The bacterial strains used in in vitro experiments included strains obtained from American Type Culture Collection (ATCC), and wild-type strains isolated from hospital surfaces. ATCC strains included S. aureus (ATCC 25923), E. coli (ATCC 25922), and P. aeruginosa (ATCC BAA-47). Hospital isolates included three strains selected for their drug resistance characteristics: S. aureus (SA2-R73), E. coli (EC-R60), and P. aeruginosa (PA-V6). Hospital isolates were collected by direct sampling of hospital surfaces with 55 mm diameter contact Rodac plates (24 cm2 surface), containing the following selective media: Baird–Parker agar (for Staphylococcus spp., cat. n. 146189), MacConkey agar (for Enterobacteriaceae spp., cat. n. 146427), and cetrimide agar (for Pseudomonas spp., cat. n. 146768) (all bacterial media were from Merck Millipore, Billerica, MA, USA). Grown colonies with morphological S. aureus, E. coli, and P. aeruginosa features were further streaked on the corresponding selective medium and incubated at 37°C for 24 hours, to isolate single pure colonies. Each individual colony was then characterized by Gram-staining, and identified by appropriate biochemical tests (API-Staph, cat. n. 20500, and API-20E, cat. n. 20100; Biomerieux, Florence, Italy).
After identification, all isolates were expanded overnight in tryptic soy broth (TSB, cat. n. 146599; Merck Millipore) at 37°C, frozen in 50% sterile glycerol, and kept at −80°C until use. Each isolate was characterized for antibiotic resistance by conventional disc-diffusion Kirby–Bauer antibiograms, using Mueller–Hinton agar plates (cat. n. 105437; Merck Millipore), testing the following antibiotics: penicillin G (cat. n. CT0043B; Oxoid, Altrincham, UK), ampicillin (Oxoid; cat. n. CT0003B; Oxoid), vancomycin (cat. n. CT0058B; Oxoid), oxacillin (cat. n. CT0040B; Oxoid), ofloxacin (cat. n. CT0446B; Oxoid), cefotaxime (cat. n. CT066B; Oxoid), cefoxitin (cat. n. CT0119B; Oxoid), gentamicin (cat. n. 9026; Liofilchem, Italy), imipenem (cat. n. CT0455B; Oxoid), aztreonam (cat. n. 9008; Liofilchem, Liofilchem, Teramo, Italy), meropenem (cat. n. 9068; Liofilchem), and colistin (cat. n. CT0017B; Oxoid). Zone inhibition diameters were interpreted according to the European Committee on Antimicrobial Susceptibility Testing breakpoint tables for interpretation of minimum inhibitory concentration (MIC) and inhibition zone diameters38 and to the Clinical and Laboratory Standards Institute manual (26th edition).39 In addition, MICs of resistant strains were also measured, accordingly to European Food Safety Authority guidelines, by using antibiotic stripes containing serial dilutions of each antibiotic (cat. n. 92003, 92033, 92006, 92066, 92141, 92009, 92054, 92085, 92099, 92015, 92102, 92057; Liofilchem).
The concentrated PCHS detergent included, as previously described,28 (link) 107/mL spores of three species of probiotics belonging to the Bacillus genus, namely Bacillus subtilis, Bacillus pumilus, and Bacillus megaterium.
After identification, all isolates were expanded overnight in tryptic soy broth (TSB, cat. n. 146599; Merck Millipore) at 37°C, frozen in 50% sterile glycerol, and kept at −80°C until use. Each isolate was characterized for antibiotic resistance by conventional disc-diffusion Kirby–Bauer antibiograms, using Mueller–Hinton agar plates (cat. n. 105437; Merck Millipore), testing the following antibiotics: penicillin G (cat. n. CT0043B; Oxoid, Altrincham, UK), ampicillin (Oxoid; cat. n. CT0003B; Oxoid), vancomycin (cat. n. CT0058B; Oxoid), oxacillin (cat. n. CT0040B; Oxoid), ofloxacin (cat. n. CT0446B; Oxoid), cefotaxime (cat. n. CT066B; Oxoid), cefoxitin (cat. n. CT0119B; Oxoid), gentamicin (cat. n. 9026; Liofilchem, Italy), imipenem (cat. n. CT0455B; Oxoid), aztreonam (cat. n. 9008; Liofilchem, Liofilchem, Teramo, Italy), meropenem (cat. n. 9068; Liofilchem), and colistin (cat. n. CT0017B; Oxoid). Zone inhibition diameters were interpreted according to the European Committee on Antimicrobial Susceptibility Testing breakpoint tables for interpretation of minimum inhibitory concentration (MIC) and inhibition zone diameters38 and to the Clinical and Laboratory Standards Institute manual (26th edition).39 In addition, MICs of resistant strains were also measured, accordingly to European Food Safety Authority guidelines, by using antibiotic stripes containing serial dilutions of each antibiotic (cat. n. 92003, 92033, 92006, 92066, 92141, 92009, 92054, 92085, 92099, 92015, 92102, 92057; Liofilchem).
The concentrated PCHS detergent included, as previously described,28 (link) 107/mL spores of three species of probiotics belonging to the Bacillus genus, namely Bacillus subtilis, Bacillus pumilus, and Bacillus megaterium.
