Multilocus sequence analysis provides basis for fast and reliable identification of Vibrio harveyi-related species and reveals previous misidentification of important marine pathogens

Vibrio harveyi and related bacteria are important pathogens responsible for severe economic losses in the aquaculture industry worldwide. Phenotypic tests and 16S rRNA gene analysis fail to discriminate species within the V. harveyi group because these are phenotypically and genetically nearly identical. This study used multilocus sequence analysis to identify 36 V. harveyi-like isolates obtained from a wide range of sources in Australia and to re-evaluate the identity of important pathogens. Phylogenies inferred from the 16S rRNA gene and five concatenated protein-coding genes (rpoA-pyrH-topA-ftsZ-mreB) revealed four well-supported clusters identified as V. harveyi, V. campbellii, V. rotiferianus and V. owensii. Results revealed that important V. campbellii and V. owensii prawn pathogens were previously misidentified as V. harveyi and also that the recently described V. communis sp. nov. is likely a junior synonym of V. owensii. Although the MLSA topologies corroborated the 16S rRNA gene phylogeny, the latter was less informative than each of the protein-coding genes taken singularly or the concatenated dataset. A two-locus phylogeny based on topA-mreB concatenated sequences was consistent with the five-locus MLSA phylogeny. Global Bayesian phylogenies inferred from topA-mreB suggested that this gene combination provides a practical yet still accurate approach for routine identification of V. harveyi-related species.     

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.     

16S ribosomal DNA sequencing confirms the synonymy of Vibrio harveyi and V. carchariae

Seventeen bacterial strains previously identified as Vibrio harveyi (Baumann et al. 1981) or V. carchariae (Grimes et al. 1984) and the type strains of V. harveyi, V. carchariae and V. campbellii were analyzed by 16S ribosomal DNA (rDNA) sequencing. Four clusters were identified in a phylogenetic analysis performed by comparing a 746 base pair fragment of the 16S rDNA and previously published sequences of other closely related Vibrio species. The type strains of V. harveyi and V. carchariae and about half of the strains identified as V. harveyi or V. carchariae formed a single, well-supported cluster designed as 'bona fide' V. harveyi/carchariae. A second more heterogeneous cluster included most other strains and the V. campbellii type strain. Two remaining strains are shown to be more closely related to V. rumoiensis and V. mediterranei. 16S rDNA sequencing has confirmed the homogeneity and synonymy of V. harveyi and V. carchariae. Analysis of API20E biochemical profiles revealed that they are insufficient by themselves to differentiate V. harveyi and V. campbellii strains. 16S rDNA sequencing, however, can be used in conjunction with biochemical techniques to provide a reliable method of distinguishing V. harveyi from other closely related species.     

Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis

We analyzed the usefulness of rpoA, recA, and pyrH gene sequences for the identification of vibrios. We sequenced fragments of these loci from a collection of 208 representative strains, including 192 well-documented Vibrionaceae strains and 16 presumptive Vibrio isolates associated with coral bleaching. In order to determine the intraspecies variation among the three loci, we included several representative strains per species. The phylogenetic trees constructed with the different genetic loci were roughly in agreement with former polyphasic taxonomic studies, including the 16S rRNA-based phylogeny of vibrios. The families Vibrionaceae, Photobacteriaceae, Enterovibrionaceae, and Salinivibrionaceae were all differentiated on the basis of each genetic locus. Each species clearly formed separated clusters with at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively. The genus Vibrio was heterogeneous and polyphyletic, with Vibrio fischeri, V. logei, and V. wodanis grouping closer to the Photobacterium genus. V. halioticoli-, V. harveyi-, V. splendidus-, and V. tubiashii-related species formed groups within the genus Vibrio. Overall, the three genetic loci were more discriminatory among species than were 16S rRNA sequences. In some cases, e.g., within the V. splendidus and V. tubiashii group, rpoA gene sequences were slightly less discriminatory than recA and pyrH sequences. In these cases, the combination of several loci will yield the most robust identification. We can conclude that strains of the same species will have at least 98, 94, and 94% rpoA, recA, and pyrH gene sequence similarity, respectively.     

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.     

Identification of vhhP2, a novel genetic marker of Vibrio harveyi, and its application in the quick detection of V. harveyi from animal specimens and environmental samples

Aims:  To investigate the species-specific prevalence of vhhP2 among Vibrio harveyi isolates and the applicability of vhhP2 in the specific detection of V. harveyi from crude samples of animal and environmental origins.
Methods and Results:  A gene ( vhhP2 ) encoding an outer membrane protein of unknown function was identified from a pathogenic V. harveyi isolate. vhhP2 is present in 24  V. harveyi strains isolated from different geographical locations but is absent in 24 strains representing 17 different non- V. harveyi species, including V. parahaemolyticus and V. alginolyticus . A simple polymerase chain reaction method for the identification of V. harveyi was developed based on the conserved sequence of vhhP2 . This method was demonstrated to be applicable to the quick detection of V. harveyi from crude animal specimens and environmental samples. The specificity of this method was tested by applying it to the examination of two strains of V. campbellii , which is most closely related to V. harveyi . One of the V. campbellii strains was falsely identified as V. harveyi .
Conclusions:  vhhP2 is ubiquitously present in the V. harveyi species and is absent in most of the non- V. harveyi species; this feature enables vhhP2 to serve as a genetic marker for the rapid identification of V. harveyi . However, this method can not distinguish some V. campbellii strains from V. harveyi .
Significance and Impact of the Study:  the significance of our study is the identification of a novel gene of V. harveyi and the development of a simple method for the relatively accurate detection of V. harveyi from animal specimens and environmental samples.     

Quorum sensing-disrupting brominated furanones protect the gnotobiotic brine shrimp Artemia franciscana from pathogenic Vibrio harveyi, Vibrio campbellii, and Vibrio parahaemolyticus isolates

Autoinducer 2 (AI-2) quorum sensing was shown before to regulate the virulence of Vibrio harveyi towards the brine shrimp Artemia franciscana. In this study, several different pathogenic V. harveyi, Vibrio campbellii, and Vibrio parahaemolyticus isolates were shown to produce AI-2. Furthermore, disruption of AI-2 quorum sensing by a natural and a synthetic brominated furanone protected gnotobiotic Artemia from the pathogenic isolates in in vivo challenge tests.     

Refined identification of Vibrio bacterial flora from Acanthasther planci based on biochemical profiling and analysis of housekeeping genes

We used a polyphasic approach for precise identification of bacterial flora (Vibrionaceae) isolated from crown-of-thorns starfish (COTS) from Lizard Island (Great Barrier Reef, Australia) and Guam (U.S.A., Western Pacific Ocean). Previous 16S rRNA gene phylogenetic analysis was useful to allocate and identify isolates within the Photobacterium, Splendidus and Harveyi clades but failed in the identification of Vibrio harveyi-like isolates. Species of the V harveyi group have almost indistinguishable phenotypes and genotypes, and thus, identification by standard biochemical tests and 16S rRNA gene analysis is commonly inaccurate. Biochemical profiling and sequence analysis of additional topA and mreB housekeeping genes were carried out for definitive identification of 19 bacterial isolates recovered from sick and wild COTS. For 8 isolates, biochemical profiles and topA and mreB gene sequence alignments with the closest relatives (GenBank) confirmed previous 16S rRNA-based identification: V. fortis and Photobacterium eurosenbergii species (from wild COTS), and V natriegens (from diseased COTS). Further phylogenetic analysis based on topA and mreB concatenated sequences served to identify the remaining 11 V harveyi-like isolates: V. owensii and V. rotiferianus (from wild COTS), and V. owensii, V. rotiferianus, and V. harveyi (from diseased COTS). This study further confirms the reliability of topA-mreB gene sequence analysis for identification of these close species, and it reveals a wider distribution range of the potentially pathogenic V. harveyi group.     

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。     

Amplification and sequence analysis of the full length toxR gene in Vibrio harveyi

This study was focused on obtaining the complete gene sequence of the toxR gene in V. harveyi by using toxR-targeted PCR to amplify 5' and 3' regions flanking the 576-bp Vibrio harveyi (NBRC 15634) toxR gene fragment previously amplified using degenerate PCR. To obtain the 5' flanking sequences, a forward PCR primer (VhtoxRpv) was designed based on known sequences upstream of toxR in V. parahaemolyticus and V. vulnificus. The reverse primer (VctoxR2R) was based on the sequence of the 576-bp Vibrio harveyi toxR fragment. The resulting 750-bp amplicon was sequenced, providing the 5' sequences of the V. harveyi (NBRC 15634) toxR gene. The 3' flanking region was amplified using a primer pair toxRS1 and toxRS2 based on V. parahaemolyticus and V. vulnificus toxR and toxS, resulting in a 900-bp amplicon that contained the remaining 3' sequences of the V. harveyi NBRC 15634 toxR. This paper reports, for the first time, a complete 882-bp nucleotide sequence for toxR in Vibrio harveyi. Sequence analysis and alignment revealed that the complete toxR gene in V. harveyi shares 87% sequence similarity with toxR of V. parahaemolyticus, 84% similarity with V. fluvialis, 83% with V. vulnificus and partial sequence of V. campbellii. The phylogenetic trees revealed wider divergence in toxR compared to 16S rRNA genes, so that V. harveyi could easily be distinguished from V. campbellii and V. parahaemolyticus.     

Multilocus sequence analysis of biocontrol fluorescent Pseudomonas spp. producing the antifungal compound 2,4-diacetylphloroglucinol

The genetic and evolutionary relationship among 2,4-diacetylphloroglucinol (Phl)-producing pseudomonads that protect plants from soil-borne pathogens were investigated by multilocus sequence typing. A total of 65 pseudomonads consisting of 58 Phl-positive biocontrol strains of worldwide origin and seven Phl-negative representatives of characterized Pseudomonas species were compared using 10 housekeeping genes (i.e. rrs, dsbA, gyrB, rpoD, fdxA, recA, rpoB, fusA, rpsL and rpsG). Multilocus sequence typing differentiated 51 strains among 58 Phl-positive pseudomonads and proved to be as discriminative as enterobacterial repetitive intergenic consensus polymerase chain reaction profiling. As phylogenetic trees inferred from each locus were rather incongruent with one another, we derived the topology from all concatenated loci, which led to the identification of six main groups of Phl-producing Pseudomonas spp. Taxonomically, these groups could correspond to at least six different species. Linkage disequilibrium analysis pointed to a rather clonal structure, even when the analysis was restricted to Phl-producing pseudomonads from a same geographic location or a same phylogenetic group. Intragenic recombination was evidenced for gyrB, rpoD and fdxA, but was shown to be a weaker force than mutation in the origin of intragenetic diversity. This is the first multilocus assessment of the phylogeny and population structure of an ecologically important bacterial group involved in plant disease suppression.     

Lateral flagellar antigen of Vibrio alginolyticus and Vibrio harveyi: existence of serovars common to the two species

The antigenicity of lateral (L-) flagella of two marine vibrios, Vibrio alginolyticus and V. harveyi, was studied, and the two species were found to have common antigenicity of their flagella. Antisera against L-flagella were prepared by immunizing rabbits with highly purified L-flagellar filaments. H-Agglutination tests with the anti-L-flagella antisera showed that four H-serovars existed in these species and that two of them were shared by the two species. Cross reactivity between H-serovars of these two species and other vibrios having lateral flagella, such as V. parahaemolyticus, V. campbellii, V. proteus, or V. fluvialis, was not observed in the H-agglutination test, although partial common antigenicity was observed in the gel diffusion test with flagellin monomers. These observations suggest that surface antigenic determinants of the lateral flagella of V. alginolyticus and V. harveyi are specific to these two species but internal antigenic determinants buried in the flagellar filaments are partially shared with other vibrio species.     

Phylogenetic analysis of the incidence of lux gene horizontal transfer in Vibrionaceae

Horizontal gene transfer (HGT) is thought to occur frequently in bacteria in nature and to play an important role in bacterial evolution, contributing to the formation of new species. To gain insight into the frequency of HGT in Vibrionaceae and its possible impact on speciation, we assessed the incidence of interspecies transfer of the lux genes (luxCDABEG), which encode proteins involved in luminescence, a distinctive phenotype. Three hundred three luminous strains, most of which were recently isolated from nature and which represent 11 Aliivibrio, Photobacterium, and Vibrio species, were screened for incongruence of phylogenies based on a representative housekeeping gene (gyrB or pyrH) and a representative lux gene (luxA). Strains exhibiting incongruence were then subjected to detailed phylogenetic analysis of horizontal transfer by using multiple housekeeping genes (gyrB, recA, and pyrH) and multiple lux genes (luxCDABEG). In nearly all cases, housekeeping gene and lux gene phylogenies were congruent, and there was no instance in which the lux genes of one luminous species had replaced the lux genes of another luminous species. Therefore, the lux genes are predominantly vertically inherited in Vibrionaceae. The few exceptions to this pattern of congruence were as follows: (i) the lux genes of the only known luminous strain of Vibrio vulnificus, VVL1 (ATCC 43382), were evolutionarily closely related to the lux genes of Vibrio harveyi; (ii) the lux genes of two luminous strains of Vibrio chagasii, 21N-12 and SB-52, were closely related to those of V. harveyi and Vibrio splendidus, respectively; (iii) the lux genes of a luminous strain of Photobacterium damselae, BT-6, were closely related to the lux genes of the lux-rib(2) operon of Photobacterium leiognathi; and (iv) a strain of the luminous bacterium Photobacterium mandapamensis was found to be merodiploid for the lux genes, and the second set of lux genes was closely related to the lux genes of the lux-rib(2) operon of P. leiognathi. In none of these cases of apparent HGT, however, did acquisition of the lux genes correlate with phylogenetic divergence of the recipient strain from other members of its species. The results indicate that horizontal transfer of the lux genes in nature is rare and that horizontal acquisition of the lux genes apparently has not contributed to speciation in recipient taxa.     

Occurrence and distribution of halophilic vibrios in subtropical coastal waters of Hong Kong.

The summer occurrence and distribution of halophilic vibrios in the subtropical coastal waters of Hong Kong were investigated. The density of vibrios in six sample sites ranged from 90 to 6,700 per ml, which made up 0.41 to 40% of the total bacterial populations of these sample sites. The sucrose-positive vibrios were found to be much more common (88% of total vibrios) than the sucrose-negative ones. A total of 48 strains belonging to six Vibrio species were fully characterized. Among these, Vibrio alginolyticus was the most frequently isolated, followed by V. parahaemolyticus, V. harveyi, V. vulnificus, V. campbellii, and V. fluvialis. The finding that eight of the nine strains of V. harveyi showed a positive Kanagawa reaction warrants further study.     

Occurrence and distribution of halophilic vibrios in subtropical coastal waters of Hong Kong

The summer occurrence and distribution of halophilic vibrios in the subtropical coastal waters of Hong Kong were investigated. The density of vibrios in six sample sites ranged from 90 to 6,700 per ml, which made up 0.41 to 40% of the total bacterial populations of these sample sites. The sucrose-positive vibrios were found to be much more common (88% of total vibrios) than the sucrose-negative ones. A total of 48 strains belonging to six Vibrio species were fully characterized. Among these, Vibrio alginolyticus was the most frequently isolated, followed by V. parahaemolyticus, V. harveyi, V. vulnificus, V. campbellii, and V. fluvialis. The finding that eight of the nine strains of V. harveyi showed a positive Kanagawa reaction warrants further study.     

霍乱弧菌和副溶血弧菌分离株的gyrB基因系统发育分析

依据gyrB基因部分编码序列构建系统发育树以分类和鉴别霍乱弧菌和副溶血弧菌,并探讨其种系发生关系。扩增并测序13株霍乱弧菌、8株副溶血弧菌、2株嗜水气单胞菌及1株类志贺邻单胞菌的gyrB基因(编码DNA促旋酶B亚单位)序列,并采用距离法与最大似然法构建系统发育树。两种方法所构建的树结构完全一致,霍乱弧菌、副溶血弧菌、嗜水气单胞菌及类志贺邻单胞菌各自形成一个独立的簇。其中,霍乱肠毒素基因(ctxA)阳性的霍乱弧菌(8株O139群与2株O1群ElTor型)聚类成一分枝;3株副溶血弧菌临床株(1株2002年流行株,2株2004年分离株)与1日本菌株及2001年1株自环境分离的毒力株聚类。系统发育分析靶分子gyrB基因可以良好区分上述4种常见病原菌。产毒O139群霍乱弧菌与产毒O1群ElTor型霍乱弧菌关系密切。副溶血弧菌环境毒力株与本地区临床主要流行株在系统发育关系上较为接近,可能是潜在的致病菌。     

Analysis of a gene (vch) encoding hemolysin isolated and sequenced from Vibrio campbellii

This study describes the amplification, localization, and sequence analysis of a hemolysin gene from type strain V. campbellii NBRC 15631--the first report of a full-length hemolysin gene for the species. An amplicon ( approximately 600 bp) of polymerase chain reaction performed using V. campbellii DNA template and primers previously designed to target a fragment of V. harveyi hemolysin gene (vhh) was shotgun-cloned and sequenced, generating 576 bp nucleotide sequences of the V. campbellii hemolysin gene. PCR primers designed based on these initial sequences were used to amplify a 551-bp V. campbellii hemolysin gene fragment that was used as probe in Southern hybridization, which localized the complete hemolysin gene within a 3.5-kb HindIII restriction fragment of the V. campbellii genomic DNA. To obtain the remaining DNA sequences upstream and downstream of the 576-bp hemolysin gene sequences, inverse PCR was performed using a self-ligated (circularized) V. campbellii HindIII restriction fragment as the template and PCR primers designed to amplify flanking regions of the 576-bp gene fragment. Nucleotide sequences from the terminal regions of the 3.1-kb product of inverse PCR provided the flanking sequences, resulting in the complete sequence for the V. campbellii hemolysin gene. A VCH PCR primer set was designed to amplify a 1.3-kb region containing the entire hemolysin gene even from other V. campbellii strains, which was sequenced to confirm the V. campbellii hemolysin gene sequence. An open reading frame (ORF) of 1,254 bp (designated as vch) was identified, sharing 79% sequence identity with V. harveyi hemolysin gene vhh, representing 262 base substitutions between V. campbellii and V. harveyi. The deduced amino acid sequence of V. campbellii hemolysin (VCH) shows homologies to the V. harveyi hemolysin (VHH), thermolabile hemolysin of V. parahaemolyticus, proteins such as phospholipase of V. vulnificus and lecithinases of V. mimicus and V. cholerae. The VCH primer set did not produce any amplicon in PCR using V. harveyi DNA, and may therefore be used to distinguish environmental strains of V. campbellii from V. harveyi.     

Phylogenetic study and identification of Vibrio splendidus-related strains based on gyrB gene sequences

Different strains related to Vibrio splendidus have been associated with infection of aquatic animals. An epidemiological study of V. splendidus strains associated with Crassostrea gigas mortalities demonstrated genetic diversity within this group and suggested its polyphyletic nature. Recently 4 species, V. lentus, V. chagasii, V. pomeroyi and V. kanaloae, phenotypically related to V. splendidus, have been described, although biochemical methods do not clearly discriminate species within this group. Here, we propose a polyphasic approach to investigate their taxonomic relationships. Phylogenetic analysis of V. splendidus-related strains was carried out using the nucleotide sequences of 16S ribosomal DNA (16S rDNA) and gyrase B subunit (gyrB) genes. Species delineation based on 16S rDNA-sequencing is limited because of divergence between cistrons, roughly equivalent to divergence between strains. Despite a high level of sequence similarity, strains were separated into 2 clades. In the phylogenetic tree constructed on the basis of gyrB gene sequences, strains were separated into 5 independent clusters containing V. splendidus, V. lentus, V. chagasii-type strains and a putative new genomic species. This phylogenetic grouping was almost congruent with that based on DNA-DNA hybridisation analysis. V. pomeroyi, V. kanaloae and V. tasmaniensis-type strains clustered together in a fifth clade. The gyrB gene-sequencing approach is discussed as an alternative for investigating the taxonomy of Vibrio species.     

Phylogenetic and evolutionary analysis of Vibrio parahaemolyticus and Vibrio alginolyticus isolates based on toxR gene sequence

The Vibrio genus contains a large number of closely related bacterial species differing, in some cases, less than 1% in 16S rRNA gene sequence. The present study evaluated the usefulness of toxR gene for phylogenetic and evolution analysis on Vibrio isolates of environmental or clinical origin belonging to the two closely related species V. parahaemolyticus and V. alginolyticus. The phylogenetic analysis based on toxR gene, contrary to 16S rRNA gene, allowed a clear differentiation of the isolates belonging to the two species and showed the presence of two separate, statistically supported clusters in V. alginolyticus (subgroup A and B). Such division, partially reflected in the biochemical features of the isolates, could not be explained by spatial and/or temporal distance in the isolation, leading to the hypothesis of two distinct, co-existing clusters in the V. alginolyticus isolates analysed. The evolutionary analysis on the toxR sequence showed that while the substitutions inferred from the alignment of V. parahaemolyticus are best explained by the negative/neutral selection model, in V. alginolyticus--and particularly in subgroup B--is acting a positive evolutionary pressure. The site detected as under diversifying selection (P164L) could be related to conformational changes of ToxR protein.     

An MLSA-based online scheme for the rapid identification of Stenotrophomonas isolates

An online scheme to assign Stenotrophomonas isolates to genomic groups was developed using the multilocus sequence analysis (MLSA), which is based on the DNA sequencing of selected fragments of the housekeeping genes ATP synthase alpha subunit (atpA), the recombination repair protein (recA), the RNA polymerase alpha subunit (rpoA) and the excision repair beta subunit (uvrB). This MLSA-based scheme was validated using eight of the 10 Stenotrophomonas species that have been previously described. The environmental and nosocomial Stenotrophomonas strains were characterised using MLSA, 16S rRNA sequencing and DNA-DNA hybridisation (DDH) analyses. Strains of the same species were found to have greater than 95% concatenated sequence similarity and specific strains formed cohesive readily recognisable phylogenetic groups. Therefore, MLSA appeared to be an effective alternative methodology to amplified fragment length polymorphism fingerprint and DDH techniques. Strains of Stenotrophomonas can be readily assigned through the open database resource that was developed in the current study (www.steno.lncc.br/).