The antagonists have as yet been detected on solid media only. They inhibited a wide range of organisms. including staphylococci and corynebacteria, and ...
ANTAGONISMS AMONGST STREPTOCOCCI ISOLATED FROM THE HUMAN ORAL CAVITY HI:LEN
MRC Dental
D.
Unit. Dental
DCINOC;HUE
School.
andJ.E.
Lower Maudlin
TYLER
Street, Bristol BSI 2LY
Summary-Antagonist production was found to be widespread amongst oral streptococci. The inhibitory agents were heterogeneous, varying in their sensitivity to heat, chloroform and catalase. They were dialysable and so presumably of low molecular weight. The antagonists have as yet been detected on solid media only. They inhibited a wide range of organisms. including staphylococci and corynebacteria, and occasionally inhibited their own producer strain. Srrcptococcus rt~uns and Streptococcus salicarius appeared to produce acid as the sole inhibitor, and only on agar with added sugar. Both lactic and acetic acids, in varying amounts and proportions. were detected in the inhibition zones around these organisms. Strep. mitior, Strep. sanguis and viridans-type streptococci produced inhibitory levels of hydrogen peroxide and, when grown on glucose-supplemented agar. they often also produced inhibitory acid. It is suggested that a sugary diet profoundly affects the inter-relationships of plaque streptococci by encouraging the production of inhibitory acid. As Strep. rmtuns is particularly aciduric. our “normal” diet may confer a selective advantage on this biotype.
INTRODUCTION It has been recognized
for several years that interactions between oral bacteria may lead to both enhancement or inhibition of growth, and that such interactions are widespread (Thompson and Shibuya, 1946; Scrivener et (II.. 1950; Rosebury, Gale and Taylor, 1954: Green and Dodd, 1956; Parker, 1970; Takazoe ?I ~11.. 1971; Donoghue, 1972: Holmberg and Hallander, 1972). Antagonistic effects have been described most frequently and have consistently been identified with the presence of alpha-haemolytic or non-haemolytic oral streptococci. This group oforganisms is recognlzed as playing a key role in the maintenance of the normal oral and pharyngeal flora and, if the delicate balance between organisms is upset in any way, pathogenic organisms such as Streptococcus pyogenrs, Strep. /"lCWtlO~kW and Stuphy1ococcu.s uureu.~ have been shown to colonize this area (Myers, 1959; Sprunt and Redman. 196X: Sanders. 1969; Johanson rt al., 1970; Crowe. Sanders and Longley, 1973). Some of the inhibitory effects are due to the accumulation of metabolic end-products such as hydrogen peroxide (Thompson and Johnson, 1951; Sprunt and Redman, 1968: Holmberg and Hallander, 1973). Some workers habe suggested that inhibition may be caused by the production of bacteriocins (Gierasimov, 196X; Kelstrup and Gibbons, 1969a; Piatkowski and Szropinska. 1971; Schlegel and Slade, 1972, 1973). It is possible that antagonisms between organisms may affect the formation of dental plaque (Bladen, Harr and Pollock. 1972). Alternatively, it has been suggested that the establishment in the mouth of organisms antagonistic to acidogenic bacteria may lead to a reduction in the level of dental caries (Rutter Ct (II.. 1961). The inhibitory phenomena amongst bacteria may thus play a fundamental role in the ecology of the mouth flora, and warrant further investigation. As previous workers had used a wide variety of bacterial strains and experimental procedures, it was thought worthwhile to examine the antagonisms of a 381
large number of oral streptococci ditions.
under standard
con-
MATERIALS AND METHODS
Bacterial strains Thirty bacterial strains, almost all streptococci. were freshly isolated from human dental plaque. Several named organisms were obtained from other workers (Table 1). These were included in an attempt to determine whether or not the production of a particular type of antagonist was species-dependent. Media Bacteria1 strains were maintained on plates of brain-heart infusion (BHI) agar (Difco Laboratories, Detroit, Michigan, U.S.A.), and subcultured at weekly intervals. Streptococcal basal medium (SBM) containing bactocasitone (Difco) 2 per cent (w/v), yeast extract (Difco) 0.5 per cent (w/v), K,HPO, 0.4 per cent (w/v). KH,P04 0.1 per cent (w/v), NaCl 0.2 per cent (w/v). and agar 1.5 per cent (w/v) (De Stoppelaar, 1971) was used for the demonstration of antagonisms. After sterilization (20 minutes at 120°C). if required, SBM was supplemented with separately sterilized (IO min at 110°C) 50 per cent (w/v) stock solutions of glucose or sorbitol, before plates were poured. Derfwrlsfratiorl ofantagonkrm Organisms to be tested for their inhibitory activity were inoculated from colonies on BHI agar on to a small area of SBM agar. Usually, six different strains were inoculated on one plate. Alternatively, individual strains of bacteria were streak-inoculated across plates. Incubation was for 2 or 3 days at 37°C aerobically or in McIntosh and Filde’s jars filled with hydrogen. After the producer organisms had grown, the macrocolonies were killed by ultraviolet light (Hanovia Lamps, Slough, Middlesex, England). Organisms to be tested for their sensitivity were suspended in soft BHI agar
H. D. Donoghue and J. E. Tyler
382
which was poured over the killed macrocolonies. After the plates had been re-incubated at 37°C for a further 2 days, antagonistic effects were shown by clear areas around the original colonies where the indicator strain had failed to grow (Fig. 1). The plates were routinely re-incubated’ aerobically and overlayered with a plaque coryneform, 071, which had been found in preliminary experiments to be very sensitive to antagonists produced by a wide range of bacteria. Except where otherwise stated, the SBM agar on which the producer strains were grown was supplemented with 0.5 per cent (w/v) glucose. In spite of determined efforts, no method could be found which resulted in the production of detectable levels of antagonist in solution. Investigation
of the inhibitory
agents
Macrocolonies of organisms producing antagonists were grown up as described above. Before ultraviolet treatment, the plates were placed in an oven at 60°C for one hr to test for antagonist heat sensitivity. The effect of chloroform was determined by placing drops in the lids of inverted plates which were left for 30 min at 37°C. Chloroform residues were evaporated by placing the inverted plates with their lids removed in an incubator at 37°C for 30 min. An estimate of the size of the antagonist molecules was gained by testing their ability to pass through dialysis membrane. Squares of sterile Visking dialysis tubing (Scientific Instrument Centre Ltd., 1 Leeke Street, London WCI, England) were placed over the producer colonies. In all these instances, after treatment, the plates were overlayered with an indicator strain in soft agar, and re-incubated, together with untreated control plates. An E.I.L. portable pH meter, model 30C (Electronic Instruments Ltd., Cardiff, Wales), with an Activion probe for measuring surface pH (Activion Glass Ltd., Kinglassie, Fife, Scotland), was used to determine the pH of the inhibition zones. Investigation of the changes in antagonist due to production conditions
composition
Bacteria were inoculated on to plates of plain SBM, and SBM containing 05 per cent (w/v) glucose or sorbitol. The effect of calcium carbonate on antagonist production was shown by inoculating bacteria on to plates of SBM with 05 per cent (w/v) glucose and duplicate plates of SBM supplemented with both 0.5 per cent (w/v) glucose and 1 per cent (w/v) calcium carbonate. After 2 days’ incubation at 37”C, the plates were overlayered with 071 and re-incubated. The antagonists on both the control and test plates were investigated as to their heat-stability and enzyme-sensitivity in selected cases. EfSect of enzyme solutions on antagonists
Organisms were streak-inoculated across SBM glucose plates and incubated for 2 days at 37°C. Plates were then cross-streaked with the test enzyme mixture and control solutions, and incubated at 37°C for 1 hr. After ultraviolet treatment, the plates were overlayered with an indicator organism and re-incubated. The antagonists were cross-streaked with trypsin (1 mg/ml) in barbitol-HCl buffer at pH 8.0, heat-inactivated trypsin, trypsin inhibitor (2 mg/ml), trypsin plus trypsin inhibitor, catalase (from bovine liver, 50 mg/ ml) in deionized water, heat-inactivated catalase, 3-
amino-1,2,4-triazole, a catalase inhibitor (Holmberg and Hallander, 1973) in deionized water (200 mg/ml) and catalase plus inhibitor (Sigma London Chemical Company Ltd., Norbiton Station Yard, Kingstonupon-Thames, Surrey KT2 7BH, England). The antagonists produced under varying experimental conditions were subsequently investigated by this method. The effect of heat or chloroform pre-treatment on the enzyme-sensitivity of antagonists was also studied. Fatty* acid analysis
Bacteria were streak-inoculated across duplicated SBM glucose plates and, after 2 days’ incubation at 37°C the organisms were killed by ultraviolet treatment. One set of plates were overlayered to demonstrate inhibition zones. The others were used for fatty acid analyses. The analyses were performed on segments of agar known to be in the inhibition zone. Volatile fatty acids were assayed by gas-phase chromatography after distillation of the agar samples by a low temperature vacuum technique (Tyler, 1971). Lactic acid was assayed by a calorimetric method (Pryce, 1969). Agar samples (0.1-0.5 g) were placed into deionized water and left overnight at 6O”C, when aliquots were removed and assayed for lactate. Uninoculated SBM glucose agar, and SBM glucose agar containing 80 mg/loO ml of lithium lactate were used as the controls. Within a single experiment, the coefficient of variation (C.V.) of the blank controls was 5.8 per cent and that of the controls containing lactate was 12.7 per cent. The C.V. of the fatty acid assay was 7.4 per cent (Tyler, 1972). RESULTS
Every bacterial strain tested was screened for its inhibitory activity against the total panel of microorganisms. The organisms varied greatly in the range of their antagonistic activity, and in their sensitivity to the antagonists. By varying the experimental conditions, antagonist production could be demonstrated in all the streptococci tested. Investigation
qf the i)lhibitorJ
ayents
All agents tested could pass through a dialysis membrane, so presumably contain low molecular weight constituents. Inhibition zones around colonies of Strep. rnutans, Strep. mitior and CHT were stable to reseeding by an indicator organism (these were the only zones tested). The pH of the inhibition zones varied according to the production conditions (Table 2). The zones were of a lower pH if produced anaerobically, and usually were of higher pH if the medium was unsupplemented, contained 0.5 per cent (w/v) sorbitol, or contained 1 per cent (w/v) calcium carbonate in addition to any added glucose. The cross-streaking experiments showed that almost all strains were producing a mixture of components, and this varied according to the production conditions used (Figs. 2 and 3). Four strains of plaque streptococci, Strep. milleri, FW75, and Strep. pneumoniue, appeared to produce a single catalase-sensitive component when grown on SBM glucose agar. Almost all the other fresh isolates, Strep. sanguis, and CHT, produced a catalase-sensitive component on this medium, and also varying amounts of a component resistant to
Antagonisms of oral streptococci Table 1. Source of bacterial Bacterial species
Strain number
Streptococcus milleri Stwp. rdiot Stwp. pneutnorliue Slwp. smguis Strq. ,mfturl.s Strep. sulinvius Stwp. IIIU~NfI.S Stl?p. tmltu11.s Swcp. n1utari,s Stwp. surl(guis
FW73 FW15 FW287 JENA 2697 NCTC 10449 NCTC 8606 C67p1 C67p25 Cl8&2 T175-I
Strrp. tfUlrallS Stwp. JiIUtutIS
BHT JC2 Kl 112 2M2 AHT BHT FA-1 HHT CHT S-5123 Ingbritt L/Sang
“cariogenic
streptococcus’
Srwp. ,sulix7hs “noncariogenic Srrrp. lnufurla
strep.”
Strep. tlluturls Srwp. ,t1utan.s Strep. sdivurius
“noncariogenic
streptococcus
swcp.,mfturl.s Strep. sur~guis
Table 2. Estimation
Producer organism
strains obtained
383
from other laboratories
Source
Reference
Dr. G. Howden, The Dental School, Bristol. England.
Colman and Williams (I 967)
Dr. J. de Stoppelaar, Dept. of Preventive Dentistry, University of Utrecht, I The Netherlands. Dr. D. B. Drucker, Dept. Bacteriology & Virology, Univ. of Manchester 1 England.
De Stoppelaar
( 197 1)
I
Drucker and Melville (I 97 1) I
Dr. A. S. Bleiweis, Univ. of Florida, Jablon and Zinner (1966) Gainesville, Florida, U.S.A. i G. H. Bowden and J. M. Hardie, Colman (1968) The London Hospital Krasse (1966) Medical College Dental School, Turner Street, London E 1.
of acetate and lactate in the inhibition zones produced by laboratory strains of plaque streptococci grown on SBM glucose agar
and freshly-isolated
Zone width
Zone
(mm)
PH
Acetate (%)
Lactate (%)
13.0 8.5 15.0 6.0 9.0 15.0
5.5 6.0 6.5 5.8 5.8 5.5
0.005 0005 0.090 0050 0.120 0260
0305 0.170 0470 0.630 0.280 0.115
9.0
6.0
0.025
0.340
aerobic anaerobic aerobic aerobic aerobic anaerobic
3.0 3.5 0.5 3-o 9.5 12.0
7.0 6.5 6.2 64 5.9 6.0
0.005 0.005 0.010 0.015 0.140 Nil
0. I 75 0.235 0.030 0.100 0.600 0.175
aerobic aerobic anaerobic aerobic aerobic
16.0 9.5 9.0 1.5 10.5
7.0 5.0 6.5 7.0 5.0
0.025 Nil Nil Nil 0070
o- I30 0.765 0.610 0.290 0.860
Oxygen tension
Strep. rmtms
10449 I 0449 BHT Ch7~~I lngbritt J
