A nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistance genes in Enterococcus species.
BACKGROUND
Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade. The most common enterococci strains present in clinical isolates are E. faecalis and E. faecium which have acquired resistant to either gentamicin or vancomycin. The conventional culture test takes 2-5 days to yield complete information of the organism and its antibiotic sensitivity pattern. Hence our present study was focused on developing a nanoplex PCR assay for the rapid detection of vancomycin and bifunctional aminoglycoside resistant enterococci (V-BiA-RE). This assay simultaneously detects 8 genes namely 16S rRNA of Enterococcus genus, ddl of E. faecalis and E. faecium, aacA-aphD that encodes high level gentamicin resistance (HLGR), multilevel vancomycin resistant genotypes such as vanA, vanB, vanC and vanD and one internal control gene.


RESULTS
Unique and specific primer pairs were designed to amplify the 8 genes. The specificity of the primers was confirmed by DNA sequencing of the nanoplex PCR products and BLAST analysis. The sensitivity and specificity of V-BiA-RE nanoplex PCR assay was evaluated against the conventional culture method. The analytical sensitivity of the assay was found to be 1 ng at the DNA level while the analytical specificity was evaluated with 43 reference enterococci and non-enterococcal strains and was found to be 100%. The diagnostic accuracy was determined using 159 clinical specimens, which showed that 97% of the clinical isolates belonged to E. faecalis, of which 26% showed the HLGR genotype, but none were vancomycin resistant. The presence of an internal control in the V-BiA-RE nanoplex PCR assay helped us to rule out false negative cases.


CONCLUSIONS
The nanoplex PCR assay is robust and can give results within 4 hours about the 8 genes that are essential for the identification of the most common Enterococcus spp. and their antibiotic sensitivity pattern. The PCR assay developed in this study can be used as an effective surveillance tool to study the prevalence of enterococci and their antibiotic resistance pattern in hospitals and farm animals.
Yean
Chan Yean
CY
Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Malaysia. yeancyn@yahoo.com
Yin
Lee Su
LS
Lalitha
Pattabhiraman
P
Ravichandran
Manickam
M
D000617:Aminoglycosides / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D024881:Drug Resistance, Bacterial; D013293:Enterococcus faecalis / Q000145:classification / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D016984:Enterococcus faecium / Q000145:classification / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D005839:Gentamicins / Q000494:pharmacology; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D014640:Vancomycin / Q000494:pharmacology
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D005839:Gentamicins; D012336:RNA, Ribosomal, 16S; D014640:Vancomycin