- Volume 43, Issue 3, 1993

The taxonomic positions of Flavobacterium sp. strain IFO 14592T (= M-73T) (T = type strain), a dextran-α-1,2-debranching enzyme producer, and Microbacterium sp. strain IFO 15204T (= H-5T), an isolate obtained from corn steep liquor, were investigated; on the basis of the results of chemotaxonomic and phenetic studies and DNA-DNA similarity data, we propose that these bacteria should be classified in the genus Microbacterium as Microbacterium dextranolyticum sp. nov. and Microbacterium aurum sp. nov., respectively. The type strain of M. dextranolyticum is strain IFO 14592, and the type strain of M. aurum is strain IFO 15204.

Twelve strains placed in the genera Flavobacterium, Pseudomonas, and Aureobacterium, including soil isolates, were characterized taxonomically. On the basis of morphological, physiological, and chemotaxonomic data, as well as DNA-DNA hybridization data, we propose that 11 of these strains should be classified in the genus Aureobacterium as new combinations or new species, as follows: Aureobacterium esteraromaticum comb. nov. (type strain, IFO 3751 [= ATCC 8091]), Aureobacterium arabinogalactanolyticum sp. nov. (type strain, IFO 14344), Aureobacterium keratanolyticum sp. nov. (type strain, IFO 13309), Aureobacterium luteolum sp. nov. (type strain, IFO 15074 [= DSM 20143]), Aureobacterium schleiferi sp. nov. (type strain, IFO 15075 [= DSM 20489]), Aureobacterium terrae sp. nov. (type strain, IFO 15300), and Aureobacterium trichothecenolyticum sp. nov. (type strain, IFO 15077 [= JCM 1358]). Whereas the peptidoglycan type of members of this genus is considered to be B2β, the new species A. keratanolyticum was shown to have a new peptidoglycan type, murein variation B2β. An emended description of the genus Aureobacterium is presented.

A numerical analysis based on phenotypic characteristics (89 enzymatic tests and 49 carbohydrate acidification tests), in which experimental strips from Biomerieux-API, La Balme les Grottes, France, were used, was performed to characterize 82 new isolates belonging or related to Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium breve. A total of 72 strains were isolated from child or adult feces, and the other strains were obtained from human vaginas and bronchi. In this study we also included 38 type and reference strains that were representative of all speices of the genus Bifidobacterium and 6 strains belonging to the genus Lactobacillus. DNA-DNA relationships between B. longum and B. infantis were determined by using 19 strains related to these species, as determined by the numerical analysis. The degree of DNA binding was determined by the S1 nuclease method. The phenotypic study revealed that there were six main clusters, which were subdivided into nine subclusters. Subcluster Va contained the type strains of B. longun and B. infantis. The DNA-DNA relatedness values of some of the new isolates were very similar to the DNA-DNA relatedness values of the type strain of B. longum. On the basis of these data, it was difficult to isolate B. infantis strains and then to define B. infantis as a single species separated from B. longum. Subclusters IVb to IVf comprised reference strains of B. breve. Cluster III and subcluster Ia were not identified.

Among the nitrogen-fixing bacteria associated with roots of Leptochloa fusca (L.) Kunth in saline-sodic soils in the Punjab of Pakistan, we repeatedly found yellow-pigmented, straight to curved, gram-negative rods. To group and identify these organisms, we examined morphological, nutritional, and biochemical features and performed polyacrylamide gel electrophoretic analyses of cellular proteins, gas chromatographic analyses of fatty acids, DNA-rRNA hybridizations, and DNA-DNA hybridizations. Our results showed that 11 isolates formed five groups distinct at the species level, with each group containing one to three members. These bacteria constituted a separate rRNA branch in rRNA superfamily III (corresponding to the beta subclass of the Proteobacteria) at a branching Tm(e) level of 67.7°C [Tm(e) is the temperature at which 50% of a hybrid is denatured under standard conditions]. On this branch, the five groups were located in two clusters with Tm(e) values of 79.4 to 80.4°C and around 71.5°C. We propose a new genus, the genus Azoarcus, for these strains. Azoarcus indigens is the type species and has a growth factor requirement; its type strain is strain VB32 (= LMG 9092). A second named species, Azoarcus communis, includes a strain obtained from French refinery oily sludge, strain LMG 5514. Bacteria of this genus have a strictly aerobic type of metabolism, fix nitrogen microaerobically, and grow well on salts of organic acids but not on carbohydrates. Swedish isolates obtained from human sources (E. Falsen group 15 strains LMG 6115 and LMG 6116), as well as “[Pseudomonas] gasotropha” LMG 7583T, were also located on this rRNA branch at a lower Tm(e) level (70.4 to 71.2°C).

Strain BV1 was isolated from the exudate of the footpad abscess of a black vulture (Coragyps atratus). The colonies had a “fried-egg” appearance consistent with that of mycoplasmal species. Electron microscopic examination of the cells revealed irregular elongated or elliptical forms and smaller circular budding processes. Profuse growth was observed in Frey medium supplemented with 20% swine serum at 37°C in a humidified atmosphere of 10% CO2 and air. Typical of mycoplasma, strain BV1 required sterol for growth and catabolized glucose but did not hydrolyze arginie or urea. The guanine-plus-cytosine content of the DNA was 28 mol%. The organism demonstrated the ability to hemolyze, absorb onto, and agglutinate the erythrocytes from several animal species. Strain BV1 was serologically unrelated by the growth inhibition test to previously established Mycoplasma, Acholeplasma, Entomoplasma, and Mesoplasma species, as well as to strains belonging to these genera but not identified to species level. Moreover, BV1 had a 16S rRNA gene with a nucleotide sequence distinct from reported sequences of other mycoplasmas. This organism represents a new species for which the name Mycoplasma corogypsi is proposed. Strain BV1 (ATCC 51148T) is the type strain of Mycoplasma corogypsi sp. nov.

DNA reassociation, a comparative analysis of whole-cell protein patterns, and indirect immunofluorescence methods were used to determine the taxonomic relationships of thermophilic Methanobacterium strains. On the basis of the results dendrograms showing degrees of similarity among the organisms were constructed. The organisms studied are members of three different groups. One group, whose members exhibit 10 to 40% cross-hybridization, includes Methanobacterium wolfeii, “Methanobacterium defluvium” ADZ, and “Methanobacterium thermoflexum” IDZ. The second group contains Methanobacterium thermophilum MT (T = type strain) and Methanobacterium thermoaggregans (levels of DNA relatedness, 30 to 45%). The third group, whose members exhibit 65 to 99% cross-hybridization, includes Methanobacterium thermoautotrophicum ΔHT, F-1, and DV; Methanobacterium thermoformicicum Z-245T; and Methanobacterium thermoalcaliphilum AC60T. The combination of three independent taxonomic methods showed that the group of strains studied is phenotypically, genotypically, and antigenically diverse. The most distinct organisms are M. wolfeii, M. thermoaggregans, M. thermophilum, “M. defluvium,”“M. thermoflexum,” and M. thermoautotrophicum. These species names should be adopted. The results of a DNA-DNA hybridization study (level of hybridization, 99%), an immunological analysis (+3 reaction), and a protein similarity study (level of similarity, 75%) indicate that M. thermoalcaliphilum should be reclassified as a synonym of M. thermoautotrophicum.

DNA-DNA hybridization experiments were carried out in order to clarify the taxonomic relationships between the F38 group of caprine mycoplasmas, the established etiologic agents of classical contagious caprine pleuropneumonia, and Mycoplasma capricolum, an organism associated with septicemia, arthritis, and mastitis in goats and sheep. The taxonomic status of the F38 group has been uncertain, principally because of the serological, genomic, and other properties which it shares with M. capricolum. Tritium-labeled DNAs from the M. capricolum type strain (California kid) and from prototype strain F38 were hybridized with unlabeled DNAs from these two strains and from four other isolates belonging to each group. The results showed consistent DNA relatedness values of about 70% between the F38 and M. capricolum groups, compared with levels of relatedness of about 90 and 85%, respectively, for the strains within each group. In addition, the results of comparisons of these 10 strains in which growth inhibition and immunofluorescence tests were used confirmed the previously reported serological relationships between the two groups and reinforced other observations concerning their shared genomic and cell membrane characteristics, indicating that there is a close taxonomic relationship. However, as the 70% DNA relatedness values between the M. capricolum and F38 groups also indicate a degree of genomic difference inconsistent with a relationship at the species level, we conclude that our findings support previous proposals for classification of the F38 group as a subspecies of M. capricolum. In view of the prospective diagnostic problems, particularly those arising from the serological similarities of two putative subspecies, we believe that further studies should be performed to define additional phenotypic and genotypic properties that would allow more rapid and specific differentiation of the F38 group mycoplasmas.

The 16S ribosomal DNA sequence of Chlamydia pneumoniae was determined and compared with the corresponding gene sequences of Chlamydia psittaci and Chlamydia trachomatis. C. pneumoniae has been reported to exhibit little chromosomal DNA homology with the other chlamydial species, and its phylogenetic relationships within the genus Chlamydia have not been described. A polymerase chain reaction was employed to determine the 16S rRNA gene sequence of C. pneumoniae. Ten primers from the C. psittaci sequences were used to amplify a C. pneumoniae template in overlapping segments of the gene. Sequence data for 1,554 bases indicated that the levels of homology of C. pneumoniae with C. psittaci and C. trachomatis were 96.19 and 94.07%, respectively. These data support the results of previous biochemical and developmental studies indicating that C. pneumoniae is more closely related to C. psittaci than to C. trachomatis.

In consideration of the criticisms of the transfer of Pseudomonas maltophilia to the genus Xanthomonas proposed by J. Swings p. De Vos, M. Van den Mooter, and J. De Ley (Int. J. Syst. Bacteriol. 33:409-413, 1983), a new generic name is created for this taxon. The name Stenotrophomonas is here proposed for the new genus, which includes a single species, Stenotrophomonas maltophilia. This proposal restores the genus Xanthomonas to its former definition (J. Bradbury p. 199-210, in N. R. Krieg and J. G. Holt, ed., Bergey’s Manual of Systematic Bacteriology, 1984) The arguments on which this proposal is based are presented.

The 16S ribosomal DNA sequence of Chlamydia pneumoniae was determined and compared with the corresponding gene sequences of Chlamydia psittaci and Chlamydia trachomatis. C. pneumoniae has been reported to exhibit little chromosomal DNA homology with the other chlamydial species, and its phylogenetic relationships within the genus Chlamydia have not been described. A polymerase chain reaction was employed to determine the 16S rRNA gene sequence of C. pneumoniae. Ten primers from the C. psittaci sequences were used to amplify a C. pneumoniae template in overlapping segments of the gene. Sequence data for 1,554 bases indicated that the levels of homology of C. pneumoniae with C. psittaci and C. trachomatis were 96.19 and 94.07%, respectively. These data support the results of previous biochemical and developmental studies indicating that C. pneumoniae is more closely related to C. psittaci than to C. trachomatis.

Restriction fragment length polymorphisms (RFLPs) of rRNA genes of Chlamydia spp. were analyzed. A Southern analysis of chromosomal DNA digests with cloned rRNA gene probes revealed the presence of one locus for rRNA genes on chromosomal DNAs in Chlamydia psittaci, Chlamydia pecorum, and Chlamydia pneumoniae and two loci in Chlamydia trachomatis. The RFLPs of rRNA genes were characteristic for each Chlamydia sp. DNA probes cloned from flanking regions of the rRNA genes of avian C. psittaci hybridized either with only C. psittaci or with only the avian and ovine abortion strains of C. psittaci. The RFLPs of rRNA genes and the flanking regions provide suitable genetic markers for differentiation of Chlamydia spp.

The 16S ribosomal DNA sequence of Taylorella equigenitalis (formerly Haemophilus equigenitalis), the causative organism of contagious equine metritis, was determined. A phylogenetic analysis of this sequence revealed a phylogenetic position of T. equigenitalis in the β subclass of the class Proteobacteria apart from the position of Haemophilus influenzae, which belongs to the γ subclass of Proteobacteria. A close phylogenetic relationship among T. equigenitalis, Alcaligenes xylosoxidans, and Bordetella bronchiseptica was detected; Spirillum volutans and Chromobacterium fluviatile (Iodobacter fluviatile) were in the same group but slightly removed. This relationship is surprising in view of the considerable differences in the G+C contents of the genomes of these bacteria.