Microbank

The following strains were used in this study: Staphylococcus aureus ATCC 6538, biofilm-producing reference strain [58 (link)], S. aureus ATCC 43300 (Methicillin resistant S. aureus-MRSA), Pseudomonas aeruginosa ATCC 9027, P. aeruginosa DSM 102273 (multidrug resistant). The strains were stored in the private collection of the Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina (Messina, Italy). They were stored at −70 °C in Microbanks™ (Pro-lab Diagnostics, Neston, UK).

The toothbrush’s head was detached and underwent five cycles of sonication (30 sec each) in 5 ml BHI (Brain Heart Infusion) medium before incubation for 24 h at 37°C with shaking. Saliva and sputum samples were treated with 0.5–1 ml Sputolysin (Calbiochem) and incubated for 40 min at 37°C with shaking. Each sample (100 µl) was plated on Columbia blood (5% sheep blood), McConkey, Mannitol Salt and Sabouraud agar plates (BD Difco), and incubated at 37°C for 48 h. All plates were kept at RT for five additional days to recover small variant colonies and slow-growing strains. Isolated colonies were identified using MALDI-TOF-MS [24] and/or Vitek2 (Biomerieux). Isolates were stored in Microbanks (ProLab Diagnostics) at – 80°C.

Isolation of Campylobacter spp. from meat and milk was conducted in compliance with the EN ISO 10272-1:2006 method with slight modification. Briefly, meat and cheese (25 g) were homogenized for 2 min in a stomacher with 225 mL buffered peptone water (Oxoid Limited, Basingstoke, United Kingdom) in the sterile plastic bags. Then, 10 mL of the homogenate solution and 10 ml of raw milk was added to 90 mL of Bolton broth containing the Bolton broth selective supplement and 5% laked horse blood (Oxoid Limited, Basingstoke, United Kingdom) and incubated at 42 °C for 48 h under microaerobic conditions (Generbox microaer-BioMerieux, Marcy l’Etoile, France). Next, the bacterial suspension was spread onto CCDA plates (Oxoid Limited, Basingstoke, United Kingdom) and then incubated at 42 °C for 48 h under microaerobic conditions. Characteristic growth from the CCDA plates was transferred to a blood plate (Columbia agar containing 5% cattle blood, Oxoid Limited, Basingstoke, United Kingdom) and incubated overnight at 42 °C. Colonies suspected as Campylobacter spp. were examined for cell morphology, motility, and oxidase reactions. Putative Campylobacter colonies were frozen at −80 in Microbanks (Pro-Lab Diagnostics, Birkenhead, United Kingdom) until species differentiation using polymerase chain reaction (PCR).