- Volume 47, Issue 3, 1997

Bacillus salexigens sp. nov. is proposed based on the characteristics of six moderately halophilic, gram-positive, rod-shaped strains isolated from salterns and hypersaline soils located in different geographical areas of Spain. These strains were motile, formed endospores, were strictly aerobic, were catalase and oxidase positive, and contained peptidoglycan of the meso-diaminopimelic acid type in their vegetative cell walls. The DNA base compositions of these strains ranged from 36.3 to 39.5 mol%, and these organisms constitute a homology group with levels of DNA-DNA homology ranging from 73 to 100%. The 16S rRNA sequence of strain C-20MoT, which was used as the representative strain of these isolates, groups with the 16S rRNA sequences of members of the genus Bacillus, and the highest level of similarity is 95.4%. The type strain is strain C-20Mo (= ATCC 700290 = DSM 11483 = CCM 4646).

Organisms with the typical characteristics of mycoplasmas were isolated from joints and lungs of crocodiles. The results of growth inhibition tests and immunobinding assays showed that the 24 mycoplasma strains isolated were identical and distinct from previously described Mycoplasma, Entomoplasma, Mesoplasma, and Acholeplasma species. These organisms represent a new species, for which the name Mycoplasma crocodyli is proposed. M. crocodyli ferments glucose and maltose, does not produce films and spots, does not hydrolyze arginine, esculin, and urea, reduces tetrazolium chloride, and possesses phosphatase activity. It lyses and adsorbs bovine, ovine, and rabbit erythrocytes. Cholesterol or serum is required for growth. The optimum growth temperature is 37°C. The G+C content of the DNA is 27.6 mol%. This organism causes exudative polyarthritis in crocodiles. The type strain of M. crocodyli is strain MP145 (= ATCC 51981).

Strain NRRL 12292T, which produces the bleomycin-like antibiotics LL-BO1208α and LL-BO1208β forms umbels consisting of chains of smooth-surface ovoid spores that are borne on verticils on the aerial mycelia, indicating that it is a member of the verticillate group of the genus Streptomyces formerly classified in the genus Streptoverticillium. This strain was compared morphologically and physiologically to 54 other verticillate Streptomyces strains. The levels of DNA relatedness between strain NRRL 12292Tand 34 other verticillate Streptomyces strains, including strains representing at least 19 genetic species clusters, were also determined. Strain NRRL 12292Tis morphologically and physiologically distinct from the other verticillate strains studied, particularly because of the straw yellow color of its aerial mycelia and spore mass. DNA hybridization data support the uniqueness of this strain, since the levels of DNA relatedness between NRRL 12292Tand the other verticillate strains used in this study were low. Our data support designation of a new species, Streptomyces stramineus, whose type strain is NRRL 12292.

A mesophilic, acetate-oxidizing, sulfur-reducing bacterium, strain NZ27T, was isolated from anoxic mud from a freshwater sulfur spring. The cells were ovoid, motile, and gram negative. In addition to acetate, the strain oxidized pyruvate, succinate, and fumarate. Sulfur flower could be replaced by polysulfide as an electron acceptor. Ferric nitrilotriacetic acid was reduced in the presence of pyruvate; however, this reduction did not sustain growth. These phenotypic characteristics suggested that strain NZ27T is affiliated with the genus Desulfuromonas. A phylogenetic analysis based on the results of comparative 16S ribosomal DNA sequencing confirmed that strain NZ27T belongs to the Desulfuromonas cluster in the recently proposed family “Geobacter-aceae” in the delta subgroup of the Proteobacteria. In addition, the results of DNA-DNA hybridization studies confirmed that strain NZ27T represents a novel species. Desulfuromonas thiophila, a name tentatively used in previous publications, is the name proposed for strain NZ27T in this paper.

Spiroplasma strain PLHS-1T from the gut of a common scorpion fly (Panorpa helena) collected in the West Virginia Allegheny Mountains was distinct from other spiroplasma species, groups, and subgroups as determined by reciprocal serological metabolism inhibition and deformation tests. However, when this strain was used as an antigen, it cross-reacted extensively with representatives of other groups. Light microscopy and/or electron microscopy of cells of strain PLHS-1T revealed helical motile cells surrounded by a single cytoplasmic membrane. The strain was resistant to penicillin, which confirmed that it had no cell wall. The organism grew well in M1D and SP-4 liquid media, in 1% serum fraction medium, and in conventional horse serum medium. The optimum temperature for growth was 30°C, at which the doubling time was 6.4 h. Multiplication occurred at temperatures from 10 to 32°C. Strain PLHS-1T catabolized glucose, hydrolyzed arginine but not urea, and required sterol for growth. The guanine-plus-cytosine content of the DNA was 31 ± 1 mol%, and the genome size was 1,465 kbp. Strain PLHS-1 (= ATCC 51752) is designated the type strain of a new species, Spiroplasma alleghenense.

Spiroplasma strain PALS-1T from the gut of the dragonfly Pachydiplax longipennis was shown to be distinct from other species, groups, and subgroups of the genus Spiroplasma as determined by reciprocal serological metabolism inhibition and deformation tests. However, this strain cross-reacted extensively with representatives of other groups when it was used as an antigen. Electron microscopy of cells of strain PALS-1T revealed cells surrounded by a single cytoplasmic membrane. Light microscopy revealed helical cells that exhibited twisting motility rather than rotatory or flexing motility. Variations in the tightness of coiling were transmitted from one end of the helix to the other. The strain was resistant to penicillin, which confirmed that no cell wall was present. The organism grew well in M1D and SP-4 liquid media under either aerobic or anaerobic conditions. Growth also occurred in 1% serum fraction medium and in conventional horse serum medium. The optimum temperature for growth was 30°C, at which the doubling time was 6.4 h. Multiplication occurred at temperatures from 10 to 32°C. Strain PALS-1T catabolized glucose and hydrolyzed arginine but not urea. The guanine-plus-cytosine content of the DNA was 29 ± 1 mol%. The genome size was 780 kbp, the smallest genome size in the genus Spiroplasma. Strain PALS-1 (= ATCC 51748) is designated the type strain of a new species, Spiroplasma platyhelix.

In order to investigate whether 16S–23S ribosomal DNA (rDNA) spacer region length polymorphisms are suitable identification of Corynebacterium strains at the species level, the 16S–23S rDNA intergenic spacer region strains belonging to 11 Corynebacterium species were studied by a PCR-based method. The lengths 16S–23S rDNA spacer regions varied from 394 to 585 bp, fragment lengths which are similar to those described for other genera. A single PCR profile was obtained for each of the following species: Corynebacterium renale, Corynebacterium urealyticum, Corynebacterium diphtheriae, Corynebacterium ulcerans, Corynebacterium pseudodiphtheriticum, and Corynebacterium kutscheri. In contrast, two and three PCR patterns were detected for Corynebacterium minutissimum, Corynebacterium striatum, Corynebacterium amycolatum, and Corynebacterium jeikeium, suggesting that genomic heterogeneity occurs in these four species. The 16S–23S rDNA spacer region length polymorphisms allowed us to discriminate among C. minutissimum, C. striatum, and C. amycolatum, three species that are frequently isolated and misidentified in clinical laboratories. Type strain Corynebacterium xerosis ATCC 373, which exhibited a PCR pattern similar to that of C. amycolatum strains classified in PCR group I, could nevertheless be discriminated from PCR group II (C. amycolatum) strains, as Well as minutissimum and C. striatum strains. Type strain C. xerosis ATCC 373 and C. amycolatum strains classified PCR group I could not be distinguished from strains belonging to C. diphtheriae, C. ulcerans, and C. pseudodiphtheriticum. The lipophilic species C. urealyticum and C. jeikeium, which are frequently encountered in clinical specimens, could be clearly distinguished from each other by this method. The use of 16S–23S spacer region length data determined by PCR-mediated amplification is suitable for identification of several Corynebacterium species. This rapid and easy method may be a useful identification tool for clinical microbiologists.

A numerically important member of marine enrichment cultures prepared with lignin-rich, pulp mill effluent was isolated. This bacterium was gram negative and rod shaped, did not form spores, and was strictly aerobic. The surfaces of its cells were covered by blebs or vesicles and polysaccharide fibrils. Each cell also had a holdfast structure at one pole. The cells formed rosettes and aggregates. During growth in the presence of lignocellulose or cellulose particles, cells attached to the surfaces of the particles. The bacterium utilized a variety of monosaccharides, disaccharides, amino acids, and volatile fatty acids for growth. It hydrolyzed cellulose, and synthetic lignin preparations were partially solubilized and mineralized. As determined by 16S rRNA analysis, the isolate was a member of the α subclass of the phylum Proteobacteria and was related to the genus Roseobacter. A signature secondary structure of the 16S rRNA is proposed. The guanine-plus-cytosine content of the genomic DNA was 65.0 mol%. On the basis of the results of 16S rRNA sequence and phenotypic characterizations, the isolate was sufficiently different to consider it a member of a new genus. Thus, a novel genus and species, Sagittula stellata, are proposed; the type strain is E-37 (= ATCC 700073).

In a previous investigation of the laboratory identification of clinical strains of the genus Peptostreptococcus, several isolates were found to be atypical. In this study, we further examined these strains by using both phenotypic and genotypic methods. Based on our findings, we describe the following three new species of the genus Peptostreptococcus from human clinical specimens: Peptostreptococcus harei, whose type strain is DSM 10020 (isolated from a sacral sore); Peptostreptococcus ivorii, whose type strain is DSM 10022 (isolated from a leg ulcer); and Peptostreptococcus octavius, whose type strain is NCTC 9810 (isolated from nasal flora). An analysis of their 16S rRNA gene sequences indicated that all three species are related to Clostridium cluster XIII, which includes most species of the genus Peptostreptococcus. The phenotypic characteristics of the new species are described.

A new gram-positive, alkaliphilic, nonsporulating, rod-shaped bacterium is described. The organism was isolated from soda soil (Lake Bogoria, Kenya) and has the following characteristics. It is nonmotile, not acid fast, halotolerant, and microaerophilic, and optimal growth occurs at pH values between 9 and 10. The peptidoglycan type is of type A4α, with lysine as the characteristic diamino acid and glutamic acid as a component of the interpeptide bridge. The major menaquinone is MK-8(H4). The polar lipids are phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, and an unknown phospholipid. 12-Methyltetradecanoic acid is the predominant fatty acid. The G+C content of the DNA is 70 mol%. The results of 16S ribosomal DNA sequence comparisons revealed that strain HKI 0088T represents a new lineage in the order Actinomycetales. Therefore, we concluded that strain HKI 0088T should be assigned to a new genus and species, for which we propose the name Bogoriella caseilytica gen. nov., sp. nov. The type strain and only strain of this genus and species is HKI 0088 (= DSM 11294).

By using gas chromatography and gas chromatography-mass spectrometry, we analyzed the mycolic acid compositions of 18 strains of Nocardia asteroides, 17 strains of Nocardia farcinica, and 17 strains of Nocardia nova classified by numerical taxonomy. These organisms had characteristic mycolic acid compositions. We calculated the peak areas of the molecular species of mycolic acids on gas chromatograms and determined the average total carbon number in each strain. The strains of N. asteroides were divided into five groups, and the type strain belonged to group C54. The strains of N. farcinica were divided into three groups, and the type strain was in group C53. On the other hand, the strains of N. nova differed distinctly from the other two species and belonged mainly to groups C56 and C57. Our detailed analysis of mycolic acids was simple and precise. Therefore, the use of this method should be encouraged more for nocardial classification in combination with DNA or RNA analysis.

Amplified ribosomal DNA restriction analysis (ARDRA) in which the almost complete 16S rRNA gene was used as the target of the PCR assay and randomly and repetitive element-primed PCR were used to characterize 67 strains belonging to the family Alcaligenaceae. Particular emphasis was placed on strains of Bordetella pertussis (13 strains), Bordetella parapertussis (10 strains), and Bordetella bronchiseptica (10 strains) (these organisms were referred to as the B. bronchiseptica group), as well as Bordetella avium (19 strains). Our data indicate that strains belonging to the B. bronchiseptica group behave as a single species comparable to strains of B. avium. ARDRA allowed us to differentiate among all other species as well but was not useful for infraspecific typing. Randomly and repetitive element-primed PCR could be used to distinguish several infraspecific types within B. avium and the B. bronchiseptica group and allowed us to differentiate these two taxa.

Fifty-three strains representing 25 species of aerobic endospore-forming bacteria, of which 11 (37 strains) belong to the genera Aneurinibacillus and Brevibacillus (Bacillus rRNA group 4 of Ash et al. [Lett. Appl. Microbiol. 13:202–206, 1991]), were characterized genotypically by amplified ribosomal DNA restriction analysis (ARDRA) and/or phenotypically by fatty acid methyl ester analysis, sodium dodecylsulfate-polyacrylamide gel electrophoresis of whole-cell proteins, pyrolysis mass spectrometry, 99 API Biotype 100 assimilation tests, and 16 other routine phenotypic tests. ARDRA revealed that Aneurinibacillus aneurinilyticus, Aneurinibacillus migulanus, and Bacillus thermoaerophilus formed a cluster quite separate from Brevibacillus species, supporting the distinction of both genera and the transfer of B. thermoaerophilus to the genus Aneurinibacillus. Two of the species, A. aneurinilyticus (the type species) and A. migulanus, are phenotypically and genotypically quite similar but can be distinguished from each other by several phenotypic characters. Phenotypic differentiation at the generic level is also described.

A strictly anaerobic thiosulfate-reducing bacterium was isolated from a corroding offshore oil well in Congo and was designated strain SEBR 4207T. Pure culture of the strain induced a very active pitting corrosion of mild steel, with penetration rates of up to 4 mm per year. This constitutes the first experimental evidence of the involvement of thiosulfate reduction in microbial corrosion of steel. Strain SEBR 4207T cells were vibrios (3 to 5 by 1 µm), stained gram negative, and possessed lateral flagella. Spores were not detected. Optimum growth occurred in the presence of 3% NaCl at pH 7.0 and 42°C. Strain SEBR 4207T utilized peptides and amino acids, but not sugars or fatty acids. It fermented serine, histidine, and Casamino Acids, whereas arginine, glutamate, leucine, isoleucine, alanine, valine, methionine, and asparagine were only used in the presence of thiosulfate. Peptides were fermented to acetate, isobutyrate, isovalerate, 2-methylbutyrate, H2, and CO2. The addition of either thiosulfate or sulfur but not sulfate increased peptide utilization, growth rate, and biomass; during growth, H2S was produced and a concomitant decrease in H2 was observed. The addition of either thiosulfate or sulfur also reversed H2 inhibition. 16S rRNA sequence analysis indicates that strain SEBR 4207T is distantly related to members of the genus Thermoanaerobacter (83% similarity). Because the phenotypic and phylogenetic characteristics cannot be assigned to any described genus, strain SEBR 4207T is designated as a new species of a new genus, Dethiosulfovibrio peptidovorans gen. nov., sp. nov. Strain SEBR 4207T has been deposited in the Deutsche Sammlung von Mikroorganismen und zellkulturen GmbH (= DSM 11002).

A non-spore-forming, coryneform bacterium, strain TA-12T, was isolated from spoiled off-flavor orange juice. Growth of this organism occurs at pH 3.2 to 7.5, and optimum growth occurs at pH values between 5.5 and 6.5. This organism produces lactic acid, propionic acid, and acetic acid from glucose. It is catalase negative. The cells are heat resistant and can withstand a temperature of 90°C for 10 min. The DNA G+C content is 66.8 mol%. This strain has an MK-9(H4) respiratory quinone system and contains meso-diaminopimelic acid in its cell wall, and ω-cyclohexyl undecanoic acid is the major cellular fatty acid. The results of a phylogenetic analysis of the 16S rRNA gene of this organism indicated that its highest level of homology is its level of homology with the representative of the classical propionibacteria, Propionibacterium freudenreichii (97.1%). Strain TA-12T is phenotypically similar to P. freudenreichii, but it produces a large amount of lactic acid and has a distinct fatty acid composition, acid tolerance, and heat resistance, which differentiate it from P. freudenreichii and other propionic acid-producing bacteria. On the basis of these findings we propose the name Propionibacterium cyclohexanicum sp. nov. for this organism. The type strain is TA-12 (= IAM 14535 = NRIC 0247).

A total of 11 strains of moderately halophilic histamine-producing bacteria isolated from fermented squid liver sauce were studied phenotypically, genotypically, and phylogenetically. These strains are considered members of the genus Tetragenococcus based on their physiological, morphological, and chemotaxonomic characteristics. A16S rRNA gene sequence analysis showed that these strains clustered with, but were separate from, Tetragenococcus halophilus. The results of DNA-DNA hybridization experiments indicated that the new isolates represent a new Tetragenococcus species, for which we propose the name Tetragenococcus muriaticus; strain X-1 (= JCM 10006) is the type strain of this species.

The inter- and intrageneric relationships of members of the genus Acinetobacter were investigated by performing a comparative sequence analysis of PCR-amplified 16S ribosomal DNAs (rDNAs) from 21 strains representing all of the DNA groups that have been described. Phylogenetic treeing confirmed that Acinetobacter spp. form a coherent cluster within the gamma subdivision of the class Proteobacteria that includes strains with overall levels of 16S rDNA sequence similarity of more than 94%. The analysis of intrageneric relationships suggested that the majority of the strains cluster in five clearly distinguishable clusters, and this conclusion was supported by the results obtained with the different methods used for phylogenetic analysis (i.e., the maximum-likelihood, parsimony, and distance matrix methods). The first cluster contains the representatives of DNA groups 2 (Acinetobacter baumannii) and TU13, whereas the second cluster comprises representatives of DNA groups 3, “Close To TU13,” and “between 1 and 3.” The representatives of closely related Acinetobacter DNA groups 8 (Acinetobacter lwoffii) and 9 belong to the third cluster, which includes the representative of DNA group 6 as well. The fourth cluster is formed by DNA groups BJ15, BJ16, and BJ17, and the fifth cluster comprises DNA groups 1 (Acinetobacter calcoaceticus), BJ14,10, and 11. Within the fifth cluster the 16S rDNA sequences of DNA group 10 and 11 strains are nearly identical. The representatives of DNA groups 4 (Acinetobacter haemolyticus), 5 (Acinetobacter junii), 7 (Acinetobacter johnsonii), 12 (Acinetobacter radioresistens), TU14, and TU15 form individual branches that are not significantly affiliated with any of the five clusters identified. Apart from the clustering of the most closely related DNA groups, the general topology of the distance dendrogram revealed some discrepancy with previous DNA-DNA hybridization data, which may point to the inadequacy of comparative 16S rDNA sequence analysis for reflecting true evolutionary relationships of closely related bacterial taxa. Important, however, was the presence of unique sequence motifs in each of the 21 different DNA groups studied, which may be useful for rapid differentiation of DNA groups of the genus Acinetobacter.

A highly motile, medium-size, saccharolytic spirochete was isolated from an advanced human periodontal lesion in medium OMIZ-Pat supplemented with 1% human serum. The growth of this organism is dependent on either glucose, maltose, starch, or glycogen. The cells contain six endoflagella, three per pole, which overlap in the central region of the cell body. On the basis of its cell morphology and enzyme activities, as well as its sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein and antigen profiles, this organism is clearly distinct from all previously cultured spirochetes. The presence of a novel species is supported by the 16S rRNA sequence of this organism, which places it in phylotype 19 of Choi et al. (B. K. Choi, B. J. Paster, F. E. Dewhirst, and U. B. Göbel, Infect. Immun. 62:1889–1895, 1994). The only isolate, strain HA2P, is designated the type strain of a novel species, for which we propose the name Treponema amylovorum.

We propose the name Pseudomonas monteilii for a new species of gram-negative, rod-shaped, motile bacteria that were nonhemolytic on blood agar and were isolated from clinical sources. The 10 strains of P. monteilii were incapable of liquefing gelatin. They grew at 10°C but not at 41°C, produced fluorescent pigments, catalase, and cytochrome oxidase, and possessed the arginine dihydrolase system. They were capable of respiratory but not fermentative metabolism. They did not hydrolyze esculin or starch and were able to use benzylamine, α-aminobutyrate, d-ribose, l-arabinose, butyrate, valerate, isovalerate, isobutyrate, inositol, phenylacetate, d-ala-nine, and amylamine. They possessed l-phenylalanine arylamidase, l-lysine arylamidase, l-alanine arylamidase, γ-glutamyl-transferase, glycyl-phenylalanine arylamidase, l-tryptophan arylamidase, glycyl-l-alanine arylamidase, esterase C4, esterase C6, esterase C8, esterase C9, esterase C10, and esterase C18. DNA relatedness studies revealed that P. monteilii strains formed a homogeneous DNA hybridization group. A total of 57 strains representing previously described or partially characterized taxa belonging to the genus Pseudomonas were 6 to 54% related to P. monteilii. The highest hybridization values were obtained with strains belonging to or related to Pseudomonas putida biovar A. The average G+C content of the DNA was 60.5 ± 0.5 mol% for four of the P. monteilii strains studied. The type strain of P. monteilii is CFML 90-60 (= CIP 104883); it was isolated from bronchial aspirate and has a G+C content of 60 mol%. The clinical significance of these organisms is not known.