Evaluation of enrichment media for improved PCR-based detection of V. cholerae and V. vulnificus from estuarine water and plankton

Abstract

Aims: Pathogenic Vibrio spp., including V. cholerae and V. vulnificus, are commonly found along the estuaries of the south‐east United States; however, it is often difficult to recover these species directly from environmental samples. Pre‐enrichment assays are commonly used to improve the detection of pathogenic vibrios from environmental sources. Here, we evaluated a novel enrichment procedure using freshly collected and autoclaved natural estuarine water amended with 1% peptone (designated as estuarine peptone water, EPW) and compared it to traditional alkaline peptone water (APW) for detection by PCR of V. cholerae and V. vulnificus. Methods and Results: Of the 50 samples collected in total, V. cholerae DNA was detected in APW 10% of the time and in EPW 40% of the time. Likewise, the cholera toxin gene (ctxA) was detected in 4 vs 18% of the samples using APW and EPW, respectively. Conversely, APW showed improved recovery for V. vulnificus relative to EPW with respective detection frequencies of 46 and 20%. Results showed similar patterns across different sample types (water and plankton). Conclusions: While enrichment in traditional APW was adequate for the recovery of Vibrio vulnificius, use of sterile estuarine water amended with peptone significantly improved the detection of V. cholerae and the virulence gene ctxA from estuarine sources.

Development of agroELgene-based species-specific multiplex polymerase chain reaction assay for simultaneous detection ofVibrio cholerae,Vibrio parahaemolyticusandVibrio vulnificus

“…Enrichment procedures can minimize the interference of the PCR inhibitors and increase the concentration of the target micro-organisms. Enrichment for 5-12 h was applied during the detection of Vibrio species from fish, fishery products, shellfish and water (Hossain et al 2011;Jeyasekaran et al 2011;Malayil et al 2011;Wong et al 2012). In this study, we extracted DNA from inoculated shellfish homogenates and sea water before and after enrichment for 5 h. Detection was possible in sea water samples before and after enrichment, but weak amplicons were observed in shellfish samples before enrichment.…”

Detection of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae with respect to seasonal fluctuations in temperature and plankton abundance

“…The limit of detection for V. cholerae (ITS region) was 10 copies per PCR reaction in the water enrichment and 65 copies per reaction in the plankton enrichments. We have previously shown that this method of enrichment followed by PCR can reliably detect as few as 5 CFU 100 ml −1 of estuarine water and 2 CFU 25 ml −1 of material from a plankton net tow (Malayil et al ., ).…”

Non-serogroup O1/O139 agglutinable Vibrio cholerae: a phylogenetically and genealogically neglected yet emerging potential pathogen of clinical relevance

“…are referred to as somatic antigen type-1/139 non-agglutinable strains (SANAS-1/139- Vc or non-serogoup agglutinating O1/O139 V. cholerae (NSA-O1/139 Vc ) (Chatterjee et al 2011 ; Sack et al 2003 ). The SANAS-1/139- Vc member does not possess cell surface polysaccharide and antigenic determinant for agglutinating somatic antgen type-1/139 as the name specifies, yet its members ( V. cholerae strains) (Shan et al 2022 ; Devi et al 2022 ; Malayil et al 2011 ) may be implicated in cholera outbreak case, wound, septicemia, gastro-enteritis and diarrhea. These Vibrio members have been in existence in the environment (water, plankton/sediment and fishes) since the discovery of the family Vibrionaceae in 1890 (Huq et al 2012 ).…”