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The production of bacteriocin-link substances by the oral bacterium Streptococcus salivarius
THE PRODUCTION OF BACTERIOCIN-LIKE BY THE ORAL BACTERIUM STREPTOCOCCUS R. P. Department
DEMPSTER
of Microbiology,
SUBSTANCES SALII/ARZUS
and J. R. TACG
University
of Otago,
Dunedin.
New Zealand
Summary---Inhibitory substances produced by six strains of Streptococcus saliwrius were isolated and partially characterized. The six prototype producer strains were selected initially on the basis of their differing spectra of inhibitory activity when tested against a set of nine standard indicator strains. Optimal production conditions were defined for each producer strain and inhibitor-containing extracts were obtained for characterization studies. All of the inhibitors appeared to be proteinaceous substances of molecular weight greater than 3500. When tested against a Streptococcus pyogenes indicator strain, one of the inhibitors was bactericidal, but the other five appeared to be bacteriostatic. Some differences between the inhibitors were observed with respect to heat and enzyme sensitivities.
INTRODUCTION
Oral streptococci have been demonstrated to be capable of producing a wide range of inhibitory compounds (Tagg, Dajani and Wannamaker, 1976; Brandis, 1978). Bacteriocin-like substances are released in vitro by strains of Streptococcus mutans (Hamada and Ooshima, 1975; Delisle, 1976; Rogers, 1976) Streptococcus sanguis (Fujimura and Nakamura. 1979) and Streptococcus mitis (Dajani, Tom and Law, 1976). Inhibitor production by Streptococcus saliuurius has seldom been reported. Donoghue and Tyler (1975) suggested that the inhibitory activity of Strep. salirurius strains may be attributable solely to their production of lactic and acetic acids. More recently, it has been demonstrated that certain strains of Strep. .sulirurius may produce a low mol. wt antibiotic, enocin. that acts by interfering with the utilization of pantothenic acid by susceptible cells (C. C. Sanders and W. E. Sanders. Abstr. No. 178, Interscience Conference on Antimicrobial Agents and Chemotherapy, 1978). In other studies (Tagg J. R. and Russell C.. unpublished; Weerkamp, Vogels and Skotnicki, 1977) inhibitory substances having bacteriocin-like properties have been reported. It has been suggested that either bacteriocins or other inhibitory substances produced by bacteria in the human oral cavity may function to protect the host against colonization by Streptococcus pneumoniae (Johanson et al., 1970) Streptococcus pyogenes (Sanders, Nelson and Sanders, 1977) and Corynebacterium diphtheriue (Thompson and Shibuya, 1946). Moreover. there is some evidence that inhibitors produced by plaque bacteria may influence the microbial composition of plaque formed in vitro (Weerkamp, Bongaerts-Larik and Vogels, 1977; Russell and Tagg, 1981) and in riro (Rutter et (I/., 1961; Rogers, Van der Hoeven and Mikx. 1979). Kelstrup et al. (1970) developed a method of “fingerprinting” oral streptococci based upon their production of, and sensitivity to, bacteriocin-like inhibitors. In the present study we utilized a similar
scheme to identify six strains of Strep. salicnrius that produced distinctive inhibitory spectra when tested against a set of nine standard indicator strains.
MATERIALS
Bacterial
AND METHODS
strains
The six prototype inhibitor-producing strains of saharius (strains 5, 6. 9, 36, MPS and 2OP3) were isolated from human saliva specimens. The initial presumptive identification of these strains as Strep. salicurius was based upon their colony appearance when saliva samples were plated on Mitis-salivarius agar. Identification was confirmed using physiological tests as outlined by Hardie and Bowden (1976). Lancefield grouping established that only strains 5 and MPS were reactive with group K antisera. The nine standard indicator strains (Ii to I,) used for producer (P)-typing the Strep. .salivarius strains, according to their production of characteristic inhibitory patterns, have been described previously (Tagg and Bannister, 1979). The indicators I, (Staphy/ococcus epidermidis strain T-l 8) and I, (Strep. pyogenes strain 71-679) were used in mode of action studies. Representative strains of various species used to further define the activity spectra of the prototype strains were obtained from the culture collection of the Department of Microbiology, University of Otago. All strains were stored at -7o’C in skimmed milk and subcultured on blood agar when in regular use. Strep.
Media
All bacteriological media were obtained from Difco Laboratories (Detroit, U.S.A.) unless otherwise stated. Liquid media tested as substrates for the production of inhibitors included Todd-Hewitt broth (THB), thioglycollate medium (TM), tryptic soy broth, brainheart infusion, veal infusion broth, cooked meat medium and APT broth (B.B.L., Cockeysville, U.S.A.). Supplements to the basic liquid media that were tested in various experiments included yeast extract (4
152
R. P. Dempster and J. R. Tagg
per cent, w/v), agar (0.05 per cent, w/v), neopeptone (3 per cent, w/v) and sucrose (1 per cent, w/v). Solid media were prepared by the addition of 1.5 per cent (w/v) agar (Davis Gelatine Ltd, N.Z.) to the liquid media. Blood agar was 5 per cent (v/v) human blood in Columbia agar base (Gibco Diagnostics, Madison, U.S.A.) poured on a base of saline agar. Detection and assay of inhibitors
Initial detection of inhibitor-producing strains was by application of the simultaneous antagonism test using I, as the indicator. “Fingerprinting” of the inhibitor-positive isolates according to their P-type was achieved using the deferred antagonism test with nine standard indicators. Both of these procedures were conducted as previously outlined (Tagg and Bannister, 1979) with the exception that the Strep. salivarius cultures were grown in a COz-enriched atmosphere (candle jar) instead of aerobically. The inhibitory titre of preparations was determined by a modification of the well-diffusion method, using constant-volume (35 ml) blood agar plates (Tagg and Wannamaker, 1978). Twofold dilutions of the inhibitory preparations were dispensed in 0.05 ml vol. into the wells. After allowing the liquid to absorb, the surface of the medium was sterilized by exposing to chloroform vapours for 20 min. After airing the plates a THB culture of Staph. epidermidis strain T-18 grown for 18 h at 37°C was swabbed evenly over the surface. Following incubation for 18 h at 37°C the highest dilution to give definite inhibition of the growth of the indicator lawn was defined as containing one arbitrary unit (AU) of inhibitor per ml. The titre of the inhibitor was the reciprocal of this dilution. Extraction of inhibitors
Attempts were made to extract active inhibitor from lawn cultures of the producers on various solid media either by freezing and thawing the medium (Tagg, Dajani and Wannamaker, 1975) or by dicing the medium and eluting at 4°C for 18 h into 8 ml vol. of either 1 M sodium chloride or 7 M urea. Supernatants obtained by centrifugation of 10 ml liquid cultures were tested for inhibitory activity and the cell pellets were divided into two portions. One half was resuspended in 0.5 ml of 7 M urea and the other in 0.5 ml of 1 M sodium chloride for 18 h at 4°C. Following removal of the cells by centrifugation the extracts were tested for inhibitor activity. Neither 7 M urea nor 1 M sodium chloride inhibited the growth of the indicator lawns under the test conditions. Characterization of inhibitors
Inhibitor preparations were partially purified by precipitation with 60 per cent ammonium sulphate, followed by dialysis in 3500mol. wt cut-off dialysis tubing (A. H. Thomas Co., Philadelphia, U.S.A.) against 100~01. of phosphate-buffered saline, pH 7.1 (PBS). These preparations were then adjusted to a titre of 4-8 AU/ml by dilution in PBS. Dialysis of inhibitor preparations against PBS for 18 h at 4°C using tubing of mol. wt cut offs 3500, 8000 and 12,000 was one method used to give an indication of the size of the inhibitory substances. In another approach, the ability to dialyse the inhibitors as pro-
duced in solid medium was tested by interposing a single sheet of dialysis membrane between the surface of a blood agar plate and 0.1 ml of blood agar medium that had been inoculated with the producer strain. As a control 0.1 ml of the inoculated blood agar was placed directly on the surface of the blood agar plate. Following incubation the dialysis sheet and blood agar drop cultures were removed. The plate was then treated with chloroform, aired and seeded with a THB culture of the indicator strain grown for 18 h at 37°C. In some experiments, the partially purified inhibitor preparations were incorporated within drops of blood agar medium and tested for their ability to dialyse using the above procedure. The partially purified preparations were tested for activity following heat treatment for 30 min at 80, 100 and 122°C and also following mixing with chloroform 10 per cent (v/v) for 30min. The preparations were also examined for susceptibility to 1 mg/ml (final concentration) of, trypsin (1:250; Difco), protease (Type V), lipase (Type 1) and cc-amylase (Sigma Chemical Co., St. Louis, U.S.A.). All enzymes were in 0.2 M (pH 7.5) TES buffer (Sigma). Incubation was at 37’C for 3 h at which time the activity of the treated preparations was compared with that of control untreated preparations. The enzymes (at 1 mg/ml) were not inhibitory to the growth of the indicator strain under the assay conditions. Mode of action of the inhibitors
The mode of action was determined by adopting a method used by Johnson, Tagg and Wannamaker (1979). Overnight THB cultures of indicators 1, and Is were diluted in warm THB to approx. lo4 colonyforming units (c.f.u.)/ml. After incubation for 2 h at 37°C these indicator cultures were then mixed with an equivalent volume of titre 8 inhibitor preparation. The mixtures were incubated at 37°C and samples taken at 0, 2. 4, 6 and 24 h were suitably diluted in PBS and the viable count determined on blood agar. RESULTS
Activity spectrum of protot)jpe inhibitors
When “fingerprinted” by the deferred antagonism procedure (producer streak cultures incubated microaerophilically on blood agar at 32°C for 18 h) the six prototype inhibitory strains of Strep. salivarius could be differentiated according to their characteristic inhibitory patterns (P-types) (Table 1). When the prototype strains were themselves used as indicators under the same test conditions (Table 2) it was evident that none of the producer strains was “autoinhibitory”. Subsequent tests indicated that for strains MPS and 36, modification of the culture conditions apparently enhanced the production of inhibitor(s). When incubated anaerobically at 37°C strain MPS produced inhibitor(s) active against all of the prototype producer strains, including itself. Strain 36 was found to produce a broader spectrum of inhibitory activity at 37°C than at 32°C. More detailed examination of the range of inhibitory activity of the prototype strains in the deferred antagonism method on blood agar revealed some basic similarities (Table 3). For this particular test the producer strains were grown in candle jars for 18 h at
153
Bacteriocin-like substances of Strep. salirarius Table
1. Standard
inhibitory
Prototype producer strain
I,
patterns of prototype strains*
Inhibition I, 13
+ + + + + (i) + + ++-++++-++--++++
5 6 9 l& 2OP3
-
inhibitor-producing
of standard indicator strain I4 I, I, I7 I, + (-) (-)
+ + (+)
+ + -~ -~
+ + + +
I9
+ + (+)
(+, (-) (-)
+
(-)
+ Indicator inhibited. - Indicator not inhibited. ( ) Weak inhibition occasionally noted. * Producer streak cultures were incubated on blood agar at 32°C for 18 h in a CO*-enriched atmosphere.
their optimal production temperatures: 37°C for strains MPS and 36 and 32°C for the other producers. Activity was particularly widespread against streptococci of Lancefield groups A, C, E and G and group B streptococci of animal origin. Group D streptococci and human strains of group B streptococci seemed generally to be resistant. Differences in the inhibitory spectra of the prototype producers became more apparent when 22 strains of “viridans” streptococci were used as indicators. The number of susceptible strains varied from three (for prototype strain 9) to 13 (for prototype strain 6). Strains of Strep. mutans were found to be relatively resistant. None of the prototype producers seemed active against either staphylococci or a variety of Gram-negative bacteria. By contrast, strains of Actinomyces viscosus and Micrococcus sp. were sensitive to all of the producers. Characterization of the prototype inhibitors As a preliminary to their characterization,
the opti-
mal conditions for the production and extraction of the inhibitory substances associated with each prototype producer strain were determined (Table 4). For each producer, the yield of inhibitory activity was determined, following growth for 18 h of cultures in a wide variety of both liquid and solid substrates (see Materials and Methods), under the conditions of optimal temperature and aeration for inhibitor production as delined by use of the deferred antagonism test on blood agar. The inhibitors were then concentrated, partially purified and adjusted to a titre of 48 AU/ml prior to investigating some of their physico-chemical properties (Table 5). With the exception of that produced by strain 36, all of the inhibitors seemed quite stable to heating at 80°C for 30min. Inhibitors from strains 5 and 2OP3 were unaffected by autoclaving (122°C) for 30 min. None of the inhibitors were inactivated when shaken with chloroform for 30 min. All of the inhibitors were found to be inactivated when treated with protease
Table 2. Inhibitory activity of prototype producers using the producers also as indicators* Prototype producer strain 5 6 9 h& 2OP3
Inhibition 5
6
-:++-_ 1+ -$ -
1+ + +
of indicator strain 9
36
MPS 2OP3
+
+
-
-
+ -
+ +
-
-
L$ -
11 -
+ Indicator inhibited: - Indicator not inhibited. * Producer streak cultures were incubated on blood agar at 32°C for 18 h in CO*-enriched atmosphere. t When strain 36 was incubated as above, but at 37°C indicators 5 and 6 were inhibited. $ When strain MPS was incubated at 37°C anaerobically indicators 5, MPS and 2OP3 were inhibited.
R. P. Dempster and J. R. Tagg
154
Table 3. Range of inhibitory
activity of prototype Number
Indicator
strain
Streptococcus: Group A (81)t Group B (human) (5) (animal) (10) Group C (21) Group D (5) Group E (3) Group F (1) Group G (12) Strep. mutans (4) Strep. salirwius sanguis (5)(10) Strep. mitior (1) Strep. pneumoniu (2) Lactohuc~illus ucidophilus (1) L. casei (1) Actinomyces tliscosus (3) A. naeslundii (1) Staphylococcus sp. (4) Corynebacterium diphtheriae (3) Micrococcus sp. (2) Gram-negative strains (12)f
producers*
of strains sensitive to prototype producer strain
5
6
9
36
MPS
2OP3
81
81
61
81
81
81
3 10 17 0 3 0 12 0
0 10 17 0 3 0 12 1
0 0 9 0 :,
0 10 14 0 3 0 6 1
0 10 12 0 3 0 3 1
1; 16 0 3 0 12 0
31 0 2 0 0 3 0 0 1 2 0
82 0 2 0 0 3 0 0 1 2 0
01 0 2 0 0 3 0 0 0 2 0
42 :
25 0 2 0 0 3 1 0 2 2 0
: 0 2 0 0 3 0 0 1 2 0
0 0
1 0 3 0 0 0 2 0
* Producer streak cultures were incubated on blood agar in a CO,-enriched atmosphere for 18 h at 32°C (37°C for strains MPS and 36). t Number of strains tested is given in parenthesis. $ Including strains of Salmonella, Proteus, Pseudomonas, Escherichia, Neisseria, Veillonella, Enterohacter and Serratia.
Table 4. Optimal
Prototype producer straint 5
conditions
for recovery of active inhibitors producers
Medium for production
6
TM + 3 per cent neopeptone Blood agar
9
Blood agar
36
MPS 2OP3
THB
THB + 3 per cent neopeptone TM + 3 per cent neopeptone
Method of inhibitor recovery Culture supernatant Freeze/thaw extract Freeze/thaw extract Cell pellet resuspended in 7 M urea at 4 C overnight Culture supernatant Culture supernatnat
from prototype
Initial inhibitor titre (AU/ml)* 8 1
1 1
2 4
* Prior to concentration and purification procedures. t The producers were incubated under the conditions of temperature and aeration found to be optimal for production on blood agar in deferred antagonism. For strains 5,6,9 and 20P3 this was at 32°C in a candle jar; for MPS at 37°C anaerobically, and for 36 at 37°C in a candle jar.
Bacteriocin-like substances of Strep. saliaaritts
155
Table 5. Susceptibility of prototype inhibitors to potential inactivating agents Titre of prototype inhibitor Treatment Nil 8O’C for 30min 100°C for 30 min 122°C for 30 min Chloroform for 30 min Protease* Trypsin* a-Amylase* Lipase*
5
6
9
36
8 8 8 8 8 0 0 4 4
8 8 4 2 8 0 0 4 4
4 4 2 1 4 0 0 2 2
4 2 0 0 4 0 0 0 2
MPS 2OP3 8 8 8 1 8 0 0 4 4
8 8 8 8 8 0 4 4 4
* Inhibitor preparations were mixed with an equivalent volume of enzyme and incubated at 37°C for 3 h. Final enzyme concentration was 1 mg/ml.
and with the exception of 2OP3 all seemed sensitive to trypsin. None of the inhibitors were lipase sensitive and only inhibitor 36 appeared sensitive to amylase. When tested for dialysability against PBS there was no apparent loss of activity with any of the inhibitory preparations. By contrast, when the inhibitors were assessed for their ability to traverse dialysis membranes set into blood agar, all six appeared to pass through membranes with mol. wt cut offs of 12,OCQ and 8000, but not 3500. Similar results were obtained when either producer cultures or the partially-purified inhibitors were incorporated within the overlying drops of blood agar. Investigation of the mode of action of the inhibitors revealed that only inhibitor 6 was bactericidal for indicator strain I, under the test conditions. This inhibitor brought about a one-thousandfold decrease in the viable count of Is after 2 h exposure. After 4 h no viable cells were detected. Each of the other inhibitor preparations was found to be bacteriostatic for I, when tested under the same conditions. All 6 inhibitors appeared to be bacteriostatic for indicator strain I,. DISCUSSION
In the present study it has been demonstrated that strains of Strep. saharius may produce a variety of bacteriocin-like inhibitory substances. Screening tests are currently being conducted in this laboratory in order to estimate the incidence of inhibitor-producing Strep. saliuarius strains in human saliva specimens. In these tests, 50 colonies of Strep. saliuarius isolated from each specimen are examined for inhibitor production against a set of indicator strains. Preliminary results have indicated that in approx. 10 per cent of individuals, more than 20 per cent of the Strep. saliuarius isolates are inhibitor positive. Moreover, longitudinal studies have demonstrated that individuals harbouring the prototype producers have retained Strep. saliuarius strains of the same P-type in approximately the same proportions for the three years of the study. The initial differentiation of the six prototype inhi-
bitor-producing strains described in this investigation was on the basis of their specific and characteristic inhibitory spectra (P-types) when tested against a set of nine standard indicator strains. With more detailed examination of the range of their activity spectra, close similarities became apparent. The high susceptibility of group A streptococcus strains is intriguing in view of the hypothesized role of “viridans” streptococci in protecting individuals against group A streptococcal pharyngitis (Sanders et a/., 1977). In a previous study it had been observed (Johanson et al., 1970) that strains of Strep. saliuarius were particularly effective inhibitors of Strep. pneumoniae and an in-uiuo role of bacterial antagonism in preventing pneumococcal colonization of the human pharynx was proposed. We also have observed that Strep. pneumonia strains are extremely sensitive to the inhibitors produced by all six prototype strains. Although there was considerable activity against some characteristic plaque organisms such as Strep. sanguis and A. uiscosus, others notably including Strep. mutans (strains UTBI, UTB2, 10449 and HS6) and Lactobacillus sp. did not seem to be susceptible. The apparent greater sensitivity of animal than human strains of group B streptococci supports a previous report of the differing bacteriocin susceptibility of these strains depending upon the source of the isolate (Tagg and Martin, 1980). Differences between the six prototype inhibitors became particularly evident when the conditions for optimal production and extraction were tested. The importance of standardization of media and incubation conditions has been emphasized previously (Rogers, 1972; Tagg et al., 1976). For strains 5, MPS and 2OP3, addition of 3 per cent (w/v) neopeptone to the basal medium was found to enhance production. This effect may have been due to the recognized antiproteinase action of neopeptone which would tend to increase the yield of biologically-active proteinaceous material in the culture supernatants (Tagg et al., 1976). In studies of bacteriocin production by strains of Strep. mutans it has been reported that yields may be increased by supplementing the basal media with
156
R. P. Dempster and J. R. Tagg
yeast extract (Rogers, 1972) or by the use of freshly prepared filter-sterilized liquid media (Kelstrup and Funder-Nielson, 1977). Neither of these approaches seemed to enhance the yield of the Strep. saliuarius inhibitors. Anaerobic incubation of strain MPS appeared to foster production of inhibitor(s) active against all of the prototype producers. Activity of bacteriocin-like inhibitors against the producing bacterium is uncommon, although it tends to occur more frequently within the Gram-positive bacteria (Tagg et al., 1976). The possibility that some additional inhibitory agent is produced in the course of anaerobic incubation cannot yet be dismissed. Nevertheless, if such is the case, then the two inhibitors seem to remain associated throughout the procedures of ammonium sulphate precipitation and dialysis against PBS. The problem of obtaining inhibitors in solution seems to be one common to most investigations involving streptococci (Tagg et al., 1976). The inhibitors produced by strains 6 and 9 could not be recovered from any of the tested liquid media, but adequate yields could be obtained by freeze-thaw extraction of cultures grown as lawns on blood agar medium. The inhibitor produced by strain 36 seemed predominantly cell-associated in broth cultures and recovery could be effected by eluting from the producer cells into 7 M urea. Some additional differences between the prototype inhibitors became apparent when the properties of the substances were examined. partially-purified Although strain 36 inhibitory activity was partially sensitive to heating at 80°C for 30min, all of the others appeared to be remarkably heat-tolerant. Such heat stability seems to be a common characteristic of low mol. wt bacteriocins produced by Gram-positive bacteria (Tagg et al., 1976). Estimation of the mol. wt of all six inhibitors, as produced during growth of the producer strains on blood agar medium, suggested they were in the range 3500 to 8000. It is not clear why the mol. wt of each of the partially purified inhibitors when tested by dialysis against PBS appeared to be greater than 12,000. It is possible that in their extracted form the inhibitors may be complexed with stabilizing medium components or, alternatively, aggregate formation by monomeric inhibitor molecules may be favoured (Barry et al., 1965). Investigation of enzyme susceptibility indicated that each of the inhibitors probably had an essential protein component as judged by sensitivity to protease. Bacteriocin sensitivity to proteases and the apparent ability of bacteriocin-sensitive strains to coexist with producers in vivo led Kelstrup and Gibbons (1969) to suggest that if bacteriocins were produced in the mouth they may be inactivated by the natural salivary proteases. By contrast, in another study (Hamada and Ooshima, 1975) it was demonstrated that plaque proteases may not necessarily be capable of inactivating bacteriocins in vivo. We have found that there is no detectable decrease in the activity of partially purified preparations of the prototype inhibitors on incubation for 4 h at 37°C with 50 per cent (v/v) fresh human saliva. At present we are attempting to determine whether the prototype inhibitors can be produced under simulated in vivo conditions.
Although it has been suggested that by definition a “classic” bacteriocin would be expected to have a bactericidal mode of action (Tagg et al., 1976), it has become clear that there are many bacteriocin-like substances that seem to have bacteriostatic action against either some or all of the sensitive bacteria (Hale and Hinsdill, 1975; Dajani et al., 1976). Indeed, the apparent mode of action may vary depending upon the choice of indicator used and the titre of inhibitor (Dajani et al., 1976). In the present study inhibitor 6 was found to be bactericidal for one indicator (I,) and bacteriostatic for another (Ii). Also, although prototype 9 appeared not to inhibit Is in the deferred antagonism test, the partially purified inhibitor 9 when concentrated to titre 8 was found to have a bacteriostatic effect against this indicator in the investigations of the mode of action. The present study has established that various strains of Strep. salivarius produce inhibitory substances with properties similar to those of the bacteriocins produced by other Gram-positive bacteria (Tagg et al., 1976). It is expected that additional inhibitors will be revealed in the course of more extensive screening of Strep. saliuarius strains. The significance to the host bacterium of the production of such substances remains obscure. Our attention has now been directed towards the evaluation of the influence exerted by inhibitory Strep. salivarius strains on the composition of the oral microbiota and the contribution of inhibitory Strep. salivarius strains to the host’s resistance to potential pathogens of the upper-respiratory tract. Acknowledgements-We thank Vicky Phillips for her technical assistance. This study was supported by the Medical Research Council of New Zealand and the New Zealand Dental
Research
Foundation. REFERENCES
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Russell C. and Tagg J. R. 1981. Role of bacteriocin during plaque formation by Streptococcus salivarius and Streptococcus sunguis on a tooth in an artificial mouth. J. uppl. Bact. so. 305-313. Rutter R. R.. Ruefenacht W. G.. Chamberlain C. R., Tho-
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Tagg J. R.. Dajam A. S. and Wannamaker L. W. 1976. Bacteriocins of gram-positive bacteria. Bacr. Rev. 40, 722-756. Tagg J. R. and Bannister L. V. 1979. “Fingerprinting” fi-haemolytic streptococci by their production of and sensitivity to bacteriocine-like inhibitors. J. med. Microbiol. 12, 397411. Tagg J. R. and Wannamaker L. W. 1978. Streptococcin A-FF22: nisin-like antibiotic substance produced by a group A streptococcus. Antimicrob. Ag. Chemother. 14. 36-39. Tagg J. R. and Martin D. R. 1980. Bacteriocin “fingerprinting” of group B streptococcus strains of bovine and human origin. Proc. Unil;. Otago Med. Sch. 58, 22-23. Thompson R. and Shibuya M. 1946. The inhibitory action of saliva on the diphtheria bacillus: the antibiotic effect of salivary streptococci. J. Bact. 51, 671.-684. Weerkamp A., Vogels G. D. and Skotnicki M. 1977. Antagonistic substances produced by streptococci from human dental plaque and their significance in plaque ecology. Caries Rex 11, 245. 256. Weerkamp A.. Bongaerts-Larik L. and Vogels G. D. 1977. Bacteriocins as factors in the in vitro interaction between oral streptococci in plaque. Infect. Immun. 16, 77X-780.
DEMPSTER
of Microbiology,
SUBSTANCES SALII/ARZUS
and J. R. TACG
University
of Otago,
Dunedin.
New Zealand
Summary---Inhibitory substances produced by six strains of Streptococcus saliwrius were isolated and partially characterized. The six prototype producer strains were selected initially on the basis of their differing spectra of inhibitory activity when tested against a set of nine standard indicator strains. Optimal production conditions were defined for each producer strain and inhibitor-containing extracts were obtained for characterization studies. All of the inhibitors appeared to be proteinaceous substances of molecular weight greater than 3500. When tested against a Streptococcus pyogenes indicator strain, one of the inhibitors was bactericidal, but the other five appeared to be bacteriostatic. Some differences between the inhibitors were observed with respect to heat and enzyme sensitivities.
INTRODUCTION
Oral streptococci have been demonstrated to be capable of producing a wide range of inhibitory compounds (Tagg, Dajani and Wannamaker, 1976; Brandis, 1978). Bacteriocin-like substances are released in vitro by strains of Streptococcus mutans (Hamada and Ooshima, 1975; Delisle, 1976; Rogers, 1976) Streptococcus sanguis (Fujimura and Nakamura. 1979) and Streptococcus mitis (Dajani, Tom and Law, 1976). Inhibitor production by Streptococcus saliuurius has seldom been reported. Donoghue and Tyler (1975) suggested that the inhibitory activity of Strep. salirurius strains may be attributable solely to their production of lactic and acetic acids. More recently, it has been demonstrated that certain strains of Strep. .sulirurius may produce a low mol. wt antibiotic, enocin. that acts by interfering with the utilization of pantothenic acid by susceptible cells (C. C. Sanders and W. E. Sanders. Abstr. No. 178, Interscience Conference on Antimicrobial Agents and Chemotherapy, 1978). In other studies (Tagg J. R. and Russell C.. unpublished; Weerkamp, Vogels and Skotnicki, 1977) inhibitory substances having bacteriocin-like properties have been reported. It has been suggested that either bacteriocins or other inhibitory substances produced by bacteria in the human oral cavity may function to protect the host against colonization by Streptococcus pneumoniae (Johanson et al., 1970) Streptococcus pyogenes (Sanders, Nelson and Sanders, 1977) and Corynebacterium diphtheriue (Thompson and Shibuya, 1946). Moreover. there is some evidence that inhibitors produced by plaque bacteria may influence the microbial composition of plaque formed in vitro (Weerkamp, Bongaerts-Larik and Vogels, 1977; Russell and Tagg, 1981) and in riro (Rutter et (I/., 1961; Rogers, Van der Hoeven and Mikx. 1979). Kelstrup et al. (1970) developed a method of “fingerprinting” oral streptococci based upon their production of, and sensitivity to, bacteriocin-like inhibitors. In the present study we utilized a similar
scheme to identify six strains of Strep. salicnrius that produced distinctive inhibitory spectra when tested against a set of nine standard indicator strains.
MATERIALS
Bacterial
AND METHODS
strains
The six prototype inhibitor-producing strains of saharius (strains 5, 6. 9, 36, MPS and 2OP3) were isolated from human saliva specimens. The initial presumptive identification of these strains as Strep. salicurius was based upon their colony appearance when saliva samples were plated on Mitis-salivarius agar. Identification was confirmed using physiological tests as outlined by Hardie and Bowden (1976). Lancefield grouping established that only strains 5 and MPS were reactive with group K antisera. The nine standard indicator strains (Ii to I,) used for producer (P)-typing the Strep. .salivarius strains, according to their production of characteristic inhibitory patterns, have been described previously (Tagg and Bannister, 1979). The indicators I, (Staphy/ococcus epidermidis strain T-l 8) and I, (Strep. pyogenes strain 71-679) were used in mode of action studies. Representative strains of various species used to further define the activity spectra of the prototype strains were obtained from the culture collection of the Department of Microbiology, University of Otago. All strains were stored at -7o’C in skimmed milk and subcultured on blood agar when in regular use. Strep.
Media
All bacteriological media were obtained from Difco Laboratories (Detroit, U.S.A.) unless otherwise stated. Liquid media tested as substrates for the production of inhibitors included Todd-Hewitt broth (THB), thioglycollate medium (TM), tryptic soy broth, brainheart infusion, veal infusion broth, cooked meat medium and APT broth (B.B.L., Cockeysville, U.S.A.). Supplements to the basic liquid media that were tested in various experiments included yeast extract (4
152
R. P. Dempster and J. R. Tagg
per cent, w/v), agar (0.05 per cent, w/v), neopeptone (3 per cent, w/v) and sucrose (1 per cent, w/v). Solid media were prepared by the addition of 1.5 per cent (w/v) agar (Davis Gelatine Ltd, N.Z.) to the liquid media. Blood agar was 5 per cent (v/v) human blood in Columbia agar base (Gibco Diagnostics, Madison, U.S.A.) poured on a base of saline agar. Detection and assay of inhibitors
Initial detection of inhibitor-producing strains was by application of the simultaneous antagonism test using I, as the indicator. “Fingerprinting” of the inhibitor-positive isolates according to their P-type was achieved using the deferred antagonism test with nine standard indicators. Both of these procedures were conducted as previously outlined (Tagg and Bannister, 1979) with the exception that the Strep. salivarius cultures were grown in a COz-enriched atmosphere (candle jar) instead of aerobically. The inhibitory titre of preparations was determined by a modification of the well-diffusion method, using constant-volume (35 ml) blood agar plates (Tagg and Wannamaker, 1978). Twofold dilutions of the inhibitory preparations were dispensed in 0.05 ml vol. into the wells. After allowing the liquid to absorb, the surface of the medium was sterilized by exposing to chloroform vapours for 20 min. After airing the plates a THB culture of Staph. epidermidis strain T-18 grown for 18 h at 37°C was swabbed evenly over the surface. Following incubation for 18 h at 37°C the highest dilution to give definite inhibition of the growth of the indicator lawn was defined as containing one arbitrary unit (AU) of inhibitor per ml. The titre of the inhibitor was the reciprocal of this dilution. Extraction of inhibitors
Attempts were made to extract active inhibitor from lawn cultures of the producers on various solid media either by freezing and thawing the medium (Tagg, Dajani and Wannamaker, 1975) or by dicing the medium and eluting at 4°C for 18 h into 8 ml vol. of either 1 M sodium chloride or 7 M urea. Supernatants obtained by centrifugation of 10 ml liquid cultures were tested for inhibitory activity and the cell pellets were divided into two portions. One half was resuspended in 0.5 ml of 7 M urea and the other in 0.5 ml of 1 M sodium chloride for 18 h at 4°C. Following removal of the cells by centrifugation the extracts were tested for inhibitor activity. Neither 7 M urea nor 1 M sodium chloride inhibited the growth of the indicator lawns under the test conditions. Characterization of inhibitors
Inhibitor preparations were partially purified by precipitation with 60 per cent ammonium sulphate, followed by dialysis in 3500mol. wt cut-off dialysis tubing (A. H. Thomas Co., Philadelphia, U.S.A.) against 100~01. of phosphate-buffered saline, pH 7.1 (PBS). These preparations were then adjusted to a titre of 4-8 AU/ml by dilution in PBS. Dialysis of inhibitor preparations against PBS for 18 h at 4°C using tubing of mol. wt cut offs 3500, 8000 and 12,000 was one method used to give an indication of the size of the inhibitory substances. In another approach, the ability to dialyse the inhibitors as pro-
duced in solid medium was tested by interposing a single sheet of dialysis membrane between the surface of a blood agar plate and 0.1 ml of blood agar medium that had been inoculated with the producer strain. As a control 0.1 ml of the inoculated blood agar was placed directly on the surface of the blood agar plate. Following incubation the dialysis sheet and blood agar drop cultures were removed. The plate was then treated with chloroform, aired and seeded with a THB culture of the indicator strain grown for 18 h at 37°C. In some experiments, the partially purified inhibitor preparations were incorporated within drops of blood agar medium and tested for their ability to dialyse using the above procedure. The partially purified preparations were tested for activity following heat treatment for 30 min at 80, 100 and 122°C and also following mixing with chloroform 10 per cent (v/v) for 30min. The preparations were also examined for susceptibility to 1 mg/ml (final concentration) of, trypsin (1:250; Difco), protease (Type V), lipase (Type 1) and cc-amylase (Sigma Chemical Co., St. Louis, U.S.A.). All enzymes were in 0.2 M (pH 7.5) TES buffer (Sigma). Incubation was at 37’C for 3 h at which time the activity of the treated preparations was compared with that of control untreated preparations. The enzymes (at 1 mg/ml) were not inhibitory to the growth of the indicator strain under the assay conditions. Mode of action of the inhibitors
The mode of action was determined by adopting a method used by Johnson, Tagg and Wannamaker (1979). Overnight THB cultures of indicators 1, and Is were diluted in warm THB to approx. lo4 colonyforming units (c.f.u.)/ml. After incubation for 2 h at 37°C these indicator cultures were then mixed with an equivalent volume of titre 8 inhibitor preparation. The mixtures were incubated at 37°C and samples taken at 0, 2. 4, 6 and 24 h were suitably diluted in PBS and the viable count determined on blood agar. RESULTS
Activity spectrum of protot)jpe inhibitors
When “fingerprinted” by the deferred antagonism procedure (producer streak cultures incubated microaerophilically on blood agar at 32°C for 18 h) the six prototype inhibitory strains of Strep. salivarius could be differentiated according to their characteristic inhibitory patterns (P-types) (Table 1). When the prototype strains were themselves used as indicators under the same test conditions (Table 2) it was evident that none of the producer strains was “autoinhibitory”. Subsequent tests indicated that for strains MPS and 36, modification of the culture conditions apparently enhanced the production of inhibitor(s). When incubated anaerobically at 37°C strain MPS produced inhibitor(s) active against all of the prototype producer strains, including itself. Strain 36 was found to produce a broader spectrum of inhibitory activity at 37°C than at 32°C. More detailed examination of the range of inhibitory activity of the prototype strains in the deferred antagonism method on blood agar revealed some basic similarities (Table 3). For this particular test the producer strains were grown in candle jars for 18 h at
153
Bacteriocin-like substances of Strep. salirarius Table
1. Standard
inhibitory
Prototype producer strain
I,
patterns of prototype strains*
Inhibition I, 13
+ + + + + (i) + + ++-++++-++--++++
5 6 9 l& 2OP3
-
inhibitor-producing
of standard indicator strain I4 I, I, I7 I, + (-) (-)
+ + (+)
+ + -~ -~
+ + + +
I9
+ + (+)
(+, (-) (-)
+
(-)
+ Indicator inhibited. - Indicator not inhibited. ( ) Weak inhibition occasionally noted. * Producer streak cultures were incubated on blood agar at 32°C for 18 h in a CO*-enriched atmosphere.
their optimal production temperatures: 37°C for strains MPS and 36 and 32°C for the other producers. Activity was particularly widespread against streptococci of Lancefield groups A, C, E and G and group B streptococci of animal origin. Group D streptococci and human strains of group B streptococci seemed generally to be resistant. Differences in the inhibitory spectra of the prototype producers became more apparent when 22 strains of “viridans” streptococci were used as indicators. The number of susceptible strains varied from three (for prototype strain 9) to 13 (for prototype strain 6). Strains of Strep. mutans were found to be relatively resistant. None of the prototype producers seemed active against either staphylococci or a variety of Gram-negative bacteria. By contrast, strains of Actinomyces viscosus and Micrococcus sp. were sensitive to all of the producers. Characterization of the prototype inhibitors As a preliminary to their characterization,
the opti-
mal conditions for the production and extraction of the inhibitory substances associated with each prototype producer strain were determined (Table 4). For each producer, the yield of inhibitory activity was determined, following growth for 18 h of cultures in a wide variety of both liquid and solid substrates (see Materials and Methods), under the conditions of optimal temperature and aeration for inhibitor production as delined by use of the deferred antagonism test on blood agar. The inhibitors were then concentrated, partially purified and adjusted to a titre of 48 AU/ml prior to investigating some of their physico-chemical properties (Table 5). With the exception of that produced by strain 36, all of the inhibitors seemed quite stable to heating at 80°C for 30min. Inhibitors from strains 5 and 2OP3 were unaffected by autoclaving (122°C) for 30 min. None of the inhibitors were inactivated when shaken with chloroform for 30 min. All of the inhibitors were found to be inactivated when treated with protease
Table 2. Inhibitory activity of prototype producers using the producers also as indicators* Prototype producer strain 5 6 9 h& 2OP3
Inhibition 5
6
-:++-_ 1+ -$ -
1+ + +
of indicator strain 9
36
MPS 2OP3
+
+
-
-
+ -
+ +
-
-
L$ -
11 -
+ Indicator inhibited: - Indicator not inhibited. * Producer streak cultures were incubated on blood agar at 32°C for 18 h in CO*-enriched atmosphere. t When strain 36 was incubated as above, but at 37°C indicators 5 and 6 were inhibited. $ When strain MPS was incubated at 37°C anaerobically indicators 5, MPS and 2OP3 were inhibited.
R. P. Dempster and J. R. Tagg
154
Table 3. Range of inhibitory
activity of prototype Number
Indicator
strain
Streptococcus: Group A (81)t Group B (human) (5) (animal) (10) Group C (21) Group D (5) Group E (3) Group F (1) Group G (12) Strep. mutans (4) Strep. salirwius sanguis (5)(10) Strep. mitior (1) Strep. pneumoniu (2) Lactohuc~illus ucidophilus (1) L. casei (1) Actinomyces tliscosus (3) A. naeslundii (1) Staphylococcus sp. (4) Corynebacterium diphtheriae (3) Micrococcus sp. (2) Gram-negative strains (12)f
producers*
of strains sensitive to prototype producer strain
5
6
9
36
MPS
2OP3
81
81
61
81
81
81
3 10 17 0 3 0 12 0
0 10 17 0 3 0 12 1
0 0 9 0 :,
0 10 14 0 3 0 6 1
0 10 12 0 3 0 3 1
1; 16 0 3 0 12 0
31 0 2 0 0 3 0 0 1 2 0
82 0 2 0 0 3 0 0 1 2 0
01 0 2 0 0 3 0 0 0 2 0
42 :
25 0 2 0 0 3 1 0 2 2 0
: 0 2 0 0 3 0 0 1 2 0
0 0
1 0 3 0 0 0 2 0
* Producer streak cultures were incubated on blood agar in a CO,-enriched atmosphere for 18 h at 32°C (37°C for strains MPS and 36). t Number of strains tested is given in parenthesis. $ Including strains of Salmonella, Proteus, Pseudomonas, Escherichia, Neisseria, Veillonella, Enterohacter and Serratia.
Table 4. Optimal
Prototype producer straint 5
conditions
for recovery of active inhibitors producers
Medium for production
6
TM + 3 per cent neopeptone Blood agar
9
Blood agar
36
MPS 2OP3
THB
THB + 3 per cent neopeptone TM + 3 per cent neopeptone
Method of inhibitor recovery Culture supernatant Freeze/thaw extract Freeze/thaw extract Cell pellet resuspended in 7 M urea at 4 C overnight Culture supernatant Culture supernatnat
from prototype
Initial inhibitor titre (AU/ml)* 8 1
1 1
2 4
* Prior to concentration and purification procedures. t The producers were incubated under the conditions of temperature and aeration found to be optimal for production on blood agar in deferred antagonism. For strains 5,6,9 and 20P3 this was at 32°C in a candle jar; for MPS at 37°C anaerobically, and for 36 at 37°C in a candle jar.
Bacteriocin-like substances of Strep. saliaaritts
155
Table 5. Susceptibility of prototype inhibitors to potential inactivating agents Titre of prototype inhibitor Treatment Nil 8O’C for 30min 100°C for 30 min 122°C for 30 min Chloroform for 30 min Protease* Trypsin* a-Amylase* Lipase*
5
6
9
36
8 8 8 8 8 0 0 4 4
8 8 4 2 8 0 0 4 4
4 4 2 1 4 0 0 2 2
4 2 0 0 4 0 0 0 2
MPS 2OP3 8 8 8 1 8 0 0 4 4
8 8 8 8 8 0 4 4 4
* Inhibitor preparations were mixed with an equivalent volume of enzyme and incubated at 37°C for 3 h. Final enzyme concentration was 1 mg/ml.
and with the exception of 2OP3 all seemed sensitive to trypsin. None of the inhibitors were lipase sensitive and only inhibitor 36 appeared sensitive to amylase. When tested for dialysability against PBS there was no apparent loss of activity with any of the inhibitory preparations. By contrast, when the inhibitors were assessed for their ability to traverse dialysis membranes set into blood agar, all six appeared to pass through membranes with mol. wt cut offs of 12,OCQ and 8000, but not 3500. Similar results were obtained when either producer cultures or the partially-purified inhibitors were incorporated within the overlying drops of blood agar. Investigation of the mode of action of the inhibitors revealed that only inhibitor 6 was bactericidal for indicator strain I, under the test conditions. This inhibitor brought about a one-thousandfold decrease in the viable count of Is after 2 h exposure. After 4 h no viable cells were detected. Each of the other inhibitor preparations was found to be bacteriostatic for I, when tested under the same conditions. All 6 inhibitors appeared to be bacteriostatic for indicator strain I,. DISCUSSION
In the present study it has been demonstrated that strains of Strep. saharius may produce a variety of bacteriocin-like inhibitory substances. Screening tests are currently being conducted in this laboratory in order to estimate the incidence of inhibitor-producing Strep. saliuarius strains in human saliva specimens. In these tests, 50 colonies of Strep. saliuarius isolated from each specimen are examined for inhibitor production against a set of indicator strains. Preliminary results have indicated that in approx. 10 per cent of individuals, more than 20 per cent of the Strep. saliuarius isolates are inhibitor positive. Moreover, longitudinal studies have demonstrated that individuals harbouring the prototype producers have retained Strep. saliuarius strains of the same P-type in approximately the same proportions for the three years of the study. The initial differentiation of the six prototype inhi-
bitor-producing strains described in this investigation was on the basis of their specific and characteristic inhibitory spectra (P-types) when tested against a set of nine standard indicator strains. With more detailed examination of the range of their activity spectra, close similarities became apparent. The high susceptibility of group A streptococcus strains is intriguing in view of the hypothesized role of “viridans” streptococci in protecting individuals against group A streptococcal pharyngitis (Sanders et a/., 1977). In a previous study it had been observed (Johanson et al., 1970) that strains of Strep. saliuarius were particularly effective inhibitors of Strep. pneumoniae and an in-uiuo role of bacterial antagonism in preventing pneumococcal colonization of the human pharynx was proposed. We also have observed that Strep. pneumonia strains are extremely sensitive to the inhibitors produced by all six prototype strains. Although there was considerable activity against some characteristic plaque organisms such as Strep. sanguis and A. uiscosus, others notably including Strep. mutans (strains UTBI, UTB2, 10449 and HS6) and Lactobacillus sp. did not seem to be susceptible. The apparent greater sensitivity of animal than human strains of group B streptococci supports a previous report of the differing bacteriocin susceptibility of these strains depending upon the source of the isolate (Tagg and Martin, 1980). Differences between the six prototype inhibitors became particularly evident when the conditions for optimal production and extraction were tested. The importance of standardization of media and incubation conditions has been emphasized previously (Rogers, 1972; Tagg et al., 1976). For strains 5, MPS and 2OP3, addition of 3 per cent (w/v) neopeptone to the basal medium was found to enhance production. This effect may have been due to the recognized antiproteinase action of neopeptone which would tend to increase the yield of biologically-active proteinaceous material in the culture supernatants (Tagg et al., 1976). In studies of bacteriocin production by strains of Strep. mutans it has been reported that yields may be increased by supplementing the basal media with
156
R. P. Dempster and J. R. Tagg
yeast extract (Rogers, 1972) or by the use of freshly prepared filter-sterilized liquid media (Kelstrup and Funder-Nielson, 1977). Neither of these approaches seemed to enhance the yield of the Strep. saliuarius inhibitors. Anaerobic incubation of strain MPS appeared to foster production of inhibitor(s) active against all of the prototype producers. Activity of bacteriocin-like inhibitors against the producing bacterium is uncommon, although it tends to occur more frequently within the Gram-positive bacteria (Tagg et al., 1976). The possibility that some additional inhibitory agent is produced in the course of anaerobic incubation cannot yet be dismissed. Nevertheless, if such is the case, then the two inhibitors seem to remain associated throughout the procedures of ammonium sulphate precipitation and dialysis against PBS. The problem of obtaining inhibitors in solution seems to be one common to most investigations involving streptococci (Tagg et al., 1976). The inhibitors produced by strains 6 and 9 could not be recovered from any of the tested liquid media, but adequate yields could be obtained by freeze-thaw extraction of cultures grown as lawns on blood agar medium. The inhibitor produced by strain 36 seemed predominantly cell-associated in broth cultures and recovery could be effected by eluting from the producer cells into 7 M urea. Some additional differences between the prototype inhibitors became apparent when the properties of the substances were examined. partially-purified Although strain 36 inhibitory activity was partially sensitive to heating at 80°C for 30min, all of the others appeared to be remarkably heat-tolerant. Such heat stability seems to be a common characteristic of low mol. wt bacteriocins produced by Gram-positive bacteria (Tagg et al., 1976). Estimation of the mol. wt of all six inhibitors, as produced during growth of the producer strains on blood agar medium, suggested they were in the range 3500 to 8000. It is not clear why the mol. wt of each of the partially purified inhibitors when tested by dialysis against PBS appeared to be greater than 12,000. It is possible that in their extracted form the inhibitors may be complexed with stabilizing medium components or, alternatively, aggregate formation by monomeric inhibitor molecules may be favoured (Barry et al., 1965). Investigation of enzyme susceptibility indicated that each of the inhibitors probably had an essential protein component as judged by sensitivity to protease. Bacteriocin sensitivity to proteases and the apparent ability of bacteriocin-sensitive strains to coexist with producers in vivo led Kelstrup and Gibbons (1969) to suggest that if bacteriocins were produced in the mouth they may be inactivated by the natural salivary proteases. By contrast, in another study (Hamada and Ooshima, 1975) it was demonstrated that plaque proteases may not necessarily be capable of inactivating bacteriocins in vivo. We have found that there is no detectable decrease in the activity of partially purified preparations of the prototype inhibitors on incubation for 4 h at 37°C with 50 per cent (v/v) fresh human saliva. At present we are attempting to determine whether the prototype inhibitors can be produced under simulated in vivo conditions.
Although it has been suggested that by definition a “classic” bacteriocin would be expected to have a bactericidal mode of action (Tagg et al., 1976), it has become clear that there are many bacteriocin-like substances that seem to have bacteriostatic action against either some or all of the sensitive bacteria (Hale and Hinsdill, 1975; Dajani et al., 1976). Indeed, the apparent mode of action may vary depending upon the choice of indicator used and the titre of inhibitor (Dajani et al., 1976). In the present study inhibitor 6 was found to be bactericidal for one indicator (I,) and bacteriostatic for another (Ii). Also, although prototype 9 appeared not to inhibit Is in the deferred antagonism test, the partially purified inhibitor 9 when concentrated to titre 8 was found to have a bacteriostatic effect against this indicator in the investigations of the mode of action. The present study has established that various strains of Strep. salivarius produce inhibitory substances with properties similar to those of the bacteriocins produced by other Gram-positive bacteria (Tagg et al., 1976). It is expected that additional inhibitors will be revealed in the course of more extensive screening of Strep. saliuarius strains. The significance to the host bacterium of the production of such substances remains obscure. Our attention has now been directed towards the evaluation of the influence exerted by inhibitory Strep. salivarius strains on the composition of the oral microbiota and the contribution of inhibitory Strep. salivarius strains to the host’s resistance to potential pathogens of the upper-respiratory tract. Acknowledgements-We thank Vicky Phillips for her technical assistance. This study was supported by the Medical Research Council of New Zealand and the New Zealand Dental
Research
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