Mha plates

We isolated 11 Streptococcus strains from collected fecal samples and tested against 13 antimicrobials (Additional file 3: Table S2 a,b). The susceptibilities of all the isolates were determined independently against beta-lactam (Penicillin 10 μg, Ampicillin 10 μg, Ceftriaxone 5 μg); Macrolides (Erythromycin 15 μg, Azithromycin 15 μg, Clarithromycin 5 μg); Lincosamide (Clindamycin 2 μg); Quinolones (Levofloxocin 5 μg, Ofloxacin 5 μg); Ansamycin (Rifampin 5 μg); Glycopeptides (Vancomycin 30 μg); Miscellaneous (Chloramphenicol 30 μg); Carbapenems (Meropenem 10 μg) using the standard Kirby–Bauer disk diffusion method [90 (link)]. Colony suspension, equivalent to a 0.5 McFarland standard, were prepared using colonies from an overnight (18-20 h) sheep blood agar plate at 35±2 °C.Antimicrobial disks (Oxoid Ltd., Basingstoke, UK) were placed on the MHA plates (Becton Dickinson, Franklin Lakes, NJ) with 5% sheep blood and incubated aerobically at 35±2 °C for 20–24 h. The inhibition zones’ diameter surrounding the Antimicrobial disks was interpreted according to Clinical and Laboratory Standards Institute guidelines (CLSI, M100, 30th Edition, 2020). Quality control for susceptibility testing was done using S. pneumoniae ATCC® 49619.

The MPC was determined with a method described by Cai et al. [9 (link)], but with modifications. Isolates AB1-5 were grown overnight on Columbia agar (Acumedia, Lansing, MI, USA) with 5 % defibrinated horse blood (Håtuna, Uppsala, Sweden) at 36 °C. Glass tubes containing 2 mL MHB (Becton Dickinson) were inoculated with bacteria from the Columbia agar and incubated overnight at 36 °C with shaking, reaching a bacterial concentration of 10⁹ CFU/mL, determined by viable count. Aliquots of 1 mL were plated on MHA-plates (Becton Dickinson) containing colistin sulphate (Sigma Aldrich) in 2-fold dilutions (0.125–128 mg/L). The MPC was defined as the lowest concentration of colistin that inhibited all visible growth of 10⁹ CFU after 48 h of incubation [7 (link)]. Colonies were isolated from the plates with the highest and the second highest colistin concentration with growth of bacteria. MaldiTof (Bruker) was used to determine that the colonies were A. baumannii, and Etest (bioMérieux) was used to determine their susceptibility to colistin. The tests were performed in duplicates to ensure reproducibility.

In vivo experiments were performed with eight S. aureus isolates, one S. pyogenes, and one S. agalactiae isolate. Except for the S. aureus ATCC reference strain, all isolates were clinical isolates. A freezer stock of approximately 1 × 10⁹ colony forming units (CFU)/mL in MHB was prepared for all isolates. At the day of infection, log-phase cultures were made from this freezer stock in fresh Mueller–Hinton Broth (MHB) and incubated for 1 or 2 h (depending on the isolate) at 37 °C, under shaking conditions. The log-cultures were diluted with MHB (thigh) or physiological saline solution (lung) to a final inoculum of approximately 10⁸ CFU/mL for infection. The number of CFU during the experiments were counted on Mueller–Hinton II agar (MHA) plates (Becton Dickinson, Olen, Belgium). Commercially available flucloxacillin (Floxapen Aurobindo Pharma B.V., Baarn, The Netherlands) was used. Dilutions of flucloxacillin were prepared in saline one hour prior to treatment and stored at 4 °C until further use.