Reisolation of Staphylococcus salivarius from the

JOURNAL OF BACTERIOLOGY, Nov. 1967, p. 1281-1286 Vol. 94, No. 5 Copyright 1967 American Society for Microbiology Printed in U.S.A. Reisolation of Staphylococcus salivarius from the Human Oral Cavity D. F. GORDON, JR. Forsyth Dental Center, Boston, Massachusetts 02115 Received for publication 3 July 1967 Twenty-four strains of gram-positive facultative cocci, arranged primarily in small clusters, were isolated from the surface of the human tongue. With the exception of 14 catalase-negative isolates, these strains were identical in cultural and biochemical characteristics and in deoxyribonucleic acid base composition. All cultures produced viscous growth in both liquid and agar media. They fermented glucose anaerobically, reduced nitrate beyond nitrite, were benzidine-positive, and failed to grow in the presence of 5% NaCl or at 45 C. In addition, they exhibited guanine plus cytosine (G + C) contents of 55.4 to 58.3%. These isolates differed from strains of pediococci, aerococci, and micrococci which were included for comparison. On the basis of G + C content, these organisms appear to be intermediate between micrococci and staphylococci; however, on the basis of anaerobic glucose fermentation, it is suggested that they be placed in the genus Staphylococcus. It is proposed that they be recognized as S. salivarius. When gram-positive facultative cocci, other than streptococci, have been isolated from the oral cavity, different investigators have assigned them to one or more of the following genera: Staphylococcus, (1, 28, 33, 35), Micrococcus (27), Gaffkya (28, 33), Sarcina (28, 33), or Pediococcus (31). Recently, Gordon and Gibbons (14) isolated from the human tongue catalase-negative cocci that were similar to micrococci or staphylococci in morphology. These catalase-negative isolates comprised about 3.5% of the predominant cultivable microorganisms on the human tongue. Preliminary findings indicated that these organisms, as well as some catalase-positive cocci also isolated from the human tongue, were similar, but not typical of any recognized species. Therefore, the present study was initiated in an attempt to clarify the taxonomic position of these organisms on the basis of cultural and biochemical characteristics and deoxyribonucleic acid (DNA) base composition. Catalase-negative strains of pediococci and aerococci were included for comparative purposes. A strain of Micrococcus varians (29) was also included when, during the present study, it was found that the oral isolates exhibited a similar DNA base composition. MATERIALS AND METHODS Five catalase-positive and four catalase-negative strains of oral cocci were obtained from the study of the predominant cultivable microflora of the human tongue (14). The remaining 15 cultures, including 1281 10 catalase-negative strains, were isolated from tongue swabs cultured on Trypticase Soy Broth (BBL) supplemented with agar (TSBA). In addition to selected oral isolates, the following cultures were included primarily for DNA base composition studies: Aerococcus viridans ATCC 11563 was obtained from J. B. Evans, North Carolina State University; Pediococcus sp. 7P1 and Pediococcus sp. SG1 were obtained from R. H. Deibel, University of Wisconsin; Micrococcus varians CCM 250 and M. conglomeratus CCM 740 were obtained from M. Kocur, Czechoslovak Collection of Microorganisms. All oral isolates, A. viridans, Pediococcus sp. 7P1, and Pediococcus sp. SGI were maintained in Trypticase Soy Broth containing 0.1% KNO3 (TSBN) and were incubated at 35 C. Addition of KNO3 enhanced the growth of the oral isolates and was added routinely. M. varians CCM 250 and M. conglomeratus CCM 740 were carried on TSBA plates incubated at room temperature. Cultural characteristics. Cultures were tested in TSBN broth for ability to grow in the presence of 5% NaCl at ph 5.0 and 9.6, and at 10 C and 45 C. In addition, the growth response was observed in a medium containing (NH4)H2PO4 as the only nitrogen source (19). Hemolytic activity was determined on Heart Infusion Agar (Difco) containing 10% defibrinated horse blood. Blood-agar containing 40% ox bile (Difco) was used to test bile tolerance. All media were incubated at least 10 days before recording final growth response. Pathogenicity for mice was determined for four catalase-negative and two catalase-positive oral strains. Cells were harvested from 18-hr TSBN cultures and washed three times with sterile physiological

1282 GORDON saline. Cell concentrations were adjusted to contain approximately 125 X 106 organisms per ml. For each organism, 0.4 ml of the cell suspension was injected intraperitoneally into each of three 1-month-old mice (Charles River strain, Charles River Mouse Farms, Inc., North Wilmington, Mass.). Sterile saline was injected into four control animals. Carbohydrate fermentation. The following basal medium was prepared and adjusted to ph 7.0: Trypticase (BBL), 0.5%; yeast extract (Difco), 0.2%; KNO3, 0.1%; and NaCI, 0.1%. Filter-sterilized carbohydrate solutions (10%) were added to autoclaved medium to give a final concentration of 0.5%. Esculin was autoclaved with the basal medium. Starch (0.5%)-agar plates were prepared with the basal medium. Filter-sterilized sodium hippurate was added at the 0.5% level to basal medium containing glucose. Carbohydrate fermentation was determined both aerobically and anaerobically (95% H2, 5% CO2 in Brewer jars). Uninoculated controls and cultures grown in basal medium without carbohydrate were included. After 5 days of incubation, the ph was measured with a glass electrode. Anaerobic fermentation of glucose was also determined, following the procedure recommended by the Subcommittee on Taxonomy of Staphylococci and Micrococci (34). Hydrolysis of esculin, starch, and hippurate were determined by procedures described by Baird-Parker (3) Ḃiochemical tests. The following determinations were made using TSBN (32), nitrate reduction, gelatin liquefaction, and production of acetoin and indole. Catalase activity was determined (32) on both TSBA and Trypticase-yeast extract-agar (34). Colonies from TSBA plates were tested for oxidase following the procedure of Kovacs (24) by using a 1% aqueous solution of N,N-dimethyl-p-phenylenediamine monohydrochloride. Cultures were tested for the presence of a cytochrome-containing respiratory system by using the benzidine test described by Deibel and Evans (10). Phosphatase activity was determined according to the method described by Baird-Parker (3) by using TSBA containing 0.0001% filter-sterilized phenolphthalein diphosphate (Sigma Chemical Co., St. Louis, Mo.). H2S production, lipolysis, urease activity, and deoxyribonuclease activity were determnined in the following media: Peptone Iron Agar (Difco), Spirit Blue Agar (Difco), Urease Test Medium (BBL), and deoxyribonuclease Test Medium (BBL), respectively. Coagulase activity was determined by using 0.2 ml of a TSBN culture according to the procedures described by Auletta and Kennedy (2). Quantitative determinations for catalase were performed on six catalase-negative and two catalasepositive strains. Cells were harvested from TSBN and washed twice with 0.01 M potassium phosphate buffer (ph 6.8). Cell-free preparations were obtained by grinding the cell paste with size 16 glass beads (Sigma Chemical Co.) in a mortar for 15 min at 4 C. Glass beads, cell debris, and remaining whole cells were removed by centrifugation. Protein determinations were performed (20) and catalase activity was determined after 1-,5-, or 10-min incubation according to the procedure described by Herbert (17). J. BACrERIOL. DNA isolation and base composition determinations. Four catalase-negative and three catalase-positive strains of oral cocci were harvested from 1 liter of TSBN after 18-hr incubation. During preliminary studies, concentrated cell suspensions could not be lysed completely by the usual chemical or mechanical means because of the thick extracellular material produced by these organisms. Treatment with NaOH made these cells susceptible to lysozyme; therefore, the oral strains were prewashed with 0.05 N NaOH before being resuspended in 60 ml of saline ethylenediaminetetraacetate (EDTA) buffer (0.15 M NaCi, 0.1 M disodium-ethylenediaminetetraacetate, ph 8.0). The strains of pediococci and aerococci were harvested from 1.5 liters of TSBN, and the micrococci were cultured on the surface of 20 TSBA plates. The cells were not treated with NaOH, but were washed once and resuspended in 40 ml of buffer. All cell suspensions were lysed by first incubating with lysozyme (0.5 mg/ml) 3 to 4 hr at 37 C. Complete lysis occurred upon addition of a 25% aqueous solution of sodium lauryl sulfate (0.8 ml per 10 ml of lysed suspension) followed by heating at 60 C for 10 min. The procedure described by Marmur (25) was used to isolate DNA. The method of Marmur and Doty (26) was used to determine mole per cent guanine plus cytosine (G + C) content with a model DU spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.) fitted with thermospacers. RESULTS Of the 24 strains of gram-positive, facultative cocci isolated from the surface of the human tongue, 14 were catalase-negative. The individual cells were 1.5 to 1.8, in diameter and arranged primarily in clusters with occasional pairs, tetrads, and short chains. Colonies on blood-agar were nonhemolytic, 1 mm in diameter, white to gray in color, round, entire, convex, mucoid, and adherent to the agar surface. Colonies were similar on TSBA but larger (1 to 3 mm). Under anaerobic conditions, colonies were smaller (0.5 mm) and very adherent to the agar surface. In broth medium containing a fermentable carbohydrate, viscous gelatinous growth developed within a few hours. This type of growth was most apparent in Trypticase Soy Broth fortified with 0.1% KNO3. Upon aging, most of the cultures developed a glutinous ring at the surface. M. varians was a facultative, gram-positive coccus, 1,u in diameter. Under aerobic or anaerobic conditions, colonies were small (0.5 mm), butyrous, cream-colored, nonhemolytic, and not adherent to agar. In broth, the growth was not viscous and tended to settle, thus forming a packed sediment which could be resuspended. Biochemical activity. The strains of oral cocci were similar in essentially all characteristics except for catalase activity and some variability in the oxidase test (Table 1). Catalase-negative

VOL. 94, 1967 TABLE 1. REISOLATION OF STAPHYLOCOCCUS SALIVARIUS Biochemical reactions of the oral isolates and Micrococcus varians" Characteristic Oral isolates 14 10 Mcro-u Strains Stra ins COCC varatss Catalase... _ + + Deoxyribonuclease.... + Nitrate reduction... + + + Benzidine test... + + + Oxidase... 10+, 4- + Production of Acetoin... + + Indole... Hydrolysis of Gelatin... + + Urea... + Esculin... + + + Growth ph 9.6... - - + loc... - - + 40% bile... - - + 5% NaCl... - - + Carbohydrate fermentationsb (aerobic and anaerobic) Galactose... + Lactose... + Melezitose... - + Salicin (aerobic)... + + a None of the organisms demonstrated coagulase, phosphatase, or lipolytic activity; produced indole or H2S; or hydrolyzed starch or hippurate. None of the cultures initiated growth at ph 5.0, 45 C, or in a medium containing (NH4)H2PO4 as the only nitrogen source. b All strains fermented glucose, sucrose, maltose, trehalose, and fructose aerobically and anaerobically. Glycerol, mannitol, ribose, arabinose, sorbose, raflinose, and xylose were not fermented. cultures were also negative on a medium without carbohydrate, an indication that acid production was probably not depressing catalase activity as had been reported for Pediococcus cerevisiae (16). Quantitative determinations of catalase activity confirmed the qualitative results. Cellfree preparations from two catalase-positive strains utilized 147 and 40,umoles, respectively, of H202 per mg of cell-free protein per min. None of the six cell-free preparations from catalasenegative strains exhibited catalase activity. Growth of the oral strains was stimulated when nitrate was added to the medium. Nitrate was 1283 reduced beyond nitrite by all the oral isolates. M. varians accumulated nitrite in the medium. A positive benzidine test was exhibited by all cultures, which indicated that these organisms probably contain cytochromes. Unlike M. varians, the oral isolates did not initiate growth at ph 9.6, in the presence of 40% bile, or in a medium containing 5% NaCl. The oral isolates failed to grow at 10 C or 45 C. M. varians grew at 10 C, but not at 45 C, and exhibited good growth at 22 C. The oral isolates grew slowly at 22 C. M. varians did not liquefy gelatin, whereas the oral cocci liquefied a medium containing 4% gelatin, but not one which contained 12% gelatin. All cultures fermented glucose both aerobically and anaerobically, and produced ph values of 4.9 to 5.6. In contrast to the present findings, Rosypal et al. (29) reported that M. varians did not ferment glucose under anaerobic conditions. The oral isolates grew poorly in the medium recommended for determining anaerobic glucose fermentation (34) and usually 10 days' incubation was required for the bromocresol purple indicator to change throughout the tube. The growth and biochemical characteristics of the pediococci and aerococci have been reported by others (8, 11, 12, 15, 36). The present study confirmed these previous findings. These organisms were catalase-negative, failed to reduce nitrate, and fermented glucose under anaerobic conditions. In addition, these strains exhibited a negative benzidine test. This finding is in contrast to a previous report for A. viridans (36). Base ratio determinations. The DNA base compositions are listed in Table 2. The catalasepositive and catalase-negative strains of the oral isolates tested exhibited guanine plus cytosine (G + C) contents ranging from 55.4 to 58.3%. This indicated that, on the basis of DNA base composition, both types of oral strains were similar. The G + C content for Pediococcus 7P1, Pediococcus SG1, A. viridans, and M. varians were 34.6, 30.5, 39.0, and 56.6%, respectively. The G + C value for M. varians was in close agreement with that previously reported (29). M. conglomeratus CCM 740 was listed by Hill (18) to have a G + C content ranging from 53.5 to 58.5%. In the present study this strain exhibited a G + C content of 70.7% and therefore was not studied further. The G + C values for pediococci and aerococci differed from those of the oral isolates. They were similar to values reported for staphylococci and Gaffkya homari (2). Although the strain of M. varians included in this study was similar to the oral isolates in DNA base composition, this strain differed in both cultural and biochemical characteristics.

1284 GORDON J. BACTERIOL. TABLE 2. DNA thermal denaturation temperature and guanine plus cytosine (G + C) content of organisms tested Organism Tempera- Moles % Tma G + C Catalase-positive strains of oral isolates 1 92.2 55.9 2 93.2 58.3 3 92.0 55.4 Catalase-negative strains of oral isolates 1 93.0 57.8 2 92.1 55.6 3 92.9 57.6 4 92.6 56.8 M. varians 92.5 56.6 A. viridans 85.3 39.0 Pediococcus 7P1 83.5 34.6 Pediococcus SG1 81.8 30.5 a Tm was reproducible to +0.4 C. DIscussIoN Oral cocci resembling staphylococci or micrococci have been studied previously (1, 27, 28, 33, 35); however, Morris (27) appears to be the only investigator to have isolated catalase-negative strains. Andrewes and Gordon (1) isolated 50 strains of staphylococci from the saliva of 17 individuals. Although these isolated organisms were not tested for catalase activity, the description of 34 of these strains is quite similar to that of the present isolates. "In broth it grows in the form of weedy gelatinous deposit at the foot of the tube, the fluid remaining clear. On agar it forms a white shining growth which is glutinous and also often adherent to the medium, so that a piece of the growth is with difficulty removed for microscopical examination." These organisms reduced nitrate, liquefied gelatin, fermented maltose and glycerin but not mannite or lactose, and they grew poorly at 22 C. These particular organisms were considered to be the type of staphylococci found most frequently in saliva and were designated Staphylococcus salivarius. The only apparent difference between these strains and the present isolates is the fermentation of glycerin. More recently, Sims (31) reported the isolation of catalase-negative pediococci from human saliva. Since only two strains were isolated from 183 saliva samples, it would seem unlikely that these organisms are indigenous to the oral cavity. These organisms grew in 40% bile and 8% NaCl at 45 C and therefore are not similar to the present cocci. In addition, the strains of Pediococcus and A. viridans included in the present study differed markedly from the oral cocci in cultural characteristics, response to tolerance tests, fermentation patterns, DNA base composition, and, most important, in exhibiting negative benzidine tests. For these reasons, the present isolates are not considered to be pediococci or aerococci. Other cocci isolated from the oral cavity have been assigned to the genera Gaffkya and Sarcina (28, 33). Since it has been recommended that the generic name Gaffkya be rejected (23) and that the genus Sarcina include only anaerobic species (22), these generic names are not considered suitable for the oral cocci. It is generally accepted that the genera Staphylococcus and Micrococcus form two distinct groups of cocci that can be differentiated on the basis of anaerobic fermentation of glucose (4, 9, 13) and DNA base composition (2, 5, 6, 29, 30). Staphylococci ferment glucose anaerobically and have a G + C content of from 30.0 to 40.0 mole %, whereas micrococci fail to ferment glucose anaerobically and G + C values are generally between 65.5 and 75.5 mole %. Auletta and Kennedy (2) reported G + C values somewhat lower for the micrococci (59 to 69%). On the basis of anaerobic fermentation of glucose, the oral isolates and M. varians 250 should be placed in the genus Staphylococcus. However, considering the G + C content (55.4 to 58.3 mole %), these organisms represent a group intermediate between staphylococci and micrococci. It is interesting that Rosypal et al. (29) reported that M. varians CCM 250 was susceptible to several staphylococcal phages; however, they found this strain unable to ferment glucose anaerobically. In the present study this strain fermented glucose anaerobically and exhibited several biochemical characteristics similar to staphylococci. Unfortunately, the natural source of this strain of M. varians is not known. With this micrococcus, other strains exhibiting G + C contents of 48 to 51% (6), and now the oral cocci, it may become necessary to recognize a new group of organisms intermediate between staphylococci and micrococci. Before this is possible, a critical evaluation is required to determine the usefulness of G + C content determinations versus physiological or other tests in placing organisms into present bacterial taxonomic schemes. Rather than suggest a new genus on the basis of DNA base composition, it is proposed that the

VOL. 94, 1967 REISOLATION OF STAPHYLOCOCCUS SALIVARIUS 1285 oral isolates be placed in the genus Staphylococcus. The oral strains differ in several respects from the recognized species of Staphylococcus (7, 21). Therefore, on the basis of source and the close similarity in description, it is proposed that the species name S. salivarius (Andrewes and Gordon, 1907) be recognized (1). This organism is a gram-positive coccus, approximately 1.5 to 1.8,u in diameter. Cells are nonmotile and are arranged in clusters and occasionally pairs, tetrads, or short chains. Surface colonies on bloodagar are round, entire, convex, gray to white in color, smooth, glistening, nonhemolytic, about 1 mm in diameter, mucoid, and very adherent to the agar surface. Catalase-variable. Facultative. Produce viscous growth in broth media. Ferment glucose, fructose, maltose, sucrose, and trehalose aerobically and anaerobically. Salicin fermented aerobically only. Glycerol, mannitol, ribose, arabinose, sorbose, raffinose, and xylose are not fermented. Starch, urea, and hippurate are not hydrolyzed. Esculin is hydrolyzed. Gelatin is liquefied. Nitrate is reduced beyond nitrite. Indole and H2S are not produced. Does not grow at 10 C or 45 C, in broth containing 5% NaCl, or at ph 5. Acetoin produced from glucose. Not pathogenic for mice. Inhibited by 40% beef bile. Does not utilize (NH4)H2PO4 as nitrogen source. Benzidine-positive. Coagulase- and phosphatase-negative. Does not exhibit lipolytic activity. Source: the oral cavity of man. ACKNOWLEDGMENIS The author wishes to thank J. B. Evans, North Carolina State University, for helpful suggestions during this study. This investigation was supported by research grant DE 01471 from the National Institute of Dental Research and by a grant from Colgate Palmolive Co. LITERATURE CITED 1. ANDREWES, M. D., AND M. H. GORDON. 1907. Report on the biological characters of staphylococci pathogenic for man. Rep. Med. Offr. Loc. Govt. Bd. (1905-1906) 7:543-560. 2. AULF-TA, A. E., AND E. R. KENNEDY. 1966. Deoxyribonucleic acid base composition of some members of the Micrococcaceae. J. Bacteriol. 92:28-34. 3. BAIRD-PARKER, A. C. 1963. A classification of micrococci and staphylococci based on physiological and biochemical tests. J. Gen. Microbiol. 30:409-427. 4. BAIRD-PARKER, A. C. 1965. The classification of staphylococci and micrococci from world wide sources. J. Gen. Microbiol. 38:363-387. 5. BOHACEK, J., M. KOCUR, AND T. MARTINEC. 1965. Deoxyribonucleic acid base composition and taxonomy of the genus Micrococcus. PubI. Fac. Sci. Univ. Brno. K35:318-322. 6. BOHACEK, J., M. KoCUR, AND T. MARTINEC. 1967. DNA base composition and taxonomy of some micrococci. J. Gen. Microbiol. 46:369-376. 7. BREED, R. S., E. G. D. MURRAY, AND N. R. SMITH. 1957. Bergey's manual of determinative bacteriology. 7th ed. The Williams & Wilkins Co., Baltimore. 8. CLAUSEN, 0. G. 1964. The discovery, isolation and classification of various a-haemolytic micrococci which resemble aerococci. J. Gen. Microbiol. 35:1-8. 9. COWAN, S. T., AND K. J. STEEL. 1964. Comparison of differentiating criteria for staphylococci and micrococci. J. Bacteriol. 88:804-805. 10. DEIBEL, R. H., AND J. B. EVANS. 1960. Modified benzidine test for the detection of cytochromecontaining respiratory systems in microorganisms. J. Bacteriol. 79:356-360. 11. DEIBEL, R. H., AND C. F. NIVEN, JR. 1960. Comparative study of Gaffkya homari, Aerococcus viridans, tetrad-forming cocci from meat curing brines, and the genus Pediococcus. J. Bacteriol. 79:175-180. 12. DEIBEL, R. H., J. H. SILLIKER, AND P. T. FAGAN. 1964. Some characteristics of an oleate-requiring, hemolytic Pediococcus. J. Bacteriol. 88:1078-1083. 13. EVANS, J. B. 1965. Current views and problems relating to the taxonomy of the Micrococcaceae. Intern. Bull. Bacteriol. Nomen. Taxon. 15:111-112. 14. GORDON, D. F., JR., AND R. J. GIBBoNs. 1966. Studies of the predominant cultivable microorganisms from the human tongue. Arch. Oral Biol. 11:627-632. 15. GUNTHER, H. L., AND H. R. WHITE. 1961. The cultural and physiological characters of pediococci. J. Gen. Microbiol. 26:185-197. 16. GUTEKUNST, R. R., E. A. DELWICHE, AND H. W. SEELEY. 1957. Catalase activity in Pediococcus cerevisiae as related to hydrogen ion activity. J. Bacteriol. 74:693-695. 17. HERBERT, D. 1955. Catalase from bacteria (Micrococcus lysodeikticus), p. 784-788. In S. P. Colowick and N. 0. Kaplan [ed.], Methods in enzymology, vol. 2. Academic Press, Inc., New York. 18. HILL, L. R. 1966. An index to deoxyribonucleic acid base compositions of bacterial species. J. Gen. Microbiol. 44:419-437. 19. HUCKER, G. J. 1924. Studies on the Coccaceae. II. A study of the general characteristics of the micrococci. N.Y. State Agr. Expt. Sta. Bull. 100. 20. ITZHAKI, R. F., AND D. M. GILL. 1964. A micro-

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