexpressió de MexXY contribueixen a la resistència d'alt nivell a aminoglucòsids. Paradoxalment, vam trobar a més que la hiperexpressió de MexXY és prescindible per al desenvolupament de resistència in vitro a aminoglucòsids, el que suggereix que aquesta hiperexpressió suposa un avantatge evolutiu in vivo. En conjunt, aquest treball demostra que l'epidemiologia clonal i l'evolució de la resistència als antibiòtics en el context de la FQ són el resultat d'una complexa interacció entre els mecanismes de resistència mutacionals, la diversificació de la població infectant i la transmissió interpaciente de ceps epidèmiques.

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S. aureus é, sem duvida, o patógeno humano mais importante entre os estafilococos. O surgimento e a disseminação progressiva da resistência a meticilina tiveram grande impacto na terapia das infecções estafilocócicas. O mecanismo de resistência a meticilina desenvolvido por S. aureus está relacionado com a alteração das proteínas ligadoras de penicilinas, as PBPs. Os Staphylococcus. aureus produzem 5 tipos de PBPs: 1,2,3,3´, e 4. As cepas de S. aureus resistentes a meticilina produzem uma nova PBP, a PBP2a, adquirida de outras cepas de estafilococos. Diversos métodos são utilizados para detecção da resistência a meticilina no S. aureus. Dentre eles, a detecção da PBP2a por meio de métodos de aglutinação em látex utilizando anticorpo monoclonal específico dirigido para o antígeno PBP2a. Na presente pesquisa, anticorpos monoclonais murinos dirigidos contra a PP2a do S. aureus resistente a meticilina foram produzidos através de fusão celular utilizando-se células de baço de camundongos BALB/c imunizados.Cinco fusões MRSA foram realizadas e os sobrenadantes de cultura foram triados por testes Elisa indireto . Foram construídos e testados 1236 híbridos e nove híbridos se mostraram reativos após o 4º. teste de Elisa indireto. Os nove híbridos foram testados frente a diferentes bactérias para observar inibição do crescimento. Este trabalho teve como foco, a produção de anticorpo monoclonal murino para uso em testes de detecção rápida
S. aureus is, without doubt, the most important human pathogen among staphylococci. The emergence and dissemination of progressive resistance to methicillin had great impact on therapy of staphylococcal infections. The mechanism of resistance to methicillin developed by S. aureus is related to the alteration of penicillin binding proteins, the PBPs. Staphylococcus. aureus produces five types of PBPs: 1,2,3,3 ', and 4. Strains of S. aureus resistant to methicillin produce a new PBP, PBP2a the acquired from other strains of staphylococci. Several methods are used for detection of methicillin resistance in S. aureus. Among them, the detection of PBP2a by latex agglutination methods using monoclonal antibody specific for the antigen directed PBP2a. In the present study, murine monoclonal antibodies directed against the PP2A methicillin resistant S. aureus were produced by cell fusion using spleen cells from immunized BALB / c mice. Five MRSA fusions were performed and the culture supernatants were screened by testing indirect ELISA. Were built and tested in 1236 hybrids and nine of them were reactive after the 4th indirect ELISA test. The nine hybrids were tested against different bacteria to observe inhibition of growth. This work focused on the production of murine monoclonal antibody for use in rapid detection tests