Abstract
BackgroundEnterococci 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.ResultsUnique 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.ConclusionThe 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.
Highlights
Enterococci have emerged as a significant cause of nosocomial infections in many parts of the world over the last decade
The vanA-type strains are resistant to high levels of both vancomycin and teicoplanin antimicrobials (MIC ≥ 64 μg/ml and >16 μg/ml, respectively). vanB-type strains are resistant to a wide range of vancomycin concentration (MIC between 4 to ≥ 1,024 μg/ml) and are susceptible to teicoplanin. vanD-type strains are resistant to moderate levels of vancomycin (MIC 128 μg/mL) and susceptible to teicoplanin, while vanC, vanE, and vanG-type strains exhibit low-level resistance to vancomycin [3,9]
High level gentamicin resistance (HLGR) phenotype is due to the expression of bifunctional aminoglycoside-modifying enzymes [AAC(6')-APH(2")] that are encoded by the aacA-aphD gene (Gentamicin Minimum inhibitory concentration (MIC) range, ≥ 100–500 μg/ ml) [10,11]
Summary
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. 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. The vanA-type strains are resistant to high levels of both vancomycin and teicoplanin antimicrobials (MIC ≥ 64 μg/ml and >16 μg/ml, respectively). High level gentamicin resistance (HLGR) phenotype is due to the expression of bifunctional aminoglycoside-modifying enzymes [AAC(6')-APH(2")] that are encoded by the aacA-aphD gene (Gentamicin MICs range, ≥ 100–500 μg/ ml) [10,11]
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