16S-23S rRNA Intergenic Spacer Research Articles

Molecular Detection of Bartonella Sp. in Wild Ruminants and Analysis of its Genetic Diversity on the Basis of 16S–23S Rrna Intergenic Spacer (ITS)

Abstract The aim of this study was to describe the state of infection of roe deer (Capreolus capreolus) and red deer (Cervus elaphus) by Bartonella sp. in North-Western Poland through PCR detection of Bartonella 16S-23S rRNA ITS region in isolates of animal tissues, and also to describe the genetic diversity of detected Bartonella species based on molecular analysis of ITS. The multiple alignment analysis of ITS sequences was carried out, and homology matrices and phylogenetic trees were constructed. The DNA of Bartonella sp. was detected in tissues of 45.6% (36/79) C. capreolus and of 50% (15/30) C. elaphus. Products of two different sizes were detected: 317 bp, characteristic for B. schoenbuchensis, and 198 bp, characteristic for B. bovis. The obtained results suggest that roe and red deer are potential reservoirs of Bartonella sp. Most of the analysed ITS sequences was not specific for one host species. In constructed phylogenetic trees, sequences obtained from roe and red deer clustered together. These results suggest a lack of host specificity of most detected B. schoenbuchensis and B. bovis intraspecies strains

Characterization of rhizobia from legumes of agronomic interest grown in semi‐arid areas of Central Spain relates genetic differences to soil properties

A study of symbiotic bacteria from traditional agricultural legumes from Central Spain was performed to create a collection of rhizobia from soils differing in physicochemical, analytical and/or agroecological properties which could be well-adapted to the environmental conditions of this region, and be used for sustainable agricultural practices. Thirty-six isolates were obtained from root-nodules of fifteen legume species (including Cicer arietinum, Lathyrus sativus, Lens culinaris, Lupinus spp., Medicago sativa, Phaseolus vulgaris, Pisum sativum, and Vicia spp.) from three agriculture areas with soils of different pHs and from a forest area with undisturbed soils. Phenotypical characterization revealed uniformity across the thirty-six isolates, with important exceptions in terms of environmental tolerance (three isolates survived at high temperatures, three at high salinity and three at acid pH). The molecular analysis of 16S rRNA gene showed a close relationship of twenty-nine isolates to Rhizobium leguminosarum, one to Rhizobium gallicum, one to Mesorhizobium ciceri, two to Sinorhizobium (Ensifer) meliloti and three to Bradyrhizobium canariense. The sequence analysis of a symbiosis-specific gene, nod C, showed a correlation with the plant host and grouped twenty-six isolates with Rhizobium leguminosarum bv. viciae, establishing the diversity in relation to legume-host. The 16S-23S rRNA intergenic spacer (IGS) region allowed for intraspecific differentiation, so that strains with equal 16S rRNA were grouped by means of their soil origin. These results indicated that phenotypical and genetically related strains may be widely distributed in this region and that soil abiotic characteristics could have a substantial bearing on the selection of the strains living in each environment.

Prevalence and different characteristics of two serotypes of Streptococcus parauberis isolated from the farmed olive flounder, Paralichthys olivaceus (Temminck and Schlegel), in Korea

The prevalence of two serotypes of Streptococcus parauberis isolated from the olive flounder, Paralichthys olivaceus, was evaluated in a total of 29 isolates between 2003 and 2010 in Korea. Streptococcus parauberis isolates were divided into two serologically distinct types (serotype 1 and serotype 2), except for one strain (S1091), using an agglutination assay with rabbit antiserum, and serotype 1 was identified as the dominant type (24 of 29 isolates) in this study. To identify the characteristics of the two serotypes of S.parauberis, we conducted a biochemical test using the API 20 Strep kit, a transmission electron microscopy (TEM) assay, sequence analysis of 16S-23S rRNA intergenic spacer region (ISR) and a pathogenicity test. In TEM, both serotypes possessed polysaccharide capsule layers around the cell surface when bacterial cells were treated with a homologous serotype of rabbit antiserum. However, we were unable to discriminate serotype-specific biochemical characteristics and genetic characteristics of 16S-23S rRNA ISR between the two serotypes. In the pathogenicity test, the serotype 1 strains induced significantly higher mortality than the serotype 2 strains in olive flounder when experimentally inoculated via the intraperitoneal route.

Leuconostoc mesenteroides subsp. suionicum subsp. nov.

Strains LMG 8159 and LMG 11499 were reclassified by a polyphasic approach, including 16S rRNA gene sequence analysis, 16S-23S rRNA intergenic spacer (IGS) sequence analysis, (GTG)(5)-PCR fingerprinting, RAPD fingerprinting, fatty acid methyl ester analysis and an analysis of phenotypic features using API 50 CH. The two strains were closely related to the type strains of the three defined subspecies of Leuconostoc mesenteroides, showing 99.7-99.9% 16S rRNA gene sequence similarity, 99.2% 16S-23S rRNA gene intergenic spacer sequence similarity, 97.1-97.4% pheS gene sequence similarity and 98.0-98.2% rpoA gene sequence similarity. Low atpA gene sequence similarity (91.4-91.7%), (GTG)(5)-PCR fingerprinting, RAPD fingerprinting, fatty acid compositions and phenotypic features allowed us to differentiate strains LMG 8159 and LMG 11499 from all established subspecies within L. mesenteroides. Based upon the data obtained in the present and previous studies, a novel subspecies is proposed within the species L. mesenteroides, Leuconostoc mesenteroides subsp. suionicum subsp. nov., with the type strain LMG 8159(T) (=ATCC 9135(T) =DSM 20241(T) =NCIMB 6992(T)).

Identification of Escherichia coli through analysis of 16S rRNA and 16S-23S rRNA internal transcribed spacer region sequences.

A bacterial strain, designated BzDS03 was isolated from water sample, collected from Dal Lake Srinagar. The strain was characterized by using 16S ribosomal RNA gene and 16S-23S rRNA internal transcribed spacer region sequences. Phylogenetic analysis showed that 16S rRNA sequence of the isolate formed a monophyletic clade with genera Escherichia. The closest phylogenetic relative was Escherichia coli with 99% 16S rRNA gene sequence similarity. The result of Ribosomal database project's classifier tool revealed that the strain BzDS03 belongs to genera Escherichia.16S rRNA sequence of isolate was deposited in GenBank with accession number FJ961336. Further analysis of 16S-23S rRNA sequence of isolate confirms that the identified strain BzDS03 be assigned as the type strain of Escherichia coli with 98% 16S-23S rRNA sequence similarity. The GenBank accession number allotted for 16S-23S rRNA intergenic spacer sequence of isolate is FJ961337.

Detection of Laribacter hongkongensis using species-specific duplex PCR assays targeting the 16S rRNA gene and the 16S-23S rRNA intergenic spacer region (ISR)

For the rapid detection of Laribacter hongkongensis, which is associated with human community-acquired gastroenteritis and traveller's diarrhoea, we developed a duplex species-specific PCR assay. Full-length of the 16S-23S rRNA intergenic spacer region (ISR) sequences of 52 L. hongkongensis isolates were obtained by PCR-based sequencing. Two species-specific primer pairs targeting 16S rRNA gene and ISR were designed for duplex PCR detection of L. hongkongensis. The L. hongkongensis species-specific duplex PCR assay showed 100% specificity, and the minimum detectable level was 2.1 × 10(-2) ng μl(-1) genomic DNA which corresponds to 5000 CFU ml(-1). The high specificity and sensitivity of the assay make it suitable for rapid detection of L. hongkongensis. This species-specific duplex PCR method provides a rapid, simple, and reliable alternative to conventional methods to identify L. hongkongensis and may have applications in both clinical and environmental microbiology.

A multiplex real-time PCR assay targeting virulence and resistance genes in Salmonella entericaserotype Typhimurium

BackgroundTyphimurium is the main serotype of Salmonella enterica subsp. enterica implicated in food-borne diseases worldwide. This study aimed to detect the prevalence of ten markers combined in a macro-array based on multiplex real-time PCR. We targeted characteristic determinants located on pathogenicity islands (SPI-2 to -5, virulence plasmid pSLT and Salmonella genomic island 1 (SGI1)) as well as a specific 16S-23S rRNA intergenic spacer sequence of definitive type 104 (DT104). To investigate antimicrobial resistance, the study also targeted the presence of genes involved in sulfonamide (sul1) and beta-lactam (blaTEM) resistance. Finally, the intI1 determinant encoding integrase from class 1 integron was also investigated.ResultsA total of 538 unrelated S. Typhimurium strains isolated between 1999 and 2009 from various sources, including food animals, food products, human and environmental samples were studied. Based on the combined presence or absence of these markers, we distinguished 34 different genotypes, including three major genotypes encountered in 75% of the studied strains, Although SPI determinants were almost always detected, SGI1, intI1, sul1 and blaTEM determinants were found 47%, 52%, 54% and 12% of the time respectively, varying according to isolation source. Low-marker patterns were most often detected in poultry sources whereas full-marker patterns were observed in pig, cattle and human sources.ConclusionThe GeneDisc® assay developed in this study madeit easier to explore variability within serotype Typhimurium by analyzing ten relevant gene determinants in a large collection of strains. This real-time multiplex method constitutes a valuable tool for strains characterization on epidemiological purposes.

Molecular Characterization of Mycoplasma gallisepticum Isolates from Jordan

Two groups of Mycoplasma gallisepticum (MG) isolates (n = 24) from Jordan were analyzed by molecular methods and compared with other Middle Eastern isolates, related international isolates, and reference strains. The first group (n = 19) was isolated from July 2004 to January 2005 (isolation period A), and the newer group (n = 5) from June 2007 to April 2008 (isolation period B). The groups of isolates are from chicken flocks from northern Jordan, but are not from the same farms. None of the flocks were vaccinated for MG. Random amplified polymorphic DNA analysis, targeted sequencing of the partial MG cytadhesin 2 (mgc2), and the MG 16S-23S rRNA intergenic spacer region (IGSR) divided the Jordanian isolates into two groups. All of the 19 isolates from time period A, in addition to two isolates from time period B, were indistinguishable from the F strain. Three of five isolates from time period B were characterized as wild types and were indistinguishable from each other. The wild-type field strain was readily distinguished from the F strain. It was 91% and 96.4% similar to the F strain based on Clustal-W alignments of sequences of mgc2 and IGSR, respectively. Sequence similarity of mgc2 gene of the Jordan wild-type strain to isolates from Israel and Egypt ranged from 96.5% to 100%, whereas for IGSR it was 99.4%-100%. We theorize that the F-strain live MG vaccine, commonly used in Jordan prior to 2007, was transmitted to nonvaccinated poultry in the region and was a predominant genotype during time period A.

Phylogeny of bradyrhizobia from Chinese cowpea miscellany inferred from 16S rRNA,atpD,glnII, and 16S–23S intergenic spacer sequences

The cowpea (Vigna unguiculata L.), peanut (Arachis hypogaea L.), and mung bean (Vigna radiata L.) belong to a group of plants known as the "cowpea miscellany" plants, which are widely cultivated throughout the tropic and subtropical zones of Africa and Asia. However, the phylogeny of the rhizobial strains that nodulate these plants is poorly understood. Previous studies have isolated a diversity of rhizobial strains from cowpea miscellany hosts and have suggested that, phylogenetically, they are from different species. In this work, the phylogeny of 42 slow-growing rhizobial strains, isolated from root nodules of cowpea, peanut, and mung bean from different geographical regions of China, was investigated using sequences from the 16S rRNA, atpD and glnII genes, and the 16S-23S rRNA intergenic spacer. The indigenous rhizobial strains from the cowpea miscellany could all be placed in the genus Bradyrhizobium , and Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense were the main species. Phylogenies derived from housekeeping genes were consistent with phylogenies generated from the ribosomal gene. Mung bean rhizobia clustered only into B. liaoningense and B. yuanmingense and were phylogenetically less diverse than cowpea and peanut rhizobia. Geographical origin was significantly reflected in the phylogeny of mung bean rhizobia. Most cowpea rhizobia were more closely related to the 3 major groups B. liaoningense, B. yuanmingense, and Bradyrhizobium elkanii than to the minor groups Bradyrhizobium japonicum or Bradyrhizobium canariense . However, most peanut rhizobia were more closely related to the 2 major groups B. liaoningense and B. yuanmingense than to the minor group B. elkanii.

Phytoplasma classification: taxonomy based on 16S ribosomal gene, is it enough?

The discovery in 1967, of a new group of plant pathogens related to bacteria led to the finding of pleomorphic, wall-less prokaryotes in the phloem of many plant species affected by yellows-type diseases. Following the application of molecular technologies the phylogeny of these prokaryotes was resolved and led to the new trivial name of “phytoplasma” and to the designation of a new taxon named ‘Candidatus phytoplasma’. The first comprehensive phytoplasma classification scheme was based on RFLP analysis of PCR-amplified 16S rDNA, providing a reliable means for the differentiation of a broad array of phytoplasmas and has become the most comprehensive and widely accepted phytoplasma classification system. This approach using RFLP analyses of PCR amplified 16S rDNA provides a simple, reliable and rapid mean for differentiation and identification of known phytoplasmas. A consensus for naming novel phytoplasmas was recommended by the IRPCM Phytoplasma/Spiroplasma Working Team-Phytoplasma Taxonomy Group that a ‘Candidatus Phytoplasma’ species description should refer to a single, unique 16S rRNA gene sequence that has <97.5% similarity to that of any previously described ‘Ca. Phytoplasma’ species. Because of the highly conserved nature of the 16S rDNA, many biologically or ecologically distinct phytoplasma strains, which may warrant designation of a new taxon may fail to meet the requirement. In this case, additional unique biological properties such as antibody specificity, host range and vector transmission specificity as well as other molecular criteria (gene) need to be included for speciation. However, because of the highly conserved nature of the 16S rDNA and of the not uncommon presence of 16S rDNA interoperon sequence heterogeneity, the classification based on ‘Candidatus’ or on 16S ribosomal group does not always provide the molecular distinction necessary for phytoplasma strain characterization. Moreover, some additional tools for phylogenetic analyses and finer strain differentiation of phytoplasmas such as rp, secY, tuf, groEL genes, and the 16S-23S rRNA intergenic spacer region sequences have been used as supplementary tools selecting those providing the most useful and reliable taxonomic information in combination with 16SrDNA.

Genospecies Classification ofAcinetobacter calcoaceticus-Acinetobacter baumanniiComplex by Restriction Fragment Length Polymorphism

Acinetobacter calcoaceticus-Acinetobacter baumannii (A. calcoaceticus-A. baumannii) complex, which includes A. calcoaceticus (genospecies 1), A. baumannii (genospecies 2), Acinetobacter genospecies 3 and 13, has been identified as A. baumannii by automated bacteria identification system. The purpose of this study is to develop rapid genospecies classification of A. calcoaceticus-A. baumannii complex by molecular techniques. Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) were determined for 4 reference strains and 80 isolates of A. calcoaceticus-A. baumannii complex from clinical sources. Four and eleven RAPD patterns were observed among the reference strains and the isolates, respectively. RAPD might be useful for genomic typing but not for genospecies classification of Acinetobacter spp. RFLP of 16S-23S rRNA intergenic spacer gene with three selected restriction enzymes (ApaLI, SwaI, and SalI) showed only four RFLP patterns in the reference and the isolates. Of 80 isolates, 10 of A. calcoaceticus (12.5%), 50 of A. baumannii (62.5%), 11 of A. genospecies 3 (13.75%), and 9 of A. genospecies 13 (11.25%) were classified by RFLP. This result suggests that RFLP of 16S-23S rRNA intergenic spacer gene of A. calcoaceticus-A. baumannii complex might be useful for genospecies classification.

Genetic Diversity ofBorrelia burgdorferiSensu Lato Isolates from Northeastern China

Thirty-two strains of Borrelia burgdorferi sensu lato were isolated from Ixodes persulcatus ticks collected from northeastern China from May to June in 2004 and 2005. Restriction fragment length polymorphism (RFLP) analysis and sequence analysis of 5S-23S rRNA intergenic spacer revealed that 29 (90.6%) belonged to Borrelia garinii, demonstrating B, C, and a unique pattern. The remaining three isolates (9.4%) were Borrelia afzelii with pattern D. The phylogenetic analysis based on 5S-23S rRNA intergenic spacer showed that B. garinii and B. afzelii genospecies clustered into two separate lineages. B. garinii strains were classified into three different branches: All the strains with RFLP pattern C were in the same branch, strain VH10 with a unique RFLP pattern clustered with strains VH9 and MDH2 with pattern B, and the rest of the strains with pattern B constitute another branch. These findings demonstrate the genetic diversity of B. burgdorferi sensu lato isolates from northeastern China.

Bradyrhizobium lablabi sp. nov., isolated from effective nodules of Lablab purpureus and Arachis hypogaea

Five strains isolated from root nodules of Lablab purpureus and Arachis hypogaea grown in the Anhui and Sichuan provinces of China were classified as members of the genus Bradyrhizobium. These strains had identical 16S rRNA gene sequences which shared 99.48 %, 99.48 % and 99.22 % similarity with the most closely related strains of Bradyrhizobium jicamae PAC68(T), Bradyrhizobium pachyrhizi PAC48(T) and Bradyrhizobium elkanii USDA 76(T), respectively. A study using a polyphasic approach, including 16S rRNA gene RFLP, IGS-RFLP, BOX-PCR, comparative sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) and the recA, atpD and glnII genes, DNA-DNA hybridization and phenotypic tests, showed that the five strains clustered into a coherent group that differentiated them from all recognized species of the genus Bradyrhizobium. Sequencing of nifH and nodC genes and cross-nodulation tests showed that the representative strains CCBAU 23086(T), CCBAU 23160 and CCBAU 61434, isolated from different plants, had identical nifH and nodC gene sequences and were all able to nodulate Lablab purpureus, Arachis hypogaea and Vigna unguiculata. Based upon these results, the name Bradyrhizobium lablabi sp. nov. is proposed for this novel species and strain CCBAU 23086(T) ( = LMG 25572(T) = HAMBI 3052(T)) is designated as the type strain. The DNA G+C mol% is 60.14 (T(m)).

High-Prevalence Borrelia miyamotoi Infection Among Wild Turkeys (Meleagris gallopavo) in Tennessee

During spring and fall 2009, 60 wild turkeys (Meleagris gallopavo) harvested by Tennessee hunters were surveyed for Borrelia spp. by sampling their blood, tissue, and attached ticks. In both seasons, 70% of turkeys were infested with juvenile Amblyomma americanum; one spring turkey hosted an adult female Ixodes brunneus. Polymerase chain reaction assays followed by DNA sequencing indicated that 58% of the turkeys were positive for the spirochete Borrelia miyamotoi, with tissue testing positive more frequently than blood (P = 0.015). Sequencing of the 16S-23S rRNA intergenic spacer indicated > or = 99% similarity to previously published sequences of the North American strain of this spirochete. Positive turkeys were present in both seasons and from all seven middle Tennessee counties sampled. No ticks from the turkeys tested positive for any Borrelia spp. This is the first report of B. miyamotoi in birds; the transmission pathways and epidemiological significance of this high-prevalence spirochetal infection remain uncertain.

Detection of five common cell culture contaminant Molllcutes using rolling circle amplification

Objective To develop a novel padlock probe and rolling circle amplification(RCA) to detect 5 common cell culture contaminant Mollicute( Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma hyorhinis, Mycomplasma orale, and Acholeplasma laidlawii ). Methods Universal primers ( SPS1, SPA2 and SPS2, SPA1) were used to amplify the Mollicute 16S-23S rRNA intergenic spacer region.Amplicon was ligate Mollicute specific padlock probe. Probe amplified and monitored using a Corbett RotorGeneTM 6000 machine. Results Five reference Mollicute strains were correctly detected by RCA method.There was no cross-reaction. RCA method can sensitive detect 10 copies templates and show strong positive signal. Sixty-two cell culture specimens were detected. Thirty-seven samples were contained single specie Mollicute and 14 samples contained two Mollicutes. Eleven samples did not contained Mollicute. RCA detection results were concordant with previously species-specific PCR. Conclusion RCA can rapid, sensitive and specific detect contamination Mollicute. Key words: Rolling circle amplification; Padlock probe

Mechanisms behind variation in the Clostridium difficile 16S–23S rRNA intergenic spacer region

Clostridium difficile infection is an increasing problem in hospitals worldwide, mainly due to the recent emergence of a hypervirulent C. difficile strain. C. difficile PCR ribotyping, based on size variation of the 16S–23S rRNA intergenic spacer region (16S–23S ISR), is widely used in Europe for molecular epidemiological investigation. The mechanism underlying the 16S–23S ISR size variations in the genome of C. difficile is currently not completely understood. To elucidate this mechanism, isolates of six different PCR ribotypes were analysed by cloning and sequencing the 16S–23S ISR. A direct repeat, IB, of 9 bp was detected up to five times in the 16S–23S ISR in all 47 clones investigated. Thirty-five clones displayed differences either by ribotype or by nucleotide sequence. The sequences of the 16S–23S ISR of C. difficile showed a uniformly organized structure, composed of a tRNAAla gene and spacers of 33 and 53 bp separated by the 9 bp direct repeat IB. The results of the study support the hypothesis that this composition is responsible for the length variations seen in the 16S–23S ISR, and indicate that these length variations result from slipped-strand mispairing and intra- and possibly interchromosomal homologous recombination.

Linking Community Profiles, Gene Expression and N-Removal in Anammox Bioreactors Treating Municipal Anaerobic Digestion Reject Water

Anaerobic ammonium oxidation (anammox) requires 60% less oxygen and no external organic carbon compared to conventional biological nitrogen removal (BNR). Nevertheless, full-scale installations of anammox are uncommon, primarily owing to the lack of well-established process monitoring and control strategies that result in stable anammox reactor performance. The overarching goal of this study was to develop and apply molecular biomarkers that link microbial community structure and activity to anammox process performance in a bioreactor fed with actual anaerobic digestion centrate from a full-scale operational wastewater treatment facility. Over long-term operation, Candidatus "Brocadia sp. 40" emerged as the dominant anammox population present in the reactor. There was good correspondence between reactor nitrogen removal performance and anammox bacterial concentrations. During the period of reactor operation, there was also a marked shift in biomass morphology from discrete cells to granular aggregates, which was paralleled by a shift also to more stable nitrogen removal and the succession and establishment of bacteria related to the Chlorobi/Bacteroidetes superfamily. Based on batch assays, hydrazine oxidoreductase (hzo) expression and concentrations of the 16S-23S rRNA intergenic spacer region (ISR) were good quantitative biomarkers of oxygen- and nitrite-mediated inhibition. When applied to a continuous anammox reactor, both molecular biomarkers show promise as monitoring tools for "predicting" reactor performance.

A panel of real-time PCR assays for specific detection of three phytoplasmas from the apple proliferation group

We report here on the development of combination of assays for fast, reliable, specific and sensitive detection and discrimination of ‘ Candidatus Phytoplasma mali’, ‘ Ca. P. prunorum’ and ‘ Ca. P. pyri’ from the 16Sr-X (apple proliferation – AP) group. These phytoplasmas are causal agents of diseases of fruit trees within the family Rosaceae, namely apple proliferation (AP), European stone fruit yellows (ESFY) and pear decline (PD). The designed panel of assays uses TaqMan ® minor groove binder probes (MGB). It comprises the same set of primers and specific probes for species-specific amplification within the 16S–23S rRNA intergenic spacer region, a set of primers and probes for amplification of the 16S ribosomal DNA region for the universal phytoplasma detection, and an additional set of primers and probe for 18S rRNA as an endogenous quality control of DNA extraction. The performance characteristics of the panel were evaluated. The advantages of new assays were shown in a comparative study with the conventional PCR, which proved their higher sensitivity combined with three-fold shorter time of testing process; and in comparison with two reported multiplex real-time PCR assays for detection of ‘ Ca. P. mali’ or ‘ Ca. P. pyri’. New panel of assays were tested on the DNA samples of ‘ Ca. P. mali’, ‘Ca. P. prunorum’, ‘ Ca. P. pyri’, other phytoplasmas and other bacteria isolated from plant material. Additionally, 198 symptomatic and asymptomatic fruit tree field samples collecting during several growing seasons were tested with new assays as well. The results of this study indicate that the combination of three specific assays may be applied in routine phytoplasma surveys and in the certification programs.

Population dynamics of Vibrio spp. associated with marine sponge microcosms

Vibrio is a diverse genus of marine-associated bacteria with at least 74 species and more expected as additional marine ecospheres are interrogated. This report describes a phylogenetic reconstruction of Vibrio isolates derived from one such unique ecosystem, marine sponges (Phylum Porifera) collected from depths of 150 to 1242 feet. 16S rRNA gene sequencing along with molecular typing of 16S-23S rRNA intergenic spacer regions clustered many sponge-associated Vibrio (spp) with current known species. That is, several benthic Vibrio species commensal with Porifera sponges seemed genetically linked to vibrios associated with coastal or shallow-water communities, signalling a panmictic population structure among seemingly ecologically disparate strains. Conversely, phylogenetic analysis provided evidence for at least two novel Vibrio speciation events within this specific sponge microcosm. Collectively, these findings earmark this still relatively unknown environment as a bastion of taxonomic and phylogenetic variability for the genus and probably other bacterial taxa.

Mycoplasma mucosicanis sp. nov., isolated from the mucosa of dogs

Fourteen Mycoplasma strains were isolated from the oral cavity and genital tract of asymptomatic dogs. Isolates had been preliminarily identified by conventional serological testing as Mycoplasma bovigenitalium, but in 16S-23S rRNA intergenic spacer PCR-RFLP assays the isolates exhibited an RFLP pattern distinct from M. bovigenitalium PG11(T). Analysis of the 16S rRNA gene placed a representative of the isolates (strain 1642(T)) in the M. bovigenitalium subcluster of the Mycoplasma bovis cluster of mycoplasmas, with the highest sequence similarities to Mycoplasma californicum ST-6(T) (96.4 %), M. bovigenitalium PG11(T) (96.3 %) and Mycoplasma phocirhinis 852(T) (96.2 %). 16S rRNA gene sequence similarities almost equidistant from three recognized species and results obtained by sequence analysis of the 16S-23S rRNA intergenic spacer region, polar lipid profiles and serological reactions indicated that this organism represents a novel species of the genus Mycoplasma for which the name Mycoplasma mucosicanis sp. nov. is proposed, with strain 1642(T) ( = ATCC BAA-1895(T) = DSM 22457(T)) as the type strain.