Real Time PCR for the Rapid Detection of vanA Gene in Surface Waters and Aquatic Macrophyte by Molecular Beacon Probe

Abstract

Enterococci serve as an "indicator" of fecal contamination for recreational water quality. The vancomycin-resistant-enterococci (VRE) are emerging environmental contaminants in the surface waters. The aim ofthis study wasto develop a rapid and specific molecular beacon probe (MBP)-based real-time PCR assay for detection of vanA gene in surface waters and aquatic macrophyte. The limit of detection (LOD) of the MBP assay was 1 CFU/mL of VRE [r = 0.943; PCR efficiency = 99.7%] in 2-fold dilution format within 2.5 h and demonstrated high specificityfor environmental enterococci isolates exhibiting VanA phenotype (n=25). VRE were detected from downstream surface waters of the rivers impacted by point sources of pollution and recreational activities.The probe detected vanA gene in rootmat associated microbiota of E. crassipes (Mart) Solms. an aquatic nuisance weed, at eutrophic sites of the surface waters (ANOVA p < 0.001). In addition, the assay enabled detection of otherwise nondetectable vanA gene concentration in the upstream sites of two Indian rivers (Student's ttest p < 0.001). The MBP assay developed can be used for sensitive and rapid detection of VRE in surface waters and identification of nonpoint sources of pollution for implementation of preventive measures to protect human health.

Enterococci in river Ganga surface waters: Propensity of species distribution, dissemination of antimicrobial-resistance and virulence-markers among species along landscape

“…Recently, Lata et al [31] reported the prevalence of vanA gene (for vancomycin resistance) in surface waters of river Ganga and its tributary and discussed the possible consequences of BPAR, its environmental carriage by plasmid maintenance systems or postsegregational killing (PSK) systems.…”

“…A panel of thirteen antimicrobials (antimicrobial abbreviation:mcg/disc) impregnated on paper discs (Himedia Ltd., India) belonging to eight different group of antimicrobials as Fluoroquinolone: Norfloxacin (Nx:10 mcg), β-lactam: Ampicillin (A:10 mcg), Oxacillin (Ox:1 mcg), PenicillinG (P:10 units), Methicillin (M:5 mcg), Aminoglycoside: Gentamicin (G:10 mcg), Streptomycin (S:10 mcg), Tetracycline: Tetracycline (T:30 mcg), Phenicol: Chloramphenicol (C:30 mcg), Macrolide: Erythromycin (E:15 mcg), Rifamycin: Rifampicin (R:5 mcg), Glycopeptides: Vancomycin (Va:30 mcg), Teicoplanin (Te:30 mcg) were used for testing the sensitivity of isolated organisms by Kirby-Bauer disc diffusion test as described by CLSI [31,44]. The diameter of zones showing inhibition were measured to the nearest mm and recorded.…”

Multiplex PCR based genotypic characterization of pathogenic vancomycin resistant Enterococcus faecalis recovered from an Indian river along a city landscape

“…vancomycin resistance and virulence markers such as gelatinase ( gelE ), collagen binding protein ( ace ), endocarditis-associated antigen ( efaA ) and enterococcal surface protein ( esp ), have been explored mostly in clinical settings worldwide (Coque et al 2002 ; Lowe et al 1995 ; Eaton and Gasson 2001 ; Ghoshal et al 2006 ; Mathur et al 2003 ). However, very little is known about the distribution of antimicrobial-resistance and virulence-markers in E. faecalis isolates recovered from river waters (Novais et al 2005 ; Moore et al 2008 ; Lata et al 2009b ). River waters in developing countries are vulnerable to contamination due to discharge of untreated or partially treated municipal and industrial wastes, domestic and wild animal fecal waste and recreational activities on the banks (Ahmed et al 2005 ; Ram et al 2007 ).…”

Persistence of wastewater antibiotic resistant bacteria and their genes in human fecal material

“…The qPCR was conducted in a StepOne™ Real-Time PCR System (Life Technologies, Carlsbad, CA) following the conditions previously described (Narciso-da-Rocha et al, 2018) for the ARGs blaTEM, blaCTX and blaOXA-A and the 16S rRNA gene. The qPCR conditions for the detection of the gene vanA were set up in 25 µL volume using the Power SYBR Green mastermix (Thermo Fisher Scientific, Austin, USA) containing 10 µM of each primer: VnF 5'-ATCGGCAAGACAATATGACAGC-3' and VnR 5'-AGCCTGATTTGGTCCACCTC-3' (Lata et al, 2009). The PCR program was initiated by a period of 5 min at 95 °C, followed by 40 cycles of 95 °C for 15 sec and 60 °C for 30 sec.…”