Diversity of Vibrio spp. isolated at ambient environmental temperature in the Eastern English Channel as determined by pyrH sequencing.

AIMS: To describe the diversity of the culturable mesophilic and potentially pathogenic vibrios isolated at 22 and 37°C on TCBS medium, in September 2009 from seawater and surface sediments. METHODS AND RESULTS: q-PCR assays previously selected for the identification of bacterial strains isolated at 37°C were used in combination with the partial sequencing of two housekeeping genes, pyrH and toxR, to identify 315 strains isolated at 22°C. The great majority of the 37°C strains was identified by q-PCR assays, (five of the six species) with the predominance of Vibrio alginolyticus (85·9%) and V. harveyi (10·7%). The human pathogens V. parahaemolyticus and V. cholerae were rarely detected (two strains each). The 22°C strains were successfully identified by the phylogeny analysis of pyrH and toxR genes, revealing 20 Vibrio species, with the predominance of the clam pathogen V. celticus (36·8%). The Splendidus and the Harveyi groups represented the main Vibrio group at 22°C (80%) and 37°C (99·5%), respectively. CONCLUSIONS: The combination of q-PCR assays and the sequencing of pyrH and toxR genes highlighted two different Vibrio communities at 22 and 37°C both dominated by pathogenic species for marine organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The sequencing of the pyrH gene revealed to be a valuable tool to identify environmental Vibrio spp. strains isolated at 22°C, as 92·3% of them were identified in this study.

Real-time PCR optimization to identify environmental Vibrio spp. strains.

AIMS: To identify Vibrio vulnificus, Vibrio cholerae and Vibrio alginolyticus using standardized DNA extraction method and real-time PCR assays, among a large number of bacterial strains isolated from marine environment. METHODS AND RESULTS: Methods for DNA extraction and real-time PCR were standardized to identify a large number of Vibrio spp. strains isolated through regular collection campaigns of environmental samples. Three real-time PCR assays were developed from a multiplex PCR, targeting V. vulnificus, V. cholerae and V. alginolyticus on the dnaJ gene. After testing their specificity, these systems were applied for the identification of 961 strains isolated at 22°C (446 strains) and 37°C (515 strains) in September 2009. The predominance of V. alginolyticus (82·6%) among the Vibrio spp. strains isolated at 37°C was shown. At 22°C, only 1·6% of the strains were identified by PCR and they were V. alginolyticus. CONCLUSIONS: Reproducible and specific real-time PCR assays combined to a DNA extraction method on microplates were used to constitute a large environmental Vibrio strains collection and to identify and detect potential human pathogenic Vibrio isolated at 37°C. For environmental strains isolated at 22°C, because of the higher species diversity, other approaches, like sequencing, should be chosen for identification. SIGNIFICANCE AND IMPACT OF THE STUDY: The protocol developed in this study provides an appropriate and rapid screening tool to identify a large number of bacterial strains routinely isolated from the environment in long-term studies.