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MMWR. Morbidity and Mortality Weekly... Oct 2010On June 24, 2010, the Spokane (Washington) Regional Health District (SRHD) was notified of two hospitalized patients under intensive care with severe dehydration whose...
On June 24, 2010, the Spokane (Washington) Regional Health District (SRHD) was notified of two hospitalized patients under intensive care with severe dehydration whose stool specimens yielded Vibrio mimicus. CDC was asked to assist with the environmental and epidemiologic investigation. Investigators learned that both persons had consumed crayfish on June 20, 2010. The previous day, live crayfish obtained from an online seafood company had been boiled and served warm at a party. The chef reported that the boiled crayfish were served out of a cooler that had contained live crayfish, and the cooler had not been cleaned before being used to serve the cooked crayfish. After the party, the remaining crayfish were refrigerated overnight in different containers and served cold as leftovers the following evening on June 20.
Topics: Animals; Astacoidea; Dehydration; Diarrhea; Food Contamination; Food Handling; Gastroenteritis; Hospitalization; Humans; Polymerase Chain Reaction; Seafood; Vibrio Infections; Vibrio mimicus; Washington
PubMed: 21030943
DOI: No ID Found -
FEMS Microbiology Letters Jul 2010This report describes Vibrio seventh pandemic island II (VSP-II) and three novel variants revealed by comparative genomics of 23 Vibrio cholerae strains and their...
This report describes Vibrio seventh pandemic island II (VSP-II) and three novel variants revealed by comparative genomics of 23 Vibrio cholerae strains and their presence among a large and diverse collection of V. cholerae isolates. Three VSP-II variants were reported previously and our results demonstrate the presence of three novel VSP-II in clinical and environmental V. cholerae marked by major deletions and genetic rearrangements. A new VSP-II cluster was found in the seventh pandemic V. cholerae O1 El Tor strain CIRS101, which is dominant (95%) among the recent (2004-2007) seven pandemic V. cholerae O1 El Tor isolates from two endemic sites, but was not found in older strains from the same region. Two other variants were found in V. cholerae TMA21 and RC385, two environmental strains from coastal Brazil and the Chesapeake Bay, respectively, the latter being prevalent among environmental V. cholerae non-O1/non-O139 and Vibrio mimicus. The results of this study indicate that the VSP-II island has undergone significant rearrangement through a complex evolutionary pathway in V. cholerae. Interestingly, one of the new VSP-II revealed the presence of 'old' and 'new' V. cholerae O1 El Tor pandemic clones circulating in some of the areas where cholera is endemic.
Topics: Brazil; Cholera; DNA, Bacterial; Environmental Microbiology; Gene Order; Genomic Islands; Genomics; Humans; Polymerase Chain Reaction; United States; Vibrio cholerae O1; Vibrio cholerae non-O1; Vibrio mimicus
PubMed: 20528940
DOI: 10.1111/j.1574-6968.2010.02008.x -
BMC Microbiology May 2010In recent years genome sequencing has been used to characterize new bacterial species, a method of analysis available as a result of improved methodology and reduced... (Comparative Study)
Comparative Study
BACKGROUND
In recent years genome sequencing has been used to characterize new bacterial species, a method of analysis available as a result of improved methodology and reduced cost. Included in a constantly expanding list of Vibrio species are several that have been reclassified as novel members of the Vibrionaceae. The description of two putative new Vibrio species, Vibrio sp. RC341 and Vibrio sp. RC586 for which we propose the names V. metecus and V. parilis, respectively, previously characterized as non-toxigenic environmental variants of V. cholerae is presented in this study.
RESULTS
Based on results of whole-genome average nucleotide identity (ANI), average amino acid identity (AAI), rpoB similarity, MLSA, and phylogenetic analysis, the new species are concluded to be phylogenetically closely related to V. cholerae and V. mimicus. Vibrio sp. RC341 and Vibrio sp. RC586 demonstrate features characteristic of V. cholerae and V. mimicus, respectively, on differential and selective media, but their genomes show a 12 to 15% divergence (88 to 85% ANI and 92 to 91% AAI) compared to the sequences of V. cholerae and V. mimicus genomes (ANI <95% and AAI <96% indicative of separate species). Vibrio sp. RC341 and Vibrio sp. RC586 share 2104 ORFs (59%) and 2058 ORFs (56%) with the published core genome of V. cholerae and 2956 (82%) and 3048 ORFs (84%) with V. mimicus MB-451, respectively. The novel species share 2926 ORFs with each other (81% Vibrio sp. RC341 and 81% Vibrio sp. RC586). Virulence-associated factors and genomic islands of V. cholerae and V. mimicus, including VSP-I and II, were found in these environmental Vibrio spp.
CONCLUSIONS
Results of this analysis demonstrate these two environmental vibrios, previously characterized as variant V. cholerae strains, are new species which have evolved from ancestral lineages of the V. cholerae and V. mimicus clade. The presence of conserved integration loci for genomic islands as well as evidence of horizontal gene transfer between these two new species, V. cholerae, and V. mimicus suggests genomic islands and virulence factors are transferred between these species.
Topics: Bacterial Proteins; Cluster Analysis; DNA-Directed RNA Polymerases; Environmental Microbiology; Evolution, Molecular; Gene Transfer, Horizontal; Genome, Bacterial; Genomic Islands; Genomics; Humans; Molecular Sequence Data; Open Reading Frames; Phylogeny; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Synteny; Vibrio; Virulence Factors
PubMed: 20507608
DOI: 10.1186/1471-2180-10-154 -
Environmental Microbiology Reports Feb 2010Vibrio mimicus is a Gram-negative bacterium, which causes gastroenteritis and is closely related to Vibrio cholerae. The environmental reservoir of this bacterium is far...
Vibrio mimicus is a Gram-negative bacterium, which causes gastroenteritis and is closely related to Vibrio cholerae. The environmental reservoir of this bacterium is far from defined. Acanthamoeba as well as Vibrio species are found in diverse aquatic environments. The present study was aimed to investigate the ability of A. castellanii to host V. mimicus, the role of bacterial protease on interaction with A. castellanii and to disclose the ability of cysts to protect intracellular V. mimicus. Co-cultivation, viable counts, gentamicin assay, electron microscopy and statistical analysis showed that co-cultivation of wild type and luxO mutant of V. mimicus strains with A. castellanii did not inhibit growth of the amoeba. On the other hand co-cultivation enhanced growth and survival of V. mimicus strains. Vibrio mimicus showed intracellular behaviour because bacteria were found to be localized in the cytoplasm of amoeba trophozoites and remain viable for 14 days. The cysts protected intracellular V. mimicus from high level of gentamicin. The intracellular growth of V. mimicus in A. castellanii suggests a role of A. castellanii as a host for V. mimicus.
PubMed: 20454692
DOI: 10.1111/j.1758-2229.2009.00129.x -
Microbiology (Reading, England) Aug 2010The discovery of non-coding RNA (ncRNA) has been mainly limited to laboratory model systems and human pathogenic bacteria. In this study, we begin to explore the ncRNA...
The discovery of non-coding RNA (ncRNA) has been mainly limited to laboratory model systems and human pathogenic bacteria. In this study, we begin to explore the ncRNA diversity in four recently sequenced environmental Vibrio species (Vibrio alginolyticus 40B, Vibrio communis 1DA3, Vibrio mimicus VM573 and Vibrio campbellii BAA-1116) by performing in silico searches using Infernal and Rfam for the identification of putative ncRNA-encoding genes. This search method resulted in the identification of 31-38 putative ncRNA genes per species and the total ncRNA catalogue spanned an assortment of regulatory mechanisms (riboswitches, cis-encoded ncRNAs, trans-encoded ncRNAs, modulators of protein activity, ribonucleoproteins, transcription termination ncRNAs and unknown). We chose to experimentally validate the identifications for V. campbellii BAA-1116 using a microarray-based expression profiling strategy. Transcript hybridization to tiled probes targeting annotated V. campbellii BAA-1116 intergenic regions revealed that 21 of the 38 predicted ncRNA genes were expressed in mid-exponential-phase cultures grown in nutrient-rich medium. The microarray findings were confirmed by testing a subset of three highly expressed (6S, tmRNA and TPP-2) and three moderately expressed (CsrB, GcvB and purine) ncRNAs via reverse transcription PCR. Our findings provide new information on the diversity of ncRNA in environmental vibrios while simultaneously promoting a more accurate annotation of genomic intergenic regions.
Topics: DNA, Intergenic; Gene Expression Profiling; Genetic Variation; Genome, Bacterial; Oligonucleotide Array Sequence Analysis; RNA, Bacterial; RNA, Untranslated; Reverse Transcriptase Polymerase Chain Reaction; Vibrio
PubMed: 20447992
DOI: 10.1099/mic.0.039149-0 -
Journal of Applied Microbiology Jul 2010Vibrio identification by means of traditional microbiological methods is time consuming because of the many biochemical tests that have to be performed to distinguish...
AIMS
Vibrio identification by means of traditional microbiological methods is time consuming because of the many biochemical tests that have to be performed to distinguish closely related species. This work aimed at evaluating the use of MALDI-TOF mass spectrometry for the rapid identification of Vibrio (V.) spp. as an advantageous application to rapidly discriminate the most important Vibrio spp. and distinguish Vibrio spp. from closely related bacterial species like Photobacterium damselae and Grimontia hollisae and other aquatic bacteria like Aeromonas spp.
METHODS AND RESULTS
Starting from sub-colony amounts of pure cultures grown on agar plates, a very simple sample preparation procedure was established and combined with a rapid and automated measurement protocol that allowed species identification within minutes. Closely related species like Vibrio alginolyticus and Vibrio parahaemolyticus or Vibrio cholerae and Vibrio mimicus could thus be differentiated by defining signatures of species-identifying biomarker ions (SIBIs). As a reference method for species designation and for determination of relationships between strains with molecular markers, partial rpoB gene sequencing was applied.
CONCLUSIONS
The MALDI-TOF MS-based method as well as the rpoB sequence-based approach for Vibrio identification described in this study produced comparable classification results. The construction of phylogenetic trees from MALDI-TOF MS and rpoB sequences revealed a very good congruence of both methods.
SIGNIFICANCE AND IMPACT OF THE STUDY
Our results suggest that whole-cell MALDI-TOF MS-based proteometric characterization represents a powerful tool for rapid and accurate classification and identification of Vibrio spp. and related species.
Topics: Aeromonas; Bacterial Typing Techniques; Biomarkers; Genes, Bacterial; Phylogeny; Sequence Analysis, DNA; Species Specificity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Vibrio
PubMed: 20059616
DOI: 10.1111/j.1365-2672.2009.04647.x -
Applied and Environmental Microbiology Feb 2010Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into...
Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of the tdh and trh genes of Vibrio parahaemolyticus and related Vibrio species.
Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. The LAMP assay was designed for both combined and individual detection of the tdh, trh1, and trh2 genes and combined detection of the trh1 and trh2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of V. parahaemolyticus, 3 strains of Grimontia hollisae, and 2 strains of Vibrio mimicus carrying the tdh, trh1, and trh2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the tdh, trh1, and trh2 genes. The sensitivities of the LAMP assay for detection of tdh-, trh1-, and trh2-carrying V. parahaemolyticus strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the tdh, trh1, and trh2 genes of V. parahaemolyticus and related Vibrio species.
Topics: Bacterial Proteins; Bacterial Toxins; Bacterial Typing Techniques; Cloning, Molecular; DNA, Bacterial; Gene Expression Regulation, Bacterial; Genes, Bacterial; Hemolysin Proteins; Nucleic Acid Amplification Techniques; Nucleic Acid Conformation; Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Temperature; Vibrio; Vibrio parahaemolyticus
PubMed: 19966027
DOI: 10.1128/AEM.02284-09 -
Journal of Applied Microbiology Jun 2010To develop a multiplex PCR targeting the gyrB and pntA genes for Vibrio species differentiation.
AIMS
To develop a multiplex PCR targeting the gyrB and pntA genes for Vibrio species differentiation.
METHODS AND RESULTS
Four pairs of primers targeting gyrB gene of Vibrios at genus level and pntA gene of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus were designed. This PCR method precisely identified 250 Vibrio species and demonstrated sensitivity in the range of 4 x 10(4) CFU ml(-1) (c. 200 CFU per PCR) to 2 x 10(3) CFU ml(-1) (c. 10 CFU per PCR). Overall, the gyrB gene marker showed a higher specificity than the dnaJ gene marker for Vibrio detection and was able to distinguish Aeromonas from Vibrio species.
CONCLUSIONS
The multiplex PCR based on combined gyrB and pntA provides a high discriminatory power in the differentiation between Vibrio alginolyticus and V. parahaemolyticus, and between V. cholerae and Vibrio mimicus.
SIGNIFICANCE AND IMPACT OF THE STUDY
This assay will be useful for rapid differentiation of various Vibrio species from clinical and environmental sources and significantly overcomes the limitations of the conventional methods.
Topics: Bacterial Proteins; DNA Gyrase; DNA Primers; DNA, Bacterial; Genes, Bacterial; NADP Transhydrogenases; Polymerase Chain Reaction; Sensitivity and Specificity; Species Specificity; Vibrio
PubMed: 19891709
DOI: 10.1111/j.1365-2672.2009.04599.x -
BMC Evolutionary Biology Oct 2009Vibrio taxonomy has been based on a polyphasic approach. In this study, we retrieve useful taxonomic information (i.e. data that can be used to distinguish different...
BACKGROUND
Vibrio taxonomy has been based on a polyphasic approach. In this study, we retrieve useful taxonomic information (i.e. data that can be used to distinguish different taxonomic levels, such as species and genera) from 32 genome sequences of different vibrio species. We use a variety of tools to explore the taxonomic relationship between the sequenced genomes, including Multilocus Sequence Analysis (MLSA), supertrees, Average Amino Acid Identity (AAI), genomic signatures, and Genome BLAST atlases. Our aim is to analyse the usefulness of these tools for species identification in vibrios.
RESULTS
We have generated four new genome sequences of three Vibrio species, i.e., V. alginolyticus 40B, V. harveyi-like 1DA3, and V. mimicus strains VM573 and VM603, and present a broad analyses of these genomes along with other sequenced Vibrio species. The genome atlas and pangenome plots provide a tantalizing image of the genomic differences that occur between closely related sister species, e.g. V. cholerae and V. mimicus. The vibrio pangenome contains around 26504 genes. The V. cholerae core genome and pangenome consist of 1520 and 6923 genes, respectively. Pangenomes might allow different strains of V. cholerae to occupy different niches. MLSA and supertree analyses resulted in a similar phylogenetic picture, with a clear distinction of four groups (Vibrio core group, V. cholerae-V. mimicus, Aliivibrio spp., and Photobacterium spp.). A Vibrio species is defined as a group of strains that share > 95% DNA identity in MLSA and supertree analysis, > 96% AAI, < or = 10 genome signature dissimilarity, and > 61% proteome identity. Strains of the same species and species of the same genus will form monophyletic groups on the basis of MLSA and supertree.
CONCLUSION
The combination of different analytical and bioinformatics tools will enable the most accurate species identification through genomic computational analysis. This endeavour will culminate in the birth of the online genomic taxonomy whereby researchers and end-users of taxonomy will be able to identify their isolates through a web-based server. This novel approach to microbial systematics will result in a tremendous advance concerning biodiversity discovery, description, and understanding.
Topics: Base Sequence; Evolution, Molecular; Genome, Bacterial; Phylogeny; Vibrio
PubMed: 19860885
DOI: 10.1186/1471-2148-9-258 -
Applied and Environmental Microbiology Sep 2009A species-specific RNA colony blot hybridization protocol was developed for enumeration of culturable Vibrio cholerae and Vibrio mimicus bacteria in environmental water...
A species-specific RNA colony blot hybridization protocol was developed for enumeration of culturable Vibrio cholerae and Vibrio mimicus bacteria in environmental water samples. Bacterial colonies on selective or nonselective plates were lysed by sodium dodecyl sulfate, and the lysates were immobilized on nylon membranes. A fluorescently labeled oligonucleotide probe targeting a phylogenetic signature sequence of 16S rRNA of V. cholerae and V. mimicus was hybridized to rRNA molecules immobilized on the nylon colony lift blots. The protocol produced strong positive signals for all colonies of the 15 diverse V. cholerae-V. mimicus strains tested, indicating 100% sensitivity of the probe for the targeted species. For visible colonies of 10 nontarget species, the specificity of the probe was calculated to be 90% because of a weak positive signal produced by Grimontia (Vibrio) hollisae, a marine bacterium. When both the sensitivity and specificity of the assay were evaluated using lake water samples amended with a bioluminescent V. cholerae strain, no false-negative or false-positive results were found, indicating 100% sensitivity and specificity for culturable bacterial populations in freshwater samples when G. hollisae was not present. When the protocol was applied to laboratory microcosms containing V. cholerae attached to live copepods, copepods were found to carry approximately 10,000 to 50,000 CFU of V. cholerae per copepod. The protocol was also used to analyze pond water samples collected in an area of cholera endemicity in Bangladesh over a 9-month period. Water samples collected from six ponds demonstrated a peak in abundance of total culturable V. cholerae bacteria 1 to 2 months prior to observed increases in pathogenic V. cholerae and in clinical cases recorded by the area health clinic. The method provides a highly specific and sensitive tool for monitoring the dynamics of V. cholerae in the environment. The RNA blot hybridization protocol can also be applied to detection of other gram-negative bacteria for taxon-specific enumeration.
Topics: Animals; Colony Count, Microbial; Copepoda; Nucleic Acid Hybridization; RNA; Sensitivity and Specificity; Vibrio cholerae; Vibrio mimicus; Water Microbiology
PubMed: 19561182
DOI: 10.1128/AEM.02007-08