- Volume 42, Issue 4, 1992

Mycoplasmas isolated from the throats of lions were shown to belong to three serotypes, all of which were serologically distinct from the previously recognized Mycoplasma and Acholeplasma spp. Eight mycoplasma colonies were cloned, including one from a leopard (strain LP), and were examined in detail for morphology, growth, and biochemical characteristics. The strains had the following properties: Guanine-plus-cytosine contents of 37 mol% (strain LXT [T = type strain]), 28 mol% (strain LL2T), and 27 mol% (strain 3L2T) and a requirement for sterol. Strain 3L2T metabolized glucose, which was not metabolized by strains LXT and LL2T. Arginine and urea were not hydrolyzed. Strain LX (= NCTC 11724) is the type strain of a new species, Mycoplasma simbae; strain LL2 (= NCTC 11725) is the type strain of a second new species, Mycoplasma leopharyngis; and strain 3L2 (= NCTC 11726) is the type strain of a third new species, Mycoplasma leocaptivus.

Strains of a new type of rapidly growing, scotochromogenic mycobacterium were isolated from sphagnum vegetation in Madagascar. These strains grew at 31 and 22°C but not at 37°C, possessed catalase, acid phosphatase, and arylsulfatase activities, split urea and pyrazinamid, hydrolyzed Tween, and produced acid from glucose, L-arabinose, fructose, mannitol, rhamnose, sorbitol, xylose, and trehalose. Furthermore, they metabolized iron and possessed putrescine oxidase activity but did not reduce nitrate. The internal similarity level of the strains, as determined by taxonomic methods, was 92.50%. The phylogenetic relationships of strain P2T (T = type strain) with members of the genus Mycobacterium, as determined by comparing the 16S rRNA primary structure of this strain with the 16S rRNA primary structure of this stain with the 16S rRNA primary structures of 41 other mycobacterial species, indicated that strain P2T belongs to a separate line of descent within a cluster that includes Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium confluentis, Mycobacterium flavescens, and Mycobacterium thermoresistibile. Hence, the new strains are considered members of a new species of nonpathogenic, rapidly growing mycobacteria, for which we propose the name Mycobacterium madagascariense. Strain P2 is the type strain; a culture of this strain has been deposited in the American Type Culture Collection as strain ATCC 49865.

A new species of rapidly growing, nonphotochromogenic mycobacteria, Mycobacterium alvei, is described. The inclusion of this organism in the genus Mycobacterium is based on its acid fastness, its mycolate pattern, and its G+C content. A study of six strains showed that they form a homogeneous group with an internal phenotypic similarity value of 97 ± 2.22%. DNA relatedness studies showed that the six M. alvei strains which we studied form a single DNA hybridization group which is less than 49% related to 14 other species of the genus Mycobacterium; the δTm values determined for the strains which exhibited higher levels of DNA homology were all greater than 7.9°C. A Iipid analysis showed that tuberculostearic acid was present. Docosanoic and tetracosanoic acid methyl esters were detected as mycolic acid cleavage products. All six isolates which we tested contained α-mycolic acids and relatively large amounts of a new kind of mycolic acid containing a methoxy group at ω-1 position, a characteristic that has not been described previously in mycobacteria. Strain CR-21 is the type strain; a culture of this strain has been deposited in the Collection Nationale de Cultures de Microorganismes de I’Institut Pasteur, Paris, France, as strain CIP 103464.

During studies of human periodontal disease, a number of bacterial strains were encountered that, on the basis of results of standard biochemical tests, appeared to be Prevotella buccalis, Prevotella denticola, Prevotella melaninogenica, or Prevotella loescheii. However, use of the standard biochemical tests, cellular fatty acid analyses, and the polyacrylamide gel electrophoresis patterns of soluble proteins resulted in conflicting identifications of these strains. The results of tests for cellobiose fermentation, inulin fermentation, and pigment production were responsible for most of the discordant results. Cellular fatty acid analyses in which the Microbial Identification System was used did not differentiate these strains from validly described species, even though separate library entries were created for them. DNA reassociation determinations in which the S1 nuclease procedure was used showed that cellobiose fermentation and pigment production are variable among strains of P. melaninogenica and P. denticola and that fermentation of xylan is not a reliable characteristic for differentiating P. buccalis from Prevotella veroralis. In contrast to previous indications, most strains of P. veroralis do not ferment xylan. These species can be differentiated by DNA-DNA reassociation and by cellular fatty acid analysis, using the Microbial Identification System, but differentiation by currently described phenotypic characteristics is not reliable. Similarly, P. loescheii and the genetically distinct (but closely related) D1C-20 group cannot be distinguished reliably from each other or from P. veroralis, P. denticola, and P. melaninogenica on the basis of currently described phenotypic tests other than cellular fatty acid composition or, for some species, electrophoretic patterns of soluble whole-cell proteins.

A total of 31 strains of Prevotella intermedia were subjected to DNA-DNA hybridization and were characterized by performing physiological tests and by performing a multilocus enzyme analysis, using malate dehydrogenase and glutamate dehydrogenase. All of the strains assigned to P. intermedia fermented glucose and sucrose, hydrolyzed starch but not esculin, and produced indole, acetic, isobutyric, isovaleric, and succinic acids as metabolic end products. The results of DNA reassociation experiments performed with the reference probe permitted separation of the strains into two well-defined homology groups. In addition, strains with DNAs that hybridized with DNA from strain ATCC 25611T (T = type strain) had high levels of peptidase activity and cleaved lipid substrates (4-methylumbelliferyl laurate and 4-methylumbellifelyl elaidate). Multilocus enzyme electrophoresis revealed two electromorphic profiles, one characteristic of strain ATCC 25611T and the other characteristic of strain ATCC 33563T. We propose that a new species, Prevotella nigrescens, should be created for the genetically distinct group of strains that hybridized with strain ATCC 33563T. Strain ATCC 33563 is designated the type strain of P. nigrescens.

The genomic diversity among 22 Nitrobacter strains was investigated by determining rRNA gene restriction patterns, DNA hybridization characteristics, and DNA base compositions. The guanine-plus-cytosine contents of the DNAs ranged from 58 to 61 mol%. As determined by DNA hybridization (S1 nuclease method), five DNA genomic groups were differentiated, and these groups formed three genomic species. Genomic species 1, which corresponded to Nitrobacter winogradskyi, was split into three subspecies. Subspecies 1a contained strain W (= ATCC 25391), the type strain of N. winogradskyi; subspecies 1b contained proposed reference strain 6R; and subspecies 1c contained a strain of “Nitrobacter agilis” (strain ATCC 14123). Genomic species 2, which has not been described previously and which contained proposed reference strain LL and four strains that were isolated from lake sediments, was distinct from N. winogradskyi and Nitrobacter hamburgensis. This species is not named in this paper since it could not be differentiated from N. winogradskyi and N. hamburgensis on the basis of phenotypic characteristics. Genomic species 3 corresponded to N. hamburgensis and was distantly related to the other genomic species.

We examined 22 previously described and newly isolated Methanosarcina strains by performing denaturing gel electrophoresis of whole-cell proteins and assigned these strains to previously described species. Methanosarcina mazei S-6T (T = type strain) and Methanosarcina frisia C 16T were very similar in terms of the electrophoresis patterns of their proteins and in their DNA sequences (the results of reassociation experiments indicated that there was 77% sequence similarity). Thus, M. frisia is a junior subjective synonym of M. mazei, and strain C 16 is a reference strain of M. mazei. M. mazei C 16 was similar to M. mazei in other characteristics that have not been reported previously, including the ability to catabolize acetate and a lack of halophily. All of the Methanosarcina strains examined, including the marine strains M. mazei C 16 (= M. frisia C 16T) and Methanosarcina acetivorans C2AT, were slightly halotolerant (rather than halophilic, as originally described). Methanosarcina sp. strain FR-1, which has gas vesicles, was more similar to Methanosarcina barkeri MST than to Methanosarcina vacuolata Z-761T in both its protein patterns and its DNA sequence (80% similarity to M. barkeri MST and 38% similarity to M. vacuolata Z-761T). Thus, the presence of gas vesicles is not an adequate taxonomic characteristic for assigning Methanosarcina strains to M. vacuolata.

On the basis of phenotypical characteristics and analysis of 16S rRNA sequence, a new species belonging to a new genus is described, and the name Marinobacter hydrocarbonoclasticus is proposed. This organism, isolated from Mediterranean seawater near a petroleum refinery, is a gram-negative, aerobic, rod-shaped bacterium. It grows at NaCl concentrations of 0.08 to 3.5 M and uses various hydrocarbons as the sole source of carbon and energy. Its DNA has a guanine-plus-cytosine content of 52.7 mol%. The 16S rRNA analysis shows a clear affiliation between M. hydrocarbonoclasticus and the gamma group of the phylum Proteobacteria. A close phylogenetic relationship appears among the species Marinomonas vaga, Oceanospirillum linum, Halomonas elongata, and Pseudomonas aeruginosa. Because of the impossibility of finding a single most closely related species, we suggest that this bacterium be assigned to a new genus, at least temporarily. The possibility of a revision of this status when new data appear is, however, not excluded. The type strain is M. hydrocarbonoclasticus SP.17 (=ATCC 49840).

New coagulase-negative staphylococci were isolated from fermented fish in Thailand. These organisms were differentiated from other Staphylococcus species on the basis of DNA relatedness and biochemical characteristics. Staphylococcus piscifermentans sp. nov. is described, and the type strain is strain SK03 (= NRIC 1817 = JCM 6057 = TISTR 824).

Eleven strains of Erwinia carotovora that were isolated mainly, but not exclusively, from slimy rot of witloof chicory and were previously designated “atypical” E. carotovora subsp. atroseptica strains were characterized and compared with strains of E. carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Erwinia chrysanthemi (including the type strains). The 11 atypical E. carotovora subsp. atroseptica strains produced a typical bananalike odor when they were inoculated onto witloof chicory leaves. DNA-DNA homology experiments, biochemical tests, tests to determine carbon utilization patterns, and tests to identify the volatile metabolites produced from rotting witloofs were performed. The volatile end products of witloof decay were analyzed by gas chromatography. Alcohols, methylketones, and ethylacetate were produced by all of the Erwinia strains which we studied, whereas propyl acetate, isobutyl acetate, isoamyl acetate, and 2-actamyl acetate were produced only by the flavoring witloof soft-rot strains. A DNA relatedness study was performed by hybridizing DNAs with a tritium-labeled DNA and estimating the δTm values (δTm is the difference between the thermal denaturation midpoint of a homoduplex and the thermal denaturation midpoint of a heteroduplex). The 11 flavoring strains constituted a tight DNA hybridization group (79 to 91% related to type strain CFBP 1878 isolated from witloof). Strains of E. carotovora subsp. carotovora were 59 to 88% related to strain CFBP 1878T (T = type strain) (δTm range, 3 to 4.5°C), indicating that they belonged to the same species but another subspecies. E. carotovora subsp. atroseptica and E. carotovora subsp. betavasculorum appeared to be less closely related to strain CFBP 1878T than E. carotovora subsp. carotovora was, exhibiting 53% homology (δTm, 7°C) and 48 to 51% homology (δTm, 8.5°C), respectively, with strain CFBP 1878T. Therefore, we propose that the 11 flavoring strains are members of a new subspecies, Erwinia carotovora subsp. odorifera. We examined 95 biochemical characteristics, API strip tests, and assimilation tests in Biotype galleries and identified nine tests which can be used for phenotypic differentiation of the new subspecies.

A mollicutes isolated from a guinea pig vagina was shown to be serologically distinct from previously recognized Mycoplasma and Acholeplasma species. Colonies isolated from 10 different guinea pigs were cloned and examined in detail. These strains were closely related and had the following properties: Guanine-plus-cytosine content of 36 mol%, no requirement for sterol, and aerobic growth. Glucose was not metabolized, and arginine and urea were not hydrolyzed. Strain GP3 (= NCTC 11727) is the type strain of a new species, Acholeplasma cavigenitalium.

A gene probe, CAP-21, which demonstrated interrelationships between the members of the Mycoplasma mycoides cluster was developed. The probe easily differentiated mycoplasmas in this cluster by clear and predictable hybridization patterns in Southern blots and separated the cluster into four groups. Strains of M. mycoides subsp. mycoides which were capable of causing contagious bovine pleuropneumonia composed one group. Strains of M. mycoides subsp. mycoides which did not cause contagious bovine pleuropneumonia together with strains of M. mycoides subsp. capri composed the second group. Mycoplasma capricolum and the F38 mycoplasmas formed a third group, while the bovine group 7 mycoplasmas composed a separate, fourth group. Further support for the above grouping of the cluster was obtained when amplified DNA analogous to the probe from one representative strain of each of the cluster members was sequenced and these data were used to construct a phylogenic tree. Contagious caprine pleuropneumonia is recognized as an important disease, and the etiological agent of this disease is now known to be the F38 mycoplasma. The CAP-21 probe did not differentiate between M. capricolum and the closely related F38 mycoplasma. A second probe, F38-12, which was capable of distinguishing these two mycoplasmas was made.

We describe three new species of the genus Peptostreptococcus which were isolated from human specimens and were tentatively identified as Peptostreptococcus prevotii. These three organisms were not homologous with previously described type strains of the genus Peptostreptococcus. A total of 12 strains that were identified biochemically as P. prevotii were divided into five independent DNA similarity groups; 10 of these strains were divided into three similarity groups which exhibited significant phenotypic differences from previously described species. Therefore, we propose the following new species: Peptostreptococcus vaginalis for group 1 strains, Peptostreptococcus lacrimalis for group 2 strains, and Peptostreptococcus lactolyticus for group 3 strains. The type strain of P. vaginalis is strain GIFU 12669 (= JCM 8138), the type strain of P. lacrimalis is strain GIFU 7667 (= JCM 8139), and the type strain of P. lactolyticus is strain GIFU 8586 (= JCM 8140).

We found that Pseudomonas syringae subsp. savastanoi strains belong to a DNA relatedness group that includes strains of P. syringae pv. glycinea and P. syringae pv. phaseolicola. This DNA group was distinct from P. syringae pv. syringae (including the type strain of P. syringae). The results of a numerical analysis were in accord with DNA hybridization data. Thus, P. syringae subsp. savastanoi (Janse) 1982 is elevated to species level as Pseudomonas savastanoi sp. nov., which includes P. savastanoi pv. savastanoi, P. savastanoi pv. glycinea, and P. savastanoi pv. phaseolicola.

The genus name Morganella was established within the family Enterobacteriaceae in 1978. Morganella morganii is the only species described thus far within this genus, and the name M. morganii has been accepted by usage in the scientific community for strains previously known as Proteus morganii. M. morganii isolates differ in their abilities to ferment trehalose and exhibit variable lysine and ornithine decarboxylase patterns, emphasizing the phenotypic heterogeneity within this species. Previous genetic studies failed to reveal separate entities within the genus Morganella. We observed some trehalose-fermenting strains with different lysine and ornithine decarboxylase patterns. Two strains were lysine and ornithine positive, 3 were lysine positive and ornithine negative, and 29 were lysine negative and ornithine positive. These strains and 25 non-trehalose-fermenting strains with different lysine and ornithine decarboxylase patterns were investigated. DNA-DNA hybridization studies and phenotypic characterizations revealed that M. morganii can be separated into three DNA relatedness groups and seven biogroups. Strains from DNA relatedness group 1 were trehalose negative, and strains from DNA relatedness groups 2 and 3 were trehalose positive. One biogroup from DNA relatedness group 2 was phenotypically indistinguishable from DNA relatedness group 3. On the basis of these studies, we propose that M. morganii be subdivided into M. morganii subsp. morganii (type strain ATCC 25830) containing biogroups A, B, C, and D (DNA relatedness group 1) and M. morganii subsp. sibonii (type strain 8103-85; =ATCC 49948) containing biogroups E, F, and G (DNA relatedness groups 2 and 3).

We studied seven strains of aerobic, marine, polarly flagellated bacteria which decompose alginate, agar, and carrageenan. The major respiratory quinone of these strains was ubiquinone-8. The G+C content of their DNA was 39.5 to 41.7 mol%. “Pseudomonas atlantica” IAM 12927 and the conspecific five isolates were concluded to constitute a single species distinguished from the other nonpigmented Alteromonas species by DNA-DNA hybridization (homology values of more than 82%) and phenotypic similarity (similarity coefficients, based on assimilation of 145 carbon compounds, were 79 to 96%). “Pseudomonas carrageenovora” IAM 12662, the sole extant strain, was distinct from “P. atlantica” and other Alteromonas species in DNA-DNA hybridization and phenotypic features. Taxonomic affinity to Alteromonas espejiana was indicated by DNA-DNA hybridization with “P. atlantica” IAM 12927 and the five conspecific isolates (39 to 55%) and with “P. carrageenovora” IAM 12662 (43 to 45%). Phenotypically, higher similarity values (79 to 89%) for assimilation of 145 carbon compounds were shared between A. espejiana IAM 12927T and the six conspecific strains, including “P. atlantica” IAM 12927. Alteromonas atlantica sp. nov. (type strain, IAM 12927, =ATCC 19262, =NCIMB 301) and Alteromonas carrageenovora (type strain, IAM 12662, =IFO 12985, =ATCC 43555, =NCIMB 302) are proposed for “P. atlantica” IAM 12927 and the conspecific five isolates and “P. carrageenovora” IAM 12662, respectively. A set of phenotypic features which differentiate the two Alteromonas species is described.

Genetic and phenotypic studies on the strains biochemically identified as Shewanella putrefaciens, which had a G+C content ranging from 52 to 54 mol% were conducted. The moles percent G+C of the type strain of S. putrefaciens is 46. Surprisingly, DNA homology experiments revealed that all these strains are genetically related to Shewanella alga (which was reported to produce tetrodotoxin), not to the type strain of S. putrefaciens. In this study, we reidentified clinical strains of S. putrefaciens which have a high range of moles percent G+C, as does S. alga. We also characterized the reidentified strains and found that the original description of S. alga (U. Simidu, K. Kita-Tsukamoto, T. Yasumoto, and M. Yotsu, Int. J. Syst. Bacteriol. 40:331-336, 1990) is insufficient to identify this strain. An emended description of S. alga is given.

Fourteen soil-inhabiting, fragmenting actinomycetes with diaminobutyric acid in their cell walls and MK-12 as their major menaquinone were characterized and assigned to two new Agromyces species; these organisms were further subdivided into four subspecies on the basis of numerical data, cell wall sugar compositions, and levels of DNA relatedness. The key characteristics which differentiated between the new species and subspecies of the genus Agromyces and Agromyces ramosus included the presence of tyvelose or fucose in the cell walls; some physiological properties, including catalase and oxidase activities; growth on media supplemented with inorganic nitrogen; reduction of nitrate; and hydrolysis of hippurate. The data which became available when we studied more strains of Agromyces species and subspecies suggested that the genus description should be emended. We suggest that the following characteristics should be included in the genus description: Catalase and oxidase activities are variable; the majority of strains grow on inorganic media; as a rule, galactose and rhamnose are present in the cell walls; some strains contain glucose, fucose, tyvelose, mannose, ribose, and/or xylose as the major cell wall sugar(s); iso and anteiso types of fatty acids predominate (94%); and the G+C contents of the DNAs are 70 to 72 mol%, as determined by the thermal denaturation method. The type strains of the new species and subspecies are as follows: Agromyces cerinus subsp. cerinus VKM Ac-1340, Agromyces cerinus subsp. nitratus VKM Ac-1351, Agromyces fucosus subsp. fucosus VKM Ac-1345, and Agromyces fucosus subsp. hippuratus VKM Ac-1352.

Staphylococcus aureus ATCC 12598 and ATCC 25923 were starved of pyridine nucleotides and precursors and then grown in a semidefined medium containing [carbonyl-14C]nicotinamide. Samples of medium from late-exponential-phase and stationary-phase cultures were analyzed for 14C-metabolites. In all cases, V factor was present primarily as NAD.