Occurrence of vancomycin resistant enterococci (VRE) in two Durban wastewater treatment plants for effluent reuse

Abstract

The presence of enterococci in improperly treated wastewater leads to pollution of the recipient water bodies which directly or indirectly affects the humans especially when antibiotic resistant strains are involved. In 2017 the World Health Organization listed vancomycin resistant enterococci (VRE) among those with highest priority for further surveillance and research, both among humans and in the receiving aquatic environment. The purpose of this study is to determine how efficient the WWTPs are in removing both vancomycin-resistant enterococci (VRE) and vancomycin-sensitive enterococci (VSE) from wastewater. One hundred (60 wastewater and 40 river) samples were collected from July 2016 to June 2017 which covered the warm and cold seasons of South Africa. Primary isolation and enumeration were carried out on Slanetz and Bartley agar supplemented with and without vancomycin (6 µg/mL) for vancomycin resistant enterococci (VRE) and total enterococci (TE) respectively. Presumptive enterococci were selected using Gram staining, growth on bile aesculin agar, catalase and pyrase tests. The presumptive enterococci isolates (202 VRE and 67 VSE) were confirmed and speciated using polymerase chain reaction (PCR). The identified Enterococcus isolates were subjected to antibiotic susceptibility testing (AST) to examine their resistance profile against fifteen antibiotics including vancomycin. Antibiotic resistance genes (van, tet and emeA) were detected by PCR. The TE and VRE counts of the two WWTPs influents ranged from 6.1 to 7.2 log10 CFU/100 mL (for TE) and 4.3 to 6.7 log10 CFU/100 mL (for VRE) while the effluent concentration of Plant II contained 1.5 to 4.4 log10 CFU/100 mL and 0.9 to 3.4 log10 CFU/100 mL for TE and VRE respectively. Neither TE nor VRE was detected in Plant I effluent. The TE and VRE counts of the recipient river samples were higher than the effluents. There were no visible seasonal effects based on the counts. The removal efficiencies in the two plants

ranged from 95 to 100%, where chlorination played a major role. Two hundred and sixty- nine (202 VRE and 67 VSE) isolates were identified by PCR as Enterococcus. The most abundant species was E. faecium followed by E. faecalis while other species include E. hirae, E. gallinarum, E. durans, E. casseliflavus and E. cecorium. MALDI-TOF and PCR were used in parallel for the identification of the isolates, which resulted in 80.1% agreement for genus identification. The AST results showed that a large percentage (39 to 98%) were resistantto all other antibiotics except amoxicillin/clavulanic acid and imipenem to which the isolates showed high sensitivity. Four van genes (vanA, vanB, vanC1, vanC2/3) and 4 tet genes (tetK, tetL, tetM, tetO), and also the multidrug efflux pump gene, emeA were detected among the 269 enterococci isolates with vanA and tetL being the most prevalent. At least one virulence gene (ace, asa1, cylA, efaA, esp, gelE and hyl) occurred in 74% (67/88) of the isolates. The result showed that the two WWTPs are efficient in removing both enterococci and VRE from their influents. Thus these effluents had little or no effect to enterococci count of their interlinked recipients. Also, a majority of the isolates are not only antibiotic resistant strains but are also virulent. They therefore pose risk to public health.