No abstract

MELLING. 1992. Gram-positive bacilli isolated during epidemiological investigations which, on the basis of conventional tests, resemble Bacillus anthracis but which fail to produce the capsule or to induce anthrax in test animals have long been dismissed in clinical and veterinary laboratories as B . cereus or simply as unidentified Bacillus spp. and thereupon discarded as inconsequential. In this study, the application of newly available DNA probe, polymerase chain reaction and specific toxin antigen detection technology has revealed that a proportion of such strains are B . anthracis which lack the plasmid carrying the capsule gene (pX02). While these techniques cannot, of course, be used to confirm the identities of strains resembling B . anthracis but which also lack the plasmid carrying the toxin genes (pXOl), the likelihood that these also are bonaJde B . anthracis becomes more acceptable. (As yet no naturally occurring pXOl-/2+ strains have been found.) At this point, the significance of the presence of such avirulent forms of B. anthracis in specimens can only be a subject for speculation, but the possibility that they may be indicators of virulent parents somewhere in the system being examined must be considered.

NOTEAn unknown, Gram-positive, ovoid-shaped bacterium isolated from the nose of a mouse was subjected to a polyphasic taxonomic analysis. Comparative 16S rRNA gene sequencing demonstrated that the unknown organism was a member of the family Micrococcaceae and possessed a specific phylogenetic association with Rothia dentocariosa and Stomatococcus mucilaginosus. Phenotypically, the bacterium closely resembled R. dentocariosa and S. mucilaginosus but could be distinguished from these species by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on both phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified in the genus Rothia, as Rothia nasimurium sp. nov. In addition, it is proposed that S. mucilaginosus be reclassified in the genus Rothia, as Rothia mucilaginosa comb. nov. Keywords : Rothia, Stomatococcus, taxonomy, phylogeny, 16S rRNARothia dentocariosa is found in the oral cavity and pharynx of man where it forms part of the normal microflora (Schaal, 1992). This species is also now recognized as an opportunistic pathogen causing septicaemia and endocarditis, as well as other serious infections (e.g. Schafer et al., 1979 ;Pape et al., 1979 ;Pers et al., 1987 ;Minato & Abiko, 1984 ; Schiff & Kaplan, 1987). The taxonomic affinities of R. dentocariosa have always been controversial. Primarily because of its cellular morphology, the species has historically been associated with Actinomyces and related genera (Schaal, 1992). In recent years it has become apparent, however, that it is only remotely related to these taxa, and is phylogenetically a member of the family Micrococcaceae (Stackebrandt et al., 1997), exhibiting a specific association with Stomatococcus mucilaginosus. Like R. dentocariosa, S. mucilaginosus is a resident of the human oral cavity and pharynx. R. dentocariosa is presently the only recognized species of the genus Rothia. There is, however, a considerable body of evidence indicating some bio- chemical and serological heterogeneity within the species (e.g. Lesher et al., 1974 ; Schofield & Schaal, 1981 ;Fotos et al., 1984 ;Kronvall et al., 1998). In addition, the existence of a second genomovar of R. dentocariosa has recently been described from humans (Kronvall et al., 1998). It is currently not known whether R. dentocariosa or similar organisms occur in other animal species (Schaal, 1992). During the course of a study of unusual Actinobacteria from animals, we have characterized a novel Gram-positive, facultatively anaerobic coccus from the nose of a healthy mouse which phenotypically resembles the genus Rothia. Based on the results of a polyphasic taxonomic study, we propose a new species, Rothia nasimurium, for this bacterium.Strain CCUG 35957 T was isolated from the nose of a healthy mouse. The unidentified organism was cultured on Columbia blood agar base supplemented with 5 % defribrinated horse blood at 37 mC in air plus 5% CO # . The strain was biochemically characterized by using the API rapid ID32 STREP, API CORYNE and...

Strain TCE1, a strictly anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), was isolated by selective enrichment from a PCE-dechlorinating chemostat mixed culture. Strain TCE1 is a gram-positive, motile, curved rod-shaped organism that is 2 to 4 by 0.6 to 0.8 μm and has approximately six lateral flagella. The pH and temperature optima for growth are 7.2 and 35°C, respectively. On the basis of a comparative 16S rRNA sequence analysis, this bacterium was identified as a new strain of Desulfitobacterium frappieri, because it exhibited 99.7% relatedness to the D. frappieri type strain, strain PCP-1. Growth with H2, formate,l-lactate, butyrate, crotonate, or ethanol as the electron donor depends on the availability of an external electron acceptor. Pyruvate and serine can also be used fermentatively. Electron donors (except formate and H2) are oxidized to acetate and CO2. When l-lactate is the growth substrate, strain TCE1 can use the following electron acceptors: PCE and TCE (to produce cis-1,2-dichloroethene), sulfite and thiosulfate (to produce sulfide), nitrate (to produce nitrite), and fumarate (to produce succinate). Strain TCE1 is not able to reductively dechlorinate 3-chloro-4-hydroxyphenylacetate. The growth yields of the newly isolated bacterium when PCE is the electron acceptor are similar to those obtained for other dehalorespiring anaerobes (e.g.,Desulfitobacterium sp. strain PCE1 andDesulfitobacterium hafniense) and the maximum specific reductive dechlorination rates are 4 to 16 times higher (up to 1.4 μmol of chloride released · min−1 · mg of protein−1). Dechlorination of PCE and TCE is an inducible process. In PCE-limited chemostat cultures of strain TCE1, dechlorination is strongly inhibited by sulfite but not by other alternative electron acceptors, such as fumarate or nitrate.

Aims: To carry out an extensive study of the microflora composition of the Labrador dog gut. Methods and Results: Faecal specimens from four Labradors were collected and plated onto growth media designed to recover total anaerobes, bacteroides, bifidobacteria, lactobacilli, clostridia, Gram-positive cocci, total aerobes and coliforms. Morphologically different isolates were collected from all agars inoculated with faeces from one canine individual (repeated four times). A total of 157 out of 171 isolates were identified using 16S rRNA gene sequencing. Sequence analysis showed that agar selectivity was poor, especially when bacteroides and Gram-positive cocci were the targets. Bifidobacteria were not detected in any of the samples analysed, indicating their presence at low or negligible levels. The gene sequences of many of the isolates (n ¼ 45, representing 29% of the total) did not correlate with known species in the Ribosomal Database Project and EMBL databases, suggesting the presence of novel gut diversity. Conclusions: Traditional culture methods fail to reflect the bacterial diversity present in Labrador dog faeces. Significance and Impact of the Study: This study has shown the value of molecular-based methodologies for determining bacterial profiles in the Labrador dog gut microbiota, but has also exposed the limitations of purportedly selective agars.

We have reported that a mouse monoclonal antibody 703D4, detects lung cancer 2 years earlier than routine chest x-ray or cytomorphology. We purified the 703D4 antigen to elucidate its role in early lung cancer biology, using Western blot detection after SDS-polyacrylamide gel electrophoresis. Purification steps included anion exchange chromatography, preparative isoelectric focusing, polymer-based C18-like, and analytical C4 reverse phase high performance liquid chromatography. After 25-50,000-fold purification, the principal immunostaining protein was > 95% pure by Coomassie staining. The NH2 terminus was blocked, so CNBr digestion was used to generate internal peptides. Three sequences, including one across a site of alternate exon splicing, all identified a single protein, heterogeneous nuclear ribonucleoprotein-A2 (hnRNP-A2). A minor co-purifying immunoreactive protein resolved at the final C4 high performance liquid chromatography step is the splice variant hnRNP-B1. Northern analysis of RNA from primary normal bronchial epithelial cells demonstrated a low level of hnRNP-A2/B1 expression, consistent with immunohistochemical staining of clinical samples, and increased hnRNP-A2/B1 expression was found in lung cancer cells. hnRNP-A2/B1 expression is under proliferation-dependent control in normal bronchial epithelial cell primary cultures, but not in SV40-transformed bronchial epithelial cells or tumor cell lines. With our clinical data, this information suggests that hnRNP-A2/B1 is an early marker of lung epithelial transformation and carcinogenesis.

The phylogenetic interrelationships of members of the Clostridium botulinum complex of species was investigated by direct sequencing of their 16S rRNA genes. Comparative analysis of the 16S rRNA sequences demonstrated the presence of four phylogenetically distinct lineages corresponding to: i) proteolytic C. botulinum types A, B, and F, and C. sporogenes, ii) saccharolytic types B, E and F, iii) types C and D and C. novyi type A, and iv) type G and C. subterminale. The phylogenetic groupings obtained from the 16S rRNA were in complete agreement with the four divisions recognised within the the 'species complex' on the basis of phenotypic criteria.

The phylogenetic interrelationships of saccharolytic C. botulinum types B, E and F together with eleven other saccharolytic clostridia were examined by 16S rRNA gene sequencing. Comparative analysis of the sequence data revealed that the saccharolytic C. botulinum types B, E and F were highly related and represent a single genetic group. Strains of C. barati and C. butyricum that produce botulinal neurotoxin revealed almost 100% 16S rRNA sequence identity with their respective non-toxigenic counterparts and were phylogenetically distinct from saccharolytic C. botulinum (types B, E and F). Proteolytic C. botulinum type F was shown to be phylogenetically remote from the saccharolytic C. botulinum group. The implications of the sequence data for the taxonomy of the C. botulinum complex are discussed.