Lipopolysaccharide structure modulates cationic biocide susceptibility and crystalline biofilm formation in Proteus mirabilis.

Chlorhexidine (CHD) is a cationic biocide used ubiquitously in healthcare settings. Proteus mirabilis, an important pathogen of the catheterized urinary tract, and isolates of this species are often described as "resistant" to CHD-containing products used for catheter infection control. To identify the mechanisms underlying reduced CHD susceptibility in P. mirabilis, we subjected the CHD tolerant clinical isolate RS47 to random transposon mutagenesis and screened for mutants with reduced CHD minimum inhibitory concentrations (MICs). One mutant recovered from these screens (designated RS47-2) exhibited ~ 8-fold reduction in CHD MIC. Complete genome sequencing of RS47-2 showed a single mini-Tn5 insert in the waaC gene involved in lipopolysaccharide (LPS) inner core biosynthesis. Phenotypic screening of RS47-2 revealed a significant increase in cell surface hydrophobicity and serum susceptibility compared to the wildtype, and confirmed defects in LPS production congruent with waaC inactivation. Disruption of waaC was also associated with increased susceptibility to a range of other cationic biocides but did not affect susceptibility to antibiotics tested. Complementation studies showed that repression of smvA efflux activity in RS47-2 further increased susceptibility to CHD and other cationic biocides, reducing CHD MICs to values comparable with the most CHD susceptible isolates characterized. The formation of crystalline biofilms and blockage of urethral catheters was also significantly attenuated in RS47-2. Taken together, these data show that aspects of LPS structure and upregulation of the smvA efflux system function in synergy to modulate susceptibility to CHD and other cationic biocides, and that LPS structure is also an important factor in P. mirabilis crystalline biofilm formation.

Investigating intracellular persistence of Staphylococcus aureus within a murine alveolar macrophage cell line.

OBJECTIVE: Staphylococcus aureus is a particularly difficult pathogen to eradicate from the respiratory tract. Previous studies have highlighted the intracellular capacity of S.aureus in several phagocytic and non-phagocytic cells. The aim of this study was to define S.aureus interaction within a murine alveolar macrophage cell line. METHODS: Cell line MH-S was infected with Newman strain. Molecular mechanisms involved in phagocytosis were explored. To assess whether S.aureus survives intracellularly quantitative (gentamicin protection assays and bacterial plating) and qualitative analysis (immunofluorescence microscopy) were performed. Bacterial colocalization with different markers of the endocytic pathway was examined to characterize its intracellular trafficking. RESULTS: We found that S.aureus uptake requires host actin polymerization, microtubule assembly and activation of phosphatidylinositol 3-kinase signaling. Time course experiments showed that Newman strain was able to persist within macrophages at least until 28.5 h post infection. We observed that intracellular bacteria are located inside an acidic subcellular compartment, which co-localizes with the late endosome/lysosome markers Lamp-1, Rab7 and RILP. Colocalization counts with TMR-dextran might reflect a balance between bacterial killing and intracellular survival. CONCLUSIONS: This study indicates that S.aureus persists and replicates inside murine alveolar macrophages, representing a privileged niche that can potentially offer protection from antimicrobial activity and immunological host defense mechanisms.

Visible, colorimetric dissemination between pathogenic strains of Staphylococcus aureus and Pseudomonas aeruginosa using fluorescent dye containing lipid vesicles.

This paper describes a biosensing concept for exotoxins secreted by Staphylococcus aureus and Pseudomonas aeruginosa based on the toxin mediated breakdown and subsequent fluorescent dye release from phospholipid vesicles (liposomes). The sensitivity of vesicles to toxins was tuned by altering the lipid and fatty acid composition of the membranes such that vesicles could be engineered to respond to toxins/enzymes from S. aureus only; P. aeruginosa only; and both S. aureus and P. aeruginosa. Nineteen types of vesicle were made with varying compositions of phosphocholine (PC), phosphoethanolamine (PE), cholesterol and the photo-polymerizable ampiphile 10,12-tricosadiynoic acid (TCDA). The selectivity of the vesicles was measured via a simple fluorescence "switch on" assay. Sensitivity of the vesicles to 40 clinically derived strains of S. aureus and P. aeruginosa was also demonstrated. This work suggests that this technology could be utilised in a diagnostic tool to discriminate between the species of S. aureus and P. aeruginosa in wound dressings.