INFECTION AND IMMUNITY, Feb. 1976, p. 619-626 Copyright © 1976 American Society for Microbiology
Vol. 13, No. 2 Printed in U.SA.
Activity of Two Streptococcus mutans Bacteriocins in the Presence of Saliva, Levan, and Dextran ALLAN L. DELISLE Department of Microbiology, University of Maryland School of Dentistry, Baltimore, Maryland 21201 Received for publication 18 August 1975
The extracellular dextrans produced from sucrose by Streptococcus mutans strains BHT and GS-5 did not prevent the synthesis or release of active bacteriocins by these two strains. In addition, several streptococci that were genetically sensitive to these bacteriocins, and that could synthesize a variety of extracellular dextrans and levans from sucrose, remained phenotypically sensitive when grown in the presence of sucrose. Bacteriocin activity was not altered by treatment with high-molecular-weight dextran or by human saliva. The bacteriocins produced by, and active against, S. mutans thus appear to be capable of acting in vivo and may play a role in regulating the bacterial ecology ofthe oral cavity. Bacteriocinogenic strains of cariogenic streptococci are known to exist in the human oral cavity (7), but whether their bacteriocins play a role in regulating the bacterial ecology of this environment is not known. It is not yet possible to quantitate, or even detect, active bacteriocins in dental plaque and saliva, so in vivo production of bacteriocins cannot be unequivocally demonstrated; however, there are no compelling a priori reasons why bacteriocins should not be produced in the oral cavity. Current research in this area has therefore focused on the question of whether bacteriocins, particularly those of Streptococcus mutans, can exert their lethal effects under conditions simulating those of the oral environment. Thus, it has been reported that some S. mutans bacteriocins appear to be inactivated by proteolytic enzymes in saliva (8) and, more recently, that bacteriocin-sensitive strains of S. mutans and S. salivarius become insensitive to bacteriocins when grown in the presence of sucrose, due to their elaboration of dextran and levan polysaccharides (11). This investigation was undertaken to determine whether two S. mutans bacteriocins presently under study in this laboratory are inactivated by enzymes in saliva and whether they can kill sensitive cells that are coated with extracellular glucan and fructan polysaccharides synthesized from sucrose.
longing to Bratthall's serological group (b) (1), produces a highly insoluble extracellular dextran from sucrose. S. mutans GS-5 was obtained from R. J. Gibbons, Forsyth Dental Center, Boston, Mass. This strain, which belongs to serological group c, is also a dextran-producing, cariogenic human isolate (5). The bacteriocins produced by these strains, which have similar physical and chemical properties (7; D. Paul and H. D. Slade, Abstr. Annu. Meet. Am. Soc. Microbiol. 1974, M267, p. 110; A. L. Delisle, J. Dent. Res. 54A:L76, 1975), are lethal to all of the indicator strains given below. Bacteriocin-sensitive indicator strains. S. mutans FA-1 was obtained from H. V. Jordon, National Institute of Dental Research, Bethesda, Md.; it is a cariogenic rat isolate (3) that produces no detectable bacteriocins but is otherwise similar to strain BHT. S. salivarius 13419 was obtained from the American Type Culture Collection (ATCC); it produces mainly a water-soluble levan from sucrose (10) and is only weakly cariogenic (12). S. sanguis 10556 (ATCC) is a noncariogenic organism (9), isolated from a patient with subacute bacterial endocarditis, that produces both dextran and levan from sucrose (13). S. bovis 9809 (ATCC) is a bovine rumen isolate that was included for comparative purposes because, although it is noncariogenic (9), it produces copious amounts of a water-soluble dextran from a sucrose. A streptomycin-resistant mutant (9809strr) of this strain was isolated after ultraviolet light mutagenesis and selection on agar containing 1 g of streptomycin per liter. The S. pyogenes culture used is a group A strain from this department collection; it produces no unique polysaccharides from sucrose. Media and chemicals. All bacteriological media were purchased from BBL, Division of Becton, DickAND METHODS MATERIALS inson & Co., Cockeysville, Md. Trypticase soy broth Bacteriocin-producing strains. S. mutans BHT (TSB) was commonly used, with 15 g of agar added was obtained from D. D. Zinner, University of per liter for bottom agar (TSA) or 7.5 g/liter added Miami Institute of Oral Biology, Miami, Fla. This for top agar overlays. To provide a substrate for strain, which is a cariogenic human isolate (14) be- synthesis of extracellular levans and dextrans, su619
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crose was added to a final concentration of 5% (wt/ vol) to TSB, TSA, or TSB top agar. Strain BHT was additionally tested on sucrose-containing media supplemented with 1.5% (wt/vol) L-arginine (Sigma Chemical Co.). One milliliter of a 1% aqueous solution of bromocresol purple (Fisher Scientific Co.) was added per liter of agar media in some experiments as a pH indicator. S. bovis 9809strr was always grown in media containing 1 g of streptomycin sulfate per liter. Bacteriological-grade sucrose was obtained from BBL. Dextran type 2000 (molecular weight, 2 x 106) was obtained from Sigma Chemical Co. Dialysis tubing with an average molecular weight cut-off pore size of 3,500 (Spectropor type 3) was obtained from Spectrum Medical Industries, Los Angeles, Calif. Phosphate buffer (0.05 M, pH 7.0) was used for dialyzing and diluting purified bacteriocin. Bacteriocin testing procedures. A modification of the classical stab overlay method was used to test the activity of and susceptibility to bacteriocins produced on agar media. Producer cultures were streaked across the surface of an agar plate with a straight inoculating needle, using TSA slant cultures as the inocula. The plates were incubated for 48 h at 37 C in Brewer Jars containing an atmosphere of 95% H2 and 5% CO2 (GasPak, BBL). Indicator cultures were grown overnight (16 to 18 h) at 37 C in TSB containing arginine and/or sucrose, and 2 to 3 drops was added to the appropriate top agar (melted and held at 42 C). The seeded top agar tubes were then immediately poured onto producer streak plates. After solidifying, the overlayed plates were incubated aerobically overnight at 37 C. Inhibition zones were measured and recorded the following morning. In each experiment the compositions of the media used for bottom agar, top agar, and indicator broth cultures were identical except for agar concentration and the addition of streptomycin. The above method was used to determine whether producer cells synthesize and release active bacteriocins when grown in sucrose and whether sucrosegrown indicator cells are sensitive to these bacteriocins. The latter was also tested in an aqueous, agarfree system by using a partially purified BHT bacteriocin preparation (described below). In this method, serial dilutions of the bacteriocin suspension were spotted onto TSB-sucrose top agar overlays seeded with TSB-sucrose-grown indicator cells. Bacteriocin destruction was revealed by a reduction in the dilution of bacteriocin that showed visible inhibition of the indicator lawn after 24 h incubation at 37 C. The effects of saliva and purified dextran on bacteriocin activity were also tested in two ways. In the first method, 0.1 ml of 1% (wt/vol) dextran 2000 or freshly collected saliva (unstimulated saliva samples from two individuals with average dental health were pooled) was spotted directly on producer streaks, either 4 h before (and held at 37 C) or immediately after overlaying with indicator cells in TSB top agar. Both untreated and sterile filtered saliva were used. After an additional 24-h incubation period, any reduction in size of the inhibition zones in the area of the spots was noted. In the second method, the cell-free BHT bacteriocin preparation
was again used. The preparation was mixed with an equal volume of dextran 2000 (1% wt/vol) or unstimulated, freshly collected saliva (either untreated or sterile filtered) and incubated for 1 h in a 37 C water bath, and then serial dilutions were spotted onto TSB top agar lawns seeded with S. pyogenes. After 4 h of incubation at 37 C, destruction of bacteriocin activity was evidenced by a reduction in the highest dilution that showed visible inhibition of the indicator lawn. Preparation of cell-free BHT bacteriocin. Strain BHT was grown overnight at 37 C in a sterile medium composed of half-strength APT broth (BBL) and 4% (wt/vol) yeast extract. The culture was sonicated for 1 min (Branson LS 75 Sonifier, 6A) and centrifuged for 10 min at 8,000 x g, and the supernatant fluid was removed and adjusted to pH 7.4 with 0.1 N NaOH. Solid (NH4)2SO4 (Sigma, enzyme grade) was then slowly added to 75% saturation, and the mixture was stirred gently for 4 h at 4 C. The precipitate from 100 ml of culture was collected by centrifuging for 10 min at 10,000 x g, redissolved in 5 ml of phosphate buffer (0.05 M, pH 7.0), dialyzed for 18 h against three 1,000-volume changes of buffer at 4 C, adjusted to pH 7.0, and then sterile filtered (Nalgene Corp., disposable membrane filters, 0.45,um pore size). The preparation used for all the experiments reported here showed detectable bacteriocin activity in spot tests at a dilution of 1:100.
RESULTS
BHT bacteriocin activity at neutral pH. To eliminate the possibility that inhibition zones might be due to the relatively high levels of lactic acid produced by S. mutans rather that to bacteriocins per se, L-arginine was added to bottom and top agar media to control the pH. Strain BHT, along with S. pyogenes and S. mutans strains belonging to Bratthall's serological group b, has a potent arginine deiminase (4; L-arginine iminohydrolase, EC 3.5.3.6); the production of ammonia from added arginine thus neutralizes the acid(s) produced during metabolism. The amount of arginine needed to maintain the pH near 7.0 was determined empirically in preliminary experiments. When 1.5% arginine was added to TSB agar, the final pH, after 24 to 48 h of growth at 37 C, was in the range of 6.8 to 7.2. Under these conditions the sensitive indicator S. pyogenes was strongly inhibited by BHT bacteriocin; the inhibition zone was, in fact, significantly larger than on unsupplemented TSB agar, where the pH could drop as low as 5.2 (Fig. 1). The larger zone at higher pH may have been due to a higher cell yield under these conditions, to stimulation of bacteriocin production, or to enhancement of bacteriocin activity. In any case, inhibition of the sensitive indicator strains by BHT was not due to acid. Bacteriocin production by sucrose-grown
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ACTIVITY OF S. MUTANS BACTERIOCINS
bacteriocinogenic strains. GS-5 and BHT were streaked on TSB-sucrose and TSB-sucrose-1.5% arginine agar media, respectively, and incubated for 48 h at 37 C in an atmosphere of 95% H2 and 5% CO2 to allow maximum synthesis of dextrans before overlaying. The plates were then overlayed with top agar (containing the same concentration of arginine and/or sucrose) seeded with an overnight TSB-sucrose broth culture of S. pyogenes (Fig. 2). In this experiment the producer cells were always coated
_~c7Lo~c
621
with extracellular dextrans but, as can be seen, no diminution of inhibition zones was evident. This result confirms the report of Rogers (11), who also found that growth of S. mutans in sucrose did not prevent the synthesis and release of bacteriocins. Effects of saliva and dextran on bacteriocins produced in agar media. Spotting freshly collected, unsterile saliva on top of 48-h producer streak colonies did not reduce the size of inhibition zones that developed on subsequent
eT _
r
n
m
FIG. 1. Inhibition of S. pyogenes by stab (lower) and streak (upper) colonies of S. mutans under acid and neutral conditions. (A) TSB agar; (B) TSB agar + 1.5% L-arginine. Bar equals 2 cm.
SC. pvOgc'c SC
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FIG. 2. Synthesis and release of active bacteriocins from producer cells grown on sucrose-containing agar. TSA-sucrose streak colonies of (A) BHT and (B) GS-5 were overlaid with TSB-sucrose top agar seeded with S. pyogenes. The bottom agar in (A) also contained arginine.
622
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incubation of plates overlayed with S. bovis 9809strr (Fig. 3). Here streptomycin was incorporated into the top agar to minimize growth of contaminants from the raw saliva. Identical results (not shown) were obtained by spotting with sterile filtered saliva and dextran 2000. Effects of saliva and dextran on cell-free BHT bacteriocin activity. When the partially purified BHT bacteriocin was incubated with saliva (either raw or sterile filtered), no reduction in bacteriocin activity could be detected by spotting serial dilutions on lawns of S. pyogenes (Fig. 4). Here the pH of the saliva-bacteriocin system could be controlled, and no difference was observed when the pH was adjusted to 6.0, 6.5, or 7.0. Again, identical results (not shown) were obtained by incubating the bacteriocin preparation with dextran 2000. Susceptibility of polysaccharide-synthesizing, sucrose-grown indicators. When genetically sensitive strains were pregrown in TSBsucrose broth, inoculated into TSB-sucrose top agar, and overlayed on 2-day-old TSB-sucrose agar streak cultures of GS-5 and BHT, clear inhibition zones developed after incubation. Moreover, the zone sizes did not differ from those on sucrose-free media (data not shown). Figure 5 shows the results with GS-5, using arginine-free media, and Fig. 6 shows the results with BHT, using arginine-containing media to control the pH. In all cases the sensitive indicator strains remained sensitive to both
bacteriocins when grown in the presence of sucrose. Under these conditions strains 13419 and 9809 synthesized copious amounts of extracellular polysaccharides, which is clearly evident from the figures. Strains FA-1 and 10556 produced tenacious dextran lawns under these conditions, but these lawns did not have the highly mucoid, "photogenic" appearance obtained with the other strains. The inhibition zones against FA-1 were quite narrow because this strain was the least sensitive of all the indicator strains. Since the relative size of the inhibition zones produced against each indicator strain, by both producers, remained constant on a wide variety of sucrose-free media (data not shown), the zone sizes reflect differences in sensitivity to the bacteriocins rather than differences in the type or quantity of extracellular polysaccharides synthesized from sucrose. The possibility that zone size differences may be due to multiple bacteriocins cannot be ruled out but is considered unlikely since single-step mutants of BHiT that had lost bacteriocin activity against S. pyogenes lost activity against all the other indicator strains at the same time (Delisle, unpublished data). When BHT cell-free bacteriocin was spotted onto TSB-sucrose top agar lawns seeded with sucrose-grown cultures of strains 13419, 9809, FA-1, and 10556, definite inhibition zones developed (after overnight incubation) at all dilutions that gave positive results in the complete
9809 StrI'
Bl.Ti
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FIG. 3. Effect ofsaliva on bacteriocin activity in agar. A streak colony ofBHT on TSA-arginine was spotted with saliva (arrow), incubated for 4 h at 3 7 C, and then overlaid with TSB-streptomycin top agar seeded with S. bovis 9809strr. After 18 h the indicator lawn shows no discernible reduction in the size ofthe inhibition zone in the region of the saliva.
VOL. 13, 1976
ACTIVITY OF S. MUTANS BACTERIOCINS
623
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C FIG. 4. Effect of untreated and sterile filtered saliva on the activity of cell-free BHT bacteriocin. Bacteriocin mixed with an equal volume of (A) sterile TSB, (B) raw saliva, or (C) sterile filtered saliva and incubated for I h at 37 C. Serial dilutions (indicated on plates) were then spotted onto fresh overlays of S. pyogenes in TSB top agar. The plates were incubated at 37 C and photographed at 4 h. was
absence of sucrose (data not shown), thus con- might enable us to control specific species in the firming that the polysaccharides synthesized by mouth by treatments that stimulate (or rethe indicators do not prevent bacteriocin ad- press) bacteriocin synthesis. sorption and activity. Some workers have claimed that the coexistence of producer and sensitive strains of S. DISCUSSION mutans at the same site is common and that The observation that inhibition zones pro- this implies that bacteriocins do not inhibit duced by BHT in the presence of arginine are sensitive strains in vivo (8, 11), otherwise sensilarger than those in the absence of arginine tive strains would be eliminated from the oral (Fig. 1), for whatever reason, suggests that the cavity; however, no data (or identification of frequent, drastic changes that occur in the oral specific strains) have been presented to support environment could greatly influence the pro- this view. These claims may therefore be based duction of bacteriocins in vivo. Information on on observations of acid sensitivity rather than the effects of these changes is needed since it on sensitivity to bacteriocins. It has recently
INFECT. IMMUN.
DELISLE
624
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.
........
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FIG. 5. Activity of GS-5 bacteriocin against sucrose-grown indicator cells on sucrose agar. Two-day-old TSA-sucrose streaks of GS-5 were overlaid with TSB-sucrose top agar seeded with overnight TSB-sucrose broth cultures of (A) S. salivarius, (B) S. bovis, (C) S. sanguis, and (D) S. mutans FA-1.
been shown, for example, that the vast majority of bacteriocin-like inhibition zones produced by oral streptococci against each other are due solely to acid (2). From the above, it could be argued that the activity of and susceptibility to GS-5 bacteriocin in the presence of saliva and polysaccharides may be reduced or even eliminated, and the results reported here are really due to acid inhibition of the various indicator strains. In the case of strain BHT, the use of argininecontaining media precludes such an argument since the amount of ammonia released by the arginine deiminase of this organism is sufficient to neutralize all of the acid produced during growth. The results reported in this study do not answer the question of whether S. mutans bacte-
riocins are produced in vivo but do clearly demonstrate that (i) saliva does not degrade all S. mutans bacteriocins, (ii) high-molecularweight dextran does not inhibit all S. mutans bacteriocins, and (iii) synthesis of extracellular levans and dextrans from sucrose by oral streptococci does not prevent bacteriocin adsorption and action. Whether saliva from all individuals or all types of in vivo-synthesized dextrans and levans behave similarly is not known, but the data suggest that if, as is generally believed, the general matrix of dental plaque consists of dextrans, bacteriocins can diffuse through this matrix in vivo and exert their lethal effects. S. mutans bacteriocins may therefore play a significant role in regulating the ecology of the oral cavity. Furthermore, since S. mutans bacteriocins are active against a rather wide spec-
VOL. 13, 1976
ACTIVITY OF S. MUTANS BACTERIOCINS
625
1.5 .1
A
FIG. 6. Activity of BHT bacteriocin against sucrose-grown indicator cells on sucrose agar at neutral pH. TSA-sucrose-arginine streak plates of BHT were overlaid with TSB-sucrose-arginine top agar seeded with overnight TSB-sucrose broth cultures of (A) S. salivarius, (B) S. bovis, (C) S. sanguis and (D) S. mutans FA1.
trum of gram-positive organisms, including S. pyogenes and Staphylococcus aureus (6, 7), they may actually be of some value in protecting the oral mucosa from infection by transient pathogens. Finally, the fact that some bacteriocins can function under conditions similar to those found in the oral cavity, as demonstrated in this study, opens the door to possible use of purified bacteriocins for the treatment and prevention of dental caries. ACKNOWLEDGMENTS This study was supported by Public Health Service research grant DE-03189 from the National Institute of Dental Research. The excellent assistance of Monica Thomas is gratefully
acknowledged. LITERATURE CITED 1. Bratthall, D. 1970. Demonstration of five serological groups of streptococcal strains resembling Streptococ-
cus mutans. Odontol. Revy 21:143-152. 2. Donoghue, H. D., and J. E. Tyler. 1975. Antagonisms amongst streptococci isolated from the human oral cavity. Arch. Oral Biol. 20:381-387. 3. Fitzgerald, R. J., H. V. Jordon, and H. R. Stanley. 1960. Experimental caries and gingival pathologic changes in the gnotobiotic rat. J. Dent. Res. 39:923-935. 4. Guggenheim, B. 1968. Streptococci of dental plaques. Caries Res. 2:147-163. 5. Gibbons, R. J., K. S. Berman, P. Knoettner, and B. Kapsimalis. 1966. Dental caries and alveolar bone loss in gnotobiotic rats infected with capsule forming streptococci of human origin. Arch. Oral Biol. 11:549-560. 6. Hamada, S., and T. Ooshima. 1975. Inhibitory spectrum of a bacteriocin-like substance (mutacin) produced by some strains of Streptococcus mutans. J. Dent. Res. 54:140-145. 7. Kelstrup, J., and R. J. Gibbons. 1969. Bacteriocins from human and rodent streptococci. Arch. Oral Biol. 14:251-258. 8. Kelstrup, J., and R. J. Gibbons. 1969. Inactivation of bacteriocins in the intestinal canal and oral cavity. J. Bacteriol. 99:888-890. 9. Krasse, B., and J. Carlsson. 1970. Various types of
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streptococci and experimental caries in hamsters. Arch. Oral Biol. 15:25-32. 10. Newbrun, E., and S. Baker. 1968. Physico-chemical characteristics of the levan produced by Streptococcus salivarius. Carbohydr. Res. 6:165-170. 11. Rogers, A. H. 1974. Bacteriocin production and susceptibility among strains of Streptococcus mutans grown in the presence of sucrose. Antimicrob. Agents Chemother. 6:547-550. 12. Rosen, S. 1969. Comparison of sucrose and glucose in
INFECT. IMMUN. the causation of dental caries in gnotobiotic rats. Arch. Oral Biol. 14:445-450. 13. Sidebotham, R. L., H. Weigel, and W. H. Bowen. 1971. Studies on dextrans and dextranases. Part IX. Dextrans elaborated by cariogenic organisms. Carbohydr. Res. 19:151-159. 14. Zinner, D. D., A. P. Aran, J. M. Jablon, M. S. Saslaw, and R. J. Fitzgerald. 1967. Induction of dental caries in gnotobiotic rats by streptococci of human origin. Nature (London) 213:200-201.
Vol. 13, No. 2 Printed in U.SA.
Activity of Two Streptococcus mutans Bacteriocins in the Presence of Saliva, Levan, and Dextran ALLAN L. DELISLE Department of Microbiology, University of Maryland School of Dentistry, Baltimore, Maryland 21201 Received for publication 18 August 1975
The extracellular dextrans produced from sucrose by Streptococcus mutans strains BHT and GS-5 did not prevent the synthesis or release of active bacteriocins by these two strains. In addition, several streptococci that were genetically sensitive to these bacteriocins, and that could synthesize a variety of extracellular dextrans and levans from sucrose, remained phenotypically sensitive when grown in the presence of sucrose. Bacteriocin activity was not altered by treatment with high-molecular-weight dextran or by human saliva. The bacteriocins produced by, and active against, S. mutans thus appear to be capable of acting in vivo and may play a role in regulating the bacterial ecology ofthe oral cavity. Bacteriocinogenic strains of cariogenic streptococci are known to exist in the human oral cavity (7), but whether their bacteriocins play a role in regulating the bacterial ecology of this environment is not known. It is not yet possible to quantitate, or even detect, active bacteriocins in dental plaque and saliva, so in vivo production of bacteriocins cannot be unequivocally demonstrated; however, there are no compelling a priori reasons why bacteriocins should not be produced in the oral cavity. Current research in this area has therefore focused on the question of whether bacteriocins, particularly those of Streptococcus mutans, can exert their lethal effects under conditions simulating those of the oral environment. Thus, it has been reported that some S. mutans bacteriocins appear to be inactivated by proteolytic enzymes in saliva (8) and, more recently, that bacteriocin-sensitive strains of S. mutans and S. salivarius become insensitive to bacteriocins when grown in the presence of sucrose, due to their elaboration of dextran and levan polysaccharides (11). This investigation was undertaken to determine whether two S. mutans bacteriocins presently under study in this laboratory are inactivated by enzymes in saliva and whether they can kill sensitive cells that are coated with extracellular glucan and fructan polysaccharides synthesized from sucrose.
longing to Bratthall's serological group (b) (1), produces a highly insoluble extracellular dextran from sucrose. S. mutans GS-5 was obtained from R. J. Gibbons, Forsyth Dental Center, Boston, Mass. This strain, which belongs to serological group c, is also a dextran-producing, cariogenic human isolate (5). The bacteriocins produced by these strains, which have similar physical and chemical properties (7; D. Paul and H. D. Slade, Abstr. Annu. Meet. Am. Soc. Microbiol. 1974, M267, p. 110; A. L. Delisle, J. Dent. Res. 54A:L76, 1975), are lethal to all of the indicator strains given below. Bacteriocin-sensitive indicator strains. S. mutans FA-1 was obtained from H. V. Jordon, National Institute of Dental Research, Bethesda, Md.; it is a cariogenic rat isolate (3) that produces no detectable bacteriocins but is otherwise similar to strain BHT. S. salivarius 13419 was obtained from the American Type Culture Collection (ATCC); it produces mainly a water-soluble levan from sucrose (10) and is only weakly cariogenic (12). S. sanguis 10556 (ATCC) is a noncariogenic organism (9), isolated from a patient with subacute bacterial endocarditis, that produces both dextran and levan from sucrose (13). S. bovis 9809 (ATCC) is a bovine rumen isolate that was included for comparative purposes because, although it is noncariogenic (9), it produces copious amounts of a water-soluble dextran from a sucrose. A streptomycin-resistant mutant (9809strr) of this strain was isolated after ultraviolet light mutagenesis and selection on agar containing 1 g of streptomycin per liter. The S. pyogenes culture used is a group A strain from this department collection; it produces no unique polysaccharides from sucrose. Media and chemicals. All bacteriological media were purchased from BBL, Division of Becton, DickAND METHODS MATERIALS inson & Co., Cockeysville, Md. Trypticase soy broth Bacteriocin-producing strains. S. mutans BHT (TSB) was commonly used, with 15 g of agar added was obtained from D. D. Zinner, University of per liter for bottom agar (TSA) or 7.5 g/liter added Miami Institute of Oral Biology, Miami, Fla. This for top agar overlays. To provide a substrate for strain, which is a cariogenic human isolate (14) be- synthesis of extracellular levans and dextrans, su619
620
INFECT. IMMUN.
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crose was added to a final concentration of 5% (wt/ vol) to TSB, TSA, or TSB top agar. Strain BHT was additionally tested on sucrose-containing media supplemented with 1.5% (wt/vol) L-arginine (Sigma Chemical Co.). One milliliter of a 1% aqueous solution of bromocresol purple (Fisher Scientific Co.) was added per liter of agar media in some experiments as a pH indicator. S. bovis 9809strr was always grown in media containing 1 g of streptomycin sulfate per liter. Bacteriological-grade sucrose was obtained from BBL. Dextran type 2000 (molecular weight, 2 x 106) was obtained from Sigma Chemical Co. Dialysis tubing with an average molecular weight cut-off pore size of 3,500 (Spectropor type 3) was obtained from Spectrum Medical Industries, Los Angeles, Calif. Phosphate buffer (0.05 M, pH 7.0) was used for dialyzing and diluting purified bacteriocin. Bacteriocin testing procedures. A modification of the classical stab overlay method was used to test the activity of and susceptibility to bacteriocins produced on agar media. Producer cultures were streaked across the surface of an agar plate with a straight inoculating needle, using TSA slant cultures as the inocula. The plates were incubated for 48 h at 37 C in Brewer Jars containing an atmosphere of 95% H2 and 5% CO2 (GasPak, BBL). Indicator cultures were grown overnight (16 to 18 h) at 37 C in TSB containing arginine and/or sucrose, and 2 to 3 drops was added to the appropriate top agar (melted and held at 42 C). The seeded top agar tubes were then immediately poured onto producer streak plates. After solidifying, the overlayed plates were incubated aerobically overnight at 37 C. Inhibition zones were measured and recorded the following morning. In each experiment the compositions of the media used for bottom agar, top agar, and indicator broth cultures were identical except for agar concentration and the addition of streptomycin. The above method was used to determine whether producer cells synthesize and release active bacteriocins when grown in sucrose and whether sucrosegrown indicator cells are sensitive to these bacteriocins. The latter was also tested in an aqueous, agarfree system by using a partially purified BHT bacteriocin preparation (described below). In this method, serial dilutions of the bacteriocin suspension were spotted onto TSB-sucrose top agar overlays seeded with TSB-sucrose-grown indicator cells. Bacteriocin destruction was revealed by a reduction in the dilution of bacteriocin that showed visible inhibition of the indicator lawn after 24 h incubation at 37 C. The effects of saliva and purified dextran on bacteriocin activity were also tested in two ways. In the first method, 0.1 ml of 1% (wt/vol) dextran 2000 or freshly collected saliva (unstimulated saliva samples from two individuals with average dental health were pooled) was spotted directly on producer streaks, either 4 h before (and held at 37 C) or immediately after overlaying with indicator cells in TSB top agar. Both untreated and sterile filtered saliva were used. After an additional 24-h incubation period, any reduction in size of the inhibition zones in the area of the spots was noted. In the second method, the cell-free BHT bacteriocin preparation
was again used. The preparation was mixed with an equal volume of dextran 2000 (1% wt/vol) or unstimulated, freshly collected saliva (either untreated or sterile filtered) and incubated for 1 h in a 37 C water bath, and then serial dilutions were spotted onto TSB top agar lawns seeded with S. pyogenes. After 4 h of incubation at 37 C, destruction of bacteriocin activity was evidenced by a reduction in the highest dilution that showed visible inhibition of the indicator lawn. Preparation of cell-free BHT bacteriocin. Strain BHT was grown overnight at 37 C in a sterile medium composed of half-strength APT broth (BBL) and 4% (wt/vol) yeast extract. The culture was sonicated for 1 min (Branson LS 75 Sonifier, 6A) and centrifuged for 10 min at 8,000 x g, and the supernatant fluid was removed and adjusted to pH 7.4 with 0.1 N NaOH. Solid (NH4)2SO4 (Sigma, enzyme grade) was then slowly added to 75% saturation, and the mixture was stirred gently for 4 h at 4 C. The precipitate from 100 ml of culture was collected by centrifuging for 10 min at 10,000 x g, redissolved in 5 ml of phosphate buffer (0.05 M, pH 7.0), dialyzed for 18 h against three 1,000-volume changes of buffer at 4 C, adjusted to pH 7.0, and then sterile filtered (Nalgene Corp., disposable membrane filters, 0.45,um pore size). The preparation used for all the experiments reported here showed detectable bacteriocin activity in spot tests at a dilution of 1:100.
RESULTS
BHT bacteriocin activity at neutral pH. To eliminate the possibility that inhibition zones might be due to the relatively high levels of lactic acid produced by S. mutans rather that to bacteriocins per se, L-arginine was added to bottom and top agar media to control the pH. Strain BHT, along with S. pyogenes and S. mutans strains belonging to Bratthall's serological group b, has a potent arginine deiminase (4; L-arginine iminohydrolase, EC 3.5.3.6); the production of ammonia from added arginine thus neutralizes the acid(s) produced during metabolism. The amount of arginine needed to maintain the pH near 7.0 was determined empirically in preliminary experiments. When 1.5% arginine was added to TSB agar, the final pH, after 24 to 48 h of growth at 37 C, was in the range of 6.8 to 7.2. Under these conditions the sensitive indicator S. pyogenes was strongly inhibited by BHT bacteriocin; the inhibition zone was, in fact, significantly larger than on unsupplemented TSB agar, where the pH could drop as low as 5.2 (Fig. 1). The larger zone at higher pH may have been due to a higher cell yield under these conditions, to stimulation of bacteriocin production, or to enhancement of bacteriocin activity. In any case, inhibition of the sensitive indicator strains by BHT was not due to acid. Bacteriocin production by sucrose-grown
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ACTIVITY OF S. MUTANS BACTERIOCINS
bacteriocinogenic strains. GS-5 and BHT were streaked on TSB-sucrose and TSB-sucrose-1.5% arginine agar media, respectively, and incubated for 48 h at 37 C in an atmosphere of 95% H2 and 5% CO2 to allow maximum synthesis of dextrans before overlaying. The plates were then overlayed with top agar (containing the same concentration of arginine and/or sucrose) seeded with an overnight TSB-sucrose broth culture of S. pyogenes (Fig. 2). In this experiment the producer cells were always coated
_~c7Lo~c
621
with extracellular dextrans but, as can be seen, no diminution of inhibition zones was evident. This result confirms the report of Rogers (11), who also found that growth of S. mutans in sucrose did not prevent the synthesis and release of bacteriocins. Effects of saliva and dextran on bacteriocins produced in agar media. Spotting freshly collected, unsterile saliva on top of 48-h producer streak colonies did not reduce the size of inhibition zones that developed on subsequent
eT _
r
n
m
FIG. 1. Inhibition of S. pyogenes by stab (lower) and streak (upper) colonies of S. mutans under acid and neutral conditions. (A) TSB agar; (B) TSB agar + 1.5% L-arginine. Bar equals 2 cm.
SC. pvOgc'c SC
-NJFl
p vCgcneS
C.S-I D I
IA
L
L
VBX + arg + Suc rs
+
FIG. 2. Synthesis and release of active bacteriocins from producer cells grown on sucrose-containing agar. TSA-sucrose streak colonies of (A) BHT and (B) GS-5 were overlaid with TSB-sucrose top agar seeded with S. pyogenes. The bottom agar in (A) also contained arginine.
622
INFECT. IMMUN.
DELISLE
incubation of plates overlayed with S. bovis 9809strr (Fig. 3). Here streptomycin was incorporated into the top agar to minimize growth of contaminants from the raw saliva. Identical results (not shown) were obtained by spotting with sterile filtered saliva and dextran 2000. Effects of saliva and dextran on cell-free BHT bacteriocin activity. When the partially purified BHT bacteriocin was incubated with saliva (either raw or sterile filtered), no reduction in bacteriocin activity could be detected by spotting serial dilutions on lawns of S. pyogenes (Fig. 4). Here the pH of the saliva-bacteriocin system could be controlled, and no difference was observed when the pH was adjusted to 6.0, 6.5, or 7.0. Again, identical results (not shown) were obtained by incubating the bacteriocin preparation with dextran 2000. Susceptibility of polysaccharide-synthesizing, sucrose-grown indicators. When genetically sensitive strains were pregrown in TSBsucrose broth, inoculated into TSB-sucrose top agar, and overlayed on 2-day-old TSB-sucrose agar streak cultures of GS-5 and BHT, clear inhibition zones developed after incubation. Moreover, the zone sizes did not differ from those on sucrose-free media (data not shown). Figure 5 shows the results with GS-5, using arginine-free media, and Fig. 6 shows the results with BHT, using arginine-containing media to control the pH. In all cases the sensitive indicator strains remained sensitive to both
bacteriocins when grown in the presence of sucrose. Under these conditions strains 13419 and 9809 synthesized copious amounts of extracellular polysaccharides, which is clearly evident from the figures. Strains FA-1 and 10556 produced tenacious dextran lawns under these conditions, but these lawns did not have the highly mucoid, "photogenic" appearance obtained with the other strains. The inhibition zones against FA-1 were quite narrow because this strain was the least sensitive of all the indicator strains. Since the relative size of the inhibition zones produced against each indicator strain, by both producers, remained constant on a wide variety of sucrose-free media (data not shown), the zone sizes reflect differences in sensitivity to the bacteriocins rather than differences in the type or quantity of extracellular polysaccharides synthesized from sucrose. The possibility that zone size differences may be due to multiple bacteriocins cannot be ruled out but is considered unlikely since single-step mutants of BHiT that had lost bacteriocin activity against S. pyogenes lost activity against all the other indicator strains at the same time (Delisle, unpublished data). When BHT cell-free bacteriocin was spotted onto TSB-sucrose top agar lawns seeded with sucrose-grown cultures of strains 13419, 9809, FA-1, and 10556, definite inhibition zones developed (after overnight incubation) at all dilutions that gave positive results in the complete
9809 StrI'
Bl.Ti
_
_~~~~~~~~- ~
FIG. 3. Effect ofsaliva on bacteriocin activity in agar. A streak colony ofBHT on TSA-arginine was spotted with saliva (arrow), incubated for 4 h at 3 7 C, and then overlaid with TSB-streptomycin top agar seeded with S. bovis 9809strr. After 18 h the indicator lawn shows no discernible reduction in the size ofthe inhibition zone in the region of the saliva.
VOL. 13, 1976
ACTIVITY OF S. MUTANS BACTERIOCINS
623
1
I
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;
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i
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C FIG. 4. Effect of untreated and sterile filtered saliva on the activity of cell-free BHT bacteriocin. Bacteriocin mixed with an equal volume of (A) sterile TSB, (B) raw saliva, or (C) sterile filtered saliva and incubated for I h at 37 C. Serial dilutions (indicated on plates) were then spotted onto fresh overlays of S. pyogenes in TSB top agar. The plates were incubated at 37 C and photographed at 4 h. was
absence of sucrose (data not shown), thus con- might enable us to control specific species in the firming that the polysaccharides synthesized by mouth by treatments that stimulate (or rethe indicators do not prevent bacteriocin ad- press) bacteriocin synthesis. sorption and activity. Some workers have claimed that the coexistence of producer and sensitive strains of S. DISCUSSION mutans at the same site is common and that The observation that inhibition zones pro- this implies that bacteriocins do not inhibit duced by BHT in the presence of arginine are sensitive strains in vivo (8, 11), otherwise sensilarger than those in the absence of arginine tive strains would be eliminated from the oral (Fig. 1), for whatever reason, suggests that the cavity; however, no data (or identification of frequent, drastic changes that occur in the oral specific strains) have been presented to support environment could greatly influence the pro- this view. These claims may therefore be based duction of bacteriocins in vivo. Information on on observations of acid sensitivity rather than the effects of these changes is needed since it on sensitivity to bacteriocins. It has recently
INFECT. IMMUN.
DELISLE
624
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A.
Ew..
.
........
S-I
V
--' ;) 4
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FIG. 5. Activity of GS-5 bacteriocin against sucrose-grown indicator cells on sucrose agar. Two-day-old TSA-sucrose streaks of GS-5 were overlaid with TSB-sucrose top agar seeded with overnight TSB-sucrose broth cultures of (A) S. salivarius, (B) S. bovis, (C) S. sanguis, and (D) S. mutans FA-1.
been shown, for example, that the vast majority of bacteriocin-like inhibition zones produced by oral streptococci against each other are due solely to acid (2). From the above, it could be argued that the activity of and susceptibility to GS-5 bacteriocin in the presence of saliva and polysaccharides may be reduced or even eliminated, and the results reported here are really due to acid inhibition of the various indicator strains. In the case of strain BHT, the use of argininecontaining media precludes such an argument since the amount of ammonia released by the arginine deiminase of this organism is sufficient to neutralize all of the acid produced during growth. The results reported in this study do not answer the question of whether S. mutans bacte-
riocins are produced in vivo but do clearly demonstrate that (i) saliva does not degrade all S. mutans bacteriocins, (ii) high-molecularweight dextran does not inhibit all S. mutans bacteriocins, and (iii) synthesis of extracellular levans and dextrans from sucrose by oral streptococci does not prevent bacteriocin adsorption and action. Whether saliva from all individuals or all types of in vivo-synthesized dextrans and levans behave similarly is not known, but the data suggest that if, as is generally believed, the general matrix of dental plaque consists of dextrans, bacteriocins can diffuse through this matrix in vivo and exert their lethal effects. S. mutans bacteriocins may therefore play a significant role in regulating the ecology of the oral cavity. Furthermore, since S. mutans bacteriocins are active against a rather wide spec-
VOL. 13, 1976
ACTIVITY OF S. MUTANS BACTERIOCINS
625
1.5 .1
A
FIG. 6. Activity of BHT bacteriocin against sucrose-grown indicator cells on sucrose agar at neutral pH. TSA-sucrose-arginine streak plates of BHT were overlaid with TSB-sucrose-arginine top agar seeded with overnight TSB-sucrose broth cultures of (A) S. salivarius, (B) S. bovis, (C) S. sanguis and (D) S. mutans FA1.
trum of gram-positive organisms, including S. pyogenes and Staphylococcus aureus (6, 7), they may actually be of some value in protecting the oral mucosa from infection by transient pathogens. Finally, the fact that some bacteriocins can function under conditions similar to those found in the oral cavity, as demonstrated in this study, opens the door to possible use of purified bacteriocins for the treatment and prevention of dental caries. ACKNOWLEDGMENTS This study was supported by Public Health Service research grant DE-03189 from the National Institute of Dental Research. The excellent assistance of Monica Thomas is gratefully
acknowledged. LITERATURE CITED 1. Bratthall, D. 1970. Demonstration of five serological groups of streptococcal strains resembling Streptococ-
cus mutans. Odontol. Revy 21:143-152. 2. Donoghue, H. D., and J. E. Tyler. 1975. Antagonisms amongst streptococci isolated from the human oral cavity. Arch. Oral Biol. 20:381-387. 3. Fitzgerald, R. J., H. V. Jordon, and H. R. Stanley. 1960. Experimental caries and gingival pathologic changes in the gnotobiotic rat. J. Dent. Res. 39:923-935. 4. Guggenheim, B. 1968. Streptococci of dental plaques. Caries Res. 2:147-163. 5. Gibbons, R. J., K. S. Berman, P. Knoettner, and B. Kapsimalis. 1966. Dental caries and alveolar bone loss in gnotobiotic rats infected with capsule forming streptococci of human origin. Arch. Oral Biol. 11:549-560. 6. Hamada, S., and T. Ooshima. 1975. Inhibitory spectrum of a bacteriocin-like substance (mutacin) produced by some strains of Streptococcus mutans. J. Dent. Res. 54:140-145. 7. Kelstrup, J., and R. J. Gibbons. 1969. Bacteriocins from human and rodent streptococci. Arch. Oral Biol. 14:251-258. 8. Kelstrup, J., and R. J. Gibbons. 1969. Inactivation of bacteriocins in the intestinal canal and oral cavity. J. Bacteriol. 99:888-890. 9. Krasse, B., and J. Carlsson. 1970. Various types of
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streptococci and experimental caries in hamsters. Arch. Oral Biol. 15:25-32. 10. Newbrun, E., and S. Baker. 1968. Physico-chemical characteristics of the levan produced by Streptococcus salivarius. Carbohydr. Res. 6:165-170. 11. Rogers, A. H. 1974. Bacteriocin production and susceptibility among strains of Streptococcus mutans grown in the presence of sucrose. Antimicrob. Agents Chemother. 6:547-550. 12. Rosen, S. 1969. Comparison of sucrose and glucose in
INFECT. IMMUN. the causation of dental caries in gnotobiotic rats. Arch. Oral Biol. 14:445-450. 13. Sidebotham, R. L., H. Weigel, and W. H. Bowen. 1971. Studies on dextrans and dextranases. Part IX. Dextrans elaborated by cariogenic organisms. Carbohydr. Res. 19:151-159. 14. Zinner, D. D., A. P. Aran, J. M. Jablon, M. S. Saslaw, and R. J. Fitzgerald. 1967. Induction of dental caries in gnotobiotic rats by streptococci of human origin. Nature (London) 213:200-201.
