Defences against oxidative stress in vibrios associated with corals

Bacteria colonizing healthy coral tissue may produce enzymes capable of overcoming the toxic effects of reactive oxygen species, including superoxide dismutase (SOD) and catalase. Significant differences in the activities of these enzymes were observed in cultures of Vibrio campbellii, Vibrio coralliilyticus, Vibrio harveyi, Vibrio mediterranei, Vibrio pelagius, Vibrio rotiferanus, Vibrio tasmaniensis, and Photobacterium eurosenbergii isolated from healthy, bleached or necrotic tropical and cold water corals. Levels of SOD in exponential phase cultures of V. coralliilyticus grown at 28 degrees C were only slightly higher than those grown at 16 degrees C whereas the levels in stationary phase cultures at 28 degrees C were 7.3 x higher than those at 16 degrees C. The increase in catalase activity of V. coralliilyticus and V. harveyi upon entry to stationary phase conferred protection against killing by oxidative stress. Increased temperature affected up-regulation of enzymes in stationary phase cultures, but pretreatment of cultures with hydrogen peroxide had no significant effect on induction of catalase or SOD. The increased activities appear to be due to up-regulation of gene expression rather than induction of different forms of the enzymes. We suggest that SOD and catalase are unlikely to be major factors in the virulence of these bacteria for corals and that their main function may be to protect against endogenous superoxide.     

Vibrio parahaemolyticus and V. harveyi cause detachment of the epithelium from the midgut trunk of the penaeid shrimp Sicyonia ingentis

Shrimp Sicyonia ingentis were either injected with Vibrio parahaemolyticus (10(4) CFU) or V. harveyi (10(6) CFU) or immersed in ASW containing either species at 10(5) CFU ml(-1). These densities were shown in preliminary experiments to kill approximately half the population by 7 d. On Day 7, surviving shrimp were classified as either diseased or apparently healthy, and their midgut trunks (MGT) were examined by light and electron microscopy. All shrimp immersed in ASW containing either species of Vibrio showed detachment of the epithelium in the MGT. In shrimp injected with either species of Vibrio, epithelial detachment was common in diseased shrimp but not in apparently healthy animals. Experiments with live shrimp were supported by in vitro experiments where MGTs were removed, tied off at both ends, and injected with either pathogenic bacteria (V. parahaemolyticus or V. harveyi), non-pathogenic bacteria (Bacillus subtilis or Escherichia coli), or ASW. After 2 h incubations in ASW at 15 degrees C, the MGTs were processed and examined. The epithelium consistently detached from isolated MGTs injected with either species of Vibrio, but not from MGTs injected with non-pathogenic bacteria or ASW. Because the MGT epithelium secretes the peritrophic membrane, loss of the epithelium eliminates 2 layers that may restrict penetration of ingested pathogens into the shrimp body and may disrupt the osmoregulatory function of the MGT. A second finding was that fixed, large-granule hemocytes associated with the basal lamina degranulated in the presence of the 2 species of Vibrio, but not with the non-pathogenic bacteria or ASW. These blood cells may help fight specific bacteria penetrating the MGT.     

Production of monoclonal antibodies for detection of Vibrio harveyi

Monoclonal antibodies (MAbs) against Vibrio harveyi were produced from mice immunized with heat-killed and SDS-mercaptoethanol-treated highly virulent V. harveyi 639. Fifteen MAbs were selected and sorted into 6 groups according to their specificity to various proteins of apparent molecular weight ranging from 8 to 49 kDa. Some antibodies were used for detection of V. harveyi at concentrations as low as 10(4) CFU ml(-1) using immunodot blots. Most of the selected MAbs did not show cross-reactivity to other Vibrio species and other gram-negative bacteria tested. Only 1 MAb (VH39-4E) showed slight cross-reactivity to Aeromonas hydrophila. Another MAb (VH24-8H) bound lightly to V. harveyi 1526 but strongly to V. harveyi 639, allowing rapid differentiation. Two of the MAb groups were used to localize V. harveyi in tissues of infected black tiger shrimp Penaeus monodon by immunohistochemistry. This study demonstrates the versatility of a highly specific immunological tool for the detection of V. harveyi in aquaculture and opens the way for further development of convenient test kits.     

A selective and differential medium for Vibrio harveyi.

A new medium, termed Vibrio harveyi agar, has been developed for the isolation and enumeration of V. harveyi. It is possible to differentiate V. harveyi colonies from the colonies of strains representing 15 other Vibrio species with this medium. This medium has been shown to inhibit the growth of two strains of marine Pseudomonas spp. and two strains of marine Flavobacterium spp. but to allow the growth of Photobacterium strains. Colonies displaying typical V. harveyi morphology were isolated from the larval rearing water of a commercial prawn hatchery with V. harveyi agar as a primary isolation medium and were positively identified, by conventional tests, as V. harveyi. This agar displays great potential as a primary isolation medium and offers significant advantages over thiosulfate-citrate-bile salts-sucrose agar as a medium for differentiating V. harveyi from other marine and estuarine Vibrio species.     

PCR detection of hemolysin (vhh) gene in Vibrio harveyi

The Vibrio harveyi hemolysin gene (vhh), which encodes for a virulence factor involved in pathogenicity to fish and shellfish species, may be targeted for species detection or strain differentiation. Primers designed for this gene were used in detection studies of V. harveyi strains from various hosts. One primer set among four tested, could amplify the expected gene fragment in PCR using templates from all 11 V. harveyi strains studied. Detection of the presence of the hemolysin gene could therefore serve as a suitable detection marker of Vibrio harveyi isolates potentially pathogenic to fish and shrimps.     

Antimicrobial activity of marine bacteria associated with sponges from the waters off the coast of South East India

Seventy-five marine bacterial strains associated with four species of sponges (Echinodictyum sp., Spongia sp., Sigmadocia fibulatus and Mycale mannarensis) were isolated from the Tuticorin coast, Gulf of Mannar region. The agar-overlay method was used to screen for antibiotic production by these strains against four bacteria, viz., Bacillus subtilis, Escherichia coli, Vibrio parahaemolyticus, and Vibrio harveyi and one fungal pathogen, viz., Candida albicans. Twenty-one per cent of the bacterial strains were found to be antibiotic producers and their activities ranged from broad spectral to species specific. A strain coded SC3 was found to be highly potent and was mass cultured. The ethyl acetate extract of the culture broth was further fractionated by reverse phase HPLC and the active fraction identified. In addition, SC3 was subjected to morphological and physiological characterization. The results of the tests showed SC3 to be a Gram-positive rod, sporulating, motile, catalase and oxidase positive. Phylogenetic analysis based on comparative analysis of sequenced 16s rRNA of the active strains indicated a preponderance of bacteria belonging to Vibrio and Bacillus genera with 95-99% sequence similarities. To our knowledge this is the first report on phylogenetic identification of antibiotic producing bacteria associated with sponges from Indian waters.     

Tryptophan biosynthesis in the marine luminous bacterium Vibrio harveyi.

Tryptophan biosynthetic enzyme levels in wild-type Vibrio harveyi and a number of tryptophan auxotrophs of this species were coordinately regulated over a 100-fold range of specific activities. The tryptophan analog indoleacrylic acid evoked substantial derepression of the enzymes in wild-type cells. Even higher enzyme levels were attained in auxotrophs starved for tryptophan, regardless of the location of the block in the pathway. A derepressed mutant selected by resistance to 5-fluorotryptophan was found to have elevated basal levels of trp gene expression; these basal levels were increased only two- to threefold by tryptophan limitation. The taxonomic implications of these and other biochemical results support previous suggestions that the marine luminous bacteria are more closely related to enteric bacteria than to other gram-negative taxa.     

Description of a bacteriocinogenic plasmid in Beneckea harveyi.

A total of 795 strains of marine Vibrio species and Beneckea harveyi, a luminescent marine bacterium, were isolated from various sources in the area of Galveston Island, Tex., and screened for the production of bacteriocin-like substances. More than 8% of the Vibrio isolates produced low-molecular-weight (dialyzable) substances, which were lethal to a test strain of V. parahaemolyticus. Approximately 5% of the B. harveyi isolates produced higher-molecular-weight (nondialyzable) substances which were lethal to a test strain of B. harveyi. One of the B. harveyi strains (strain SY) produced a nondialyzable substance which was lethal to two of 39 strains of B. harveyi. The substance showed no activity toward 17 test strains drawn from the Vibrionaceae and Enterobacteriaceae. Strain SY showed no sensitivity to its own lethal agent and was shown by agarose gel electrophoresis and electron microscopy to harbor a single plasmid of 38 x 10(6) daltons. Variants of strain SY lacking the plasmid were produced by growth in the presence of the antibiotic novobiocin. These variants lacked both the ability to produce the lethal substance and the ability to survive in its presence. The lethal agent produced by strain SY is the first bacteriocin reported in marine bacteria. The term "harveyicin" is proposed to name this lethal substance.     

The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule

Many bacteria control gene expression in response to cell population density, and this phenomenon is called quorum sensing. In Gram-negative bacteria, quorum sensing typically involves the production, release and detection of acylated homoserine lactone signalling molecules called autoinducers. Vibrio harveyi, a Gram-negative bioluminescent marine bacterium, regulates light production in response to two distinct autoinducers (AI-1 and AI-2). AI-1 is a homoserine lactone. The structure of AI-2 is not known. We have suggested previously that V. harveyi uses AI-1 for intraspecies communication and AI-2 for interspecies communication. Consistent with this idea, we have shown that many species of Gram-negative and Gram-positive bacteria produce AI-2 and, in every case, production of AI-2 is dependent on the function encoded by the luxS gene. We show here that LuxS is the AI-2 synthase and that AI-2 is produced from S-adenosylmethionine in three enzymatic steps. The substrate for LuxS is S-ribosylhomocysteine, which is cleaved to form two products, one of which is homocysteine, and the other is AI-2. In this report, we also provide evidence that the biosynthetic pathway and biochemical intermediates in AI-2 biosynthesis are identical in Escherichia coli, Salmonella typhimurium, V. harveyi, Vibrio cholerae and Enterococcus faecalis. This result suggests that, unlike quorum sensing via the family of related homoserine lactone autoinducers, AI-2 is a unique, 'universal' signal that could be used by a variety of bacteria for communication among and between species.     

Evidence for a signaling system in Helicobacter pylori: detection of a luxS-encoded autoinducer

Helicobacter pylori possesses a homolog of the luxS gene, initially identified by its role in autoinducer production for the quorum-sensing system 2 in Vibrio harveyi. The genomes of several other species of bacteria, notably Escherichia coli, Salmonella enterica serovar Typhimurium, and Vibrio cholerae, also include luxS homologs. All of these bacteria have been shown to produce active autoinducers capable of stimulating the expression of the luciferase operon in V. harveyi. In this report, we demonstrate that H. pylori also synthesizes a functional autoinducer (AI-2) that can specifically activate signaling system 2 in V. harveyi. Maximal activity is produced during early log phase, and the activity is diminished when cells enter stationary phase. We show that AI-2 is not involved in modulating any of the known or putative virulence factors in H. pylori and that a luxS null mutant has a two-dimensional protein profile identical to that of its isogenic parent strain. We discuss the implications of having an AI-2-like quorum-sensing system in H. pylori and suggest possible roles that it may play in H. pylori infection.     

Sequence analysis of partial toxR gene from Philippine Vibrio isolates and design of toxR-targeted primers for detection

Vibriosis in penaeid species cultured in the Philippines results in massive mortalities and consequently in severe economic losses in the shrimp industry. Rapid and accurate detection of the causative agent of the disease is imperative. In this study, toxR gene sequence analysis of ten Vibrio isolates (from several provinces of the Philippines) implicated in disease affecting the penaeid shrimp (Penaeus monodon) was performed in order to develop a toxR-targeted PCR detection of similar strains of shrimp pathogens. Analysis of the partial toxR gene revealed 97-100% sequence similarity among the ten Philippine Vibrio isolates. Distinct sequence variation of the toxR gene, however, was observed between the Philippine Vibrio isolates and the type strains, with the Philippine isolates exhibiting only 92-93% and 74-75% sequence similarity with the type strain V. campbellii (NBRC 15631T) and V. harveyi (NBRC 15634T), respectively. The use of a PCR primer set that was designed based on toxR sequences of the Philippine Vibrio isolates amplified the expected 226-bp toxR fragment using templates from all ten Philippine Vibrio isolates. No amplified product was observed in PCR using templates from type strains of V. harveyi, V. campbellii, and other non-target bacteria, suggesting that the primers were specific for the Philippine Vibrio isolates. The toxR-targeted PCR primers reported in this study could be useful in the detection of Philippine Vibrio isolates associated with mortalities in the shrimp industry, which could not be detected in PCR using primers designed for type strains of V. harveyi and V. campbellii.     

大黄鱼病原哈维氏弧菌单克隆抗体的制备及其应用

用甲醛灭活哈维氏弧菌（Vibrio harveyi）GYC1108-1制备免疫原免疫8周龄雌性BALB/c小鼠,利用淋巴细胞杂交瘤技术,用间接酶联免疫吸附试验（ELISA）对杂交瘤进行筛选,阳性克隆经2－3次亚克隆后,共获得5株针对GYC1108-1的单克隆抗体。选取1B7制备小鼠腹水抗体,其细胞上清及腹水效价分别为1∶3200和1∶32000。同样用灭活的哈维氏弧菌免疫新西兰大白兔,5次免疫后颈动脉采血,离心取血清,并用饱和硫酸铵法进行纯化,制备了兔抗哈维氏弧菌多克隆抗体,其ELISA效价为1∶51200。利用1B7单克隆抗体和兔抗哈维氏弧菌多克隆抗体以及山羊抗小鼠HRP酶标二抗,建立了检测哈维氏弧菌的三抗体夹心酶联免疫吸附试验（TAS-ELISA）方法。该方法对哈维氏弧菌的最小检出浓度为1×104个/mL。用该TAS-ELISA方法检测大黄鱼（Pseudosciaena crocea）样品,54尾患病大黄鱼中有45尾检出哈维氏弧菌,而14尾健康大黄鱼都没有检出哈维氏弧菌。由此可见,本试验建立的TAS-ELISA方法,可以用于患病大黄鱼哈维氏弧菌的快速诊断。     

不同大菱鲆(Scophthalmus maximus)个体肠道菌群结构差异研究

目的:肠道微生物能够帮助宿主完成多种生理生化功能,对宿主的健康生长也起到非常重要的作用。大菱鲆(Scophthalmus maximus)是我国北方最重要的海水养殖品种之一。然而,细菌性疾病的频发造成大规模的鱼类死亡。此外,许多大菱鲆致病菌也被证实是人类潜在的致病菌。因此鉴定成年养殖大菱鲆肠道中所包含的核心菌群,并筛选可能与大菱鲆或人类健康密切相关的细菌尤为重要。方法:构建三个大菱鲆个体肠道16S rRNA文库,利用限制性片段长度多态性分析(RFLP)及生物信息学技术研究三个大菱鲆个体肠道菌群结构及多样性。结果:共得到758个阳性克隆,16个OTU类型。分类分析显示,变形菌门在大菱鲆肠道中占优势地位。进一步将细菌按属来分类显示,弧菌属所占的比例最高,约为95.3%。韦恩图显示大菱鲆三个个体共有的OTU类型数量为3,分别占三个大菱鲆个体肠道文库中阳性克隆总数的94.9%、96.7%以及93.5%。分类结果显示它们分别属于条件致病菌哈氏弧菌、副溶血弧菌和创伤弧菌。结论:大菱鲆的核心肠道菌群为弧菌属,它的主要成员哈氏弧菌、副溶血弧菌和创伤弧菌对大菱鲆及人类的健康起到关键的作用。在水产养殖过程中,通过监控这些弧菌种类的浓度变化能够及时对养殖环境进行评价,并及时调整温度、水质等因素,这为预防大菱鲆细菌性疾病,保证人类健康提供重要的理论依据。     

Development of monoclonal antibodies that identify Vibrio species commonly isolated from infections of humans, fish, and shellfish.

Monoclonal antibodies (MAbs) against Vibrio species that infect humans, fish, and shellfish were developed for application in rapid identifications. The pathogens included Vibrio alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus, and V. vulnificus. Three types of MAbs were selected. The first important group included MAbs that reacted with only a single species. A second group comprised a number of MAbs that reacted with two, taxonomically closely related Vibrio species. For example, of 22 MAbs raised against V. alginolyticus, 6 recognized a 52-kDa flagellar H antigen common to both V. alginolyticus and V. parahaemolyticus; V. anguillarum and V. ordalii also shared antigens. A third group included three genus-specific MAbs that reacted with almost all Vibrio species but did not react with other members of the family Vibrionaceae (e.g., members of the Aeromonas, Photobacterium, and Plesiomonas genera) or a wide range of gram-negative bacteria representing many genera. This last group indicated the possible existence of an antigenic determinant common to Vibrio species. Two of these three genus-specific MAbs reacted with heat-stable antigenic determinants of Vibrio species as well as lipopolysaccharide extracted from Vibrio species. The use of the MAbs in blind tests and diagnosis of clinical isolates indicated that three different types of bacteria, viz., live, formalin-fixed, and sodium azide-killed bacteria, were detected consistently. Overall, it was found that the genus-specific MAbs were very useful for rapidly identifying vibrios in the screening of acute infections, while the species-specific MAbs and others were useful for completing the diagnosis.     

Development of monoclonal antibodies that identify Vibrio species commonly isolated from infections of humans, fish, and shellfish.

Monoclonal antibodies (MAbs) against Vibrio species that infect humans, fish, and shellfish were developed for application in rapid identifications. The pathogens included Vibrio alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus, and V. vulnificus. Three types of MAbs were selected. The first important group included MAbs that reacted with only a single species. A second group comprised a number of MAbs that reacted with two, taxonomically closely related Vibrio species. For example, of 22 MAbs raised against V. alginolyticus, 6 recognized a 52-kDa flagellar H antigen common to both V. alginolyticus and V. parahaemolyticus; V. anguillarum and V. ordalii also shared antigens. A third group included three genus-specific MAbs that reacted with almost all Vibrio species but did not react with other members of the family Vibrionaceae (e.g., members of the Aeromonas, Photobacterium, and Plesiomonas genera) or a wide range of gram-negative bacteria representing many genera. This last group indicated the possible existence of an antigenic determinant common to Vibrio species. Two of these three genus-specific MAbs reacted with heat-stable antigenic determinants of Vibrio species as well as lipopolysaccharide extracted from Vibrio species. The use of the MAbs in blind tests and diagnosis of clinical isolates indicated that three different types of bacteria, viz., live, formalin-fixed, and sodium azide-killed bacteria, were detected consistently. Overall, it was found that the genus-specific MAbs were very useful for rapidly identifying vibrios in the screening of acute infections, while the species-specific MAbs and others were useful for completing the diagnosis.     

Identification of intestinal bacteria from Japanese flounder (Paralichthys olivaceus) and their ability to digest chitin

AIMS: The aim of the present study was to clarify the taxonomic status of intestinal bacteria isolated from Japanese flounder (Paralichthys olivaceus) and describe their ability to digest chitin. METHODS AND RESULTS: Phylogenetic analysis based on 16S ribosomal DNA (rDNA) sequences showed that 82 representative isolates were closely related to three major species of marine vibrios, Vibrio scophthalmi-Vibrio ichthyoenteri group (41 isolates), Vibrio fischeri (39 isolates) and Vibrio harveyi (two isolates), with similarities of 97.2-99.8%, 96.4-100% and 98.6-99.5% respectively. These findings indicate that V. scophthalmi-V. ichthyoenteri group is indigenous to the intestinal tract of Japanese flounder. Moreover, the ability of 82 isolates to digest chitin was examined using the agar plate method and PCR amplification of the chiA gene. The two V. harveyi isolates and 36 of 41 V. scophthalmi-V. ichthyoenteri isolates digested chitin and were chiA PCR positive, whereas all 39 V. fischeri isolates digested chitin but were chiA PCR negative. CONCLUSIONS: Intestinal bacteria from Japanese flounder were mainly composed of Vibrio scophthalmi-V. ichthyoenteri group and V. fischeri. Taken together, the results showed that 81 of 82 isolates could digest chitin. However, only 38 of these isolates possessed a chiA homologue which could be identified by PCR. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study shows that Japanese flounder harbours bacteria of the V. scophthalmi-V. ichthyoenteri group, and these results are similar to what has been found for turbot (Scophthalmus maximus).     

Three parallel quorum-sensing systems regulate gene expression in Vibrio harveyi

In a process called quorum sensing, bacteria communicate using extracellular signal molecules termed autoinducers. Two parallel quorum-sensing systems have been identified in the marine bacterium Vibrio harveyi. System 1 consists of the LuxM-dependent autoinducer HAI-1 and the HAI-1 sensor, LuxN. System 2 consists of the LuxS-dependent autoinducer AI-2 and the AI-2 detector, LuxPQ. The related bacterium, Vibrio cholerae, a human pathogen, possesses System 2 (LuxS, AI-2, and LuxPQ) but does not have obvious homologues of V. harveyi System 1. Rather, System 1 of V. cholerae is made up of the CqsA-dependent autoinducer CAI-1 and a sensor called CqsS. Using a V. cholerae CAI-1 reporter strain we show that many other marine bacteria, including V. harveyi, produce CAI-1 activity. Genetic analysis of V. harveyi reveals cqsA and cqsS, and phenotypic analysis of V. harveyi cqsA and cqsS mutants shows that these functions comprise a third V. harveyi quorum-sensing system that acts in parallel to Systems 1 and 2. Together these communication systems act as a three-way coincidence detector in the regulation of a variety of genes, including those responsible for bioluminescence, type III secretion, and metalloprotease production.     

Differential detection of vibrios pathogenic to shrimp by multiplex PCR

The research was focused on the multiplex polymerase chain reaction (PCR) differential detection of shrimp pathogens Vibrio harveyi, Vibrio campbellii and isolates from a variant strain of Vibrio (referred to as Philippine Vibrio isolates in this study) exhibiting characteristics distinct from these two species. Sequence alignment of the hemolysin gene from type strains Vibrio harveyi (NBRC 15634) and Vibrio campbellii (NBRC 15631), as well as 10 variant Philippine Vibrio isolates, was performed in order to design a set of hemolysin-targeted primers for the specific detection of the Philippine Vibrio isolates. Primer PNhemo amplified a 320-bp hemolysin gene fragment of the Philippine Vibrio isolates in PCR using 65 degrees C annealing temperature, but did not amplify the target gene fragment in type strains V. harveyi and V. campbellii. Another new primer (VcatoxR) targeting the toxR gene was designed for the specific detection of type strain V. campbellii under stringent 65 degrees C annealing temperature. PCR using VcatoxR primer resulted in the specific amplification of a 245-bp V. campbellii toxR fragment. The simultaneous use of three primer sets in PCR, including PNhemo and VcatoxR (the two new primers designed in this study), and a primer VhtoxR (previously reported for the specific detection of V. harveyi), resulted in differential profiles with 390-bp, 245-bp, and 320-bp amplicons for V. harveyi, V. campbellii, and variant Philippine Vibrio isolates, respectively. Presence of all three types of Vibrio shrimp pathogens in the sample could be detected with a multiplex PCR profile containing all the expected size amplicons.