Detection of vanA and vanB genes in vancomycin-resistant enterococci (VRE) from groundwater using multiplex PCR analysis

A total of 22 groundwater samples were randomly collected from three rural communities in the Mafikeng area. Bile esculin agar was used for selective isolation of enterococci. Standard preliminary tests (Gram staining, oxidase test, catalase test) and confirmatory tests (Prolex™ Streptococcal Grouping Rapid Latex Agglutination test kit) were used to determine the identities of presumptive enterococci. The antibiotic sensitivity test was performed on all positively identified enterococci; percentage resistance and multiple antibiotic resistance (MAR) phenotypes were generated. Multiplex polymerase chain reaction (PCR) was performed to detect vanA and vanB genes vancomycin-resistant enterococci (VRE). A total of 179 enterococci were positively identified and the proportion of isolates from Dibate (62.5%) was higher compared to those from Majemantsho and Motlhabeng (22.3 and 15.0, respectively). A large proportion (81.5 to 100%) of the isolates from Dibate, Motlhabeng and Majemantsho were resistant to ampicillin, vancomycin and penicillin G. Two main MAR phenotypes, PG-VA-Ap-A-OX and PG-VA-Ap-OX, were identified. Multiplex PCR analysis of 50 VRE indicated that 17 (34%) were positive for vanA and vanB genes. This highlights the need to determine the cause of vancomycin resistance in enterococci in the sampled sites and suggests that sequence analysis be used to confirm the identities of these amplicons.