Rozprawy doktorskie na temat „Antibiotic resistance transmission”

Antibiotic resistance is a growing threat to human health and is defined by the World Health Organization as a crisis that must be managed with the utmost urgency. Antibiotic resistant bacteria increase both mortality and morbidity and have a great impact on the global economy. Resistance is not confined to human health care, but is present also among animals and in our environment at large. Indeed, resistant bacterial strains have now been found in virtually all parts of the world, even in locations without direct human contact. The human gastrointestinal tract is populated by a complex, dynamic, diverse and highly interactive collection of microorganisms, including bacteria, archaea, fungi, yeasts and viruses, which constitutes our gastrointestinal microbiota. This microbiota is an important reservoir of resistance genes (our gastrointestinal resistome) and a “melting pot” for transfer of resistance genes between microbes, including potential pathogens. In this thesis I investigated the prevalences of two clinically important kinds of antibiotic resistance: extended-spectrum β-lactamases (ESBL) and vancomycin-resistant enterococci (VRE), as well as asymptomatic carriage of potential enteropathogens among healthy preschool children in Uppsala. Fecal samples from unidentified, individual diapers were collected in 2010 (125+313 samples) and in 2016 (334 samples). In addition, 204 environmental samples from the children’s preschools were collected in autumn 2016. A prevalence of 2.9% ESBL-producing Enterobactericeae was demonstrated in the first samples from 2010. No VRE were found and the occurrence of enteropathogens were reassuringly low. Results on ESBL prevalence in 2016 and transmission of resistance between children will be presented when the manuscript is published and at the dissertation.

Anthropozoonotic diseases, defined as infectious diseases caused by pathogens transmitted from humans to wildlife, pose a significant health threat to wildlife populations. Many of these pathogens are also able to move from wildlife reservoirs to humans, termed zoonotic diseases, creating the possibility for bi-directional transmission between humans and wildlife. Recent studies show that a significant proportion of emerging infectious diseases in humans originate in wildlife reservoirs and that the frequency of emergence is increasing, yet the specific transmission pathways still remain speculative in most cases. Human fecal waste is persistent across human-modified landscapes and has been identified as a potential source of disease exposure for wildlife populations living near humans. As part of a long-term study of banded mongoose (Mungos mungo) that live in close association with humans and human fecal waste I used Escherichia coli and banded mongoose (Mungos mungo) for evaluating exchange of fecal waste-borne microorganisms at the human-wildlife interface. Antibiotic resistance was found in 57.5% ° 10.3% (n=87) of mongoose fecal samples and 37.2% ° 5.9% of isolates (n=253). Multidrug resistance was detected in 13.8% ° 4.2% of isolates (n=253). Mongoose and human fecal waste isolates consistently clustered together in phylogenetic analyses and statistical analysis of genetic variation showed no significant differences (p=0.18) between E. coli from human and mongoose populations. These results suggest that human fecal waste contamination is an important mechanism for the transmission of pathogens to both humans and animals, including the spread of antibiotic resistance in the environment, an emerging global health threat.
Master of Science

Whole genome sequencing (WGS) has transformed molecular infectious diseases epidemiology in the last five years, and represents a high resolution means by which to catalogue the genetic content and variation in bacterial pathogens. This thesis utilises WGS to enhance our understanding of antimicrobial resistance in two clinically important members of the Enterobacteriaceae family of bacteria, namely Escherichia coli and Klebsiella pneumoniae. These organisms cause a range of clinical infections globally, and are increasing in incidence. The rapid emergence of multi-drug resistance in association with infections caused by them represents a major threat to the effective management of a range of clinical conditions. The reliability of sequencing and bioinformatic methods in the analysis of E. coli and K. pneumoniae sequence data is assessed in chapter 4, and provides a context for the subsequent study chapters, investigating resistance genotype prediction, outbreak epidemiology in two different contexts, and population structure of an important global drug-resistant E. coli lineage, ST131 (5-8). In these, the advantages (and limitations) of short-read, high-throughput, WGS in defining resistance gene content, associated mobile genetic elements and host bacterial strains, and the relationships between them, are discussed. The overarching conclusion is that the dynamic between all the components of the genetic hierarchy involved in the transmission of important antimicrobial resistance elements is extremely complicated, and encompasses almost every imaginable scenario. Complete/near-complete assessment of the genetic content of both chromosomal and episomal components will be a prerequisite to understanding the evolution and spread of antimicrobial resistance in these organisms.

[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.

L’émergence et la dissémination des bactéries résistantes aux antibiotiques sont préoccupantes. L’infection causée par les bactéries multi-résistantes (BMR) aggrave le pronostic des malades infectés et augmente les dépenses liées à leur prise en charge. Parmi les BMR, les bactéries à Gram négatif (BGN), plus particulièrement les entérobactéries productrices de béta-lactamase à spectre étendu (E-BLSE) sont les plus fréquemment isolées. La résistance aux antibiotiques pourrait avoir un impact sur la morbidité et la mortalité dans les pays à revenu faible ou intermédiaire (PRFI) en raison du potentiel d’émergence et de diffusion de bactéries résistantes aux antibiotiques, et du fardeau des infections bactériennes dans ces pays. Cependant, les données sur la résistance bactérienne sont rares et très majoritairement hospitalières dans les PRFI. De plus, dans ces pays, les infections bactériennes néonatales sévères (septicémies, pneumonies et méningites) représentent encore les principales causes de décès chez les nouveau-nés. Les entérobactéries sont majoritairement responsables de ces infections néonatales. Ainsi, investiguer la transmission d'E-BLSE chez le nouveau-né permettrait de proposer des stratégies de prévention. Ce travail de recherche s’est appuyé sur le programme BIRDY (Bacterial Infections and antibiotic Resistant Diseases among Young children in low-income countries). Le premier objectif était d’estimer la prévalence de la colonisation par des E-BLSE chez les femmes enceintes à Madagascar ainsi que les facteurs favorisant cette colonisation. Les résultats ont montré une prévalence globale de colonisation de 18.5% [IC à 95% 14.5-22.6]. Des facteurs reflétant un niveau socioéconomique plus élevé comme l’accès privatif à l’eau de boisson et avoir une maison individuelle sont associés à la colonisation. Le second objectif de ce travail était d'étudier l'incidence de la première colonisation par des E-BLSE chez les nouveau-nés en milieu communautaire et d'identifier les facteurs de risque d'acquisition. Les résultats révèlent une incidence globale d'acquisitions d'E-BLSE de 10.4 pour 1000 nouveau-nés-jours [IC à 95% : 8.0; 13.4]. Par ailleurs, nous avons mis en évidence que le faible poids à la naissance HR ajusté 2.7 [IC à 95% 1.2 ; 5.9], l'accouchement par césarienne HR ajusté 3.4 [IC à 95% 1.7 ; 7.1], la prise maternelle d'antibiotique à l'accouchement HR ajusté 2.2 [IC à 95% 1.1 ; 4.5] étaient des facteurs de risque d'acquisition d'E-BLSE. Le troisième objectif était de documenter les infections néonatales. Nous avons trouvé une incidence d'infections néonatales de 30.6 cas pour 1000 naissances vivantes [IC à 95%: 23.4 ; 40.1].Nos résultats montrent que les mesures de santé publique devraient axer sur l’amélioration de la prise en charge de la grossesse et sur le diagnostic précoce des infections néonatales
The emergence and spread of antibiotic-resistant bacteria is a concern. Infection caused by multidrug-resistant bacteria (MDR) worsens the prognosis of infected patients and increases the costs associated with their management. Among the MDRs, Gram-negative bacteria (GNB), especially extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-PE) are the most frequently isolated. Antibiotic resistance may have an impact on morbidity and mortality in low- and middle-income countries (LMICs) because of the potential for emergence and spread of antibiotic-resistant bacteria, and the burden of bacterial infections in these countries. However, data on bacterial resistance are scarce or came from the hospital, for the great majority, in LMICs. In these settings, severe neonatal bacterial infections (sepsis, pneumoniae and meningitis) still represent the leading causes of death in newborns. Enterobacteriaceaeare responsible for a great part of these neonatal infections. Thus, investigating the transmission of ESBL-PE in newborns would make it possible to propose prevention strategies. This work was based on the BIRDY program (Bacterial Infections and Antibiotic Resistant Diseases among Young Children in Low-Income Countries). The first objective was to estimate the prevalence of colonization by ESBL-PE in pregnant women in Madagascar as well as the risk factors of this colonization. The results showed an overall colonization prevalence of 18.5% [95% CI 14.5-22.6]. Factors reflecting a higher socioeconomic level such as private access to drinking water and having a house are associated with colonization. The second objective of this work was to study the incidence of ESBL-PE colonization in community-based infants and to identify acquisition risk factors. The results reveal an overall incidence of ESBL-PE acquisition of 10.4 per 1000 newborn-days [95% CI: 8.0; 13.4]. In addition, we found that low birth weight adjusted HR 2.7 [95% CI 1.2; 5.9], cesarean section delivery adjusted HR 3.4 [95% CI 1.7; 7.1], maternal intake of antibiotic at delivery adjusted HR 2.2 [95% CI 1.1; 4.5] were risk factors for the acquisition of ESBL-PE. The third objective was to document neonatal infections. We found an incidence of neonatal infections of 30.6 cases per 1000 live births [95% CI: 23.4; 40.1]. Our results suggest that public health measures should focus on the improvement of pregnancy follow-up and early diagnosis of neonatal infections

Comprendre l'émergence et la propagation des maladies infectieuses chez les animaux sauvages est devenue une priorité en santé publique et en conservation. En combinant la collecte de données et le développement de modèles épidémiologiques, cette thèse s'est focalisée sur la compréhension de deux phénomènes clés: (i) étudier comment l'hétérogénéité au niveau des individus, des groupes, de la population et de l'environnement influence la propagation de parasites et (ii) quantifier la transmission de bactéries résistantes aux antibiotiques depuis l'homme vers les animaux sauvages dans trois aires protégées d'Afrique (Tsaobis NP- Namibie, Lopé NP-Gabon et Dzanga-Ndoki NP- République Centrafricaine). Les principaux résultats de ce travail montrent que : (1) De multiples facteurs incluant la température, la pluie, l'utilisation du territoire, le genre, l'âge et la condition physique influencent la richesse spécifique de parasites gastro-intestinaux chez le babouin chacma, (2) Les contacts entre animaux autour des points d'attractions de l'habitat peuvent influencer de manière importante la propagation spatio-temporelle d'une maladie, (3) La transmission d'entérobactéries résistantes semble avoir lieu depuis les humains ou le bétail vers les animaux sauvages dans des zones où le contact entre ces populations est élevé, (4) Le gradient de densité de gorilles produit par la chasse peut générer une augmentation de prévalence d'un parasite avec la distance au point d'introduction. Les conclusions de ce travail ouvrent de nouvelles possibilités pour l'étude des maladies émergentes chez les animaux sauvages
Understanding the emergence and spread of infectious disease in wild animal populations has become an important priority for both public health and animal conservation. Combining the collection of empirical data with the development of epidemiological models, this thesis focuses on understanding two key issues of wildlife epidemiology: (i) how heterogeneity at the individual, group, population and landscape level affects parasite spread (ii) investigating whether transmission of antibiotic resistant bacteria from humans to wildlife is occurring within three protected areas of Africa (Tsaobis NP-Namibia, Lope NP-Gabon and Dzanga-Ndoki NP-Central African Republic). The main findings of this work indicated that: (1) multiple-scale factors including temperature, rainfall, home range use, sex, age and body condition influence gastro-intestinal parasite richness among wild baboons; (2) animal contacts around ‘habitat hotspots' can substantially influence the spatio-temporal dynamics of a disease; (3) antibiotic resistant enterobacteria seem to be spreading from humans/livestock to wildlife when the territory overlap between these two populations is expected to be high; (4) gradients in gorilla density created by bushmeat hunting can reverse the expected pattern of decreasing parasite prevalence with distance to human-spillover. The conclusions of this work open new possibilities for studying the mechanisms explaining the spread of emerging infectious diseases among wild animals

Among infectious diseases, two large groups have great clinical relevance: parasitic and bacterial infections. Belonging to the first category is malaria, caused by the parasite Plasmodium falciparum. Transmission of Plasmodium parasites between humans and Anopheles mosquitoes is one of the most important contributors to the global impact of malaria and to the difficulties encountered in eliminating this parasite1 . Gametocytogenesis, the process by which merozoites switch from asexual replication to produce male and female gametocytes, represents a critical step in malaria transmission and Plasmodium genetic diversity. Still too little is known about the biochemical events that regulate gametocytogenesis and there are few existing drugs able of inhibiting the gametocytes development and block malaria transmission2 . To encourage drug discovery and research, the non-profit foundation Medicines for Malaria Venture (MMV) has provided a library of 400 compounds that present antimalarial activity in the micromolar range, but their molecular targets and mode of action are not necessarily known3 . Here we describe the medchem investigation of one of the most promising hit compound included in this library, MMV019918. MMV019918 has been highlighted in several in vitro studies for its promising antigametocyte activity coupled to activity against schizonts. On the basis of its structure, the synthesis of a new series of compounds with transmission-blocking activity has been designed. It has been found very interesting the activity of one derivative NF2350 which resulted active also in the standard membrane feeding assay (SMFA), to measure subsequent mosquito infection, and which has an improved toxicological profile compared to MMV019918. A further investigation of NF2350 will allow us to optimize the transmission-blocking activity and to indentify its putative target. Regarding bacterial infections, a critical issue to be addressed is bacterial resistance to antibiotics and in particular to β-lactams. Metallo-β-lactamases (MBL) are a family of enzymes involved in the widespread mechanisms of resistance to beta-lactam antibiotics, The diffusion of MBL-producing isolates of Pseudomonas aeruginosa, a bacterial pathogen of primary relevance for both nosocomial and chronic infections of the respiratory tracts in cystic fibrosis patients, is notably increasing in some specific settings. No clinically useful MBLs inhibitors are currently available in therapy3 Here we will present the design, synthesis, and biological investigation of a series of functionalized 2-arylfuran compounds with sub-micromolar antiplasmodial activity, against both asexual and sexual stages. Furthermore, appropriate decoration of this molecular scaffold allowed to obtain activity against some isoforms of MBL (including IMP-1and NDM-1).

Dissertação de Mestrado Integrado em Medicina Veterinária
ABSTRACT - Objective: This study aims to characterize the dynamics of antibiotic resistance gene transmission in dogs with skin and soft tissue infections (SSTI) and human beings co habiting with them. We also aim to evaluate the gut colonization of these individuals for the presence of extended spectrum beta-lactamases (ESBL’s) in Enterobacteriaceae and to assess the effect of antibiotherapy on the selection of MDR bacteria from human and canine gut microbiota. Methods: Two types of biological samples were gathered in a teaching veterinary hospital in Portugal, at the dermatology department, from twelve dogs diagnosed with SSTI and their household members. Collections included a swab from the infection site (ISS) and a faecal sample (FS). Gathering of samples was performed at two different times. The ISS were cultured and an AST was performed. The FS was also cultured, and the bacteria isolated subjected to molecular analysis. Antibiotic resistance patterns were obtained by disk diffusion antimicrobial susceptibility’s testing and Enterobactereaceae ESBL’s production was confirmed by amplification of the specific gene by PCR and sequencing. Results: High levels of Methicillin resistant Staphylococcus pseudintermedius (MRSP) were isolated, and high levels of other multidrug resistant bacteria (MDR) as well. One family was suspected of carrying the same E. coli clone, shared by two humans and one dog of the same household, with a blaCTX-M-15 gene. All of the isolated Enterobactereaceae displayed susceptibility to carbapenems. The most common ESBL genes found were from the blaCTX-M group, followed by blaOXA-1 and then blaTEM, no gene from the blaSHV gene was found. Conclusions: There is a high prevalence of ESBL-producing E. coli. Interspecies transmission of antimicrobial resistance is real. This issue should be addressed with introduction of antimicrobial stewardship strategies on a wider scale and better use of antimicrobials like chlorohexidine, especially in SSTI.
RESUMO - Esta dissertação enquadra-se nos objetivos de um projeto muito maior e ainda mais ambicioso, chamado PetRisk. Pretende-se analisar o impacto e as interações dos genes de resistência aos antibióticos, percebendo de que forma as barreiras interespécie podem ser ultrapassadas. Assim sendo, o estudante integrou o núcleo desse mesmo empreendimento em Portugal, o Laboratório de Resistência aos Antibióticos da FMV-UL, liderado pela Professora Doutora Constança Pomba. Objetivo: Este estudo teve como objetivo principal compreender a dinâmica de transmissão de genes de resistência aos antibióticos, entre cães com infeção de pele e tecidos moles e as pessoas com quem vivem em regime de co-habitação. Pretendeu-se avaliar a colonização do tubo digestivo desses mesmos indivíduos quanto à presença de Enterobactereacea produtoras de beta-lactamases de largo expectro e verificar o efeito da antibioterapia na seleção de estirpes multiresistentes da microbiota intestinal canina e humana. Outro obejtivo da realização deste trabalho é o de aumentar os recursos biológicos – com isto subentende-se, bactérias de diferentes agregados familiares e o seu perfil de resistência aos antimicrobianos – para o projeto mãe, o PetRisk. Métodos: Para tal, recolheram-se dois tipos de amostras biológicas no departamento de dermatologia do hospital veterinário escolar da FMV-ULisboa. A amostra inclui doze cães com infeções de pele e tecidos moles, assim como os restantes membros do seu agregado familiar. Os materiais recolhidos foram zaragatoas do local de infeção (ZLI) e amostras fecais (AF). As colheitas decorreram em dois tempos diferentes. Às ZLI após cultura microbiológica realizaram-se testes de suscetibilidade aos antibióticos de rotina. Os isolados provenientes das AF foram sujeitos a cultura microbiológica e análise molecular. Os padrões de resistência aos antibióticos foram obtidos pelo método de difusão de discos e a confirmação da produção das beta-lactamases de largo expectro pelas Enterobactereaceae por amplificação do respetivo gene por PCR e sequenciação. Resultados: Foram encontrados elevados níveis de Staphylococcus resistentes à meticilina, assim como a resistência a multiplos antibióticos. Também as nas Escherichia coli provenientes das AF foram encontrados elevados nívreis de resistência a múltiplos antibióticos. Uma familia foi suspeita de partilhar o mesmo clone de E.coli (duas pessoas e um cão) com o mesmo filogrupo e o gene blaCTX-M-15. Todas as Enterobactereaceae isoladas demonstraram suscetibilidade aos carbapenemos. Os genes de beta-lactamases de largo expectro detetados foram (da maior para a menor frequência): blaCTX-M , blaOXA-1 e blaTEM, com nenhum registo de blaSHV. Conclusões: Existe uma elevada prevalência de E. Coli produtoras de beta-lactamases de largo expetro. A transmissão de informação genética inter-espécies é uma realidade. A prática clínica beneficiaria de uma administração mais prudente e integrada de antibióticos, com uma equipa a trabalhar apenas para a gestão destes recursos num hospital, assim como maior atenção ao uso de antisséticos como a clorohexidina, especialmente em infeções de pele e tecidos moles onde os produtos disponivéis são de fácil aplicação.
N/A

A reutilização de águas residuais tratadas tem sido incentivada em todo o mundo, em particular para irrigação agrícola, porque pode ser uma fonte de água confiável e importante, principalmente em países sob estresse hídrico. Ainda se sabe pouco sobre os riscos associados à utilização de águas residuais tratadas, designadamente no que se refere à disseminação de bactérias resistentes a antibióticos (ARB) e genes de resistência a antibióticos (ARGs). Uma das principais consequências da disseminação através da irrigação agrícola utilizando águas residuais tratadas é a contaminação da cadeia alimentar humana. Atualmente, o conhecimento sobre a transmissão de ARB e ARGs para seres humanos a partir de fontes ambientais é escasso. No entanto, a reutilização de águas residuais surge como uma fonte potencial de transmissão direta ou indireta, principalmente através da cadeia alimentar humana. Esta tese explorou estas questões com base em pesquisa bibliográfica e em abordagens experimentais. O estudo foi planeado com o objetivo de inferir se as bactérias que sobrevivem nas águas residuais serão capazes de colonizar as plantas e se ARB isoladas de águas residuais e os respectivos ARGs poderiam persistir na presença da microbiota fecal autóctone de um indivíduo saudável. As hipóteses propostas para este trabalho foram as de que: 1) ARB e ARGs isoladas de águas residuais tratadas podem colonizar plantas e, eventualmente, estabelecer-se como endófitos; 2) ARGs de ARB isoladas de água residual tratada podem persistir na presença da microbiota fecal de um humano saudável. O argumento que suporta esta duas hipóteses é o de que, se ingeridas através de vegetais, as ARB e ARGs poderiam persistir no intestino humano. Utilizaram-se três abordagens principais para testar as hipóteses referidas: a) foi realizada pesquisa bibliográfica com termos de busca relacionados com bactérias endófitas, filtrada para variedades agrícolas edíveis e normalmente consumidas cruas, e que pertencessem a grupos bacterianos também reportados em águas residuais e no microbioma humano; b) analisou-se a presença de ARB e ARGs em isolados bacterianos em vegetais edíveis (agrião e morango prontos para consumo); e c) foi avaliada a persistência de ARGs transportados por ARB isoladas de efluentes na presença de microbiota fecal de uma criança saudável. A investigação baseada na literatura teve como objetivo explorar a diversidade de bactérias endofíticas associadas a diferentes habitats (plantas, águas residuais e microbioma Resumo x humano) e inferir a probabilidade de que as ARB e ARGs possam ser transferidos das águas residuais para as plantas e depois para os seres humanos (Capítulo 3). Este estudo bibliográfico permitiu compilar uma lista de bactérias endofíticas distribuídas por mais de 20 filos reportadas em mais de 45 variedades de vegetais. Como o estudo procurava explorar as possíveis vias de transmissão para humanos, a lista de espécies agrícolas encontradas na literatura foi reduzida apenas a plantas que podem ser consumidas cruas, como sejam alface, cenoura, rabanete, pepino e tomate. Com o objetivo de avaliar a probabilidade dessas plantas captarem bactérias que podem atuar como vetores de resistência a antibióticos, a presença dos grupos bacterianos identificados como endófitos foi também investigada em comunidades microbianas de águas residuais e em microbiomas humanos. Para grupos bacterianos, cuja ocorrência foi relatada nos três tipos de ambiente, endófitos de plantas edíveis/cruas, águas residuais e microbioma humano, foi realizada uma análise exploratória com base em literatura e bases de dados públicas, de genes de resistência a antibióticos e de virulência. Este estudo sugere que bactérias relacionadas com géneros como Enterobacter, Acinetobacter, Pseudomonas e Staphylococcus, cuja relação taxonómica com agentes patogénicos oportunistas e portadores de resistência a antibióticos, é conhecida, podem ser encontrados como endófitos em variedades agrícolas comestíveis, sugerindo que podem ser veiculados por águas residuais e ser transmitidos para o microbioma humano. Esses resultados sustentam a hipótese de que as bactérias transportadas pelas águas residuais, com propriedades fisiológicas e ecológicas semelhantes a bactérias endófitas, podem ser captadas por variedades agrícolas edíveis e, assim, se ingeridas, podem eventualmente colonizar o corpo humano. Deste modo, sugere-se que a irrigação com águas residuais pode aumentar os riscos de transmissão de bactérias clinicamente relevantes do meio ambiente para os seres humanos, através da cadeia alimentar. Com recurso a métodos dependentes da cultura, avaliou-se a diversidade de bactérias cultiváveis e resistentes a antibióticos que podem ser encontradas em associação com variedades edíveis, especificamente o agrião e morangos prontos para o consumo (capítulo 4). Os agriões prontos para consumo foram adquiridos em supermercados locais, enquanto as amostras de morangos foram colhidas ao longo de uma cadeia de processamento de frutas de uma empresa que produz preparados de frutas para a indústria alimentar. Estudou-se a fração microbiana das folhas de agrião e as bactérias associadas ao morango (comunidade epífita e endofítica). Os isolados de agrião (n = 68) e morango (n = 52) foram caracterizados fenotipicamente de acordo com seu perfil de resistência a antibióticos. Destes, selecionou-se uma coleção de isolados que exibia fenótipo de resistência a pelo menos três classes de Resumo xi antibióticos, para identificação com base na análise da sequência do gene 16S rRNA e caracterização por reação em cadeia da polimerase (PCR) convencional, quanto à presença do gene da integrase de intregrões de classe 1, intl1, os ARGs sul1, blaTEM, blaCTX-M, blaOXA-A e blaSHV, e os grupos de incompatibilidade de plasmídeos com base nos tipos de origem de replicação FIA, FIB, FIC, HI1, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, e FIIA. Além disso, testou-se se as bactérias isoladas de morango e agrião poderiam adquirir ARGs por transformação. Observou-se que o agrião pronto a consumir apresentava contagens de heterotróficos totais na ordem de 3,5 x 107 ± 4,9 x 107 CFU/mL e 5,4 x 105 ± 6,2 x 104 CFU/mL de solução de lavagem de agrião para os supermercados A e B, respectivamente, enquanto o morango processado apresentava contagens de bactérias cultiváveis na ordem de 9,1 x 105 ± 1,8 x 105 CFU/g de peso seco de morango, com alguns dos isolados a apresentarem fenótipos de resistência a antibióticos. Algumas destas bactérias foram identificadas como membros dos géneros Pseudomonas, Stenotrophomonas, Erwinia, Rahnella, Methylobacterium e Chryseobacterium. Alguns destes grupos bacterianos foram anteriormente identificados com endófitos e géneros como Pseudomonas e Stenotrophomonas estão proximamente relacionados com patogénicos oportunistas que podem ser transportadores de ARGs adquiridos. A pesquisa de ARGs, intI1 e grupos de incomatibilidade de plasmídeos revelou que estes elementos genéticos foram encontrados numa pequena fração das bactérias analisadas (2 isolados). Além disso, resultados preliminares sugeriram que os isolados de agrião e morango podem ser suscetíveis a adquirir resistência a antibióticos e, assim, estar envolvidos na disseminação da resistência a antibióticos. O terceiro estudo baseou-se no argumento de que bactérias de águas residuais ou os seus ARGs poderiam ser rapidamente eliminados em presença da complexa comunidade da microbiota fecal. Este estudo foi realizado em ensaios de microcosmo com material fecal (FMAs) inoculado com bactérias isoladas de água residual, portadoras de genes de resistência a antibióticos conhecidos (Capítulo 5). Os isolados de águas residuais inoculados foram Escherichia coli (estirpe A2FCC14) e Enterococcus faecium (estirpe H1EV10), portadores dos ARGs blaTEM, blaCTX, blaOXA-A e vanA, respectivamente. O efeito de condições como a presença ou ausência de oxigénio ou os antibióticos cefotaxima ou vancomicina, na composição da comunidade microbiana do material fecal e na persistência dos ARGs exógenos foram investigados. Os FMAs foram monitorizados nos tempos 0, 1, 3 e 7 dias de incubação, com recurso a métodos de cultivo, PCR quantitativo (qPCR) e análises da comunidade bacteriana com base Resumo xii na sequência nucleotídica de amplicões do gene para o rRNA 16S. Na comunidade bacteriana do material fecal predominavam membros dos filos Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria e Verrucomicrobia. Esta comunidade sofreu aumento na abundância relativa de Proteobacteria, quando os FMA foram incubados na presença de oxigénio. A presença de doses sub-inibitórias de antibióticos não esteve associada a variações relevantes na composição da comunidade microbiana. Os ARGs das ARB inoculadas persistiram durante o período de 7 dias em todos os ensaios e, ao fim de 30 dias, podiam ainda ser quantificados, tanto em condições de aerobiose como de anaerobiose. A adição de doses sub-inibitórias de antibióticos não se correlacionou com variações significativas na persistência das ARB ou ARGs testados, quando comparados os respetivos controlos em antibióticos. Este estudo sugeriu que ARB que vivem em águas residuais podem persistir pelo menos por uma semana na presença de microbiota fecal complexa e, mesmo quando essas ARB estão abaixo do limite de detecção cultivável, os seus ARGs eram quantificáveis por qPCR. Esses resultados apoiam a hipótese de que, se as bactérias das águas residuais, por acaso, atingirem o intestino humano, poderá haver uma colonização bem-sucedida, um tema que merece mais investigação. No geral, a investigação realizada nesta tese sugere que as águas residuais tratadas usadas para irrigação podem conter bactérias com relevância clínica que, por essa via, podem estabelecer associação estável com culturas edíveis, como endófitos. Se, como consequência desse fato, essas bactérias atingirem o intestino humano, há indícios de que terão capacidade para ali persistir. Embora os presentes estudos evidenciem a complexidade desta questão, a tese apresentada sustenta que as águas residuais, as variedades agrícolas edíveis e os humanos podem estar interligados na cadeia de transmissão de ARB e ARGs. Assim, esta tese abre também caminhos para novas investigações neste domínio.
The reuse of treated wastewater has been encouraged worldwide, in particular for agricultural irrigation, because it can be a reliable and important water source, mainly in countries under water stress. The recycling of wastewater poses still not evaluated risks concerning, among other, the dissemination of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) across distinct One-Health compartments. One of the main concerns associated with such dissemination is the contamination of the human food-chain through crops irrigation with treated wastewater. Currently, the knowledge about the transmission of ARB and ARGs to humans through environmental sources is sparse. However, wastewater reuse emerges as a potential source for direct or indirect transmission, mainly through the human food-chain. This project addressed these issues, through literature- and experiment-based investigation about potential risks of antibiotic resistance transmission to humans emerging from treated wastewater reuse. The study was planned aiming to infer if bacteria surviving in wastewater would be able to colonize plants and if wastewater ARB and the respective ARGs would be able to persist in the presence of the autochthonous fecal microbiota of a healthy human. The research hypotheses of this work were: 1) ARB and ARGs present in treated wastewater are capable of colonizing plants, and establish as endophytes; 2) ARB and ARGs are able to persist in the presence of the autochthonous human gut microbiota. The rationale behind these two hypotheses was that if ingested through vegetables, ARB and ARGs could be able to persist in the human gut. To test the study hypotheses were used three major approaches: a) it was made a literature search seeking to investigate the diversity of endophytic bacteria that belong to groups that can also be found in wastewater and in the human microbiota as well as in edible crops, normally consumed raw - this approach offered a predictor of the likelihood of a crop behave as a possible mode of transmission of ARB to humans; b) it was analysed the presence of potential ARB and ARGs in isolates from ready-to-eat watercress and strawberry; and c) it was assessed the persistence of ARGs harboured by wastewater ARB in the presence of fecal microbiota of a healthy child. The literature-based investigation aimed to explore the diversity of endophytic bacteria associated with different habitats (plant, wastewater, and human microbiota), and infer the probability that ARB and ARGs might be transferred from wastewater to plants and then to humans (Chapter 3). This literature survey permitted to shortlist of more than 20 phyla of Abstract ii endophytic bacteria distributed over more than 45 crop varieties. Because the major focus was on potential links of transmission to humans, the array of crops found in the literature was narrowed to plants that can be consumed raw, such as lettuce, carrot, radish, cucumber and tomato. In order to assess the likelihood that these plants uptake bacteria that might be antibiotic resistance vectors, the bacterial groups identified as endophytes were also investigated as potential wastewater and human microbiome member communities. Finally, for bacterial groups, whose occurrence was reported in the three types of environments, the previous description of antibiotic resistance and virulence genes was surveyed from public databases and literature. The search revealed that members of genera such as Enterobacter, Acinetobacter, Pseudomonas, and Staphylococcus, with known taxonomic proximity to human opportunistic pathogens and harbour of acquired antibiotic resistance may be found as endophytes in edible crops as well as in wastewater or the human microbiota. These results support the hypothesis that bacteria transported by wastewater, with physiological and ecological properties similar to endophytes, may be uptake by edible crops and being ingested may eventually colonize the human body. It is suggested that wastewater irrigation may raise the risks of transmission of clinically relevant bacteria from the environment to humans, via the food-chain. Plant associated bacteria were also searched on ready-to-eat watercress and strawberry, based on culture-dependent methods (Chapter 4). Ready-to-eat watercress were purchased at local supermarkets and strawberries samples were collected along a fruit processing chain of a company that produces fruits preparations for the food industry. Bacteria adsorbed onto watercress leaves as well as strawberry-associated bacteria (epiphytic and endophytic community) were isolated and analysed. The antibiotic resistance phenotype of watercress (n=68) and strawberry (n=52) isolates was characterized. Isolates exhibiting resistance to at least three antibiotics classes were selected for identification based on the 16S rRNA gene sequence analysis, and the presence of class 1 integron integrase gene intl1, the ARGs sul1, blaTEM, blaCTX-M, blaOXA-A and blaSHV and the plasmid incompatibility groups FIA, FIB, FIC, HI1, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA by conventional Polymerase Chain Reaction (PCR) were screened. Some isolates were also tested for the capacity to acquire ARGs by transformation assay. Ready-to-eat watercress presented counts of total heterotrophs in the order of 3.5 x 107 ± 4.9 x 107 and 5.4 x 105 ± 6.2 x 104 CFU/mL of watercress washing solution for supermarket A and B, respectively, whilst processed strawberry presented counts of total heterotrophs in the order of 9.1 x 105 ± 1.8 x 105 CFU/g of strawberry dry weight, with some isolates exhibiting antibiotic resistance phenotypes. The Abstract iii bacteria identified belong to the genera Pseudomonas, Stenotrophomonas, Erwinia, Rahnella, Methylobacterium, and Chryseobacterium. These bacterial taxa were previously identified as endophytes and some, such as members of the genera Pseudomonas and Stenotrophomonas are closely related to human opportunistic pathogens that can harbor acquired ARGs. The screening of ARGs, int1 and plasmid incompatibility groups revealed that these genetic elements were present within a minority group (2 isolates) of the examined plant-associated bacterial isolates. Additionally, preliminary results suggested that watercress and strawberry isolates could be susceptible to acquire antibiotic resistance and thus be involved in the dissemination of antibiotic resistance. The third study was designed based on the arguments that wastewater bacteria as well as their harboured ARGs would be rapidly lost in the complex community of fecal microbiota. This study was performed using fecal microcosm assays (FMAs) inoculated with wastewater ARB isolates harbouring known ARGs (Chapter 5). The inoculated wastewater isolates were Escherichia coli (strain A2FCC14) and Enterococcus faecium (strain H1EV10), harbouring the ARGs blaTEM, blaCTX, blaOXA-A and vanA, respectively. The effect of variables such as the presence or absence of oxygen or sub-inhibitory dose of the antibiotic cefotaxime or vancomycin on the feces microbial community composition and on the persistence of the exogenous ARGs were investigated. FMAs were monitored at time 0, 1, 3 and 7 days of incubation based on cultivation methods, quantitative PCR (qPCR) and 16S rRNA gene sequence amplicon community analyses. The fecal bacterial community was characterized by the predominance of members of the phyla Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Verrucomicrobia. The presence of oxygen was associated with an increase in the relative abundance of Proteobacteria, while antibiotics did not lead to consistent microbial community composition variations. The ARGs harboured by the inoculated ARB persisted over the 7 days incubation period in all the assays and could still be quantified at least for one month, under both aerobic and anaerobic conditions. Sub-inhibitory concentrations of antibiotics were not correlated with significant variations on the persistence of the tested ARB or ARGs, when compared with antibiotic-free microcosms. This microcosm-based investigation suggested that wastewater ARB can persist at least for a week in the complex fecal microbiota and, even when these ARB are below the culture detection limit, their ARGs were quantifiable by qPCR. These results support the hypothesis that, if wastewater bacteria by chance can reach the human Abstract iv gut, a successful colonization cannot be disregarded, a topic that deserves further investigation. Overall, the investigation conducted in this thesis suggests that bacteria with potential clinical relevance that may occur in treated wastewater used for irrigation, may establish stable association with edible crops, as endophytes. If, as consequence of this fact, these bacteria reach the human gut, they may be capable of persisting there. Although this investigation put in evidence the complexity of this issue, showing that further studies are required, the thesis presented is that the wastewater, edible crops and humans may be part of the same transmission link chain of ARB and ARGs.

Les infections nosocomiales sont causées par des germes opportunistes souvent résistants aux antibiotiques et persistants sur les surfaces, représentant une source constante de risque d’infection en milieu hospitalier. Dans ce contexte, l’isolement et la caractérisation de bactériophages s’attaquant spécifiquement aux bactéries nosocomiales telles que Staphylococcus aureus résistant (SARM), Enterococcus résistant (ERV), Pseudomonas aeruginosa et Acinetobacter baumanii, pourraient fournir une alternative bactéricide naturelle contre la transmission de ces infections. Des phages isolés des eaux usées, ont été sélectionnés selon leur capacité d’amplification, leur profil génomique et leur potentiel lytique envers différentes souches bactériennes cliniques. Les meilleurs ont été caractérisés en détail pour s’assurer de leur spécificité, sécurité, stabilité et efficacité préalablement à leur utilisation in vivo. Sept phages contre SARM et trois contre Acinetobacter baumanii ont été caractérisés. Quatre phages SARM s’avèrent être de bons candidats potentiels et pourraient être testés en milieu hospitalier comme agents désinfectants dans le but de lutter contre les infections nosocomiales.
Nosocomial infections are directly related to opportunistic germs, which are often resistant to antibiotics and persistent on surfaces, representing a high infectious risk in hospitals. In this context, the isolation and characterization of bacteriophages specifically targeting nosocomial bacteria such as resistant Staphylococcus aureus (MRSA), resistant Enterococcus (VRE), Pseudomonas aeruginosa and Acinetobacter baumanii, could provide a natural bactericidal alternative against the transmission of these infections. Phages, isolated from waste water, were selected according to their capacity of amplification, their genomic profile and lytic potential towards various bacterial clinical strains. The best ones were characterized in detail to primarily ensure their specificity, safety, stability and effectiveness, before studying their in vivo usage. Seven phages against MRSA and three against Acinetobacter baumanii were characterized. Four MRSA phages proved to be good potential candidates and could be tested in hospitals as disinfectant agents with the aim of fighting nosocomial infections.

This diploma thesis is focused on the evaluation of six selected zoonoses with the occurrence in South Bohemia (campylobacteriosis, salmonellosis, listeriosis, tick-borne meningoencephalitis, Lyme borreliosis, tularemia) between the years 2003-2013. Campylobacteriosis and salmonellosis were evaluated the most common zoonoses in the South Bohemian Region during the monitored period. Their common feature is the alimentary transmission. Between 2003 to 2008 salmonellosis recorded the highest number of reported cases, on the contrary, campylobacteriosis has reported the highest number of occurrences since 2008. District of Czech Budweis showed the highest incidence of both zoonoses during the monitored period (4,139 cases of salmonellosis and 4,924 cases of campylobacteriosis). Another but not less important zoonotic is tick-borne meningoencephalitis, which incidence had the highest number in South Bohemian Region of all the regions of Czech Republic. The second part of the thesis is based on questionnaire studies focused on awareness of zoonoses and the issue of resistance and overuse of antibiotics. In total 479 questionnaires were evaluated. The survey findings may be equally described as interesting and positive, since the respondents had considerable awareness of the issue of antibiotic resistance.