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Bacteriocin-producing probiotic Streptococcus salivarius M18 offers beneficial modulatory capabilities within the oral microbiome, apparently through potent inhibitory activity against potentially deleterious bacteria, such as Streptococcus pyogenes. The oral cavity persistence of S. salivarius M18 was investigated in 75 subjects receiving four different doses for 28 days. Sixty per cent of the subjects already had some inhibitor-producing S. salivarius in their saliva prior to probiotic intervention. Strain M18's persistence was dependent upon the dose, but not the period of administration. Culture analysis indicated that in some individuals the introduced strain had almost entirely replaced the indigenous S. salivarius, though the total numbers of the species did not increase. Selected subjects showing either high or low probiotic persistence had their salivary populations profiled using Illumina sequencing of the V6 region of the 16S rRNA gene. Analysis indicated that while certain bacterial phenotypes were markedly modulated, the overall composition of the oral microbiome was not modified by the probiotic treatment. Megaplasmids encoding bacteriocins and adhesion factors were transferred in vitro to generate a transconjugant S. salivarius exhibiting enhanced antimicrobial production and binding capabilities to HEp-2 cells. Since no widespread perturbation of the existing indigenous microbiota was associated with oral instillation and given its antimicrobial activity against potentially pathogenic streptococci, it appears that application of probiotic strain M18 offers potential low impact alternative to classical antibiotic prophylaxis. For candidate probiotic strains having relatively poor antimicrobial or adhesive properties, unique derivatives displaying improved probiotic performance may be engineered in vitro by megaplasmid transfer.

Streptococcus salivarius is naturally a predominant member of the human oropharynx and the commercial probiotic strain K12 has been consumed for more than a decade. The present study examines the health responses of human volunteers to oral ingestion of high doses of S. salivarius K12. A randomized group of 53 subjects received a dose of 1 × 10(10)cfu S. salivarius K12 (N=25) or placebo (N=28) for 28 days, followed by a 28-day wash out period. Blood, urine and saliva samples were collected at baseline and following treatment and analyzed, while the oral and gastrointestinal tolerance of the subjects to the dosing regimen was determined by use of questionnaires. Adverse events (AE)s were recorded for both groups. No statistically significant differences between the probiotic and placebo treated groups were detected in either the blood clinical chemistry or hematology results (P>0.05). The questionnaire responses of the subjects indicated that both treatments were well tolerated. The frequency and intensity of AEs was similar in the two groups. This data demonstrates that the daily ingestion of S. salivarius K12 over a 28-day period does not adversely affect the human host and supports the safety of its oral delivery in a food-based carrier., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Indigenous microbes are known to influence human health outcomes and various approaches are now being made to positively modulate these microbe-induced outcomes via the administration of probiotics. The application of probiotics that are specific to the oral cavity is a relatively undeveloped field, and their emergence has largely occurred as a reasoned follow-up to initial studies in which probiotics that had already been developed and obtained regulatory approval for intestinal applications were then also evaluated for their putative influence on oral microbiota functionality. These attempts to extend the application of existing probiotics were probably at least in part motivated by recognition of the substantial safety and regulatory hurdles that must be overcome prior to the introduction of a novel probiotic agent. Nevertheless, from an efficacy perspective it appears more logical to develop microbes of oral origin as the specific providers of probiotic solutions for oral diseases, rather than attempting to adapt intestinally-derived strains for this role. Oral bacteria and their bioactive molecules have evolved to operate optimally in this environment and in some cases are known to persist only in oral sites. Amongst the bacteria of more than 700 species now identified within the human oral microbiota, it is the streptococci that are numerically predominant. Although this review highlights the development of the oral cavity bacterium Streptococcus salivarius as an oral probiotic, a number of other streptococcal species have also been shown to have considerable potential as probiotic candidates.

Salivaricin 9 (Sal9) is a 2560 Da lantibiotic having just 46 % amino acid identity with its closest known homologue, the Streptococcus pyogenes lantibiotic SA-FF22. The Sal9 locus (designated siv) in Streptococcus salivarius strain 9 was partially sequenced and localized to an approximately 170 kb megaplasmid, which also harbours the locus for the lantibiotic salivaricin A4. The entire locus was fully characterized in the draft genome sequence of S. salivarius strain JIM8780 and shown to consist of eight genes, having the following putative functions: sivK, sensor kinase; sivR, response regulator; sivA, Sal9 precursor peptide; sivM, lantibiotic modification enzyme; sivT, ABC transporter involved in the export of Sal9 and concomitant cleavage of its leader peptide; and sivFEG, encoding lantibiotic self-immunity. Intriguingly, in contrast to strain 9, the siv locus was chromosomally located in strain JIM8780--the first lantibiotic locus shown not to be exclusively plasmid-associated in S. salivarius. Sal9-containing extracts specifically induced lantibiotic production in both strain 9 and strain JIM8780, indicating that Sal9 functions as a signal peptide for upregulation of its own biosynthesis. Screening representative strains of three streptococcal species (S. salivarius, S. pyogenes and S. mitis) for sivA indicated that it was present only in S. salivarius, with 12 of 28 tested S. salivarius positive. Since Sal9 was inhibitory to all tested S. pyogenes strains it appears to have potential as an important component of the bacteriocin armoury of S. salivarius probiotics intended to control S. pyogenes infections of the human oral cavity.

A powder preparation of the oral probiotic Streptococcus salivarius K12 has been given to 19 young otitis media-prone children following a 3-day course of amoxicillin administered as a preliminary to ventilation tube placement. In two subjects, the use of strain K12 appeared to effect the expansion of an indigenous population of inhibitory S. salivarius. In other children, strain K12 colonisation extended beyond the oral cavity to also include the nasopharynx or adenoid tissue. The relatively low proportion (33%) of subjects that colonised was attributed to failure of the amoxicillin pre-treatment to sufficiently reduce the indigenous S. salivarius populations prior to dosing with strain K12 powder.

Streptococcus salivarius is an early colonizer of human oral and nasopharyngeal epithelia, and strain K12 has reported probiotic effects. An emerging paradigm indicates that commensal bacteria downregulate immune responses through the action on NF-kappaB signaling pathways, but additional mechanisms underlying probiotic actions are not well understood. Our objective here was to identify host genes specifically targeted by K12 by comparing their responses with responses elicited by pathogens and to determine if S. salivarius modulates epithelial cell immune responses. RNA was extracted from human bronchial epithelial cells (16HBE14O- cells) cocultured with K12 or bacterial pathogens. cDNA was hybridized to a human 21K oligonucleotide-based array. Data were analyzed using ArrayPipe, InnateDB, PANTHER, and oPOSSUM. Interleukin 8 (IL-8) and growth-regulated oncogene alpha (Groalpha) secretion were determined by enzyme-linked immunosorbent assay. It was demonstrated that S. salivarius K12 specifically altered the expression of 565 host genes, particularly those involved in multiple innate defense pathways, general epithelial cell function and homeostasis, cytoskeletal remodeling, cell development and migration, and signaling pathways. It inhibited baseline IL-8 secretion and IL-8 responses to LL-37, Pseudomonas aeruginosa, and flagellin in epithelial cells and attenuated Groalpha secretion in response to flagellin. Immunosuppression was coincident with the inhibition of activation of the NF-kappaB pathway. Thus, the commensal and probiotic behaviors of S. salivarius K12 are proposed to be due to the organism (i) eliciting no proinflammatory response, (ii) stimulating an anti-inflammatory response, and (iii) modulating genes associated with adhesion to the epithelial layer and homeostasis. S. salivarius K12 might thereby ensure that it is tolerated by the host and maintained on the epithelial surface while actively protecting the host from inflammation and apoptosis induced by pathogens.

Aims: To determine whether dosing with bacteriocin-producing Streptococcus salivarius following an antimicrobial mouthwash effects a change in oral malodour parameters and in the composition of the oral microbiota of subjects with halitosis., Materials and Results: Twenty-three subjects with halitosis undertook a 3-day regimen of chlorhexidine (CHX) mouth rinsing, followed at intervals by the use of lozenges containing either S. salivarius K12 or placebo. Assessment of the subjects' volatile sulphur compound (VSC) levels 1 week after treatment initiation showed that 85% of the K12-treated group and 30% of the placebo group had substantial (>100 ppb) reductions. The bacterial composition of the saliva was monitored by culture and PCR-denaturing gradient gel electrophoresis (PCR-DGGE). Changes in the PCR-DGGE profiles occurred in most subjects following K12 treatment. In vitro testing showed that S. salivarius K12 suppressed the growth of black-pigmented bacteria in saliva samples and also in various reference strains of bacteria implicated in halitosis., Conclusions: Administration of bacteriocin-producing S. salivarius after an oral antimicrobial mouthwash reduces oral VSC levels., Significance and Impact of the Study: The outcome of this preliminary study indicates that the replacement of bacteria implicated in halitosis by colonization with competitive bacteria such as S. salivarius K12 may provide an effective strategy to reduce the severity of halitosis.

Background & Objectives: The mutans streptococci (MS) are a group of 7 species of dental cariesassociated bacteria of which Streptococcus mutans and Streptococcus sobrinus are the most important in humans. Many MS produce bacteriocin-like inhibitory substances (BLIS), some of which have been characterised as small peptides capable of inhibiting the growth of closely-related species. These peptides have most commonly been referred to as mutacins. S. mutans strains N and UA140 appear to have closely similar BLIS activities. Both produce mutacins that seem to target the same species of bacteria. On closer analysis however, these two strains have been shown to produce distinctly different mutacins, known as mutacin N and mutacin I respectively. In the present study the mutacin N structural gene (mutN) was cloned and compared with the mutacin I structural gene (mutA)., Methods: Cloning and sequencing of S. mutans N was done. The distribution of mutN using DNA from 216 streptococcal strains was determined by dot blotting., Results: Mut N was cloned and sequenced from an 1800 bp Bam HI/Eco RI fragment. PCR with the mutN primers mutNF and mutNR on the four mutN-positive strains identified identical bands to S. mutans N. The location of mutN differs significantly from that of mutA in that it is directly upstream of comC, a gene encoding a putative competence stimulating factor., Interpretation & Conclusion: The close upstream proximity of mutN to comC suggests a link between mutacin N production and competence development. Further studies need to be done to detect competence-related genes in S. mutans strain N.

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.

The normal bacterial flora of the upper airways provides an important barrier to invading pathogens. This study investigated the production of bacteriocin-like inhibitory substances (BLIS) by streptococci isolated from the nasopharyngeal flora of children who either do or do not experience recurrent acute otitis media (AOM). Twenty children with recurrent AOM and 15 controls were tested. Swabs from the nasopharynx were evaluated for streptococci having BLIS activity against two representative strains of each of the AOM pathogens Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenzae and Moraxella catarrhalis. Streptococci displaying strong BLIS activity were characterized further and tested for known streptococcal bacteriocin structural genes. Sixty-five per cent of children had nasopharyngeal streptococcal isolates that were inhibitory to strains of one or more of the AOM pathogens. Six children (17 %) had streptococci that demonstrated strong BLIS activity against strains of at least three of the pathogenic species. Three of these inhibitory isolates were Streptococcus salivarius, two were S. pneumoniae and one was S. pyogenes. The inhibitory S. salivarius and S. pyogenes were shown to have structural genes for known streptococcal bacteriocins. No statistically significant difference was found between the two groups of children with respect to the presence of inhibitory streptococci in their nasopharyngeal floras. The finding of S. salivarius with strong inhibitory activity against several AOM pathogens in the nasopharyngeal flora of children is unique. Although there is no clear evidence from the present study that these organisms protect against AOM, their low pathogenicity and strong in-vitro BLIS production capability indicate that they should be incorporated in future trials of bacteriotherapy for recurrent AOM.

Streptococcus salivarius 20P3 produces a 22-amino-acid residue lantibiotic, designated salivaricin A (SalA), that inhibits the growth of a range of streptococci, including all strains of Streptococcus pyogenes. Lantibiotic production is associated with the sal genetic locus comprising salA, the lantibiotic structural gene; salBCTX genes encoding peptide modification and export machinery proteins; and salYKR genes encoding a putative immunity protein and two-component sensor-regulator system. Insertional inactivation of salB in S. salivarius 20P3 resulted in abrogation of SalA peptide production, of immunity to SalA, and of salA transcription. Addition of exogenous SalA peptide to salB mutant cultures induced dose-dependent expression of salA mRNA (0.2 kb), demonstrating that SalA production was normally autoregulated. Inactivation of salR encoding the response regulator of the SalKR two-component system led to reduced production of, and immunity to, SalA. The sal genetic locus was also present in S. pyogenes SF370 (M type 1), but because of a deletion across the salBCT genes, the corresponding lantibiotic peptide, designated SalA1, was not produced. However, in S. pyogenes T11 (M type 4) the sal locus gene complement was apparently complete, and active SalA1 peptide was synthesized. Exogenously added SalA1 peptide from S. pyogenes T11 induced salA1 transcription in S. pyogenes SF370 and in an isogenic S. pyogenes T11 salB mutant and salA transcription in S. salivarius 20P3 salB. Thus, SalA and SalA1 are examples of streptococcal lantibiotics whose production is autoregulated. These peptides act as intra- and interspecies signaling molecules, modulating lantibiotic production and possibly influencing streptococcal population ecology in the oral cavity.

A new selective medium (CNA-P) that reduces or eliminates the inhibitory activity of bacteriocin-producing Streptococcus salivarius against beta-hemolytic streptococci has been developed and compared with sheep blood agar (SBA) for the sensitive detection of small numbers of beta-hemolytic streptococci in clinical specimens. CNA-P has as its basis a commercial medium (Difco Columbia CNA agar) supplemented with 5% (vol/vol) sheep blood, and the CNA is further modified by addition of 100 mM PIPES buffer [piperazine-N,N'-bis(2-ethanesulfonic acid)] (pH 7.5) to maintain cultures at an alkaline pH during incubation. CNA-P was shown to inhibit the production and/or release of four different types of S. salivarius bacteriocins or bacteriocin-like inhibitory molecules. The efficacies of CNA-P and SBA for detection of beta-hemolytic streptococci in 1,352 pharyngeal samples from 376 children were compared. The beta-hemolytic streptococcal isolates recovered from the samples included 314 group A (S. pyogenes), 61 group G, 33 group B, and 5 group C streptococci. Of 314 samples that yielded S. pyogenes, 300 were positive on CNA-P (96%) and 264 (86%) were positive on SBA. A significantly greater number of S. pyogenes isolates from these samples were recovered only on CNA-P (50 of 314) compared with the number of isolates recovered only on SBA (14 of 314). In addition, the degree of positivity, a measure of the total numbers of S. pyogenes isolates on the plate, was significantly higher on CNA-P than on SBA (2.40 versus 2.07; P < 0.001). Interestingly, CNA-P was also found to enhance the hemolytic activity of streptolysin O, allowing detection of streptolysin S-deficient S. pyogenes strains which might otherwise go undetected on SBA and other isolation media.

Twenty-one oral Streptococcus isolates of known interbacterial coaggregation groups were tested against one another (as both producers and indicators) to detect bacteriocin-like inhibitory activity. In agar-based antagonism tests, seven strains produced small inhibitory zones (< or = 3 mm diameter) but in liquid medium, only strain Streptococcus gordonii DL1 (Challis) produced a detectable antibacterial action (bacteriocin STH1). Five strains were sensitive to bacteriocin STH1, but neither the production of nor the sensitivity to any of the antagonistic agents correlated with coaggregation groupings. Four strains (C219, 903, 118 and Wicky) developed stable resistance in response to the bacteriocin, whereas one isolate (strain 34) remained sensitive following repeated bacteriocin exposure. With one exception (strain 903), bacteriocin STH1-sensitive strains were competent for genetic transformation, but not all competent strains were bacteriocin-sensitive. Bacteriocin-resistant derivatives of transformable strains exhibited decreased competence (80-90% reduction) compared with their parent strains.

Electron microscopy of zoocin A-treated sensitive streptococcus cells revealed cytoplasmic disruption and ultimately complete rupture of the cell wall. Culture viability and optical density were shown to decrease rapidly and simultaneously in Streptococcus pyogenes FF22 but less quickly in the relatively more resistant Streptococcus mutans 10449. Zoocin A was shown to cleave hexaglycine in a colorimetric cell-free microtiter assay system, and it is concluded that the killing action of zoocin A, like that of lysostaphin, is most probably the result of direct cleavage of the peptidoglycan cross-links in the cell wall. The relationship between sensitivity to zoocin A and the peptidoglycan cross-linkage structure of Streptococcus zooepidemicus, Lactococcus spp., S. pyogenes, Streptococcus gordonii, Streptococcus oralis, S. mutans, and Streptococcus rattus has been evaluated.

A bacteriocin-like inhibitory substance, salivaricin A, was purified from cultures of Streptococcus salivarius 20P3 and was shown by ion spray mass spectrometry to have a molecular mass of 2,315 +/- 1.1 Da. Amino acid composition analysis demonstrated the presence of lanthionine, indicating that salivaricin A may be a member of the lantibiotic class of antibiotic substances. The sequence of eight amino acids at the N terminus of the molecule was determined by Edman degradation, and mixed oligonucleotide probes based on part of this sequence (GSGWIA) were used to detect the salivaricin A structural gene. A 6.2-kb EcoRI fragment of chromosomal DNA from strain 20P3 that hybridized with the probes was cloned, and the hybridizing region was further localized to a 379-bp DraI-AluI fragment. Analysis of the nucleotide sequence of this fragment indicated that salivaricin A is synthesized as a 51-amino-acid prepeptide that is posttranslationally modified and cleaved to give a biologically active 22-residue peptide containing one lanthionine and two beta-methyllanthionine residues. The secondary structure of presalivaricin A was predicted to be similar to that of type A lantibiotics, with a hydrophilic alpha-helical leader sequence and a propeptide region with potential for beta-turn formation and a lack of alpha-helicity. The sequence around the cleavage site of presalivaricin A differed from that of other type A lantibiotics but was similar to that of several bacteriocin-like inhibitory substances produced by lactic acid bacteria.

Genotypic and phenotypic analysis of 42 strains of Streptococcus uberis isolated from mammary secretions of 17 cows collected at different periods of the lactation cycle and from episodes of clinical mastitis were performed. Seventeen restriction endonuclease fingerprint (REF) patterns and 12 bacteriocin-like inhibitory substance (BLIS) fingerprints were observed. REF identified and differentiated closely related strains of S. uberis isolated from mammary secretions collected from the same cow at different periods of the lactation cycle and from episodes of clinical mastitis. BLIS fingerprinting of S. uberis complemented REF results. REF and BLIS fingerprinting provided evidence concerning persistence of infection in the same quarter or different quarters of the mammary gland over different periods of the lactation cycle, and occurrence of infection with similar and dissimilar strains of S. uberis. Biochemical profiles could not identify closely related strains nor did they complement REF results. Antibiotic resistance patterns alone were of little value in differentiating closely related strains, but were identical with isolates having same REF pattern. None of the S. uberis strains was found to carry plasmids. REF and BLIS fingerprinting can be utilized effectively to differentiate closely related and unrelated strains of S. uberis isolated from bovine mammary secretions.

A typing scheme based on detection of the production of bacteriocin-like inhibitory substances (BLIS) was a useful tool in a series of epidemiological studies of the spread and survival on surfaces of salivary streptococci and staphylococci. The organisms survived for longer periods on glass, plastic, stainless steel and latex surfaces than on unpainted wood or paper. The presence of saliva as a suspending medium prolonged the viability of Staphylococcus aureus C55 and Streptococcus pyogenes FF22 but not of Strep. salivarius Min 5. Young children were shown to carry Strep. salivarius of identical BLIS-types on their fingers and in their saliva. BLIS typing of Strep. salivarius strains obtained from telephone mouthpieces and from the tongue-sealed flap of envelopes was used as a method of tracing the human source of the salivary deposits.

Many of the strategies that have been formulated to decrease the incidence of dental caries have focused upon attempting to reduce either the numbers or the acidogenic activities in dental plaque of the mutans streptococci. In recent years there has been a growing interest in a new method of caries control which depends upon exploiting the ability of certain bacteria to produce antibiotics called bacteriocin-like inhibitory substances (BLIS) with killing activity for mutans streptococci. The approaches under investigation include either the regular oral administration of the purified BLIS or the genetic modification of existing plaque bacteria such that they produce the antimutans BLIS in situ.

A longitudinal study of the distribution of oral beta-haemolytic streptococci in 103 children (ages 5-6 y) attending three Dunedin schools was initiated in February 1987. A total of 858 paired pharyngeal and saliva specimens were obtained in nine principal sampling sessions, the last being in May 1989. The detection rate of Lancefield group A streptococci in directly plated pharyngeal cultures (19.8%) was considerably higher than in the corresponding saliva cultures (5.1%). By contrast, Lancefield group F and serologically non-groupable beta-haemolytic Streptococcus anginosus tended to be more prevalent in saliva (10.9% positive) than in pharyngeal specimens (7.1%). Group A streptococci were recovered at least once from 59 (57.3%) of the subjects. Representative beta-haemolytic streptococcus isolates were typed according to their production of bacteriocin-like inhibitory substances (BLIS) and by traditional serological techniques. The epidemiological value of BLIS typing was particularly evident in that both BLIS-positive and BLIS-negative strains could be differentiated within serotypes M1, M12 and M22 and also the serologically not typable group A streptococcus isolates. Ten children consistently had small numbers of group A streptococci present in their pharyngeal cultures, for periods of at least 12 months. In six of these subjects the carried strain was M-type 4/BLIS-type 655. This study has shown that in young schoolchildren long-term carriage of a variety of species of beta-haemolytic streptococci with little associated clinical evidence of infection may be common.

Inhibitory substances produced by six strains of Streptococcus salivarius 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.

A scheme for the "fingerprinting" of streptococci according to their production of (P typing) and sensitivity to (S typing) bacteriocine-like inhibitory substances has been developed. P typing of 450 beta-haemolytic streptococci by their action on a set of nine standard indicator strains revealed that 80% of strains produced one or more detectable inhibitors, and that 17 different P types could be recognised. Production of some inhibitors seemed to be a property of strains of a particular serological group or type. Bacteriocine-like substances were produced by streptococci of serological groups, A, B, C, D, E, F and G. Nine strains were selected as standard producers for S typing. These strains differed in their spectra of inhibition, but all seemed to be active only against gram-positive bacteria. One producer, a group-F streptococcus, specifically inhibited group-A streptococci. The conditions of incubation were critical for demonstration of inhibitor production. A requirement for blood and for incubation at 32 degrees C were important factors. None of the inhibitors was induced by ultraviolet irradiation. The observed inhibitory effects were not attributable to either hydrogen peroxide or low pH, but to the production of a variety of substances having diverse physicochemical properties and production requirements. Most of the inhibitors do not seem to be produced in liquid media. The "fingerprinting" procedure is simple and inexpensive, and provides a reliable means of subdividing streptococcal strains that may find application as a supplement to the existing serological typing schemes.

A previously described inhibitor typing scheme for hemolytic streptococci has been utilized to test 12 well-characterized strains of group E streptococci. These strains were differentiated into five inhibitor production (P) types and four inhibitor sensitivity (S) types: seven different strain "fingerprints" (combinations of P-type and S-type) being demonstrated. Inhibitor production by group E streptococci was increased under conditions of anaerobic incubation. The inhibitor fingerprints were stable on repeated testing of strains and it is suggested that the scheme is of value both for the labelling of serologically untypable strains and for subdividing strains belonging to existing serotypes.

Spontaneous, low-frequency loss of ability to produce streptococcin A-FF22 (SA) by group A streptococcus strain FF22 was observed. The proportion of non-SA-producing (SA(-)) derivatives occurring in strain FF22 cultures grown in Todd Hewitt broth supplemented with 1% of yeast extract (THBY) was increased on treatment with ethidium bromide, acriflavin, or rifampin. The highest incidence of SA(-) organisms, however, was found in untreated THBY cultures that had been aging by incubation at 37 degrees C for several months. The possibility of selective effects in these experiments, operating to enhance the apparent frequency of SA(-) bacteria, was discounted. The survival of SA(-) derivatives in association with populations of SA(+) bacteria was dependent upon the use of culture conditions inimical to SA activity, since a consistent finding was that the loss of ability to produce SA was associated with loss of immunity to the killing action of this bacteriocin. Whereas selective killing of SA(-) derivatives was evident in mixed cultures of SA(+) and SA(-) strains in tryptic soy broth, no such effect was demonstrable in THBY. In these experiments, elimination of SA(-) cells seemed directly related to the presence of active SA. Purified clones of SA(-) substrains did not seem revertible to SA production, either spontaneously or on treatment with nitrosoguanidine. It is suggested that the property of production of SA by group A streptococcus strain FF22, together with that of host cell immunity to the homologous bacteriocin, may be mediated by plasmid-borne genetic determinants.

Seven beta-hemolytic Streptococcus salivarius isolates produced bacteriocin-like inhibitory activity in deferred antagonism tests using a set of nine indicator bacteria (I1-I9). Five of these S. salivarius strains (KWF, TOVE-R, K17, K21, and K26) were inhibitory to indicators I2, I5, I6, and I7. Mutated non-hemolytic derivatives showed concomitant loss of inhibitory activity against I2, I5, and I6, but retained activity against I7. Inhibitory activity against I2, I5, and I6 was restored in beta-hemolytic revertants of such mutants. Strain 3638 was inhibitory to all of the indicator organisms except I3, and this pattern of inhibitory activity was retained by non-hemolytic derivatives. It appeared that strain 3638 produced an additional broadly-active inhibitory agent, since a mutant (strain 3638A), which was apparently defective in the production of this inhibitor, retained both the beta-hemolytic and I2-, I5-, I6-, and I7-inhibitory activities. Non-hemolytic derivatives of strain 3638A were inhibitory only to I7. Strain 3638, therefore, appeared to produce at least three inhibitory agents: one active only on I7; another acting on I2, I5, and I6 (and associated with beta-hemolytic activity); and a third apparently active on all of the indicators other than I3. S. salivarius strain JH inhibited all nine indicator strains and possessed a beta-hemolytic character which differed from that of the other strains in being readily eliminated on treatment with the plasmid-curing agent novobiocin. Non-hemolytic derivatives of JH retained inhibitory activity against the complete set of indicators.(ABSTRACT TRUNCATED AT 250 WORDS)

An inhibitor "fingerprinting' technique was used to test 120 group B and 50 group C streptococcus strains for production of inhibitory activity. The incidence of inhibitor production was low. Five of 70 group B streptococci of animal origin and one of 50 from human sources consistently produced inhibitory activity. Six of 20 animal strains and three of 30 human strains of group C streptococci were inhibitor producers. These included two Streptococcus dysgalactiae, three S. equisimilis and four S. zooepidemicus. The temperature of incubation and species of origin of the blood used in the culture medium were critical determinants of inhibitor production. Investigation of inhibitors for their spectrum of activity, heat stability, sensitivity to trypsin, dialysability and mode of action indicated that five of the group B and seven of the group C streptococci produced inhibitors that had bacteriocin-line properties. Three bovine strains of group B streptococci produced very similar inhibitory activity. A greater variety of bacteriocin-like substances was detected in the group C streptococcus strains. Two of the S. zooepidemicus strains were each found to produce more than one inhibitory substance.

Inhibitor production (P)-typing was used as a strain marker in epidemiological studies of Streptococcus salivarius. 43 per cent of 180 adult subjects had inhibitory Strep. salivarius strains as components of their oral microbiota. Strains of 13 different P-type patterns were detected and strains of different P-types often co-existed in the same subject. Adults from whom inhibitor-producing Strep. salivarius strains had been isolated retained their characteristic bacteriocinogenic strains over a 3-yr period. A specific Strep. salivarius-inhibitor screening method was used to study oral acquisition of Strep. salivarius by 14 newborn babies. Initially the babies were colonized by a wide variety of strains, many of which were not detected in the mothers' mouths. By the fifth day of life, strains with P-types identical with those in the mother had often become established as quantitatively prominent members of the babies' Strep. salivarius population.

A bacteriocin, streptococcin sal-P, was isolated by freeze-thaw elution from cultures of Streptococcus salivarius strain P grown on tryptic soy agar plus 1% neopeptone. The inhibitor could also be extracted with either 7 M urea of 1 M NaCl from cells grown on this medium, but little activity was recovered from cells grown in liquid media of from the supernatants of these cultures. Streptococcin sal-P was found to be a proteinaceous substance of molecular weight approximately 8000. It was remarkably stable at extremes of pH or temperature and appeared to adsorb nonspecifically to both sensitive and resistant bacterial cells and also to cellulose membranes. The range of its inhibitory activity was almost entirely against Gram-positive bacteria, particularly streptococci, including strains of S. pyogenes and S. sanguis, but not S. mutans or group D streptococci. Streptococcin sal-P was bactericidal for actively metabolizing susceptible strains.

The production of bacteriocin-like inhibition by two of 135 strains of group B streptococci was demonstrated during the growth of these organisms on solid nutrient media. Although a variety of gram-positive organisms was susceptible, none of a wide range of different gram-negative strains was inhibited. The active substance produced by one of these strains was extracted from cultures grown on solid media (Todd-Hewitt agar) and was shown to be bactericidal in action. This bacteriocin (streptocin B(1)) was partially purified by ammonium sulfate precipitation followed by gel filtration and ion-exchange chromatography. Production of the bacteriocin was repressed by addition of certain fermentable carbohydrates to the basic medium and enhanced by the addition of yeast extract. The bacteriocin was shown to exist in two distinct and interconvertible physical forms: a basic unit of molecular weight 10,000 and an aggregate having a molecular weight of over 200,000. The bacteriocin was inactivated by proteolytic enzymes and, although labile in alkali, it was stable to boiling in mild acids.

Strains of group-G streptococci were tested by a "fingerprinting" method for the production of (P typing) and sensitivity to (S typing) inhibitory agents, and were biotyped. In the standard P-typing test, 28 of 50 strains of human origin, but none of 30 strains of animal origin, showed inhibitory activity. Of the human strains, 12 formed a bacteriocin that was active on group-A streptococci, including three (strains I2, I5 and I8) of the four streptococci of this group among the indicator strains. Sixteen other human strains inhibited the fourth group-A indicator (strain I7), and to a lesser extent strain I2, by lowering the pH of the typing medium. This acid-mediated inhibition was eliminated by testing on a medium containing calcium carbonate 0.5%; the 16 strains were then completely non-inhibitory, and the bacteriocin-forming strains, the typing pattern of which had originally been I2, I5, I7, I8, showed only inhibition attributable to the action of the bacteriocin. Nearly all group-A streptococci were sensitive to the group-G bacteriocin. The indicator strain I7 and several other members of M-type 28 were exceptions, but their resistance was not associated with the presence of R-antigen 28. Fifteen inhibitor-sensitivity patterns and 12 biotypes were identified among the strains; some of these tended to be associated with either a human or an animal origin. Neither S type nor biotype appeared to correlate with inhibitor production.

A typing scheme was used to test 15 strains of Streptococcus uberis according to their production of (P-type) and sensitivity to (S-type) bacteriocin-like inhibitory substances. Twelve of the strains were inhibitor producers and nine different P-types were detected. All of the strains were typable according to inhibitor sensitivity, ten different S-types being distinguished. Both the P-type and S-type designations of the strains were reproducible on repeated testing. By combination of P-typing and S-typing, highly discriminatory inhibitor 'fingerprints' of the strains could be obtained. This scheme would appear to have considerable potential for typing isolates of Str. uberis as an aid to investigations into the epidemiology of Str. uberis mastitis in dairy cattle.

We assembled an international collection of strains from sporadic and epidemic human infection with Streptococcus zooepidemicus (Lancefield group C) for laboratory study. Cultural and physiological characteristics of the isolates were determined, including biotyping with the API 20 STREP test kit and susceptibility testing with penicillin, erythromycin and tetracycline. The strains were examined for bacteriocin production and sensitivity and typed with a specially developed group-C streptococcal bacteriophage system incorporating a panel of 14 phages. Results of these tests gave useful discrimination between many of the strains: differences were shown between each of the major outbreak strains, including those complicated by post-streptococcal glomerulonephritis. Serious group C streptococcal infection may be caused by S. zooepidemicus and isolates should be identified to species level; the application of a typing scheme such as this may help to distinguish epidemiological patterns of infection.

A bacteriocin, streptocin A, was isolated from the supernatant fluid of tryptic soy broth cultures of Group A streptococcus strain FF-22. Evidence was obtained which supports the view that the failure to recover active streptocin A after growth of the producer strain in certain fluid media is due to the inactivation of the bacteriocin by concomitantly synthesized streptococcal proteinase. The bacteriocin was purified 139-fold and the active product appeared to be of uniform size, having a molecular weight of approximately 8,000. Streptocin A was bactericidal, but not lytic, for a susceptible Group A streptococcus and the lethal effect was markedly temperature dependent. The bacteriocin inhibited the synthesis of DNA, RNA, and protein, and also prevented the uptake and incorporation of glucose by the sensitive cells. Degradation of RNA occurred, but appeared to be less than that produced by a staphylococcal bacteriocin. This effect may be due to differences in the killing potency of the two bacteriocins in preparations having similar inhibitory activity when measured by lawn culture assays.

Mutans streptococci (MS) are known to be causative agents of dental caries. It has been suggested that these cariogenic bacteria could be eliminated from dental plaque by application of bacteriocins or bacteriocin-like inhibitory substances (BLIS). In the present study 272 bacterial strains of the genera Streptococcus, Enterococcus and Staphylococcus were tested for their production of BLIS activity against MS by use of a deferred antagonism test on agar media supplemented with either whole blood or yeast extract. Although only 14.3% of the test strains displayed anti-MS activity, the inhibitory agents produced by these strains were characterised by considerable diversity in the range of their inhibitory action against both MS and other common oral streptococcal species. It is suggested that combinations of relatively specifically targeted anti-MS BLIS may have potential application to the prevention of dental caries. [ABSTRACT FROM AUTHOR]