Vibrio parahaemolyticus is a leading cause of seafood-related gastroenteritis and is also an autochthonous member of marine and estuarine environments worldwide. One-hundred seventy strains of V. parahaemolyticus were isolated from water and plankton samples collected along the Georgian coast of the Black Sea during 28 months of sample collection. All isolated strains were tested for presence of tlh, trh, and tdh. A subset of strains were serotyped and tested for additional factors and markers of pandemicity. Twenty-six serotypes, five of which are clinically relevant, were identified. Although all 170 isolates were negative for tdh, trh, and the Kanagawa Phenomenon, 7 possessed the GS-PCR sequence and 27 the 850 bp sequence of V. parahaemolyticus pandemic strains. The V. parahaemolyticus population in the Black Sea was estimated to be genomically heterogeneous by rep-PCR and the serodiversity observed did not correlate with rep-PCR genomic diversity. Statistical modeling was used to predict presence of V. parahaemolyticus as a function of water temperature, with strongest concordance observed for Green Cape site samples (Percent of total variance = 70, P < 0.001). Results demonstrate a diverse population of V. parahaemolyticus in the Black Sea, some of which carry pandemic markers, with increased water temperature correlated to an increase in abundance of V. parahaemolyticus.
Microbial safety of recreational water is one of the major human public health issues in developing countries. Three water bodies, the Tbilisi Sea, Kumisi and Lisi lakes, in the South Caucasus region near Tbilisi, Georgia, were monitored in 2006-2009 to determine microbiological quality using standard methods. Microbial pollution indicators were determined in parallel with phytoplankton abundance and measurement of a number of physical-chemical parameters. Kumisi Lake, a brackish water body in an active agricultural area, appeared to be the most polluted, whereas the Tbilisi Sea, a freshwater reservoir was the least polluted. High values for fecal indicators in all three lakes in summer and early autumn were revealed. In our study, total enterococci counts (TEC) appeared to be a better indicator than either fecal or total coliform counts for the evaluation of fresh and brackish microbial water quality. We found significant correlation between total Vibrio counts and TEC for all three water bodies. Prevalence of somatic coliphages and V. cholerae-specific phages as additional water pollution indicator significantly correlated with abundance of the host bacteria. Particular phytoplankton groups in the lakes responded to the changes of fecal indicators; however, no correlation was observed between dominant zooplankton taxonomic groups and microbial parameters.
Although horizontal gene transfer plays a pivotal role in bacteriophage evolution, many lytic phage genomes are clearly shaped by vertical evolution. We investigated the influence of minor genomic deletions and insertions on various phage-related phenotypic and serological properties.
The Ibis T5000 is a novel diagnostic platform that couples PCR and mass spectrometry. In this study, we developed an assay that can identify all known pathogenic Vibrio species and field-tested it using natural water samples from both freshwater lakes and the Georgian coastal zone of the Black Sea. Of the 278 total water samples screened, 9 different Vibrio species were detected, 114 (41%) samples were positive for V. cholerae, and 5 (0.8%) samples were positive for the cholera toxin A gene (ctxA). All ctxA-positive samples were from two freshwater lakes, and no ctxA-positive samples from any of the Black Sea sites were detected.
Three freshwater lakes, Lisi Lake, Kumisi Lake and Tbilisi Sea, near Tbilisi, Georgia, were studied from January 2006 to December 2007 to determine the presence of Vibrio cholerae employing both bacteriological culture method and direct detection methods, namely PCR and direct fluorescent antibody (DFA). For PCR, DNA extracted from water samples was tested for presence of V. cholerae and genes coding for selected virulence factors. Vibrio cholerae non-O1/non-O139 was routinely isolated by culture from all three lakes; whereas V. cholerae O1 and O139 were not. Water samples collected during the summer months from Lisi Lake and Kumisi Lake were positive for both V. cholerae and V. cholerae ctxA, tcpA, zot, ompU and toxR by PCR. Water samples collected during the same period from both Lisi and Kumisi Lake were also positive for V. cholerae serogroup O1 by DFA. All of the samples were negative for V. cholerae serotype O139. The results of this study provide evidence for an environmental presence of toxigenic V. cholerae O1, which may represent a potential source of illness as these lakes serve as recreational water in Tbilisi, Georgia.
We describe the small-scale, laboratory-based, production and quality control of a cocktail, consisting of exclusively lytic bacteriophages, designed for the treatment of Pseudomonas aeruginosa and Staphylococcus aureus infections in burn wound patients. Based on successive selection rounds three bacteriophages were retained from an initial pool of 82 P. aeruginosa and 8 S. aureus bacteriophages, specific for prevalent P. aeruginosa and S. aureus strains in the Burn Centre of the Queen Astrid Military Hospital in Brussels, Belgium. This cocktail, consisting of P. aeruginosa phages 14/1 (Myoviridae) and PNM (Podoviridae) and S. aureus phage ISP (Myoviridae) was produced and purified of endotoxin. Quality control included Stability (shelf life), determination of pyrogenicity, sterility and cytotoxicity, confirmation of the absence of temperate bacteriophages and transmission electron microscopy-based confirmation of the presence of the expected virion morphologic particles as well as of their specific interaction with the target bacteria. Bacteriophage genome and proteome analysis confirmed the lytic nature of the bacteriophages, the absence of toxin-coding genes and showed that the selected phages 14/1, PNM and ISP are close relatives of respectively F8, phiKMV and phage G1. The bacteriophage cocktail is currently being evaluated in a pilot clinical study cleared by a leading Medical Ethical Committee.
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