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P38 Isolation frequency of extended-spectrum beta-lactamases producing Escherichia coli, Klebsiella pneumoniae or Proteus mirabilis and metallo-beta-lactamases producing Pseudomonas aeruginosa from 72 centres in Japan, 2007
Friday, June 19, 2009 ampicillin-resistant E. faecium clones. In contrast, E. faecium strains with aph(2 )-Ie were classified into newly assigned STs (ST426 or its single locus variant ST427). Conclusion: HLGR gene aac(6 )-Ie-aph(2 )-Ia was distributed to E. faecalis with various genetic lineages and E. faecium with lineages in CC17 mostly. The aph(2 )-Ie was carried by E. faecium from a few limited lineages. P37 Genetic diversity of the low-level vancomycin resistance gene vanC in enterococci and identification of a novel subtype (vanC4) in Enterococcus casseliflavus N. Kobayashi1 *, D. Qui˜ nones Perez2 , S. Watanabe1 . 1 Sapporo Medical University, Sapporo, Japan, 2 Instituto de Medicina Tropical “Pedro Kourí”, Havana, Cuba Objective: Enterococcus gallinarum and Enterococcus casseliflavus are nosocomial pathogens and are intrinsically resistant to vancomycin. These bacterial species possess vanC gene which confers lowlevel resistance to vancomycin. The vanC in E. gallinarum and E. casseliflavus are classified into subtypes vanC-1 and vanC-2/vanC-3, respectively. In the present study, genetic diversity of vanC genes were analyzed for clinical isolates. Methods: We analyzed 6 E. gallinarum clinical strains, and 13 E. casseliflavus strains including four standard strains and seven clinical strains. Clinical strains were isolated in two Japanese hospitals during a period from 1997 to 2003. The vanC, and vancomycin resistance gene cluster containing four genes associated with expression of vancomycin resistance, and atpA gene for ATP synthase alpha chain were sequenced by PCR and direct sequencing. Obtained sequence data were analyzed phylogenetically. Results: The vanC1 from the clinical isolates and E. gallinarum prototype strain BM4174 were genetically highly close to each other with the identity of 99.4 100%. The vanC-2/vanC-3 from ten E. casseliflavus strains including prototype strains of these genes showed extremely high sequence identity (98.4% or more). In contrast, vanC genes from three clinical isolates exhibited slightly lower identities (93 95%) to the “classical” vanC-2/vanC-3 gene sequences, although higher identity (96 98%) was observed among them. The vanC from the three strains were considered to represent a new subtype of vanC, and was designated as vanC4. The vanXYc, vanSc, and vanRc of strains with the vanC-2/vanC-3 were genetically distinct from those with the vanC4. The atpA genes of the classical vanC-2/vanC-3 were highly conserved (more than 98%), but exhibited lower identities (93 96%) to strains with vanC4. Conclusion: The vanC1 of E. gallinarum was genetically almost identical. In contrast, genetic diversity was found in vanC-2/vanC-3 in E. caseliflavus, and novel vanC subtype vanC4 which is discriminated from the classical vanC-2/vanC-3 was identified. P38 Isolation frequency of extended-spectrum beta-lactamases producing Escherichia coli, Klebsiella pneumoniae or Proteus mirabilis and metallo-beta-lactamases producing Pseudomonas aeruginosa from 72 centres in Japan, 2007 Y. Ishii1 *, A. Ohno1 , K. Yamaguchi1 . 1 Toho University, Tokyo, Japan Objectives: In Gram-negative pathogens, beta-lactamase production remains the most important contributing factor to beta-lactam resistance. Extended-spectrum beta-lactamases (ESBLs) and carbapenemases including metallo-beta-lactamases (MBLs) are major clinical concerns. They greatly reduce therapeutic options for infected patients. In this study, we investigated the frequency of ESBL- and MBL-producing of clinical isolates in Japan. Methods: In a 2007 study, 72 centers collected consecutive nonduplicate isolates of Escherichia coli (743), Klebsiella pneumoniae (663), Proteus mirabilis (547) and 1,262 isolates of Pseudomonas aeruginosa to determine the frequency of ESBL and MBL producers. CLSI methods were used for ESBL screening. MBL production in
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P. aeruginosa was investigated with tests involving 200 mg/L of the dipicolinic acid in combination with ceftazidime or imipenem. The types of ESBL or MBLs were confirmed by PCR or DNA sequencing. Results: Isolation frequency of ESBL producers in E. coli, K. pneumoniae or P. mirabilis was 8.6% (from 39 centers), 5.3% (from 19 centers) and 10.8% (from 30 centers), respectively. The most frequent subtype of ESBL in E. coli or K. pneumoniae was CTX-M-9 group or CTX-M-1 group, respectively. All ESBLs from P. mirabilis were CTX-M-2. Isolation frequencies of MBL producing P. aeruginosa from urinary tract and respiratory tract were 5.6% (from 19 centers) and 2.2% (from 7 centers), respectively. IMP-1 subgroup MBLs were produced by 94% of MBL-positive P. aeruginosa. Only 3 isolates produced VIM-2 group MBL. Most of MBL-producing P. aeruginosa isolates were resistant to not only b-lactams but also aminoglycosides and fluoroquinolones. Conclusions: In this study 8.1% of E. coli, K. pneumoniae and P. mirabilis produced ESBLs and 3.8% of P. aeruginosa produced MBLs such as IMP-1 subgroup or VIM-2 subgroup. Present data indicated that isolation frequency of ESBL-produced Enterobacteriaceae increased compared with data of 2004. On the other hand, MBL-positive P. aeruginosa isolates did not increase. P39 Multi-locus sequencing analysis of conjugative IncF plasmids with blaCTX-M-15 Z. Zong1 *, S. Partridge2 , J. Iredell2 . 1 University of Sydney, Sydney, Australia, 2 The University of Sydney, Sydney, Australia Objective: blaCTX-M-15 has emerged as the most widespread ESBL gene, including in western Sydney. The global and local spread of blaCTX-M-15 is largely mediated by IncF plasmids. A comprehensive and robust approach is required to investigate the relatedness of IncF plasmids carrying blaCTX-M-15 . A number of IncF plasmids have been completely sequenced and can be used as references to develop a DNA sequencingbased scheme for plasmid characterization. Methods: Conjugative plasmids carrying blaCTX-M-15 were obtained from local clinical E. coli isolates by mating (filter and broth) and were identified as IncF (FII, FII+FIA or FII+FIA+FIB) by PCR-based replicon typing. Plasmid sizes were estimated using nuclease S1 pulsefield gel electrophoresis (PFGE). Plasmids prepared by alkaline lysis were restricted with HpaI. Individual genes or regions containing multiple genes responsible for basic plasmid functions, i.e., replication (RepFIIA, RepFIA, RepFIB), maintenance (partitioning: F-like sopA-BC and R100-like stbC-A-B; post-segregation killing: sok/hok, srnCB’-B and ccdA-B) and conjugation (traA, traD, traI) plus psiA-B (encoding SOS inhibition) were chosen as targets. and amplicons were sequenced. Results: Thirteen conjugative IncF plasmids carrying blaCTX-M-15 were obtained, ranging from 70 to 240 kb and containing various combinations of IncF replicons (2 FIIA, 6 FIIA+FIA; 5 FIIA+FIA+FIB). They were found to belong to 6 sequence types (ST). Two plasmids (ST1) appear to share highly-similar backbones with pC15 1a (from Canada) except for a minor variation in traD, suggesting a possible international spread of blaCTX-M-15 mediated by pC15 1a-like plasmids. Three plasmids (ST2) are likely hybrids of a R1-like plasmid and pU302L (from the USA), while three plasmids (ST3) have features in common with both pC15 1a and pU302L. Three plasmids (ST 4) appear closely related to pRSB107 (from Germany) but with slightly different RepFIB and additional tra genes. One plasmid (ST5) has a mosaic composition with components from various sources, while another plasmid (ST6) is closely-related to pIP1206 (from Belgium). Conclusions: A multi-locus sequencing scheme was successfully developed and is a useful tool for investigating plasmid relatedness and origins, thus contributing to our understanding of the spread of resistance genes. A variety of IncF plasmids seem to be involved in the dissemination of blaCTX-M-15 ,
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P. aeruginosa was investigated with tests involving 200 mg/L of the dipicolinic acid in combination with ceftazidime or imipenem. The types of ESBL or MBLs were confirmed by PCR or DNA sequencing. Results: Isolation frequency of ESBL producers in E. coli, K. pneumoniae or P. mirabilis was 8.6% (from 39 centers), 5.3% (from 19 centers) and 10.8% (from 30 centers), respectively. The most frequent subtype of ESBL in E. coli or K. pneumoniae was CTX-M-9 group or CTX-M-1 group, respectively. All ESBLs from P. mirabilis were CTX-M-2. Isolation frequencies of MBL producing P. aeruginosa from urinary tract and respiratory tract were 5.6% (from 19 centers) and 2.2% (from 7 centers), respectively. IMP-1 subgroup MBLs were produced by 94% of MBL-positive P. aeruginosa. Only 3 isolates produced VIM-2 group MBL. Most of MBL-producing P. aeruginosa isolates were resistant to not only b-lactams but also aminoglycosides and fluoroquinolones. Conclusions: In this study 8.1% of E. coli, K. pneumoniae and P. mirabilis produced ESBLs and 3.8% of P. aeruginosa produced MBLs such as IMP-1 subgroup or VIM-2 subgroup. Present data indicated that isolation frequency of ESBL-produced Enterobacteriaceae increased compared with data of 2004. On the other hand, MBL-positive P. aeruginosa isolates did not increase. P39 Multi-locus sequencing analysis of conjugative IncF plasmids with blaCTX-M-15 Z. Zong1 *, S. Partridge2 , J. Iredell2 . 1 University of Sydney, Sydney, Australia, 2 The University of Sydney, Sydney, Australia Objective: blaCTX-M-15 has emerged as the most widespread ESBL gene, including in western Sydney. The global and local spread of blaCTX-M-15 is largely mediated by IncF plasmids. A comprehensive and robust approach is required to investigate the relatedness of IncF plasmids carrying blaCTX-M-15 . A number of IncF plasmids have been completely sequenced and can be used as references to develop a DNA sequencingbased scheme for plasmid characterization. Methods: Conjugative plasmids carrying blaCTX-M-15 were obtained from local clinical E. coli isolates by mating (filter and broth) and were identified as IncF (FII, FII+FIA or FII+FIA+FIB) by PCR-based replicon typing. Plasmid sizes were estimated using nuclease S1 pulsefield gel electrophoresis (PFGE). Plasmids prepared by alkaline lysis were restricted with HpaI. Individual genes or regions containing multiple genes responsible for basic plasmid functions, i.e., replication (RepFIIA, RepFIA, RepFIB), maintenance (partitioning: F-like sopA-BC and R100-like stbC-A-B; post-segregation killing: sok/hok, srnCB’-B and ccdA-B) and conjugation (traA, traD, traI) plus psiA-B (encoding SOS inhibition) were chosen as targets. and amplicons were sequenced. Results: Thirteen conjugative IncF plasmids carrying blaCTX-M-15 were obtained, ranging from 70 to 240 kb and containing various combinations of IncF replicons (2 FIIA, 6 FIIA+FIA; 5 FIIA+FIA+FIB). They were found to belong to 6 sequence types (ST). Two plasmids (ST1) appear to share highly-similar backbones with pC15 1a (from Canada) except for a minor variation in traD, suggesting a possible international spread of blaCTX-M-15 mediated by pC15 1a-like plasmids. Three plasmids (ST2) are likely hybrids of a R1-like plasmid and pU302L (from the USA), while three plasmids (ST3) have features in common with both pC15 1a and pU302L. Three plasmids (ST 4) appear closely related to pRSB107 (from Germany) but with slightly different RepFIB and additional tra genes. One plasmid (ST5) has a mosaic composition with components from various sources, while another plasmid (ST6) is closely-related to pIP1206 (from Belgium). Conclusions: A multi-locus sequencing scheme was successfully developed and is a useful tool for investigating plasmid relatedness and origins, thus contributing to our understanding of the spread of resistance genes. A variety of IncF plasmids seem to be involved in the dissemination of blaCTX-M-15 ,