Comparison of primers for the pathogenicity factors <i>vvhA</i> and <i>rpoS</i> in <i>Vibrio vulnificus</i> environmental isolates from the Texas Coastal Bend region of the Gulf of Mexico

The gram-negative marine bacterium Vibrio vulnificus may cause necrotizing fasciitis in persons with predisposing conditions such as type II diabetes and immunosuppression. Inhabitants of the Texas Coastal Bend have a high prevalence of such conditions and are at risk from contracting vibriosis upon flooding of marine and estuarine waters during storms, leading to increased risk of morbidity and mortality. While digital droplet polymerase chain reaction (ddPCR) and other methods may quickly identify pathogenic bacteria with more sensitivity and specificity, the time to optimize the assay, cost and dependence on uninterrupted power supplies make this method less useful in locations with minimal resources. This investigation undertook a longitudinal study from 2009, 2014, and 2021 to determine if primers for the cytolysin-hemolysin gene, vvhA , and the alternative sigma factor, rpoS , could be consistently used in polymerase chain reaction (PCR) assays to identify V. vulnificus strains taken from local waters. Inconsistencies in expected results led to the hypothesis that inability to reproduce PCR assays resulted from technical concerns or the vvhA locus exhibited instability or genome plasticity. Whole genome sequences of V. vulnificus ATCC 27562 were retrieved on a high-performance cluster through NCBI Primer Blast and used to design additional vvhA and rpoS primers. In-silico PCR detecting a 265 bp amplicon of vvhA was performed for 12,410 sequences to determine specificity and sensitivity, producing amplicons from 17 of 32 V. vulnificus strains (53%) but not any other species within the genus Vibrio , demonstrating sensitivity. No members of other gram-negative or gram-positive bacteria yielded amplicons, indicating specificity. New rpoS primers were designed to produce 393 bp amplicons. Due to loss of strains from hurricane outages, the number of Coastal Bend V. vulnificus isolates studied decreased from 2009 to 2021, but 21 of 22 V. vulnificus strains (96%) showed vvhA amplicons and 22 of 23 strains (96%) revealed rpoS amplicons. Primers for vvhA were analyzed within versions of the vvhA sequence for single nucleotide variants (SNVs). Two SNVs were seen in the area bound by the forward primer and four within the annealing region of the reverse primer, but SNV analysis reveals that these mutations may affect primer binding; however, further research is warranted. Limitations of the study were small sample sizes. These findings support the hypothesis that technical issues, rather than inherent genetic instability of the vvhA locus, caused the lack of PCR reproducibility, and confirm vvhA as a target for detecting pathogenic V. vulnificus in coastal waters of the Texas Coastal Bend region. Future goals will be to analyze larger numbers of south Texas V. vulnificus isolates to allow monitoring for Vibrio infections that may occur from post-hurricane floods resulting from climate change.