Comparison of Biofilm Formation between Major Clonal Lineages of Methicillin Resistant <i>Staphylococcus aureus</i>

<div><p>Objectives</p><p>Epidemic methicillin-resistant <i>S. aureus</i> (MRSA) clones cause infections in both hospital and community settings. As a biofilm phenotype further facilitates evasion of the host immune system and antibiotics, we compared the biofilm-forming capacities of various MRSA clones.</p><p>Methods</p><p>Seventy-six MRSA classified into 13 clones (USA300, EMRSA-15, Hungarian/Brazilian etc.), and isolated from infections or from carriers were studied for biofilm formation under static and dynamic conditions. Static biofilms in microtitre plates were quantified colorimetrically. Dynamic biofilms (Bioflux 200, Fluxion, USA) were studied by confocal laser-scanning and time-lapse microscopy, and the total volume occupied by live/dead bacteria quantified by Volocity 5.4.1 (Improvision, UK).</p><p>Results</p><p>MRSA harbouring SCC<i>mec</i> IV produced significantly more biomass under static conditions than SCC<i>mec</i> I–III (P = 0.003), and those harbouring SCC<i>mec</i> II significantly less than those harbouring SCC<i>mec</i> I or III (P<0.001). In the dynamic model, SCC<i>mec</i> I–III harbouring MRSA were significantly better biofilm formers than SCC<i>mec</i> IV (P = 0.036). Only 16 strains successfully formed biofilms under both conditions, of which 13 harboured SCC<i>mec</i> IV and included all tested USA300 strains (n = 3). However, USA300 demonstrated remarkably lower percentages of cell-occupied space (6.6%) compared to the other clones (EMRSA-15 = 19.0%) under dynamic conditions. Time-lapse microscopy of dynamic biofilms demonstrated that USA300 formed long viscoelastic tethers that stretched far from the point of attachment, while EMRSA-15 consisted of micro-colonies attached densely to the surface.</p><p>Conclusions</p><p>MRSA harbouring SCC<i>mec</i> types IV and I–III demonstrate distinct biofilm forming capacities, possibly owing to their adaptation to the community and hospital settings, respectively. USA300 demonstrated abundant biofilm formation under both conditions, which probably confers a competitive advantage, contributing to its remarkable success as a pathogen.</p></div

Live/dead BacLight staining and CLSM on 72-hour old MRSA biofilms under shear flow (Bioflux system).

<p>A) 3-dimensional representations. B) 2-dimensional projections, thin white lines in the xy plane depict the location of the section plane on the z axis. White bars have a length of 10 µm.</p

Performance of MRSA clones in the static and shear flow assay.

<p>A) In the static assay: biomass production was subdivided into weak, moderate and strong. B) In the shear flow assay biofilm formation was assessed as positive or negative.</p

Space occupied by live (green) or dead (red) or total (grey) cells by different epidemic MRSA clones in a biofilm.

<p>Error bars denote standard deviations were calculated using 1 (USA300, EMRSA-15), 2 (USA600, Hungarian/Brazilian, Iberian) or 3 (USA500) independent z-stacks.</p

Overview of MRSA clone characteristics, site of isolation, and biofilm formation in static and shear flow assays.

<p>Overview of MRSA clone characteristics, site of isolation, and biofilm formation in static and shear flow assays.</p

Beneficial Effects of Anti-Interleukin-6 Antibodies on Impaired Gastrointestinal Motility, Inflammation and Increased Colonic Permeability in a Murine Model of Sepsis Are Most Pronounced When Administered in a Preventive Setup - Fig 2

<p><b>Effect of</b><u><b><i>preventive</i></b></u><b>treatment with anti-IL-6 antibodies on sepsis-induced clinical signs of disease 24h (A) and 48h (B) following CLP or sham-surgery, percentage of weight loss at day 1 (C), percentage of gastric emptying (D), geometric center of GI transit (E) and colonic permeability as measured by the <i>Evans blue</i> method (F).</b> Two-way ANOVA followed by One-way ANOVA and SNK <i>post-hoc</i> testing when appropriate, or its non-parametric equivalent for ordinal data; n = 7–10/group for A, B and C; n = 9–12/group for D; *p ≤ 0.05, ***p ≤ 0/001, # significant effect of CLP, § significant effect of anti-IL-6. CDS: clinical disease score; CLP: caecal ligation and puncture; GC: geometric center; %GE: percentage of gastric emptying; GI: gastrointestinal.</p

Kaplan-Meier survival analysis.

<p>Kaplan-Meier curve displaying survival of sham and CLP-mice treated with anti-IL-6 antibodies or IgG isotype control up until 48h following the CLP-procedure. Log rank test p = 0.011.</p

Cytokine levels in serum (A) and supernatants of homogenized colons (B) measured by CBA, and determined by RT-PCR in colon (C) during the <i>curative</i> set-up (administration of anti-IL-6 antibodies 24h following the CLP- or sham-procedure).

<p>Cytokine levels in serum (A) and supernatants of homogenized colons (B) measured by CBA, and determined by RT-PCR in colon (C) during the <u><i>curative</i></u> set-up (administration of anti-IL-6 antibodies 24h following the CLP- or sham-procedure).</p

Box-whisker plot of biofilm formation by MRSA clones in the static assay.

<p>Boxes depict the 95% CI, black horizontal lines the average OD492 value, and the whiskers the OD492 value range (lowest and highest values) for each clone.</p

Cytokine levels in serum (A) and supernatants of homogenized colons (B) measured by CBA or ELISA (IL-1α), and determined by RT-PCR in colon (C) during the <i>preventive</i> set-up (administration of anti-IL-6 antibodies simultaneously with the CLP- or sham-procedure).

<p>Cytokine levels in serum (A) and supernatants of homogenized colons (B) measured by CBA or ELISA (IL-1α), and determined by RT-PCR in colon (C) during the <u><i>preventive</i></u> set-up (administration of anti-IL-6 antibodies simultaneously with the CLP- or sham-procedure).</p