Polynucleotide sequence relationships among Japanese and American strains of Vibrio parahaemolyticus

Polynucleotide sequence relationships between two reference Vibrio parahaemolyticus strains isolated from Japanese and American gastroenteritis patients were investigated by use of (32)P-DNA/DNA reassociation in free solution. In addition, these strains were similarly compared with 22 other strains of estuarine and marine vibrios, including 11 strains previously identified as V. parahaemolyticus (2 Japanese, 1 of unknown location, and 8 American strains obtained from diverse geographical locations and sources in North America), 3 strains of V. alginolyticus, and 8 of Vibrio spp. Deoxyribonucleic acid (DNA) from the Japanese and American gastroenteritis isolates showed high relative levels of intraspecific duplex formation (92 to 93%) when reassociated, reciprocally, at 60 C. Heterologous DNA duplexes exhibited thermal elution midpoint [Tm(e)] values comparable to those obtained from homologous duplexes (88.0) when thermally eluted from hydroxyapatite, thus indicating high base-pair complementarity. Other V. parahaemolyticus strains showed DNA homologies of 85% or greater, with correspondingly high Tm(e) values (86.0 to 88.0) for the heteroduplexes formed. DNA of two of three V. alginolyticus strains (ATCC 17749 and 166-70) was 55 to 60% homologous to reference V. parahaemolyticus DNA preparations; Vibrio sp. strain 5144 (originally classified as V. parahaemolyticus biotype 2 and subsequently as V. alginolyticus strain 5144) showed only 24 to 26% DNA homology to the same reference DNA. These data provide evidence that Vibrio sp. strain 5144 is genetically distinct from the other V. alginolyticus strains used in this study. Three bioluminescent strains thought to be closely related to V. parahaemolyticus demonstrated only 24 to 31% DNA homology to the reference V. parahaemolyticus DNA. These data firmly establish the existence in some Atlantic and Gulf Coast estuaries of organisms genetically very similar to V. parahaemolyticus, the causative agent of "shirasu" food poisoning in Japan.     

Ecological relationship between Vibrio parahaemolyticus and agar-digesting vibrios as evidenced by bacteriophage susceptibility patterns.

Twenty bacteriophages active against Vibrio parahaemolyticus and agar-digesting vibrios, isolated from oysters (Crassostrea gigas) and Dungeness crab (Cancer magister) and by induction of a lysogenic agar digester, were tested as to their host range. These phages were specific for V. parahaemolyticus and various agar-digesting vibrios, and interspecies lysis occurred only between these two groups. V. alginolyticus, V. anguillarum and related species, V. cholerae, and a group of marine psychrophilic and psychrotrophic vibrios were not affected. No correlation was observed between the O and K serotypes of V. parahaemolyticus strains and bacteriophage susceptibility patterns, and 7 of 28 strains of V. parahaemolyticus were not lysed by any of the phages. Only two of the phage isolates were capable of lysing all susceptible V. parahaemolyticus strains. No correlation was observed between the inter-and intraspecies genetic relatedness (DNA homologies) of V. parahaemolyticus and agar-digesting vibrios and susceptibility patterns to different bacteriophages. Some of the phages were capable of plaque formation on V. parahaemolyticus as well as on some strains of agar-digesting vibrios that were separated by 70 to 80% differences in their DNA homologies. The possible ecological significance of these vibrio bacteriophages, particularly those having a wide host range, is discussed.     

VHH/TLH溶血素基因在海洋弧菌中分布的研究

哈维氏弧菌(V.harveyi)的VHH溶血素是对海水养殖鱼类的潜在致病因子。哈维氏弧菌的VHH溶血素基因与副溶血弧菌(V.parahaemolyticus)的TLH热不稳定性溶血素基因具有高度相似性,其氨基酸序列的相似性达到85.6 %。根据哈维氏弧菌vhhA溶血素基因序列,合成一个地高辛标记的VHH基因探针,利用其进行Southern Blot ,检测VHH溶血素基因在57株弧菌(包括26株国际标准菌株,20株哈维氏弧菌,11株副溶血弧菌)中的分布情况。结果显示,VHH基因探针与13株弧菌标准菌株有强杂交信号,包括2株溶藻胶弧菌(V.alginolyticus) ,2株哈维氏弧菌以及1株霍氏格里蒙菌(Grimontia hollisae) ,坎贝氏弧菌(V.campbellii) ,辛辛那提弧菌(V.cincinatiensis) ,费氏弧菌(V.fischeri) ,拟态弧菌(V.mimicus) ,飘浮弧菌(V.natriegens) ,副溶血弧菌,解蛋白弧菌(V.proteolyticus)和火神弧菌(V.logei)。与6株弧菌标准菌株有弱杂交信号,包括鳗弧菌(V.anguillarum) ,河口弧菌(V.aestuarianus) ,美人鱼发光杆菌(Photobacterium damselae subsp.damselae) ,河弧菌(V.fluvialis) ,弗尼斯弧菌(V.furnissii)和创伤弧菌(V.vulnificus) ,而另外7株弧菌标准菌株中无杂交信号。所有的哈维氏弧菌菌株至少含有一条杂交带,其中菌株VIB645 , VIB 648和SF-1分别含有2条杂交带。11株副溶血弧菌中均含有一条杂交带。上述数据表明,vhh/tlh溶血素基因广泛分布于弧菌中,尤其是哈维氏弧菌相关菌株和费氏弧菌相关菌株中。另外对鳗弧菌VIB 72 ,坎贝氏弧菌VIB 285 ,飘浮弧菌VIB 299和哈维氏弧菌VIB 647的vhh/tlh溶血素基因进行克隆并测序,其氨基酸序列与VHH溶血素和TLH溶血素氨基酸序列的同源性分别为67 %~99 %和69 %~91 %。对vhh/tlh溶血素基因在弧菌中的分布研究,将有助于进一步确定这类溶血素基因在病原弧菌致病性中的作用。     

Note: Molecular cloning of chitinase genes from Vibrio anguillarum and V. parahaemolyticus

Chitinase genes from Vibrio anguillarum KV9001 and V. parahaemolyticus ATCC17802 were cloned into Escherichia coli. Open reading frames of chitinase genes from V. anguillarum (vac) and V. parahaemolyticus (vpc) are 1755 bp and 1890 bp, respectively. The deduced amino acid sequences of these genes have 71·6% identity. There are two consensus sequence regions in the VAC and VPC proteins. The vac gene was highly prevalent in V. anguillarum , and the DNA probe of the vac gene hybridized to V. alginolyticus and Beneckea proteolytica DNA. The DNA probe of the vpc gene hybridized to V. alginolyticus, V. harveyi and V. ordalii DNA.     

Decarboxylase activity involved in methyl ketone production by Staphylococcus carnosus 833, a strain used in sausage fermentation

Outbreaks of Vibrio parahaemolyticus gastroenteritis in the United States (Texas, New York and Pacific Northwest) in 1997-98 emphasized the need to develop molecular methods for identification and differentiation of these organisms. When outbreak isolates were analyzed for the enterobacterial repetitive intergenic consensus sequences, the Texas and New York outbreak isolates had a specific 850-bp DNA fragment that was absent in Pacific Northwest isolates. The 850-bp polymerase chain reaction (PCR) product was found in isolates of serovar O3:K6, which have an unusual potential to spread and cause infections. To develop a specific molecular detection method for serovar O3:K6, the nucleotide sequence of the 850-bp product was determined. The GenBank blast analysis did not show homology with any known Vibrio spp. gene sequences. Two PCR primers were designed to specifically amplify the unique sequences from serovar O3:K6 isolates. Genomic DNA from 10 Texas, eight New York, and seven Pacific Northwest outbreak isolates of V. parahaemolyticus was assayed by PCR. Texas and New York isolates were positive in the PCR assay, giving a 327-bp PCR product as predicted; however, Pacific Northwest isolates were negative, indicating the absence of the target gene. Texas and New York isolates were all serovar O3:K6; the Pacific Northwest isolates were not. The primers were tested with other Vibrio spp. and other closely related species and no amplification of the 327-bp PCR product was found. The PCR method can be used to specifically identify O3:K6 V. parahaemolyticus isolates in less than 6 h.     

Deoxyribonucleic acid homology in bacterial taxonomy: effect of incubation temperature on reaction specificity

Parameters affecting deoxyribonucleic acid duplex (DNA-DNA) formation on membrane filters were evaluated. The reference strains used were Cytophaga succinicans strain 8, which has a guanine plus cytosine (GC) content of 38%, and Myxococcus xanthus strain FB, which has a GC content of 70%. Both organisms are gliding bacteria classified among the myxobacteria. Among the parameters evaluated, the incubation temperature used during duplex formation was found to be the most important in terms of the physical nature of the reaction product. When an incubation temperature 25 C below the melting point (T(m)) of the native DNA was used, homologous duplexes exhibited a thermal stability similar to that of native DNA. At 35 C below the T(m), a considerable proportion of the duplexes were of much lower stability; at 40 C below the T(m), most of the duplexes were of much lower stability. Similar duplexes of low stability were also formed between DNA molecules from morphologically and nutritionally diverse organisms, provided the GC percentages of the DNA preparations were similar. Competition between unlabeled and labeled DNA fragments for binding sites on immobilized DNA was also greatly influenced by the incubation temperature. Heterologous DNA-DNA complexes exhibited thermal stabilities which correlated with measurements of DNA homology in experiments involving competition. In addition, the difference in thermal stabilities of heterologous and homologous DNA complexes (DeltaT'(m)) may provide a measure of divergence in nucleotide sequences.     

Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia

Previous studies have shown that Vibrio alginolyticus and Vibrio parahaemolyticus can be isolated from similar types of marine samples. In this report, the results of an examination of 567 V. alginolyticus and V. parahaemolyticus strains, isolated from seawater in Jakarta Bay and from more than 30 types of seafood from markets in Jakarta, Indonesia, are presented. Most isolates were from mackerel, shrimp, or squid. Numerical taxonomic analyses clustered 337 isolates and three V. alginolyticus reference strains at S greater than or equal to 80%. These strains produced acid from sucrose, but only approximately 80% produced acetoin or grew in the presence of 10% NaCl. The frequency of occurrence of V. alginolyticus in seawater samples ranged from 0% (in February and March 1972) to 100% (in September and December 1972) and was highest in seafood samples from August to December 1972. A second cluster of 230 isolates and seven V. parahaemolyticus reference strains was observed at S greater than or equal to 82%. These strains did not produce acetoin or acid from sucrose, and approximately 20% grew in the presence of 10% NaCl. V. parahaemolyticus was detected in seawater samples each month, with the highest frequency of occurrence (83.3%) in May 1972. Twenty-nine K antigen serotypes were demonstrated in V. parahaemolyticus isolates, and another 40% were untypable. The modal antibiotic resistance pattern for each species included five drugs. Only 12% of the V. parahaemolyticus strains were Kanagawa positive, and 10% elicited fluid accumulation in ligated rabbit ileal loops. All of the 7 V. alginolyticus strains and 94 (70%) of the V. parahaemolyticus strains tested killed mice when inoculated intraperitoneally.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia.

Previous studies have shown that Vibrio alginolyticus and Vibrio parahaemolyticus can be isolated from similar types of marine samples. In this report, the results of an examination of 567 V. alginolyticus and V. parahaemolyticus strains, isolated from seawater in Jakarta Bay and from more than 30 types of seafood from markets in Jakarta, Indonesia, are presented. Most isolates were from mackerel, shrimp, or squid. Numerical taxonomic analyses clustered 337 isolates and three V. alginolyticus reference strains at S greater than or equal to 80%. These strains produced acid from sucrose, but only approximately 80% produced acetoin or grew in the presence of 10% NaCl. The frequency of occurrence of V. alginolyticus in seawater samples ranged from 0% (in February and March 1972) to 100% (in September and December 1972) and was highest in seafood samples from August to December 1972. A second cluster of 230 isolates and seven V. parahaemolyticus reference strains was observed at S greater than or equal to 82%. These strains did not produce acetoin or acid from sucrose, and approximately 20% grew in the presence of 10% NaCl. V. parahaemolyticus was detected in seawater samples each month, with the highest frequency of occurrence (83.3%) in May 1972. Twenty-nine K antigen serotypes were demonstrated in V. parahaemolyticus isolates, and another 40% were untypable. The modal antibiotic resistance pattern for each species included five drugs. Only 12% of the V. parahaemolyticus strains were Kanagawa positive, and 10% elicited fluid accumulation in ligated rabbit ileal loops. All of the 7 V. alginolyticus strains and 94 (70%) of the V. parahaemolyticus strains tested killed mice when inoculated intraperitoneally.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and Relatedness of Marine Vibrios Pathogenic to Fish: Physiology, Serology, and Epidemiology

Cultural characteristics and serological relationships of pathogenic marine vibrios isolated from fish in the Pacific Northwest were studied. These organisms were compared with cultures of Vibrio anguillarum, a known fish pathogen. On the basis of morphological and cultural characteristics, the Pacific Northwest strains of Vibrio were found to be closely related to V. anguillarum. Serological analyses of thermostable antigens served to distinguish three serotypes among the vibrios. Serotype 1 was composed of organisms isolated from Northwest salmonids; serotype 2 of strains of V. anguillarum from European waters; and serotype 3 of organisms isolated from Pacific herring. The epidemiology of vibrio disease among populations of fish in the Pacific Northwest is discussed.     

Characterization of Vibrio anguillarum and closely related species isolated from farmed fish in Norway.

A total of 264 bacterial strains tentatively or definitely classified as Vibrio anguillarum were examined. The strains were isolated from diseased or healthy Norwegian fish after routine autopsy. With the exception of five isolates from wild saithe (Pollachius virens), the strains originated from nine different species of farmed fish. The bacteria were subjected to morphological, physiological, and biochemical studies, numerical taxonomical analyses, serotyping by slide agglutination and enzyme-linked immunosorbent assay, DNA-plasmid profiling, and in vitro antimicrobial drug susceptibility testing. The results of the microbiological studies were correlated to anamnestic information. The bacterial strains were identified as V. anguillarum serovar O1 (n = 132), serovar O2 (n = 89), serovar O4 (n = 2), serovar O8 (n = 1), and not typeable (n = 1) as well as Vibrio splendidus biovar I (n = 36) and biovar II (n = 1), Vibrio tubiashii (n = 1), and Vibrio fischerii (n = 1). V. anguillarum serovar O1 or O2 was isolated in 176 out of 179 cases of clinical vibriosis in Atlantic salmon (Salmo salar). V. anguillarum serovar O1 was the only serovar isolated from salmonid fish species other than Atlantic salmon, while V. anguillarum serovar O2 was isolated from all marine fish suffering from vibriosis. A 48-Mda plasmid was isolated from all V. anguillarum serovar O1 isolates examined. Serovar O2 isolates did not harbor any plasmids. Resistance against commonly used antibiotic compounds was not demonstrated among V. anguillarum isolates. Neither V. splendidus biovar I nor other V. anguillarum-related species appeared to be of clinical importance among salmonid fish.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleic Acid Homologies Among Oxidase-Negative Moraxella Species

The deoxyribonucleic acid (DNA) base composition and DNA homologies of more than 40 strains of oxidase-negative Moraxella species were determined. These bacteria have also been identified as belonging to the Mima-Herellea-Acinetobacter group and the Bacterium anitratum group, as well as to several other genera including Achromobacter and Alcaligenes. The DNA base content of these strains ranged from 40 to 46% guanine plus cytosine. DNA-DNA competition experiments distinguished five groups whose members were determined by showing 50% or more homology to one of the reference strains: B. anitratum type B5W, Achromobacter haemolyticus var. haemolyticus, Alcaligenes haemolysans, Achromobacter metalcaligenes, and Moraxella lwoffi. A sixth group comprised those strains showing less than 50% homology to any of the reference strains. Negligible homology was found between strains of oxidase-negative and oxidase-positive Moraxella species in DNA-DNA competition experiments. However, evidence of a distant relationship between the two groups was obtained in competition experiments by using ribosomal ribonucleic acid.     

Antigenic and molecular characterization of Vibrio ordalii strains isolated from Atlantic salmon Salmo salar in Chile

Biochemical, serological and molecular properties of a group of 14 Vibrio ordalii strains isolated from cultured Atlantic salmon Salmo salar in Chile in recent years were studied. The characteristics of isolates were compared with the type strain V. ordalii ATCC 33509T. The Chilean V. ordalii represented a biochemically homogenous group; however, some minor differences with the type strain were observed. The serological relationships among isolates, as well as the study of their antigenic determinant (LPS) revealed a strong reaction with antisera raised against Atlantic salmon strains and the antiserum raised against Listonella anguillarum serotype O2. However, LPS electrophoretic patterns were completely different from the V. ordalii type strain, regardless of the serum employed, suggesting the possibility that the Chilean strains constitute a new serological subgroup within this bacterial species. Genetic analyses by PFGE, RAPD, REP-PCR and ERIC-PCR demonstrated that all V. ordalii strains were genetically homogenous, displaying similar DNA patterns, regardless of the techniques used. Moreover, the analysis of DNA banding patterns generated by ERIC-PCR and REP-PCR also clearly separated the type strain from the Chilean strains. This is the first report of characterization of V. ordalii strains from the Southeastern Pacific area, the results of which should facilitate the development of vaccines for protecting cultured Atlantic salmon against vibriosis in this area.     

Rapid detection and identification of Vibrio anguillarum by using a specific oligonucleotide probe complementary to 16S rRNA.

Partial 16S rDNA from Vibrio collection type strains and recent isolates of Vibrio-related strains were sequenced and compared with previously published sequences. A 24-base DNA oligonucleotide (VaV3) was designed and used as a specific probe for detection and identification of Vibrio anguillarum. Its specificity was tested against collection type strains and environmental isolates and no cross-reaction was found. The probe detected 8 of the 10 V. anguillarum serovars. It was applied to screen different Vibrio-related strains isolated from marine hatcheries and fish farms. The detection limit in DNA-DNA slot blot hybridization was 150 pg.     

Clostridium sporogenes isolates and their relationship to C. botulinum based on deoxyribonucleic acid reassociation.

Sixty-two isolates of Clostridium sporogenes from canned foods were examined for cultural properties, heat resistance and DNA-DNA homology to Clostridium botulinum type A190. Sporulation was observed in most of 21 umbonate and rhizoidal colony-forming strains (colony-type I strains), but not in most of the 41 strains with convex and circular or crenate colonies with a mat to semi-glossy surface (colony-type II strains). More than half of the latter strains showed much higher heat resistance than the rhizoidal colony-forming strains. The DNA isolated from colony-type II strains was 81% or more homologous to C. botulinum A190 DNA, forming duplexes which had thermostabilities similar to homologous duplexes of strain A190 DNA. Colony-type I strains differed from C. botulinum by 30 to 40% DNA homology and the DNA duplexes formed between these strains and strain A190 showed deltaT m(e) values of 7-0 degrees C when compared with the T m(e) of homologous DNA duplexes of strain A190.     

An investigation of aquatic bacteria capable of utilizing chitin as the sole source of nutrients

R. OSAWA AND T. KOGA. 1995. A total of 48 bacterial strains capable of utilizing chitin (a polymer of N -acetyl-D-glucosamine [NAG]) as a sole source of nutrients were isolated from river and marine waters in Tokushima. These bacteria were identified as Vibrio fluvialis, V. parahaemolyticus, V. alginolyticus, V. mimicus, Listonella anguillarum and Aeromonas hydrophila . All strains were found positive for chitinase and chitobiase activities, and capable of utilizing NAG as a sole source of carbon and nitrogen.     

Conjugation of drug-resistance plasmids from vibrio anguillarum to Vibrio parahaemolyticus

Drug-resistant strains of Vibrio anguillarum, a fish pathogen, were isolated from diseased fish in culture ponds. In investigations of these strains, the transfer of resistance to chloramphenicol, tetracycline, and sulfanilamide from multiple resistant organisms to laboratory recipients was observed. The plasmid from V. anguillarum was stably maintained in both recipient strains of Vibrio parahaemolyticus and Escherichia coli. The plasmids isolated from Vibrio anguillarum belong to incompatibility group C. The molecular weight of these plasmids determined by electron microscopic observation was about 103 to 113 megadaltons.     

DNA homologies between the rainbow trout, chum salmon and coho salmon

The thermal stabilities of hybrid duplexes between the DNAs from three salmonid fish species were monitored as measures of DNA homology. The chum salmon, Oncorhynchus keta, and coho salmon, Oncorhynchus kisutch, had more DNA homology with each other than either had with the rainbow trout, Salmo gairdnerii. Morphological, ecological and protein similarities between the coho salmon and the rainbow trout may be due to parallel or convergent evolution.     

香港养殖海鲷弧菌致病菌药物敏感性及耐药质粒研究

从发病海鲷(Sparus sarba)中共分离到51株弧菌(\%Vibrio)\%,经API20E细菌快速鉴定系统及Alsina和Blanch关键生理生化特性分析鉴定为7个种,它们分别是：溶藻胶弧菌(\%V.alginolyticus)(24株),创伤弧菌(V.vulnificus)(12株)和副溶血弧菌(V.parahaemolyticus)(7株),火神弧菌(V.logei)(4株),远洋弧菌Ⅱ菌(V.pelagius Ⅱ)(2株),河弧菌(V.fluvialis)(1株)和地中海弧菌(V.mediterranei)(1株)\%。其中3种优势菌溶藻胶弧菌创伤弧菌和副溶血弧菌证实对海鲷有致病性。另外采用平板稀释法检测了51株菌对16种抗菌素的敏感性。发现所有菌株对ceftriaxone,链霉素,萘啶酮酸和利福霉素敏感,几乎所有菌株对ceftazidime, netilimicin,氯霉素和sulfamethoxazole敏感.大部分菌株对氨苄青霉素 (60.8%),cefuroxime(667%),丁胺卡那霉素(55%),卡那霉素(588%)和三甲氧苄氨嘧啶(765%)等具有较强的耐药性。通过对菌株中所含有的耐药质粒进行分析,发现15株菌株含有1～4个质粒,分子量范围为9～123kb之间,对12株既含有较大分子量质粒又具有耐药性的菌株进行了质粒转化试验,结果其中9株菌的质粒具有转化能力,转化率为１０－１１～１０－９,表明所分离的菌株的抗药性是由于细菌染色体相关突变造成的。     

Numerical taxonomy of Vibrionaceae isolated from cultured amberjack (Seriola dumerili) and surrounding water

A numerical taxonomic study was performed on 148 isolates of Gram-negative, heterotrophic, facultative anaerobic bacteria isolated from amberjack (Seriola dumerili) and its surrounding culture water. The study included 30 type and reference strains belonging to genera Vibrio, Listonella, and Photobacterium. The strains were characterized by 109 morphological, biochemical, physiological, and nutritional tests. Cluster analysis of similarity matrices obtained with S(SM) and S(J) coefficients was carried out. UPGMA (unweighted pair group mathematical average) analysis defined 11 phena at S(SM) values > or = 86%. Nine phena were identified as Vibrio alginolyticus, V. fischeri, V. harveyi, V. carchariae, V. mediterranei, V. splendidus, V. furnissii, V. parahaemolyticus, and Photobacterium damselae subsp. damselae. The two latter comprised strains isolated from diseased fish.     

Plasmids mediating iron uptake in Vibrio anguillarum strains isolated from turbot in Spain

Vibrio strains isolated from diseased turbot in an experimental fish farm on the Atlantic coast of northwest Spain were identified as Vibrio anguillarum. The isolates shared many biochemical characteristics with V. anguillarum strains obtained from other sources, and harboured a plasmid species that showed extensive homology with plasmid pJM1, carried by V. anguillarum strain 775 isolated from an epizootic in North America. Restriction endonuclease analysis showed that the two plasmids were very similar albeit not identical. The presence of the plasmid in the turbot isolates was associated with their ability to cause disease in fish. Plasmid-carrying bacteria could also grow under conditions of iron limitation. Two outer membrane proteins, of 86 and 79 kDal, were induced, and a similar siderophore activity to that produced by V. anguillarum 775 was also detected under these conditions. The 86 kDal outer membrane protein cross-reacted immunologically with antiserum raised against the outer membrane protein OM2 produced by strain 775. Nonvirulent plasmidless derivatives were unable to grow under iron-limiting conditions, and were also unable to produce either siderophore activity or the 86 kDal outer membrane protein, suggesting the plasmid-mediated nature of these components.