Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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.

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.

In isolated skeletal muscle arterioles, increasing transmural pressure causes an increase in constriction. This active myogenic response varies with age and fiber type. Increased transmural pressure activates both Ca2+-activated (KCa) potassium channels and voltage-dependent (Kv) potassium channels; these channels have a role in the negativefeedback pathways that modulate depolarization and myogenic constriction. We tested the hypothesis that increased KCa channel and Kv channel activity contribute to reduced myogenic responsiveness in skeletal muscle arterioles of aged rats. 1A arterioles were isolated from soleus, an oxidative muscle, and superficial gastrocnemius, a glycolytic muscle, of young (4 mos) and aged (24 mos) Fischer 344 rats. Myogenic responses were assessed by increasing intraluminal pressure (0-140 cm H2O) in increments of 20cm H2O. Vasoconstrictor response were determined in response to increasing concentrations of the KCa channel blocker, charybdotoxin (CTX; 10-10 to 10-7 M) and the Kv channel blocker, 4-Aminopyridine (4-AP; 10-5 to 10-2 M). To determine the role of potassium channels in modulating the myogenic response, cannulated arterioles from soleus and gastrocnemius were incubated with CTX (50 nM) and 4-AP (5mM) for 15 minutes prior to evaluation of the myogenic response. Increased Kv channel activity contributes to reduced myogenic constriction in soleus and gastrocnemius muscle arterioles from aged rats. In soleus muscle arterioles, KCa channel activity opposes myogenic tone in young but not old rats. In gastrocnemius muscle arterioles, treatment with CTX did not eliminate age-related differences in the myogenic response, and the KCa channel contribution to myogenic tone was, in fact, greater arterioles from young as compared to old rats. Kv channels contribute to greater myogenic constriction in soleus arterioles, KCa channels appear to be more active in gastrocnemius muscle arterioles as compared to soleus muscle arterioles. Therefore Kv and KCa channels are tonically active in skeletal muscle arterioles, contributing to a hyperpolarizing force that opposes myogenic constriction. Furthermore, increased Kv channel activity contributes to the age-related reduction of myogenic constriction in soleus and gastrocnemius muscle arterioles.

Purpose: Despite its many health benefits, moderate exercise can induce joint discomfort when done infrequently or too intensely even in individuals with healthy joints. This study was designed to evaluate whether NEM (R) (natural eggshell membrane) would reduce exercise-induced cartilage turnover or alleviate joint pain or stiffness, either directly following exercise or 12 hours post exercise, versus placebo. Patients and methods: Sixty healthy, postmenopausal women were randomly assigned to receive either oral NEM 500 mg (n=30) or placebo (n=30) once daily for two consecutive weeks while performing an exercise regimen (50-100 steps per leg) on alternating days. The primary endpoint was any statistically significant reduction in exercise-induced cartilage turnover via the biomarker C-terminal cross-linked telopeptide of type-II collagen (CTX-II) versus placebo, evaluated at 1 and 2 weeks of treatment. Secondary endpoints were any reductions in either exercise-induced joint pain or stiffness versus placebo, evaluated daily via participant questionnaire. The clinical assessment was performed on the per protocol population. Results: NEM produced a significant absolute treatment effect (TEabs) versus placebo for CTX-II after both 1 week (TEabs - 17.2%, P=0.002) and 2 weeks of exercise (TEabs - 9.9%, P=0.042). Immediate pain was not significantly different; however, rapid treatment responses were observed for immediate stiffness (Day 7) and recovery pain (Day 8) and recovery stiffness (Day 4). No serious adverse events occurred and the treatment was reported to be well tolerated by study participants. Conclusion: NEM rapidly improved recovery from exercise-induced joint pain (Day 8) and stiffness (Day 4) and reduced discomfort immediately following exercise (stiffness, Day 7). Moreover, a substantial chondroprotective effect was demonstrated via a decrease in the cartilage degradation biomarker CTX-II.

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.