The dnaJ gene as a novel phylogenetic marker for identification of Vibrio species

The utility of the dnaJ gene for identifying Vibrio species was investigated by analyzing dnaJ sequences of 57 type strains and 22 clinical strains and comparing sequence homologies with those of the 16S rDNA gene and other housekeeping genes (recA, rpoA, hsp60). Among the 57 Vibrio species, the mean sequence similarity of the dnaJ gene (77.9%) was significantly less than that of the 16S rDNA gene (97.2%), indicating a high discriminatory power of the dnaJ gene. Most Vibrio species were, therefore, differentiated well by dnaJ sequence analysis. Compared to other housekeeping genes, the dnaJ gene showed better resolution than recA or rpoA for differentiating Vibrio coralliilyticus from Vibrio neptunius and Vibrio harveyi from Vibrio rotiferianus. Among the clinical strains, all 22 human pathogenic strains, including an atypical strain, were correctly identified by the dnaJ sequence. Our findings suggest that analysis of the dnaJ gene sequence can be used as a new tool for the identification of Vibrio species.     

基于16S rRNA和HSP60基因序列分析粘细菌孢囊杆菌亚目形态分类的种属之间的亲缘关系

[目的]利用16S rRNA和HSP60基因分子标记分析鉴定形态分类特征不稳定的粘细菌种属.[方法]利用粘细菌的传统分离纯化方法从土壤中分离粘细菌,根据菌株的形态特征进行分类,PCR方法扩增菌株的16S rRNA和HSP60基因序列并进行系统发育关系分析.[结果]根据形态特征,分离得到的15株粘细菌菌株归入孢囊杆菌亚目(Cystobacterineae)的2个科3个属.其中11株粘细菌具有典型的所在种属的子实体结构,而菌株0085-4、0121-3、NM03和Myx9736的子实体结构发生了不同程度退化.15株粘细菌的16S rRNA基因序列的相似性在95.4%到99.5%之间.而HSP60基因序列差异较大.[结论]在属水平上,粘细菌形态分类特征和16S rRNA基因系统进化关系具有很好的一致性;在揭示粘细菌种间系统发育关系中,HSP60基因序列更为适用.     

Lactobacillus Strain Diversity Based on Partial hsp60 Gene Sequences and Design of PCR-Restriction Fragment Length Polymorphism Assays for Species Identification and Differentiation

A phylogenetic tree showing diversities among 116 partial (499-bp) Lactobacillus hsp60 (groEL, encoding a 60-kDa heat shock protein) nucleotide sequences was obtained and compared to those previously described for 16S rRNA and tuf gene sequences. The topology of the tree produced in this study showed a Lactobacillus species distribution similar, but not identical, to those previously reported. However, according to the most recent systematic studies, a clear differentiation of 43 single-species clusters was detected/identified among the sequences analyzed. The slightly higher variability of the hsp60 nucleotide sequences than of the 16S rRNA sequences offers better opportunities to design or develop molecular assays allowing identification and differentiation of either distant or very closely related Lactobacillus species. Therefore, our results suggest that hsp60 can be considered an excellent molecular marker for inferring the taxonomy and phylogeny of members of the genus Lactobacillus and that the chosen primers can be used in a simple PCR procedure allowing the direct sequencing of the hsp60 fragments. Moreover, in this study we performed a computer-aided restriction endonuclease analysis of all 499-bp hsp60 partial sequences and we showed that the PCR-restriction fragment length polymorphism (RFLP) patterns obtainable by using both endonucleases AluI and TacI (in separate reactions) can allow identification and differentiation of all 43 Lactobacillus species considered, with the exception of the pair L. plantarum/L. pentosus. However, the latter species can be differentiated by further analysis with Sau3AI or MseI. The hsp60 PCR-RFLP approach was efficiently applied to identify and to differentiate a total of 110 wild Lactobacillus strains (including closely related species, such as L. casei and L. rhamnosus or L. plantarum and L. pentosus) isolated from cheese and dry-fermented sausages.     

Analysis of gyrB and toxR gene sequences of Vibrio hollisae and development of gyrB- and toxR-targeted PCR methods for isolation of V. hollisae from the environment and its identification

Isolation of Vibrio hollisae strains, particularly from the environment, is rare. This may be due, in part, to the difficulty encountered when using conventional biochemical tests to identify the microorganism. In this study, we evaluated whether two particular genes may be useful for the identification of V. hollisae. The two genes are presumed to be conserved among the bacterial species (gyrB) or among the species of the genus Vibrio (toxR). A portion of the gyrB sequence of V. hollisae was cloned by PCR using a set of degenerate primers. The sequence showed 80% identity with the corresponding Vibrio parahaemolyticus gyrB sequence. The toxR gene of V. hollisae was cloned utilizing a htpG gene probe derived from the V. parahaemolyticus htpG gene, which is known to be linked to the toxR gene in V. hollisae. The coding sequence of the cloned V. hollisae toxR gene had 59% identity with the V. parahaemolyticus toxR coding sequence. The results of DNA colony hybridization tests using the DNA probes derived from the two genes of V. hollisae indicated that these gene sequences could be utilized for differentiation of V. hollisae from other Vibrio species and from microorganisms found in marine fish. PCR methods targeting the two gene sequences were established. Both PCR methods were shown to specifically detect the respective target sequences of V. hollisae but not other organisms. A strain of V. hollisae added at a concentration of 1 to 10(2) CFU/ml to alkaline peptone water containing a seafood sample could be detected by a 4-h enrichment incubation in alkaline peptone water at 37 degrees C followed by quick DNA extraction with an extraction kit and 35-cycle PCR specific for the V. hollisae toxR gene. We conclude that screening of seafood samples by this 35-cycle, V. hollisae toxR-specific PCR, followed by isolation on a differential medium and identification by the above htpG- and toxR-targeted PCR methods, can be useful for isolation from the environment and identification of V. hollisae.     

Genotypic diversity of Haemophilus parasuis field strains

Haemophilus parasuis is the cause of Gl?sser's disease and other clinical disorders in pigs. It can also be isolated from the upper respiratory tracts of healthy pigs, and isolates can have significant differences in virulence. In this work, a partial sequence from the 60-kDa heat shock protein (Hsp60) gene was assessed as an epidemiological marker. We analyzed partial sequences of hsp60 and 16S rRNA genes from 103 strains of H. parasuis and other related species to obtain a better classification of the strains and examine the correlation with virulence. The results were compared with those obtained by enterobacterial repetitive intergenic consensus PCR. Our results showed that hsp60 is a reliable marker for epidemiological studies of H. parasuis and that the analysis of its sequence is a better approach than fingerprinting methods. Furthermore, the analysis of the hsp60 and 16S rRNA gene sequences revealed the presence of a separate lineage of virulent strains and indicated the occurrence of lateral gene transfer among H. parasuis and Actinobacillus strains.     

干酪乳杆菌的近缘种及亚种部分看家基因的系统发育分析

【背景】16S rRNA基因序列分析已广泛应用于细菌的分类鉴定,但是存在一定局限性,而使用看家基因作为分子标记在近缘种及亚种间的系统发育分析中具有其独特的优势。【目的】研究16S rRNA、uvr C (核酸外切酶ABC,C亚基)和mur E (UDP-N-乙酰胞壁酰三肽合酶)基因序列对干酪乳杆菌的近缘种及亚种的区分能力。【方法】采用分离自传统发酵乳中的6株干酪乳杆菌为研究对象,选取uvr C和mur E基因片段,通过PCR扩增、测序,结合已公布的干酪乳杆菌的近缘种或亚种的相应序列计算遗传距离、构建系统发育树,并与16S rRNA基因序列分析技术进行比较。【结果】研究发现Lactobacilluscasei及相近种间的uvr C、mur E和联合基因(uvr C-mur E)构建的系统发育树拓扑结构与16S rRNA基因结果基本一致,区别在于相似性的不同,其分别为79.00%-99.16%、89.08%-99.20%、76.56%-99.69%和99.58%-100%。基于16S rRNA基因不能区分干酪乳杆菌的近缘种及亚种,而看家基因uvr C和mur E基因序列能够很好地区分干酪乳杆菌的近缘种及亚种,并且将uvr C和mur E基因串联使用后,试验菌株与参考菌株的分类关系更加清晰。【结论】联合基因(uvr C-mur E)可作为16SrRNA基因的辅助工具用于干酪乳杆菌的近缘种及亚种的快速准确鉴定。     

两株对虾幼体弧菌病病原的分离和鉴定

从患弧菌病的凡纳滨对虾（Litopenaeus vannamei）幼体中分离到两株病原菌zouA和zouB,常规形态和生理生化试验表明均为弧菌属菌种,弧菌编码鉴定系统分别鉴定为溶藻弧菌（Vibrioatginolyticus）和副溶血弧菌（V.parahaemolyticus）。副溶血弧菌R72H序列检测结果进一步证实菌株zouB为副溶血弧菌。对菌株zouA的16S rRNA基因序列分析表明该菌株与溶藻弧菌、副溶血弧菌等弧菌的相似性均高于98％,相互间不能区分;HSP60基因序列分析表明该菌株与溶藻弧菌相似性达98％以上,而与所有其它弧菌的相似性不到92％。结合表型和分子特征的鉴定结果,菌株zouA和zouB分别被鉴定为溶藻弧菌和副溶血弧菌。     

两株对虾幼体弧菌病病原的分离和鉴定

从患弧菌病的凡纳滨对虾(Litopenaeus vannamei)幼体中分离到两株病原菌zouA和zouB, 常规形态和生理生化试验表明均为弧菌属菌种, 弧菌编码鉴定系统分别鉴定为溶藻弧菌(Vibrio alginolyticus)和副溶血弧菌(V. parahaemolyticus)。副溶血弧菌R72H序列检测结果进一步证实菌株zouB为副溶血弧菌。对菌株zouA的16S rRNA基因序列分析表明该菌株与溶藻弧菌、副溶血弧菌等弧菌的相似性均高于98%, 相互间不能区分; HSP60基因序列分析表明该菌株与溶藻弧菌相似性达98%以上, 而与所有其它弧菌的相似性不到92%。结合表型和分子特征的鉴定结果, 菌株zouA和zouB分别被鉴定为溶藻弧菌和副溶血弧菌。     

粪肠、屎肠球菌及相近种部分持家基因的系统发育分析

【目的】利用16S rRNA、clpX和recA基因分子标记研究Enterococcus faecalis、Enterococcus faecium及相近种间的种系发育关系,并比较这些基因序列对E.faecalis、E.faecium及相近种的区分能力。【方法】以分离自传统乳制品中的9株E.faecium和1株E.durans分离株为研究对象,以clpX和recA基因片段为标记,通过PCR扩增、测序,结合已公布的近缘种相应序列构建系统发育树并与16S rRNA基因进行比较。【结果】在基于clpX和recA基因的进化树中,10株试验菌株与E.faecalis始终处于同一分支。与该物种这两个基因的平均相似性为99.6%和98.6%,与另一分支的Faecium-group(E.durans和E.faecium)的平均相似性仅为61.5%和33.5%。相近种E.durans和E.hirae间这两个基因的差异性为20.3%和39.0%;在基于16S rRNA基因的进化树中,试验菌株与Faecium-group(E.lactis、E.faecium、E.durans、E.hirae)处于同一分支。与这些成员间该基因的相似性大于99.6%,与E.faecalis基因的平均相似性可达98.4%。相近种间该基因相似性无明显差异。【结论】按照10株试验菌株clpX和recA基因的分析结果可将由传统生理生化和16S rRNA基因序列鉴定的9株E.faecium和1株E.durans归类为E.faecalis,clpX和recA基因可用于部分相近种的分类鉴定。     

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.     

Genotypic characterization of Lactic acid bacteria in gut microbiome of freshwater fish

The present study focused on identification and genotypic characterization of Lactic acid bacteria (LAB) in the intestine of freshwater fish. 76 strains of LAB were isolated and identified by 16S rRNA gene sequences and hsp60 gene sequences as different strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus brevis, Lactobacillus reuteri, Lactobacillus salivarius, Pediococcus pentosaceus, Pediococcus acidilactici, Weissella paramesenteroides, Weissella cibaria, Enterococcus faecium, and Enterococcus durans. The hsp60 gene showed a higher level of sequence variation among the isolates examined, with lower interspecies sequence similarity providing more resolutions at the species level than the 16S rRNA gene. Phylogenetic tree derived from hsp60 gene sequences with higher bootstrap values at the nodal branches was more consistent as compared to phylogenetic tree constructed from 16S rRNA gene sequences. Closely related species L. plantarum and L. pentosus as well as species L. delbrueckii subsp. bulgaricus and L. fermentum were segregated in different cluster in hsp60 phylogenetic tree whereas such a distribution was not apparent in 16S rRNA phylogenetic tree. In silico restriction analysis revealed a high level of polymorphism within hsp60 gene sequences. Restriction pattern with enzymes AgsI and MseI in hsp60 gene sequences allowed differentiation of all the species including closely related species L. plantarum and L. pentosus, E. faecium and E. durans. In general, hsp60 gene with higher evolutionary divergence proved to be a better phylogenetic marker for the group LAB.     

Sequence Variation in Superoxide Dismutase Gene of Toxoplasma gondii among Various Isolates from Different Hosts and Geographical Regions

Toxoplasma gondii, an obligate intracellular protozoan parasite of the phylum Apicomplexa, can infect all warm-blooded vertebrates, including humans, livestock, and marine mammals. The aim of this study was to investigate whether superoxide dismutase (SOD) of T. gondii can be used as a new marker for genetic study or a potential vaccine candidate. The partial genome region of the SOD gene was amplified and sequenced from 10 different T. gondii isolates from different parts of the world, and all the sequences were examined by PCR-RFLP, sequence analysis, and phylogenetic reconstruction. The results showed that partial SOD gene sequences ranged from 1,702 bp to 1,712 bp and A + T contents varied from 50.1% to 51.1% among all examined isolates. Sequence alignment analysis identified total 43 variable nucleotide positions, and these results showed that 97.5% sequence similarity of SOD gene among all examined isolates. Phylogenetic analysis revealed that these SOD sequences were not an effective molecular marker for differential identification of T. gondii strains. The research demonstrated existence of low sequence variation in the SOD gene among T. gondii strains of different genotypes from different hosts and geographical regions.     

Nucleotide sequence of the gyrA gene of Arcobacter species and characterization of human ciprofloxacin-resistant clinical isolates

The nucleotide sequence of the gyrA gene of Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter cibarius, and Arcobacter skirrowii was determined. The deduced GyrA proteins are closely related to those of Wolinella succinogenes and Helicobacter pullorum, whereas those of Campylobacter species showed less sequence identity. The phylogenetic analysis of GyrA sequences provides a result similar to 16S rRNA gene sequence phylogenetic analysis and allows the discrimination among A. butzleri species. In addition, a Thr-->Ile mutation at amino acid 85 in the quinolone resistance-determining region was associated with ciprofloxacin resistance for two A. butzleri and one A. cryaerophilus ciprofloxacin-resistant strains.     

Bombiscardovia coagulans gen. nov., sp. nov., a new member of the family Bifidobacteriaceae isolated from the digestive tract of bumblebees

One hundred and eighty-seven fructose-6-phosphate phosphoketolase positive strains were isolated from the digestive tract of three different bumblebee species. Analyses of the partial 16S rRNA gene sequences of the representative strains showed only 92.8% and 92.5% similarity to Bifidobacterium coryneforme YIT 4092(T) and Bifidobacterium indicum JCM 1302(T), 92.2% similarity to Alloscardovia omnicolens CCUG 18650 and slightly reduced similarity of 91% to other members of the family Bifidobacteriaceae. On the other hand, analyses of the partial heat-shock protein 60 (hsp60) gene sequence revealed that the proposed type strain BLAPIII-AGV(T) was affiliated only to the 60 kDa chaperonin sequence of uncultured bacteria from human vagina (79-80%) and the hsp60 gene sequence of A. omnicolens CCUG 31649(T) (75.5%). The peptidoglycan type was A4α with an l-Lys-d-Asp interpeptide bridge. The polar lipids contained diphosphatidylglycerol, an unknown phospholipid, six glycolipids and two phosphoglycolipids. The major fatty acids were C(18:1), C(20:0) and C(18:0). These and other analyses indicated that the isolates represented a new genus within the family Bifidobacteriaceae. This observation was further substantiated by determination of the DNA G+C contents (46.1-47.1 mol%). Affinity of the strains to some scardovial genera (Aeriscardovia, Alloscardovia and Metascardovia) was also confirmed by their ability to grow under aerobic conditions. Besides the above mentioned differences, Bombiscardovia coagulans was found to differ from all scardovial genera in the ability to grow at temperatures as low as 5°C, which was another major phenotypically different characteristic of this new member of the family Bifidobacteriaceae. Hence, on the basis of phylogenetic analyses using partial 16S rRNA and hsp60 gene sequence data, and the temperature related phenotypic difference, we propose a novel taxa, B. coagulans gen. nov., sp. nov. (type strain=BLAPIII-AGV(T)=DSM 22924(T)=ATCC BAA-1568(T)).     

Population genetics of the nomenspecies Enterobacter cloacae

The genetic heterogeneity of the nomenspecies Enterobacter cloacae is well known. Enterobacter asburiae, Enterobacter cancerogenus, Enterobacter dissolvens, Enterobacter hormaechei, Enterobacter kobei, and Enterobacter nimipressuralis are closely related to it and are subsumed in the so-called E. cloacae complex. DNA-DNA hybridization studies performed previously identified at least five DNA-relatedness groups of this complex. In order to analyze the genetic structure and the phylogenetic relationships between the clusters of the nomenspecies E. cloacae, 206 strains collected from 22 hospitals, a veterinarian, and an agricultural center in 11 countries plus all 13 type strains of the genus and reference strain CDC 1347-71(R) were examined with a combination of sequence and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses of the three housekeeping genes hsp60, rpoB, and hemB as well as ampC, the gene of a class C beta-lactamase. Based on the neighbor-joining tree of the hsp60 sequences, 12 genetic clusters (I to XII) and an unstable sequence crowd (xiii) were identified. The robustness of the genetic clusters was confirmed by analyses of rpoB and hemB sequences and ampC PCR-RFLPs. Sequence crowd xiii split into two groups after rpoB analysis. Only three strains formed a cluster with the type strain of E. cloacae, indicating that the minority of isolates identified as E. cloacae truly belong to the species; 13% of strains grouped with other type strains of the genus, suggesting that the phenotypes of these species seem to be more heterogeneous than so far believed. Three clusters represented 70% of strains, but none of them included a type or reference strain. The genetic clustering presented in this study might serve as a framework for future studies dealing with taxonomic, evolutionary, epidemiological, or pathogenetic characteristics of bacteria belonging to the E. cloacae complex.     

Nitric oxide synthase sequences in the marine fish Stenotomus chrysops and the sea urchin Arbacia punctulata, and phylogenetic analysis of nitric oxide synthase calmodulin-binding domains

The phylogenetic distribution and structural diversity of the nitric oxide synthases (NOS) remain important and issues that are little understood. We present sequence information, as well as phylogenetic analysis, for three NOS cDNAs identified in two non-mammalian species: the vertebrate marine teleost fish Stenotomus chrysops (scup) and the invertebrate echinoderm Arbacia punctulata (sea urchin). Partial gene sequences containing the well-conserved calmodulin (CaM)-binding domain were amplified by RT-PCR. Identical 375-bp cDNAs were amplified from scup brain, heart, liver and spleen; this sequence shares 82% nucleic acid and 91% predicted amino acid identity with the corresponding region of human neuronal NOS. A 387-bp cDNA was amplified from sea urchin ovary and testes; this sequence shares 72% nucleic acid identity and 65% deduced amino acid identity with human neuronal NOS. A second cDNA of 381 bp was amplified from sea urchin ovary and it shares 66% nucleic acid and 57% deduced amino acid identity with the first sea urchin sequence. Together with earlier reports of neuronal and inducible NOS sequences in fish, these data indicate that multiple NOS isoforms exist in non-mammalian species. Phylogenetic analysis of these sequences confirms the conserved nature of NOS, particularly of the calmodulin-binding domains.     

Partial sequencing of sodA gene and its application to identification of Streptococcus dysgalactiae subsp. dysgalactiae isolated from farmed fish

AIMS: To investigate the difference between Lancefield group C Streptococcus dysgalactiae (GCSD) strains isolated from diseased fish and animals by sequencing and phylogenetic analysis of the sodA gene. METHODS AND RESULTS: The sodA gene of Strep. dysgalactiae strains isolated from fish and animals were amplified and its nucleotide sequences were determined. Although 100% sequence identity was observed among fish GCSD strains, the determined sequences from animal isolates showed variations against fish isolate sequences. Thus, all fish GCSD strains were clearly separated from the GCSD strains of other origin by using phylogenetic tree analysis. In addition, the original primer set was designed based on the determined sequences for specifically amplify the sodA gene of fish GCSD strains. The primer set yield amplification products from only fish GCSD strains. CONCLUSIONS: By sequencing analysis of the sodA gene, the genetic divergence between Strep. dysgalactiae strains isolated from fish and mammals was demonstrated. Moreover, an original oligonucletide primer set, which could simply detect the genotype of fish GCSD strains was designed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that Strep. dysgalactiae isolated from diseased fish could be distinguished from conventional GCSD strains by the difference in the sequence of the sodA gene.