Investigation of the Prevalence of vanA and vanB genes in vancomycin resistant enterococcus (VRE) by Taq Man real time PCR Assay

Abstract

Objective: Enterococci are important nosocomial agents and due to their potential antimicrobial resistance they have a significant role in the dissemination of resistance genes. Currently, these species are described as healthcare concern. The aim of this study was to determine vanA and vanB genes in vancomycin resistant enterococci (VRE) strains isolated from the various clinical samples in the hospitals in Iran. Methods: Susceptibility of 235 strains to vancomycin was screened as minimum inhibitory concentration (MIC) by E-test. The genes encoding modifying vancomycin precursor's dipeptide termini named as vanA and vanB genes were targeted by Taq Man real time PCR assay in vancomycin resistant and vancomycin intermediate resistant Enterococcus faecalis and Enterococcus faecium strains. Results: A total of 235 enterococci were isolated from the clinical specimens. One hundred and ninety three (82.1%) of them were defined as E. faecalis, 33 (14.0%) E. faecium, 1/235 (0.4%) E. avium, 1/235 (0.4%) E. raffinosus and 7/235 (3.0%) E. galinarium. The prevalence of vancomycin resistance was 13.6% (32/235) consisting of 18/235 (7.7%) E. faecalis and 6.0% (14/235) E. faecium. Among the 32 VRE strains, a total of 36 vanA and vanB genes were detected (some isolates had both vanA and vanB genes). These resistance genes were not detected in 5 out of 32 (15.6%) isolates. Conclusion: E. faecalis was more common in clinical samples and vanA (58.3%) gene was the predominant gene among the VRE isolates. The current study showed that Taq Man real time PCR assay is the useful, precise and rapid detection of vancomycin resistance genes.

Keywords

• VRE, Taq Man real time PCR, vanA, vanB, Enterococcus faecalis

Investigation of the Prevalence of vanA and vanB genes in vancomycin resistant enterococcus (VRE) by Taq Man real time PCR Assay

Öz

Amaç: Enterokoklar önemli hastane enfeksiyonu etkenidirler ve antimikrobiyal direnç potansiyelleri nedeniyle rezistans genlerinin yayılmasında önemli rolleri bulunmaktadır. Günümüzde bu mikroorganizmalar önemli bir halk sağlığı sorunu olarak tanımlanmıştır. Bu çalışmada İran’daki bazı hastanelerde değişik klinik örneklerden izole edilen vankomisin dirençli enterokok (VRE)’lar arasında vanA ve vanB genlerinin sıklığının araştırılması amaçlandı. Yöntemler: Suşların vankomisin duyarlılıkları E test yöntemiyle araştırılarak minimum inhibitör konsantrasyon (MİK) değerleri belirlendi. Vankomisin dirençli ve vankomisine orta düzeyde dirençli Enterococcus faecalis ve Enterococcus faecium suşlarında Taq Man real time PCR assay ile vankomisin direncini kodlayan vanA ve vanB genleri hedeflendi. Bulgular: Çalışma süresince klinik örneklerden toplam 235 enterokok suşu izole edildi. Bu şuşlardan 193’ü (% 82,1) E. faecalis, 33’ü (% 14,0) E. faecium, 1’i (% 0,4) Enterococcus avium, 1’i (% 0,4) Enterococcus raffinosus ve 7’si (% 3,0) Enterococcus galinarium olarak tanımlandı. Toplam 235 enterokok suşunda 32’si (% 13,6) VRE idi. Toplam 235 suştan 18 (% 7,7)’i E. faecalis ve 14’ü (% 6,0) E. faecium idi. Bu suşların 27 (% 84,4)’sinde vanA ve vanB genleri belirlendi (bazı izolatlar hem vanA ve hem vanB genlerine sahip idi). Suşların 5’inde (% 15,6) direnç geni belirlenemedi.Sonuçlar: Araştırmamızda klinik VRE suşları arasında E. faecalis suşlarının sıklıkta olduğu ve vanA geninin vankomisin direncine sebep olan en sık gen olduğu gözlendi. Bu çalışma Taq Man real time PCR assay testinin vankomisin direnç genlerinin gösterilmesinde kullanılabilen, kesin ve hızlı bir test olduğunu göstermektedir

Anahtar Kelimeler

• VRE, Taq Man real time PCR, vanA, vanB, Enterococcus faecalis, Enterococcus faecium

References

1. Murray P, Rosenthal K, Pfaller M. Medical Microbiology, 6th edn. Philadelphia, 2009:243-246.
2. Kacmaz B, Aksoy A. Antimicrobial resistance of enterococci in Turkey. Int J Antimicrob Agents 2005;25:535-538.
3. Malathum K, Murray BE. Vancomycin-resistant enterococci: recent advanCes in genetics, epidemiology and therapeutic options. Drug Resist Updat 1999;2:224-243.
4. Japoni A, Farshad S, Ziyaeyan M, Ziaian S. Detection of Van- positive and negative vancomycin resistant entrococci and their antibacterial susceptibility patterns to the newly intro- duced antibiotics. Pak J Biol Sci 2009;12:844-851.
5. Pangallo D, Drahovská H, Harichová J, et al. Assessment of environmental enterococci: bacterial antagonism, pathogen- ic capacity and antibiotic resistance. Antonie Van Leeuwen- hoek 2008;94:555-562.
6. Emaneini M, Aligholi M, Aminshahi M. Characterization of glycopeptides, aminoglycosides and macrolide resistance among Enterococcus faecalis and Enterococcus faecium iso- lates from hospitals in Tehran. Pol J Microbiol 2008;57:173- 178.
7. Palladino S, Kay ID, Costa AM, Lambert EJ, Flexman JP. Real- time PCR for the rapid detection of vanA and vanB genes. Diagn Microbiol Infect Dis 2003;45:81-84.
8. Feizabadi MM, Sayadi S, Shokrzadeh L, Parvin M, Yadegaryn- ia D. Increase in prevalence of vancomycin resistant isolates of Enterococcous faecium at Labbafinejad hospital. Iranian J Clin Infect Dis 2008;3:73-77.

9. Arbeur N, Weirich A. Real time detection for VREs. Spartan Bioscience, 2008;1-3.
10. Forbes BA, Sahmm DF, Weisfeld A. Catalase negative gram positive cocci. Baily and Scotts Diagnostic Microbiology ,10th edn. St. Louis, Missouri, USA; Mosby, 1998:620-635.
11. Betty A, Daniel F, Alice S. Baily and Scotts Diagnostic Micro- biology, 10th edn. 2007;17:265-278.
12. Clinical and Laboratory Standards Institute (CLSI). Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria; approved guide- line. M100-S20. Clinical and Laboratory Standards Institute, Wayne, PA 2010;30:20.
13. Sharifi Y, Hasani A, Ghotaslou, R. Survey of virulence deter- minants among vancomycin resistant Enterococcus faecalis and Enterococcus faecium isolated from clinical specimens of hospitalized patients of North West of Iran. Open Micro J 2012;6:34-43.
14. Salem-Bekhit MM, Moussa IM, Muharram MM, Alanazy FK, Hefni HM. Prevalence and antimicrobial resistance pattern of multidrug-resistant enterococci isolated from clinical speci- mens. Indian J Med Microbiol 2012;30:44-51.
15. Giridhara Upadhyaya PM, Ravikumar KL, Umapathy BL. Re- view of virulence factors of Enterococcus: an emerging Nos- ocomial pathogen. Indian J Med Microbiol 2009;27:301-305.
16. Laupland KB, Bagshaw SM, Gregson DB, Kirkpatrick AW, Ross T, Church DL. Intensive Care Unit-Acquired Tract Infec- tion in a regional critical care system. Crit Care 2005;9:R60- 65.
17. Pérez-Hernández X, Méndez-Alvarez S, Delgado T, et al. Low prevalence of vancomycin-resistant enterococci in clini- cal samples from hospitalized patients of the Canary Islands, Spain. Int Microbiol 2002;5:117-120.
18. Arthur M, Quintiliani R Jr. Regulation of VanA- and VanB-type glycopeptide resistance in enterococci. Antimicrob Agents Chemother 2001;45:375-381.
19. vanDen Braak N, Ott A, van Belkum A, et al. Prevalence and determinants of fecal colonization with vancomycin-resistant Enterococcus in hospitalized patients in The Netherlands. Infect Control Hosp Epidemiol 2000;21:520-524.
20. Kim WJ, Weinstein RA, Hayden MK. The changing molecular epidemiology and establishment of endemicity of vancomy- cin resistance in enterococci at one hospital over a 6-year period. J Infect Dis 1999;179:163-171.
21. Woodford N, Chadwick PR, Morrison D, Cookson BD. Strains of glycopeptide-resistant Enterococcus faecium can alter their vanGenotypes during an outbreak. J Clin Microbiol 1997;35:2966-2968.
22. Patel R, Uhl JR, Kohner P, Hopkins MK, Cockerill FR 3rd. Multiplex PCR detection of vanA, vanB, vanC-1, and vanC- 2/3 genes in enterococci. J Clin Microbiol 1997;35:703-707.
23. Grayson ML, Grabsch EA, Johnson PD, et al. Outcome of a screening program for vancomycin-resistant enterococci in a hospital in Victoria. Med J Aust 1999 2;171:133-136.