Introduction: In this decade, the CTX-M family of enzymes are considered to be the most common type of extended-spectrum β-lactamases (ESBLs). The production of these Class A β-lactamases are noted to be most prevalent in the Enterobacteriaceaeae family. Many global reports indicated that CTX-M-15, of the CTX-M-1 group, is a growing concern, causing resistance to different classes of antibiotics. Worrisome trends of the spread of this enzyme have been indicated in nosocomial and community settings worldwide. Moreover, the same predicament is faced along the Middle East area, especially in the absence of restricted antibiotic usage policies. Many reports from Kuwait indicated the spread of a multi-drug resistant (MDR) blaCTX-M-15 gene in different hospitals. blaCTX-M-15 genes are often known to be carried on large transferable plasmids. Usually, the mobilization of these plasmid-encoded enzymes is carried out by insertion sequence like ISEcp1. Aims: This work aims to investigate the distribution of blaCTX-M genes in five major hospitals in Kuwait and to study and analyse the genetic environment of the described blaCTX-M genes. Materials and methods: One hundred and seven isolates of E. coli (84) (78.50%) and K. pneumoniae (23) (21.49%) were collected between 2006 and 2010 from five distantly located hospitals in Kuwait. All of the collected isolates were identified as ESBL-producers using Vitek 2 system. The production of cefotaximases was detected using disc diffusion with cefotaxime and clavulanic acid according to Clinical and Laboratory Standards Institute (CLSI) criteria. Conformation of CTX-M production was maintained by PCR amplification and further sequencing. The minimum inhibitory concentrations (MICs) of the collected isolates were determined by the double dilutions agar method described by the CLSI. Four different classes of antibiotics were used (aminoglycosides, different generations of cephalosporins, fluoroquinolones, and 3 different carbapenems). The genotypic relatedness of the collected strains was assessed by the use of an enhanced Pulsedfield gel electrophoresis (PFGE) method. Further amplifications with primer walking and simplex PCR were done to seek the genetic context of the MDR strains. S1 nuclease was used to size plasmids carrying the described blaCTX-M genes and conjugation studies were implemented to detect the transferability of the plasmids carrying the reported blaCTX-M genes. Results: All of the collected strains showed to be ESBL-producers and in particular cefotaximases-producers. Upon amplification, CTX-M-1 group was the only CTX-Mgroup present in the collected strains. When sequenced, blaCTX-M-15 was found to be the most prevalent. In addition, strains carrying the blaCTX-M-3 gene were identified, these have previously been found in the Middle East; however, this thesis has the first descriptions of blaCTX-M-28, blaCTX-M-55, and blaCTX-M-117 in this region. After the determination of the MICs of the collected strains, 94 (87.85%) were resistant to cefepime, 107 (100%) to cefotaxime, 48 (44.85%) to cefoxitin, 78 (72.89%) ciprofloxacin, and 71 (66.35%) to gentamicin. All of the strains were susceptible to carbapenems. Twenty-eight strains (26.2%) showed MDR pattern. With the enhanced PFGE method, only 22 isolates exhibited banding patterns that allowed grouping them into 10 distinct PFGE clusters. Notably, strains sharing ≥85% were from the same hospitals (isolates 1 with 2, 21 with 22, and 91 with 92 from the maternity hospital (M), 52 with 53 from Kuwait Oil Company hospital (KOC), 78 with 79 and 83 with 84 from Infectious Diseases Hospital (IDH), 97 with 98 and 95 with 96 from Al-Amiri hospital(A) ). Primer walking and simplex PCR experiments used for the genetic environment studies yielded 7 different genetic constructions for the described blaCTX-M genes. All of the described blaCTX-M genes were carried on plasmids ranging in size from 60 – 271 kb. Only 3 of the selected strains were of IncFII and the rest wereindeterminate. Possibly, two blaCTX-M-15 genes are likely to be carried on the chromosome. All of the described blaCTX-M genes are considered to be transferable except for blaCTX-M-28. The sizes of the conjugative plasmids and incompatibility groups are the same as their parental plasmids. Conclusion: In conclusion, blaCTX-M-15 is the most common ESBL gene found in Kuwaiti hospitals. It is also causing a MDR pattern with resistance to 3 different generations of cephalosporins and to two other classes (aminoglycosides and gentamicin), but sensitive to carbapenems. This led to restricting the treatment option into carbapenems. Antibiotic selective pressure could have played a major role in the development of blaCTX-M-15 derivatives such as blaCTX-M-28, blaCTX-M-55, and blaCTX-M-117. The probable explanation of the spread of blaCTX-M-15 is horizontal gene transfer carried by ISEcp1 and the conjugative properties of the plasmids carrying blaCTX-M-15. Variability of the genetic environments obtained explains the nonconditional existence of ISEcp1 to the ‘’W’’ region. Absence of the ISEcp1 in one of the reported structures of blaCTX-M-15 genetic contexts is noted. Therefore, the existence of blaCTX-M-15 could be due to the presence of another insertion sequences downstream or as a part of a larger gene cassette.

No description available.

La Tesi descriu la prevalença de les soques portadores de betalactamases d'espectre ampliat (BLEA), cefamicinases i carbapenemases, principals mecanismes de resistència als antibiòtics betalactàmics, no només en l'àmbit clínic [mostres clíniques i malalts atesos al Servei d'Urgències (portadors fecals)] sinó en altres ecosistemes com en els subsòls de granges d'animals (aus, porcs i conills), en aigües residuals humanes, en aliments, i en portadors fecals afectats per un brot alimentari. A més a més s'han caracteritzat aquests enzims per tal de determinar la freqüència de cadascun dels tipus de betalactamases presents: tipus TEM, SHV i CTX-M com a BLEA, CMY, DHA o ACC com a cefamicinases, o VIM, IMP i altres com a carbapenemases. La conclusió del treball mostra com aquests enzims es poden trobar en tots els ecosistemes avaluats i, per tant, com la selecció de soques portadores d'aquests no es dóna només en l'àmbit clínic sinó que roman en tot l'entorn humà. Per altra banda, arran d'aquest treball s'han descrit dues noves betalactamases, la CTX-M-14b, i la CTX-M-51, ambdues derivades de la CTX-M-9. El fet que les soques portadores d'aquests enzims s'hagin trobat en tots els àmbits és causa de greu preocupació. Cal, doncs, intentar evitar la selecció d'aquestes soques, així com esbrinar quin o quins són els mecanismes que han permès aquesta expansió. L'estudi epidemiològic dels mecanismes de resistència, és de gran interès, no només en l'àmbit clínic sinó també veterinari, perquè coneixent la prevalença i el tipus d'enzims implicats es pot actuar oportunament dissenyant polítiques d'antibiòtics correctes. / This thesis describes the prevalence of strains carrying extended spectrum betalactamase (ESBL), plasmid-mediated AmpC and carbapenemase. There are the main mechanism of betalactam resistance, not only in clinic environment [clinical samples and patients attending the emergency room (faecal carriers)] also in others environments such as the floor of animal farms (poultry, pig and rabbit), the urban sewage, food, and human faecal carriers in the context of food-borne disease outbreaks. We have characterized these enzymes to know the frequency of each type of betalactamase: TEM, SHV and CTX-M type as ESBL, CMY, DHA and ACC as plasmid-mediated AmpC, or VIM, IMP and others as carbapenemase. The work's conclusion show's that these enzymes can be found in all environments evaluated. Then the selection of strains carrying this kind of enzymes is due not only to clinic environments otherwise to all human environments. In the other hand, we have described two new betalactamase, CTX-M-14b and CTX-M-51, both deriving from CTX-M-9.The finding of strains carrying these enzymes in all the environments is of great concerning. We need not to select these strains, and to know what are the mechanisms of expansion of these enzymes. The epidemiologic study of the resistance mechanism is of great interest, not only in hospitals also in veterinarian environment, because knowing the prevalence and the type of enzymes, we can design specific antibiotic policy.

Escherichia coli and Klebsiella pneumoniae cause a wide range of infections. Multidrug-resistance strains carrying extended-spectrum β-lactamases (ESBLs) has become a growing problem worldwide. The CTX-M type ESBLs has emerged distinctly, especially in Escherichia coli and Klebsiella pneumoniae. CTX-M type has been associated with many outbreaks of infections both in the hospitals and community. CTX-M-15 is now identified as the most predominantly distributed CTX-M enzyme. Clonal outbreaks of CTX-M-15 producing Enterobacteriaceae have been described in many countries including the United Kingdom, and Escherichia coli is the most commonly involved species. A total of 100 isolates were received in 2010 from London St George’s hospital, England, 50 Escherichia coli, 17 Klebsiella spp, 9 Enterobacter spp, 13 Proteus spp, 6 Lactose fermenting coliforms, 2 Pantoea spp, one Serratia marcescens, one Morganella morganii, and one Hafnia alvei. The antimicrobial susceptibility results showed that 5 Escherichia coli and one Klebsiella pneumoniae isolates were found to be resistance to cefotaxime, ceftazidime, ceftriaxone, cefotaxime, ciprofloxacin, and gentamicin, making them multi-drug resistant bacteria. None of the isolates showed resistance to imipenem, ertapenem, or morepenem, thus making carbapenems the drug of choice for the treatment of these infections due to multi-resistant isolates. The overall frequency of CTX-M-15 type ESBL-producers detected in this study was 6 (6%) most of them 5/6 (83%) were from Escherichia coli and one was (17%) Klebsiella pneumoniae isolates. The 6 CTX-M-positive isolates were typed by PFGE, only two strains of Escherichia coli showed more than 85% similarity, owing to clonal homology for both strains. The rest strains showed less than 85% similarity. S1 nuclease plasmid profiles were obtained for ESBL-producers isolates. A total of one to three plasmids per isolate, ranging from approximately 78.0 to 152.0 kb, were observed. The plasmids from most isolates were assigned to be IncFA and IncFB replicons. Analysis of phylogenetic groups showed group A and group B2. The method of phylogenetic classification of exteraintestinal pathogenic Escherichia coli depends on examine and combination of two preserved genes (chuaA and yjaA) and the DNA fragment TSP. Primer walking and PCR experiments were used for the genetic environment studies which showed 5 different genetic constructions for the described blaCTX-M-15 genes. Conjugation studies were used to detect the transferability of the plasmids harbouring the reported blaCTX-M-15 genes. Three isolates were found transferable by conjugation. In conclusion, this study reports the presence of hospital highly resistant blaCTX-M-15 in St George’s hospital. The spread of blaCTX-M-15 is probably due to horizontal gene transfer harbouring ISEcp1 and the conjugative properties of plasmids carrying blaCTX-M-15.

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Em nosso meio, a resistência às cefalosporinas de amplo espectro em enterobactérias tem sido associada à produção de β-lactamases de espectro ampliado (ESβL). Porém, nos últimos anos, a produção de cefalosporinases do tipo AmpC mediadas por genes plasmídiais (pAmpC) também tem sido responsabilizada pela resistência a essas drogas. O objetivo deste estudo foi avaliar a presença de pAmpCs entre amostras de enterobactérias isoladas de pacientes internados no Hospital São Paulo que apresentaram infecção de corrente sangüínea, entre janeiro e julho de 2006. Foram estudadas 133 amostras de enterobactérias identificadas como K. pneumoniae (65 amostras), K. oxytoca (5 amostras), E. coli (41 amostras), P. mirabilis (18 amostras) e Salmonella spp. (4 amostras). O perfil de sensibilidade aos antimicrobianos selecionados foi avaliado pela técnica de diluição em ágar, segundo as recomendações do CLSI. A detecção fenotípica da produção de pAmpC foi realizada pelos testes tri-dimensional e de Hodge modificado. Adicionalmente, foi realizada a detecção fenotípica da produção de ESβL. A pesquisa e identificação dos genes que codificam pAmpC foram realizados pela técnica de reação em cadeia da polimerase (PCR) e seqüenciamento. Entre os antimicrobianos testados, imipenem (99,2%) apresentou a maior taxa de sensibilidade, seguido pela cefoxitina (80,5%). De forma geral, os isolados de K. pneumoniae apresentaram alto grau de resistência aos β- lactâmicos e os isolados de P. mirabilis apresentaram a menor taxa de sensibilidade para ciprofloxacino (27,8%). Entre as 133 amostras estudadas, 59 (44,4%) foram classificadas fenotipicamente como produtoras de ESβL pela metodologia de disco aproximação. Foi observada discordância entre os resultados dos testes fenotípicos para detecção de pAmpC. A produção de pAmpC foi observada em seis isolados de acordo com o teste tri-dimensional, enquanto, o método de Hodge modificado classificou 19 isolados como possíveis produtores de pAmpC. Além disso, resultados duvidosos foram observados em ambas as técnicas. Um único isolado de K. pneumoniae foi identificado como produtor de CMY-2. Este isolado foi corretamente identificado como produtor de pAmpC por ambos métodos fenotípicos empregados. / The broad-spectrum cephalosporins are the main therapeutic options to Enterobacteriaceae infections and the resistance to these agents has been associated to ESβL production. However, plasmid-mediated AmpC β-lactamases (pAmpC) have been associated with this resistance phenotype. The aim of this study was to determine the occurrence of pAmpC among clinical isolates recovered from bloodstream from patients hospitalized at a Brazilian teaching hospital, collected between January and July 2006. A total of 133 non-repetitive Enterobacteriaceae per patients (65 K. pneumoniae, 41 E. coli, 18 P. mirabilis, 05 K. oxytoca and 04 Salmonella spp.) were studied. The antimicrobial susceptibility profile was determined by CLSI agar dilution method. ESβL phenotype was detected by doubledisk diffusion method while pAmpC production was evaluated by two phenotypic methods: modified three-dimensional and modified Hodge tests and the presence of pAmpC genes was confirmed by PCR and sequencing. Among tested antimicrobial, imipenem showed the highest susceptibility rate (99.2%), followed by cefoxitin (80.5%). K. pneumoniae presented high resistance to β-lactams. P. mirabilis isolates showed the lower susceptibility rate to ciprofloxacin (27.8%). Fifty-nine (44.4%) of studied isolates were phenotypically classified as ESβL producers. Modified threedimensional and Hodge methods classified 06 and 19 Enterobacteriaceae strains as pAmpC producers, respectively. Discordant results were observed between phenotypic pAmpC detection methods. Of those, a single K. pneumoniae isolate was confirmed as CMY-2 producer. / TEDE / BV UNIFESP: Teses e dissertações

Les taux de multi-résistance aux antibiotiques chez les entérobactéries sont croissants en milieu hospitalier en Tunisie. Cette évolution remet notamment en cause l'efficacité de certaines molécules d'importance clinique majeure telles que les ß-lactamines.Par ailleurs, la résistance aux ß-lactamines n'est pas restreinte aux bactéries responsables d'infections humaines, elleest également rencontrée chez les bactéries issues du milieu vétérinaire. C'est dans ce cadre que s'est inscrit ce travail de thèse, qui avait pour objectif d'identifier les mécanismes de résistance aux céphalosporines de dernière génération chez des souches bactériennes isolées des deux contextes (humain et animal), et de faire une caractérisation moléculaire des plasmides responsables dans le but de contribuer à une meilleure connaissance de l'épidémiologie comparée Homme/animal.La caractérisation génotypique a montré que, dans les deux contextes, l'enzyme CTX-M est largement dominante parmi les?-lactamases à spectre étendu (BLSE) identifiées. Plus spécifiquement, le plasmide CTX-M-1/IncI1/ST3, déjà bien implanté chez l'animal en Europe, est également très prévalent chez l'animal en Tunisie, qu'il s'agisse d'animaux de production ou de compagnie. Chez l'Homme, les souches hospitalières de K. pneumoniaeproduisent principalement l'enzyme CTX-M-15, dont le gène codant est porté par des plasmides de type IncFII.Egalement, une carbapénémase très récemment décrite (OXA-204) a été identifiée dans notre hôpital, associée à un plasmide du type IncA/C. Des gènes de résistance plasmidique aux fluoroquinolones ont également été décrits.Ces résultats d'épidémiologie moléculaire sont essentiels pour une meilleure compréhension des risques de transmission croisée entre l'Homme et l'animal, ainsi qu'entre différents contextes de soins (communauté et hôpital) en Tunisie / Multidrug resistance in Enterobacteriaceae is rising up dramatically at hospital in Tunisia. Such an evolution impairs the clinical efficacy of antibiotics for humans, in particular of the widely used ß-lactams.On the other hand, resistance to ß-lactamsis not restricted to bacteria affecting humans but has also been abundantly recognized in bacteria isolated in veterinary medicine. The aimof this thesis was to identify the resistance mechanisms to broad-spectrum cephalosporins in bacteria isolated from humans and animals and to fully characterize the plasmids carrying those genes in order to contribute to a better knowledge of the molecular epidemiology of resistance in both contexts.Genotypic characterization showed that the CTX-M was the Extended-Spectrum Beta-Lactamases (ESBL) dominant type in both cases. In particular, the CTX-M-1/IncI1/ST3 plasmid, which was already found to circulate in animals in Europe, was also found very prevalent both in food- and companion animals in Tunisia. On the other hand, in humans at hospital, we highlighted the dominance ofK. pneumoniaeisolates producing CTX-M-15 associated with the spread of IncFIIk-type plasmids. We also reported the recently described blaOXA-204carbapenemasegene located on an IncA/C-type plasmid. In addition, plasmid-mediated fluoroquinolone resistance genes were reported.Those data are essential for a better understanding of the comparative molecular epidemiology of resistance to ß-lactams (in particular to broad-spectrum cephalosporins) in Tunisia in order to accurately assess the risk of inter-transmission between humans and animals

The genus Acinetobacter currently contains 34 species, the vast majority of which are not regularly implicated in causing infection. However, incidences of hospitalacquired infection with Acinetobacter species are increasing, mainly due to the rise in the number of infections caused by the species Acinetobacter baumannii in immunocompromised patients particularly in intensive care units (ICUs). Due to high levels of resistance in A. baumannii to many classes of antibiotic, the carbapenems have been portrayed as the ‘drugs of choice’ for treating infections with this species. However, the activity of the carbapenems against A. baumannii has come under threat with the identification of four groups of class D β-lactamases carried by members of the species. Of these, the OXA-51-like enzymes have been suggested to be ubiquitous and intrinsic enzymes within A. baumannii. This presents the worrying scenario of the potential for all A. baumannii to become carbapenem resistant, leaving few treatment options available for this species. This project aimed to investigate the epidemiological spread of the OXA-51-like β-lactamases, examine the diversity within these enzymes, and whether this diversity may have implications for their ability to confer resistance to the carbapenems. A functional map showing the amino-acid similarities between the OXA-51-like enzymes demonstrated that the enzymes fall into distinct closely-related groups, with notable clusters surrounding OXA-66, OXA-69 and OXA-98. PCR and sequencing analysis of a geographically diverse group of 64 A. baumannii isolates demonstrated that isolates forming specific sequence groups (SGs) as defined by Turton et al (2007) also contained the same or closely related blaOXA-51-like gene. Higher minimum inhibitory concentrations (MICs) of carbapenems were found in association with acquired carbapenemases, or with the presence of ISAba1 upstream of the blaOXA-51- like gene. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates did not demonstrate relatedness between isolates which formed the same sequence group. Multilocus sequence typing (MLST) of a subset of 44 isolates grouped isolates more consistently with the SGs and blaOXA-51-like alleles, indicating that PFGE is unreliable for use with A. baumannii unless studying a short time period, and that blaOXA-51-like alleles are a good epidemiological marker. Mutation studies using meropenem with five A. baumannii isolates encoding different OXA-51-like enzymes, while resulting in an increase in meropenem MICs of between 8- and 128-fold, did not result in a nucleotide substitution in the blaOXA- 51-like genes or a change in the upstream region of the genes in any isolate suggesting that the carbapenems may not be producing a strong selective pressure on the blaOXA- 51-like genes. Analysis of πN/πS ratios for the blaOXA-51-like genes, MLST genes and the TEM, SHV and CTX-M β-lactamase families showed the blaOXA-51-like genes to be under less positive selection than these other β-lactamases, though under less purifying selection than the MLST genes. Phylogenetic analysis of the MLST genes and the blaOXA-51-like genes indicates that the blaOXA-51-like genes have been evolving within A. baumannii since its speciation, and that different groups of blaOXA-51-like genes have been evolving at different rates corresponding to different rates of evolution within their parent lineages. Structural modelling studies based upon the published crystal structure for OXA-40 indicated that amino-acid variation at particular sites in the OXA-51-like enzymes are likely to have an effect of enzyme function. Alterations at amino-acid position 167 change the shape of the entrance to the active site which may affect hydrolysis by accommodating the antibiotic differently, or may affect the substrate profile of the enzyme. The substitution of glutamine for proline at position 194 may significantly alter the shape of the enzyme thereby affecting substrate hydrolysis. This project found that specific groups of blaOXA-51-like genes are associated with specific A. baumannii lineages and that these genes could serve as convenient epidemiological markers. Most of the diversity within the OXA-51-like enzymes is due to their continued evolution within A. baumannii since the species’ emergence. However, certain amino-acid changes may play a role in altering the rate of hydrolysis or substrate profile of these enzymes.