Indian J Med Res 119 (Suppl) May 2004, pp 13-16
Prevention of streptococcal pharyngitis by anti-Streptococcus pyogenes bacteriocin-like inhibitory substances (BLIS) produced by Streptococcus salivarius J.R. Tagg Department of Microbiology, University of Otago, P.O. Box 56, Dunedin, New Zealand Received August 6, 2003 Background & objectives: Streptococcus salivarius is a numerically prominent member of the human oral microbiota that produces a variety of bacteriocin-like inhibitory substances (BLIS) having in vitro inhibitory activity against S. pyogenes. Our previous studies of S. salivarius isolates from children using a deferred antagonism BLIS production (P)-typing scheme showed that the 9 per cent of children having large populations of P-type 677 S. salivarius experienced fewer S. pyogenes acquisitions than either the 11 per cent of children having predominant P-type 226 populations or the 60 per cent of children with largely non-inhibitory (P-type 000) S. salivarius. Amongst the other BLIS P-types detected were a number of strongly-inhibitory (P-type 777) S. salivarius. In the present study the inhibitory agents produced by prototype strains of P-types 226, 677 and 777 S. salivarius are compared. Methods: The prototype BLIS-producing S. salivarius strains SN, 20P3, and K12 were isolated from tongue swabbings. BLIS P-typing was done using standard procedures. The BLIS molecules were purified and characterized. Results: S. salivarius SN (P-type 226) produces a heat-labile muramidase. S. salivarius 20P3 (P-type 677) produces the 2315 Da lantibiotic salivaricin A and S. salivarius K12 (P-type 777) produces two lantibiotics; salivaricin A2 (2368 Da) and salivaricin B (2733 Da). Interpretation & conclusion: The P-type 777 S. salivarius strain produced salivaricin A2 and salivaricin B. The combined production of two anti-S. pyogenes BLIS activities by this strain indicates that it could be adopted as a colonizing strain in bacterial interference trials. Key words Bacteriocin - bacteriocin-like inhibitory substance - lantibiotic - replacement therapy - Streptococcus salivarius
Streptococcus pyogenes is a major cause of acute infections and of serious delayed sequelae, particularly in children. At present, the most effective strategy available to deal with S. pyogenes is treatment of acute infections by administration of therapeutic doses of a broad-spectrum antibiotic like penicillin. Also, since there is currently no anti-S. pyogenes immunization available, the only means of protecting at risk individuals is antibiotic prophylaxis. However, these approaches have many inherent problems: the cost of the antibiotics, the
possibility of adverse host reactions, severe disruption of the indigenous microbiota and bacterial resistance development. The implementation of bacterial interference may offer a relatively specifically-targeted alternative means of preventing the development of acute S. pyogenes infections. Since commensal streptococci are numerically predominant in the oral cavity, it seems reasonable to speculate that they may be central to any naturally occurring interference with colonization or infection by S. pyogenes. Children who acquire 13
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INDIAN J MED RES (SUPPL) MAY 2004
S. pyogenes have been shown to have a lower proportion of throat cultures containing bacteria inhibitory or bacteriocidal for S. pyogenes than those who do not become colonised1. The pantothenic acid antagonist enocin, produced by S. salivarius, was later speculated to contribute to protection against S. pyogenes infections 2. Grahn and Holm 3 established that the prevalence of oral alpha-haemolytic streptococci having inhibitory activity against S. pyogenes was lower in those children who became infected during an outbreak of streptococcal tonsillitis. This group later recommended dosing with a mixture of alpha haemolytic streptococci as a supplementary treatment of recurrent streptococcal tonsillitis4. Of all the bacterial species known to regularly inhabit the human oral microbiota in large numbers S. salivarius is perhaps the most innocuous. There appear to be no reports of this species causing infections within the oral cavity and the rare instances of its association with bacteraemia or meningitis appear to have occurred in immunologically-compromised patients or following trauma to the patients tissues5. Since S. salivarius is common, not only on the dorsum of the tongue but also on the oropharyngeal mucosa6 , it is well positioned to directly repel invasion by S. pyogenes. Our previous studies7,8 have demonstrated that approximately 45 per cent of S. salivarius strains inhibit the growth of one or more members of a set of 9 strains used routinely as indicators of streptococcal BLIS. The pattern of inhibition of these indicators, when expressed in code form, is referred to as the BLIS production (P)-type of a bacterium. Subsequent studies of streptococcal pharyngitis in populations of school-aged children showed that some children were seldom infected. Many of these rarely-infected children were found to harbour large populations of S. salivarius producing anti-S. pyogenes BLIS activity. A study of 780 Dunedin school children9 found that there were two major types of BLIS activities produced by the S. salivarius, the corresponding P-type patterns being referred to as 226 (11% of children positive) and 677 (9% positive). A further 20 per cent of the children had S. salivarius of various other P-type designations, including some isolates producing particularly strong (P-type 777) BLIS activity. The present study compares the BLIS activities produced by S. salivarius strains SN, 20P3 and K12, the prototypes of BLIS P-types 226, 677 and 777 respectively.
Material & Methods Bacterial strains and BLIS P-typing: The prototype BLIS-producing S. salivarius strains SN (P-type 226), 20P3 (P-type 667)10 and K12 (P-type 777) were isolated on Mitis Salivarius agar (Difco Laboratories, Detroit) from tongue swabbings of human subjects. The 9 standard indicators and the standard procedure used for the BLIS P-typing of streptococci have been described previously7. Isolation and characterization of BLIS molecules: The BLIS produced by S. salivarius strain SN was purified from 18 h 35C Todd Hewitt broth (Difco) culture supernatants using the procedures previously devised for the similar BLIS, zoocin A, produced by S. equi subsp. zooepidemicus11. The procedures for isolation of the lantibiotic peptides salivaricin A2 and salivaricin B from S. salivarius strain K12 were those applied previously to the purification of salivaricin A from S. salivarius strain 20P3 10 . Amino acid compositions, mass spectrometry and N-terminal amino acid sequencing were done by the Protein Microchemistry Facility, Department of Biochemistry, University of Otago. To enable Edman degradation to proceed through blockages caused by the presence of dehydro amino acids, these residues were first modified by addition of thiol groups12. Results A muralytic enzyme, salivaricin SN, was isolated from the supernatants of Todd Hewitt broth cultures of S. salivarius strain SN. Tests of the inhibitory spectrum of strain SN using the deferred antagonism test established that 18 of 20 S. pyogenes were susceptible, but none of a variety of other streptococci, including representatives of Lancefield groups B, C, D, F and G, S. salivarius, S. sanguinis and S. mutans. The purified protein was inactivated on heating at 80º C for 30 min. The N-terminal sequence of salivaricin SN was DINGGANTPGAYD……. S. salivarius strain 20P3 has been shown previously10 to produce the lantibiotic salivaricin A (Table). Also shown in the Table is the sequence of salivaricin A1, the homologous lantibiotic produced by the serotype M4 S. pyogenes strain 2000613,14.
TAGG : APPLICATION OF STREPTOCOCCUS SALIVARIUS BLIS
S. salivarius strain K12 was found to produce two anti-S. pyogenes peptides (Table). The detection of lanthionine in amino acid analyses and stability to heating at 100ºC for 30 min indicated that both are of the lantibiotic class of bacteriocins. One of these was a T4S/ F7I variant of salivaricin A, named salivaricin A2. The second was quite unrelated to salivaricin A and is referred to as salivaricin B.
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in 15 probe-positive S. pyogenes revealed consistent codon differences indicative of conservative amino acids changes in positions 2 (K instead of R) and 7 (F instead of I) of the salivaricin A propeptide. This variant form, called salivaricin A1, appears to be expressed as a functional product only in serotype M4 strains of S. pyogenes but is sufficiently similar to effect induction of salivaricin A production in salA-positive strains of S.salivarius.
Discussion The present study shows that the anti-S. pyogenes inhibitory activities previously shown to be relatively commonly produced in vitro by S. salivarius are of three markedly different molecular types. Strain SN, the prototype of the P-type 226 S. salivarius produced a heat labile lytic enzyme, salivaricin SN, the N-terminus of which was completely different from that of the previously-described streptococcal muralysin, zoocin A (ATYTRPLDTG…). Zoocin A has been shown to be a domain-structured enzyme similar to lysostaphin, with the N-terminus responsible for catalysis and the C-terminal domain effecting target recognition15. Unlike zoocin A, which displays strong activity against S. mutans in addition to S. pyogenes, the S. salivarius agent appears relatively specifically active against S. pyogenes. Cloning of the salivaricin SN structural gene is in progress and when completed will facilitate direct comparison between the two enzymes for evidence of domain sharing. Our previous studies 13,14 have shown that the structural gene salA, which encodes the lantibiotic salivaricin A, is the usual form detected in S. salivarius, PCR amplification and sequencing of the salA-like gene
In the present study, yet another variant of salivaricin A has been found to be produced by S. salivarius strain K12. Interestingly this variant, salivaricin A2, which differs in two conservative amino acid substitutions from both salivaricin A and salivaricin A1, appears commonly to be produced together with salivaricin B in P-type 777 S. salivarius (data not shown). The P-type 777 S. salivarius strain K12 was shown to produce in addition to salivaricin A2 an unrelated lantibiotic salivaricin B. The combined production of two anti-S. pyogenes BLIS activities by strain K12 was one consideration leading to its adoption as a colonizing strain in bacterial interference trials in populations of school children and young adults. Acknowledgment The financial support from the Marsden Fund, Royal Society of New Zealand and by the Health Research Council of New Zealand is acknowledged.
References 1.
Crowe CC, Sanders WE Jr, Longley S. Bacterial interference. II. Role of the normal throat flora in prevention of colonization by group A streptococcus. J Infect Dis 1973; 128: 527-32.
Table. Primary sequence and molecular mass of lantibiotic salivaricins Salivaricin
Amino acid sequence
Mass (Da)
A*
KRGSGWIATITDDCPNSVFVCC
2315
A1*
KKGSGWFATITDDCPNSVFVCC
2327
A2
KRGTGWFATITDDCPNSVFVCC
2368
B
GGGVIQTISHECRMNSWQFLFTCCS
2733
* The data for salivaricin A10 and salivaricin A113 have been previously published
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INDIAN J MED RES (SUPPL) MAY 2004 Sanders CC, Sanders WE Jr. Enocin: an antibiotic produced by Streptococcus salivarius that may contribute to protection against infections due to group A streptococci. J Infect Dis 1982; 146 : 683-90.
3.
Grahn E, Holm SE. Bacterial interference in the throat flora during a streptococcal tonsillitis outbreak in an apartment house area. Zentralbl Bakteriol Mikrobiol Hyg (A) 1983; 256: 72-9.
4.
Roos K, Holm SE, Grahn E, Lind L. Alpha-streptococci as supplementary treatment of recurrent streptococcal tonsillitis: a randomized placebo controlled study. Scand J Infect Dis 1993; 25: 31-5.
5.
Carley NH. Streptococcus salivarius bacteremia and meningitis following upper gastrointestinal endoscopy and cauterization for gastric bleeding. Clin Infect Dis 1992; 14 : 947-8.
6.
Frandsen EV, Pedrazzoli V, Kilian M. Ecology of viridans streptococci in the oral cavity and pharynx. Oral Microbiol Immunol 1991; 6: 129-33.
7.
Tagg JR, Bannister LV. “Fingerprinting” ß-haemolytic streptococci by their production of and sensitivity to bacteriocine-like inhibitors. J Med Microbiol 1979; 12 : 397-411.
and streptococcal diseases entering the new millenium. New Zealand: Securacopy; 2000 p. 81-5. 10. Ross KF, Ronson CW, Tagg JR. Isolation and characterization of the lantibiotic salivaricin A and its structural gene salA from Streptococcus salivarius 20P3. Appl Environ Microbiol 1993; 59: 2014-21. 11. Simmonds RS, Naidoo J, Jones CL, Tagg JR. The streptococcal bacteriocin-like inhibitory substance, zoocin A, reduces the proportion of Streptococcus mutans in an artificial plaque. Microb Ecol Health Dis 1995; 8: 281-92. 12. Navaratna MA, Sahl HG, Tagg JR. Two-component antiStaphylococcus aureus lantibiotic activity produced by Staphylococcus aureus C55. Appl Environ Microbiol 1998; 64 : 4803- 8. 13. Simpson WJ, Ragland NL, Ronson CW, Tagg JR. A lantibiotic gene family widely distributed in Streptococcus salivarius and Streptococcus pyogenes. Dev Biol Stand 1995; 85 : 639-43.
8.
Dempster RP, Tagg JR. The production of bacteriocin-like substances by the oral bacterium Streptococcus salivarius. Arch Oral Biol 1982; 27: 151-7.
14. Upton M, Tagg JR, Wescombe P, Jenkinson HF. Intraand interspecies signaling between Streptococcus salivarius and Streptococcus pyogenes mediated by SalA and SalA1 lantibiotic peptides. J Bacteriol 2001; 183: 3931-8.
9.
Dierksen KP, Tagg JR. The influence of indigenous bacteriocinproducing Streptococcus salivarius on the acquisition of Streptococcus pyogenes by primary school children in Dunedin, New Zealand. In: Martin DR, Tagg JR, editors. Streptococci
15. Lai AC, Tran S, Simmonds RS. Functional characterization of domains found within a lytic enzyme produced by Streptococcus equi subsp. zooepidemicus. FEMS Microbiol Lett 2002; 215: 133.
Reprint requests: Dr J.R. Tagg, Professor, Department of Microbiology, University of Otago, P.O. Box 56, Dunedin, New Zealand e-mail:
[email protected]
