- Volume 47, Issue 4, 1997

Two groups of strains isolated from sows were shown to belong to new sublines in the genus Streptococcus. Based on phenotypic and phylogenetic analyses, we propose that these bacteria should be classified as two new species, Streptococcus hyovaginalis sp. nov. and Streptococcus thoraltensis sp. nov. These two species are found in the genital tract, but the capnophilic species S. thoraltensis may also occur in the intestinal tract of pigs. The type strain of S. hyovaginalis is SHV515 (= LMG 14710), and S69 (= LMG 13593) is the type strain of S. thoraltensis.

Spiroplasma strain TALS-2T from the viscera of the striped horsefly, Tabanus lineola, collected in Georgia was serologically distinct from other Spiroplasma species, groups, putative groups, and subgroups. Light and electron microscopy of cells of strain TALS-2T revealed helical motile cells surrounded only by a single cytoplasmic membrane. The organism grew in M1D and SP-4 liquid media. Growth also occurred in 1% serum fraction medium and in conventional horse serum medium. Growth in liquid media was serum dependent. The strain passed through 220-nm filter pores, but was retained in filters with 100-nm pores. The optimum temperature for growth was 30°C. Multiplication occurred at temperatures from 20 to 37°C, with a doubling time at the optimum temperature of 5.6 h in M1D broth. Strain TALS-2T catabolized glucose but hydrolyzed neither arginine nor urea. The guanine-plus-cytosine content of the DNA was 25 ± 1 mol%. The genome size was 1,390 kbp. Six isolates serologically similar to strain TALS-2T were obtained from the same host in coastal Georgia. Three strains closely related to strain TALS-2T were isolated from the horsefly Poeciloderas quadripunctatus in Costa Rica. Strain TALS-2T (= ATCC 51749), a representative of group XXVII, is designated the type strain of a new species, Spiroplasma lineolae (Mollicutes: Entomoplasmatales).

Fourteen strains of a hitherto unknown catalase-positive, aerobic, gram-positive coryneformlike organism were isolated from the milk of sheep with subclinical mastitis from different regions of Spain. The strains phenotypically closely resembled one another and biochemically were similar to Corynebacterium urealyticum and Corynebacterium afermentans subsp. lipophilum. The results of chemotaxonomic investigations were consistent with membership in the genus Corynebacterium, and comparative 16S rRNA gene sequencing studies showed that the unknown bacterium from sheep was indeed a member of the genus Corynebacterium. Within the genus Corynebacterium the new bacterium formed a distinct subline that exhibited >4% sequence divergence with other species. Based on both phenotypic and phylogenetic findings, a new species, Corynebacterium mastitidis, is proposed for the organisms from mastitic sheep. The type strain of C. mastitidis is CECT 4843 (= S-8).

The genetic structure of Bradyrhizobium isolates recovered from three Lupinus species (Lupinus campestris, Lupinus montanus, and Lupinus exaltatus) grown in Mexico was examined. Among 41 Bradyrhizobium isolates, 18 electrophoretic types (ETs) were distinguished by multilocus enzyme electrophoresis of five metabolic enzymes. The mean genetic diversity, 0.64, indicated that there was great genetic diversity in the population sampled. Most isolates (63%) fell into two closely related clusters (clusters I and II) and were the types most frequently isolated from the root nodules of L. montanus and L. campestris. ET cluster III isolates were frequent nodule occupants of L. exaltatus. The isolates also were assigned to three main groups by using Curie point pyrolysis mass spectrometry. In general, the multilocus enzyme electrophoretic data and pyrolysis mass spectrometric data agreed. We determined the 16S rRNA sequences of representative Lupinus isolates and of Bradyrhizobium japonicum USDA 6T and found that the lupine isolates were highly related to the B. japonicum type strain, although not all B. japonicum type strains (subcultures maintained in different bacterial collections) had identical small-subunit rRNA.

We studied two coryneform strains from clinical specimens. These strains had type IV and corynemycolic acids in their cell walls and also had phenotypic characteristics, such as urease activity and fermentation of glucose and sucrose but not trehalose, which did not permit assignment to any previously recognized taxon. According to DNA-DNA hybridization data, these two strains are members of the same species (level of DNA similarity, 86%). Phylogenetic analysis based on comparisons of almost complete small-subunit ribosomal DNA sequences revealed that these strains are closely related to Corynebacterium minutissimum, but DNA relatedness experiments clearly showed that they constitute a distinct new species with a level of DNA relatedness to the C. minutissimum type strain of less than 40%. This new species can be differentiated from C. minutissimum strains by its enzymatic activities and carbon source utilization, and the name Corynebacterium singulare is proposed for it. The type strain is strain IBS B52218 (= CCUG 37330), which was isolated from a semen specimen.

The phylogenetic positions of four rhizobial strains obtained from nodules of common bean plants (Phaseolus vulgaris L.) grown in an Austrian soil and of the Mexican bean isolate FL27 are described. Analysis of the 16S rRNA genes revealed sequences almost identical to that of the Rhizobium gallicum type strain, R602sp, with a maximum of two nucleotide substitutions. Comparison of the 16S rRNA gene sequences with those from other bacteria indicated highest similarity to Rhizobium sp. strain OK-50, Rhizobium leguminosarum IAM 12609, and Rhizobium etli. DNA homology determined by DNA-DNA hybridization was high among the Austrian isolates and R602spT (45 to 90%) and ranged from 21 to 65% with FL27, but hybridization analysis revealed very low homology to the recognized common bean-nodulating species, R. leguminosarum bv. phaseoli, R. etli, and Rhizobium tropici. Ribosomal gene organization was studied by Southern hybridization with the 16S rRNA gene and temperature gradient gel electrophoresis, indicating identical organizations and the presence of three identical 16S rRNA copies in the genome of this species. The six strains investigated showed different plasmid profiles based on their geographical origins. We propose that the Austrian isolates and the Mexican strain FL27 are members of the species R. gallicum.

A new Corynebacterium species, Corynebacterium durum, was isolated from respiratory tract specimens of five human patients. The strains of this species exhibited similar morphologic and biochemical features that differentiated them from all recognized species. Notably, all of these strains developed irregular and strongly adherent colonies under aerobic conditions and produced acid from mannitol and galactose. The cells are long pleomorphic rods with some filaments. This species has characteristics of the genus Corynebacterium, such as 55 mol% guanine plus cytosine in the DNA and the presence of corynomycolic acids, meso-diaminopimelic acid, arabinose, and galactose in the cell wall. These isolates formed a homogeneous group in which the DNA-DNA similarity values (as determined by an SI nuclease procedure) compared with reference strain IBS G15036T (T = type strain) ranged from 71 to 100%. The analysis of the nearly complete 16S rRNA gene sequence of IBS G15036T indicated that this new species represents a distinct taxon within the genus Corynebacterium. This new species can be identified on the basis of its colony morphology, fermentation of sugars, and enzymatic activities. Strain IBS G15036 (= CCUG 37331) is the type strain of C. durum.

Among the three main species of Borrelia burgdorferi sensu lato associated with Lyme borreliosis, B. burgdorferi sensu stricto (B. burgdorferi) is the sole species present both in North America and in Europe, where Borrelia garinii and Borrelia afzelii also occur. The greater genetic diversity together with the greater clinical polymorphism observed in the Old World suggests that this is the birthplace of the complex B. burgdorferi sensu lato. However, the genetic proximity of some North American and European B. burgdorferi strains is quite mystifying. A previous study of the whole genome (M. Foretz, D. Postic, and G. Baranton, Int. J. Syst. Bacteriol. 47:11-18, 1997) compared the diversity of North American and European B. burgdorferi strains. To further investigate the geographical origin and the migration of B. burgdorferi, we have focused on the study of the single variable and highly adaptive gene ospC. Both approaches demonstrated the greater diversity of North American strains and the close relatedness between European strains and between some isolates from the two areas. We discuss the significance of these features and suggest that they might be evidence of the anteriority of North American B. burgdorferi strains.

A new thermophilic, anaerobic rod-shaped bacterium, strain BI429T was isolated from the gills of a deep-sea vent hydrothermal mussel, Bathymodiolus brevior, from the Lau Basin (Southwestern Pacific Ocean). Pheno-typically, this isolate exhibited characteristics similar to those described for members of the order Thermotogales. This organism was identified as a member of the genus Thermosipho on the basis of the presence of the typical outer sheath-like structure (toga), its 16S rRNA sequence, and its ability to grow on carbohydrates (sucrose, starch, glucose, maltose, lactose, cellobiose, and galactose). The cells of this organism were gram negative and rod shaped and generally occurred singly or in pairs, rarely occurring as chains with a maximum of five rods. At the optimum temperature for growth (70°C), optimum pH (6.5), and optimum salinity (30 g of NaCl per liter), the doubling time was 100 min. In spite of the high percentage of similarity of its 16S rRNA sequence with that of Thermosipho africanus (98.6%), the weak level of DNA-DNA reassociation with this strain (2%) and particular physiological characteristics allowed us to differentiate this new organism from the sole species of the genus Thermosipho previously described (T. africanus). On the basis of these observations, we propose that the new organism should be described as a new species, Thermosipho melanesiensis. The type strain of T. melanesiensis is BI429.

A mesophilic, gram-negative, vibrio-shaped, marine, acetate-oxidizing sulfate reducer (strain B54) was isolated from a water-oil separation system on a North Sea oil platform. The optimum conditions for growth were 33°C, pH 6.8 to 7.0, and concentrations of NaCI and MgCl2 6H2O of at least 1 and 0.3%, respectively. Of various organic acids tested, only acetate was used as an electron and carbon source. The presence of 2-oxoglutarate:dye oxidoreductase suggests acetate oxidation via an operative citric acid cycle. Even though growth of most Desulfobacter strains (including strain B54) did not occur on hydrogen, hydrogenase was detected at low activity. The growth yields were 4.6, 13.1, and 9.6 g of (dry weight) cells per mol of acetate oxidized with sulfate, sulfite, and thiosulfate, respectively, as electron acceptors. Strain B54 was able to fix dinitrogen. Desulforubidin and cytochromes of the c and b types were present. The G+C content of the DNA was 47 mol%. Strain B54 is most closely related to Desulfobacter latus, with a 16S rDNA sequence similarity of 98.1%. The DNA-DNA relatedness between them was 40.5%. On the basis of differences in genotypic, pheno-typic, and immunological characteristics, we propose that strain B54 is a member of a new species, D. vibrioformis. It can be easily identified and distinguished from other Desulfobacter species by its large, vibrio-shaped cells.

A novel actinomycete was isolated from compost soil and was studied taxonomically and phylogenetically. Cells of this organism were gram positive, not acid fast, nonmotile, nonsporulating, irregular coccoid to short rod shaped, and microaerophilic. The cell wall peptidoglycan contained lysine and was cross-linked via an l-Lys←.-Ser←D-Asp interpeptide bridge. The major menaquinone was MK-8(H4). The polar lipids were phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, and two unknown phospholipids. Mycolic acids were absent The cellular fatty acid profile was complex, with large amounts of saturated and monounsaturated straight-chain acids and smaller amounts of iso and anteiso branched-chain acids. The G+C content of the DNA was 66 mol%. Comparative 16S ribosomal DNA studies revealed that strain HKI0089T represents a novel lineage within Actinobacteria (32) distinct from all previously described genera and most closely related to members of the genera Kytococcus, Dermacoccus, and Dermatophilus of the family Dermatophilaceae. On the basis of our results, we suggest that strain HKI 0089 should be classified in a new genus and species, for which we propose the name Demetria terragena. The type strain and the only strain of the genus and species is HKI 0089 (DSM 11295).

Almost complete 16S ribosomal DNA (rDNA) sequences were determined for the type strains of nine species belonging to the genus Desulfotomaculum and for seven strains described as strains of this genus. The sequences were compared with previously published 16S rDNA and rRNA sequences of the type strains of the other species of the genus. The majority of the species form a phylogenetically coherent cluster within the Clostridium-Bacillus subphylum of gram-positive bacteria. The cluster consists of phylogenetically well-separated lineages containing (i) Desulfotomaculum nigrificans, Desulfotomaculum aeronauticum, and Desulfotomaculum ruminis, (ii) Desulfotomaculum geothermicum, Desulfotomaculum thermosapovorans, and Desulfotomaculum sapomandens, (iii) Desulfotomaculum kuznetsovii, Desulfotomaculum australicum, and Desulfotomaculum thermocisternum, (iv) Desulfotomaculum thermobenzoicum and Desulfotomaculum thermoacetoxidans, and (v) Desulfotomaculum acetoxidans. Some as-yet-undescribed Desulfotomaculum strains are phylogenetically well-separated from strains of the described species. Desulfotomaculum guttoideum shares extremely high 16S rDNA similarity with certain Clostridium species (e.g., Clostridium sphenoides and Clostridium celerecrescens) and is most likely a misidentified species. Desulfotomaculum orientis represents a new genus which branches most closely to the genus Desulfitobacterium. The name Desulfosporosinus orientis gen. nov., comb. nov., is proposed for this taxon.

An intracellular symbiotic bacterium was isolated from the hemolymph of Triatoma infestans and cultured in an Aedes albopictus cell line. 16S ribosomal DNA sequence analysis revealed that the bacterium was a member of the γ-3 subgroup of the class Proteobacteria, having 96.2% sequence identity with the most closely related bacterium, Arsenophonus nasoniae, the causative agent of the son-killer trait in the parasitoid wasp Nasonia vitripennis. These bacteria share morphological features and a common tissue distribution and transmission mode. The A. nasoniae-T, infestans symbiont branch represents a lineage of insect symbionts which may be capable of horizontal transmission between phylogenetically distant host insects. We propose that the intracellular symbiont from T. infestans be classified as “Candidatus Arsenophonus triatominarum.” The bacterium found in the hemocytes of T. infestans is designated the type strain of this species.

All living organisms fall into discrete clusters of closely related individuals on the basis of gene sequence similarity. Evolutionary genetic theory predicts that in the bacterial world, each sequence similarity cluster should correspond to an ecologically distinct population. Indeed, surveys of sequence diversity in protein-coding genes show that sequence clusters correspond to ecological populations. Future population surveys of protein-coding gene sequences can be expected to disclose many previously unknown ecological populations of bacteria. Sequence similarity clustering in protein-coding genes is recommended as a primary criterion for demarcating taxa.

The recently reported chemotaxonomic and genotypic description of two well-separated subgroups (I and II) in Aeromonas eucrenophila and their affiliation to Aeromonas encheleia and the unnamed Aeromonas DNA hybridization group (HG) 11 (G. Huys, M. Altwegg, M.-L. HÄnninen, M. Vancanneyt, L. Vauterin, R. Coopman, U. U. Torck, J. Lüthy-Hottenstein, P. Janssen, and K. Kersters, Syst. Appl. Microbiol. 19:616-623, 1996) has questioned the original species descriptions of A. eucrenophila and A. encheleia. In order to elucidate the uncleartaxonomic status of these taxa in the genus Aeromonas, we have further investigated a collection of 14 reference strains and 14 related isolates encompassing the taxa A. eucrenophila subgroups I and II, A. encheleia, and HG11 by DNA-DNA hybridization (on 17 of the 28 strains) and phenotypic characterization (on all 28 strains). Genotypically, the investigated strains could be grouped into two DNA hybridization groups that exhibited between-group homologies ranging from 42 to 52%. The members of DNA homology group I (DNA binding, 76 to 100%) were strains of A. eucrenophila subgroup I, including the type strain LMG 3774, and two A. eucrenophila-like isolates, leading to the conclusion that these strains should be considered true representatives of the species A eucrenophila. The strains of A eucrenophila subgroup II, HG11, and A. encheleia, on the other hand, were closely joined in DNA homology group II (DNA binding, 74 to 105%) together with two presumptive A. encheleia isolates. The fact that strain LMG 16330T A. encheleia was the only type strain residing in DNA homology group I1 implies that HG11 and A. eucrenophila subgroup II should be classified in the species A. encheleia. Except for the somewhat aberrant phenotypic positions of HG11 strains LMG 13075 and LMG 13076, the establishment of DNA homology groups I and II was supported by the delineation of phena 1 and 2 (level of correlation, 90%), respectively, as revealed by numerical analysis of 136 phenotypic test results. These data indicate A. eucrenophila and A encheleia are phenotypically highly related but can be easily separated by testing the production of acid from d-cellobiose. and lactose and the assimilation of d-cellobiose. Extended descriptions of both species are given.

We examined the taxonomic position of seven Aeromonas isolates, recovered from Flemish and Scottish drinking water production plants and reservoirs, which were previously recognized by numerical analysis of genomic AFLP fingerprints as members of an unknown Aeromonas taxon that most closely resembled the species Aeromonas bestiarum (DNA hybridization group [HG] 2). The new phenotypic and DNA-DNA hybridization data obtained in this study show that the A. bestiarum-like strains constitute a separate Aeromonas species, for which the name Aeromonas popoffii sp. nov. is being proposed. The new species exhibited an internal DNA relatedness ranging from 79 to 100% and was 22 to 63% related to the type or reference strains of other Aeromonas spp. The highest DNA binding values were determined with A. bestiarum (51 to 63%), followed by Aeromonas hydrophila sensu stricto (HG1; 50 to 60%) and Aeromonas salmonicida (HG3; 39 to 55%). Although fingerprints generated by ribotyping and cellular fatty acid analysis often were highly similar, minor differences between the respective fingerprints were of significance for the differentiation of A. popoffii from its closest taxonomic neighbors, HG1, HG2, and HG3. Phenotypically, all seven strains of A. popoffii were positive for acid and gas production from d-glucose and glycerol, growth in KCN broth, arginine dihydrolase, DNase, Voges-Proskauer reaction, and resistance to vibriostatic agent O/129 and ampicillin but displayed negative reactions for production of urease, tryptophan deaminase, ornithine decarboxylase, and lysine decarboxylase (LDC). None of the strains displayed strong hemolytic activity. The lack of d-sucrose fermentation and LDC production and the ability to utilize dl-lactate as the sole energy and carbon source were useful characteristics for the biochemical separation of A. popoffii from A. bestiarum. Other Aeromonas spp. could be differentiated phenotypically from the new species by at least two features. The chromosomal G+C content of A. popoffii ranges from 57.7 to 59.6 mol%. Strain LMG 17541 is proposed as the type strain.

The results of investigations on the morphology, physiology, pigment composition, light-harvesting antenna and reaction center organization, and electron carriers of five Erythromicrobium representatives, and on phylogenetic relations among them, are summarized. On the basis of clear phenotypic differences and distinct phylogenetic positions shown by 16S ribosomal DNA analysis, the tentative species “Erythromicrobium sibiricum” and “Erythromicrobium ursincola” are formally described as the type species of two new genera: Sandaracinobacter sibiricus gen. nov., sp. nov., and Erythromonas ursincola gen. nov., sp. nov., respectively. The genus Erythromicrobium is at present composed of the type species, E. ramosum, and two species, “E. hydrolyticum” and “E. ezovicum,” whose nomenclature is yet to be validated. All species studied group within the α-4 subclass of Proteobacteria.

AFLP is a novel genomic fingerprinting method based on the selective PCR amplification of restriction fragments. The usability of this method for the differentiation of genomic species in the genus Acinetobacter was investigated. A total of 151 classified strains (representing 18 genomic species, including type, reference, and field strains) and 8 unclassified strains were analyzed. By using a single set of restriction enzymes (HindIII and TaqI) and one particular set of selective PCR primers, all strains could be allocated to the correct genomic species and all groups were properly separated, with minimal intraspecific similarity levels ranging from 29 to 74%. Strains belonging to genomic species 8 (Acinetobacter lwoffii sensu stricto) and 9 grouped together in one cluster. The closely related DNA groups 1 (Acinetobacter calcoaceticus), 2 (Acinetobacter baumannii), 3, and 13TU (sensu Tjernberg & Ursing 1989) were clearly distinguishable, with intraspecific linkage levels above 50%. Strains of the independently described genomic species 13BJ (sensu Bouvet & Jeanjean 1989) and 14TU linked together at a relatively low level (33%). Although a previous DNA-DNA hybridization study seemed to justify the unification of these genomic species, AFLP analysis actually divides the 13BJ-14TU group into three well-separated subgroups. Finally, four unclassified strains obtained from diverse sources and origins grouped convincingly together, with a similarity linkage level of approximately 50%. These strains showed no similarities in their AFLP patterns with any of the other 155 strains studied and may represent a thus-far-undescribed Acinetobacter species. Based on these results, AFLP should be regarded as an important auxiliary method for the delineation of genomic species. Furthermore, because AFLP provides a detailed insight into the infraspecific structure of Acinetobacter taxa, the method also represents a highly effective means for the confirmation of strain identity in the epidemiology of acinetobacters.

We performed an integrated genotypic and phenotypic analysis of 128 strains of the genera Burkholderia, Ralstonia, and Pseudomonas in order to study the taxonomic structure of Burkholderia cepacia and its relationships with other Burkholderia species. Our data show that presumed B. cepacia strains isolated from cystic fibrosis patients belong to at least five distinct genomic species, one of which was identified as Burkholderia vietnamiensis. This group of five phenotypically similar species is referred to as the B. cepacia complex. The name Burkholderia multivorans is proposed for one of these genomic species, which was formerly referred to as B. cepacia genomovar II; the remaining B. cepacia groups are referred to as genomovars I, III, and IV, pending additional differential phenotypic tests. The role and pathogenic potential of each of these taxa, particularly in view of the potentially fatal infections in cystic fibrosis patients, need further evaluation. The data presented also demonstrate that Pseudomonas glathei and Pseudomonas pyrrocinia should be reclassified as Burkholderia species.