Production and characterization of salivaricin MPS-like inhibitory activity from streptococcus uberis strain NY42

Abstract

Streptococcus salivarius, a predominant bacterial species found in the human oral cavity, is increasingly studied for its production of bacteriocin-like activity. Much of the interest in the study of S. salivarius is driven by the perceived potential probiotic applications. The preliminary characterization of salivaricin MPS (salMPS), a bacteriocin produced by S. salivarius strain MPS revealed a large antimicrobial molecule with specific inhibitory activity against Streptococcus pyogenes, a potential human pathogen.

In the present study, the production of salivaricin MPS was evaluated from its original producer strain MPS using various growth media. Contrary to previous findings, it was almost impossible to detect any inhibitory activity when the producer was grown in liquid THB (Todd-Hewitt broth) medium, with or without supplement. The additional inhibitory activity of strain MPS towards non-S. pyogenes indicators and presumed two-component inhibitor system (from two salMPS genes) further complicated the production scenario. In comparison, Streptococcus uberis strain NY42 which was selected from the screening study as an alternative salMPS producer demonstrated a more specific anti-S. pyogenes salMPS-like inhibitor activity while containing only one of the two salMPS genes found in strain MPS.

Recovery of salMPS-like activity from strain NY42 was mainly achieved by freeze thaw method extraction of cultures grown on blood agar. The biggest challenge during the enrichment of the inhibitory activity using ammonium sulfate precipitation was the interference by large amounts of haemoglobin from the blood agar that associated with the inhibitory activity. Attempts at removing the haemoglobin from the inhibitory activity using guanidine hydrochloride and urea did not succeed. Fractionation of the NY42 freeze thaw sample using gel permeation HPLC (high pressure liquid chromatography) did indicate the presence of the putative salMPS-like inhibitor within the active fractions, supported by the SDS-PAGE (sodium dodecyl sulphate-polyacylamide gel electrophoresis) and mass spectrometry analysis results, although data from online database searching was less significant due to the lack of suitable library entries.

The effect of blood and saliva as well as uv-irradiation on the production of inhibitory activity by several P-type 226 producer strains was also investigated in this study. In addition, the possibility of using S. pyogenes cells to bind salMPS-like activity, followed by an attempt to release the inhibitory activity was also assessed.

Despite all the difficulties with the original producer S. salivarius strain MPS, the study of strain NY42 discovered an alternative producer capable of producing a similar but more specific inhibitory activity compared to strain MPS. Purification of salMPS-like inhibitor using HPLC, combined with 1D (one dimension) -PAGE and mass spectrometry analysis further demonstrated the possibility of using strain NY42 as a favorable salMPS producer.