Molekulare Methoden zum Nachweis des oral-probiotischen Stammes Streptococcus salivarius ssp. salivarius K12 und Anwendung in vitro und in vivo
S. salivarius belongs to the viridans group streptococci and is a predominant inhabitant of the oral cavity. It produces bacteriocin-like inhibitory sustances (BLIS) - antimicrobial peptides that inhibit the growth of closely related bacteria. S. salivarius ssp. salivarius strain K12 is highly active against S. pyogenes with lantibiotic salivaricin A2. S. pyogenes, which is ß-hemolytic and in serogroup A, causes pharyngeal infections, particularly in children aged four to twelve, and occasionally complications as acute rheumatic fever and acute glomerulonephritis. To profit by the benefits of oral probiotic strain K12, it is important to be able to monitor K12 in a product or the environment with a highly sensitive and specific method. For this reason, first of all a species specific PCR on basis of the gene, encoding for dextranase, was tested in this study. 72 percent of the tested S. salivarius strains were detected. Furthermore, a strain specific method for detection of the industrial strain S. salivarius ssp. salivarius K12 was developed. The method was tested in vitro and in vivo by colonization of a single representative individual with BLIS K12 Throat Guard. Based on salivaricin A operon information for S. salivarius 20P3, conserved primers for amplification and sequencing of gene salA and salB of S. salivarius K12 were designed. With the aid of the generated sequence fragment, three K12 strain specific PCR methods were developed: a conventional PCR, a nested PCR and a quantative real-time (RTQ-)PCR. Our data showed that conventional PCR offers a method for detection of S. salivarius K12 during phases of active colonization. In addition, patients can be tested on existing colonization with strain K12 as high quantaty is guarenteed. To increase sensitivity, a strain specific nested PCR was developed. This double round PCR is able to detect even a small number of K12 cells while specifity and reproducability are warranted. Avoidance of contamination has to be assured in the course of all laboratory processes. Futhermore, a strain specific real-time (RTQ-) PCR was established. The advantages in contrast to nested PCR are quantative determination of salivaricin A amplificats by using standard curves and safer working procedures in regard of contamination. Using nested PCR and real-time PCR, a colonization of the pharynx, the dorsum of the tongue and the saliva of the test person with S. salivarius ssp. salivarius strain K12 could be shown for as long as 22 days. In conclusion, repeated administration of S. salivarius ssp. salivarius strain K12 should be required at least every three weeks to achieve optimal probiotic effects against colonization and infection of S. pyogenes.
info:eu-repo/classification/ddc/610, Medizin, Streptococcus salivarius, Probiotikum, Pharyngitis, probiotic
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