Effect of transformation with <i>sesC</i> on <i>in vitro</i> and <i>in vivo</i> catheter colonization and <i>in vivo</i> organ colonization.

<p>(<b>A</b>) Seven mm catheter fragments were inoculated with 8325–4 or its <i>sesC</i>-expressing transformant. After 2 h incubation at 37°C, catheters were rinsed and adherent bacteria were detached by sonication and the numbers of bacteria recovered from each catheter fragments were quantified by quantitative cultures. (<b>B</b>) <i>In vivo</i>, catheterized animals were inoculated through the catheter lumen with 8325–4 strain or its 8325–4 (pCN<i>sesC</i>) transformant. At day 5 post-infection, the numbers of bacteria attached to the catheters or recovered from organs were quantified by quantitative cultures. The <i>sesC</i>-expressing transformant 8325–4, (pCN<i>sesC</i>) has a higher rate of colonization of different organs up to 100-fold compare to its parental strain. *: <i>P</i><0.05</p

SEM images of biofilms formed by 8325–4, its <i>sesC</i>-expressing transformants and BH1CC as controls.

<p>Biofilm growth on glass disks was allowed during overnight incubation at 37°C in BHI supplemented with 1% glucose. The next day, samples were fixed and sputter coated with platinum. The images show bacteria attached on the surface of disks at 1000x, 15000x magnification.</p

Effect of transformation with <i>sesC</i> on biofilm formation by the biofilm-forming isogenic <i>srtA</i> mutants of 8325–4.

<p>Effect of <i>srtA</i> mutation on biofilm formation of PIA-dependent biofilm-forming strain 8325–4, (Atl/FnBP)-mediated biofilm-forming strain BH1CC, the <i>sesC</i>-tranformed 8325–4 <i>srtA</i>::<i>tet</i> strain and complementation of 8325–4 <i>srtA</i>::<i>tet</i> (pCN<i>sesC</i>) with <i>srtA</i>, was evaluated using the quantitative microtiter plate assay. (SM: sodium metaperiodate, PK: proteinase K; error bars mean standard deviation)</p

Effect of αSesC-IgGs on catheter colonization and infection rate by 8325–4 and its <i>sesC</i>-expressing transformant.

<p>Catheterized animals were inoculated with bacteria pre-incubated with αSesC-IgGs or pre-immune IgGs for 2 h at 4°C. The next day, animals were sacrificed and the bacteria were recovered from catheter and organs and quantified by quantitative cultures. Pre-incubation with αSesC-IgGs significantly reduced the rate of catheter and organ colonization by 8325–4 (pCN<i>sesC</i>) from 10 to 10000-fold. *p<0.05</p

Effect of transformation of <i>S</i>. <i>aureus</i> strains with <i>sesC</i> or <i>sesK</i> on the biofilm formation.

<p>Using a semi-quantitative microtiter plate assay the level of biofilm formation in different media, the phenotype of biofilms and effect of αSesC-IgG antibodies on biofilm formation of different strains were identified. Biofilm formation in medium supplemented with NaCl or the effect of dispersal agents were used to discriminate the phenotype of biofilms. <b>(A</b>) Expression of <i>sesC</i>, <i>sesK</i>, <i>sasF</i> and <i>icaA</i> in cDNA of transformed strains was evaluated using the gel-based reverse transcription-PCR assay. <b>(B</b>) Detection of SesC production in <i>S</i>. <i>epidermidis</i> 10b, 8325–4, 8325–4 (pCN<i>sesC</i>) and 8325–4 <i>ica</i>::<i>tet</i> (pCNsesC) 8325–4 <i>srt</i>::<i>tet</i> (pCNsesC)) in soluble and insoluble fractions via Western blot assay. (<b>C</b>) The effect of <i>S</i>. <i>aureus</i> 8325–4 transformation with <i>sesC</i>. Introduction of a plasmid carrying <i>sesC</i> but not the mock plasmid changed the biofilm formation of transformants in BHI-NaCl and also the effects of PK and SM were opposite. (<b>D</b>) Microtiter plate assay demonstrating the effect of the inducers of biofilm formation (glucose and NaCl) and dispersal agents on biofilm formation of strains <b>(E)</b> quantification of PIA production using PIA non-specific immunoblot assay for biofilm in BHI-Glu. (<b>F</b>) Effect of transformation with <i>sesK</i> on the biofilm formation of strain 8325–4 and also the effect of <i>sesC</i> on the biofilm formation of strain MSSA4 in comparison with MRSA strain BH1CC. (SM: sodium metaperiodate, PK: proteinase K; error bars mean standard deviation)</p