Parashewanella tropica sp. nov., a mesophilic bacterium isolated from a marine sponge from Chuuk lagoon, Federated States of Micronesia, and emended description of the genus Parashewanella.

A mesophilic, straight-rod-shaped, non-flagellated bacterium, designated MEBiC05444T, was isolated from a marine sponge collected from Chuuk lagoon, Federated States of Micronesia. The strain was Gram-negative, catalase- and oxidase-positive, and facultative anaerobic. The isolate aerobically grew at 8-38 °C (optimum, 24-32 °C), pH 4.0-10.0 (pH 7.0-7.5) with an absolute requirement for Na+ up to 6 % (w/v) NaCl (2 %). Phylogenetic analyses based on 16S rRNA gene sequences revealed that MEBiC05444T belonged to the family Shewanellaceae, within the class Gammaproteobacteria. Strain MEBiC05444T showed highest 16S rRNA gene sequence similarity to Parashewanella curva C51T, followed by [Shewanella] irciniae UST040317-058T and Parashewanella spongiae HJ039T (98.9 %, 97.2 and 95.7 %, respectively). In the phylogenetic tree based on the 16S rRNA gene sequences, MEBiC05444T formed a cluster with P. curva C51T, but the average nucleotide identity value between the two strains was 82 %, thus confirming their separation at species level. The major fatty acids were iso-C15 : 0 (19.7 %), summed feature 3 (composed of C16 : 1 ω7c and/or C16 : 1ω6c; 16.1 %) and C17 : 1ω8c (10.2 %). The only detected respiratory quinone was ubiquinone Q-8. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, three unidentified aminoglycolipids, two unidentified glycolipids, an unidentified aminoglycophospholipid and an unidentified lipid. The genomic DNA G+C content of strain MEBiC05444T was 40.8 mol%. Based on the results of polyphasic analysis, the strain represents a novel species of the genus Parashewanella, distinct from P. curva C51T, [Shewanella]irciniae UST040317-058T and P. spongiae HJ039T for which the name Parashewanellatropica sp. nov. is proposed with type strain MEBiC05444T (=KCCM 43304T=JCM 16653T).

Agreement, precision, and accuracy of epifluorescence microscopy methods for enumeration of total bacterial numbers.

To assess interchangeability of estimates of bacterial abundance by different epifluorescence microscopy methods, total bacterial numbers (TBNs) determined by most widely accepted protocols were statistically compared. Bacteria in a set of distinctive samples were stained with acridine orange (AO), 4'-6-diamidino-2-phenylindole (DAPI), and BacLight and enumerated by visual counting (VC) and supervised image analysis (IA). Model II regression and Bland-Altman analysis proved general agreements between IA and VC methods, although IA counts tended to be lower than VC counts by 7% on a logarithmic scale. Distributions of cells and latex beads on polycarbonate filters were best fitted to negative binomial models rather than to Poisson or log-normal models. The fitted models revealed higher precisions of TBNs by the IA method than those by the VC method. In pairwise comparisons of the staining methods, TBNs by AO and BacLight staining showed good agreement with each other, but DAPI staining had tendencies of underestimation. Although precisions of the three staining methods were comparable to one another (intraclass correlation coefficients, 0.97 to 0.98), accuracy of the DAPI staining method was rebutted by disproportionateness of TBNs between pairs of samples that carried 2-fold different volumes of identical cell suspensions. It was concluded that the TBN values estimated by AO and BacLight staining are relatively accurate and interchangeable for quantitative interpretation and that IA provides better precision than does VC. As a prudent measure, it is suggested to avoid use of DAPI staining for comparative studies investigating accuracy of novel cell-counting methods.

Modular evolution of glutathione peroxidase genes in association with different biochemical properties of their encoded proteins in invertebrate animals.

BACKGROUND: Phospholipid hydroperoxide glutathione peroxidases (PHGPx), the most abundant isoforms of GPx families, interfere directly with hydroperoxidation of lipids. Biochemical properties of these proteins vary along with their donor organisms, which has complicated the phylogenetic classification of diverse PHGPx-like proteins. Despite efforts for comprehensive analyses, the evolutionary aspects of GPx genes in invertebrates remain largely unknown. RESULTS: We isolated GPx homologs via in silico screening of genomic and/or expressed sequence tag databases of eukaryotic organisms including protostomian species. Genes showing strong similarity to the mammalian PHGPx genes were commonly found in all genomes examined. GPx3- and GPx7-like genes were additionally detected from nematodes and platyhelminths, respectively. The overall distribution of the PHGPx-like proteins with different biochemical properties was biased across taxa; selenium- and glutathione (GSH)-dependent proteins were exclusively detected in platyhelminth and deuterostomian species, whereas selenium-independent and thioredoxin (Trx)-dependent enzymes were isolated in the other taxa. In comparison of genomic organization, the GSH-dependent PHGPx genes showed a conserved architectural pattern, while their Trx-dependent counterparts displayed complex exon-intron structures. A codon for the resolving Cys engaged in reductant binding was found to be substituted in a series of genes. Selection pressure to maintain the selenocysteine codon in GSH-dependent genes also appeared to be relaxed during their evolution. With the dichotomized fashion in genomic organizations, a highly polytomic topology of their phylogenetic trees implied that the GPx genes have multiple evolutionary intermediate forms. CONCLUSION: Comparative analysis of invertebrate GPx genes provides informative evidence to support the modular pathways of GPx evolution, which have been accompanied with sporadic expansion/deletion and exon-intron remodeling. The differentiated enzymatic properties might be acquired by the evolutionary relaxation of selection pressure and/or biochemical adaptation to the acting environments. Our present study would be beneficial to get detailed insights into the complex GPx evolution, and to understand the molecular basis of the specialized physiological implications of this antioxidant system in their respective donor organisms.

Diversity and seasonality of bioluminescent Vibrio cholerae populations in Chesapeake Bay.

Association of luminescence with phenotypic and genotypic traits and with environmental parameters was determined for 278 strains of Vibrio cholerae isolated from the Chesapeake Bay during 1998 to 2000. Three clusters of luminescent strains (A, B, and C) and two nonluminescent clusters (X and Y) were identified among 180 clonal types. V. cholerae O1 strains isolated during pandemics and endemic cholera in the Ganges Delta were related to cluster Y. Heat-stable enterotoxin (encoded by stn) and the membrane protein associated with bile resistance (encoded by ompU) were found to be linked to luminescence in strains of cluster A. Succession from nonluminescent to luminescent populations of V. cholerae occurred during spring to midsummer. Occurrence of cluster A strains in water with neutral pH was contrasted with that of cluster Y strains in water with a pH of >8. Cluster A was found to be associated with a specific calanoid population cooccurring with cyclopoids. Cluster B was related to cluster Y, with its maximal prevalence at pH 8. Occurrence of cluster B strains was more frequent with warmer water temperatures and negatively correlated with maturity of the copepod community. It is concluded that each cluster of luminescent V. cholerae strains occupies a distinct ecological niche. Since the dynamics of these niche-specific subpopulations are associated with zooplankton community composition, the ecology of luminescent V. cholerae is concluded to be related to its interaction with copepods and related crustacean species.

RNA colony blot hybridization method for enumeration of culturable Vibrio cholerae and Vibrio mimicus bacteria.

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.

Incidence of Staphylococcus aureus and associated risk factors in Nham, a Thai fermented pork product.

Staphylococcus aureus is one of the most prevalent bacterial pathogens causing food-borne disease worldwide. Staphylococcal food poisoning is caused by ingestion of staphylococcal enterotoxins (SEs) pre-formed in the implicated food. In this study, the incidences of S. aureus and classical SEs (SEA-SEE) contamination in 'Nham', a traditional Thai fermented pork product, were determined. Among 155 Nham samples tested, as high as 39.35% of the samples were positive for S. aureus (2-3500 MPN/g), but none were positive for the SEs. The risk factors for S. aureus contamination were highly correlated with the manufacturer and the pH of the product. A predictive model determined the probability of the presence of S. aureus to be < or = 0.24 at the pH < or = 4.6. During the fermentation process, the number of S. aureus slightly increased in the first day and decreased afterward. S. aureus counts continued to decrease when Nham was stored refrigerated. The negative result for enterotoxins and low counts of S. aureus in Nham surveyed in this study, and reduction of the pathogen counts during fermentation and storage suggested that there is very low risk of staphylococcal food poisoning from consuming properly fermented Nham.

The microbiome and antibiotic resistance in integrated fishfarm water: Implications of environmental public health.

Aquaculture, the production of farm-raised fish, is a major industry that employs and feeds millions of individuals across the globe, but which may also be a nexus of emerging public health threats. This study examined potential health risks associated with integrated aquaculture operations by with One Health approach using a suite of tools to study water contamination sources, pathogens, antibiotic resistant bacteria, and bacterial community in the water from fishponds. Water samples from 27 fishponds across 9 villages were collected in Jiangmen City, China. Microbial source tracking, pathogens (including Salmonella and Arcobacter), toxin-producing Microcystis, and antibiotic resistant bacteria (ARB) resistant to tetracycline, sulfonamide, and carbapenem were quantified with qPCR. Bacterial community was determined with next-generation sequencing. All ponds exceeded E. coli single-day maximum criteria of US, and 67% ponds exceeded World Health Organization (WHO) waste-fed aquaculture guidelines for protecting consumers and pond workers, representing a high degree of fecal contamination and potential pathogen risks in these ponds. The majority of the ponds were positive for human- (84%) and pig- (41%) associated fecal contamination. Salmonella and microcystin-producing Microcystis were detected in 37% and 15% of the ponds, respectively, while Arcobacter was not detected in any ponds. ARB were highly prevalent. Among the measured factors, canonical correspondence analysis and network analysis demonstrated that secchi depth, temperature and conductivity were the major environmental elements impacting the bacterial community structure, while Proteobacteria and Actinobacteria were the major biological factor. This study demonstrated the presence of intersecting health risk factors in aquaculture facilities and can lay the foundation for addressing these risks in aquaculture management in rural China, with potential applicability in other developing regions dependent on aquaculture.

Dynamics of biogenic amines and bacterial communities in a Thai fermented pork product Nham.

A traditional Thai fermented pork, nham, is a product popularly consumed in Thailand. Fermentation of the protein-rich product by uncontrolled bacterial community can result in high amounts of hazardous biogenic amines (BA). This study aimed to unveil dynamics of microbial community and its relation to BA accumulation in nham. Three batches of nham were analyzed for pH, lactic acid bacteria population, concentrations of organic acids and BA. Bacterial communities were analyzed by pyrosequencing of 16S rRNA gene amplicons. In all batches, pH dropped to the quality standard of nham (≤4.6) within 3-5 days by production of lactic acid and acetic acid. Initial BA levels varied batch-by-batch and increased with fermentation time. In the highest quality batch, levels of histamine, tyramine, and total BA were within the recommended safety limits (200, 100 and 1000 mg/kg, respectively) throughout the 10-days study. However, in other batches, unsafe levels of tyramine and total BA were found after 5 days of fermentation. The results indicated that over-fermentation and inferior conditions of ingredients increased risk due to high levels of BA. Lactobacillus, Lactococcus, Pediococcus and Weissella were prevalent and comprised >90% of total bacteria during fermentation. Weissella was predominant in the batch with low BA while Lactobacillus and Pediococcus were predominant in the higher BA batches. A negative correlation between Weissella dominance and total BA was observed (r = -0.90, p = .003). A 10% increase in dominance of Weissella was associated with 75-170 mg/kg decrease in total BA. W. hellenica was the species prevalent only in low BA batch. Therefore, W. hellenica isolates were suggested as subjects for future study to develop efficient starter culture securing safety of nham.

Determination of clonality and relatedness of Vibrio cholerae isolates by genomic fingerprinting, using long-range repetitive element sequence-based PCR.

A high-throughput method which is applicable for rapid screening, identification, and delineation of isolates of Vibrio cholerae, sensitive to genome variation, and capable of providing phylogenetic inferences enhances environmental monitoring of this bacterium. We have developed and optimized a method for genomic fingerprinting of V. cholerae based on long-range PCR. The method uses a primer set directed to enterobacterial repetitive intergenic consensus sequences, a high-fidelity DNA polymerase, and analysis via conventional agarose gel electrophoresis. Long ( approximately 10 kb), highly reproducible amplicons were generated from V. cholerae isolates, including those from different geographical locations and historical strains isolated during the period 1931-2000. The amplicons yielded reduced variability in their densitometric band patterns to /=90% similarity, discriminating O serotypes and biotypes (classical versus El Tor) as well as pathogenic and nonpathogenic strains. Compared to genome similarity measured by DNA-DNA hybridization, the results showed good correlation (r = 0.7; P < 0.001), with five times less measurement error and without bias. The method permits both phylogenetic inference and clonal differentiation of individual V. cholerae strains, enables robust, high-throughput analysis, and does not require specialized equipment to perform. With access to a curated public database furnished with appropriate analytical software applications, the method should prove useful in large-scale multilaboratory surveys, especially those designed to detect specific pathogens in the natural environment.

Covariability of Vibrio cholerae microdiversity and environmental parameters.

Fine-scale diversity of natural bacterial assemblages has been attributed to neutral radiation because correspondence between bacterial phylogenetic signals in the natural environment and environmental parameters had not been detected. Evidence that such correspondence occurs is provided for Vibrio cholerae, establishing a critical role for environmental parameters in bacterial diversity.

A simple binomial test for estimating sequencing errors in public repository 16S rRNA sequences.

Sequences in public databases may contain a number of sequencing errors. A double binomial model describing the distribution of indel-excluded similarity coefficients (S) among repeatedly sequenced 16S rRNA was previously developed and it produced a confidence interval of S useful for testing sequence identity among sequences of 400-bp length. We characterized patterns in sequencing errors found in nearly complete 16S rRNA sequences of Vibrionaceae as highly variable in reported sequence length and containing a small number of indels. To accommodate these characteristics, a simple binomial model for distribution of the similarity coefficient (H) that included indels was derived from the double binomial model for S. The model showed good fit to empirical data. By using either a pre-determined or bootstrapping estimated standard probability of base matching, we were able to use the exact binomial test to determine the relative level of sequencing error for a given pair of duplicated sequences. A limitation of the method is the requirement that duplicated sequences for the same template sequence be paired, but this can be overcome by using only conserved regions of 16S rRNA sequences and pairing a given sequence with its highest scoring BLAST search hit from the nr database of GenBank.

Further Evidence of Increasing Diversity of Plasmodium vivax in the Republic of Korea in Recent Years.

BACKGROUND: Vivax malaria was successfully eliminated from the Republic of Korea (ROK) in the late 1970s but re-emerged in 1993. Two decades later as the ROK enters the final stages of malaria elimination, dedicated surveillance of the local P. vivax population is critical. We apply a population genetic approach to gauge P. vivax transmission dynamics in the ROK between 2010 and 2012. METHODOLOGY/PRINCIPAL FINDINGS: P. vivax positive blood samples from 98 autochthonous cases were collected from patients attending health centers in the ROK in 2010 (n = 27), 2011 (n = 48) and 2012 (n = 23). Parasite genotyping was undertaken at 9 tandem repeat markers. Although not reaching significance, a trend of increasing population diversity was observed from 2010 (HE = 0.50 ± 0.11) to 2011 (HE = 0.56 ± 0.08) and 2012 (HE = 0.60 ± 0.06). Conversely, linkage disequilibrium declined during the same period: IAS = 0.15 in 2010 (P = 0.010), 0.09 in 2011 (P = 0.010) and 0.05 in 2012 (P = 0.010). In combination with data from other ROK studies undertaken between 1994 and 2007, our results are consistent with increasing parasite divergence since re-emergence. Polyclonal infections were rare (3% infections) suggesting that local out-crossing alone was unlikely to explain the increased divergence. Cases introduced from an external reservoir may therefore have contributed to the increased diversity. Aside from one isolate, all infections carried a short MS20 allele (142 or 149 bp), not observed in other studies in tropical endemic countries despite high diversity, inferring that these regions are unlikely reservoirs. CONCLUSIONS: Whilst a number of factors may explain the observed population genetic trends, the available evidence suggests that an external geographic reservoir with moderate diversity sustains the majority of P. vivax infection in the ROK, with important implications for malaria elimination.

Stratified distribution of nutrients and extremophile biota within freshwater ice covering the surface of Lake Baikal.

Biological entities and gradients of selected chemicals within the seemingly barren ice layers covering Lake Baikal were investigated. Ice cores 40-68 cm long were obtained from in shore and offshore sites of Southern Lake Baikal during the cold period of a year (March-April) in 2007 and 2008. In microscopic observations of the melted ice, both algae and bacteria were found in considerable numbers (>10(3) cells/L and >10(4) cells/ml, respectively). Among all organisms found, diatom was generally the most predominant taxon in the ice. Interestingly, both planktonic and benthic algae were present in considerable numbers (2-4×10(4) cells/L). Dominant phototrophic picoplankton were comprised of small green algae of various taxa and cyanobacteria of Synechococcus and Cyanobium. The bacterial community consisted mostly of short rod and cocci cells, either free-living or aggregated. Large numbers of yeast-like cells and actinomycete mycelium were also observed. Concentrations of silica, phosphorus, and nitrate were low by an order of magnitude where biota was abundant. The profile of the ice could be interpreted as vertical stratification of nutrients and biomass due to biological activities. Therefore, the organisms in the ice were regarded to maintain high activity while thriving under freezing conditions. Based on the results, it was concluded that the freshwater ice covering the surface of Lake Baikal is considerably populated by extremophilic microorganisms that actively metabolize and form a detritus food chain in the unique large freshwater ecosystem of Lake Baikal.

Reductive divergence of enterobacterial repetitive intergenic consensus sequences among Gammaproteobacteria genomes.

Enterobacterial repetitive intergenic consensus (ERIC) sequence is a transcription-modulating, nonautonomous, miniature inverted-repeat transposable element. Its origin and the mechanism of highly varying incidences, limited to Enterobacteriaceae and Vibrionaceae, have not been identified. In this study, distribution and divergence of ERICs along bacterial taxonomie units were analyzed. ERICs were found among five families of gammaproteobacteria, with the copy numbers varying with exponential increments. The variability was explained by genus (45%) and species (36%) affiliations, indicating that copy numbers are specific to sub-family taxa. ERICs were interspersed in genomes with considerable divergences. Locations of ERICs in a genome appeared to be strongly conserved in a strain, moderately in a species or a genus, and weakly in a family. ERICs in different species of a genus were from the identical population of sequences while ERICs in different genera of a family were nearly identical. However, ERICs in different families formed distinct monophylectic groups, implying vertical transmission of diverging population of sequences. In spite of large difference in copy numbers, overall intra-genome evolutionary distances among ERICs were similar among different species, except for a few genomes. The exceptions substantiated hypotheses of genetic drifts and horizontal gene transfers of mobility capacity. Therefore, the confined, variable distribution of ERIC could be explained as a two-step evolution: introduction and proliferation of ERIC in one of the progenitors of gammaproteobacteria, followed by vertical transmission under negative selection. Deterioration of sequences and reduction in copy number were concluded to be the predominant patterns in the evolution of ERIC loci.

Biochemical properties of a novel cysteine protease of Plasmodium vivax, vivapain-4.

BACKGROUND: Multiple cysteine proteases of malaria parasites are required for maintenance of parasite metabolic homeostasis and egress from the host erythrocyte. In Plasmodium falciparum these proteases appear to mediate the processing of hemoglobin and aspartic proteases (plasmepsins) in the acidic food vacuole and the hydrolysis of erythrocyte structural proteins at neutral pH. Two cysteine proteases, vivapain (VX)-2 and VX-3 have been characterized in P. vivax, but comprehensive studies of P. vivax cysteine proteases remain elusive. FINDINGS: We characterized a novel cysteine protease of P. vivax, VX-4, of which orthologs appears to have evolved differentially in primate plasmodia with strong cladistic affinity toward those of rodent Plasmodium. Recombinant VX-4 demonstrated dual substrate specificity depending on the surrounding micro-environmental pH. Its hydrolyzing activity against benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA was highest at acidic pH (5.5), whereas that against Z-Arg-Arg-MCA was maximal at neutral pH (6.5-7.5). VX-4 preferred positively charged amino acids and Gln at the P1 position, with less strict specificity at P3 and P4. P2 preferences depended on pH (Leu at pH 5.5 and Arg at pH 7.5). Three amino acids that delineate the S2 pocket were substituted in VX-4 compared to VX-2 and VX-3 (Ala90, Gly157 and Glu180). Replacement of Glu180 abolished activity against Z-Arg-Arg-MCA at neutral pH, indicating the importance of this amino acid in the pH-dependent substrate preference. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. VX-4 showed maximal activity against actin at neutral pH, and that against P. vivax plasmepsin 4 and hemoglobin was detected at neutral/acidic and acidic pH, respectively. CONCLUSION: VX-4 demonstrates pH-dependent substrate switching, which might offer an efficient mechanism for the specific cleavage of different substrates in different intracellular environments. VX-4 might function as a hemoglobinase in the acidic parasite food vacuole, a maturase of P. vivax plasmepsin 4 at neutral or acidic pH, and a cytoskeleton-degrading protease in the neutral erythrocyte cytosol.

Genomic profiles of clinical and environmental isolates of Vibrio cholerae O1 in cholera-endemic areas of Bangladesh.

Diversity, relatedness, and ecological interactions of toxigenic Vibrio cholerae O1 populations in two distinctive habitats, the human intestine and the aquatic environment, were analyzed. Twenty environmental isolates and 42 clinical isolates were selected for study by matching serotype, geographic location of isolation in Bangladesh, and season of isolation. Genetic profiling was done by enterobacterial repetitive intergenic consensus sequence-PCR, optimized for profiling by using the fully sequenced V. cholerae El Tor N16961 genome. Five significant clonal clusters of haplotypes were found from 57 electrophoretic types. Isolates from different areas or habitats intermingled in two of the five significant clusters. Frequencies of haplotypes differed significantly only between the environmental populations (exact test; P < 0.05). Analysis of molecular variance yielded a population genetic structure reflecting the differentiating effects of geographic area, habitat, and sampling time. Although a parameter confounding the latter differences explained 9% of the total molecular variance in the entire population (P < 0.01), the net effect of habitat and time could not be separated because of the small number of environmental isolates included in the study. Five subpopulations from a single area were determined, and from these we were able to estimate a relative differentiating effect of habitat, which was small compared with the effect of temporal change. In conclusion, the resulting population structure supports the hypothesis that spatial and temporal fluctuations in the composition of toxigenic V. cholerae populations in the aquatic environment can cause shifts in the dynamics of the disease.