Tryptone soy agar tsa

Each swab was cultured in Buffered Peptone Water (BPW) (Oxoid Ltd., Basingstoke, UK) at 37 °C for 24 h. The culture was then transferred onto Kanamycin Aesculin Azide Agar (KAAA) plates and aerobically incubated at 42 °C for 24 h. A single isolated colony from each sample was subcultured on Tryptone Soy Agar (TSA) (Oxoid Ltd.) to obtain pure cultures and further processed. The isolates were subjected to species identification using the API 20Strep^® (Biomerieux, Marcy l’Etoile, France) in strict accordance with the manufacturer’s instructions. Isolated strains were stored in Brain–Heart Infusion (BHI) broth (Oxoid Ltd.), with the addition of 30% glycerol as a cryoprotectant, at −80 °C.

The reference laboratory strains Klebsiella pneumoniae (ATCC BAA-1706), P. aeruginosa (ATCC 27853), S. aureus (ATCC 33591), and S. epidermidis (ATCC 35984) were used for the study. For the preparation of stock cultures, bacterial strains were grown in Tryptone Soy Broth (TSB) (Oxoid, Basingstoke, UK) until mid-log phase, subdivided in aliquots and stored at −80°C. For the colony-forming units (CFU) count, serially diluted bacterial suspensions were plated on Tryptone Soy Agar (TSA) (Oxoid) and incubated for 24 h at 37°C.

The following bacterial species/strains were used for the study: Staphylococcus aureus (ATCC 33591), Staphylococcus epidermidis (ATCC 35984), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC BAA-1706) and the clinical isolate Enterococcus faecium VanR 1. For liquid culture, bacteria were grown in Luria Bertani broth (LB), in Mueller Hinton Broth (MHB) or in Tryptone Soy Broth (TSB) (Oxoid, Basingstoke, UK) at 37 °C with shaking depending on the type of experiment. Enumeration of colony-forming units (CFU) was performed by serially diluting bacterial suspensions and plating them on Tryptone Soy Agar (TSA) (Oxoid). After an incubation of 24 h at 37 °C, CFU were counted.

P. aeruginosa strains were isolated from sputum of CF patients at the Microbiology Unit of the University Hospital of Pisa, Italy. MALDI-TOF (Bruker Daltonics, Bremen, Germany) was used for the identification of the bacterial species and VITEK^® 2 system (bioMérieux, Bagno a Ripoli, Italy) for the definition of the antimicrobial susceptibility profile, according to The European Committee on Antimicrobial Susceptibility Testing (EUCAST Clinical Breakpoints http://http://www.eucast.org/clinical_breakpoints/). Five clinical isolates of P. aeruginosa (PaNM01, PaNM02, PaM01, PaM02, and PaM03) and the reference strain P. aeruginosa ATCC 27853 were used in the study. P. aeruginosa strains were qualitatively evaluated regarding their ability to express a mucoid phenotype. To this aim, bacterial suspensions were streaked onto the surface of MacConkey agar (bioMérieux, Marcy-l′Étoile, France) and Cetrimide agar (Sigma-Aldrich, St. Louis, MO, USA) plates and incubated at 37 °C for 48 h. The development of colonies with a slimy phenotype was rated as positive for the presence of mucoid, alginate-producing cells. The strains were grown with shaking in TSB (Oxoid, Basingstoke, UK) or in Luria Bertani broth (LB) at 37 °C for liquid cultures and on Tryptone soy agar (TSA) (Oxoid, Hampshire, UK) for 48 h at 37 °C for CFU determination.

Six Escherichia coli (E. coli) strains were used in this study, i.e. E. coli ATCC 25922, ATCC 4157, K12, LMC500, MC4100 and 506 (O78K80), of which the latter was used as the reference strain in all experiments31 (link)32 (link). All bacterial strains were grown in Tryptone Soy Broth (TSB; Oxoid Limited, Hampshire, UK) and on Tryptone Soy Agar (TSA; Oxoid Ltd). For all experiments bacteria were inoculated and grown overnight in TSB at 37 °C. The next day bacteria were transferred to a fresh TSB tube and grown to mid-logarithmic phase.

The reference strains Staphylococcus epidermidis ATCC 35984 and Pseudomonas aeruginosa ATCC 27853 were used for the study. For the preparation of stock cultures, bacterial strains were grown in Muller Hinton broth (MHB) or Tryptone Soy Broth (TSB) (Oxoid, Basingstoke, Hampshire RG24 8PW, UK) until mid-log phase, subdivided in aliquots and kept frozen at −80 °C until use. For colony forming unit (CFU) count, serially diluted bacterial suspensions were plated in duplicate on Tryptone Soy agar (TSA)(Oxoid) and incubated at 37 °C for 24 h.