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Diversité génétique et phénotypique d'une collection de souches de Streptococcus salivarius : étude des éléments intégratifs et des capacités d’adhésion aux cellules des écosystèmes digestif et buccal

Abstract : Streptococcus salivarius is a commensal bacterium present in the oral cavity and all along the digestive tract (Eckburg et al., 2005; Hao & Lee, 2004). It is frequently found attached to tongue or to stomach and intestinal walls within biofilms (Cvitkovitch et al., 2003). In a biofilm, the cellular density implies a physical proximity between bacteria of different species which is favorable for genetic exchange between them. These horizontal transfers permit the bacteria to acquire genes of adaptation such as virulence and antibiotic resistance genes (Kolenbrander et al., 2010; Molin & Tolker-Nielsen, 2003). Largely present in the bacterial genomes, although not widely studied, the integrative conjugative elements (ICEs) and the integrative mobilizable elements (IMEs) probably play a major role in these transfers and in the adaptation of strains to their environment. The objective of this thesis is to study within S. salivarius the integrative elements (prevalence, diversity and transfer of one ICE) as well as the adhesion capacity to the cells of the oral and digestive ecosystems. A MLST study (MultiLocus Sequence Type) has allowed the classification of 212 strains of the S. salivarius collection from the DynAMic laboratory in 96 Sequence Types and to reveal the diversity of these strains. Among these, 138 were selected as representative of the diversity of the collection for ulterior studies. PCR research of integrative elements of these strains has shown that more than 70% of these strains present at least one integrative element carrying at least one antibiotic resistance gene. Therefore the diversity of the antibiotic resistance phenotypes observed were able to be correlated to the diversity of the integrative elements found (Tn916, Tn3872, Tn6002, Tn2009, MEGA) or described for the first time (IQ element) in S. salivarius. The diversity and prevalence of the ICE and IME in S. salivarius were characterized within the completely sequenced genomes of 19 strains. This analysis has allowed the detection of 27 ICE belonging to 2 superfamilies (Tn916 and Tn5252) and 4 distinctive families (ICESt3, Tn916, Tn1549 and TnGBS2) which are integrated into 12 different integration sites. The analysis also allowed the detection of 37 IME which are integrated in 7 different sites as well as 10 MEGA elements integrated in two different genes. The study of the ICE of the ICESt3 family which are described for the first time in S. salivarius in this work showed that they are integrated in the site already described (at the 3’ end of the fda gene), but also in 2 new sites (at the 3’ end of the rpsl and rpmG genes). In parallel to these studies, the adhesion capacity of S. salivarius strains to saliva and gastro-intestinal epithelial cells has been studied. The results have shown that adhesion capacity to the saliva of 24 strains are much diversified and strain dependent. Likewise, the capacity of adhesion to gastro-intestinal epithelial cell lines Caco2 and HT29-MTX of 14 of these strains is strain dependent but also cellular line dependent. These results have allowed the selection of the F6-1 strain of which the adhesion phenotype to HT29-MX and HT29-CL16E cells is particularly high and dependent on the secretion of mucus. The study of mutants of this strain has shown that its adhesion capacity is dependent both on some of their surface proteins and on the gastro-intestinal epithelial cell lines. Finally, conjugative transfer experiments of the ICESt3 F6-1 rpsl were performed under different conditions. Its excision capacity has been shown and an in silico analysis indicates that this ICE could be functional. However, no transconjugant has been obtained whatever the tested conditions
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Fanny Chaffanel. Diversité génétique et phénotypique d'une collection de souches de Streptococcus salivarius : étude des éléments intégratifs et des capacités d’adhésion aux cellules des écosystèmes digestif et buccal. Bactériologie. Université de Lorraine, 2016. Français. ⟨NNT : 2016LORR0070⟩. ⟨tel-01752352⟩

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