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.

Gram-negative bacteria are able to cause many infections including blood stream infections (BSI).These bacteria may become resistant to antibiotics, often by acquiring genes in the presence of antibiotic selection pressure. Multi drug resistant Gram-negative bacteria have become an increasing problem worldwide. A study of antibiotic resistance in Gram-negative bacteria isolated from blood cultures from patients in the New Royal Infirmary of Edinburgh (NRIE) was performed. In addition, a study was performed on isolates from patients in an intensive care unit in Egypt. All isolates were investigated for susceptibility to an extensive range of antibiotics. Gram-negative bacteria from Edinburgh found to be resistant to either cefotaxime or ceftazidime were investigated further. Among the cefotaxime/ceftazidime resistant isolates, Polymerase Chain Reaction (PCR) analysis revealed the presence of CTX-M- β-lactamases. Seven E.coli isolates were found to have CTX-M-15 β-lactamases while the CTX-M-14 β-lactamase was detected in six Enterobacter cloacae. The insertion sequence ISEcp1 was detected upstream of the blaCTX-M-15 gene in some isolates while IS26 was found truncating the ISEcp1 in other isolates. Conjugation experiments found the blaCTX-M-15 gene was transferable to E. coli J62-2. All the isolates had detectable plasmids, a plasmid ~260kb carried the blaCTX-M-15 gene. Analysis of the -containing isolates by PFGE shows that those carrying the CTX-M-14 β-lactamase were identical indicating cross infection within the hospital. The CTX-M- 15 β-lactamase-containing isolates showed four isolates had ≥85% similarity but the others were diverse. Class 1 integrons were found in eight of the CTX-M β-lactamase containing isolates with the associated gene cassette and sul1 gene. The isolates from Egypt were found to be resistant to carbapenem, which is the final mainstream antibiotic option in the treatment of multidrug resistant Gram-negative bacteria. Further analysis revealed all carried the CTX-M-14 β-lactamase and two additionally carried the VIM-4 metallo β-lactamase, which accounted for the resistance to the carbapenems. Furthermore, the insertion sequence ISEcp1 was found upstream of the blaCTX-M-14 gene in two of the isolates. The blaVIM-4 gene was found to be part of the gene cassette in the class 1 integron associate with complex ISCR1. Two of the Egyptian isolates had a detectable plasmid, ~300kb in size, which carried both blaCTX-M-14 and blaVIM-4 genes. All the blood culture isolates were examined to ascertain the persistence of sulphonamide resistance despite the long-term prescribing reduction on this antibacterial. PCR was performed to detect sul1, sul2 and sul3 genes in all the isolates. Of the sulphonamide resistant isolates 25 carried the sul1, 27 carried the sul2 and none carried the sul3 genes. Eight isolates had both the sul1 and sul2 genes. Most of the isolates carried sul1 had Int1 as part of the same class 1 integron. Interestingly three isolates were PCR negative for sul1 but positive for sul2 and int1. Int2 and 3 were found in 3 and 2 isolates respectively. The class 1 integron contained different insert gene cassettes; dfrA (dfrA17, dfrA16, dfrA15), aadA (aadA5, aadA2, aadA1) and blaOXA-1 families in addition to the resident sul gene. In conclusion this thesis shows the diversity of the genetic environment and carriers of the CTX-M β-lactamases within the same hospital. Sulphonamide resistance in Gram-negatives persists despite the prescribing reduction of this antibacterial in a Scottish hospital and the recommended constraint on the use of sulphonamide.

No description available.

303 gesunde, deutsche Freiwillige, die in 53 verschiedene Länder (meist nach Asien, Afrika und Südamerika) reisten, wurden in eine prospektive Kohortenstudie aufgenommen. Stuhlproben und Daten über potenzielle reiseassoziierte Risikofaktoren (wie Reisestil, Essgewohnheiten, Auftreten von Gastroenteritis, Hygienemaßnahmen) wurden vor und nach der Reise mittels Fragebogen gesammelt. Mittels Selektivmedien (CHROMagar™ ESBL/KPC-Platten) wurden eine Untersuchung der Stuhlproben auf extended-spectrum beta-lactamase bildende Enterobacteriaceae (ESBL-PE) und Carbapenemase bildende Enterobacteriaceae (CPE) durchgeführt. Isolate mit bestätigtem ESBL-Phänotyp wurden auf das Vorhandensein von blaCTX-M-, blaTEM-, blaSHV-, blaVIM-, blaNDM-, blaKPC- und blaOXA-48-Genen mit Hilfe von PCR-Amplifikation und -Sequenzierung getestet. Bei den Antibiotikaempfindlichkeitstests wurde mit konventioneller Mikrobouillonverdünnung gearbeitet. Die Auswertung von 205 kompletten Teilnahmen zeigte vor Reiseantritt eine ESBL-PE Prävalenzrate von 6,8% (14/205). Unter den 191 Teilnehmern, die vor der Reise ESBL-negativ getestet wurden, waren nach Reiserückkehr 58 (30,4%) mit ESBL-bildenden Escherichia coli kolonisiert und 5 Reisende (8,6%) waren zusätzlich mit ESBL-produzierenden Klebsiella pneumoniae besiedelt. Carbapenem-resistente Enterobacteriaceae wurden nicht nachgewiesen. Die molekulargenetische ESBL-Typisierung zeigte, dass 52/54 (96,6%) der E. coli und 4/4 (100%) der K. pneumoniae-Stämme, die für die Sequenzierung verfügbar waren, CTX-M-Enzyme produzierten, und zwar überwiegend CTX-M-15 (33/56, 58,9%), und 2/54 (3,7%) der E. coli-Stämme SHV-12-Enzyme bildeten. Die Reisenden nach Indien wiesen die höchste Kolonisationsrate mit ESBL-PE (11/15, 73,3%: p=0.015) auf, gefolgt von Reisenden nach Südostasien (22/46, 47,8%; p=0.038). Die Auswertung der reiseassoziierten Risikofaktoren ergab allein für das Auftreten einer Gastroenteritis eine statistische Signifikanz (p=0.011). Strikte Händehygiene und ausschließliches Konsumieren abgepackter Getränke zeigten keinen protektiven Effekt. Die ESBL-PE-Persistenzrate nach 6 Monaten lag bei 8,6% (3/35). Daraus schlossen wir, dass weltweite Anstrengungen notwendig sind, um die weitere Ausbreitung von ESBL-PE in der Bevölkerung anzugehen. Eine aktive Überwachung und Kontaktisolation ist bei Aufnahme in eine medizinische Einrichtung speziell für Patienten, die innerhalb der letzten 6 Monate nach Indien und Südostasien gereist waren, empfehlenswert.

Entre as ß-lactamases, as enzimas CTX-M têm despertado atenção especial pela alta incidência e grande capacidade de propagação. Eventos como recombinação gênica, transferência plasmideal e multirresistência podem ser a razão da manutenção e da ampla disseminação dos genes blaCTX-M. Este é um trabalho retrospectivo que teve como objetivo caracterizar genes blaCTX-M presentes em Klebsiella spp. Foram estudadas 27 linhagens de Klebsiella pneumoniae e 8 linhagens de Klebsiella oxytoca, produtoras de ?-lactamase de espectro estendido, isoladas de pacientes hospitalizados no período de janeiro a junho de 2000. A detecção e identificação dos genes blaCTX-M, assim como dos elementos relacionados com a mobilização destes genes, foi realizada por PCR e seqüenciamento. A localização genética e a mobilidade dos genes blaCTX-M foram pesquisadas por análise plasmideal e hibridação e por conjugação. Os perfis de sensibilidade das linhagens estudadas e das linhagens transconjugantes foram comparados pela determinação da concentração inibitória mínima de antibióticos das classes das cefalosporinas, cefamicinas, aminoglicosídeos e quinolonas. Foram encontrados genes blaCTX-M em plasmídeos conjugativos em 13 (37%) linhagens estudadas: blaCTX-M-9 em 4 K. oxytoca, e blaCTX-M-2 em 9 K. pneumoniae. Os genes blaCTX-M-9 estavam associados ao elemento de inserção ISEcp1, enquanto os genes blaCTX-M-2 estavam associados a integrons de classe I contendo ISCR1. O genes blaCTX-M-2, carreado por plasmídeo, pode estar relacionado com disseminação horizontal entre vários clones de K. pneumoniae, enquanto o gene blaCTX-M-9 foi encontrado sendo carreado por um único clone de K. oxytoca. Este estudo determinou a incidência e a diversidade de enzimas CTX-M no período estudado, além de fornecer dados epidemiológicos que podem explicar a sua prevalência no mundo e contribuir para o entendimento e controle da disseminação deste tipo de resistência. / CTX-M enzymes, the world\'s most prevalent ß-lactamases disseminate very easily. Genetic recombination, plasmid transference and multiresistance could be responsible for the wide spread of blaCTM-X genes. This retrospective study aims to characterize blaCTX-M genes found in Klebsiella spp. The strains were isolated in hospital patients from January to June 2000 and consisted of 27 ESBL-producing Klebsiella pneumoniae and 8 ESBL-producing Klebsiella oxytoca. PCR and sequencing were used in the detection and identification of blaCTX-M genes and genetic elements associated with their mobilization. Determination of genetic localization and mobility of blaCTX-M genes was by plasmid analyses, hybridization and transfer assays. The minimal inhibitory concentrations (MICs) of cephalosporins, cefamicins, aminoglycosides and quinolone antimicrobials evaluated the antibiotic susceptibility profile of transconjugants and strains in the study. The blaCTX-M genes were found in 13 strains (37%): blaCTX-M-9 in 4 K. oxytoca and blaCTX-M-2 in 9 K. pneumoniae. The insertion sequence ISEcp1 was associated with blaCTX-M-9 and blaCTX-M-2 was found in a class I integron bearing ISCR1. Plasmid blaCTX-M-2 genes dissemination was due to horizontal transfer among many K. pneumoniae clones, while blaCTX-M-9 dissemination was associated with a particular clone of K. oxytoca. The study characterized incidence and diversity of CTX-M enzymes during the period studied. Moreover it showed epidemiological data, which may explain CTX-M prevalence worldwide and contribute for the understanding and control of the resistance spread.

O uso excessivo de antimicrobianos em medicina veterinária e humana tem contribuído para o surgimento e seleção de bactérias multirresistentes (MR) em animais de produção, sendo que a presença de resíduos de antibióticos em alimentos derivados constitui uma predisposição para o estabelecimento de reações alérgicas para o consumidor, e altera as características organolépticas dos alimentos. No Brasil, embora a presença de Escherichia coli produtora de β-lactamase de espectro estendido (ESBL) em avicultura, bubalinocultura e suinocultura tenha sido documentada recentemente, não há informações sobre sua prevalência em bovinocultura, que é um setor importante do agronegócio no país. Assim, o presente estudo teve como objetivo investigar a presença de linhagens de Escherichia coli produtoras de ESBLs na microbiota intestinal de bovinos de leite, em uma fazenda comercial com prática de uso rotineiro de cefalosporinas na forma terapêutica. Amostras de suabe retal foram coletadas em 95 ruminantes (lactantes e adultos saudáveis), na presença de um regime de tratamento com ceftiofur terapêutico, em uma fazenda no estado de São Paulo. As amostras foram triadas para a presença de bactérias Gram-negativas produtoras de ESBL, onde os isolados positivos foram identificados por MALDI-TOF, com posterior identificação dos genes codificando variantes ESBL, por PCR e sequenciamento. A presença de cepas de E. coli multirresistentes (resistência a >= 3 classes de antibióticos) foi confirmada em 45 ruminantes (47,3%). Adicionalmente, foram identificadas 68 cepas de E. coli produtoras de ESBL (71,5%), sendo 7 portadoras do gene blaCTX-M-2, 11 portadoras do gene blaCTX-M-8, e 50 portadoras do gene blaCTX-M-15. As cepas de E. coli produtoras de CTX-M-15 foram clonalmente relacionadas por ERIC-PCR e PFGE, confirmando-se a presença de linhagens clonais em diferentes animais, na mesma propriedade. Quatro cepas representativas deste clone tiveram seus genomas sequenciados, demonstrando pertencer ao complexo clonal 23 (ST90), mundialmente reportado em animais e humanos. Estes resultados denotam uma alta prevalência de E. coli produtora de ESBL do tipo CTX-M na microbiota intestinal do gado leiteiro, com predominância de clones de importância clínica humana e veterinária, que hipoteticamente possuem uma genética que favorece sua adaptação (provavelmente favorecida pela pressão seletiva de antibióticos) na interface animal-humana, o que representa um potencial zoonótico de importância para a saúde pública. / The excessive use of antimicrobials in veterinary medicine has contributed to the emergence and selection of multiresistant (MR) bacteria in livestock animals, and the presence of residues of antibiotics in food products is a predisposition for the establishment of allergic reactions to the consumer, changing the organoleptic characteristics of food. In Brazil, although the presence of Escherichia coli-producing extended-spectrum β-lactamase (ESBL) in poultry, buffalo and swine farming has been documented recently, there is no information on its prevalence in cattle breeding, which is an important sector of agribusiness in the country. Thus, the present study aimed to investigate the presence of Escherichia coli strains producing ESBLs in the intestinal microbiota of dairy cattle in a commercial farm with routine practice of cephalosporins in the therapeutic form. Rectal swab samples were collected from 95 ruminants (healthy calf and adults) in the presence of a treatment regimen with ceftiofur therapeutic, on a farm in the state of São Paulo. The samples were screened for the presence of ESBL-producing Gram-negative bacteria, where positive isolates were identified by MALDI-TOF, with subsequent identification of genes encoding ESBL variants, by PCR and sequencing. The presence of multiresistant strains of E. coli (resistance to >= 3 classes of antibiotics) was confirmed in 45 ruminants (47.3%). In addition, 68 ESBL-producing E. coli strains (71.5%) were identified, 7 of which were carriers of the blaCTX-M-2 gene, 11 carriers of the blaCTX-M-8 gene, and 50 carriers of the blaCTX-M-15. The E. coli strains producing CTX-M-15 were clonally related by ERIC-PCR and PFGE, confirming the presence of clonal lineages in different animals, on the same property. Four representative strains of this clone had their genomes sequenced, demonstrating belonging to the clonal complex 23 (ST90), worldwide reported in animals and humans. These results indicate a high prevalence of E. coli producing CTX-M ESBL in the intestinal microbiota of dairy cattle, with a predominance of clones of human and veterinary clinical importance, which hypothetically have a genetics that favors their adaptation (probably favored by the selective pressure of antibiotics) at the animal-human interface, which represents a zoonotic potential of public health importance.

O uso excessivo de antimicrobianos em medicina veterinária e humana tem contribuído para o surgimento e seleção de bactérias multirresistentes (MR) em animais de produção, sendo que a presença de resíduos de antibióticos em alimentos derivados constitui uma predisposição para o estabelecimento de reações alérgicas para o consumidor, e altera as características organolépticas dos alimentos. No Brasil, embora a presença de Escherichia coli produtora de β-lactamase de espectro estendido (ESBL) em avicultura, bubalinocultura e suinocultura tenha sido documentada recentemente, não há informações sobre sua prevalência em bovinocultura, que é um setor importante do agronegócio no país. Assim, o presente estudo teve como objetivo investigar a presença de linhagens de Escherichia coli produtoras de ESBLs na microbiota intestinal de bovinos de leite, em uma fazenda comercial com prática de uso rotineiro de cefalosporinas na forma terapêutica. Amostras de suabe retal foram coletadas em 95 ruminantes (lactantes e adultos saudáveis), na presença de um regime de tratamento com ceftiofur terapêutico, em uma fazenda no estado de São Paulo. As amostras foram triadas para a presença de bactérias Gram-negativas produtoras de ESBL, onde os isolados positivos foram identificados por MALDI-TOF, com posterior identificação dos genes codificando variantes ESBL, por PCR e sequenciamento. A presença de cepas de E. coli multirresistentes (resistência a >= 3 classes de antibióticos) foi confirmada em 45 ruminantes (47,3%). Adicionalmente, foram identificadas 68 cepas de E. coli produtoras de ESBL (71,5%), sendo 7 portadoras do gene blaCTX-M-2, 11 portadoras do gene blaCTX-M-8, e 50 portadoras do gene blaCTX-M-15. As cepas de E. coli produtoras de CTX-M-15 foram clonalmente relacionadas por ERIC-PCR e PFGE, confirmando-se a presença de linhagens clonais em diferentes animais, na mesma propriedade. Quatro cepas representativas deste clone tiveram seus genomas sequenciados, demonstrando pertencer ao complexo clonal 23 (ST90), mundialmente reportado em animais e humanos. Estes resultados denotam uma alta prevalência de E. coli produtora de ESBL do tipo CTX-M na microbiota intestinal do gado leiteiro, com predominância de clones de importância clínica humana e veterinária, que hipoteticamente possuem uma genética que favorece sua adaptação (provavelmente favorecida pela pressão seletiva de antibióticos) na interface animal-humana, o que representa um potencial zoonótico de importância para a saúde pública. / The excessive use of antimicrobials in veterinary medicine has contributed to the emergence and selection of multiresistant (MR) bacteria in livestock animals, and the presence of residues of antibiotics in food products is a predisposition for the establishment of allergic reactions to the consumer, changing the organoleptic characteristics of food. In Brazil, although the presence of Escherichia coli-producing extended-spectrum β-lactamase (ESBL) in poultry, buffalo and swine farming has been documented recently, there is no information on its prevalence in cattle breeding, which is an important sector of agribusiness in the country. Thus, the present study aimed to investigate the presence of Escherichia coli strains producing ESBLs in the intestinal microbiota of dairy cattle in a commercial farm with routine practice of cephalosporins in the therapeutic form. Rectal swab samples were collected from 95 ruminants (healthy calf and adults) in the presence of a treatment regimen with ceftiofur therapeutic, on a farm in the state of São Paulo. The samples were screened for the presence of ESBL-producing Gram-negative bacteria, where positive isolates were identified by MALDI-TOF, with subsequent identification of genes encoding ESBL variants, by PCR and sequencing. The presence of multiresistant strains of E. coli (resistance to >= 3 classes of antibiotics) was confirmed in 45 ruminants (47.3%). In addition, 68 ESBL-producing E. coli strains (71.5%) were identified, 7 of which were carriers of the blaCTX-M-2 gene, 11 carriers of the blaCTX-M-8 gene, and 50 carriers of the blaCTX-M-15. The E. coli strains producing CTX-M-15 were clonally related by ERIC-PCR and PFGE, confirming the presence of clonal lineages in different animals, on the same property. Four representative strains of this clone had their genomes sequenced, demonstrating belonging to the clonal complex 23 (ST90), worldwide reported in animals and humans. These results indicate a high prevalence of E. coli producing CTX-M ESBL in the intestinal microbiota of dairy cattle, with a predominance of clones of human and veterinary clinical importance, which hypothetically have a genetics that favors their adaptation (probably favored by the selective pressure of antibiotics) at the animal-human interface, which represents a zoonotic potential of public health importance.

Entre as ß-lactamases, as enzimas CTX-M têm despertado atenção especial pela alta incidência e grande capacidade de propagação. Eventos como recombinação gênica, transferência plasmideal e multirresistência podem ser a razão da manutenção e da ampla disseminação dos genes blaCTX-M. Este é um trabalho retrospectivo que teve como objetivo caracterizar genes blaCTX-M presentes em Klebsiella spp. Foram estudadas 27 linhagens de Klebsiella pneumoniae e 8 linhagens de Klebsiella oxytoca, produtoras de ?-lactamase de espectro estendido, isoladas de pacientes hospitalizados no período de janeiro a junho de 2000. A detecção e identificação dos genes blaCTX-M, assim como dos elementos relacionados com a mobilização destes genes, foi realizada por PCR e seqüenciamento. A localização genética e a mobilidade dos genes blaCTX-M foram pesquisadas por análise plasmideal e hibridação e por conjugação. Os perfis de sensibilidade das linhagens estudadas e das linhagens transconjugantes foram comparados pela determinação da concentração inibitória mínima de antibióticos das classes das cefalosporinas, cefamicinas, aminoglicosídeos e quinolonas. Foram encontrados genes blaCTX-M em plasmídeos conjugativos em 13 (37%) linhagens estudadas: blaCTX-M-9 em 4 K. oxytoca, e blaCTX-M-2 em 9 K. pneumoniae. Os genes blaCTX-M-9 estavam associados ao elemento de inserção ISEcp1, enquanto os genes blaCTX-M-2 estavam associados a integrons de classe I contendo ISCR1. O genes blaCTX-M-2, carreado por plasmídeo, pode estar relacionado com disseminação horizontal entre vários clones de K. pneumoniae, enquanto o gene blaCTX-M-9 foi encontrado sendo carreado por um único clone de K. oxytoca. Este estudo determinou a incidência e a diversidade de enzimas CTX-M no período estudado, além de fornecer dados epidemiológicos que podem explicar a sua prevalência no mundo e contribuir para o entendimento e controle da disseminação deste tipo de resistência. / CTX-M enzymes, the world\'s most prevalent ß-lactamases disseminate very easily. Genetic recombination, plasmid transference and multiresistance could be responsible for the wide spread of blaCTM-X genes. This retrospective study aims to characterize blaCTX-M genes found in Klebsiella spp. The strains were isolated in hospital patients from January to June 2000 and consisted of 27 ESBL-producing Klebsiella pneumoniae and 8 ESBL-producing Klebsiella oxytoca. PCR and sequencing were used in the detection and identification of blaCTX-M genes and genetic elements associated with their mobilization. Determination of genetic localization and mobility of blaCTX-M genes was by plasmid analyses, hybridization and transfer assays. The minimal inhibitory concentrations (MICs) of cephalosporins, cefamicins, aminoglycosides and quinolone antimicrobials evaluated the antibiotic susceptibility profile of transconjugants and strains in the study. The blaCTX-M genes were found in 13 strains (37%): blaCTX-M-9 in 4 K. oxytoca and blaCTX-M-2 in 9 K. pneumoniae. The insertion sequence ISEcp1 was associated with blaCTX-M-9 and blaCTX-M-2 was found in a class I integron bearing ISCR1. Plasmid blaCTX-M-2 genes dissemination was due to horizontal transfer among many K. pneumoniae clones, while blaCTX-M-9 dissemination was associated with a particular clone of K. oxytoca. The study characterized incidence and diversity of CTX-M enzymes during the period studied. Moreover it showed epidemiological data, which may explain CTX-M prevalence worldwide and contribute for the understanding and control of the resistance spread.

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.

Klebsiella spp. are opportunistic pathogens that cause hospital and community acquired infections such as pneumonia, urinary tract infection, septicaemia, soft tissue infections, liver abscess, and meningitis. Multidrug-resistant strains possessing extended-spectrum β-lactamases (ESBLs) has become an increasing problem worldwide. The over use and, in some cases, misuse of antibiotics in humans and in animal husbandry has been cited as a responsible factor in the development of drug resistance in all bacterial species. The advancing age; female gender, hospital crossinfection, the food chain trade and human migrations have contributed to increase the risk for community-acquired ESBL. A total of 223 isolates collected in 2006 and 2007 at Royal Infirmary of Edinburgh, Scotland, 219 K. pneumoniae, 2 K. oxytoca, 1 Enterobacter cloacae, and one isolate Salmonella enterica were identified by API 20E and confirmed genotypically with gyrA PCR-RFLP method. The antimicrobial susceptibility results showed that 34 (15.2%), 36 (16.1%), 35 (15.7%), 45 (20.2%), 30 (13.5%) and 55 (24.7%) of these strains were found to be resistant to cefotaxime, ceftazidime, ceftriaxone, naladixic acid, ciprofloxacin and cefoxitin. None of the isolates were found resistant to meropenem keeping carbapenems the drug of choice for the treatment of multiresistant isolates. The overall frequency of ESBL producers observed in this study was 35 (15.7%) most of them 32/35 (91.4%) were from K. pneumoniae. The genetic analysis showed that SHV β-lactamases were detected in 32, whereas TEM and CTX-M were detected in 24 and 16 respectively. From the ESBL-producing isolates, molecular methods identified nine strains possessing ESBL-SHV genes (1 strain blaSHV-5, 1 strain blaSHV-80 and 8 strains blaSHV-12), whereas the remaining were from the “non-ESBL” producing strains. Conjugation methods demonstrated that 29/32 isolates harboured transferable blaSHV genes. The large SHV transposon-borne promoters were amplified from only one non-transferable blaSHV-11, 15 isolates produced the small SHV transposon-borne promoters. Furthermore, the IS26 was found 73bp upstream of the blaSHV gene in all small SHV transposon-borne promoters. A new blaLEN gene was identified from K. pneumoniae (KpII) phylogenetic group but remained susceptible to all cephalosporins. Sixteen (7.3%) of K. pneumoniae isolates were found to be producers of the CTX-M- 15 ESBL, of which two isolates (12.5%) were reported to be from communityacquired infections. The insertion sequence ISEcp1 was detected by sequencing 48 nucleotides upstream of blaCTX-M-15 in all isolates but one. Five different clones of CTX-M-15-producing isolates were identified by PFGE. The findings indicated a higher prevalence of qnr genes than in previous studies but still low in general. By PCR, 18 (8%) (11 qnrB1, 2 qnrB6 and 5 qnrA1) genes were identified from K. pneumoniae isolates. Also, the findings indicated the frequent coexpression of fluoroquinolones and ESBLs resistance in the same isolate. Two K. oxytoca strains were isolated from urine and blood specimens of hospitalized patients. Both strains were positive for the blaOXY-2 gene. One strain showed resistance to pencillins, monbactams, cephalosporins including cefotaxime and ceftazidime but was not inhibited by clavulanic acid. It differed by an amino acid substitution Ala237→Thr, which enhances the binding of cefotaxime. S1-nuclease plasmid profiles were obtained for some isolates. A total of one to two plasmids, ranging in size from approximately 40 to 210 kb, were observed per strain. The plasmids from 24 ESBL K. pneumoniae strains were assigned to be IncN or IncFII replicons. Analysis of phylogenetic groups showed that the majority of K. pneumoniae isolates were belonged to KpI-type. Both K. oxytoca strains were assigned to be KoII phylogenetic group based on rpoB and gyrA sequencing. Integrons are capable of capturing and mobilizing genes called gene cassettes which play an important role in the dissemination of antimicrobial resistance through horizontal transmission. In fact, the present study indicated a high frequency of occurrence of class 1 integrons among ESBL-positive K pneumoniae. Three isolates positive for class 1 integrons were found positive for class 2 integrons as well. Class 1 integrons including dfr, aadA and ereA2 gene cassettes have been identified by sequencing, which confer resistance to trimethoprim, streptomycin/spectinomycin and erythromycin respectively. In conclusion, the results from this thesis report the emergence of hospital and community-acquired highly resistant CTX-15 β-lactamase in the Edinburgh, Scotland. The prevalence of ESBL-producing isolates in Scotland is still much lower than in many other European countries. The dissemination of SHV- and TEM- β- lactamase types in this study is more predominate than CTX-M-15.