Microbial Diversity and Phage-Host Interactions in the Georgian Coastal Area of the Black Sea Revealed by Whole Genome Metagenomic Sequencing.

Viruses have the greatest abundance and highest genetic diversity in marine ecosystems. The interactions between viruses and their hosts is one of the hot spots of marine ecology. Besides their important role in various ecosystems, viruses, especially bacteriophages and their gene pool, are of enormous interest for the development of new gene products with high innovation value. Various studies have been conducted in diverse ecosystems to understand microbial diversity and phage-host interactions; however, the Black Sea, especially the Eastern coastal area, remains among the least studied ecosystems in this regard. This study was aimed at to fill this gap by analyzing microbial diversity and bacteriophage-host interactions in the waters of Eastern Black Sea using a metagenomic approach. To this end, prokaryotic and viral metagenomic DNA from two sampling sites, Poti and Gonio, were sequenced on the Illumina Miseq platform and taxonomic and functional profiles of the metagenomes were obtained using various bioinformatics tools. Our metagenomics analyses allowed us to identify the microbial communities, with Proteobacteria, Cyanobacteria, Actinibacteria, and Firmicutes found to be the most dominant bacterial phyla and Synechococcus and Candidatus Pelagibacter phages found to be the most dominant viral groups in the Black Sea. As minor groups, putative phages specific to human pathogens were identified in the metagenomes. We also characterized interactions between the phages and prokaryotic communities by determining clustered regularly interspaced short palindromic repeats (CRISPR), prophage-like sequences, and integrase/excisionase sequences in the metagenomes, along with identification of putative horizontally transferred genes in the viral contigs. In addition, in the viral contig sequences related to peptidoglycan lytic activity were identified as well. This is the first study on phage and prokaryote diversity and their interactions in the Eastern coastal area of the Black Sea using a metagenomic approach.

Microbial water quality of recreational lakes near Tbilisi, Georgia.

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.

New Bacteriophages with Podoviridal Morphotypes Active against Yersinia pestis: Characterization and Application Potential.

Phages of highly pathogenic bacteria represent an area of growing interest for bacterial detection and identification and subspecies typing, as well as for phage therapy and environmental decontamination. Eight new phages-YpEc56, YpEc56D, YpEc57, YpEe58, YpEc1, YpEc2, YpEc11, and YpYeO9-expressing lytic activity towards Yersinia pestis revealed a virion morphology consistent with the Podoviridae morphotype. These phages lyse all 68 strains from 2 different sets of Y. pestis isolates, thus limiting their potential application for subtyping of Y. pestis strains but making them rather promising in terms of infection control. Two phages-YpYeO9 and YpEc11-were selected for detailed studies based on their source of isolation and lytic cross activity towards other Enterobacteriaceae. The full genome sequencing demonstrated the virulent nature of new phages. Phage YpYeO9 was identified as a member of the Teseptimavirus genus and YpEc11 was identified as a member of the Helsettvirus genus, thereby representing new species. A bacterial challenge assay in liquid microcosm with a YpYeO9/YpEc11 phage mixture showed elimination of Y. pestis EV76 during 4 h at a P/B ratio of 1000:1. These results, in combination with high lysis stability results of phages in liquid culture, the low frequency of formation of phage resistant mutants, and their viability under different physical-chemical factors indicate their potential for their practical use as an antibacterial mean.

Phenotypic and Genetic Characterization of Aeromonas hydrophila Phage AhMtk13a and Evaluation of Its Therapeutic Potential on Simulated Aeromonas Infection in Danio rerio.

Phage therapy can be an effective alternative to standard antimicrobial chemotherapy for control of Aeromonas hydrophila infections in aquaculture. Aeromonas hydrophila-specific phages AhMtk13a and AhMtk13b were studied for basic biological properties and genome characteristics. Phage AhMtk13a (Myovirus, 163,879 bp genome, 41.21% CG content) was selected based on broad lytic spectrum and physiologic parameters indicating its lytic nature. The therapeutic potential of phage AhMtk13a was evaluated in experimental studies in zebrafish challenged with A. hydrophila GW3-10 via intraperitoneal injection and passive immersion in aquaria water. In experimental series 1 with single introduction of AhMtk13a phage to aquaria water at phage-bacteria ratio 10:1, cumulative mortality 44% and 62% was registered in fish exposed to phage immediately and in 4 h after bacterial challenge, correspondingly, compared to 78% mortality in the group with no added phage. In experimental series 2 with triple application of AhMtk13a phage at ratio 100:1, the mortality comprised 15% in phage-treated group compared to the 55% in the control group. Aeromonas hydrophila GW3-10 was not detectable in aquaria water from day 9 but still present in fish at low concentration. AhMtk13a phage was maintained in fish and water throughout the experiment at the higher concentration in infected fish.

Identification of pathogenic Vibrio species by multilocus PCR-electrospray ionization mass spectrometry and its application to aquatic environments of the former soviet republic of Georgia.

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.

Occurrence and Diversity of Clinically Important Vibrio Species in the Aquatic Environment of Georgia.

Among the more than 70 different Vibrio species inhabiting marine, estuarine, and freshwater ecosystems, 12 are recognized as human pathogens. The warm subtropical climate of the Black Sea coastal area and inland regions of Georgia likely provides a favorable environment for various Vibrio species. From 2006 to 2009, the abundance, ecology, and diversity of clinically important Vibrio species were studied in different locations in Georgia and across seasons. Over a 33-month period, 1,595 presumptive Vibrio isolates were collected from the Black Sea (n = 657) and freshwater lakes around Tbilisi (n = 938). Screening of a subset of 440 concentrated and enriched water samples by PCR-electrospray ionization/mass spectrometry (PCR-ESI/MS) detected the presence of DNA from eight clinically important Vibrio species: V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. alginolyticus, V. harveyi, V. metschnikovii, and V. cincinnatiensis. Almost 90% of PCR/ESI-MS samples positive for Vibrio species were collected from June through November. Three important human-pathogenic Vibrio species (V. cholerae, V. parahaemolyticus, and V. vulnificus) were detected in 62.8, 37.8, and 21.4% of samples testing positive for Vibrios, respectively. The results of these activities suggest that natural reservoirs for human-pathogenic Vibrios exist in Georgian aquatic environments. Water temperature at all sampling sites was positively correlated with the abundance of clinically important Vibrio spp. (except V. metschnikovii), and salinity was correlated with species composition at particular Black Sea sites as well as inland reservoirs.

Molecular diversity and predictability of Vibrio parahaemolyticus along the Georgian coastal zone of the Black Sea.

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.

Detection of toxigenic Vibrio cholerae O1 in freshwater lakes of the former Soviet Republic of Georgia.

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.