Biomedical Science Letters (대한의생명과학회지)

The Korean Society for Biomedical Laboratory Sciences (대한의생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Simple and Rapid Detection of Vancomycin-Resistance Gene from Enterococci by Loop-Mediated Isothermal Amplification

  • Baek, Yun Hee (Department of Microbiology, Chungbuk National University College of Medicine) ;
  • Hong, Seung Bok (Department of Clinical Laboratory Science, Chungbuk Health & Science University) ;
  • Shin, Kyeong Seob (Department of Laboratory Medicine, Chungbuk National University Hospital)
  • Received : 2020.05.26
  • Accepted : 2020.08.10
  • Published : 2020.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

We developed a simple and rapid method for detecting vancomycin resistance genes, such as vanA and vanB, using loop-mediated isothermal amplification (LAMP). To identify not only vancomycin resistance genes, but also the genus Enterococcus, primers were designed for vanA, vanB, and 16S rRNA. Screening for vancomycin susceptibility in Enterococcus was performed using Etest (bioMérieux Inc). The results of the LAMP assay were compared to those of real-time RT-PCR. The optimal conditions for the LAMP assay were 65℃ for 60 min. The detection limits of the LAMP assay for vanA, and vanB were 2 × 102 copies/reaction. Compared to RT-PCR, the sensitivities and specificities of LAMP for 16S rRNA, vanA, and vanB were 100/100%, 100/100%, and 100/100%, respectively. The vanA genotype-vanB phenotype accounted for 57.5% (46/80) of the vancomycin-resistant Enterococci samples collected from 2016 to 2019. In conclusion, the LAMP assay developed in this study showed high sensitivity and specificity for vancomycin-resistant genes. Moreover, due to the simplicity and rapidity of the LAMP assay, its use can be very useful in clinical microbiology laboratories.

Keywords

References

  1. Ballard SA, Grabsch EA, Johnson PD, Grayson ML. Comparison of three PCR primer sets for identification of vanB gene carriage in feces and correlation with carriage of vancomycinresistant enterococci: interference by vanB-containing anaerobic bacilli. Antimicrob Agents Chemother. 2005. 49: 77- 81. https://doi.org/10.1128/AAC.49.1.77-81.2005
  2. Bonten MJ, Willems RJ, Weinstein RA. Vancomycin-resistant enterococci: why are they here, and where do they come from? Lancet Infect Dis. 2001. 1: 314025.
  3. Cetinkaya Y, Falk P, Mayhall CG. Vancomycin-resistant enterococci. Clin Microbiol Reviews. 2000. 13: 686-707. https://doi.org/10.1128/CMR.13.4.686
  4. Chiang H-Y, Perencevich NE, Nair R, Nelson RE, Samore M, Khader K, et al. Incidence and outcomes associated with infections caused by vancomycin-resistant enterococci in the United States: systematic literature review and meta-analysis. Infect Control Hosp Epidemiol. 2017. 38: 203-215. https://doi.org/10.1017/ice.2016.254
  5. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing-Twenty-eighth Informational Supplement: M100-S28. CLSI, Wayne, PA, USA, 2018.
  6. Delmas J, Robin F, Schweitzer C, Lesens O, Bonnet R. Evaulation of a new chromogenic medium, chromID VRE, for detection of vancomycin-resistant Enterococci in stool samples and rectal swabs. J Clin Microbiol. 2007. 45: 2731-2733. https://doi.org/10.1128/JCM.00448-07
  7. Graham M, Ballard SA, Grabsch EA, Johnson PD, Grayson ML. High rates of fecal carriage of nonenterococcal vanB in both children and adults. Antimicrob Agents Chemother. 2008. 52: 1195-1197. https://doi.org/10.1128/AAC.00531-07
  8. Hara-Kudo Y, Yoshino M, Kojima T, Ikedo M. Loop-mediated isothermal amplification for the rapid detection of Salmonella. FEMS Microbiol Lett. 2005. 253: 155-161. https://doi.org/10.1016/j.femsle.2005.09.032
  9. Huang QQ, Liu BB, Zhu HF, Ma JJ, Tsoi M, Yao BQ, et al. Rapid and sensitive detection of the vanA resistance gene from clinical Enterococcus faecium and Enterococcus faecalis isolates by loop-mediated isothermal amplification. J Global Antimicrob Resist. 2019. 16: 262-265. https://doi.org/10.1016/j.jgar.2018.10.012
  10. Jung MK, Ahn SH, Lee WG, Lee EH. Molecular epidemiology of vancomycin-resistant enterococci isolated from non-tertiarycare and tertiary-care hospitals in Korea. Epidemiol Infect. 2014. 142: 2372-2377 https://doi.org/10.1017/S0950268813003543
  11. Kim HJ, Kim YJ, Yong DE, Lee K, Park JH, Lee JM, et al. Loop-mediated isothermal amplification of vanA gene enables a rapid and naked-eye detection of vancomycin-resistant enterococci infection. J Microbiol Methods. 2014. 104: 61-66. https://doi.org/10.1016/j.mimet.2014.05.021
  12. Lauderdale TL, McDonald LC, Shiau YR, Chen PC, Wang HY, Lai JF, Ho M. Vancomycin-resistant enterococci from humans and retail chickens in Taiwan with unique VanB phenotypevanA genotype incongruence. Antimicrob Agents Chemother. 2002. 46: 525-527. https://doi.org/10.1128/AAC.46.2.525-527.2002
  13. Lee WG, Hur JY, Cho SR, Lim YA. Reduction in glycopeptide resistance in vancomycin-resistant enterococci as a result of vanA cluster rearrangements. Antimicrob Agents Chemother. 2004. 48: 1379-1381. https://doi.org/10.1128/AAC.48.4.1379-1381.2004
  14. Li Y, Fan P, Zhou S, Zhang L. Loop-mediated isothermal amplification (LAMP): a novel rapid detection platform for pathogens. Microb Pathog. 2017. 107: 54-61. https://doi.org/10.1016/j.micpath.2017.03.016
  15. Misawa Y, Yoshida A, Saito R, Yoshida H, Okuzumi K, Ito N, et al. Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures. J Infect Chemother. 2007. 13: 134-140. https://doi.org/10.1007/s10156-007-0508-9
  16. Niederhausern SD, Bondi M, Messi P, Iseppi R, Sabia C, Manicardi G, et al. Vancomycin-resistant transferability from vanA Enterococci to Staphylococcus aureus. Current Microbiology. 2011. 62: 1363-1367. https://doi.org/10.1007/s00284-011-9868-6
  17. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acid Res. 2000: 28: e63. https://doi.org/10.1093/nar/28.12.e63
  18. Palladino S, Kay ID, Costa AM, Lambert EJ, Flexman JP. Realtime PCR for the rapid detection of vanA and vanB genes. Diagn Microbiol Infect Dis. 2003. 45: 81-84. https://doi.org/10.1016/S0732-8893(02)00505-9
  19. Simonsen GS, Myhre MR, Dahl KH, Olsvik O, Sundsfjord A. Typeability of Tn1546-like elements in vancomycin-resistant enterococci using long-range PCRs and specific analysis of polymorphic regions. Microb Drug Resist. 2000. 6: 49-57. https://doi.org/10.1089/mdr.2000.6.49
  20. Uttley AH, Collins CH, Naidoo J, George RC. Vancomycinresistant enterococci. Lancet. 1988. 1: 57-58. https://doi.org/10.1016/S0140-6736(01)18851-3
  21. Yamazaki W, Seto K, Taguchi M, Ishibashi M, Inoue K. Sensitive and rapid detection of cholera toxin-producing Vibrio cholerae using a loop-mediated isothermal amplification. BMC Microbiol. 2008. 8: 94. https://doi.org/10.1186/1471-2180-8-94