A major outbreak involving an Escherichia coli strain that was resistant to expanded-spectrum cephalosporins occurred in Toronto and surrounding regions in 2000 to 2002. We report the complete sequence of a plasmid, pC15-1a, that was found associated with the outbreak strain. Plasmid pC15-1a is a circular molecule of 92,353 bp consisting of two distinct regions. The first is a 64-kb region that is essentially homologous to the non-R-determinant region of plasmid R100 except for several point mutations, a few small insertions and deletions, and the absence of Tn10. The second is a 28.4-kb multidrug resistance region (MDR) that has replaced the R-determinant region of the R100 progenitor and consists mostly of transposons or partial transposons and five copies of the insertion element IS26. All drug resistance genes found in pC15-1a, including the beta-lactamase genes bla CTX-M-15 , bla OXA-1 , and bla TEM-1 , the tetracycline resistance gene tetA, and aminoglycoside resistance genes aac(6)-Ib and aac(3)-II, are located in the MDR. The bla CTX-M-15 gene was found downstream of ISEcp1as part of a transposition unit, as determined from the surrounding sequence. Examination of the plasmids from CTX-M-15-harboring strains isolated from hospitals across Canada showed that pC15-1a was found in several strains isolated from a site in western Canada. Comparison of pC15-1a and pCTX15, found in an E. coli strain isolated in India in 1999, revealed that the plasmids had several features in common, including an R100 backbone and several of the resistance genes, including bla CTX-M-15 , bla TEM-1 , bla OXA-1 , tetA, and aac(6)-Ib.Plasmid-mediated extended-spectrum beta-lactamase (ESBL) enzymes are most commonly of the TEM, SHV, or CTX-M type (8). To date more than 120 TEM enzymes, more than 50 SHV enzymes, and more than 30 CTX-M enzymes have been reported (www.lahey.org/studies/). Members of these groups are class A enzymes and, for the most part, are inhibited by clavulanic acid.The CTX-M-type beta-lactamases are increasingly found in enterobacterial species throughout the world; more than half have been reported within the last 4 years (7, 28). They are generally most active against cefotaxime and show little activity against ceftazidime. Phylogenetically, they are grouped into five clusters based on their amino acid identities: the CTX-M-1 cluster etc.), the CTX-M-2 cluster etc.), the CTX-M-8 cluster (CTX-M-8), the CTX-M-25 cluster , and the CTX-M-9 cluster

This report describes a study carried out to gain baseline information on the molecular characteristics of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella spp. in Canada. A total of 29,323 E. coli and 5,156 Klebsiella sp. isolates were screened at 12 participating sites. Of these, 505 clinically significant, nonrepeat isolates displaying reduced susceptibility to the NCCLS-recommended beta-lactams were submitted to a central laboratory over a 1-year period ending on 30 September 2000. A total of 116 isolates were confirmed to be ESBL producers. PCR and sequence analysis revealed the presence of TEM-11 (n ‫؍‬ 1), TEM-12 (n ‫؍‬ 1), TEM-29 (n ‫؍‬ 1), TEM-52 (n ‫؍‬ 4), CTX-M-13 (n ‫؍‬ 1), CTX-M-14 (n ‫؍‬ 15), CTX-M-15 (n ‫؍‬ 11), SHV-2 (n ‫؍‬ 2), SHV-2a (n ‫؍‬ 12), SHV-5 (n ‫؍‬ 6), SHV-12 (n ‫؍‬ 45), and SHV-30 (n ‫؍‬ 2). Five novel beta-lactamases were identified and designated TEM-115 (n ‫؍‬ 2), TEM-120 (n ‫؍‬ 1), SHV-40 (n ‫؍‬ 2), SHV-41 (n ‫؍‬ 4), and SHV-42 (n ‫؍‬ 1). In addition, no molecular mechanism was identified for five isolates displaying an ESBL phenotype. Macrorestriction analysis of all ESBL isolates was conducted, as was restriction fragment length polymorphism analysis of plasmids harboring ESBLs. Although a "clonal" distribution of isolates was observed at some individual sites, there was very little evidence suggesting intrahospital spread. In addition, examples of identical or closely related plasmids that were identified at geographically distinct sites across Canada are given. However, there was considerable diversity with respect to plasmid types observed.Infections caused by aerobic gram-negative bacilli are common in hospitalized patients and result in serious infections, such as bacteremia and the majority of cases of nosocomial pneumonia (8, 32). These infections also are associated with high rates of mortality; for example, sepsis is one of the most common causes of death in intensive-care-unit patients (32,35). The emergence of gram-negative bacilli that contain extended-spectrum beta-lactamases (ESBLs) and AmpC cephalosporinases has compounded this problem and has become a worldwide concern (7).The first ESBL was identified in Germany in 1983; since then, over 200 variants of the clavulanic acid-inhibited form of the enzyme have been described worldwide. The most common extended-spectrum phenotypes arise from point mutations in the bla TEM , bla SHV , or bla CTX gene resulting in alterations of the primary amino acid sequence of the enzyme (7). Since these genes are generally found on plasmids, many of the organisms that harbor ESBLs also are resistant to other classes of antibiotics, such as aminoglycosides, fluoroquinolones, tetracyclines, chloramphenicol, and sulfonamides (24).In this study, we examined the molecular characteristics of ESBLs isolated over a 1-year period in 12 Canadian hospitals. MATERIALS AND METHODSSurveillance network. Established in 1995, The Canadian Nosocomial Infection Surveillance Program (CNISP) is a collaborative effort in...

Salmonella serotyping remains the gold-standard tool for the classification of Salmonella isolates and forms the basis of Canada’s national surveillance program for this priority foodborne pathogen. Public health officials have been increasingly looking toward whole genome sequencing (WGS) to provide a large set of data from which all the relevant information about an isolate can be mined. However, rigorous validation and careful consideration of potential implications in the replacement of traditional surveillance methodologies with WGS data analysis tools is needed. Two in silico tools for Salmonella serotyping have been developed, the Salmonella in silico Typing Resource (SISTR) and SeqSero, while seven gene MLST for serovar prediction can be adapted for in silico analysis. All three analysis methods were assessed and compared to traditional serotyping techniques using a set of 813 verified clinical and laboratory isolates, including 492 Canadian clinical isolates and 321 isolates of human and non-human sources. Successful results were obtained for 94.8, 88.2, and 88.3% of the isolates tested using SISTR, SeqSero, and MLST, respectively, indicating all would be suitable for maintaining historical records, surveillance systems, and communication structures currently in place and the choice of the platform used will ultimately depend on the users need. Results also pointed to the need to reframe serotyping in the genomic era as a test to understand the genes that are carried by an isolate, one which is not necessarily congruent with what is antigenically expressed. The adoption of WGS for serotyping will provide the simultaneous collection of information that can be used by multiple programs within the current surveillance paradigm; however, this does not negate the importance of the various programs or the role of serotyping going forward.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen that has disseminated throughout Canadian hospitals and communities. Pulsed-field gel electrophoresis of over 9,300 MRSA isolates obtained from the Canadian Nosocomial Infection Surveillance Program has identified 10 epidemic strain types in Canada (CMRSA1 to CMRSA10). In an attempt to determine specific genetic factors that have contributed to their high prevalence in community and/or hospital settings, the genomic content of representative isolates for each of the 10 Canadian epidemic types was compared using comparative genomic hybridizations. Comparison of the community-associated Canadian epidemic isolates (CMRSA7 and CMRSA10) with the hospital-associated Canadian epidemic isolates revealed one open reading frame (ORF) (SACOL0046) encoding a putative protein belonging to a metallo-beta-lactamase family, which was present only in the community-associated Canadian epidemic isolates. A more restricted comparison involving only the most common hospital-associated Canadian epidemic isolates (CMRSA1 and CMRSA2) with the community-associated Canadian epidemic isolates did reveal additional factors that might be contributing to their prevalence in the community and hospital settings, which included ORFs encoding potential virulence factors involved in capsular biosynthesis, serine proteases, epidermin, adhesion factors, regulatory functions, leukotoxins, and exotoxins.

Background: Oma1 is a conserved membrane-bound protease that forms a high molecular mass complex. Results: Oma1 activity is induced by stress stimuli and required for survival. The activation is linked to changes in Oma1 oligomer stability and involves its C-terminal region. Conclusion: Oma1 function is activated by mitochondrial stress and is important for stress tolerance. Significance: Novel insights into Oma1 function and a potential stress activation mechanism are provided.