INFECTION AND IMMUNITY, Feb. 1983, p. 609-6140019-9567/83/020609-06$02.00/0Copyright C 1983, American Societv for Microbiology

Vol. 39, No. 2

Production, Purification, and Properties of a Bacteriocin fromStaphylococcus aureus Isolated from Saliva

TEKESHI NAKAMURA,* NOBUO YAMAZAKI, HIROO TANIGUCHI, AND SETSUO FUJIMURA

Department of Oral Microbiology, Matsumoto Dental College, Shiojiri-City, Nagano-Prefecture,Japan 399-07

Received 8 September 1982/Accepted 1 November 1982

Staphylococci from samples of human saliva were isolated on staphylococcal-selective agar plates. These strains were tested for the inhibition of the growth ofStaphylococcus aureus FDA209P. The frequency of inhibitory strains among allof the staphylococcal isolates was 5.2%. Strain IYS2, which formed the biggestinhibitory zone against the growth of the indicator strain, was used as theproducer of bacteriocin. IYS2 was identified to be S. aureus, based on itsbiological properties. The bacteriocin was purified by sequential procedures,including ammonium sulfate precipitation, fractionation with ethanol, ion-ex-change chromatography, and gel filtration. Its molecular weight was determinedto be 5,000. The isoelectric point was 10.0. In amino acid composition, lysine,histidine, aspartic acid, valine, and phenylalanine were predominant. The bacte-riocin was heat stable but inactivated by proteases or peptidase. The bacteriocinhad a bactericidal effect on susceptible cells. An analysis of the inhibitoryspectrum among typical oral indigenous bacteria showed that Streptococcussalivarius, Propionibacterium acnes, Corynebacterium parvulum, and Actinomy-ces israelii were susceptible to the bacteriocin. Streptococcus mutans, Strepto-coccus sanguis, Streptococcus mitis, Actinomyces viscosus, Actinomyces naes-lundii, Fusobacterium nucleatum, Bacterionema matruchotii, and Bacteroidesmelaninogenicus were resistant. The majority of S. aureus tested were suscepti-ble, and all Staphylococcus epidermidis strains tested were resistant.

In studies of the production and characteriza-tion of bacteriocin among oral bacteria, strepto-cocci, which are the predominant microorga-nisms in oral flora, have attracted much interest(9, 12, 22, 23, 27, 28). Although staphylococcalbacteriocins have been relatively intensivelystudied (4, 10, 11, 15-17, 25), little information isavailable on bacteriocins produced by oralstrains of staphylococci. The aim of this studywas to isolate S. aureus which produces a sub-stance inhibitory to bacteria and to examine itsproperties using the purified material.

MATERIALS AND METHODSIsolation of staphylococci from saliva and testing of

growth inhibition. Samples of saliva were collected insterile test tubes from 52 students of our college. A0.5-ml amount of each saliva sample was inoculatedonto a selective medium for staphylococci (Staphylo-coccus no. 110 agar; Eiken Chemical Co., Tokyo,Japan) and incubated aerobically for 2 days at 37°C.Replicas of each plate were made by the method ofLederberg and Lederberg (18). Then, to detect growthinhibition, the initial plates were overlaid with thesame agar containing about 1 x 10i cells of S. aureusFDA209P as an indicator, followed by incubation in

the same way. After this procedure, assuming that theinhibitory zone was developed by the action of bacte-riocin produced by the organisms, we chose the colo-ny which formed the biggest inhibitory zone around itfor the producer of bacteriocin, and this was designat-ed IYS2.

Identification of IYS2. IYS2 was taken from thereplica plate and passaged weekly on an agar slantcontaining 3.7% brain heart infusion (Difco Labora-tories, Detroit, Mich.) and 0.2% yeast extract (Difco).Work to identify this strain was conducted by theprocedures of Bergey's Manual ofDeterminative Bac-teriology (3) and those of Faddin (5). The properties ofIYS2 were as follows: colonies on solid medium wereround, smooth, glistening, and yellowish. Under lightmicroscopic observations, the organism was grampositive, spherical, and arranged in irregular clusters.It grew in a medium containing 15% NaCl. Acids wereproduced from glucose and mannitol under eitheraerobic or anaerobic conditions. The production ofcoagulase and DNase was positive. It liquefied gelatin.The Voges-Proskauer reaction, nitrate reduction, andarginine hydrolysis were positive. Oxidase was nega-tive.

Assay of bacteriocin activity. The activity of thebacteriocin was measured by the diffusion method onagar plates as described earlier (20). The number ofbacteriocin units per milliliter was defined as thereciprocal of the highest dilution that gave clear zones

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FIG. 1. Demonstration of bacteriocin activity inculture supernatant of IYS2. A sterilized culture su-pernatant with a membrane filter (Millipore) was dilut-ed in sterile 0.05 M phosphate buffer (pH 7.0), and 0.25ml of each sample was poured into wells in a platecontaining S. aureus FDA209P. The plate was incubat-ed overnight at 37°C. The numbers indicate the folddilution of the culture supernatant.

of growth inhibition of the indicator strain, as in thecase of colicin (13).

Cultivation of organism. The above-described medi-um for stock culture lacking agar was used for theproduction of the bacteriocin. The cultivation of IYS2was carried out with shaking for 10 h at 37°C. Theeffect of mitomycin C on the production of the bacte-riocin was examined by the method of Fujimura et al.(6, 7).

Estimation of molecular weight of the bacteriocin. (i)Sodium dodecyl sulfate-polyacrylamide gel electropho-resis. Using the method of Weber and Osborn (32), wedetermined the molecular weight of the bacteriocin.The concentration of polyacrylamide was 15%. Thestandard markers used were trypsin inhibitor, cyto-chrome c, approtinin, and insulin B-chain.

(ii) Sephadex G-50 gel filtration. A mixture of thepurified bacteriocin, chymotrypsinogen A, cyto-chrome c, and insulin was applied to a Sephadex G-50column (2.0 by 80 cm) and equilibrated with 0.05 Mphosphate buffer (pH 7.0) containing 0.15 M NaCl.The column was eluted with this buffered saline. Themolecular weight was determined by the method ofAndrews (2).

Isoelectric focusing. The pl value was estimated withisoelectric focusing (110-ml column; LKB-Produkter,Stockholm, Sweden) by the method of Vesterberg etal. (31). The sample was dialyzed with Spectrapormembrane tubing Por. 6 (Spectrum Medical Indus-tries, Inc. Los Angeles, Calif.) against 1% glycinesolution overnight at 4°C before use. Thereafter, thismembrane tubing was used for dialysis throughout thestudy. Ampholite (LKB) concentration was 1%, andthe pH range was from 9 to 11. Focusing was done for40 h under constant voltage (600 V), and the columnwas maintained at about 4°C with a circulating bath.Chemical analysis. Protein was estimated by the

method of Lowry et al. (19) with bovine serum albu-min as a reference. Carbohydrate content was deter-mined with anthrone (9,10-dihydro-9-oxyanthracene;Nakarai Chemical Ltd., Kyoto, Japan) (29) with glu-cose as a standard. For amino acid analysis, a sampleof the purified bacteriocin was dialyzed against deion-ized water exhaustively and lyophilized. The powderproduced was dissolved in 6 N HCl and hydrolyzed at

110°C for 24 h in a sealed, evacuated tube. Amino acidcomposition was determined with a JEOL automaticamino acid analyzer, model JLC-6AH.Enzyme treatment. The bacteriocin (128 U/ml) was

Aincubated separately with various enzymes at 1 mg ofenzyme per ml at 37°C for 2 h, and the residual activityof the bacteriocin was assayed. Pronase (KakenChemical Co., Tokyo, Japan), trypsin (Sigma Chemi-cal Co., St. Louis, Mo.), and lipase (Sigma) weredissolved in 0.05 M Tris-hydrochloride buffer (pH 8.0)containing 0.02 M CaCl2. a-Chymotrypsin (Sigma),papain (E. Merck AG, Darmstadt, West Germany),peptidase (Sigma), lysozyme (Sigma), catalase (Sig-ma), DNase (Sigma), and RNase (Sigma) were dis-solved in 0.1 M phosphate buffer (pH 7.2).Adsorption of the bacteriocin to indicator cells. The

demonstration of the adsorption of the bacteriocin wascarried out essentially by the method of Dajani andWannamaker (4). To 1.0 g (wet weight) of bacterialcells, 5 ml of the purified bacteriocin (128 U/ml) inphosphate buffer (pH 7.0) was added. The mixture wasincubated at 37°C for 2 h with shaking at intervals of 30min. After incubation, the mixture was centrifuged at10,000 x g for 10 min, and the supernatant wasexamined for the titration of the activity.

Determination of inhibitory spectrum. The solutionof purified bacteriocin was sterilized with a membranefilter (0.45-jLm pore size; Millipore Corp., Bedford,Mass.) and streaked diagonally across the surface ofbrain heart infusion agar. The culture of the test strainfor susceptibility to the bacteriocin was initiallystreaked at right angles to the line of the bacteriocinand then turned at right angles near the bacteriocin. Inthe test for Bacteroides melaninogenicus, blood agarcontaining hemin and menadione (21, 26) was usedinstead of a brain heart infusion agar. The plates wereincubated anaerobically in a glove box (8, 21) at 37°C,but staphylococcal plates were incubated aerobically.After incubation, strains whose growth was inhibitedwere judged to be sensitive to the bacteriocin.

RESULTSDetection of inhibitory staphylococci from sali-

va. From 52 plates of Staphylococcus no. 110agar, each of which was inoculated with 0.5 mlof a different saliva, a total of 9,072 staphylococ-cal colonies were obtained. The average numberof staphylococci obtained was calculated to be0.35 x 103/ml. Although the numbers of theinhibitory active colonies in each plate variedfrom one saliva sample to another, the averageratio between the number of inhibitory coloniesand the total colonies in each sample was 5.2%.In a particular sample, however, nearly 40% ofthe isolates were active.

Production of the bacteriocin in liquid medium.It was demonstrated that the growth of theindicator strain was inhibited by the culturesupernatant of IYS2 (Fig. 1). Among the follow-ing three media, brain heart infusion plus yeastextract, Trypticase (BBL Microbiology Sys-tems, Cockeysville, Md.) soy broth, and nutri-ent broth, the production of the bacteriocin wasmost intensive in the first medium. Therefore,

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BACTERIOCIN FROM S. AUREUS 611

TM (HOUS)

FIG. 2. Growth and production curves of bacterio-cin. Symbols: 0, activity; 0, growth.

this medium was used to produce the bacterio-cin. Bacteriocin activity in the culture superna-tant appeared in the logarithmic phase ofgrowth, and the production continued for about6 h (Fig. 2). When the production ceased, thebacteriocin accumulated maintained its full ac-tivity without inactivation. In the stationaryculture, about one-fourth of the activity ob-tained in the shaking culture was produced. Theaddition of mitomycin C to the culture at a

concentration of 0.1, 0.5, 1.0, or 5.0 ,ug/ml at an

early stage of the logarithmic phase caused thelysis of the cells. However, it did not stimulatethe production of the bacteriocin.The cells were collected at 7 and 10 h after

inoculation and then disrupted with ultrasonictreatment. In neither cell-free extract was anysignificant amount of the bacteriocin activitydetected.

Purification. All subsequent procedures were

conducted at 4°C, if not otherwise specified.Strain IYS2 was cultured for 10 h, and theculture supematant (2,000 ml) was obtained b)centrifugation at 10,000 x g for 10 min. Solidammonium sulfate was added to the culturesupernatant with stirring to give 80o saturationThe mixture was stirred for a further 2 h. Theresultant precipitate was collected by centrifugation at 15,000 x g for 20 min, dissolved in 120 mof 0.05 M phosphate buffer (pH 7.0), and fol

lowed by dialysis against the same buffer for 24 hwith several changes of the buffer. Cold (-20°C)ethanol was added drop by drop to the dialyzedmaterial in an ice water bath until the ethanolconcentration reached 75% (vol/vol), and themixture was stirred gently for 4 h. The resultingprecipitate was removed by centrifugation at15,000 x g for 20 min. The supernatant solutionwas subjected to evaporation in vacuo at 30°C toremove the ethanol. After the volume decreasedto the level at which the ethanol had been added,the solution was dialyzed against 0.05 M phos-phate buffer (pH 7.0). This material was appliedto a column (2.6 by 33 cm) of carboxymethylcellulose, previously equilibrated with 0.05 Mphosphate buffer (pH 7.0), and the column waswashed thoroughly with this buffer. The columnwas then eluted with a linear gradient of NaCl atconcentrations from 0 to 0.8 M. The bacteriocineluted between 0.2 and 0.3 M NaCl (Fig. 3). Theactive fractions were pooled, concentrated byevaporation, and dialyzed against 0.05 M phos-phate buffer (pH 7.0) containing 0.15 M NaCl.The concentrated active fractions from the CM-cellulose column were applied to gel filtration ona Sephadex G-50 column (2.6 by 96 cm), previ-ously equilibrated with the dialysis-buffered sa-line. In the gel ifitration, the bacteriocin elutedas a single peak of activity, and the elutionposition coincided with that of the UV-lightabsorbance peak. The active fractions were col-lected and dialyzed against 0.05 M phosphatebuffer (pH 7.0). This was the final purifiedsample of the bacteriocin. Through these proce-dures, the specific activity (number of the bacte-riocin units per milligram of the protein) in-creased 1,450-fold compared with the crudeculture supematant. The recovery of the activitywas 23%. Polyacrylamide gel electrophoresis of

t the purified sample showed a single-stained

I

Jo

FIG. 3. Chromatography of the bacteriocin. Afterethanol fractionation, the bacteriocin preparation was

applied to a carboxymethyl cellulose column andeluted with a linear gradient of NaCl. The conditionsare described in the text. Symbols: 0, activity; 0,absorbance at 230 nm; -------, concentration of NaCl.

rII

bI

I

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1111

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FIG. 4. Polyacrylamide gel electrophoresis of thepurified bacteriocin. A gel was stained for protein(left). Another unstained gel was briefly rinsed withsterile 0.05 M phosphate buffer (pH 7.0) and embeddedin an agar plate containing S. aureus FDA209P andthen incubated to determine the location of the bacte-riocin activity in the gel.

band, which corresponded to the activity inmobility (Fig. 4).

Molecular weight. Electrophoresis on a sodi-um dodecyl sulfate-polyacrylamide gel gave avalue of 5,000 for the molecular weight. Thesame value was obtained by Sephadex G-50 gelfiltration.

Isoelectric point. The profile of isoelectric fo-cusing showed that the bacteriocin activity wasfound in a single peak with a pl value of 10.0.

Chemical composition. The bacteriocin wasfree from carbohydrates. In amino acid compo-sition (Table 1), lysine, histidine, aspartic acid,

valine, and phenylalanine were predominant. Arather high pl value of the bacteriocin may beattributed to the high content of lysine andhistidine.

Stability. When the bacteriocin (128 U/ml) washeated at 100°C for 15 min, no reduction of theactivity was observed. Heating at 120°C for 15min (in an autoclave), however, resu4ed in a75% loss of activity. Treatment with trypsin, (x-chymotrypsin, papain, and peptidase causedcomplete inactivation. Pronase was also effec-tive; 75% of the activity was lost when thebacteriocin was treated with this enzyme. Lyso-zyme, catalase, lipase, RNase, and DNase hadno effect.

Bactericidal effect of the bacteriocin. Treat-ment with the bacteriocin of S. aureus FDA209Pgrown in exponential phase caused a rapid de-crease in the number of CFUs (Fig. 5). The cellswere almost completely killed within 2 h. How-ever, the optical density at 600 nm of the cellsuspension treated with the bacteriocin re-mained constant. These results indicate that thebacteriocin acted on susceptible cells bactericid-ally without bacteriolytic effect.

Adsorption. The incubation of bacteriocinwith cells of S. aureus FDA209P at 37°C for 2 hresulted in the disappearance of the bacteriocinactivity from the supernatant of the mixture.When a resistant strain of S. aureus was usedinstead of FDA209P, the initial activity (128U/ml) was recovered in the supernatant. Thesefindings suggest that the adsorption of the bacte-riocin to susceptible cells occurred. However, itwas not seen in resistant cells.

10O

106TABLE 1. Amino acid composition of bacteriocinAmino acid Contenta

Lysine ..................................Histidine ................................Arginine ................................Aspartic acid ............................Threonine ...............................Serine .................................Glutamic acid............................Proline ..................................Glycine .................................Alanine .................................Cystine .................................Valine ..............Methionine ..............................Isoleucine ...............................

199145Tr103

895640

69130

1081533

Leucine ................................. 2Tyrosine ................................ 9Phenylalanine............................ 137

i

1 42

D 1 0

2

10°,

IJCUBATION TIME (HOURS)

FIG. 5. Bactericidal effect of the bacteriocin oncells of S. aureus FDA209P. Symbols: 0, control (nobacteriocin); 0, 128 U of bacteriocin per ml. Thebacteriocin was added at zero time.

a Values express the numbers of residues per 1,000amino acids.

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BACTERIOCIN FROM S. AUREUS 613

Inhibitory spectrum. Various bacterial spe-

cies, primarily of typical oral indigenous spe-

cies, were tested for their susceptibility to thebacteriocin. The results show that the bacterio-cin inhibited the growth of S. salivarius ATCC9759; P. acnes ATCC 6919, ATCC 11827, andCN 8; C. parvulum ATCC 11829; and A. israeliiATCC 12102. The following bacteria were resist-ant: S. mutans Ingbritt, S. sanguis ATCC 10557,S. mitis ATCC 9811, A. viscosus ATCC 19246,A. naeslundii ATCC 12104, F. nucleatum ATCC25286, B. matruchotii ATCC 14266, and B. me-

laninogenicus NM2. Out of 20S. aureus strains,15 were found to be susceptible. The producerstrain IYS2 was resistant. All S. epidermidistested (20 strains) were resistant.

DISCUSSIONHsu and Wiseman reported (14) that 4.9% of

1,065 strains of coagulase-positive staphylococ-ci produce a substance inhibitory to S. aureus.

This value is quite close to our result (5.2%),observed in staphylococci isolated from humansaliva.The production of bacteriocin commenced in

the logarithmic phase and continued in the sta-tionary phase. After the production stopped, thebacteriocin maintained full activity for at least 24h. The stimulation of the production of severalstaphylococcal extracellular enzymes and toxinsby mytomicin C has been studied in relation tothe induction of prophage (6, 7). The stimulationof the production of this bacteriocin was notobserved with this antibiotic.

Recently, Sahl and Brandis purified an anti-staphylococcal agent (Pep 5) produced by S.epidermidis (25). They found a molecular weightof 6,000 and a pI of 10.5. These values resemblethose of our bacteriocin. Similarities betweenthe two bacteriocins were also found in heatstability, sensitivity to proteases, and the lack ofeffect by mitomycin C on production. As formolecular weight, Hale and Hinsdill also found a

low value (9,000) in a bacteriocin from S. aureus

(10). However, the molecular weight of staphy-lococcin 1580 produced by S. epidermidis hasbeen reported to be high (17). It has beenreported to be between 150,000 and 400,000 andto consist of 20,000 molecular weight subunits.It is unlikely that the molecular weight of 5,000of our bacteriocin represents a subunit of thepolymerized molecule, because the gel filtrationmethod gave the same value as did sodiumdodecyl sulfate-polyacrylamide gel electropho-resis.The bacteriocin acted bactericidally on the

susceptible indicator cells. The bactericidal ef-

fect of staphylococcal bacteriocins has also beenreported by Jetten and Vogels (15, 16) and

Dajani and Wannamaker (4). Their bacteriocins

were found to adsorb susceptible cells as in ourcase. Bacteriostatic activity was demonstratedby Hale and Hinsdill in staphylococcin 462 (11).They demonstrated further that no adsorption ofthis bacteriocin to the susceptible cells occurred(11).The evidence as to whether bacteriocins play

a significant ecological role in vivo remainsinconclusive. Howevet, Aly et al. (1) and Shine-field et al. (30) demonstrated that a certain strainof S. aureus controls outbreaks of staphylococ-cal infection in nurseries and treats patients withrecurrent staphylococcal fumculosis. Further-more, since bacteriocins produced by oral bacte-ria are thought to play an important role in theestablishment of the ecosystem (24, 33), it maybe possible that the bacteriocin presented here isresponsible for a part of the control mechanismof microbial ecology.

LITERATURE CITED

1. Aly, R., H. I. Maibach, H. R. Shinefield, A. Mandel, andW. G. Strauss. 1974. Bacterial interference among strainsof Staphylococcus aureus in man. J. Infect. Dis. 129:720-724.

2. Andrews, P. 1964. Estimation of molecular weights ofprotein by Sephadex gel filtration. Biochem. J. 91:222-233.

3. Baird-Parker, A. C. Genus H, Staphylococcus Rosenbach1884, 18 nom. cons. Opin. 17 Jud. Comm. 1958, 153, p.483-489. In R. E. Buchanan and N. E. Gibbons (ed.),Bergey's manual of determinative bacteriology, 8th ed.The Williams & Wilkins Co., Baltimore.

4. Dajani, A. S., and L. W. Wannamaker. 1973. Kineticstudies on the interaction of bacteriophage type 71 staphy-lococcal bacteriocin with susceptible bacteria. J. Bacteri-ol. 114:738-742.

5. Faddin, J. F. M. 1980. Gram-positive bacteria, p. 345-370. In Biochemical tests for identification of medicalbacteria, 2nd ed. Williams & Wilkins Co., Baltimore.

6. Fujimura, S., T. Makino, and T. Hayashi. 1970. Effect ofmitomycin C on the production of staphylokinase, nucle-ase, and hemolysin by Staphylococcus aureus. Jpn. J.Bacteriol. 25:316-320. (In Japanese).

7. Fujimura, S., T. Makino, and T. Hayashi. 1970. Stimula-tion of staphylokinase production by mitomycin C. Natur-wissenschaften 57:394-395.

8. Fujimura, S., and T. Nakamura. 1978. Purification andproperties of a bacteriocin-like substance (acnecin) of oralPropionibacterium acnes. Antimicrob. Agents Chemo-ther. 14:893-898.

9. Fujimura, S., and T. Nakamura. 1979. Sanguicin, a bacte-riocin of oral Streptococcus sanguis. Antimicrob. AgentsChemother. 16:262-265.

10. Hale, E. M., and R. D. Hinsdill. 1973. Characterization ofbacteriocin from Staphylococcus aureus strain 462. Anti-microb. Agents Chemother. 4:634-640.

11. Hale, E. M., and R. D. HInsdill. 1975. Biological activityof staphylococcin 462: bacteriocin from Staphylococcusaureus. Antimicrob. Agents Chemother. 7:74-81.

12. Hamada, S., and T. Ooshima. 1975. Production and prop-erties of bacteriocins (mutacins) from Streptococcus mu-

tans. Arch. Oral Biol. 20:641-648.13. Herschmsan, H. R., and D. R. Helinski. 1967. Purification

and characterization of colicin E2 and E3. J. Biol. Chem.242:5360-5368.

14. Hsu, C.-Y., and G. M. Wiseman. 1967. Antibiotic sub-stances from staphylococci. Can. J. Microbiol. 13:947-955.

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16. Jetten, A. M., and G. D. Vogels. 1972. Model of action ofa Staphylococcus epidermidis bacteriocin. Antimicrob.Agents Chemother. 2:456-463.

17. Jetten, A. M., G. D. Vogels, and F. De Windt. 1972.Production and purification of Staphylococcus epidermi-dis bacteriocin. J. Bacteriol. 112:235-242.

18. Lederberg, J., and E. M. Lederberg. 1952. Replica platingand indirect selection of bacterial mutants. J. Bacteriol.63:399-406.

19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and J.Randall. 1951. Protein measurement with the Folin phenolreagent. J. Biol. Chem. 193:265-275.

20. Nakamura, T., Y. Sugunaka, T. Obata, N. Obata, and N.Yamazaki. 1977. Bacteriocin-like activities of human den-tal plaque flora against oral anaerobic microorganisms.Bull. Tokyo Dent. Coll. 18:217-229.

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22. Paul, D., and H. Slade. 1975. Production and properties ofan extracellular bacteriocin from Streptococcus mutansbactericidal for group A and other streptococci. Infect.Immun. 12:1375-1385.

23. Rogers, A. H. 1976. Bacteriocinogeny and the propertiesof some bacteriocins of Streptococcus mutans. Arch. OralBiol. 21:99-104.

24. Rogers, A. H., J. S. Van der Hoeven, and F. H. M. Mikx.1979. Effect of bacteriocin production by Streptococcusmutans on the plaque of gnotobiotic rats. Infect. Immun.23:571-576.

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26. Sawyer, S. J., J. B. Macdonald, and R. J. Gibbons. 1962.Biochemical characteristics of Bacteroides melaninogeni-cus. A study of thirty-one strains. Arch. Oral Biol. 7:685-691.

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