Atcc 12228

S. epidermidis reference isolates ATCC^® 14990 and ATCC^® 12228 (American Type Culture Collection (ATCC), Manassas, VA, USA), and clinical S. epidermidis isolates cultured from PD effluent (C015 to C019) were provided by PathWest Laboratory Medicine, Western Australia. Identities were confirmed by MALDI-TOF using a MALDI Biotyper Reference Library (Bruker Daltonics, Bremen, Germany) prior to use. Bacteria were grown on 5% sheep blood agar (BA) plates at 37°C/5% CO₂, and a single colony chosen for expansion overnight in Luria-Bertani broth (LB; LB-Miller, BD Difco^™, Cat. No. 244620) at 37°C at 200 rpm. Standardised bacterial suspensions were prepared to a density 1.0–1.5 x 10⁸ colony forming units (cfu)/mL using the approximation 0.1 OD₆₀₀ = 1 x 10⁸ cfu/mL using a spectrophotometer (NanoPhotometer^™, Implen, Munich, Germany), or to 0.5 McFarland Standard (~1.5 x 10⁸ cfu/mL) using a Sensititre^™ Nephelometer (Thermo Fisher Scientific). Viable counts were determined by serial dilution in phosphate buffered saline (PBS) and plating on BA plates.

Due to the limitation on biofilm production of laboratory strains, S. epidermidis and C. tropicalis were isolated from blood samples of patients from the King Chulalongkorn Memorial Hospital (Bangkok, Thailand) with the approval by the ethical institutional review board (610/2564), Faculty of Medicine, Chulalongkorn University according to the Declaration of Helsinki with written informed consent. Additionally, the laboratory standard for S. epidermidis (ATCC 12228; American Type Culture Collection, Manassas, VA, USA) was used in some experiments.

In this study, three bacterial strains were tested for various responses to the exposure of selected metallic nanoparticles. They included gram-negative Escherichia coli (ATCC^® 25922™) and gram-positive Bacillus cereus (ATCC^® 11778™), and Staphylococcus epidermidis (ATCC^® 12228™) strains purchased from the American Type Culture Collection (ATCC). E. coli was maintained using Bacto™ Tryptic Soy Broth (cat. 211825; pancreatic digest of casein 17.0 g L⁻¹, papaic digest of soybean 3.0 g L⁻¹, dextrose 2.5 g L⁻¹, sodium chloride 5.0 g L⁻¹, dipotassium phosphate g L⁻¹); however, B. cereus and S. epidermidis were passaged in Difco™ Nutrient Broth (cat. 234000; beef extract 3.0 g L⁻¹, peptone 5.0 g L⁻¹).
All these strains were exposed to four types of nanoparticles (NPs): Ag-NPs (cat. 576832), Cu-NPs (cat. 774081), ZnO-NPs (cat. 677450) obtained from Sigma-Aldrich company and TiO₂-NPs (cat. US1019F) acquired from US Research. The size of Ag-NPs, Cu-NPs and ZnO-NPs ranged in <100 nm, 25 nm and <50 nm, respectively, while TiO₂-NPs were 20 nm in size. All NPs were characterised by 97–99.5% purity. Before starting the actual experiment, the stock solutions of NPs in sterile Millipore Water were sonicated (Vibra-Cell™, 20 kHz) for 10–20 min to avoid their aggregation/agglomeration.

The MRSA strains (including ATCC 43,300 and USA300), MSSA strains (including ATCC 25,923 and MW2) and Staphylococcus epidermidis RP62A and ATCC 12,228 were purchased form American Type Culture Collection or BNCC BeNa Culture Collection (Beijing, China). Other type strains of Escherichia coli ATCC 25,923, Enterococcus faecalis ATCC 29,212, Klebsiella pneumoniae ATCC 700,603, Acinetobacter baumannii ATCC 19,606 and Pseudomonas aureginosa PAO1 were provided by Luo Juncai (Tiandiren Biotech, Changsha, China). Clinical strains of MRSA were isolated from the Third Xiangya Hospital of Central South University, and identified by Matrix-Assisted Laser Desorption Ionization (BD, Germany). S. aureus and S. epidermidis were grown in Tryptic Soy Broth (TSB), E. faecalis was grown in Brain Heart Infusion (BHI). Other Gram-negative strains were grown in Luria-Bertani (LB) broth. All the culture mediums were purchased form Solarbio (Beijing, China). Chemicals of AKBA and other antibiotics were purchased from the MedChem Express (New Jersey, USA) and dissolved in dimethyl sulfoxide (DMSO) or deionized water.

The strain used was S. epidermidis strain ATCC12228 obtained from the American Type Culture Collection, Manassas, VA, USA. Medium used throughout was trypticase soy yeast extract (TSY) medium consisting of (g/L): tryptone, 15; soytone, 5; NaCl, 5; yeast extract, 3; K₂HPO₄, 2.5; glucose, 2.5; and final pH 7. For semisolid plates, agar was added to TSY at 15.0 g/L. As appropriate, the antibiotic rifampicin (Rif; Sigma–Aldrich) was added to TSY at a final concentration of 5 μg/mL.

All of the bacterial strains and plasmids used in the study are shown in Table 4. The biofilm-positive strain S. epidermidis ATCC 35984 (RP62A; GenBank accession number NC_002976) and non-biofilm-forming strain ATCC 12228 (GenBank accession number NC_004461) were purchased from the American Type Culture Collection (ATCC; Manassas, VA) (50 (link)). S. epidermidis 1457 (SE1457) and S. aureus RN4220 were provided by Gao Fu from the University of Hong Kong. The S. epidermidis strains and S. aureus RN4220 were cultured in tryptic soya broth (TSB; Oxoid, Basingstoke, UK) at 37°C with shaking at 220 rpm. Glucose was added to the TSB medium at a concentration of 0.5% for the detection of biofilm formation. Electroporation was used for plasmid transformation, and B2 medium (1% casein hydrolysate, 2.5% yeast extract, 0.5% glucose, 2.5% NaCl, 0.1% K₂HPO₄, pH 7.5) was used for the recovery of bacteria. The antibiotics used in this study were purchased from Sigma Chemical Co. (Los Angeles, CA, USA) and used at concentrations of 10 mg/liter for chloramphenicol, 100 mg/liter for ampicillin, 50 ng/ml for anhydrotetracycline, and 10 mg/liter for erythromycin.