- Volume 47, Issue 1, 1997

Boirelia burgdorferi sensu lato is present all over the world. However, most species belonging to this complex have a limited distribution; the only exception is B. burgdorferi sensu stricto, which is encountered both in Europe and in the United States. The aim of this study was to clarify the way that B. burgdorferi sensu stricto migrated. To do this, we compared the genetic polymorphism of 33 B. burgdorferi sensu stricto strains isolated in Europe and 28 strains isolated in the United States by using both pulsed-field gel electrophoresis and arbitrarily primed PCR. Phylogenetic analysis of each of the two sets of data was performed by a genetic distance method and a parsimony method. The results were consistent and revealed that the American strains were more heterogeneous than the European strains. The hypothesis that B. burgdorferi sensu stricto was introduced to Europe from America rather than vice versa has to be considered.

A blue-pigmented colony that had a metallic copper-colored sheen was isolated in 1973 from a standard spread plate count preparation of oxidation pond sediment. Over the next 11 years, an additional 12 strains of blue-pigmented bacteria were isolated from freshwater samples and compared to several reference strains of bacteria. Morphological and biochemical tests revealed that these 13 isolates were very similar to [Pseudomonas] indigofera ATCC 19706T (T = type strain) and ATCC 14036. A numerical analysis (in which simple matching similarity coefficients were clustered by the unweighted pair group mathematical averaging method) of morphological and biochemical characteristics revealed 90.0% relatedness between the 13 isolates and [P.] indigofera ATCC 19706T and ATCC 14036 and 73.6% relatedness between the 13 isolates and a cluster containing Burkholderia cepacia ATCC 25416T, Janthinobacterium lividum ATCC 12473T, and the Pseudomonas species tested. A phylogenetic analysis, in which both 5S rRNA and 16S rRNA were used, also revealed that the 13 isolates were closely related to each other and to strains ATCC 19706T and ATCC 14036. In addition, both 5S rRNA and 16S rRNA analyses demonstrated that the isolates and strains ATCC 19706T and ATCC 14036 were members of the beta subdivision of the Proteobacteria and were closely related to Chromobacterium violaceum ATCC 12742T but sufficiently distinct to warrant placement in a new genus. Accordingly, we propose that the 13 isolates and strains ATCC 19706T and ATCC 14306 be placed in the genus Vogesella gen. nov., which is named in honor of Otto Voges, who first isolated and described this blue-pigmented eubacterium in 1893. We also propose that [P.] indigofera be renamed Vogesella indigofera comb. nov. and designated the type species of the genus; strain ATCC 19706 is the type strain of this species.

A number of Arococcus-like organisms were recently recognized as human pathogens. Five Aerococcus-like strains were proposed as members of the new species Aerococcus urinae (with type strain E2 [= NCTC 12142]) on the basis of the results of a 16S rRNA sequence analysis. The intraspecies phenotypic and genetic relatedness of 22 selected A. urinae strains was investigated, and a hitherto unrecognized esculin hydrolysis-positive biotype was identified. A total of 14 of the 15 more common esculin-negative strains exhibited very high DNA relatedness as determined by the hydroxyapatite method (the levels of relatedness were greater than 90% in 55 and 70°C reactions, with 1.5% or less divergence in related sequences). The DNA relatedness among the six esculin-positive strains was more heterogeneous, and two DNA hybridization subgroups were formed. Our results are compatible with the hypothesis that both biotypes are members of the single species A. urinae, which contains two or more genetic subspecies. The putative subspecies have not been formally proposed since they cannot be definitively differentiated. The inclusion of A. urinae in the genus Aerococcus is supported by the results of 16S rRNA sequencing. The rRNA sequence data also is compatible with placing both biotypes in a single species.

The Colorado potato beetle, Leptinotarsa decemlineata, is a major holarctic pest of solanaceous crops. Presumably, this insect spread from Solanum species in central America to the Mexican plateau, and this was followed by multiple invasions of North America and Europe. Attempts are being made to control this beetle by using a genetically modified spiroplasma that occurs naturally in its gut. In the current study, spiroplasmas isolated from beetles collected in North America and Poland exhibited serologic (spiroplasma motility inhibition test) and genomic (restriction fragment length polymorphism) profiles that suggest that there were multiple spiroplasma introductions. Two serovars were identified; one is found in northern North America and at high elevations in Poland, and the other is found in southern North America and at low elevations in Poland. The patterns of genovar distribution coincide with the serovar patterns. The existence of such biovars—intraspecific taxal units reflected by serologic and genomic differences—should be taken into consideration when taxonomies are developed and strains are chosen for biocontrol.

A 5.0-kb region containing the hsp70 (dnaK) gene was cloned from Mycoplasma capricolum and sequenced. In addition to the hsp70 gene, this sequence region also contained the complete sequences for the grpE and orfA genes and partial sequences for the clpB and dnaj genes. The order of the above gene sequences in the cloned fragment was found to be clpB-orfA-grpE-hsp70-dnaJ. which is similar to the order seen in various other gram-positive groups of bacteria. The Hsp70 homologs from two mycoplasma species, Mycoplasma capricolum and Mycoplasma genitalium. contain a number of sequence signatures, including the absence of a large insert in the N-terminal quadrant, that are characteristics of the homologs from gram-positive bacteria and archae-bacteria. A detailed phylogenetic analysis based on Hsp70 sequences was also performed. In neighbor-joining and parsimony trees based on Hsp70 sequences, both mycoplasma species branched with the low-G+C-content gram-positive group of bacteria (e.g., Lactobacillus and Erysipelothrix species) in 87% and 96% of the bootstrap replicates, respectively, indicating their close evolutionary relationship to this group. The phylogenetic trees based on Hsp70 sequences show a polyphyletic branching of archaebacteria with the gram-positive species, which is statistically strongly favored.

A systematic phylogenetic analysis of the genus Actinomyces was performed. The 16S rRNA gene sequences of 13 Actinomyces species, an unnamed Actinomyces strain (ATCC 49338), and an Actinomyces-like isolate from sea mammals were determined. Comparative sequence analysis with closely related taxa revealed phylogenetic diversity and internal structure within the genus Actinomyces. In addition, some members of other genera (viz., the genera Arcanobacterium, Mobiluncus, and Rothia) were shown to be phylogenetically intermixed with the Actinomyces species. It was evident from both distance and tree topology considerations that the genus Actinomyces is in urgent need of taxonomic revision and requires subdivision into several genera. Based on the results of the present study it is proposed that Actinomyces bernardiae and Actinomyces pyogenes be assigned to the genus Arcanobacterium as Arcanobacterium bernardiae comb. nov. and Arcanobacterium pyogenes comb. nov., respectively. In addition, a new species, Arcanobacterium phocae, is proposed for Actinomyces-like bacteria isolated from seals.

The primary structures of the 16S rRNA genes of the type strains of Lactobacillus casei and related taxa were determined by PCR DNA-sequencing methods. The sequences of Lactobacillus casei, Lactobacillus zeae, Lactobacillus paracasei, and Lactobacillus rhamnosus were different. The K nuc values ranged from 0.0040 to 0.0126. On the basis of the K nuc values and the levels of DNA-DNA relatedness among the strains of these species, the L. casei-related taxa should be classified in the following three species: L. zeae, which includes the type strains of L. zeae and L. casei; a species that includes the strains of L. paracasei and L. casei ATCC 334; and L. rhamnosus.

Six strains isolated from the intestines of juvenile turbot in a fish hatchery in the north of Spain were found to be phenotypically members of the genus Vibrio. However, the phenotypic traits of these organisms did not place them in any of the currently known Vibrio species. These isolates formed an homogeneous group in which the DNA-DNA similarity values (the differences between the thermal denaturation midpoints of the homologous and heterologous duplexes) with reference strain A089T (T = type strain) ranged from 0 to 1.7°C. The results of a 16S rRNA sequence analysis of A089T placed this strain in the genus Vibrio in the gamma subclass of the Proteobacteria. The closest relative is Vibrio aestuarianus, with a sequence similarity of 97.8%. This group of strains can be easily differentiated from the other Vibrio species by their clear phenotype. We propose the name Vibrio scophthalmi sp. nov. for these strains; the type strain is strain A089 (= CECT 4638).

The heterogeneous bacterial group known as oral streptococci was screened for the presence of cellular polyglycerolphosphate-containing lipoteichoic acid. This compound was detected in phenol extracts of lyophilized cells by an immunoassay in which polyglycerolphosphate-specific monoclonal antibody was used. Polyglycerolphosphate-containing lipoteichoic acid occurred in all 86 strains of oral streptococci examined except the Streptococcus mitis and Streptococcus oralis strains. This confirms the findings of Rosan (B. Rosan, Science 201:918–920, 1978) and Hamada et al. (S. Hamada, J. Mizuno, S. Kotani, and M. Torii, FEMS Microbiol. Lett. 8:93–96, 1980), is consistent with the results of the taxonomic study of oral streptococci performed by Kilian et al. (M. Kilian, L. Mikkelsen, and J. Henrichsen, Int. J. Syst. Bacteriol. 39:471–484, 1989), who emended the descriptions of Streptococcus sanguis, S. oralis, and S. mitis, and reflects the phylogenetic relationship among S. mitis, S. oralis, and Streptococcus pneumoniae.

A new Treponema species, for which we propose the name Treponema medium, was isolated from subgingival plaque from an adult with periodontal disease. The morphological characteristics, differential biochemical characteristics, and protein profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of this organism are described. The guanine-plus-cytosine content of the DNA of T. medium is 51 mol%. The levels of DNA-DNA relatedness of the new species to other Treponema species, including Treponema denticola, Treponema vincentii, Treponema socranskii, Treponema pallidum, and Treponema phagedenis, are less than 30%. A phylogenetic analysis based on 16S rRNA sequences distinguished the new Treponema strain from strains belonging to previously described Treponema species. The type strain of T. medium is strain G7201.

Strains arg-1T (T = type strain) and arg-2T, two new strains of extremely halophilic archaea, were isolated from the soils of the Argentine salt flats. The taxonomic features of arg-1T were similar to, but distinct from, those of the type strain of Haloarcula vallismortis and other Haloarcula species. On the 16S rRNA phylogenetic tree, strain arg-1T formed a cluster together with Haloarcula species. Strain arg-2T differed in its glycolipid composition but still was more closely related to the genus Haloarcula than to other established genera. We propose that strain arg-1T be classified as a member of a new species, Haloarcula argentinensis, and that strain arg-2T be classified as a member of Haloarcula mukohataei sp. nov., although arg-2T may belong to a new genus or a subgenus of the genus Haloarcula. The type strain of H. argentinensis is strain arg-1 (= JCM 9737), and the type strain of H. mukohataei is strain arg-2 (= JCM 9738).

Spiroplasma strain DU-1T (T = type strain), which was isolated from hemolymph of the corn rootworm Diabrotica undecimpunctata (Coleoptera: Chrysomelidae), was serologically distinct from other spiroplasma species, groups, and subgroups. Cells of strain DU-1T were shown by light microscopy to be helical motile filaments. Electron microscopy revealed cells bounded by a single cytoplasmic membrane, with no evidence of a cell wall. The organism was not sensitive to 500 U of penicillin per ml. Strain DU-1T grew well in SM-1, M1D, and SP-4 liquid media, in broth supplemented with 1% bovine serum fraction or conventional horse serum, and under both aerobic and anaerobic conditions. This organism did not appear to have a sterol requirement for growth, as has been reported for several other Spiroplasma species or strains. Optimal growth occurred at 32°C, with a doubling time of 0.9 h; strain DU-1T multiplied at 10 to 41°C but failed to grow at 5 or 43°C. It produced acid from glucose but hydrolyzed neither arginine nor urea. The results of reciprocal serologic tests in which antigens or antisera to established Spiroplasma species, groups, subgroups, and putative groups were used indicated that strain DU-1T was serologically distinct. This organism has a DNA guanine-plus-cytosine content of 25 ± 1 mol% and a genome size of 1,350 kbp. Strain DU-1T is a member of a cluster of fast-growing insect-associated spiroplasmas, as determined by sequence analysis of 16S rRNA. On the basis of the results of this study and previously published data, strain DU-1 (= ATCC 43210) is designated the type strain of a new species, Spiroplasma diabroticae.

Whole-cell protein electrophoretic analysis of the type strain of Streptococcus difficile (LMG 15799) revealed that this organism was indistinguishable from Streptococcus agalactiae strains. Although LMG 15799T (T = type strain) was originally described as serologically untypeable, we found that this strain was a group B streptococcus belonging to the capsular polysaccharide antigen type Ib group. The biochemical reactivity of S. difficile, which differed from the biochemical reactivity of typical S. agalactiae strains mainly by being less versatile, is similar to the biochemical reactivity of other group B, type Ib streptococci isolated from Poikilothermic animals, such as fish and frogs.

A new rapidly growing, scotochromogenic mycobacterium was isolated from urine. This strain is thermophilic (it grows at 65°C), tolerates 5% NaCl, and was unable to utilize any of the sugars tested or citrate or to take up iron. The isolate splits benzamide, urea, nicotinamide, and pyrazinamide and is sensitive to streptomycin, ethambutol, cycloserine, ciprofloxacin, and clarithromycin but resistant to isoniazid, rifampin, and prothionamide. These characteristics clearly place this organism in a new mycobacterial species, which was confirmed by the unique 16S rRNA nucleotide sequence. The high level of similarity between this rapid grower and Mycobacterium xenopi is surprising. For this new rapidly growing scotochromogenic and thermophilic mycobacterium we propose the name Mycobacterium hassiacum sp. nov.

Over a 5-year period, six isolates of a previously unknown nonlipophilic coryneform bacterium were isolated from human clinical specimens. The most characteristic phenotypic reactions of these isolates included slow fermentative acid production from glucose but no acid production from maltose and sucrose and a strongly positive CAMP reaction. Chemotaxonomic investigations revealed that meso-diaminopimelic acid and mycolic acids were present, that palmitic, oleic, and stearic acids were the predominant cellular fatty acids, and that the G+C content was 62 to 64 mol%, characteristics which are consistent with assignment to the genus Corynebacterium. Phenotypically, the unknown coryneform bacterium could be readily differentiated from all other Corynebacterium species. A 16S rRNA gene sequence analysis and quantitative DNA-DNA hybridization experiments demonstrated unambiguously that the unknown coryneform bacterium is a member of the genus Corynebacterium and is genotypically distinct from all other members of this genus. Based on phenotypic and genorypic findings, a new species, Corynebacterium coyleae sp. nov., is proposed. The type strain of C. coyleae is strain DSM 44184 (= CCUG 35014).

Actinomycete strain YU127-1T (T = type strain), which produces zoospores, was isolated from a soil sample. The aerial mycelium of this organism at maturity forms short chains of spores. The hyphae form coils, and sporangia are not observed. Strain YU127-1T contains glutamic acid, glucosamine, glycine, alanine, and meso-diaminopimelic acid in its cell wall (wall chemotype II), 3-O-methylmannose, mannose, xylose, and glucose as whole-cell sugars, menaquinone 10(H4), and glycolyl cell wall polysaccharides and has a guanine-plus-cytosine content of 69.0 mol%. Mycolic acids are absent. Phosphatidylinositol and phosphatidylethanolamine are diagnostic phospholipids. The chemotaxonomic data, except for the lack of arabinose in the whole-cell sugars, indicate that this strain belongs to the family Micromonosporaceae. The morphological and physiological characteristics and chemotaxonomic and phylogenetic data for this strain differ from those of the previously described actinomycetes. We therefore propose a new genus, Spirilliplanes, for this organism; the type species of the genus is Spirilliplanes yamanashiensis sp. nov., and the type strain of S. yamanashiensis is strain YU127-1 (= IFO 15828).

Fusobacterium nucleatum forms part of the resident microbiota in both oral and extraoral sites in humans and animals. It is also involved in infections in such sites. Despite the genetic heterogeneity within the species, it has been divided into five subspecies, the validities of which have been questioned. In the present study, 44 F. nucleatum isolates were examined at 21 enzyme loci by using the allozyme electrophoretic technique to establish an accurate genetic framework for taxonomic purposes. Three distinct genetic clusters were identified; one cluster consisted exclusively of extraoral isolates, another cluster consisted predominantly of human oral isolates, and the third cluster consisted of a single human oral isolate. Our results highlight the urgent need for extensive biochemical, immunological, and epidemiological studies to accurately define the systematics of the genus Fusobacterium based on the framework derived in this study by using 21 independent genetic characteristics.

Twenty-one strains of Saccharomonospora azurea, Saccharomonospora cyanea, Saccharomonospora glauca, Saccharomonospora viridis, and “Saccharomonospora caesia” were examined to evaluate the discriminatory value of 16S ribosomal DNA (rDNA) fingerprints. The 16S rDNAs were amplified by PCR by using oligonucleotide primers complementary to 16S rRNA genes. A restriction fragment length polymorphism (RFLP) analysis of the 16S rDNAs was performed with SmaI and MluI. The four validly described Saccharomonospora species could be differentiated on the basis of their characteristic 16S rDNA restriction patterns. The strains of “S. caesia” gave a restriction pattern identical to that of S. azurea K161T (T = type strain). This result was anticipated from the previous report that S. azurea K161T and the strains of “5. caesia” have identical 16S rRNA sequences. We found that purification of amplified 16S rDNA products following PCR was necessary for our RFLP analysis.

Six Moraxella-like strains that formed a phenotypically homogeneous group were isolated from the nasal flora of healthy goats. Total genomic DNA-DNA hybridization, rRNA gene restriction pattern, DNA base composition, and genetic transformation studies were performed to determine the relationships of these bacteria to species belonging to the genus Moraxella and other fastidious gram-negative species. The new group of isolates was very homogeneous, as shown by rRNA gene restriction fragment length patterns (ribotyping), and these organisms displayed high relative binding ratios (RBRs) to each other in DNA-DNA hybridization experiments (RBRs, = 58%) but distinctly lower levels of DNA homology with all other species investigated. However, the RBRs obtained with species of the genus Moraxella were higher than the RBRs obtained with all other gram-negative strains examined. Although the new strains had most of the Moraxella bovis phenotypic characteristics except nitrate reduction, quantitative and qualitative genetic transformation data led to the conclusion that they belong to a distinct new cluster in the genus Moraxella. The results of this study, combined with the general morphological and phenotypic profiles of the new strains, are consistent with the creation of a new Moraxella species, for which the name Moraxella boevrei is proposed. Strain 88365 (= ATCC 700022 = CCUG 35435 = NCTC 12925 = CIP 104716) is the type strain of M. boevrei.