Bacterial strains and biofilm growth conditions
Strains used in this study included S. aureus isolates ATCC 29213, ATCC 25923, 209P, TT-UA-1, MK99–1, MK99–2, MK99–3, MK99–4, MK99–5, MK99–6, MK99–7, and MK99–8; and P. aeruginosa isolates ATCC 33348, ATCC 27853, E7, 16–45, 10–49, 14–57, 27–08, 27–122, 31–56, 4–37, 4–6, and 1–95. Prior to use, strains were stored at −80 °C. All strains were first subcultured and then diluted to 103 colony-forming units (CFU)/mL in Mueller-Hinton broth (MHB; Difco, BD Biosciences, San Jose, CA, USA) for subsequent cultivation with catheter tubes at 35 °C in a horizontal shaking incubator. Each polyvinyl chloride catheter tube (diameter, 5 mm; Terumo, Tokyo, Japan) was cut into 10-mm-long round slices and sterilized prior to biofilm growth. Mature biofilms were obtained after 7 days of incubation (data not shown) and initial inoculation of 1.5 × 108 CFU to 5.0 × 108 CFU per tube.
Chemical disinfectant preparation
Each disinfectant was diluted with sterile water according to manufacturers’ instructions: 0.3% PAA (6% Acecide; Saraya Co., Ltd., Osaka, Japan), 0.55% OPA (undiluted Disopa; Johnson & Johnson, Pittsburgh, PA, USA), 2.0% GA (20% Sterihyde; Maruishi Pharmaceutical Co., Ltd., Osaka, Japan) for endoscopy HLD, or 0.1% sodium hypochlorite solution (NaClO; 1% Yakulax; Yakuhan Pharmaceutical Co., Ltd., Hokkaido, Japan); chlorine levels that are safe for environmental disinfection at comparable standard formulations were used. Sterile distilled water (SDW) was used as a positive control for diluted disinfectant testing.
In vitro biofilm models
Prior to disinfection, laboratory-grown mature biofilms accumulated in plastic tubing were transferred to 5-mL sterile test tubes, followed by transfer of 2 mL of disinfectant into each test tube for gentle mixing. Test samples were exposed for 1, 2, 5, 10, 15, 30, and 60 min; immediately after treatment, biofilms were carefully rinsed twice with phosphate-buffered saline (PBS; pH 7.4) to inactivate the drug and remove floating bacteria. The bactericidal effects were assessed after incubating each tubing sample at 35 °C for 2 days and visually determining the presence of a bacterial suspension. Bacterial growth was confirmed by agar-plate incubation. Every experiment included a positive control biofilm culture and non-inoculated MHB as a negative control, and was performed five times.
Transmission electron microscopy (TEM)
S. aureus 209P and P. aeruginosa E7 strains were selected for microscopic observation, because they produced more biofilm matrix than other cultures in this study. As previously described, biofilms were exposed to 0.3% PAA and 2.0% GA for 5 min and 30 min. Immediately after treatment, S. aureus and P. aeruginosa biofilms were rinsed with PBS (pH 7.4) and 0.1 M cacodylic acid buffer solution (pH 7.2), respectively. Samples were fixed overnight with 2.5% electron microscope-grade GA and 0.1% uranium acetate, followed by fixation with 1.0% osmium tetroxide and 1.0% tryptophan for 5 h. Samples were then washed with 0.1% tannic acid buffer, dehydrated in a graded ethanol series, and embedded in Epon 812 resin by polymerization at 60 °C. Samples were cut with an ultra-microtome fitted with a glass knife, stained with 5.0% uranium acetate for 15 min and 0.1% lead citrate for 5 min at 20 °C, coated with carbon, and observed under a JEM-1200EX TEM (Jeol Ltd., Tokyo, Japan) at 80 kV.
Scanning electron microscopy (SEM)
S. aureus 209P and P. aeruginosa E7 biofilms were prepared following the same protocol as described for TEM observation. Samples were fixed for 2 h at room temperature, first with 2.5% electron microscope-grade GA and then with 1.0% osmium tetroxide. Samples were then washed with 0.1% tannic acid buffer, dehydrated in a graded ethanol series, and dried in a critical point dryer with isoamyl acetate and carbon dioxide. Samples were set on a stand using carbon tape coated with platinum and observed under a S-4500 SEM (Hitachi, Tokyo, Japan) at 15 kV to 20 kV (at least 10 fields/biofilm).
Statistical analysis
Complete bactericidal activity was defined as the absence of bacterial suspensions in all five experiments for each exposure time. A Student’s t test was performed to compare exposure times for bactericidal effectiveness between disinfectants and the control. P < 0.05 was considered significant, and the results were analyzed using Microsoft Excel (Microsoft, Co., Redmond, WA, USA).