To ensure practical relevance, clinical MRSA isolates, as well as American Type Culture Collection (Manassas, VA, USA; ATCC®) control strains, were tested in this study. Clinical isolates were recovered as follows: screening swab samples were inoculated on Columbia 5% sheep blood agar plate (BD Diagnostics, Sparks, USA) and chromogenic plates for MRSA detection (ChromAgar MRSA II, BD) and incubated under aerobic conditions for 48 h at 36 °C. If growth on chromogenic plates was detected, identification by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Bremen, Germany) was performed . Agglutination with Pastorex® StaphPlus (Alere, Jena, Germany) was performed to confirm S. aureus growth. Susceptibility testing was performed by VITEK2 (bioMérieux) and results were interpreted according to EUCAST breakpoints. Biofilm-forming capacities of each isolate were determined by the crystal violet staining technique (data not shown). Six representative isolates with significant biofilm-forming capacity compared to the standard disinfectant efficacy test isolate ATCC® 6538™, according to ATCC® product sheet, were selected and used for further testing.
Preparation of antimicrobials and neutralizer
For standardization of experimental conditions, in each experiment, water of standardized hardness (WSH) was prepared with a total hardness of 300 ppm (CaCO3) with 0.119 g/l magnesium chloride (Carl Roth, Karlsruhe, Germany), 0.277 g/l calcium chloride (Carl Roth), and 0.28 g/l sodium hydrogen carbonate (Carl Roth). WSH at a pH of 7.0 ± 0.2 at 25 °C was used as a diluent. Tryptic soy broth (TSB) containing lecithin, Tween 80, histidine, and sodium thiosulfate neutralizing agent (all from Merck Millipore, Darmstadt, Germany) (LTHTh) was used in both treatment and control groups, immediately following disinfection according to the manufacturer’s instructions. The media and neutralizers used in this study were approved for effective neutralization of the applied disinfectants prior to the experiments (data not shown).
Biofilm viability assay
The MRSA isolates were cultured on Columbia blood agar plates at 37 °C for 12 h. Bacterial killing in biofilms was determined as reduction [%] in metabolic activity using a kinetic biofilm viability assay as previously described . Briefly, the test substances were diluted in WSH at 25 °C at the standard concentration as well as half the standard concentration to demonstrate the dilution effects in a practical setting. For testing of antimicrobial effects on the bacterial metabolic activity in biofilms, the antimicrobial substances were diluted in WSH at standard working concentrations 0.05% and 0.1% (w/v) for OCT (TCI, Eschborn, Germany), 1% and 2% (w/v) for CHG (Sigma Aldrich, Taufkirchen, Germany), 0.02% and 0.04% (w/v) for POL (Fagron, Barsbuettel, Germany), 0.12% and 0.24% (w/v) for CLO (Sigma Aldrich), and 1% and 2% (w/v) for MUP (Fagron). Each preparation was applied to the prepared biofilms for different exposure times of 15 s, 1, 3, 5, 10 and 20 min at 37°Cfor the OCT, CHG, POL, and CLO solutions. Prolonged exposure times of up to 3.5 h were used for MUP to analyze the different mode of action of this substance. To take into account the different mode of action of MUP compared to disinfectants, extended exposure times of up to 3.5 h were used for this substance adapted to simplified testing protocols for determination of bactericidal activity on Staphylococcus aureus isolates as previously described . After exposure, the remaining metabolic activity in the biofilm was measured. Biofilms exposed to WSH alone (without supplements) served as a control for 100% viability or 0% inhibition. The bacterial killing by the disinfectants in the biofilms was determined as the reduction [%] in metabolic activity as compared to the untreated controls.
Live/dead staining of biofilms
To analyze the killing effects on the bacterial cells in biofilms, biofilms were cultured on glass coverslips (Carl Roth). After incubation, biofilms were washed twice in a 0.9% NaCl solution. Then, 100 μL M63 minimal medium consisting of 0.015 M ammonium hydrogen sulfate (Carl Roth), 0.1 M potassium dihydrogen sulfate (Sigma-Aldrich, Steinheim, Germany), 1.8 μM iron sulfate heptahydrate (Carl Roth), 1 mM magnesium sulfate heptahydrate, 2 ml/L glycerol (VWR Chemicals, Darmstadt, Germany) and 1 g/L casein hydrolysate standard (Carl Roth)containing disinfectants in different concentrations was added, and the biofilms were incubated for up to 2 h at 37 °C. After incubation, biofilms were washed twice in 0.9% NaCl solution and stained using the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Leiden, Netherlands) according to the manufacturer’s instructions. Stained biofilms were analyzed after mounting on object slides by using a BZ 8100 fluorescence microscope (Keyence, Neu-Isenburg, Germany) in the green fluorescent band for Syto 9 staining of dead and living bacterial cells, and in red fluorescent band for selective propidium iodide staining of dead cells or cells with disturbed cell integrity; therefore, yellow color in the overlay image is indicative of dead bacteria in the biofilm.
Determination of bactericidal activity
For determination of bactericidal effects, the antimicrobial substances were diluted in WSH at standard working concentrations 0.1% (w/v) for OCT, 2% for CHG, 0.04% (w/v) for POL, 0.24% (w/v) for CLO, and 2% (w/v) for MUP, and at least 1x109 bacterial cells were added for different exposure times. Each sample was tested for the surviving bacterial count by membrane filtration of the disinfectant solution through a 0.45-μm nitrocellulose membrane (Sartorius Stedim, Göttingen, Germany) followed by rinsing three times using 100 ml of a NaCl-peptone solution (Becton Dickinson, Heidelberg, Germany) for removal of remaining disinfectant. Afterwards, the filters were transferred to Caso Agar containing LTHTh as a neutralizer (Merck Millipore, Darmstadt, Germany). The neutralization and washing steps were validated for effective neutralization of the tested disinfectants prior to this study (data not shown). The media were incubated for 48 h at 37 °C and then checked for microbial growth. Colony forming units (CFUs) were counted and the Log10 reduction factor (LRF10) was calculated compared to the untreated controls as a measure of the bactericidal effect.
For descriptive purposes, arithmetic mean value, standard deviation, median, interquartile range, and cumulative frequencies were calculated as appropriate. P values of ≤ .05 were considered statistically significant. Statistical analysis was performed using the SPSS ver. 21.0 statistical package (SPSS, Chicago, IL).