Brain heart infusion broth

At the time of sampling, the species of the potential contaminants were unknown. Therefore, a general medium was used to culture the bacteria, optimized for the detection of GMM (the initial goal of the sampling), as described elsewhere (Fraiture et al., 2021 (link)). For each sample, 1 g of the food/feed matrix was mixed with 250 ml of Brain-Heart Infusion broth (Sigma-Aldrich, Missouri, USA) for overnight aerobic incubation at 30°C (Fraiture et al., 2021 (link)). A total of 100 μl of the culture was then plated on nutrient agar (Sigma-Aldrich, Missouri, USA) for overnight aerobic incubation at 30°C. In case bacterial growth was observed, two bacterial isolates were randomly selected (not considering morphology) and subcultured for subsequent identification and characterization with whole-genome sequencing.

Four FE products in solid form were collected from the market (Table 1). One gram of each batch tested from each FE product was added to 250 mL of Brain–Heart Infusion broth (Sigma-Aldrich) in the presence of Kanamycin (50 µg/mL; Sigma-Aldrich) for incubation overnight at 30 °C. An amount of 100 μL of the culture was plated on nutrient agar (Sigma-Aldrich) in the presence of Kanamycin (50 µg/mL; Sigma-Aldrich) for incubation overnight at 30 °C. Based on real-time PCR analysis (see Section 2.2), isolates of the GM B. velezensis producing protease were selected for genomic analysis. For each batch tested from each FE product, two isolates were selected, to investigate the presence of potentially different strains, as well as to serve as a back-up against potential loss of plasmids from the isolates.

The flagellin proteins were isolated as described previously [9 (link)] with a few modifications. In brief, S. Enteritidis stock culture was inoculated into Brain Heart Infusion Broth (Sigma-Aldrich, MO) and grown for 48 hours at 37°C without shaking. The bacterium was washed two times with PBS (pH 7.4) by centrifugation at 4,000 ×g for 40 minutes. The bacterial cell pellet was treated with 3M Potassium Thiocyanate in PBS for 2 hours at room temperature with magnetic stirring. The cell suspension was ultracentrifuged at 35,000 ×g for 30 minutes. The supernatant containing the flagellin protein-enriched extract was dialyzed against PBS (pH 7.4) followed by dialysis in Milli-Q water. The protein concentration was assessed and freeze-dried using 5% sucrose as a cryoprotectant.

Bacteria were grown in brain-heart infusion broth (Sigma Aldrich Milan, Italy). When bacteria were in the log phase of growth, the suspension was centrifuged at 1000 x g for 15 min, the supernatant discarded, and the bacteria were resuspended and diluted into sterile saline to achieve a concentration of approximately 5x10⁷ CFU/ml.

All bacterial stains used were obtained from ATCC. Methicillin-resistant S. aureus strain USA300 (ATCC®-BA1717™), P. aeruginosa (ATCC®-27107™), S. pneumoniae (ATCC®-6303™), and S. typhimurium (ATCC®-14028™). S. aureus and P. aeruginosa were cultured overnight in tryptic soy broths, diluted at 1:100 and grown exponentially. In general, OD_A600 = 1 corresponded to 1.5 × 10⁹ CFU per ml for S. aureus and 7.5 × 10⁸ CFU per ml for P. aeruginosa. Bacteria were used at their exponential growth phase (OD_A600 = ~0.6). S. pneumoniae and S. typhimurium were grown in Brain-Heart infusion broth (Sigma, 53286).

Fourth instar SENN and SENN-DDT larvae as well as three-day old non-blood fed adults were dissected in sterile phosphate buffered saline to remove the midgut. The midguts of 25 mosquitoes of each strain were inoculated into Brain Heart Infusion broth (Sigma Aldrich: 53286) and incubated for 16 hours at 37 °C. After the incubation, 20 µl of the inoculum was streaked onto either McConkey agar, blood agar or chocolate agar and was incubated at 37 °C for 16 hours. Individual colonies were selected for Matrix-assisted laser desorption time of flight (MALDI TOF). Individual colonies were selected for MALDI TOF mass spectrometry analysis based on colony morphology. Specimens were analysed with a MALDI Biotyper System with a benchtop microflex LT/SH mass spectrometer. Results were analysed using MALDI biotyper 4.0 system software^{29 (link)}.

To show that live bacterial challenge causes the release of EVs, mice were infected with Haemophilus influenzae (H. influenzae). Briefly, Hib Eagan strain was a kind gift from P. Langford (Faculty of Medicine, St Mary's Hospital, Imperial College London). Bacteria were cultured at 37°C in 5% CO₂ in Brain heart Infusion broth (OXOID) supplemented with 10 µg/ml of both Hemin (10ug/ml) and Nictinamide adenine dinucleotide (NAD) (Sigma-Aldrich, UK) or on BHI agar (OXOID) supplemented with 4% Levinthals when agar was <50°C. Levinthals was made by adding 50% horse blood (TCS Biosciences) to BHI broth and heating to 70°C for 45 minutes. On cooling to 50°C, 0.7 mg/ml NAD was added and the supernatant stored at –80°C. Bacteria were cultured to an OD600 of 0.3 (approximately 1×10⁹ CFU/ml) and stored at −80°C in 10% glycerol as single use aliquots.
Groups of mice (n = 4–5 per group) were infected i.n. with 1×10⁷ colony forming units of H. influenzae serotype b (strain Eagan) in sterile phosphate buffered saline (PBS). Terminal anaesthesia was induced at 6, 24 and 72 hours after challenge. Mice were culled and BALF samples were collected at 6, 24 and 48 hours. Presence of EVs was assessed as detailed above.