- Volume 24, Issue 1, 1974

Previous studies by us and our colleagues suggested three distinctive deoxyribonucleic acid (DNA) homology groups among the bifidobacteria; these were provisionally named “catenulatum, “dentium,” and “angulatum.” One hundred eighty-four strains isolated from sewage, in addition to many of the strains from the previous study, were investigated and their DNA homology relationships were assessed using 23 reference systems. Strains in the catenulatum group were found not to differ significantly from those of Bifidobacterium adolescentis Reuter in their main physiological characters, such as sugars fermented and temperature, pH, and oxygen relationships; however, their DNA reciprocal homology is only some 50%, their guanine plus cytosine values were 54.7±0.2 and 59.4±0.4 mol %, respectively, and there were some morphological differences between them. The DNA of the dentium group has only about 45% homology with the DNA of B. adolescentis and is even less related to other members of the genus. The dentium strains can also be distinguished from other bifidobacteria by means of their sugar fermentations. The DNA of the angulatum group has little or no homology with that of any other bifidobacteria; the angulatum group also has a distinctive pattern of sugar fermentation and a unique morphology, resembling that of the coryneform bacteria. The three groups are named and described as new species of the genus Bifidobacterium: B. catenulatum, B. dentium, and B. angulatum. The type strains of these species are B669 (= ATCC 27539), B764 (= ATCC 27534), and B677 (= ATCC 27535), respectively. DNA-DNA homology relationships are basic to currently proposed species concepts, and data are presented confirming the reliability of critical experimental parameters influencing filter-bound DNA and thus the final relative homology values (e.g., temperature and time of incubation and annealing of DNA in the presence of homologous and heterologous competitive or nonspecific DNA, and the replicability of homology values using different homologous DNA preparations with single DNA competitor and reference DNA).

Twenty-two strains of bifidobacteria from the feces of chickens, rats, and rabbits, and from sewage, formed a single deoxyribonucleic acid (DNA) homology group displaying complete homology with Bifidobacterium longum subsp. animalis Mitsuoka biotype a strain R 101-8 (=ATCC 25527). The DNA relatedness of this group of strains to the known species of the genus Bifidobacterium ranged from 5 to 40%. Therefore, we propose that B. longum Reuter subsp. animalis Mitsuoka biotype a should be elevated to species rank as Bifidobacterium animalis (Mitsuoka) comb. nov. The type strain is ATCC 25527. Also, a small number of strains isolated from sewage are suggested as probably being distinctive bifidobacteria and are placed in two new groups designated “ minimum” and “subtile” The reference strains for these groups are F392 (=ATCC 27538) and F395 (=ATCC 27537), respectively.

Among 130 strains of bifidobacteria isolated from the feces of rabbits, 26 were distinctive in: (i) having large cellular dimensions (4 to 5 by 17 to 20 μm); (ii) requiring an initial pH of 5.3 to 5.5 for optimum growth; and (iii) having a need for Tween 80 for optimal development in Trypticase-phytone-yeast extract-glucose medium. The other characters of these 26 strains, such as their temperature and oxygen relationships, catalase production, nitrate reduction, end products from glucose fermentation, enzymes of the hexose catabolic pathway, the percent of guanine plus cytosine in the deoxyribonucleic acid (DNA), etc., are similar to those of most bifidobacteria. However, DNA-DNA hybridization studies revealed little or no polynucleotide sequence similarity to any other members of the genus Bifidobacterium. We therefore regard these 26 strains as constituting a new species, for which we propose the name Bifidobacterium magnum. The type strain of this new species is RA3 (= ATCC 27540).

Ninety-five strains of Bacteroides hypermegas Harrison and Hansen and of related organisms were isolated from the feces of humans, pigs, dogs, and chickens and were compared with three authentic strains of B. hypermegas. All isolates shared certain features in common: They were gram-negative, nonspore-forming, nonmotile, strictly anaerobic, stout rods which fermented a wide range of carbohydrates, had low final pH values in glucose broth (4.1 to 4.8), and were markedly stimulated by the presence of glucose in the medium. On the basis of a number of additional features, two distinct groups could be differentiated: 52 strains isolated from humans, chickens, and dogs were identical with B. hypermegas; the remaining 43 strains isolated from humans and pigs were considered to form a new species, for which the name Bacteroides multiacidus was proposed. B. multiacidus differs from B. hypermegas in that the former has the following: Smaller size of cell (0.8 to 1.5 μm by 3.0 to 20.0 μm); positive nitratase activity; low final pH value (4.1 to 4.3) in glucose broth; ability to produce major amounts of lactic and acetic acids with moderate amounts of succinic acid but no propionic acid from glucose; ability to grow in the presence of sodium propionate (15 mg/ml) as well as to ferment melezitose, dextrin, and starch; and higher guanine plus cytosine content in the deoxyribonucleic acid (56 to 58 mol %). The type strain of B. multiacidus is A 405-1 (= ATCC 27723 = NCTC 10934).

Ribosomes from erwiniae, pectobacteria, and other Enterobacteriaceae were compared by two criteria: (i) disc gel electrophoresis of 50S subunit proteins, and (ii) Ouchterlony gel diffusion of intact ribosomes. There were no significant differences in the electrophoretic protein profiles of strains of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, or Enterobacter agglomerans (synonym: Erwinia herbicola). The only signficant profile differences occurred with the faster-migrating proteins of enterobacteria grouped according to plant or animal source. Several non-enterobacteria also had similar over-all profiles; however, specific differences were evident. Immuno-diffusion data were effective in establishing relatedness at the subspecies level. A single antigenic determinant was responsible for the specificity and was found to differ in its sensitivity to treatment with trypsin, ribonuclease, or heat.

Gray and Thornton’s strain (ATCC 17041) of Mycobacterium coeliacum, here designated as the type strain of this species (which was later transferred to the genus Nocardia as Nocardia coeliaca), is a strain of M. rhodochrous (tentative generic assignment). The strain producing the antibiotic nocardin and similar strains, however, have been mistakenly labeled N. coeliaca. This report presents some of the characteristics of 27 of these mislabeled strains and demonstrates their close relationship to the type strain of N. autotrophica (Takamiya and Tubaki) Hirsch. The nocardin-producing strain and others like it are, therefore, assigned to N. autotrophica.

The serological relationship between 125 strains of Nocardia and Mycobacterium was analyzed by means of the comparative immunodiffusion technique. Two reference precipitation systems, N. corallina and “M.” pellegrino, were utilized. Most of the tested strains had one to three precipitinogens in common with the N. corallina and the “M.” pellegrino reference strains. Most of the mycobacterial and most of the nocardia strains examined seemed to be serologically related to the two reference strains to an equally large extent. Eight strains, either designated N. corallina, N. rubra, or “M.” rhodochrous, revealed a close serological relationship with the N. corallina reference strain, and so it might be justified to consider these strains as belonging to one taxonomic group. Ten other strains, designated N. rubra, “M.” pellegrino, or “M.” rhodochrous, displayed a close serological relationship with the “M.” pellegrino reference strain, and they may therefore be regarded as belonging to another taxonomic group. Eight strains with the designations N. corallina, N. rubra, or “M.” rhodochrous showed little serological similarity to either of the two reference strains. Thus, the present taxonomy of a number of strains of Nocardia and Mycobacterium seems questionable since some strains were found to be serologically different, although referred to the same species, whereas some strains, referred to different species, were serologically closely related. The classification and nomenclature of such strains should, therefore, be reconsidered.

Fifteen Acholeplasma strains, including some belonging to A. laidlawii, A. axanthum, A. granularum, or A. modicum, incorporated radioactivity into their lipids when grown in a medium containing [1-14C] acetate. The majority of the radioactivity was found in the glycolipid (43%) and the polar lipid (55%) fractions. The amount of radioactivity incorporated into the lipids (disintegrations per minute per milligram of lipid) of strains of 6 Mycoplasma species was less than 1% of the amount found in the 15 Acholeplasma strains. The ability to synthesize lipids from acetate is a useful criterion for differentiating the genus Acholeplasma from Mycoplasma.

Micrococci were commonly isolated from the skins of people living in various regions of the United States. Not all micrococci isolated in this investigation could be identified with the currently recognized species of Micrococcus, viz., M. luteus, M. varians, or M. roseus, and these micrococci therefore became the subject of further taxonomic study. As a result of this study, two new species are proposed: Micrococcus lylae and M. kristinae. The type strains of these species are ATCC 27566 and ATCC 27570, respectively. Numerous strains were isolated that were similar to M. sedentarius or M. nishinomiyaensis, species that were previously represented by only single strains. (ZoBell’s strain 541 [ATCC 14392; CCM 314] is designated herein as the type strain of M. sedentarius.) A few micrococci were left unclassified. A variety of morphological, physiological, biochemical, and genetic characters were examined for their use as taxonomic criteria, and key characters, many of which can be determined by simple laboratory procedures, were selected for species differentiation. The more sophisticated studies of aliphatic hydrocarbons and cell-wall peptidoglycans were also very useful in the taxonomy of the micrococci. The predominant micrococci found on human skin were M. luteus and M. varians.

The properties of an extremely thermophilic bacterium isolated from water at a Japanese hot spring and previously named Flavobacterium thermophilum are described. The cells are gram-negative, nonsporulating, aerobic rods containing yellow pigment. The optimum temperature for growth is between 65 and 72 C, the maximum being 85 C and the minimum being 47 C. The guanine plus cytosine content of the deoxyribonucleic acid of the thermophile is 69 mol %. This microorganism is sensitive to various antibiotics including those which are known to be rather ineffective against gram-negative bacteria. Spheroplast-like bodies are formed upon treating intact cells with egg-white lysozyme at 60 C. The spheres are osmotically more stable than mesophile protoplasts, and their rupture under hypotonic conditions is not complete unless 0.5% Brij 58 is added to the suspension. Bulk protein extracted from this thermophile is much more stable to heat than mesophile proteins, and only about 10% of the total protein is denatured by heating at 110 C for 5 min. Nevertheless, the amino acid composition of the bulk protein is similar to that of mesophile proteins. As the properties of this organism are similar to those of Thermus aquaticus (Brock and Freeze) and inasmuch as Flavobacterium is a poorly defined genus, this thermophilic microorganism is transferred to the genus Thermus as T. thermophilus (Yoshida and Oshima) comb. nov. The type strain is HB8 (=ATCC 27634).

A study of the tricarboxylic acid cycle enzymes of Brevibacterium leucinophagum ATCC 13809, the type strain of this species, together with morphological, physiological, and electron microscopy studies indicated that, contrary to its present classification, B. leucinophagum is gram negative and probably is better classified in the genus Acinetobacter.

Unfixed fruiting bodies of myxobacters have been viewed in great detail with a scanning electron microscope. Specimens of species of the genera Myxococcus, Chondrococcus, Archangium, Stelangium, Melittangium, Cystobacter, Polyangium, Stigmatella, and Chondromyces were examined. The desirability of using the scanning electron microscope for the study of the gross morphology of myxobacter structures has been clearly demonstrated.

Five glucose-fermenting Mycoplasma strains were isolated from the pharynx of dogs. Biological and serological studies demonstrated that this group of mycoplasmas was different from the established canine species and from other glucose-fermenting Mycoplasma species of animal or human origin. The isolates appear to constitute a new species, for which the name Mycoplasma molare is proposed (L. adj. molaris, millst one-like; intended to refer to the heavy film reaction produced by the strains which resembles the pattern on the surface of a millstone). The type strain of M. molare is H 542 (=ATCC 27746; = NCTC 10144).

Sixty-two cultures representing 59 Mycoplasma strains from goats and sheep were purified, biochemically characterized, and serologically identified. All of the strains were identified as belonging to Mycoplasma agalactiae subsp. agalactiae (Wroblewski) Freundt by the metabolic-inhibition test. The serological reactions of the strains were specific and reproducible, and their biochemical characteristics were identical. The antigenic relationships among the strains investigated, other caprine and ovine mycoplasmas, and M. mycoides subsp. mycoides are discussed.

Fifty-seven bacterial strains, including corynebacteria, “plant pathogenic corynebacteria,” mycobacteria, and nocardias, were examined (aerobically and anaerobically) for their capacity to produce a glucan phosphorylase (α-1,4-glucan:orthophosphate glucosyl transferase, EC 2.4.1.1), which synthesizes an iodinophilic starch-like polysaccharide from glucose-1-phosphate. Only Corynebacterium diphtheriae, C. belfanti, C. ulcerans, C. ovis, C. kutscheri, C. minutissimum, and two (out of eight) strains of C. renale yielded positive results. The presence of glucose-1-phosphate-induced starch-like material appears to be a valid additional means of distinguishing most true corynebacteria from mycobacteria and nocardias.