Vitek device

Microbiological assays were performed in accordance with the ISO 7932:2005 method. In brief, 10 g of each sample and 90 mL (1:10 (w/w)) of sterilized buffered peptone water (BPW; Oxoid, UK) were placed in a sterile stomacher bag and homogenized for 3 min at 230 rpm using a peristaltic homogenizer (BagMixer^®400 P, Interscience, Saint Nom la Bretèche, France). Subsequently, the homogenized samples were submitted to ten-fold serial dilutions in BPW followed by spread-plating on Mannitol Egg Yolk Polymyxin Agar (MYP; Thermo Scientific, Waltham, MA, USA) and incubated at 30 °C for 48 h. Suspected colonies were counted and randomly picked out (up to five isolates) for the evaluation of hemolytic activity on Trypticase Soy Agar 5% sheep blood (Biolife, Monza, Italy). Confirmatory biochemical assessment of the isolates was carried out by means of a Vitek device (bioMerieux, France), according to the manufacturer’s instructions. Harvested colonies were then subcultured in nutrient broth (Biolife, Monza, Italy), and incubated at 30 °C for 4 h for subsequent real-time PCR.

A swab from the middle meatus was collected guided by nasal endoscopy and was seeded on agar plates (sheep blood, MacConkey, and mannitol salt) and incubated at 37°C for 24 hours for microbial identification in the automated VITEK^® device (BioMérieux). Complementary tests were performed to characterize genus and species whenever necessary. For planktonic bacteria, the antimicrobial susceptibility profile was determined by the VITEK^® 2 card system (BioMérieux, AST-P612, AST-GN), as well as minimum inhibitory concentration (MIC) was determined by the E-test^® method (BioMérieux) for the following antibiotics: amoxicillin, amoxicillin/clavulanic acid (AMX-CLAV), clarithromycin, and levofloxacin. The determination of MIC breakpoints followed the guidelines of the Clinical Laboratory Standards Institute (CLSI) (CLSI, 2018 ). Bacterial strains from positive cultures were stocked at -70°C in tryptic soy broth with 20% glycerol until further testing involving biofilms.