Most streptococcal species are essentially non-pathogenic and are part of the normal commensal microflora of the mouth, skin, intestine, and upper respiratory tract of humans (and other animals). Streptococcus salivarius is a predominant oral streptococcal species in humans that is thought to play an important role in the maintenance of a healthy oral microbial ecosystem. A notable characteristic of this species is that they commonly produce Bacteriocin-like Inhibitory Substances (BLIS). Streptococcus salivarius strain Pirie resembles strain K12, the prototype BLIS-producing S. salivarius, in that it inhibits all nine standard indicators when tested in a streptococcal bacteriocin ;;fingerprinting” scheme. Another similarity to strain K12 is the presence of the class I bacteriocins salivaricin A genes in strain Pirie. However, strain Pirie differs from strain K12 in having a broader spectrum of inhibitory activity against other members of the commensal oral microflora, including most strains of S. salivarius. Preliminary studies indicated that strain Pirie may produce multiple BLIS-like activities, including some heat-labile anti-S. salivarius activities, a characteristic considered uncommon for S. salivarius BLIS. The aim of the present study was to gain further insight into the basis for the broad inhibitory spectrum of strain Pirie, especially the anti-S. salivarius activities, as a precursor to possible practical applications for this strain as a probiotic for the human oral cavity. Through the application of deferred antagonism tests, the thermostability of the multiple BLIS activities produced by strain Pirie was evaluated by comparison to the known BLIS activities of other S. salivarius. This indicated that there were multiple BLIS produced by strain Pirie. The BLIS produced by strain Pirie appeared to share similarities in their activity with that of some other S. salivarius including strains #36, J01, JH, 193, GR and Sub-5. However, strain Pirie also expresses a unique heat-labile anti-Streptococcus constellatus and anti-S. salivarius BLIS activity. By application of the salivaricin A (SalA) induction assay it was established that strain Pirie does express SalA activity. The optimal growth conditions for BLIS production were also assessed to determine what temperature/nutrients/aeration factors could enhance the level of BLIS produced. The broad inhibitory spectrum of strain Pirie was evident on a variety of growth media. The BLIS production appeared to be consistent even when strain Pirie was grown anaerobically or when the incubation temperature was decreased to 32°C. Interestingly, no detectable BLIS activity was evident in cultures grown in liquid media or with use of the simultaneous antagonism test. The latter finding may indicate that strain Pirie produces its BLIS activity in the stationary growth phase. With use of a biphasic overlayer system, yeast extract (YE) was found to enhance strain Pirie BLIS production, especially against Streptococcus pyogenes and S. salivarius, and blood was required to be present in the liquid growth phase to enhance BLIS recovery. The BLIS expression of strain Pirie was also found to be subject to carbon catabolite repression (CCR), since BLIS activity was decreased when fermentable sugars were present. Attempts were made to extract the BLIS activities produced by strain Pirie, especially those responsible for the anti-S. salivarius activity. Freeze-thaw extraction and elution of agar-grown cultures with 7 M urea yielded the highest levels of inhibitory activity. The presence of blood components appeared essential for BLIS recovery. The fact that the heat-labile BLIS activity could not be extracted directly by suspending strain Pirie cells in various solvents indicated that most of the heat-labile BLIS was released extracellularly. This is uncommon, as most previously-studied large, heat-labile BLIS are typically found bound at least partially to the producer cell surface. Further evaluations of the BLIS-containing extracts were performed to increase understanding of the nature of the inhibitory substances produced, which indicated that the anti-S. salivarius BLIS activities precipitated at 50% saturation with ammonium sulphate and dialysis experiments indicated the size was larger than 8,000 Da.During attempts to obtain a plasmid-negative derivative of strain PiriestrR by incubating brain heart infusion cultures at elevated temperature, rough variant (RV) derivatives were obtained that retained their characteristic inhibitory activity and also their sensitivity to the BLIS produced by S. salivarius strains K12, M18 and NR. ERIC-PCR established that the RV strain was clonally related to the parent strain. Similar RV derivatives were obtained under these same growth conditions from S. salivarius strain M18. It was concluded that S. salivarius (similarly to the previously-studied strain Tove-R) may have the ability to produce a different array of extracellular polymers to enhance their survival when exposed to certain environmental stresses. Since S. salivarius strains JH and 193, both of which have similar activity spectra to strain Pirie, have previously been shown to produce bacteriocin-like peptide (BLP), the presence of the BLP transporter gene was screened for in strain Pirie. No amplicons having high homology were detected however.The major difficulty relating to strain Pirie BLIS recovery was the apparent requirement for blood components, and it proved difficult to separate the blood components from the heat-labile BLIS activity. However, the present study has increased knowledge about the multiple BLIS produced by strain Pirie, and progress has been made in understanding the extraction and purification requirements. Further approaches to the extraction and purification of the large BLIS produced by strain Pirie such as SDS-PAGE and column chromatography should help define the characteristics of these novel antimicrobials. Follow up studies of Pirie RV could include comparison of the growth kinetics of the parent and RV strains when incubated at different temperatures. Strain Pirie RV could also be compared with the parent strain Pirie for relative adhesive capability on enamel surfaces to determine if it may preferentially colonise the tooth surface as a component of dental plaque.
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The unusual BLIS activities of Streptococcus salivarius strain Pirie
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