Castro-Alarcón N, Alonso-Morales A, Silva-Sánchez J, Armenta-Solís A

Language: Spanish
References: 27
Page: 165-174
PDF size: 269.43 Kb.

ABSTRACT

Enterobacter cloacae is an important opportunistic pathogen known to cause nosocomial infection. Reports of multidrug-resistant isolates have increased during the last decade. An increasing number of clonal outbreaks caused by Enterobacter cloacae are being reported. For the detection of clonality, pulsed-field gel electrophoresis (PFGE) is considered the golden standard, but PCR-based methods are cheaper, easier to performed, and provide faster results. The objective of this study was to validate the results from the molecular typing of Enterobacter cloacae isolates by two variants of amplification technique: repeated sequences (rep-PCR) compared with the results of pulsed-field gel electrophoresis (PFGE) patterns. Twenty-six isolates were analyzed, which ones a screening was performed to confirm extended-spectrum β-lactamases (ESBL) producing, isoelectric points from β-lactamases were identified too. The strains were typing by PFGE using the enzyme XbaI, to perform the rep-PCR was used two sets of initiators related to REP and ERIC sequences. The ESBL isolates producer showed multidrug resistance, the most frequent isoelectric points were 5.4 (88.4%) and 8.2 (76.9%). Epidemiologically, all isolates were clearly discriminated by both methods. The isolates were grouped in 18 clonal groups through variants rep-PCR, whereas the PFGE grouped into 20 clonal groups. The power of discrimination showed for the two variants, rep-PCR was 0.97, while the power of discrimination for PFGE was 0.98. The rep-PCR is proposed as an alternative molecular tool in the microbiology laboratory for typing of E. cloacae clinical isolates in the investigating of outbreaks.

REFERENCES

1. Healy M, Huong J, Bittner T, Lising M, Frye S, Raza S, et al. Microbiol DNA typing by automated repetitive-sequence-based PCR. J Clin Microbiol. 2005; 43: 199-207.

2. Coll P, Coque MT, Dominguez MA, Vazquez J, Vila J. Métodos moleculares de tipificación epidemiológica en bacteriología. En: Procedimientos en Microbiología Clínica. 2a ed. España: Seimc: 2005: 1-72.

3. Singh A, Goering RV, Simjee S, Foley SL, Zervos MJ. Application of molecular techniques to the study of hospital infection. Clin Microbiol Rev. 2006; 19: 512-30.

4. Miranda G, Kelly C, Solorzano F, Leanos B, Coria, Patterson JE. Use of pulsed-field gel electrophoresis typing to study an outbreak of infection due to Serratia marcescens in a neonatal intensive care unit. J Clin Microbiol. 1996; 34: 3138-41.

5. Versalovic J, Koeuth T, Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application to finger printing of bacterial genomes. Nucleic Acids Res. 1991; 25: 6823.

6. Spigaglia P, Mastrantonio P. Evaluation of repetitive element sequence-based PCR as a Molecular Typing Method for Clostridium difficile. J Clin Microbiol. 2003; 41: 2454-7.

7. Johnson JR, O´Bryan T. Improved repetitive-element PCR fingerprinting for resolving pathogenic and nonpathogenic phylogenetic groups within Escherichia coli. Clin Diagn Lab Immunol. 2000; 7: 265-73.

8. Olive DM, Bean P. Principles and applications of methods for DNA-based typing of microbiol organisms. J Clin Microbiol. 1999; 37: 1661-9.

9. Matín-Lozano D, Cisneros JM, Becerril B, Cuberos L, Prados T, Ortíz-Leyba C, et al. Comparison of a repetitive extragenic palindromic sequence-based PCR method and clinical and microbiological methods for Acinetobacter baumannii bacteremia. J Clin Microbiol. 2002; 40: 4571-5.

10. Sanders WE Jr, Sanders CH. Enterobacter spp: pathogens poised to flourish at the turn of the century. Clin Microbiol Rev. 1997; 10: 220-41.

11. Haertl R, Bandlow G. Epidemiological fingerprinting of Enterobacter cloacae by small-fragment restriction endonuclease analysis and pulsed-field gel electrophoresis of genomic restriction fragments. J Clin Microbiol. 1993; 31: 128-33.

12. Jiang X, Ni Y, Jiang Y, Yuan F, Han L, Li M, et al. Outbreak of infection caused by Enterobacter cloacae producing the novel VEB-3 beta-lactamase in China. J Clin Microbiol. 2005; 43: 826-31.

13. Paauw A, Verhoef J, Fluit AC, Titia EM, Hopmans EM, Troelstra A, et al. Failure to control an outbreak of qnrA1-positive multidrug-resistant Enterobacter cloacae infection despite adequate implementation of recommended infection control measure. J Clin Microbiol. 2007; 45: 1420-5.

14. CLSI/NCCLS. Performance Standard for antimicrobial disk susceptibility test. 8th edition. Phyladelphia, USA: Approved standard; 2005.

15. Mattew H, Harris Am, Marshall MJ, Ross GW. The use of analytical isoelectric focusing for detection and identification of betalactamases. J Gen Microbiol. 1975; 88: 169-78.

16. Chung M, Lencastre H, Matthews P, Tomasz A, Adamsson I, Aires de Sousa M, et al. Molecular typing of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis: comparison of results obtained in a multilaboratory effort using identical protocols and MRSA strains. Microb Drug. 2000; 6: 189-98.

17. Snelling AM, Gernet-smidt P, Hawkey PM, Heritage J, Parnell P, Porter C, et al. Validation of use of whole-cell repetitive extrangenic palindromic sequence-based PCR (RER-PCR) for typing strains belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii complex and application of the method to the investigation of a hospital outbreak. J Clin Microbiol. 1996; 34: 1193-202.

18. Mantilla JR, García I, Espinal PA, Valenzuela EM. Estandarización y evaluación de tres sistemas de rep-PCR para la tipificación de Klebsiella pneumoniae. Rev Col Cienc Quím Farm. 2004; 33: 48-58.

19. Hunter PR, Gaston MA. Numerical index of the discriminatory ability of tiping systems: an application of simpson’s index of diversity. J Clin Microbiol. 1988; 26: 2465-6.

20. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol. 1995; 33: 2233-9.

21. Van Nierop WH, Duse AG, Stewart RG, Bilgeri YR, Koornhof HJ. Molecular epidemiology of an outbreak of Enterobacter cloacae in the neonatal intensive care unit of a provincial hospital in Gauteng, South Africa. J Clin Microbiol. 1998; 36: 3085-7.

22. Manzur A, Tubau F, Pujol M, Calatayud L, Dominguez MA, Peña C, et al. Nosocomial outbreak due to extended-spectrum-beta-lactamase-producing Enterobacter cloacae in a cardiothoracic intensive care unit. J Clin Microbiol. 2007; 45: 2365-9.

23. Bell JM, Turnidge JD, Jones RN, SENTRY Asia-Pacific Participants. Prevalence of extended-spectrum-beta-lactamase-producing Enterobacter cloacae in the Asia-pacific region: Result from SENTRY Antimicrobial surveillance program 1998-2001. Antimicrobial agent Chemother. 2003; 47: 3989-93.

24. Silva J, Cano M, Contreras J, Donis J, Martinez G, Morfin R, et al. Prevalence and molecular typing of extended-spectrum beta-lactamases (ESBLs) producing Escherichia coli and Enterobacter cloacae causing nosocomial infections in Mexico. Intersci Conf Antimicrob Agents Chemother. 2002; 42: abstract no. C2-1873.

25. Woods CR, Versalovic J, Koeuth T, Lupski JR. Whole-cell repetitive element sequence-based polymerase chain reaction allows rapid assessment of clonal relationships of bacterial isolates. J Clin Microbiol. 1993; 31: 1927-31.

26. Shen W, Hohn B. DMSO improves PCR amplification of DNA with complex secondary structure. Trends Genet. 1992; 8: 227.

27. Ross TL, Merz WE, Farkosh M, Carroll KC. Comparison of an automated repetitive sequence-based PCR microbial typing system to pulsed-field gen electrophoresis for analysis of outbreaks of methicillin-resistant Staphylococcus aureus. J Clin Microbiol. 2005; 43: 5642-7.