Virulence gene expression of three hypervirulent S. pyogenes M1T1 and membrane vesicles isolation

The genus Steptococcus [Rosenbach, 1844] consists of catalase-negative, Gram-positive cocci which are arranged in pairs and chains and are usually facultatively anaerobic. Streptococci are a diverse collection of species inhabiting many body sites and they are both commensals and pathogens. In particular, nonpathogenic streptococci, are the most abundant bacterial species at the oropharyngeal level, and they have been found to exert an important role in the protection against pathogenic agents causing inflammation and infections [Tagg JR et al., 2003]. Much attention has recently been devoted to the analysis of the oral microbiota to develop bacteriotherapy focused on prevention and/or treatment of upper respiratory tract infections. In this regard, a key species is Streptococcus salivarius, a lactic acid bacterium that is mainly encountered in the mouths of human beings. It is the first commensal bacterium that appears in the oral cavity of newborns where it colonizes the upper respiratory tract [Aas JA et al., 2005] and persists there as a predominant member of the native microbiota throughout the life of its human and sole natural host [Favier CF et al., 2002]. Many LAB (Lactic Acid Bacteria) strains, including S.salivarius, are prolific producers of bacteriocins, which are an abundant and diverse group of ribosomally synthesized antimicrobial peptides produced by bacteria that kill or inhibit species closely related to the producer bacterium. Furthermore, according to several studies, large populations of S.salivarius efficiently adhere to oral epithelial cells, especially the papillary surface of the tongue that is a strategic location to carry out a population surveillance within the oral microbiota [Tagg JR et al., 1983; Wescombe PA et al., 2010 ]. The presence of an adhesion system such as pili, fibrils, saliva-binding proteins and host-cell-binding proteins, together with its high competition rate, helps this species to stay in the human mouth [Nobbs AH et al., 2009]. In our laboratory, during my PhD studies, we characterized one strain, S.salivarius 24SMBc, isolated from one healthy child that showed excellent inhibitory activity against S.pneumoniae and S.pyogenes and a potent capacity of adhesion to HEp-2 cells. The main aim of this study was to evaluate the clinical evidence of a probiotic application of S.salivarius 24SMBc for the prevention or reduction of recurrent medium otite (OM) children [Santagati M et al., 2014]. Therefore, this strain was included in a randomized, placebo-controlled, double-blind paediatric trial that involved 100 otitis prone children. This preliminary study showed a reduction of OM episodes in children who received the intranasal administration of this probiotic with respect to children treated with placebo [Santagati M et al., 2014]. The study of S.salivarius 24SMBc ended with the production and marketing of a new medical device, the Rinogermina nasal spray, in collaboration with D.M.G s.r.l Italy. Streptococci, as mentioned above, include both nonpathogenic and pathogenic bacteria. In particular, Streptococcus pyogenes (group A streptococci, GAS) is an exclusive human bacterial pathogen. The virulence potential of this species is tremendous. Interactions with humans range from asymptomatic carriage over mild and superficial infections of skin and mucosal membranes up to systemic purulent toxic-invasive disease manifestations [Fiedler T et al., 2015]. Simultaneously with the study of S.salivarius 24SMBc, my PhD project focalized on global regulation of virulence expression genes of three hypervirulent strains of Streptococcus pyogenes [Santagati M et al., 2014] and their eventual production of membrane vesicles like new delivery system of virulence-associated components