Production of the Lantibiotic Salivaricin A and Its Variants by Oral Streptococci and Use of a Specific Induction Assay To Detect Their Presence in Human Saliva

Abstract

Salivaricin A (SalA), the first Streptococcus salivarius lantibiotic to be characterized, appears to be inhibitory to most Streptococcus pyogenes strains. A variant of the SalA structural gene (salA1) is present in more than 90% of S. pyogenes strains, but only strains of M serotype 4 and T pattern 4 produce the biologically active peptide. The present study identifies four additional variants (salA2 to salA5) of the SalA structural gene and demonstrates that each of the corresponding inhibitory peptides (SalA2 to SalA5) is produced in vitro. These variants appear to be similar to SalA and SalA1 in their inhibitory activity against Micrococcus luteus and in their ability to act as inducers of SalA production. It had previously been shown that S. pyogenes strain SF370 had a deletion (of approximately 2.5 kb) in the salM and salT genes of the salA1 locus. In the present study, several additional characteristic deletions within the salA1 loci were identified. S. pyogenes strains of the same M serotype all share the same salA1 locus structure. Since S. salivarius is a predominant member of the normal oral flora of healthy humans, strains producing anti-S. pyogenes lantibiotics, such as SalA, may have excellent potential for use as oral probiotics. In the present study, we have used a highly specific SalA induction system to directly detect the presence of SalA in the saliva of humans who either naturally harbor populations of SalA-producing S. salivarius or who have been colonized with the SalA2-producing probiotic S. salivarius K12.

Analysis of the Inhibitory Potential of Streptococcus Salivarius Isolated From Adult Saliva and the Tongue Dorsum for the Growth of Candida Albicans

“…In some concentrations, lantibiotic production is triggered [11]. According to Wescombe et al [8], salivaricin A production can be detected in individuals' saliva in varying concentrations because there is a variety of salivary flow rates that dissolve salivaricin A and allow adsorption in normal cell flora [8]. SDS-PAGE results in the current study showed that from six S. salivarius samples, only four subjects produced protein with the same profiles, containing one band located inline.…”

“…A centrifuge tube (15 ml, 50 ml), cytobrush, 1000 ml tipped pipet, 200 ml tipped pipet, micropipette, Eppendorf tube, sterilized reaction tubes, 96-well microplate, incubator, anaerobic jar, vortex, mini centrifuge, autoclave, centrifuge, orbital shaker, enzyme-linked immunosorbent assay plater reader, analytic scale, water bath, round end ose berujung bulat, light lamp, Petri dishes, thermal cycler T100, electrophoresis, Gel Doc, Thermoblock, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) chamber, SDS-PAGE tank, and AxioCam ERc5s (Zeiss) microscope were the tools used to conduct this study. Additional materials included a brain-heart infusion (BHI) broth, BHI agar, sabouraud dextrose agar, bakto agar (Biomatic), mitis salivarius agar (MSA), sterilized aquadest, aquabides, cotton, aluminum foil, 70% alcohol, gas mixture (N 2 , CO 2 : 10%; H 2 : 10%), phosphate buffer saline, primer Sal-AUS (5'-GTAGAAAATATTTACTACATACT) [8], primer Sal-ADS (5'-GTTAAAGTATTCGTAAAACTGATG) [8], nucleus-free water, Dream Taq, Agarose, gel red, Tris-acetate-ethylenediaminetetraacetic acid buffer, 100 bp ladder, 10 mM Tris-HCl pH 7.4 liquid, 0.1 M Tris-HCL pH 6.8 liquid, phenyl methyl sulfonyl fluoride, glycerol, Coomassie brilliant blue, albumin protein standard, acrylamide 40%, stacking gel buffer, resolving gel buffer, SDS 10%, ammonium persulfate, tetra methyl ethylene diamine, marker protein (Peqlab), MiliQ water, Laemmli buffer, β-mercaptoethanol, SDS running buffer, C. albicans ATCC 10231 glycerol stocks, and S. salivarius ATCC 13419 glycerol stocks.…”

Analysis of the Inhibitory Potential of Clinical Streptococcus Salivarius Isolates From Saliva and Dorsal of Tongue of Adults on Enterococcal Faecalis Growth

“…Then, E. faecalis were dissolved and incubated for 24 hrs. After that, the formed inhibition zone was measured [12].…”

Analysis of the Inhibition Potential of Streptococcus Salivarius Isolated From the Saliva and Dorsum of the Tongue of Adults on the Growth of Streptococcus Mutans

“…Identification of colony morphology was conducted by observing the size and consistency of the surface (big, soft, smooth, and mucous-like colony that looks like a gum drop) [12]. Colonies considered as S. salivarius were taken and confirmed using the polymerase chain reaction (PCR) technique with SalAUS primers (5'-GTAGAAAATATTTACTACATACT) and SalADS (5'-GTTAAAGTATTCGTAAAACTGATG) [13]. PCR amplification results were analyzed using 1% agarose running on electrophoresis for 30 minutes (100V) [13].…”

“…Colonies considered as S. salivarius were taken and confirmed using the polymerase chain reaction (PCR) technique with SalAUS primers (5'-GTAGAAAATATTTACTACATACT) and SalADS (5'-GTTAAAGTATTCGTAAAACTGATG) [13]. PCR amplification results were analyzed using 1% agarose running on electrophoresis for 30 minutes (100V) [13]. DNA strands that were 118 bp indicated that the colonies identified on the MSA medium were S. salivarius.…”