A practical scheme for identification of the most numerous oral gram negative anaerobic rods

Arch. oral Biol. Vol.10,pp.723-725,1965. PergamcmPressLtd. Printedin Ct. Britain.

A PRACTICAL SCHEME FOR IDENTIFICATION OF THE MOST NUMEROUS ORAL GRAM NEGATIVE ANAEROBIC RODS W. J.

LOESCHE”

and R. J.

GIBBONS

Forsyth Dental Center, Harvard School of Dental Medicine, Boston, Massachusetts, U.S.A. STRICTLY anaerobic gram negative, nonsporulating, catalase negative rods comprise approximately 16 per cent of the total cultivable gingival crevice flora (GIBBONS et al., 1963). They would be expected to represent the genera Bacteroides, Fusobacterium, Vibrio and Spirillum, as these are the only genera possessing species with the above characteristics which have been considered indigenous to man (ROSEBURY, 1962). Recent characterization of the “anaerobic” vibrios have demonstrated that Vibrio sputorum is a microaerophile, since it will grow in the presence of 5% oxygen (LOESCHE,GIBBONSand SOCRANSKY, 1965). However these organisms were included in this study because they have been historically considered as strict anaerobes, and because they fail to grow in air. A total of seventy-five anaerobic gram negative rods were isolated from high dilutions of human gingival debris. Using the scheme described in Fig. 1, it was found possible to assign genus and species designation to almost all strains. Colonies were originally isolated on anaerobically incubated blood menadione agar plates as previously described (GIBBONS et al., 1963). Subsequent biochemical tests were performed in thioglycollate medium without added dextrose (BBL) which was supplemented with 0.2% yeast extract, 0.5% glucose, Spg/ml haemin, 0.25 pg/ml menadione and 0.1% KNO,. The haemin and menadione permitted growth of B. melaninogenicus; haemin stimulated growth of some B. oraiis strains, and the KNO, enhanced growth of V. sputorum. B. melaninogenicus was readily separated from other organisms by its black pigmented colony. The identity of this organism was confirmed as indicated in Fig. 1 in accordance with the findings of SAWYER,MACDONALD and GIBBONS(1962). The remaining nonpigmented organisms could be separated on the basis of terminal pH in glucose broth into nonfermenters, weak fermenters and strong fermenters. All nonfermenters had the characteristics of Vibrio sputorum (LOESCHEet al., 1965) and were further characterised by their failure to produce detectable acid end products, and ability to grow in the presence of 5% oxygen and reduce nitrate. Only a few cells in young cultures displayed active motility.

THE

* Present address: Department of Nutrition, Bldg. 16-129, MassachusettsInstitute of Technology, Cambridge,Massachusetts. 723

W. J.

724

LOESCHE AND

R. J.

GIBBONS

FIG. 1. A practical scheme for the identification negative rods isolated from the oral cavity of man.

of seventy-five anaerobic

gram

Gram negative, anaerobic rods Growth on blood menadione agar plates

I

Nonpig’mented colonies

Black-pigmented colonies

B.

I

Terminal pH in glucose broth carbohydrate fermentation

I

I I I

melnninogenicus

Confirmatory Tests:

6&O I

Nonfermenters

I

6&2 I

5%5’5 strong fermenters

Weak fermenters

1

I

F. nuc~eafum v. spuilorum Acid end products Nonmbtile Butyric I Bacteroides Acetic Propionic Confirmatory Tests: Confirmatory Tests: Formic I Acid end prpducts No detectable I B. orah Unclassified acid end products ?%: Indole pas. Formic Indole neg. Confirmatory Tests: Indolevar. Lactic Gelatin pos.

NO; pos. H,S pos.

NO; neg.

HeS pos.

Somemotility

Ferments Glucose Galactose

Fructose sucrosevar.

Acid end

products Succinic Acetic

Lactic Formic

in young cultures Growth in 5 % 0,

Indole pas. H,S var. NO, neg.

Indole neg. HIS neg. NO7 neg.

M&e S. sputigenum

Confirmatory Tests:

Acid end products Propionic

H,S var.

Acetic

NO; neg.

Indoleneg. NO; pas. H,S neg.

The weak fermenters possessed a fusiform morphology and corresponded with Fusobacterium nucleatum as defined by OMATA and BRAUNBERG (1960) or the Fusobacterium fisiforme of ROSEBURY (1962). They fermented glucose, galactose and occasionally sucrose to pH 64-6~2 and fructose to pH 5.7. Butyric acid was a major end product. The strong fermenters acidified glucose broth to pH 5.0-55 and could be separated into Bacteroides and Spirillum strains on the basis of motility. The majority of the Bacteroides were found to be B. oralis (LOESCHE, SOCRANSKY and GIBBONS, 1964). These were active against a wide variety of carbohydrates, forming succinate as an important acid end product. Nonmotile acidogenic, saccharolytic strains which were variable in fermentation pattern and acid end products were considered as unclassified Bacteroides. The Spirillum strains were active against a wide range of carbohydrates, and reduced nitrate. They possessed characteristics consistent with Spirillum sputigenum strains (MACDONALD, 1953). Acid end products for S. sputigenum have not been reported previously. They were found to consist of propionic and acetic acids in the ratio of 1.6: 1.0, and resembled those of some strains of Selenomonas ruminantium (BRYANT, 1956) further suggesting the similarity of these species. Motility, though important in the differentiation of Spirillum strains, was not reliable in the identification of the Vibrio strains. Newly isolated Vibrio strains have

IDENTIFICATION OF ANAEROBICRODS

725

been shown to be nonmotile in most artificial media and the majority of cells lacked flagella, as determined by electron microscopy (LOESCHE et al., 1965). On microscopic examination they could not be readily distinguised from the Bacteroides. Motility, thus, is a poor criterion for the identification of oral vibrios. This taxonomic analysis permitted the separation of the predominant cultivable anaerobic, gram negative rods of the gingival crevice area into their component genera. The taxonomic boundaries of each genus are sufficiently distinct and the parameters tested so characteristic that all seventy-five strains could be given generic status. Remarkably little variation was observed within each genus as all twentyeight Vibrio strains proved to be V. sputorum, all thirteen Fusobacterium strains were F. nucleatum, twenty-two of thirty-two Bacteroides strains were B. oralis, five were B. melaninogenicus, and five could not be classified. Two Spirillum strains were S. sputigenum. Thus, in most cases, species designation could be given. Acknowledgements-This work was supported in part by Public Health Service research grant D 1471 from the National Institute of Dental Research, and in part by a grant from the Colgate Palmolive Company. One of the authors (RJG) was supported by a Public Health Service career development award from the National Institutes of Health. REFERENCES BRYANT,

M. P. 1956. The characteristics of strains of Selenomonus isolated from bovine rutnen contents. J. Bacterial. 72, 162-167. GIBBONS, R. J., SOCRANSKY, S. S., SAWYER, S., KAPSIMALIS,B. and MACDONALD, J. B. 1963. The microbiota of the gingival crevice area of man II. The predominant cultivable organisms. Arch. oral Biol. 8, 281-289. LOESCHE,W. J., GIBBONS,

R. J., and SOCRANSKY, S. S. 1965. Biochemical characteristics of Vibrio to Vibrio bubulus and Vibrio fetus. J. Bacterial. 89, 1109-l 116. LOESCHE.W. J., SOCRANSKY, S. S. and GIBBONS, R. J. 1964. Bacteroides oralis, proposed new species isolated from the oral cavity of man. J. Bucteriol. 88, 1329-1337. MACDONALD, J. B. 1953. The motile non-sporulating anaerobic rods of the oral cavity. Thesis. University of Toronto, 95 pp. sputorum and relationship

OMATA, R. R. and BRAUNBERG, R. C. 1960. Oral fusobacteria. J. Bacterial. 80, 737-740. ROSEBURY, T. 1962. Microorganisms Indigenous to Man, 144-185. McGraw-Hill, New York. SAWYER, S. J., MACDONALD, J. B. and GIBBONS,R. J. 1962. Biochemical characteristics of Buc-

teroides melaninogenicus.

A study of thirty-one strains.

Arch. oral Biol. 7, 685-691.