Sequence-based differentiation of strains in the Mycobacterium avium complex.

The complete 16S-23S rDNA internal transcribed spacer (ITS) was sequenced in 35 reference strains of the Mycobacterium avium complex. Twelve distinct ITS sequences were obtained, each of which defined a "sequevar"; a sequevar consists of the strain or strains which have a particular sequence. ITS sequences were identified which corresponded to M. avium (16 strains, four ITS sequevars) and Mycobacterium intracellulare (12 strains, one ITS sequevars). The other seven M. avium complex strains had ITS sequences which varied greatly from those of M. avium and M. intracellulare and from each other. The 16S-23S rDNA ITS was much more variable than 16S rDNA, which is widely used for genus and species identification. Phylogenetic trees based on the ITS were compatible with those based on 16S rDNA but were more detailed and had longer branches. The results of ITS sequencing were consistent with the results of hybridization with M. avium and M. intracellulare probes (Gen-Probe) for 30 of 31 strains tested. Serologic testing correlated poorly with ITS sequencing. Strains with the same sequence were different serovars, and those of the same serovar had different sequences. Sequencing of the 16S-23S rDNA ITS should be useful for species and strain differentiation for a wide variety of bacteria and should be applicable to studies of epidemiology, diagnosis, virulence, and taxonomy.     

Outer membrane efflux protein (OMEP) is a suitable molecular marker for resolving the phylogeny and taxonomic status of closely related cyanobacteria

Taxonomy of Cyanobacteria, the oldest phototrophic prokaryotes, is problematic for many years due to their simple morphology, high variability and adaptability to diverse ecological niches. After introduction of the polyphasic approach, which is based on the combination of several criteria (molecular sequencing, morphological and ecological), the whole classification system of these organisms is subject to reorganization. The aim of this study was to evaluate whether the outer membrane efflux protein (OMEP) sequences can be used as a molecular marker for resolving the phylogeny and taxonomic status of closely related cyanobacteria. We have performed phylogenetic analyses based on the amino acid sequences of the OMEP and the DNA sequences of the 16S rRNA gene from 86 cyanobacterial species/strains with completely sequenced genomes. Phylogenetic trees based on the OMEP showed that most of the cyanobacterial species/strains belonging to different genera are clustered in separate clades supported by high bootstrap values. Comparing the OMEP trees with the 16S rDNA tree clearly showed that the OMEP is more suitable marker in resolving phylogenetic relationships within Cyanobacteria at generic and species level.     

Analysis of denitrification genes and comparison of nosZ, cnorB and 16S rDNA from culturable denitrifying bacteria in potato cropping systems

Bacterial denitrification in agricultural soils is a major source of nitrous oxide, a potent greenhouse gas. This study examined the culturable bacterial population of denitrifiers in arable field soils in potato (Solanum tuberosum L.) production and denitrification genes (nir, nor and nos) and 16S rDNA in those isolates. Enrichments for culturable denitrifiers yielded 31 diverse isolates that were then analysed for denitrification genes. The nitrous oxide reductase (nosZ) gene was found in all isolates. The majority of isolates ( approximately 90%) contained the cnorB nitric oxide reductase gene, with the remainder containing the qnorB gene. Nitrite reductase genes (nirS and nirK) were amplifiable from most of the isolates, and were segregated between species similar to previously isolated denitrifiers. Isolated strains were preliminarily identified using fatty acid methyl ester analysis and further identified using 16S rDNA sequencing. The majority of isolates (21) were classified as Pseudomonas sp., with smaller groups of isolates being most similar to Bosea spp. (4), Achromobacter spp. (4) and two isolates closely related to Sinorhizobium/Ensifer spp. Phylogenetic trees were compared among nosZ, cnorB and 16S rDNA genes for a subset of Pseudomonas strains. The trees were mostly congruent, but some Pseudomonas sp. isolates grouped differently depending on the gene analysed, indicating potential horizontal gene transfer of denitrification genes. Although Bosea spp. are known denitrifiers, to the best of our knowledge this is the first report of isolation and sequencing of denitrification genes from this bacterial genus.     

Analysis of 16S rRNA gene mutations in a subset of Aeromonas strains and their impact in species delineation

Characterization of 999 Aeromonas strains using a published 16S rDNA RFLP identification method showed that 8.1% of the strains produced unexpected (hereafter called "atypical") restriction patterns, making their identification uncertain. Atypical patterns were due to the presence of nucleotide polymorphisms among the rrn operons of the 16S rRNA gene (so-called microheterogeneities). Double sequencing signals at certain positions revealed the nucleotide composition was responsible for the microheterogeneities. Although the number of microheterogeneities was relatively low (0.06-0.66%), trees inferred from the 16S rRNA gene led either to a misidentification or to an inconclusive result for the majority of these strains. Strains with atypical patterns were, however, correctly identified using the rpoD gene sequences, as belonging to Aeromonas caviae, A. veronii, and A. media. All of them, but particularly the two former species, are associated with human disease. Microheterogeneities in 16S rRNA gene sequence were significantly (P 0.01) more prevalent in clinical than in environmental strains. This work also analyzed the effects of these microheterogeneities on the taxonomic position of the investigated strains. The results suggest the need for recording microheterogeneities in the 16S rRNA gene.     

Population genomics reveals structure at the individual,host‐tree scale and persistence of genotypic variants of the undomesticated yeast Saccharomyces paradoxus in a natural woodland

Genetic diversity in experimental, domesticated and wild populations of the related yeasts, Saccharomyces cerevisiae and Saccharomyces paradoxus, has been well described at the global scale. We investigated the population genomics of a local population on a small spatial scale to address two main questions. First, is there genomic variation in a S. paradoxus population at a spatial scale spanning centimetres (microsites) to tens of metres? Second, does the distribution of genomic variants persist over time? Our sample consisted of 42 S. paradoxus strains from 2014 and 43 strains from 2015 collected from the same 72 microsites around four host trees (Quercus rubra and Quercus alba) within 1 km² in a mixed hardwood forest in southern Ontario. Six additional S. paradoxus strains recovered from adjacent maple and beech trees in 2015 are also included in the sample. Whole‐genome sequencing and genomic SNP analysis revealed five differentiated groups (clades) within the sampled area. The signal of persistence of genotypes in their microsites from 2014 to 2015 was highly significant. Isolates from the same tree tended to be more related than strains from different trees, with limited evidence of dispersal between trees. In growth assays, one genotype had a significantly longer lag phase than the other strains. Our results indicate that different clades coexist at fine spatial scale and that population structure persists over at least a one‐year interval in these wild yeasts, suggesting the efficacy of yearly sampling to follow longer term genetic dynamics in future studies.     

Description of Gibbsiella quercinecans gen. nov., sp. nov., associated with Acute Oak Decline

Gram-negative, facultatively anaerobic bacterial strains were consistently isolated from oak trees displaying symptoms of extensive stem bleeding. In Britain, this disorder is called Acute Oak Decline (AOD). A similar condition has been noted on species of Mediterranean oak in Spain. The identity of bacterial isolates from symptomatic trees in both countries was investigated using molecular techniques and phenotypic assays. 16S rRNA gene sequencing indicated that the strains were most closely related to the genera Serratia, Kluyvera, Klebsiella and Raoultella (all>97%). Phylogenetic analysis revealed that the strains formed a distinct lineage within the family Enterobacteriaceae, which was confirmed by both gyrB- and rpoB-gene sequencing. DNA-DNA hybridization confirmed that the strains belonged to a single taxon which could also be differentiated phenotypically from its closest phylogenetic neighbours. The phylogenetic and phenotypic data both demonstrated that the strains isolated from oak represented a novel genus and species within the family Enterobacteriaceae for which the name Gibbsiella quercinecans gen. nov., sp. nov. (type strain=FRB 97(T)=LMG 25500(T)=NCPPB 4470(T)) is proposed.     

Origins of Bradyrhizobium nodule symbionts from two legume trees in the Philippines

Aim Geographic affinities were analysed for nodule bacteria (Bradyrhizobium sp. Jordan) associated with two legume trees indigenous to the Philippines: Pterocarpus indicus (Papilionoideae) and Wallaceodendron celebicum (Mimosoideae). Location Nodule bacteria from Luzon, the Philippines, were compared with reference strains from Central America, eastern North America, Japan, Korea, China and Australia. Methods Two PCR assays targetting length polymorphisms in the rRNA region were carried out on 96 Philippine bacterial isolates. A 496‐bp portion of the 23S rRNA gene was sequenced in 14 representative isolates. Eight strains were analysed in greater depth by sequencing portions of four other genes (16S rRNA [1410 bp], dnaK [603 bp], nifD [822 bp], recA [512 bp]), and phylogenetic trees were constructed by maximum parsimony, neighbour joining and maximum likelihood methods. Results Most of the Philippine Bradyrhizobium strains showed greater similarity to reference strains from Central America than to strains from other source regions included in the analysis. However, phylogenetic trees for the five genes had significantly conflicting topologies, suggesting that lateral gene transfer events had altered genealogical relationships at different loci. In particular, two Philippine strains resembled Bradyrhizobium strains from Central America or China for 16S rRNA, dnaK and recA sequences, but had nifD sequences that clustered with Australian strains (with bootstrap support values of 90–96%). Main conclusions The Philippines have been colonized by Bradyrhizobium strains from multiple source regions. Subsequent lateral gene transfer has resulted in the evolution of Bradyrhizobium strains that combine DNA segments of different geographic origin.     

Isolation of Enterobacter cowanii from Eucalyptus showing symptoms of bacterial blight and dieback in Uruguay

Aims:  This study was performed to identify bacterial strains isolated simultaneously with Pantoea species from Eucalyptus trees showing symptoms of bacterial blight and dieback in Uruguay.
Methods and Results:  Several molecular techniques including 16S rRNA and rpoB gene sequencing and DNA–DNA hybridization were used to characterize the Gram-negative, facultatively anaerobic, slime-producing bacterial strains isolated along with Pantoea species from Eucalyptus . Hypersensitivity reactions (HR) and pathogenicity tests were performed on tobacco and Eucalyptus seedlings, respectively. The isolates clustered closely with the type strain of Enterobacter cowanii in both phylogenetic trees constructed. The DNA–DNA similarity between the isolates and the type strain of Ent. cowanii ranged from 88% to 92%. A positive HR was observed on the tobacco seedlings, but no disease symptoms were visible on the inoculated Eucalyptus seedlings.
Conclusions:  Enterobacter cowanii was isolated from trees with symptoms of bacterial blight although strains of this bacterial species do not appear to be the causal agent of the disease.
Significance and Impact of the Study:  This study provides the first report of Ent. cowanii isolated from Eucalyptus . Its presence in Eucalyptus tissue suggests that it is an endophyte in trees showing symptoms of blight.     

Elucidation of the taxonomic status of industrial strains of thermophilic lactic acid bacteria by sequencing of 16S rRNA genes

Phenotypic characteristics and results of PCR tests for the presence of species-specific genes indicate that a number of strains of thermophilic lactic acid bacteria previously considered as belonging to Streptococcus thermophilus are actually closely related to enterococci. In the present study, partial (over 500 nucleotides) sequencing of 16S rRNA genes from 12 strains of thermophilic lactic acid bacteria used as starters for manufacturing sour milk products on the territory of the Commonwealth of Independent States (CIS) has been performed. According to the results of the sequencing, seven of the strains have been classified with Enterococcus durans. The earlier classification (based on PCR tests) of two of the strains as S. thermophilus and three of the strains as E. faecium has been confirmed. The data obtained demonstrate that the enterococci E. durans and E. faecium are widely used as thermophilic starters for manufacturing sour milk products on the territory of the CIS.     

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。     

Phylogenetic relationships and coaggregation ability of freshwater biofilm bacteria

Nineteen numerically dominant heterotrophic bacteria from a freshwater biofilm were identified by 16S ribosomal DNA gene sequencing, and their coaggregation partnerships were determined. Phylogenetic trees showed that both distantly related and closely related strains coaggregated at intergeneric, intrageneric, and intraspecies levels. One strain, Blastomonas natatoria 2.1, coaggregated with all 18 other strains and may function as a bridging organism in biofilm development.     

16S～23S RDNA间区在链球菌和流感嗜血杆菌分类中的应用

利用16S~23S rDNA间区（intergenic spacer regions,ISR）在不同细菌中拷贝数、碱基排列、序列长度及所含tRNA基因种类和数目的差异,对15株链球菌和流感嗜血杆菌进行属、种、型和株系的分类鉴定。在16S rDNA的3′端和23S rDNA的5′端的保守区中合成引物,PCR扩增16S~23S rDNA ISR序列,对多态片段切胶纯化直接测序。在GenBank上查找对应细菌的ISR序列。用DNAMAN软件进行系统进化分析。链球菌属为单拷贝16S~23Sr RNA ISR、有一个tRNAAla基因编码区、分子大小在269~446bp之间,序列分成4个保守区和4个可变区,可变区碱基排列方式和数目的不同是种分类的依据。7株链球菌的同源率在78%~88%。同种异株的差异反映在碱基的插入和缺失上。流感嗜血杆菌各生物型均为2个拷贝的ISR,小片段为514~519bp,编码1个tRNAGlu基因,有3个狭窄可变区。大片段富含A T碱基,在I、II和IV型中分别是868、848和856bp,编码一个tRNAIle基因和一个tRNAAla基因。不同生物型小分子ISR与标准菌株比较,同源性在97.3%~99.6 %之间。 ISR作为细菌分类的目的基因具有属、种、型和株特异性与灵敏性。简单的基因分离分析技术为认识病原微生物提供了更多的机会。 Abstract:To facilitate species level identification of bacteria without the requirement of presumptive identification,the paper describes a rapid identification method of bacteria by amplification and direct sequencing 16S~23S rDNA intergenic spacer regions (ISR) of the pathogens which cause the upper respiratory tract infective disease by Streptococcus and Haemophilus.Three pairs of primer targeting conserved sequences flanking the 3′ end of 16S and the 5′end of 23S rRNA were used to amplify 16S~23S rRNA ISR of 7 streptococcus strains and 8 Haemophilus strains.The PCR products were separated by 1% agarose gel electrophoresis and the polymorphisms fragments were purified with the Wizard PCR Min-Prep Kit (Promega) and Protocol-SK131(Sangon).The nucleotide sequences of ISR inserts were determined by using the XEQTM DTCS Kit——Terminator Cycle Sequencing and a CEQTM 2000XL DNA Analysis system (Backman Coulter) automatic DAN sequencer.Then those sequences were compared with known seqnences on the GenBank.The alignment of nucleotide sequence,evolutionary distances and phylogenetic tress were analyzed by software DANMAN version 4.0.The PCR products were showed polymorphism patterns with agarose gel.One band was contained in streptococcus genus.The significant variation was found among the spacer sequences of different species in Streptococcus with the lengths of the spacer varying from 269 to 446bp.All the ISR of the streptococcal species had a tRNA Ala gene in the spacer and the sequence identities varied from 78 to 88% within genera.It was found that some spacer sequence blocks were highly conserved between operons of a genome,whereas the presence of others was variable,three regions showed significant spatial variation.Most of the differences between the sequences came from several bases insertions/deletions and substitutions.There are two major bands in the Haemophilus biotypes(515 and 884bp),the small ISR amplicon contained one tDNA coding for tRNAGlu.In contrast to the large one contained two tRNA genes coding for tRANAla and tRNAIle.Two regions of repeating motifs with only A or T were present in higher copy numbers between tRANAla and tRNAIle.The phylogenetic trees varied from 97.5 to 98.8%.The PCR and direct sequencing of 16S~23S rRAN ISR were successful in the pathogen species identification.     

Genetic diversity of bacterial strains isolated from soils, contaminated with polycyclic aromatic hydrocarbons, by 16S rRNA gene sequencing and amplified fragment length polymorphism fingerprinting

In order to study microbial diversity in a polycyclic aromatic hydrocarbon-impacted soil, 14 bacterial strains were analyzed by 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis. Bacterial strains isolated from two different hydrocarbon-polluted sites were identified to the species level by 16S rRNA full-gene sequencing using MicroSeq 16S rRNA gene sequencing. Their genome was subsequently analyzed by high-resolution genotyping with AFLP analysis, in order to monitor species variability and to differentiate closely related strains. Cluster analysis based on AFLP fingerprinting showed intra-specific polymorphism, even among strains with 100% 16S rRNA gene sequence identity. The results show that AFLP is a powerful, highly reproducible and discriminatory tool for revealing genetic relationships in bacterial populations. The ability to differentiate and track related closely microbes is fundamental for studying structure and dynamics of microbial communities in contaminated ecosystems.     

Variations in the 16S-23S rRNA internal transcribed spacer of fibrolytic Butyrivibrio isolates from the reindeer rumen

Strains of Butyrivibrio are principal cellulytic bacteria in the rumen of the High Arctic Svalbard reindeer ( Rangifer tarandus platyrhynchus ). According to phylogenetic analysis based on 16S rRNA gene sequencing, Butyrivibrio can be divided into three subgroups within the Clostridia class of the phylum Firmicutes, but the current phenotypic and genotypic differentiation within the family Lachnospiraceae is insufficient. This current study describes the sequence diversity of the 16S-23S rRNA intergenic transcribed spacer (ITS) region of Butyrivibrio isolates from reindeer. A total of 17 different ITS sequences with sizes between 449 and 784 nt were obtained. Genes encoding tRNA(Ile) and tRNA(Ala) were identified in four of the sequences. Phylogenetic neighbor-joining trees were constructed based on the ITS sequence and compared with a phylogenetic neighbor-joining tree based on 16S rRNA gene sequences previously obtained for the same isolates. These comparisons indicated a better differentiation between strains in the ITS sequence than the 16S rRNA gene based tree. Through this study, a better means for identifying and tracking fibrolytic and potentially probiotic Butyrivibrio strains in reindeer and other ruminants has been provided.     

Phylogenetic Relationships and Coaggregation Ability of Freshwater Biofilm Bacteria

Nineteen numerically dominant heterotrophic bacteria from a freshwater biofilm were identified by 16S ribosomal DNA gene sequencing, and their coaggregation partnerships were determined. Phylogenetic trees showed that both distantly related and closely related strains coaggregated at intergeneric, intrageneric, and intraspecies levels. One strain, Blastomonas natatoria 2.1, coaggregated with all 18 other strains and may function as a bridging organism in biofilm development.     

Near-full length sequencing of 16S rDNA and RFLP indicates that Rhizobium etli is the dominant species nodulating Egyptian winter Berseem clover (Trifolium alexandrinum L.)

Egyptian winter Berseem clover (EWBC) is one of the main important forage legume crops in Egypt that is used for animal feeding in winter and it occupies about 2.5 million feddans (Feddan = 4200 m²) in winter agricultural rotation systems. Forty-eight rhizobial isolates that nodulated this legume host from different geographical regions within Egypt were isolated. RFLP analyses of 16S rDNA (1.5 kb) and whole ribosomal DNA (5 kb), the sequencing of 16S rDNA, and the sequencing of nodC, nifH and house keeping genes were used to identify these isolates. The RFLP analysis of 16S rDNA (1.5 kb) among 15 representative strains with three enzymes generated two genotypes. The largest genotype was similar to Rhizobium etli CFN42T (93.33%) except for strain 902 that failed to re-nodulate EWBC. RFLP analysis of complete ribosomal DNA (5 kb) produced five genotypes. The majority of tested strains shared the genotype with R. etli CFN42T (53.33%). Only one strain (1002) shared the genotype with Rhizobium leguminosarum sv. trifolii 3023. The other four strains were comprised of two unique genotypes. Phylogenetic analysis of 16S rDNA sequences revealed that seven representative strains could be divided into two genetic clusters sharing the ancestral clad with R. etli CFN42T. A phylogenetic tree based on nodC gene sequence confirmed that all the examined strains shared the genetic lineage with R. leguminosarum sv. trifolii WSM1325. The phylogenetic trees of house keeping genes are supported strongly the identification of majority of strains as a novel symbiovar of R. etli with new lineages.     

Horizontal transfer of segments of the 16S rRNA genes between species of the Streptococcus anginosus group

The nature in variation of the 16S rRNA gene of members of the Streptococcus anginosus group was investigated by hybridization and DNA sequencing. A collection of 708 strains was analyzed by reverse line blot hybridization. This revealed the presence of distinct reaction patterns representing 11 different hybridization groups. The 16S rRNA genes of two strains of each hybridization group were sequenced to near-completion, and the sequence data confirmed the reverse line blot hybridization results. Closer inspection of the sequences revealed mosaic-like structures, strongly suggesting horizontal transfer of segments of the 16S rRNA gene between different species belonging to the Streptococcus anginosus group. Southern blot hybridization further showed that within a single strain all copies of the 16S rRNA gene had the same composition, indicating that the apparent mosaic structures were not PCR-induced artifacts. These findings indicate that the highly conserved rRNA genes are also subject to recombination and that these events may be fixed in the population. Such recombination may lead to the construction of incorrect phylogenetic trees based on the 16S rRNA genes.     

Diversity and relationships of bradyrhizobia from legumes native to eastern North America

DNA sequencing and polymerase chain reaction (PCR) assays with lineage-specific primers were used to analyze the diversity of 276 isolates of Bradyrhizobium sp. nodule bacteria associated with 13 native legumes species in the northeastern United States, representing eight genera in six legume tribes. A PCR screen with two primer pairs in the rRNA region indicated that seven of the legume species were exclusively associated with strains having markers resembling Bradyrhizobium elkanii, while the remaining six host species harbored strains related to both B. elkanii and Bradyrhizobium japonicum. Sequence analysis of 22 isolates for portions of 16S rRNA and 23S rRNA yielded congruent phylogenetic trees and showed that isolates from different legume genera often shared similar or identical sequences. However, trees inferred from portions of two other genes (alpha-ketoglutarate dioxygenase gene (tfdA), the alpha-subunit of nitrogenase (nifD)) differed significantly from the rRNA phylogeny. Thus, for Bradyrhizobium populations in this region, lateral gene transfer events appear to have altered genealogical relationships of different portions of the genome. These results extend the number of likely cases of gene transfer between divergent taxa of Bradyrhizobium (from members of the B. elkanii lineage to the B. japonicum group) and suggest that transfers have also occurred among separate subgroups of the B. elkanii lineage.     

Cytidine deamination assay to differentiate Staphylococcus aureus from other staphylococci

AIMS: Adoption of the property of cytidine (cytosine-beta-d-riboside) deamination in staphylococci to distinguish Staphylococcus aureus from other staphylococci. METHODS AND RESULTS: A total of 560 staphylococcal strains were examined. The test demonstrated a sensitivity of 97.1% and a specificity of 98.8%. Of the 249 S. aureus strains (115 oxacillin-resistant) 58 strains were coagulase-negative S. aureus and another 16 strains were clumping factor-negative S. aureus. The 74 deficient S. aureus strains were identified by 16S rRNA gene sequencing and further investigated by spa typing and 13 spa types were found. CONCLUSIONS: The cytidine deaminase test (CDT) is useful especially for distinguishing coagulase- and clumping factor-negative S. aureus from other staphylococci and the results correlated well with 16S rRNA sequencing and the polymerase chain reaction (PCR) amplification of the nuc gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Cytidine deamination assay differentiates S. aureus from other staphylococci. This method is fast (6 h) and reliable in distinguishing between non-S. aureus and the defective (coagulase-negative, clumping factor-negative) S. aureus isolates which could have major consequences for therapy.     

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.