Genes pyrH Research Articles

Vibrio harveyi in a Caribbean spiny lobster (Panulirus argus) with hepatopancreas necrosis.

A Caribbean spiny lobster, Panulirus argus, was submitted for necropsy after a number of species-specific fatalities in a public aquarium. The hemolymph was opaque and did not clot and the hepatopancreas had multiple foci of necrosis centered on gram-negative bacteria. Pure cultures of Vibrio harveyi were isolated, identified initially by matrix laser desorption ionization-time of flight mass spectrometry, and confirmed by multilocus sequencing of the gyrB, recA, rpoA, and pyrH genes. As Caribbean spiny lobsters continue to be used for consumption and displayed in public aquariums, chronicling potential pathogens is warranted to inform differential diagnoses and to develop management strategies to reduce incidence of infectious disease in captive populations.

Prevalence, Antibiotics Resistance and Plasmid Profiling of Vibrio spp. Isolated from Cultured Shrimp in Peninsular Malaysia.

Vibrio is the most common bacterium associated with diseases in crustaceans. Outbreaks of vibriosis pose a serious threat to shrimp production. Therefore, antibiotics are commonly used as preventative and therapeutic measures. Unfortunately, improper use of antibiotics leads to antibiotic resistance. Nevertheless, information on the occurrence of Vibrio spp. and antibiotic use in shrimp, particularly in Malaysia, is minimal. This study aimed to provide information on the occurrence of Vibrio spp., its status of antibiotic resistance and the plasmid profiles of Vibrio spp. isolated from cultured shrimp in Peninsular Malaysia. Shrimp were sampled from seven farms that were located in different geographical regions of Peninsular Malaysia. According to the observations, 85% of the shrimp were healthy, whereas 15% were unhealthy. Subsequently, 225 presumptive Vibrio isolates were subjected to biochemical tests and molecular detection using the pyrH gene. The isolates were also tested for antibiotic susceptibility against 16 antibiotics and were subjected to plasmid profiling. Eventually, 13 different Vibrio spp. were successfully isolated and characterized using the pyrH gene. They were the following: V. parahaemolyticus (55%), V. communis (9%), V. campbellii (8%), V. owensii (7%), V. rotiferianus (5%), Vibrio spp. (4%), V. alginolyticus (3%), V. brasiliensis (2%), V. natriegens (2%), V. xuii (1%), V. harveyi (1%), V. hepatarius (0.4%) and P. damselae (3%). Antibiotic susceptibility profiles revealed that all isolates were resistant to penicillin G (100%), but susceptible to norfloxacin (96%). Furthermore, 16% of the isolates revealed MAR of less than 0.2, while 84% were greater than 0.2. A total of 125 isolates harbored plasmids with molecular weights between 1.0 and above 10 kb, detected among the resistant isolates. The resistant isolates were mediated by both chromosomal and plasmid factors. These findings support the use of surveillance data on the emerging patterns of antimicrobial-resistance and plasmid profiles of Vibrio spp. in shrimp farms. The findings from this study can be used to develop a better disease management strategy for shrimp farming.

Rapid screening of marine bacterial symbionts using MALDI-TOF MS.

Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) is a rapid, cost-effective and high-throughput method for bacteria characterization. However, most previous studies focused on clinical isolates. In this study, we evaluated the use of MALDI-TOF MS as a rapid screening tool for marine bacterial symbionts. A set of 255 isolates from different marine sources (corals, sponge, fish and seawater) was analyzed using cell lysates to obtain a rapid grouping. Cluster analysis of mass spectra and 16S rRNA showed 18 groups, including Vibrio, Bacillus, Pseudovibrio, Alteromonas and Ruegeria. MALDI-TOF distance similarity scores ≥ 60% and ≥ 70% correspond to ≥ 98.7% 16S rRNA gene sequence similarity and ≥ 95% pyrH gene sequence similarity, respectively. MALDI-TOF MS is a useful tool for Vibrio species groups' identification.

Possible transmission routes of Vibrio spp. in tropical cage-cultured marine fishes.

This study investigates the possible transmission routes of Vibrio spp. in a tropical cage-cultured marine fishes. Samplings of cultured Asian seabass, red snapper, hybrid grouper, wild fish, trash fish, fish fry, water and sediment samples were conducted from December 2016 to August 2017. All fish were dissected insitu and swabs were taken aseptically from the skin, eye, liver and kidney for bacterial isolation and identification. Bacterial isolation and identification from water, sediment and trash fish were also made. A total of 261 Vibrio spp. isolates recovered from the cultured, wild and fry fish, as well as from the sediment and water of the farm environment were analysed. Sequences of the pyrH gene were used to investigate the degree of relatedness and possible transmission routes existing between the isolated Vibrio spp. The population tree revealed the existence of selected Vibrio spp. that possibly transmitted between the newly introduced fish fry and wild fish into the cultured fish, while water also might possibly serves as natural transmission medium of certain Vibrio spp. in this fish farm. SIGNIFICANCE AND IMPACT OF THE STUDY: The source of transmission of Vibrio spp. into farmed marine fish remains unclear. This study highlights the possible transmission routes of Vibrio into cage-cultured marine fishes via newly introduced fish fry and wild fish. Understanding the routes of transmission of Vibrio spp. might help in controlling the disease in the near future.

Two Rapid Methods to Identify Three Species of Pathogenic Vibrio in Penaeus vannamei

Vibrio species frequently infect Penaeus vannamei causing great economic losses to the Whiteleg shrimp industry. Rapid detection of pathogenic Vibrio infection would improve the fight against these diseases. In this study, single and multiple polymerase chain reaction (PCR) methods were developed to detect three species of pathogenic Vibrio: Vibrio fluvialis, Vibrio anguillarum, and Vibrio alginolyticus. Specific primers were designed for the toxR gene of V. fluvialis, the flaA gene of V. anguillarum, and the pyrH gene of V. alginolyticus. The bacteria were used as templates to establish a 25 μL reaction system for PCR amplification. The results showed that single and specific PCR amplification products of expected sizes were obtained (228bp, 1665bp, and 383bp, respectively). The lowest concentration detected for the three Vibrio species were 5.21×102 , 2.70×104 , and 2.48×102 colony forming units (cfu)/mL, respectively. We also developed a multiplex PCR method to identify the three Vibrio species accurately, and with improved identification efficiency. In addition, quantitative real-time PCR (qPCR) was used to identify the minimum detectable DNA concentration for the three Vibrio species (1.0×10-6 nmol/L for V. fluvialis, 1.0×10-7 nmol/L for V. anguillarum, and 1.0×10- 8 nmol/L for V. alginolyticus). Technical requirements for ordinary PCR are low, therefore PCR is a feasible technique to detect and diagnose Penaeus vannamei bacterial disease.

Quantitative Detection of Active Vibrios Associated with White Plague Disease in Mussismilia braziliensis Corals.

Over recent decades several coral diseases have been reported as a significant threat to coral reef ecosystems causing the decline of corals cover and diversity around the world. The development of techniques that improve the ability to detect and quantify microbial agents involved in coral disease will aid in the elucidation of disease cause, facilitating coral disease detection and diagnosis, identification and pathogen monitoring, pathogen sources, vectors, and reservoirs. The genus Vibrio is known to harbor pathogenic strains to marine organisms. One of the best-characterized coral pathogens is Vibrio coralliilyticus, an aetilogic agent of White Plague Disease (WPD). We used Mussismilia coral tissue (healthy and diseased specimens) to develop a rapid reproducible detection system for vibrios based on RT-QPCR and SYBR chemistry. We were able to detect total vibrios in expressed RNA targeting the 16S rRNA gene at 5.23 × 106 copies/μg RNA and V. coralliilyticus targeting the pyrH gene at 5.10 × 103 copies/μg RNA in coral tissue. Detection of V. coralliilyticus in diseased and in healthy samples suggests that WPD in the Abrolhos Bank may be caused by a consortium of microorganism and not only a single pathogen. We developed a more practical and economic system compared with probe uses for the real-time detection and quantification of vibrios from coral tissues by using the 16S rRNA and pyrH gene. This qPCR assay is a reliable tool for the monitoring of coral pathogens, and can be useful to prevent, control, or reduce impacts in this ecosystem.

PHENOTYPIC CHARACTERIZATION AND MOLECULAR IDENTIFICATION OF A LUMINESCENT MARINE BACTERUM ISOLATED FROM THE NW SHELF OF CUBA

Light emitted by luminescent bacteria is sensitive to several toxic compounds; therefore, some luminous species have been used to evaluate the quality of water environments. In the present study we carried out the phenotypic characterization and molecular identification of a marine luminescent isolate (CBM-784) from the NW Cuban coast. The identification of the CBM-784 luminous isolate was based on phenotypic and genotypic characteristics. Phenotypically, the CBM-784 strain revealed the following characteristics: Gram negative, positive to oxidase and catalase reactions,bioluminescent, and facultative anaerobic respiration. The bacterial strain produces enzymes with gelatinase, lysine carboxylase and amylase activity. Taken together, these assays indicated that CBM-784 showed a high phenotypic similarity to the Vibrio harveyi ATCC 14126 strain. On the basis of 16S rRNA gene sequencing, CBM-784 was closely related to Vibrio harveyi and Vibrio rotiferianus (94% similarity). Sequence analysis of gyrB gene, has shown that CBM-784 shares taxonomic position with Vibrio campbellii and Vibrio harveyi isolates, with 95% of bootstrap value. In addition, sequence analysis of pyrH gene, grouped this isolate to the Vibrio harveyi cluster with a strong bootstrap support (99%). The multilocus sequence analysis and phenotypic characterization of CBM-784 indicated that this strain have a strong relation to Vibrio harveyi.

Thaumasiovibrio occultus gen. nov. sp. nov. and Thaumasiovibrio subtropicus sp. nov. within the family Vibrionaceae, isolated from coral reef seawater off Ishigaki Island, Japan

Two phylogenetically distinct Vibrionaceae strains C4II189T and C4V358T isolated from reef seawater off Ishigaki Island, Japan, in 2014 were studied with advanced genome-based taxonomy approaches. All aspects of phylogenetic (16S rRNA phylogeny, MLSA), phenotypic and genetic (ANI, DDH, AAI, and the number of core genes) cohesions between the two identified species were high enough to propose them as members of a new genus within the family Vibrionaceae. Consequently, an eighth genus Thaumasiovibrio gen. nov. is proposed that contains two new species Thaumasiovibrio occultus sp. nov. strain C4II189T (=DSM 101554T=JCM 31629T) (type species) and Thaumasiovibrio subtropicus sp. nov. strain C4V358T (=DSM 101555T=JCM 31630T). Thaumasiovibrio species were phylogenetically distinct from the other Vibrionaceae species based on pyrH gene sequences. The combination of catalase negative, sensitivity to vibriostatic agent O/129, and green colony formation on TCBS for the phylogenetically affiliated strains was the diagnostic features for the current tentative identification of this genus.

In-depth sequence analysis of highly-conserved pyrH gene to study distributions of oral treponemes in periodontal disease versus health

ABSTRACTBackground: More than 75 species/species-level phylotypes of oral treponeme bacteria inhabit the oral cavity. However, their respective genomic compositions and clinical distributions remain poorly understood.Objectives: To compare distributions of phylogroup 1 and 2 oral treponemes in subjects with various periodontal health conditions, via sequence analysis of a highly-conserved treponeme ‘housekeeping’ gene.Methods: Subgingival plaque samples were collected from Chinese subjects with chronic periodontitis (n=5), aggressive periodontitis (n=4), gingivitis (n=5), and healthy controls (n=4). Samples were analyzed by a PCR/plasmid clone sequencing-based approach, using primer sets targeting the pyrH gene. Data was analyzed using various computational/bioinformatic approaches.Results: 1,227 quality-filtered pyrH gene sequences were obtained (mean 66.2±9.6 sequences per subject), which were assigned to 33 ‘pyrH genotypes’ (97% identity cut-off). 538 pyrH sequences (17 pyrH genotypes) corresponded to phylogroup 1 treponemes (including ‘T. vincentii’, Treponema medium, and ‘Treponema sinensis’ taxa). 689 pyrH sequences (16 pyrH genotypes) corresponded to phylogroup 2 taxa. Correlations between pyrH genotype distributions and disease status were complex. One pyrH genotype, which was phylogenetically-related to T. denticola GM-1/MS25 strains, was highly prevalent: being detected in 17/18 subjects.Conclusions: Both healthy and periodontally-diseased subjects harbor multiple genetic lineages corresponding to the same treponeme species/phylotype within their subgingival niches.

Isolation of Vibrio tapetis from two native fish species (Genypterus chilensis and Paralichthys adspersus) reared in Chile and description of Vibrio tapetis subsp. quintayensis subsp. nov.

A group of seven Chilean isolates presumptively belonging to Vibrio tapetis was isolated from diseased fine flounders (Paralichthys adspersus) and red conger eel (Genypterus chilensis) experimentally reared in Quintay (Chile). All isolates were confirmed as members of V. tapetis on the basis of matrix-assisted laser desorption ionization time-of-flight MS, 16S rRNA gene sequencing, DNA-DNA hybridization values and G+C content. The ERIC-PCR and REP-PCR patterns were homogeneous among those isolates recovered from the same host (red conger or fine flounders), but distinct from the type strains V. tapetis subsp. tapetis CECT 4600T and V. tapetis subsp. britannicus CECT 8161T. On the basis of atpA, rpoA, rpoD, recA and pyrH gene sequence similarities (99.7-100 %) and clustering in the phylogenetic trees, the red conger isolates (Q20, Q047, Q48 and Q50) were confirmed as representing V. tapetis subsp. tapetis. However, they differed from V. tapetis subsp. tapetis CECT 4600T in their lipase, alpha quimiotripsin and non-acid phosphatase production. On the other hand, the fine flounder isolates (QL-9T, QL-35 and QL-41) showed rpoD, recA and pyrH gene sequence similarities ranging from 91.6 to 97.7 % with the type strains of the two V. tapetis subspecies (CECT 4600T and CECT 8161T) and consistently clustered together as an independent phylogenetic line within V. tapetis. Moreover, they could be differentiated phenotypically from strains CECT 4600T and CECT 8161T by nine and three different biochemical tests, respectively. In conclusion, the presence of V. tapetis in diseased red conger eel and fine flounder was demonstrated, extending the known host range and geographical location for this pathogen. Furthermore, this study demonstrates that the three isolates from fine flounder represent a novel subdivision within V. tapetis, for which the name V. tapetis subsp. quintayensis subsp. nov. is proposed and with QL-9T (=CECT 8851T=LMG 28759T) as the type strain. Although QL-9T was isolated from kidney of diseased fine flounder specimens, the challenge assays showed that it was non-pathogenic for this species.

The First Temporal and Spatial Assessment of Vibrio Diversity of the Surrounding Seawater of Coral Reefs in Ishigaki, Japan

Coral reefs perform a major role in regulating marine biodiversity and serve as hotspot for highly dynamic and diverse microbiomes as holobionts. Corals around Ishigaki, however, are at risk due to tremendous stressors including elevation of seawater temperature, eutrophication and so on. However, no information is currently available on how Vibrio diversity fluctuates spatially and temporally due to environmental determinants in Ishigaki coral reef ecosystems. The aim of this study is to elucidate spatiotemporal Vibrio diversity dynamic at both community and population levels and to assess the environmental drivers correlated to Vibrio abundance and diversity. The Vibrio community identified based on pyrH gene phylogeny of 685 isolates from seawater directly connecting to Ishigaki coral holobionts consisted of 22 known and 12 potential novel Vibrionaceae species. The most prominent species were V. hyugaensis, V. owensii and V. harveyi followed by V. maritimus/V. variabillis, V. campbellii, V. coralliilyticus, and Photobacterium rosenbergii. The Vibrio community fluctuations, assessed by PCoA with UniFrac distance and clustering with Euclidiean distance were varied less not only by year but also by site. Interestingly, significant positive correlation was observed between rising seawater temperature and the abundance of V. campbellii (r = 0.62; P < 0.05) whereas the opposite was observed for V. owensii (r = -0.58; P < 0.05) and the C6 group of V. hyugaensis (r = -0.62; P < 0.05). AdaptML-based microhabitat differentiation revealed that V. harveyi, V. campbellii, P. rosenbergii, and V. coralliilyticus populations were less-ecologically distinctive whereas V. astriarenae and V. ishigakensis were ecologically diverse. This knowledge could be important clue for the future actions of coral conservation.

An MLSA approach for the taxonomic update of the Splendidus clade, a lineage containing several fish and shellfish pathogenic Vibrio spp.

A multilocus sequence analysis was undertaken in order to redefine the Splendidus clade of the genus Vibrio, a large group of species containing several pathogenic members that affect fish and shellfish, and are difficult to identify through both phenotypic and genotypic approaches. The study included analysis of partial sequences of recA, gyrB, mreB, rpoD and pyrH genes, as well as the 16S rRNA gene. Seventeen type strain species were included that were complemented with other reference strains and a collection of isolates tentatively identified as members of this clade, as well as a set of other Vibrio species. The clade was well defined and stable in all analyses, and was confirmed to contain V. celticus, V. atlanticus, V. artabrorum, V. toranzoniae and V. hemicentroti, in addition to the twelve previously recognized species. While some species were well-defined members (e.g. Vibrio cyclitrophicus, V. chagasii) others formed tight groups that were related by sequence similarities and lineage topology, which suggested a synonymy among their members, particularly the V. splendidus-V. hemicentroti pair. Most of the isolates were related to two major groups: the V. celticus-V. crassostreae-V. gigantis subclade that contained all isolates from oysters sampled in the cold season, and V. chagasii that included oyster isolates from warm months. This suggested a sharp seasonal occurrence for these species. None of the single genes were able to mimic the resolving power of the five-gene MLSA and none worked well for the identification of the whole group of species in the clade.

Advanced Microbial Taxonomy Combined with Genome-Based-Approaches Reveals that Vibrio astriarenae sp. nov., an Agarolytic Marine Bacterium, Forms a New Clade in Vibrionaceae.

Advances in genomic microbial taxonomy have opened the way to create a more universal and transparent concept of species but is still in a transitional stage towards becoming a defining robust criteria for describing new microbial species with minimum features obtained using both genome and classical polyphasic taxonomies. Here we performed advanced microbial taxonomies combined with both genome-based and classical approaches for new agarolytic vibrio isolates to describe not only a novel Vibrio species but also a member of a new Vibrio clade. Two novel vibrio strains (Vibrio astriarenae sp. nov. C7T and C20) showing agarolytic, halophilic and fermentative metabolic activity were isolated from a seawater sample collected in a coral reef in Okinawa. Intraspecific similarities of the isolates were identical in both sequences on the 16S rRNA and pyrH genes, but the closest relatives on the molecular phylogenetic trees on the basis of 16S rRNA and pyrH gene sequences were V. hangzhouensis JCM 15146T (97.8% similarity) and V. agarivorans CECT 5085T (97.3% similarity), respectively. Further multilocus sequence analysis (MLSA) on the basis of 8 protein coding genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) obtained by the genome sequences clearly showed the V. astriarenae strain C7T and C20 formed a distinct new clade protruded next to V. agarivorans CECT 5085T. The singleton V. agarivorans has never been included in previous MLSA of Vibrionaceae due to the lack of some gene sequences. Now the gene sequences are completed and analysis of 100 taxa in total provided a clear picture describing the association of V. agarivorans into pre-existing concatenated network tree and concluded its relationship to our vibrio strains. Experimental DNA-DNA hybridization (DDH) data showed that the strains C7T and C20 were conspecific but were separated from all of the other Vibrio species related on the basis of both 16S rRNA and pyrH gene phylogenies (e.g., V. agarivorans CECT 5085T, V. hangzhouensis JCM 15146T V. maritimus LMG 25439T, and V. variabilis LMG 25438T). In silico DDH data also supported the genomic relationship. The strains C7T also had less than 95% average amino acid identity (AAI) and average nucleotide identity (ANI) towards V. maritimus C210, V. variabilis C206, and V. mediterranei AK1T, V. brasiliensis LMG 20546T, V. orientalis ATCC 33934T, and V. sinaloensis DSM 21326. The name Vibrio astriarenae sp. nov. is proposed with C7 as the type strains. Both V. agarivorans CECT 5058T and V. astriarenae C7T are members of the newest clade of Vibrionaceae named Agarivorans.

Metabolic and genetic factors affecting the productivity of pyrimidine nucleoside in Bacillus subtilis.

BackgroundCytidine and uridine are produced commercially by Bacillus subtilis. The production strains of cytidine and uridine were both derivatives from mutagenesis. However, the exact metabolic and genetic factors affecting the productivity remain unknown. Genetic engineering may be a promising approach to identify and confirm these factors.ResultsWith the deletion of the cdd and hom genes, and the deregulation of the pyr operon in Bacillus subtilis168, the engineered strain produced 200.9 mg/L cytidine, 14.9 mg/L uridine and 960.1 mg/L uracil. Then, the overexpressed prs gene led to a dramatic increase of uridine by 25.9 times along with a modest increase of cytidine. Furthermore, the overexpressed pyrG gene improved the production of cytidine, uridine and uracil by 259.5%, 11.2% and 68.8%, respectively. Moreover, the overexpression of the pyrH gene increasesd the yield of cytidine by 40%, along with a modest augments of uridine and uracil. Lastly, the deletion of the nupC-pdp gene resulted in a doubled production of uridine up to 1684.6 mg/L, a 14.4% increase of cytidine to 1423 mg/L, and a 99% decrease of uracil to only 14.2 mg/L.ConclusionsThe deregulation of the pyr operon and the overexpression of the prs, pyrG and pyrH genes all contribute to the accumulation of pyrimidine nucleoside compounds in the medium. Among these factors, the overexpression of the pyrG and pyrH genes can particularly facilitate the production of cytidine. Meanwhile, the deletion of the nupC-pdp gene can obviously reduce the production of uracil and simultaneously improve the production of uridine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0237-1) contains supplementary material, which is available to authorized users.

Multilocus sequence analysis of putative Vibrio mediterranei strains and description of Vibrio thalassae sp. nov.

A multilocus sequence analysis based on partial gyrB, mreB, rpoD and pyrH genes was undertaken with 61 putative Vibrio mediterranei/V. shilonii strains from different hosts (mussels, oysters, clams, coral, fish and plankton) or habitat (seawater and sediment) and geographical origins (Mediterranean, Atlantic and Pacific). A consistent grouping was obtained with individual and concatenated gene sequences, and the clade, comprising 54 strains, was split into three subclades by all methods: subclade A (40 strains, including AK1, the former type strain of Vibrio shilonii), subclade B (8 strains) corresponding to the species V. mediterranei, and subclade C (six strains) representing a new species, V. thalassae sp. nov., with strain MD16T (=CECT 8203T=KCTC 32373T) as the proposed type strain.Average nucleotide identity (ANI) values, determined as a measure of genomic similarity, confirmed these assignments, and supported that strains in subclade C were a different species from V. mediterranei, with ANIb and ANIm figures lower than 90.0%. The synonymy of V. shilonii and V. mediterranei was also stressed by both MLSA and ANI determinations (97.0% between both type strains). No connection was found between geographic origin or sample type and MLSA grouping.

Vibrio madracius sp. nov. isolated from Madracis decactis (Scleractinia) in St Peter & St Paul Archipelago, Mid-Atlantic Ridge, Brazil.

Three novel isolates (A-354(T), A-328, and A-384) were retrieved from apparently healthy scleractinian Madracis decactis in the remote St Peter & St Paul Archipelago, Mid-Atlantic Ridge, Brazil. The novel isolates formed a distinct lineage based on the phylogenetic reconstruction using the 16S rRNA and pyrH gene sequences. They fell into the Mediterranei clade and their closest phylogenetic neighbour was V.mediterranei species, sharing upto 98.1% 16S rRNA gene sequence similarity. Genomic analysis including in silico DDH, MLSA, AAI and genomic signature distinguished A-354(T) from V.mediterranei LMG 19703 (=AK1) with values of 33.3, 94.2, 92%, and 11.3, respectively. Phenotypically, the novel isolates can be differentiated from V.mediterranei based on the four following features. They do not grow at 8% NaCl; use D-gluconic acid but not L-galactonic acid lactone as carbon source; and do not have the fatty acid C18:0. Differentiation from both the other Mediterranei clade species (V.maritimus and V.variabilis) is supported by fifteen features. The novel species show lysine decarboxylase and tryptophan deaminase, but not gelatinase and arginine dihydrolase activity; produce acetoin; use α-D-lactose, N-acetyl-D-galactosamine, myo-Inositol, D-gluconic acid, and β-hydroxy-D,L-butyric acid; and present the fatty acids C14:0 iso, C15:0 anteiso, C16:0 iso, C17:0 anteiso, and C17:1x8c . Whole-cell protein profiles, based on MALDI-TOF, showed that the isolates are not clonal and also distinguished them from the closes phylogenetic neighbors. The name Vibrio madracius sp. nov. is proposed to encompass these novel isolates. The G+C content of the type strain A-354(T) (=LMG 28124(T)=CBAS 482(T)) is 44.5mol%.

Outbreak of a novel disease associated with Vibrio mimicus infection in fresh water cultured yellow catfish, Pelteobagrus fulvidraco

An outbreak of a novel disease characterized by serious and regularly shaped ulcers in the skin was recorded in cultured yellow catfish (Pelteobagrus fulyidraco) in Guangdong and Guangxi provinces of South-China in 2011. Several bacterial strains that exhibited the same phenotypic traits and biochemical characteristics were isolated from the lesions of diseased fish. One bacterial isolate named XQ was identified as Vibrio mimicus according to biochemical tests and phylogenetic analysis derived from 16S rDNA, rpoA, recA and pyrH gene sequencing. In challenge trials, the bacterial isolate XQ exhibited high virulence in yellow catfish with an intra-peritoneal LD50 value of 3.39 x 10(6) CFU per fish. In addition, skin ulcers occurred in all challenged fish groups whether they were treated with the bacterial isolate XQ by intraperitoneal injection, oral delivery or bath exposure, suggesting that the V. mimicus is the etiological agent of the enzootic and could infect the host fish via more than one route. Furthermore, histopathology of diseased fish infected with V. mimicus showed destruction of the epidermis and fibrinoid necrosis in skin; renal tubular epithelial cell necrosis in kidney; hepatocyte swelling and serious hemorrhage in the liver; macrophage aggregations and necrosis of the lymphoid tissue in spleen; severe necrosis and shedding of the intestinal mucosal villi in intestines; and, edema and exfoliation of epithelial cells in gill secondary lamellae. (C) 2014 Elsevier B.V. All rights reserved.

Identification and characterization of Vibrio harveyi associated with diseased abalone Haliotis diversicolor

Mass mortality of farmed small abalone Haliotis diversicolor occurred in Fujian, China, from 2009 to 2011. Among isolates obtained from moribund abalones, the dominant species AP37 exhibited the strongest virulence. After immersion challenge with 106 CFU ml-1 of AP37, abalone mortalities of 0, 53 and 67% were induced at water temperatures of 20°C, 24°C, and 28°C, respectively. Following intramuscular injection, AP37 showed a low LD50 (median lethal concentration) value of 2.9 × 102 CFU g-1 (colony forming units per gram abalone wet body weight). The LT50 (median lethal time) values were 5.2 h for 1 × 106 CFU abalone-1, 8.4 h for 1 × 105 CFU abalone-1, and 21.5 h for 1 × 104 CFU abalone-1. For further analysis of virulence, AP37 was screened for the production of extracellular factors. The results showed that various factors including presence of flagella and production of extracellular enzymes, such as lipase, phospholipase and haemolysin, could be responsible for pathogenesis. Based on its 16S rRNA gene sequence, strain AP37 showed >98.8% similarity to Vibrio harveyi, V. campbellii, V. parahaemolyticus, V. alginolyticus, V. natriegens and V. rotiferianus, so it could not be identified by this method. However, multi-locus sequence analysis (MLSA) of concatenated sequences, including the rpoD, rctB, gyrB, toxR and pyrH genes, identified strain AP37 as V. harveyi. Phenotypic characters of AP37 were identified by API 20E. In antibiotic susceptibility tests, strain AP37 exhibited susceptibility to 7 antibiotics and resistance to 13. This is the first report of a V. harveyi-related species being linked with the mass mortality of adult abalone H. diversicolor in southern China.

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

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. 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. 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. 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.

Cloning, Expression, Purification and Characterization of UMP Kinase from Staphylococcus aureus

Uridine monophosphate kinase (UMPK) an enzyme of de novo biosynthesis catalyses the formation of UDP and it is involved in cell wall and RNA biosynthesis. In the present study UMPK of Staphylococcus aureus ATCC12600 was characterized. Analysis of purified UMPK by gel filtration chromatography on Sephadex G-200 indicated a molecular weight of 150kDa and exhibited monomeric form with molecular weight of 25kDa in SDS-PAGE confirming homohexamer nature of UMPK in solution. The enzyme kinetics of UMPK showed K(m) of 2.80±0.1μM and Vmax 51.38±1.39μM of NADH/min/mg. The enzyme exhibited cooperative kinetics with ATP as substrate, as GTP decreased this cooperativity and increased affinity for ATP. The UMPK gene was amplified, sequenced (Accession number: FJ415072), cloned in pQE30 vector and overexpressed in Escherichia coli DH5α. The purified recombinant UMPK showed similar properties of native UMPK. The UMPK gene sequence showed complete homology with pyrH gene sequence of all S. aureus strains reported in the database, the 3D structure of S. aureus UMPK built from the deduced amino acid sequence was super imposed with human UMPK (PDB ID: 1TEV) to find out the structural identity using the MATRAS programme gave an RMSD value 4.24Å indicating very low homology and extensive structural variations with human UMPK structure. Thus, UMPK may be a potential drug target in the development of antimicrobials.