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Seven isolates of a slowly growing, non-chromogenic Mycobacterium species were obtained from sputum and bronchial lavage fluid samples from elderly patients in different regions of Japan. These isolates were distinguished from related non-tuberculous species by colony morphology, positive results for Tween hydrolysis, catalase at 68 °C, nitrate reductase and pyrazinamidase and negative results for semi-quantitative catalase, urease and arylsulfatase. The mycolic acid pattern obtained by HPLC revealed a single cluster of late-eluting mycolic acids similar to but different from those of Mycobacterium malmoense ATCC 29571(T). The 16S rRNA gene, 16S-23S internal transcribed spacer (ITS), rpoB and hsp65 sequences were unique in comparison with those of other mycobacteria. Comparison of 16S rRNA gene sequences showed that the isolates were most closely related to Mycobacterium tuberculosis H37Rv(T) (21 base differences in 1508 bp; 98.6 % 16S rRNA gene sequence similarity). A representative strain, GTC 2738(T), showed 91.9 % rpoB sequence similarity with Mycobacterium marinum strain M, 95 % hsp65 sequence similarity with Mycobacterium kansasii CIP 104589(T) and 81.1 % 16S-23S ITS sequence similarity with Mycobacterium gordonae ATCC 14470(T). Phylogenetic analysis of concatenated sequences of the 16S rRNA, rpoB and hsp65 genes showed that strain GTC 2738(T) was located on a distinct clade adjacent to M. tuberculosis, M. ulcerans and M. marinum, with bootstrap values of 81 %. DNA-DNA hybridization demonstrated less than 70 % reassociation with type strains of genetically related species and supported the novel species status of the isolates. On the basis of this evidence, a novel species with the name Mycobacterium shinjukuense sp. nov. is proposed. The type strain, isolated from a sputum sample, is strain GTC 2738(T)( = JCM 14233(T) = CCUG 53584(T)).

A strain isolated from pleural fluid of a patient with suppurative pleuritis (strain GTC 3021(T)) was characterized in terms of its phenotypic and biochemical features, cellular fatty acid profile and phylogenetic position based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that the isolate was a member of the genus Prevotella. The isolate was related to Prevotella enoeca ATCC 51261(T) with about 92 % 16S rRNA gene sequence similarity. The strain was an obligately anaerobic, non-pigmenting, non-spore-forming, non-motile, Gram-negative rod. Although the phenotypic and biochemical characteristics of the strain were similar to those of P. enoeca JCM 12259(T), the cellular fatty acid composition of the isolate was significantly different from that of P. enoeca JCM 12259(T) (C(18 : 1) omega 9c and anteiso-C(15 : 0) fatty acid content). Based on these data, we propose a novel Prevotella species, Prevotella pleuritidis sp. nov., with the type strain GTC 3021(T) (=JCM 14110(T) =CCUG 54350(T)). The G+C content of the type strain is 45.4 mol%.

The interrelationships of 27 Aeromonas strains were investigated using dnaJ sequences and DNA-DNA hybridization. dnaJ sequence similarities showed a stronger relationship with DNA-DNA relatedness values than did 16S rRNA gene sequence similarities. Additionally, dnaJ sequence analysis, with interspecies divergence over 5.2 % in most cases, gave better resolution than 16S rRNA gene sequences for the differentiation of strains at the species level. Relationships among Aeromonas species were therefore elucidated on the basis of dnaJ sequences and DNA-DNA reassociation. Strains of Aeromonas encheleia and Aeromonas sp. HG11 were unquestionably grouped in the same genetic species, since they shared 98.7 % dnaJ sequence similarity and 82-85 % genomic relatedness. The phylogenetically close relationships obtained from dnaJ sequence analysis (1.7-3.3 % genetic distance) were corroborated by high DNA-DNA relatedness (73-97 %) to support the previous suggestion that Aeromonas culicicola and Aeromonas allosaccharophila are later heterotypic synonyms of Aeromonas veronii. Our findings will contribute to the clarification of controversial relationships in the genus Aeromonas and also demonstrate that analysis of dnaJ sequences can be a powerful tool for interspecies study of the genus.

In the last few years, many attempts have been made to use conserved gene sequences for identification and for phylogenetic studies of Staphylococcus species. In an effort to identify a more reliable approach, a dnaJ gene sequence-based database was created. In this study, an approximately 883 bp portion of the dnaJ gene sequence from 45 staphylococcal type strains was compared with 16S rRNA and other conserved gene (hsp60, sodA and rpoB) sequences available in public databases. Nucleotide sequence comparisons revealed that the staphylococcal dnaJ gene showed higher discrimination (mean similarity 77.6 %) than the 16S rRNA (mean similarity 97.4 %), rpoB (mean similarity 86 %), hsp60 (mean similarity 82 %) and sodA (mean similarity 81.5 %) genes. Analysis of the dnaJ gene sequence from 20 Staphylococcus isolates representing two clinically important species showed <1 % sequence divergence. Phylogenetic data obtained from the dnaJ gene sequence were in general agreement with those of 16S rRNA gene sequence analysis and DNA-DNA reassociation studies. In conclusion, the dnaJ gene sequence-based assay is an effective alternative to currently used methods, including 16S rRNA gene sequencing, for identification and taxonomical analysis of Staphylococcus species.

The genetic relationship between Streptococcus agalactiae and Streptococcus difficilis was studied. S. difficilis was originally described as serologically non-typable but was later reported to be a group B, type Ib streptococcus. Upon comparative analysis of five gene sequences, it was found that S. agalactiae and S. difficilis are closely related. Sequence similarity values between these two species were 100.0 % for 16S rRNA, 99.6 % for gyrB, 98.6 % for sodA, 99.5 % for gyrA and 99.8 % for parC genes. These data strongly suggest that S. agalactiae and S. difficilis are synonyms. The biochemical characteristics of S. difficilis, which differ slightly from those of typical S. agalactiae, are similar to those of other group B, type Ib streptococci isolated from fish and frogs. Whole genome DNA-DNA hybridization values between the type strains of both species were greater than 78.6 %. On the basis of these data, it is proposed that S. difficilis is a later synonym of S. agalactiae.