Role of Ionic Strength in Staphylococcal Cell Aggregation.

Cell aggregation plays a key role in biofilm formation and pathogenesis of Staphylococcus species. Although the molecular basis of aggregation in Staphylococci has already been extensively investigated, the influence of environmental factors, such as ionic strength, remains poorly understood. In this paper, we report a new type of cellular aggregation of Staphylococci that depends solely on ionic strength. Seven strains out of 14, all belonging to staphylococcal species, formed large cell clusters within minutes in buffers of ionic strength ranging from 1.5 to 50 mM, whereas isolates belonging to other Gram-positive species did not display this phenotype. Atomic force microscopy (AFM) with chemically functionalized tips provided direct evidence that ionic strength modulates cell surface adhesive properties through changes in cell surface charge. The optimal ionic strength for aggregation was found to be strain dependent, but in all cases, bacterial aggregates formed at an ionic strength of 1.5-50 mM were rapidly dispersed in a solution of higher ionic strength, indicating a reversibility of the cell aggregation process. These findings suggest that some staphylococcal isolates can respond to ionic strength as an external stimulus to trigger rapid cell aggregation in a way that has not yet been reported.

Staphylococcus epidermidis Affinity for Fibrinogen-Coated Surfaces Correlates with the Abundance of the SdrG Adhesin on the Cell Surface.

Staphylococcus epidermidis is a world-leading pathogen in healthcare facilities, mainly causing medical device-associated infections. These nosocomial diseases often result in complications such as bacteremia, fibrosis, or peritonitis. The virulence of S. epidermidis relies on its ability to colonize surfaces and develop thereupon in the form of biofilms. Bacterial adherence on biomaterials, usually covered with plasma proteins after implantation, is a critical step leading to biofilm infections. The cell surface protein SdrG mediates adhesion of S. epidermidis to fibrinogen (Fg) through a specific "dock, lock, and latch" mechanism, which results in greatly stabilized protein-ligand complexes. Here, we combine single-molecule, single-cell, and whole population assays to investigate the extent to which the surface density of SdrG determines the ability of S. epidermidis clinical strains HB, ATCC 35984, and ATCC 12228 to bind to Fg-coated surfaces. Strains that showed enhanced adhesion on Fg-coated polydimethylsiloxane (PDMS) were characterized by increased amounts of SdrG proteins on the cell surface, as observed by single-molecule analysis. Consistent with previous reports showing increased expression of SdrG following in vivo exposure, this work provides direct evidence that abundance of SdrG on the cell surface of S. epidermidis strains dramatically improves their ability to bind to Fg-coated implanted medical devices.

Forces driving the attachment of Staphylococcus epidermidis to fibrinogen-coated surfaces.

Cell surface proteins of bacteria play essential roles in mediating the attachment of pathogens to host tissues and, therefore, represent key targets for anti-adhesion therapy. In the opportunistic pathogen Staphylococcus epidermidis , the adhesion protein SdrG mediates attachment of bacteria to the blood plasma protein fibrinogen (Fg) through a binding mechanism that is not yet fully understood. We report the direct measurement of the forces driving the adhesion of S. epidermidis to Fg-coated substrates using single-cell force spectroscopy. We found that the S. epidermidis -Fg adhesion force is of ~150 pN magnitude and that the adhesion strength and adhesion probability strongly increase with the interaction time, suggesting that the adhesion process involves time-dependent conformational changes. Control experiments with mutant bacteria lacking SdrG and substrates coated with the Fg ß(6-20) peptide, instead of the full Fg protein, demonstrate that these force signatures originate from the rupture of specific bonds between SdrG and its peptide ligand. Collectively, our results are consistent with a dynamic, multi-step ligand-binding mechanism called "dock, lock, and latch".

On-site comparison of an enzymatic detergent and a non-enzymatic detergent-disinfectant for routine manual cleaning of flexible endoscopes.

Background and study aims Flexible endoscopes are potential vectors of pathogen transmission to patients that are subjected to cleaning and high-level disinfection after each procedure. Efficient manual cleaning is a prerequisite for effective high-level disinfection. The goal of this study was to demonstrate the impact of the cleaning chemistry in the outcome of the manual cleaning of endoscopes. Materials and methods Twelve endoscopes were included in this study: four colonoscopes, four gastroscopes, two duodenoscopes and two bronchoscopes. This study was designed with two phases; in each of them, the manual cleaning procedure remained identical, but a different detergent was used: a non-enzymatic detergent-disinfectant (NEDD) and an enzymatic detergent (ED). Biopsy and suction channels of endoscopes were sampled using 10 mL of physiological saline at two points: before and after manual cleaning, and adenosine triphosphate (ATP) was measured on each sample. In total, 208 procedures were analyzed for the NEDD phase and 253 for the ED phase. Results For each endoscope type, cleaning endoscopes with ED resulted in larger median decrease in ATP than with NEDD: respectively 99.43 % and 95.95 % for bronchoscopes ( P  = 0.0007), 99.28 % and 96.93 % for colonoscopes ( P  < 0.0001) and 98.36 % and 95.36 % for gastroscopes ( P  < 0.0001). In addition, acceptability rates of endoscopes based on defined post-manual cleaning ATP thresholds (200, 150, 100 or 50 relative light units) for all endoscope types were significantly higher with ED compared to NEDD. Conclusions With all other parameters of manual cleaning remaining unchanged, the enzymatic chemistry of ED provided more consistent and improved cleaning of endoscopes compared to NEDD. Therefore, choice of the detergent for endoscope cleaning has an impact on the outcome of this process.

Synergistic Removal of Static and Dynamic Staphylococcus aureus Biofilms by Combined Treatment with a Bacteriophage Endolysin and a Polysaccharide Depolymerase.

Staphylococcus aureus is an important pathogen and biofilm former. Biofilms cause problems in clinics and food production and are highly recalcitrant to antibiotics and sanitizers. Bacteriophage endolysins kill bacteria by degrading their cell wall and are therefore deemed promising antimicrobials and anti-biofilm agents. Depolymerases targeting polysaccharides in the extracellular matrix have been suggested as parts of a multi-enzyme approach to eradicate biofilms. The efficacy of endolysins and depolymerases against S. aureus biofilms in static models has been demonstrated. However, there is a lack of studies evaluating their activity against biofilms grown under more realistic conditions. Here, we investigated the efficacy of the endolysin LysK and the poly-N-acetylglucosamine depolymerase DA7 against staphylococcal biofilms in static and dynamic (flow cell-based) models. LysK showed activity against multiple S. aureus strains, and both LysK and DA7 removed static and dynamic biofilms from polystyrene and glass surfaces at low micromolar and nanomolar concentrations, respectively. When combined, the enzymes acted synergistically, as demonstrated by crystal violet staining of static biofilms, significantly reducing viable cell counts compared to individual enzyme treatment in the dynamic model, and confocal laser scanning microscopy. Overall, our results suggest that LysK and DA7 are potent anti-biofilm agents, alone and in combination.

Surveillance of Endoscopes: Comparison of Different Sampling Techniques.

OBJECTIVE To compare different techniques of endoscope sampling to assess residual bacterial contamination. DESIGN Diagnostic study. SETTING The endoscopy unit of an 1,100-bed university hospital performing ~13,000 endoscopic procedures annually. METHODS In total, 4 sampling techniques, combining flushing fluid with or without a commercial endoscope brush, were compared in an endoscope model. Based on these results, sterile physiological saline flushing with or without PULL THRU brush was selected for evaluation on 40 flexible endoscopes by adenosine triphosphate (ATP) measurement and bacterial culture. Acceptance criteria from the French National guideline (<25 colony-forming units [CFU] per endoscope and absence of indicator microorganisms) were used as part of the evaluation. RESULTS On biofilm-coated PTFE tubes, physiological saline in combination with a PULL THRU brush generated higher mean ATP values (2,579 relative light units [RLU]) compared with saline alone (1,436 RLU; P=.047). In the endoscope samples, culture yield using saline plus the PULL THRU (mean, 43 CFU; range, 1-400 CFU) was significantly higher than that of saline alone (mean, 17 CFU; range, 0-500 CFU; P<.001). In samples obtained using the saline+PULL THRU brush method, ATP values of samples classified as unacceptable were significantly higher than those of samples classified as acceptable (P=.001). CONCLUSION Physiological saline flushing combined with PULL THRU brush to sample endoscopes generated higher ATP values and increased the yield of microbial surveillance culture. Consequently, the acceptance rate of endoscopes based on a defined CFU limit was significantly lower when the saline+PULL THRU method was used instead of saline alone. Infect Control Hosp Epidemiol 2017;38:1062-1069.