Rozprawy doktorskie na temat „Gram Negative Baterial Infections”

Urinary tract infections (UTIs) are among the most common infections caused by both Gram-negative and Gram-positive bacteria, acquired in the community and hospitals. There are three main groups of UTIs: (i) lower UTIs that affect the urethra or the bladder, (ii) upper UTIs that affect the kidneys, or (iii) asymptomatic bacteriuria (ABU). Group B Streptococcus (GBS) is a Gram-positive bacteria known to cause a variety of infections in neonates, pregnant women, the elderly or immunocompromised individuals. GBS has been estimated to cause 1-2% of all single organism UTIs. GBS has been shown to form biofilms, on abiotic and biotic surfaces, protecting it from killing by antibiotics or host immune cells and promotes host colonisation. Various factors have been shown to affect the biofilm forming ability of GBS. Here we determined that LB supplemented with glucose was the optimal media for biofilm formation by a strong biofilm forming strain. We then investigated the biofilm forming phenotype of 292 clinical GBS isolates that presented with asymptomatic, acute, or recurrent infection. We found that there was no significant difference in the biofilm forming ability across the clinical presentations. We also showed a significant reduction in the biofilm forming ability of a strong biofilm forming strain and its isogenic maeK and maeE mutants in LB supplemented with 1% glucose. A multiplex PCR screen for genes encoding bsaB, pil1, pil2a, and pil2b found that there was no significant difference in the number of strains that had the right sized fragments for all four genes across the three clinical presentations. We also found that there was a significant difference in the proportion of strains that had the right sized fragments for the pil genes across the three different levels of biofilm activity under shaking conditions. High biofilm forming strains had the lowest proportion of strains that possessed all four genes, compared to low and medium biofilm formers. Lastly, we assessed the haemolytic activity of the strains by growing them on tryptic soy agar plates supplemented with 5% horse blood and found that asymptomatic strains had a significantly higher number of strains with high haemolytic activity compared to acute and recurrent strains.
Thesis (Masters)
Master of Medical Research (MMedRes)
School of Medical Science
Griffith Health
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Nas últimas décadas, a incidência de infecções hospitalares causadas por bactérias Gramnegativas multidrogarresistentes (MDR) vem crescendo de maneira vertiginosa em todo o mundo, de modo que a Organização Mundial de Saúde (OMS) recentemente reconheceu essas infecções como uma preocupação mundial devido ao seu impacto negativo sobre as taxas de mortalidade intra-hospitalar e dos custos da assistência à saúde, afetando tanto os países desenvolvidos quanto os em desenvolvimento. Atualmente considera-se que a higienização das mãos, o uso racional de antimicrobianos e o isolamento de contato são as principais medidas disponíveis para contenção desse avanço. Porém, elas são apenas parcialmente efetivas e de implementação trabalhosa e onerosa. Assim, considera-se necessário o desenvolvimento de formas mais simples e eficientes paralidar com esse problema. No presente estudo, nos propusemos a avaliar o impacto da administração de um produto simbiótico a pacientes colonizados e/ou infectados por bactérias Gram-negativas MDR sobre as taxas de descolonização desses patógenos no trato digestivo. Trata-se de um ensaio clínico randomizado, duplamente cego, controlado com placebo, envolvendo 101 pacientes hospitalizados com colonização prévia por bactérias Gram-negativas MDR, demonstrada por meio de cultura seletiva de swab retal, cuja intervenção consistiu na administração oral ou enteral diária de 1010 unidades de Lactobacillus bulgaricus e 1010 unidades de Lactobacillus rhamnosus associados a fruto-oligossacarídeos durante (FOS) 7 dias. O desfecho primário do estudo foi a descolonização completa do trato digestivo posterior à intervenção, que, na análise do tipo \"intenção de tratar modificada\" foi de 16,7% (8/48) no grupo experimental e 20,7% (11/53) no grupo controle (p=0,600). Na análise \"per protocol\", a descolonização completa do trato observada foi de 18,9% (7/37) no grupo experimental e 23,3% (7/30) no grupo controle (p=0,659). Em uma análise multivariada por meio de modelo de regressão logística o uso do simbiótico não influenciou significativamente o risco de descolonização completa do trato digestivo (OR= 0,80, IC 95%= 0,28-2,27, p= 0,678). A ocorrência de eventos adversos de natureza leve a moderada foi semelhante entre os grupos: 7,55% no grupo que utilizou placebo e 6,25% no grupo sob intervenção (p= 1,000). Nenhum evento adverso grave potencialmente relacionado às medicações de estudo foi observado. Nas condições estudadas, os dados obtidos pelo estudo nos levam à conclusão de que o simbiótico estudado demonstrou-se inefetivo na descolonização do trato digestivo de pacientes previamente colonizados por bactérias Gram-negativas MDR.
In recent decades the incidence of multidrug resistant (MDR) Gram-negative nosocomial infections has been dramatically raising in the whole world. The World Health Organization (WHO) recently recognized nosocomial infections due to MDR pathogens as a global concern due to its negative impact on patients, health-care workers and health-care institutions, affecting developed countries as well as developing ones. They negatively impact in-hospital mortality and health-care related costs. Hand hygiene promotion, antibiotic stewardship and contact precautions are the main available measures to control such MDR Gram-negative organisms in hospitals. However, they are only partially effective as well as difficult to be implemented and expensive. Therefore, simpler and more effective actions are thought to be helpful and urgent. In the present study, we analyzed the impact of the administration of a symbiotic product on patients harboring Gram-negative multidrug-resistant bacteria upon the subsequent rates of decolonization of these pathogens from the gastro-intestinal tract.This is a double-blinded and placebo controlled randomized clinical trial evaluating the oral/enteral daily administration of 1010 units of Lactobacillus bulgaricusplus 1010 units of Lactobacillus rhamnosus associated with fructo-oligosacharide (FOS), or placebo, for 7 days, to 101 patients previously colonized by MDR Gram-negative bacteria, identified through selective culture of rectal swab. The primary study outcome was the rate of complete decolonization of the MDR microorganism from the gastro-intestinal tract following the intervention. In the \"modified intention to treat\" analysis, decolonization rates observed were 16.7% (8/48) in the experimental group and 20.7% (11/53) in the placebo group (p=0,600). In the \"per protocol\" analysis, decolonization rates were 18.9% (7/37) in the experimental group and 23.3% (7/30) in the placebo group (p=0,659). In a logistic regression model, symbiotic use did not produce any impact on the chance of decolonization (OR=0.80, CI95%=0.28-2.27, p=0.678). Mild to moderate adverse events occured similarly in both the placebo (7.55%) and the experimental group (6.25%), (p=1,000). No severe adverse event potentially related to the medications was detected during the study period. In the present study conditions, the results obtained lead to the conclusion that the studied symbiotic proved to be ineffective to decolonize patients harboring multidrug resistant Gram-negative bacilli.

Antimicrobial resistance is a worldwide problem that has deleterious long-term effects as the development of drug resistance outpaces the development of new drugs. Plants have been used for many generations for healing purposes, and screening of extracts of these plants has often yielded positive outcomes. This study was aimed at isolating and characterizing the major active antimicrobial compounds present in the stem bark of C. molle, in a bid to identify potential sources of cheap starting materials for the synthesis of new drugs. Various solvents (hexane, ethyl acetate, dichloromethane, acetone, ethanol and methanol) were used for extraction. The agar well diffusion technique was used to screen for antimicrobial activity of C. molle extracts against Streptococcus pyogenes ATCC 49399, Plesiomonas shigelloides ATCC 51903, Pseudomonas aeruginosa ATCC 15442, Helicobacter pylori ATCC 43526 and Helicobacter pylori 252C (clinical isolate); minimum inhibition concentration (MIC) of the most active extracts was determined by the broth dilution method. Fractionation of acetone extract was done by thin layer chromatography (TLC) and bioautography to determine the compounds present and their antimicrobial activity respectively. The acetone extract was purified by column chromatography and their MIC determined. The most potent fraction (EA4) was subjected to Gas chromatography- Mass spectrometry (GC-MS) and High performance liquid chromatography (HPLC) for identification of the active compounds. Results were analyzed by the Fisher‟s exact test. All the extracts tested demonstrated antimicrobial activity with zone diameters of inhibition ranging from 0–32 mm. Acetone was the most potent extract with its MIC ranging from 0.078–5.0 mg/mL. Seventeen fractions were collected from column chromatography and the most active fraction against all the organisms was EA 4 (eluted with 100 percent ethyl acetate), with its MIC ranging from 0.078 - 2.5mg/mL. There was no statistically significant difference (P>0.05) in the potency of the xii four extracts (acetone, methanol, ethanol and ethyl acetate) and antibiotic (ciprofloxacin) on the different bacterial strains tested, likewise the crude extract and the fractions. No compound was detected by GC-MS whereas numerous peaks were identified by HPLC implying that the active compounds in this plant are non volatile. We could not identify the compounds thereby proposing further studies using Nuclear magnetic resonance to identify the compounds. The study revealed that the acetone extract of C. molle was the most active against all the test organisms and therefore justifies the use of this plant in traditional medicine.

P. aeruginosa est une bactérie pathogène de l'homme, responsable d'infections nosocomiales chez les patients immunodéprimés. Bien que son évolution au sein d'un patient soit bien décrite, son évolution génomique globale au cours de sa propagation dans un hôpital est très mal connue. Le clone à haut-risque ST395 multirésistant aux antibiotiques a diffusé dans le Centre Hospitalier Regional Universitaire de Besançon entre 1997 et 2008 en infectant ou colonisant plus de 300 patients. Une approche WGS a été utilisée afin d'identifier l'origine de l'épidémie, les caractéristiques ayant aidé à son installation à l'hôpital ainsi que celles à l'origine de sa disparition. Les génomes de 54 isolats représentatifs de l'épidémie ont été séquencés. L’arbre phylogénétique a mis en évidence deux clusters distincts indiquant la présence de deux épidémies parallèles. La datation d'un ancêtre commun en 1979, date de début de la construction de l'hôpital, indiquerait une contamination précoce du réseau d'eau de l'hôpital. Cette hypothèse est soutenue par la présence d'un îlot génomique spécifique de ST395 portant les gènes codant 6 transporteurs du cuivre et associée à une résistance phénotypique à ce métal constituant les tuyaux du réseaux de distribution d'eau potable. Les isolats tardifs présentaient des signatures génomiques d'adaptation à l’infection chronique (altération du lipopolysaccharide et de la porine OprD – objectivées phénotypiquement, et extinction de la surproduction de la pompe d’efflux MexAB-OprM – contrôlée par RT-qPCR) suite à des mutations indépendantes. Certaines de ces mutations ont été associées à une perte de fitness bactérien. Nous émettons l’hypothèse que l’émergence indépendante d’isolats adaptés à l’infection chronique, et ainsi l’accumulation de culs-de-sac épidémiologiques, a participé à l’épuisement de l’épidémie hospitalière de P. aeruginosa ST395
P. aeruginosa is an opportunistic pathogen responsible of hospital-acquired infections in immunocompromised patients. Although in-host evolution of P. aeruginosa is well documented, little is known about this pathogen evolution during its spread on a hospital scale. The high-risk multidrug resistant clone ST395 spread among more than 300 patients in the University Hospital of Besançon between 1997 and 2008. We used a WGS approach to identify the origin of the outbreak, the features that could have helped its implantation in our hospital and those associated with the end of the epidemics. The genomes of 54 representative isolates were fully sequenced. The phylogenetic tree indicated two distinct clusters corresponding to two parallel outbreaks. The ancestor of the ST395 clone possibly contaminated our hospital water network during its construction in 1979. This hypothesis is supported by the fact that the ST395 strain had a specific genomic island carrying 6 copper transporter genes implicated in copper resistance, correlated with the resistance to this metal which water supply network is made of. The late isolates displayed independent genomic signatures of chronic adaptation in patients (altered LPS and porin OprD, and extinction of MexAB-oprM efflux pump overproduction). Some of these mutations were associated with a decreased in vitro fitness. We hypothesize that the independent emergence of isolates adapted to chronic infection, and thus the accumulation of epidemiological dead-ends, participated to the end of the hospital outbreak of P. aeruginosa ST395

Gram-negative infection has been linked to hospital water although few studies have examined whether water systems are reservoirs of nosocomial pathogens. This study investigated longitudinal recovery of the opportunistic pathogens Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Acinetobacter baumannii from water outlets of a haematology unit and evaluated Point-Of-Use Filtration (POU-F) as a control measure. In a two-year double cross-over trial, water samples and swabs were taken weekly from 39 showers/taps on the unit. Four study phases alternated between non-filtered (Phases 1 & 3), and filtered outlets (Phases 2 & 4) using Pall AquasafeTM 14-day filters. In Phases 1 & 3; 99% of 1396 samples yielded bacterial growth, with colonies generally too numerous to count. Target species were isolated from 22% of water samples (P. aeruginosa 14%; S. maltophilia 10%) and 10% of swabs. P. aeruginosa was particularly associated with handwash stations and S. maltophilia with showers. A. baumannii was not isolated. With POU-F; 22% of 1242 samples yielded bacterial growth (mean CFU/100ml ,4.6). S. maltophilia was isolated only once from water but never from outlet swabs. PCR typing identified clusters of isolates colonizing different outlets over time but no clear association between water and patient isolates was identified. The incidence of Gram negative infections remained low throughout the study. Without POU-F, water from taps/showers represented a source of bacteria including the target species. POU-F substantially reduced the frequency and number of target species from every outlet, and merits further investigation as an intervention to protect immunocompromised patients from opportunistic pathogens.

Quarenta e nove amostras EPEC típica (tEPEC) e atípica (aEPEC) pertencentes a diferentes sorotipos, isoladas de humanos e animais (cães, gatos, bovinos, ovinos, coelhos e sagüis) foram investigadas quanto ao perfil de virulência pela PCR e similaridade clonal por Multilocus Sequence Typing (MLST) e Pulsed-Field Gel Electrophoresis (PFGE). O objetivo deste estudo foi verificar se animais atuam como reservatório e fonte infecção de aEPEC para humanos. Os marcadores de virulência analisados revelaram que cepas aEPEC isoladas de animais possuem potencial para causar diarréia em humanos. As técnicas MLST e PFGE revelaram que amostras isoladas de animais e humanos compartilham relações clonais próximas ou idênticas. Estes resultados indicam que os animais estudados atuam como reservatório de aEPEC e representam fonte de infecção para humanos. Pelo fato de humanos, também atuarem como reservatório de aEPEC, ciclos de infecção cruzada animal-humano não podem ser descartados, pois a dinâmica de transmissão entre reservatórios de aEPEC não é muito bem compreendida.
Forty-nine typical and atypical EPEC strains belonging to different serotypes, isolated from humans, pets (cats and dogs), farm (bovines, sheep and rabbits) and wild animals (monkeys) were investigated for virulence markers and clonal similarity by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The virulence markers analyzed revealed that atypical EPEC strains isolated from animals have the potential to cause diarrhea in humans. Close clonal relationship between human and animal isolates was found with MLST and PFGE. These results indicate that these animals act as atypical EPEC reservoirs and may represent sources of infection for humans. Since humans also act as a reservoir of atypical EPEC strains, the cycle of mutual infection of atypical EPEC between animals and humans, mainly pets and their owners, cannot be ruled out, since the transmission dynamics between the reservoirs are not yet clearly understood.

La surexpression des pompes d’efflux (PE) appartenant à la famille Resistance-Nodulation-Division (RND) est l’un des contributeurs majeurs de la multirésistance (MDR) et la pathogénicité des bactéries Gram-négatives. Ces transporteurs sont capables d'expulser à l’extérieur de la cellule bactérienne différentes classes d'antibiotiques, ce qui contribue de manière significative à l'échec thérapeutique du traitement des maladies infectieuses. Dans ce contexte, les PEs sont des cibles intéressantes pour la découverte de nouveaux antimicrobiens. Afin de combattre ce mécanisme de résistance, des inhibiteurs des pompes d’efflux (EPIs) sont développés comme adjuvants d'antibiotiques dans le but de restaurer ou d'améliorer leur activité. L'archétype AcrAB-TolC est particulièrement répandu chez les espèces d’Enterobacter pertinentes en clinique (pathogènes « ESKAPE »). Cette étude décrit une stratégie basée sur des analogues des fluoroquinolones pour le drug design des EPIs, contre la pompe AcrB chez E. aerogenes. Ainsi, la synthèse et l'évaluation microbiologique des dérivés de quinazoline-4(3H)-one ont été effectuées. Les propriétés structurales et moléculaires des composés testés (i.e. rigidité et flexibilité) ont également été étudiées. Pour cela, de nouveaux scaffolds ont été évaluées. Plusieurs molécules ont montré une augmentation de la sensibilité des bactéries à la norfloxacine et au chloramphénicol. Les résultats obtenus, appuyés par la modélisation moléculaire, suggèrent que la flexibilité moléculaire et la nature des fonctions chimiques des EPIs jouent un rôle essentiel dans l'amélioration de l'activité et la sélectivité vis-à-vis des fluoroquinolones
Overexpression of Resistance-Nodulation-Division (RND) efflux pumps (EP) is a major contributor in multidrug resistance (MDR) and pathogenicity in Gram-negative bacteria. These transporters are able to expel out of the bacterial cell clinically important antibiotic classes, contributing in a significant manner to the treatment failure of infectious diseases. With the worrying levels of bacterial resistance reported worldwide and the continuous spreading of MDR pathogens, EPs are interesting targets for the discovery of new antimicrobial drugs. Therefore, to overcome this mechanism, efflux pump inhibitors (EPIs) are being developed as adjuvants in order to restore or improve the activity of usual antibiotics. The AcrAB-TolC archetype is particularly widespread in Enterobacter spp. presenting clinical relevance (ESKAPE pathogens). In this study, we described the drug design strategy based on fluoroquinolone antibiotic analogs, against the AcrB pump of E. aerogenes. Thus, synthesis and microbiological evaluation of quinazolin-4(3H)-one derivatives were performed. The structural and molecular properties of the tested compounds (i. e. rigidity and flexibility) were also investigated. In this purpose, a scaffold hopping of the quinazolinone core to homologous benzoquinazolinones and precursors benzamides were carried out. Several molecules increased the bacterial susceptibility towards norfloxacin and chloramphenicol. The obtained results, supported by molecular modeling, suggest that molecular flexibility and the nature of chemical functions play a critical role to improve activity and selectivity on fluoroquinolone potentiation targeting AcrB efflux pump

Infecção do Trato Urinário (ITU) é o segundo tipo de infecção bacteriana mais comum em crianças. Nesse estudo amostras de urina de 6012 pacientes pediátricos foram analisadas, a prevalência de ITU foi determinada, os uropatógenos foram identificados e o perfil antimicrobiano dos mesmos foi determinado. Os resultados mostraram que a prevalência de ITU varia de acordo com o sexo e a idade do paciente. Bactérias Gram-negativas foram responsáveis por 89 % de todos os casos de ITU e Escherichia coli foi a espécie mais prevalente. Os uropatógenos foram resistentes a ampicilina 63 %, a nitrofurantoina 37 % e ao trimethoprim-sulfamethoxazole 28 %. Todavia, 99 % deles foram sensíveis a cefalexina e 96 % ao cloranfenicol. Resultados obtidos com a caracterização de 90 isolados de E. coli, mostraram que todas as amostras foram positivas para os marcadores fimA e fimH, 53 % para pap, 32 % para sfa, 10 % para o marcador genético da toxina pic e 29 % foram capazes de produzir hemolisina-a. Esses isolados se distribuíram entre os grupos filogenéticos da seguinte maneira: B2 42 %, D 25 %, A 21 % e B1 11 %. Dessas amostras 19 % não foram tipáveis (ONT), 15,56 % pertenceram ao sorogrupo O2 e 12,22 % aos sorogrupos O6 e OR. A maioria dos isolados de E. coli aderiu às células epiteliais, poliestireno e PVC.
Urinary Tract Infection (UTI) is the second most common type of bacterial infection in children. In this study, 6012 urine samples from pediatric patients were analyzed, the prevalence of UTI was determined, the uropathogens were identified and their antimicrobial profile was determined. The results have shown that the prevalence of UTI varies according to the sex and age of the patient. Gram negative bacteria were responsible for 89 % of all cases of UTI and E. coli was the most prevalent species. The uropathogens were resistant to: ampicillin 63 %, nitrofurantoin 37 % and trimethoprim-sulfamethoxazole 28 %. However, 99 % of them were sensitive to cephalexin and 96 % to chloramphenicol. Results obtained with the characterization of 90 isolates of E. coli showed that all of them were positive for fimA and fimH, 53 % were positive for pap, 32 % were positive for sfa, 10 % were positive for the genetic marker of pic and 29 % were able to produce hemolysin-a. These isolates were distributed between the phylogenetic groups as follows: B2 42 %, D 25 %, A 21 % and B1 11 %. Nineteen percent of these samples were untypeable (ONT), 15.56 % belonged to O2 serogroup and 12,22 % belonged to the O6 and OR serogroups. Most E. coli isolates were able to adhere to epithelial cells, polystyrene and PVC.

L’augmentation et la dissémination de la résistance aux β-lactamines chez les bacilles à Gram négatif, particulièrement les Entérobactéries, les bactéries du genre Pseudomonas et Acinetobacter, représentent un problème majeur de santé publique. Les infections nosocomiales causées par ces bactéries multi-résistantes (BMR) ont conduit à une augmentation de la mortalité, de la morbidité et du coût de traitement. L’utilisation abusive et non contrôlée de ces antibiotiques a grandement contribué à la large diffusion de cette résistance. Ainsi, face à cette préoccupation mondiale et suite à de nombreuses recommandations, plusieurs études épidémiologiques et moléculaires ont été rapportées afin de contrôler et de surveiller la diffusion et la dissémination des BMR. Contrairement à de nombreuses régions dans le monde, il existe peu d’informations concernant la caractérisation moléculaire des gènes de résistance aux β-lactamines des bacilles à Gram négatif isolés en Tunisie et surtout en Libye. C’est dans cette optique que ce projet de Thèse de Doctorat s’articule avec comme objectifs: (i) mettre en évidence la prévalence des bacilles à Gram négatifs multi-résistants isolés aux niveaux des hôpitaux tunisiens et libyens (ii) identifier le support génétique de la résistance aux β-lactamines de ces souches cliniques (iii) étudier la diversité clonale des souches multi-résistantes par typage moléculaire
The increase and spread of β-lactam resistance in gram negative bacteria especially Enterobacteriaceae, Pseudomonas and Acinetobacter (E.P.A) species have become a major concern worldwide. The hospital-acquired infections caused by MDR bacteria have led to an increase in mortality, morbidity and cost of treatment. The frequent misuse of antibiotic drug has greatly contributed to worldwide dissemination of antibiotics resistance. Front of this worldwide concern, and various recommendations, several epidemiological and molecular studies have been reported in order to control the spread and the dissemination of these MDR. Unlike many parts of the world, there is little information concerning the molecular characterization of the β-lactam resistance genes of Gram-negative bacilli isolated in Tunisia and especially in Libya. Therefore, it is in this context that the project of this thesis was conducted with essential objectives: (i) highlight the prevalence of multi-resistant Gram negative bacilli isolated in Tunisian and Libyan hospitals (ii) identify the genetic support of resistance to β-lactams of these clinical strains (iii) study the clonal diversity of the multi-resistant strains by molecular typing (iii) study the molecular epidemiology of these BMRs in these countries in order to control the decision-making process of the treatment and the rapid identification of epidemics by implementing appropriate control measures for the spread of infections and especially developing new tools and software for the diagnosis and monitoring of potential MDR bacteria in Mediterranean countries

La détection, la surveillance et la diffusion de la résistance des bactéries aux antibiotiques est un enjeu majeur au niveau mondial depuis la découverte et la diffusion de bactéries multi résistantes, en particulier la résistance aux carbapénèmes, spécifiquement chez les Entérobactéries et les bactéries du genre Pseudomonas et Acinetobacter. L’émergence et la dissémination des pathogènes Gram- résistants aux carbapénèmes est un contributeur significatif de la morbidité et la mortalité du patient. Malgré les efforts radicaux dans le contrôle de l’infection et les améliorations dans le diagnostique moléculaire, les bacilles Gram- résistants aux carbapénèmes demeurent une formidable menace vue que quelques agents antimicrobiens sont actifs et très peu devraient être disponibles dans le futur proche.L’origine et la source des gènes de résistance dans le monde sont mal connues et des travaux récents suggèrent que les animaux domestiques et sauvages, l’environnement mais également le tube digestif des mammifères et des humains pourraient représenter un réservoir et une source importante de gènes de résistance susceptibles d’être transmissibles à l’homme. C’est dans cette optique que ce projet de thèse s’articule avec comme objectifs : (i) la réalisation d’études épidémiologiques moléculaires d’isolats cliniques et animales résistants aux carbapénèmes isolés dans le basin Méditerranéen et la caractérisation des supports moléculaires de cette résistance ; (ii) la description de nouveaux mécanismes de résistance a l’imipenème ; et enfin (iii) le séquençage de génomes d’isolats cliniques résistants aux carbapénèmes et l’analyse de ces derniers
The detection, monitoring and dissemination of bacterial resistance to antibiotics are a major issue worldwide since the discovery and spread of multi-resistant bacteria, in particular resistance to carbapenems, specifically among Enterobacteriaceae and bacteria of the genus Pseudomonas and Acinetobacter.The emergence and dissemination of carbapenem-resistant Gram-negative pathogens is a significant contributor to patient morbidity and mortality. Despite radical efforts in infection control and improvements in molecular diagnostics, carbapenem-resistant Gram-negative bacilli remain a formidable threat as few antimicrobial agents are reliably active and very little is expected to be available in the near future.The origin and source of resistance genes in the world are not well known and recent works suggest that domestic and wild animals, the environment (soil, water, rivers ..) but also the digestive tract of mammals and humans could represent a reservoir and an important source of resistance genes that may be transmissible to humans.It is in this context that this thesis project articulates with the following objectives: (i) The achievement of molecular epidemiological studies on carbapenem-resistant clinical and animal isolates collected from countries in the Mediterranean basin (Lebanon, Libya, France) and the characterization of the genetic determinants of this resistance; (ii) the description of new resistance mechanisms to imipenem; and finally (iii) The genome sequencing of clinical isolates resistant to carbapenems, the analysis of these genomes and the identification of mechanisms and genetic supports of the resistance to carbapenems and other antibiotics

Helicobacter pylori is a Gram-negative microaerophilic bacteria known to infect as much as 80% of some populations with an average morbidity range of around 50% of the world population. It has been recognized as a definitive carcinogen (Type I). H. pylori shows extraordinary genetic diversity and this property is critical to its success as a human pathogen. High genetic diversity and interstrain variations seen in H. pylori is attributed to its remarkable ability to take foreign DNA by natural transformation. Natural transformation in H. pylori is governed to some extent by the presence of Restriction-Modification systems (R-M systems). Three types of DNA methylation are associated with R-M systems in bacteria, N6-adenine (m6A), C5-cytosine (m5C) and N4-cytosine (m4C). Recent studies in pathogenic bacteria have shown the epigenetic roles of m6A in virulence, gene regulation and genetic evolution of the organism. This is in contrast to eukaryotes where m5C is known to be the epigenetic signal. In mammals and plants DNA cytosine methyltransferases epigenetically regulate the gene expression through the precise epigenetic modification of certain cytosine residues with a methyl group. Moreover, aberrant methylation patterns are embryonic lethal in mammals, and can also lead to diseases including cancer. In plant it can result in pleiotropic morphological defects. The role of cytosine methylation in bacteria is not very well known. A recent study has shown that the loss of m5C in H. pylori strains alters the expression of genes involved in motility, adhesion, and virulence. Another study in E. coli has shown the role of m5C in stationary phase stress regulation. However, no physiological role of the other form of cytosine methylation (m4C), aside restriction protection is known in bacteria. Genome sequences of various strains of H. pylori reveal an abundance of R-M systems. Typically 25-34 R-M systems are present in different H. pylori strains. Methylome analysis of H. pylori 26695 strain has revealed the presence of numerous m6A and m5C methyltransferases. H. pylori 26695 strain harbors a phase variable type IIS HpyAII R-M system. This R-M system is composed of two exocyclic methyltransferases, M1.HpyAII (m6A) and M2.HpyAII (m4C) and one type IIS phase variable endonuclease (HpyAII). HpyAII recognizes the sequence 5' GAAGA 3' / 3' CTTCT 5' and cleaves eight bp downstream on the top strand and seven bp downstream on the bottom strand. HpyAII is a novel phase-variable restriction endonuclease containing multiple repetitive stretches of adenine residues in the ORF. M1.HpyAII methylates the final adenine residue of GAAGA sequence, whereas M2.HpyAII methylates the first cytosine of the complementary TCTTC sequence. M2.HpyAII is the only N4-cytosine (m4C) MTase present in H. pylori strain 26695. The aim of the present study is to understand the potential epigenetic role of m4C modification by understanding the roles of HpyAII R-M system in virulence, gene expression and natural transformation of H. pylori. Understanding the biochemical properties of the novel phase variable HpyAII endonuclease can reveal critical information about the regulation of natural transformation in H. pylori. Bioinformatics analysis shows that HpyAII is an HNH catalytic motif containing endonuclease. The biochemical study on HpyAII indicates that the enzyme prefers two-site substrate over a one-site substrate for maximal activity. A strong preference for two-sites was observed with supercoiled plasmid and oligonucleotide duplex DNA. Cofactor analysis revealed the preference of R.HpyAII for transition metals (Ni2+, Cd2+, and Co2+) over alkaline earth metals (Mg2+, Ca2+) for maximal cleavage activity. Mutational analysis of the conserved residues of the HNH motif in HpyAII confirmed the presence of functional HNH motif. Interestingly, mutation of first His residue (general acid) of the HNH motif to Ala does not abolish the enzymatic activity but instead causes loss of fidelity compared to wild type HpyAII. The H328A mutant displayed promiscuous DNA cleavage activity on different DNA substrates. The novelty of this observation lies in the fact that mutation of first His residue (general acid) of the HNH motif in other known HNH motif containing enzymes has always abolished enzymatic activity. Mutation at a single amino acid residue leading to the loss of fidelity provides insights into the regulation of fidelity and evolution of restriction enzymes by point mutation.

Patients who experience multiple hospitalizations over short periods of time may be at greater risk of hospital-acquired infections (HAIs). While it is known that prior hospitalizations are associated with HAIs, there is a gap in knowledge regarding which factors of prior hospitalizations have an impact on the risk of HAIs in subsequent hospitalizations. HAIs caused by gram-negative bacteria (GNB) are of particular concern due to their propensity to develop drug resistance and the limited antibiotics available to treat them. The aims of this dissertation are to: 1) examine clinical and patient risk factors associated with acquiring at least one gram-negative hospital-acquired infection in adult patients with multiple hospitalizations; 2) systematically review the literature assessing the association between repeat gram-negative bacterial infections and changes in antibiotic susceptibility patterns; and 3) assess the association between repeat infections with three common gram-negative pathogens and risk of subsequent drug resistant infections with the same species among patients with multiple hospitalizations. A retrospective cohort study was conducted to identify risk factors from prior hospitalizations associated with incident HAIs caused by three common GNB. Of the 129,372 patients with multiple hospitalizations, 1,672 (1.3%) acquired K. pneumoniae, 1,127 (0.9%) acquired P. aeruginosa, and 262 (0.2%) acquired A. baumannii infections. In survival analyses, older age, mechanical ventilation, history of chronic diseases, and increasing days of use of antibiotics decreased the time to infection for all 3 pathogens. This study highlights potential modifiable risk factors for infection control. Patients with multiple hospitalizations are also inherently at greater risk for repeat HAIs which may result in decreased antibiotic susceptibility, making them more difficult to treat. A systematic review was conducted to evaluate if there is an association between repeat GNB HAIs and drug resistance. From 2000 to 2015, only seven studies explicitly examined repeat GNB HAIs and change in antibiotic susceptibility, five of which reported decreased susceptibility in later infections. The association between repeat GNB HAIs and risk of drug resistance among patients with multiple hospitalizations was then investigated with available electronic medical record data. The risk of a drug-resistant K. pneumoniae HAI increased by 1.14 times (95%CI: 1.04-1.24) with each prior K. pneumoniae HAI, after adjusting for potential confounders and antibiotic use. Similarly, patients with repeat P. aeruginosa infections had a 1.23 times increased risk of a subsequent drug-resistant infection (95%CI: 1.12-1.36) with each prior P. aeruginosa HAI as compared to patients with only one infection. Repeat A. baumannii infections were not analyzed due to limited sample size. The studies in this dissertation demonstrate that patients with multiple hospitalizations are a high-risk population for GNB HAIs. Prevention of GNB HAIs in this group is critical in order to reduce complications to medical care and limit transmission of infections to others in healthcare facilities and the community. Patient medical history can be used for infection risk assessment and to guide future medical care to reduce risk of infection in patients with multiple hospitalizations.

The Gram-negative bacterium Shigella flexneri is the causative agent of Shigellosis, also known as bacillary dysentery, responsible for 5-15% of the global burden of diarrhoeal disease. Despite over 50 years of Shigellosis research, the search for a commercially viable vaccine is ongoing. A thorough understanding of bacterial pathogenesis is paramount for the development of an effective vaccine. To this end the aim of this thesis is to further our understanding of S. flexneri pathogenesis by characterizing novel virulence factors and to develop a new animal model of shigellosis. The first aim of this study is to investigate the role of L-asparaginase (AnsB) and gamma-glutamyltranspeptidase (GGT) in S. flexneri pathogenesis. Using a reverse genetic approach I found that AnsB and GGT are required for bacterial adherence to host cells. In vivo studies in both the Caenorhabditis elegans and the murine pulmonary model of shigellosis revealed that AnsB and GGT contribute to S. flexneri virulence. Differential in-gel electrophoresis (DIGE) showed that ansB and ggt mutations exert pleiotropic effects on the expression of a number of S. flexneri genes, including prominent bacterial outer membrane proteins, OmpA and YaeT. This is the first report in S. flexneri where the functions of AnsB and GGT have been found to extend beyond their canonical metabolic roles. The requirement of AnsB and GGT for the virulence of S. flexneri makes these genes attractive candidates for designing new Shigella vaccine strategies. The second aim of this study is to identify bacteriophage genes that contribute to S. flexneri virulence. To date in S. flexneri, the O-antigen modifying genes are the only bacteriophage genes that have been linked to host virulence. Here the S. flexneri phage SfII was isolated from a highly prevalent S. flexneri serotype 2a strain and completely sequenced to identify novel bacteriophage-encoded genes. In parallel, uncharacterized genes in the S. flexneri phage, SfV were studied to identify potential phage-encoded virulence factors. A mutant lysogenic strain lacking five phage genes identified as expressed in the host, orf28-32, was generated. In vitro and in vivo virulence studies indicate that genes within the SfV orf28-32 cluster play a role in host virulence. This is the first reported study to identify bacteriophage-encoded virulence factors outside of the O-antigen modifying cluster in S. flexneri. The third aim of this study is to characterize C. elegans as a small animal model of shigellosis. In recent years the use of C. elegans as an animal model for several microbial diseases has been gaining momentum. Using electron microscopy, I have shown that virulent strains of S. flexneri are ingested by C. elegans and that S. flexneri cells invade the nematode intestinal cells. DIGE was used to compare the proteomes of nematodes infected with S. flexneri and control strains to successfully identify nematode responses to S. flexneri. These findings are significant as they provide further evidence supporting the use of C. elegans as a viable model to study shigellosis.

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2017
One of the most concerning health problems present in the twenty-first century is antibiotic-resistant infections. The growing number of multi-resistant infections has led to complicated public health problems that require the most consideration. Simultaneously there are economic and regulatory barriers. Having all of these difficulties in mind, some researchers are trying to find new therapeutic classes that may became reliable alternatives to the therapeutics use nowadays. One of these new classes may be antimicrobial peptides. AMPs have demonstrated antimicrobial activity which makes them a good choice for research. Their cationic amino acids confer the molecule an attraction towards some components of the bacteria, this leads to the possibility of destroying the microorganism due to membrane rupture. The AMP studied on this work was BP214, which was characterised in some previous studies. BP214 has shown a similar antimicrobial activity to colistin (a last-line therapeutic) and a reduced hemolytic activity compared to other AMPs. Therefore, on this study, eleven different BP214s analogues were synthesised and studied in order to discover which amino acids are essential to its antimicrobial and hemolytic activities. Each of these analogues differs from BP214 only in one amino acid that was replaced by an alanine. The results demonstrated that only the peptides that had a lysine or an arginine replaced showed an improved antimicrobial activity but also an increase in their hemolytic activity. The remaining replacements resulted in a loss of antimicrobial and hemolytic activities of the peptide. It was also possible to associate the loss of activity to the low hydrophobicity of the molecule, which resulted from the amino acids replacement. The peptides where the lysine or the arginine were replaced, were also the ones that demonstrated the higher hydrophobicity. Both amino acids are basic and possess high values of pKa. This characteristic can be closely related with higher antimicrobial activity.
Um dos maiores problemas do século vinte e um tem sido a resistência aos antibióticos. O crescente número de infeções multirresistentes desencadeou um problema de saúde pública, o que requer uma enorme preocupação por parte da sociedade. Aliados a esta problemática estão os obstáculos a nível económico e regulatório. Tendo todas estas dificuldades em consideração, alguns investigadores estão a tentar encontrar novas classes terapêuticas que possam conferir alternativas às terapêuticas existentes de modo a podermos combater estas infeções com armas inovadoras e contra as quais os patogénicos ainda não possuam resistências. Uma das novas classes emergentes podem ser os péptidos antimicrobianos. Estes péptidos têm demonstrado uma atividade antimicrobiana que faz deles uma boa opção contra as bactérias resistentes. Os seus aminoácidos catiónicos conferem-lhes uma atração específica a determinados componentes das bactérias, levando depois à possibilidade de as eliminar por rotura da membrana. Estes péptidos têm também atividades imunomodulatórias em que são capazes de aumentar a resposta imunitária contra o patogénico, aumentar a quimioatração e ativar a resposta inata e adaptativa, antibiofilme em que conseguem impedir a sua formação mesmo abaixo da sua concentração mínima inibitória e anticancerosas pela sua atividade citolítica específica para tecido tumoral que, tal como as bactérias está carregado negativamente. Entre as várias vantagens dos péptidos antimicrobianos está a facilidade de síntese. Através de uma síntese bastante simples é possível manipulá-los aminoácido por aminoácido de modo a obter um híbrido o mais potente possível em comparação com o péptido original. Deste modo, é viável controlar a sua relação estrutura-atividade. De uma maneira tão simples, é também possível adicionar novos aminoácidos à sequência ou mutar o péptido de modo a torná-lo mais estável ou menos agressivo para o ser humano. Esta síntese pode ser feita em pequena escala no laboratório, mas também pode ser transferida para ser produzida numa grande escala de modo automatizado. Apesar de entre si poderem ser bastante diferentes, os péptidos antimicrobianos possuem algumas características que são transversais a todos eles. Por exemplo, possuem um comprimento médio de menos de 60 aminoácidos, são geralmente carregados positivamente (com carga entre +2 a +9) e as suas estruturas são flexíveis e anfipáticas, ou seja, parte da molécula é hidrofóbica e outra é hidrofílica. Vai ser esta última característica que vai permitir ao péptido passar da sua conformação em solução para a conformação que permite a sua entrada na membrana da bactéria. Esta particularidade vem dos seus aminoácidos catiónicos e hidrofóbicos. São estes aminoácidos catiónicos que desencadeiam uma atração electroestática para as moléculas aniónicas da membrana da bactéria, como os lipopolissacáridos das bactérias gram-negativas e os ácidos lipoteicóicos das gram-positivas. É também através desta peculiaridade que mantém um elevada especificidade para as células procariotas ao invés das eucariotas. A estrutura catiónica vai então levar a uma interação do péptido com a membrana da bactéria e também com os lípidos da membrana citoplasmática, ambos carregados negativamente. A sua carga positiva vai então estabilizar a carga dos fosfolípidos da membrana e causar a sua perturbação. A estrutura anfipática vai depois permitir a sua inserção na bicamada da membrana resultando na sua disrupção e posterior morte da bactéria. O mecanismo exato de como esta disrupção acontece ainda não é totalmente conhecido, sendo que existem diversas teorias para o explicar mas ainda nenhuma foi globalmente aceite. As teorias existentes até ao momento são o modelo “barrel-stove” (i) em que se assume que a inserção do péptido se dá com a orientação das regiões hidrofóbicas no core dos lípidos, levando à formação de um poro que causa a disrupção da membrana; o modelo “toroidal-pore” (ii) que supõe que a inserção do péptido na bicamada leva a que esta se dobre e forme um poro que permite a associação do péptido às cabeças polares dos fosfolípidos; o modelo “aggregate” (iii) em que se crê que vai ocorrer a formação de agregados péptido-lípido, agregados esses que levam a flutuações na condutância e translocações de péptidos na membrana; e, por último, o modelo “carpet” (iv) em que se supõe que a conformação anfipática do péptido leva à sua acumulação na membrana da bactéria formando uma espécie de carpete, causando a disrupção (Figura 1). Para além desta capacidade de disrupção da membrana da bactéria que os péptidos antimicrobianos possuem, eles possuem ainda alvos intracelulares. Apesar de precisarem de estar a uma determinada concentração para serem capazes de causar a lise das bactérias, com apenas concentrações muito baixas conseguem atingir os seus alvos no interior da bactéria, provando assim que estes fenómenos acontecem por mecanismos distintos. Estes péptidos vão então ser capazes de inibir o material genético da bactéria ao nível do ADN, do ARN, da síntese proteica e também da atividade citosólica das suas enzimas. O péptido estudado neste trabalho foi o BP214 que já foi estudado em trabalhos anteriores e que demonstrou uma atividade antimicrobiana semelhante à colistina (uma terapêutica de última linha para o tratamento de infeções bacterianas multirresistentes) e uma atividade hemolítica reduzida quando comparado com outros péptidos antimicrobianos, tendo assim menos efeitos indesejados. Tendo esses estudos em conta, neste trabalho foram sintetizados onze análogos do péptido BP214 e, posteriormente, estudados ao nível da atividade antimicrobiana e hemolítica. Este trabalho foi realizado no sentido de poder descobrir quais os aminoácidos da sequência deste péptido que são essenciais para a sua atividade antimicrobiana e hemolítica e quais podem vir a ser alterados de modo a podermos aumentar a atividade antimicrobiana mas diminuir a atividade hemolítica, de modo a torna-los viáveis ao nível comercial. Cada um dos análogos vai diferir do BP214 apenas num único aminoácido que é consecutivamente substituído por uma alanina, um aminoácido bastante simples. Os resultados demonstraram que apenas os péptidos em que uma lisina ou uma arginina foram substituídas resultaram num aumento da atividade antimicrobiana mas também num aumento da atividade hemolítica. As restantes substituições levaram à perda de atividade antimicrobiana e também hemolítica. Foi também possível associar essa perda de atividade à diminuição do carácter hidrofóbico da molécula, resultado da substituição que foi feita. Este parâmetro foi avaliado pela percentagem de eluente B, o eluente hidrófobo, utlizado no HPLC. Os péptidos em que se trocaram a lisina ou a arginina eram também aqueles que tinham maior hidrofobicidade e nos quais ocorreu um aumento das atividades, quer hemolítica quer antimicrobiana. Quer a lisina como a arginina são aminoácidos básicos que conferiram um carácter mais básico à molécula. Esta característica pode estar intimamente relacionada com o aumento das suas atividades antimicrobianas e hemolíticas.

A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Master of Pharmacy Johannesburg, 2018.
The increased prevalence of multi-drug resistant (MDR) Gram-negative infections in critically ill patients has resulted in the re-introduction of colistin as rescue therapy. Various guidelines for colistin administration have led to confusion in establishing the appropriate dose which has potential for adverse consequences including treatment failure or toxicity. Colistin, also known as Polymixin E, is a concentration-dependent bactericidal antibiotic considered to be highly nephrotoxic and neurotoxic. Colistin is used either intravenously to treat life threatening systemic infections or by nebulisation for the treatment of respiratory tract infections. Although colistin resistance has been documented in South Africa, there is no local evidence as to why and how colistin is used in hospitals and similarly compliance with current dosing guidelines is unknown. This study aimed to evaluate the utilization of colistin in order to identify stewardship opportunities regarding its’ appropriate use in the future. A retrospective electronic record review of adult patients treated with intravenous (IV) and aerosolised colistin therapy in four Gauteng private hospitals was conducted between 1 September 2015 - 30 June 2016. The following data were collected on a standardized template; patient demographics including: age, gender, weight and hospital location; laboratory indicators including: renal function markers of creatinine and estimated Glomerular Filtration Rate (eGFR), as well as, culture specimens taken and their corresponding results. With regards to the colistin therapy: the indication for use, admitting diagnosis, the prescribed dose, frequency and route of administration, duration of treatment and if prescribed in combination with another Gram-negative antibiotic was considered. The following stewardship principles were monitored in addition to appropriate dose and duration; if a culture was taken prior to the initiation of treatment, if therapy was de-escalated and if a loading dose was prescribed. Outcome measures included overall in-hospital mortality, intensive care unit length of stay and overall hospital length of stay. Furthermore, compliance to two local colistin dosing guidelines was measured and a colistin stewardship bundle was developed, including nine process measures, to enhance the appropriate use of IV colistin. A total of 237 patients were included in the study of which 212 received colistin IV and, 25 via nebulisation. The results of patients who received IV colistin therapy demonstrated an 81.2% overall compliance to the proposed colistin stewardship bundle developed from this study. Non-compliance was mainly due to incorrect maintenance doses prescribed (50%), ‘hang time’ (66%) and poor de-escalation practices (69%). Significantly shorter durations of treatment were found in patients who received higher loading doses (p=0.040) and in those that received maintenance doses of 4.5 Million Units (MU) twice daily vs 3 MU three times daily (p=0.0027). In addition, more of the patients that demised received the 3 MU three times daily maintenance doses, compared to those who survived (p=0.0037). Aerosolised colistin was only prescribed in one of the four hospitals studied. Of those patients who received aerosolised colistin, 13 were for cystic fibrosis and 12 for other nosocomial lower respiratory tract infections (LRTI’s). Compliance to appropriate dose for the cystic fibrosis patients was good at 92.3%, however, for other LRTI’s was poor at only 41.7%. This study demonstrated that there is noteworthy prevalence of MDR Gram-negative infections in South African hospitals which requires the use of colistin. In addition, the study identified many stewardship related opportunities to improve appropriate colistin utilization in particular relating to dose for both routes of administration. The implementation of a colistin stewardship bundle is necessary, as a matter of urgency, to preserve the efficacy of this last resort antibiotic.
LG2018