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Phylogenetic evidence that the gram-negative nonsporulating bacterium Tissierella (Bacteroides) praeacuta is a member of the Clostridium subphylum of the gram-positive bacteria and description of Tissierella creatinini sp. nov.

The 16S rRNA gene sequence of the type strain of Tissierella praeacuta (formerly Bacteroides praeacutus) was determined by PCR direct sequencing. A comparative sequence analysis showed that T. praeacuta is a member of the Clostridium subphylum of the gram-positive bacteria and has a close phylogenetic affinity with the species that form Clostridium cluster XII (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994). Although T. praeacuta is gram negative and does not produce endospores, 16S rRNA sequence data showed that it is closely related genealogically (level of sequence similarity, 99.9%) to Clostridium hastiforme. On the basis of our results and the results of previous studies, a second species of Tissierella, Tissierella creatinini sp. nov., is described.

Amino Acids↗

Tissierella creatinophila sp. nov., a gram-positive, anaerobic, non-spore-forming, creatinine-fermenting organism.

A strictly anaerobic, Gram-positive, non-spore-forming bacterium was isolated from sewage sludge which grew on creatinine as sole source of carbon and energy. This new isolate, designated strain KRE 4T, totally degraded creatinine via creatine, sarcosine and glycine to the products acetate, monomethylamine, ammonia and carbon dioxide. Growth on creatinine or creatine was selenium-dependent and stimulated by formate, indicating the involvement of a creatine reductase, sarcosine reductase and/or glycine reductase. This was substantiated by the fact that creatine, sarcosine and glycine were reduced by cell-free extracts. Growth on creatinine or creatine was also possible in the absence of formate, but with an increase in doubling time. The new bacterium occurred as rod-shaped cells, which exhibited an angular form (2-6 microns long and 0.7-1.1 microns wide) and showed motility by means of peritrichous flagella. The G+C content of the DNA was 30 mol %. Comparative 16S rRNA sequence analysis demonstrated that strain KRE 4T represents a new subline within the genus Tissierella. Due to its very restricted substrate spectrum and the inability of whole cells to utilize sarcosine and glycine as intermediates of creatine breakdown, this organism can be readily separated from currently described species of Tissierella. Therefore, based on the phenotypic and phylogenetic distinctiveness of the new isolate, it si proposed that the bacterium be classified as a new species of the genus Tissierella, Tissierella creatinophila sp. nov. The type strain is KRE 4 (= DSM 6911T).

Base Sequence↗

Clostridium hastiforme is a later synonym of Tissierella praeacuta.

The previously proposed species Clostridium hastiforme and Tissierella praeacuta appear to be similar from their published descriptions. Accordingly, the aim of the current study was to perform phenotypic and genetic analyses of the type strains of both species, in order to clarify their taxonomic positions. The type strains of C. hastiforme (DSM 5675(T)) and T. praeacuta (NCTC 11158(T)) exhibited identical biochemical profiles and their 16S rRNA gene sequences displayed 99.9 % similarity. DNA-DNA hybridization was also estimated to be 96.5 %. Thus, it was concluded that C. hastiforme and T. praeacuta are synonyms, where T. praeacuta has priority. An emended description of the genus Tissierella is also given.

Clostridium↗

Sarcosine reductase of Tissierella creatinophila: purification and characterization of its components.

Sarcosine reductase is the only reductase system present in Tissierella creatinophila when grown on creatinine plus formate. The acetyl-phosphate-forming component protein C was purified to homogeneity. SDS-PAGE of the purified protein revealed two protein bands with apparent mol. masses of 62 and 50 kDa. The N-terminal amino acid sequence of the two subunits was determined. Antibodies raised against each of the subunits of protein C from Eubacterium acidaminophilum cross-reacted with the corresponding protein present in T. creatinophila, Clostridium litorale and Clostridium sporogenes. The arsenate-dependent hydrolysis of acetyl phosphate catalyzed by protein C was partly inhibited by antibodies directed against the large subunit. Antibodies raised against the small subunit were twice as effective, which indicates that this subunit is the primary site of acetyl transfer from acetyl phosphate. The protein A component of the sarcosine reductase of T. creatinophila was purified to homogeneity by cochromatography with thioredoxin reductase on DEAE-Sephacel, hydroxylapatite, Q-Sepharose, and Sephacryl 100-HR. Protein A had an apparent mol. mass of 21 kDa. Its N-terminal amino acid sequence showed high similarities to that of other proteins A. Initial steps for the purification and preliminary characterization of the sarcosine-specific, substrate-binding protein Bsarcosine component of T. creatinophila indicated the involvement of a 50-kDa protein.

Amino Acid Oxidoreductases↗

Isolation and identification of anaerobic bacteria from ovine foot rot in Spain.

A microbiological study was made of 125 Merino sheep showing clinical signs of foot rot. A total of 435 strictly anaerobic strains were isolated, belonging to the following genera: Bacteroides, Peptostreptococcus, Tissierella, Fusobacterium, Megasphaera, Eubacterium, Acidaminococcus, Clostridium, Peptococcus and Propionibacterium. Of the 35 species obtained, the following were found in more than 10 per cent of animals sampled: Bacteroides nodosus, B putredinis, B buccae, B ruminicola subspecies brevis, Tissierella praeacuta, Peptostreptococcus anaerobius and Megasphaera elsdenii. Six culture media were used for isolation. Agar brucella and agar brucella enriched with G-N anaerobe supplement proved to be the most efficient for isolating anaerobic bacteria.

Animals↗

Obligately anaerobic bacterial species isolated from foot-rot lesions in goats.

Lesions showing clinical signs of foot-rot from 120 goats were cultured on six selective media during October 1987 to November 1988. A total of 582 strictly anaerobic microorganisms belonging to 50 different species was isolated and identified. The anaerobes most frequently isolated belonged to the following genera: Bacteroides (80%), Peptostreptococcus (63.6%), Megasphaera (40%), Fusobacterium (29.2%), Clostridium (22.5%), Propionibacterium (12.5%), Eubacterium (11.7%) and Leptotrichia (10.8%). Percentages for Acidaminococcus, Peptococcus, Tissierella, Wolinella and Veillonella were below 10%. The following species were identified in 10% or more of cases: Peptostreptococcus anaerobius (61.7%), Bacteroides buccae (51.7%), Bacteroides nodosus (42.5%), Megasphaera elsdenii (40%), Bacteroides ruminicola subsp. brevis (22.5%), Fusobacterium necrophorum (19.2%), Leptotrichia buccalis (11.7%) and Clostridium perfringens (10%). Lower percentages were obtained for the remaining species. The efficiency and selectivity of the six culture media used for isolation are discussed.

Animals↗

Fast purification of thioredoxin reductases and of thioredoxins with an unusual redox-active centre from anaerobic, amino-acid-utilizing bacteria.

Thioredoxin reductase and thioredoxin are primarily involved in catabolic metabolism as important electron carriers in anaerobic, amino-acid-degrading bacteria. A general and fast procedure was developed for the purification of thioredoxin reductase and thioredoxin from Eubacterium acidaminophilum, Clostridium litorale, C. sticklandii, C. sporogenes, C. cylindrosporum and 'Tissierella creatinophila' based upon their properties: the binding to 2',5'-AMP-Sepharose by thioredoxin reductase and the inability of thioredoxins to bind to a DEAE-Sephacel column. The consensus sequence at the active site of thioredoxins (-WCGPC-) was found to be modified in all of these anaerobes: Trp-31 (Escherichia coli nomenclature) was replaced by Gly or Ser, Gly-33 by Val or Glu. None of these thioredoxins reacted with thioredoxin reductase of E. coli or vice versa, but they did interact with the thioredoxin reductases obtained from the other anaerobes studied. Based upon their distinguishing features it is suggested that these thioredoxins might form an evolutionarily separate group.

Amino Acid Sequence↗

Multicentre survey of the in-vitro activity of seven antimicrobial agents, including ertapenem, against recently isolated Gram-negative anaerobic bacteria in Greece.

The in-vitro activities of penicillin, ticarcillin-clavulanic acid, cefoxitin, imipenem, ertapenem, metronidazole and clindamycin were evaluated against 138 Gram-negative anaerobic isolates (82 Bacteroides fragilis group, 17 non-fragilis Bacteroides spp., 31 Prevotella spp., four Fusobacterium spp., two Veillonella spp., one Porphyromonas sp. and one Tissierella praeacuta) collected from six general hospitals in Athens, Greece. Overall rates of non-susceptibility (both resistant and intermediately-resistant) to penicillin and ticarcillin-clavulanic acid were 81.8% and 2.3%, respectively. The rates of non-susceptibility to cefoxitin and clindamycin were 30.3% and 31.1%, respectively, and that for metronidazole was 4.3% (four Prevotella spp. isolates, one Porphyromonas sp. isolate and one B. fragilis isolate). Only the single B. fragilis isolate was nim-positive by PCR. Only one B. fragilis isolate was resistant to both carbapenems tested, while six more Bacteroides spp. isolates were imipenem-susceptible and ertapenem-non-susceptible. The MIC range, MIC(50) and MIC(90) values were comparable for imipenem and ertapenem, although ertapenem MIC(90)s were one or two two-fold dilutions higher.

Anti-Bacterial Agents↗