Genetic Environments of bla_CTX-M Genes Located on Conjugative Plasmids of Enterobacteriaceae Nosocomial Isolates Collected in Russia within 2003-2007

The study showed that bla_CTX-M genes were present in the genomes of 71% of сephаlosporin resistant Enterobacteriaceae nosocomial isolates (n=833) collected in Russian hospitals within 2003-2007, including 91% of E.coli, 90% of Klebsiella spp., 38% of Enterobacter spp., 31 % of Citrobacter spp. (n=9), and 36% of the other Enterobacteriaceae species. The genes belonging to the following subtypes (clusters) were identified: bla_CΎX__M_₁ (529 bla_CTX__M_₁₅ genes; 25 bla_CTX__M_₃ genes; 1 bla_CTX__M_₂₂ gene, 1 bla_CTX-M-23 gene and 1 bla_CTX-M-34 gene); bla_CTX-M-2 (1 blaCTX-M-2 gene and 4 bla_CTX-M-5 genes), and blaCTX-M-9 (2 blaCTX-_M_9 genes, and 28 bla_CTX__M_₁₄ genes). It was shown that bla_CTX-M genes were located on high-molecular weight (60-160 bp) conjugative plasmids belonging mainly to the incompatibility groups IncF, IncL/M and IncA/C (bla_CTX-M-15 gene); IncL/M (bla_CTX-M-3 gene); and IncF, IncL/M and IncIl-ly (_CTX-M-14 gene). The gene environments of bla_CTX-M genes were shown specific for the subtype of the genes. A mobile genetic element lSEcp1 (in some cases deleted or inserted by IS26, IS1, IS10, resTn2, or resTn3 sequences, in direct or reverse position) were detected upstream of bla_CTX-M-3, bla_CTX-M-14, and bla_CTX-M-15 genes. A special characteristic was the sequence between ISEcpl and bla_CTX-M gene: 48 bp for bla_CTX-M-15 (except 1 E.coli isolate having such a sequence deleted by 3 bp); 127 bp for bla_CTX-M-3; 42 bp for bla_CTX-M-14. Downstream of bla_CTX-M and bla_CTX-M-15 genes in the major bacterial isolates orf477 mucA and Aorf477-AmucA sequences were detected respectively. Two isolates had additional Aorf3 insertion inside of Aorf477-AmucA sequence. Insertion sequence IS903 (intact or deleted) was detected downstream of bla_CTX-M-14 gene. Unlike the others, bla_CTX-M-2 and bla_CTX-M-9 genes were located inside of IS CR1 mobile element, downstream of class 1 integron and orf513 sequence.

bla_CTx_-M, ISEcpl, IS26, IS903, orf513, ISCRl, bla_CTX-M, ISEcpl, IS26, IS903, orf513, ISCRl

1. Nicaido H. Multidrug resistance in bacteria. Annu Rev Biochem 2009; 78: 119-146.

2. Canton R., Coque T.M. The CTX-M /З-lactamase pandemic. Curr Opinion Microbiol 2006; 9: 466-475.

3. http://www.lahey.org/Studies/other.asp#table1

4. Rossolini G. M., D'Andrea M. M., Mugnaioli C. The spread of CTX-M-type extended-spectrum beta-lactamases. Clin Microbiol Infect 2008; 14: 1: 33-41.

5. Coque T. M., Novais Â., Carattoli A. et al. Dissemination of clonally related Escherichia coli strains expressing extended-spectrum /З-lacta-mase CTX-M-15. Emerging Infectious Diseases 2008; 14: 2: 195-200.

6. Rodriguez-Bano J, Pascual A. Clinical significance of extended-spectrum beta-lactamases. Expert Rev Antiinfect Ther 2008; 6: 5: 671-683.

7. Hawkey P.M., Jones A.M. The changing epidemiology of resistance. J Antimicrob Chemother 2009; 64: 1: i3-10.

8. Barlow M., Fatollahi J., Salverda M. Evidence for recombination among the alleles encoding TEM and SHV /З-lactamases. J Antimicrob Chemother 2009; 63: 256-259.

9. Gazoli M., TzelepiE., Sidorenko S. V., Tzouvelekis L. S. Sequence of the gene encoding a plasmid-mediated cefotaxime-hydrolyzing class A /З-lactamase (CTX-M-4): involvement of serine 237 in cephalosporin hydrolysis. Antimicrob Agents Chemother 1998; 42: 5: 1259-1262.

10. Edelstein M., Pimkin M., Palagin I. et al. Prevalence and molecular epidemiology of CTX-M extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in Russian hospitals. Antimicrob Agents Chemother 2003; 47: 12: 3724-3732.

11. Fursova N., Abaev I., Pryamchuk S. et al. Variability of Inc group of conjugative plasmids and CTX-M gene environments in Enterobacteriaceae nosocomial strains isolated from Russia. Clin Microbiol Infect 2008; 15: 4: 38-39.

12. NCCLS/CLSI. 2006. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 5th ed. Document M7-A5. Clinical and Laboratory Standards Institute, Wayne, PA.

13. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/ Disk_test_documents/ EUCAST_breakpoints_v1.0_20091221.pdf

14. Kado C. I., Liu S. T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol 1981; 145: 1365-1373.

15. Carattoli A., Bertini A., Villa L. et al. Identification of plasmids by PCR-based replicon typing. J Microbiol Meth 2005; 63: 219-228.

16. Eckert C., Gautier V., Arlet G. DNA sequence analysis of the genetic environment of various blaCTX-M genes. J Antimicrob Chemother 2006; 57: 14-23.

17. Saladin M., Cao V. T., Lambert T. et al. Diversity of CTX-M beta-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals. FEMS Microbiol Lett 2002; 209: 2: 161-168.

18. Machado E., Canton R., Baquero F. et al. Integron content of extended-spectrum-beta-lactamase-producing Escherichia coli strains over 12 years in a single hospital in Madrid, Spain. Antimicrob Agents Chemother 2005; 49: 5: 1823-1829.

19. Skurnik D., Le Menach A., Zurakowski D. et al. Integron-associated antibiotic resistance and phylogenetic grouping of Escherichia coli isolates from healthy subjects free of recent antibiotic exposure. Antimicrob Agents Chemother 2005; 49: 7: 3062-3065.

20. Livermore D. M., Canton R., Gniadkowski M. et al. CTX-M: changing the face of ESBLs in Europe. J Antimicrob Chemother 2007; 59: 2: 165-174.

21. Coque T. M., Baquero F., Canton R. Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. Eurosurveillance 2008; 13: 47: 1-11.

22. Poirel L., Naas T., Nordmann P. Genetic support of extended-spectrum /З-lactamases. Clin Microbiol Infect. 2008; 14: Suppl. 1: 75-81.