Camhb

Prior to experiments, A. baumannii AB090342 was subcultured on nutrient agar plates and incubated for 16–18 h at 37°C. A single colony was then inoculated into 10 mL of cation-adjusted Mueller-Hinton broth (CaMHB, Oxoid) and incubated in a shaking water bath at 180 rpm and 37°C for 18 h. The overnight culture was then diluted by 1:100 into four different reservoirs with 100 mL fresh CaMHB and grown to an optical density at 600 nm (OD₆₀₀) of 0.50 ± 0.02 to achieve the starting inoculum at ∼10⁸ cfu/mL at early logarithmic growth phase. The bacterial culture was treated with colistin (1 mg/L), aztreonam (128 mg/L), and the combination of colistin and aztreonam (1 mg/L and 128 mg/L, respectively) for 1, 4 and 24 h; concentrations of colistin and aztreonam were chosen based on their MICs, pharmacokinetics in patients, and in vitro static time-kill results to ensure sufficient bacterial cells for the metabolomics study. The untreated bacterial culture was served as the control and five biological replicates were prepared independently from different colonies of AB090342 on different days.

12-bis-THA was tested to determine their Minimum Inhibitory Concentration (MIC) against B. thailandensis E264 and B. thailandensis E555 using the broth microdilution method in accordance with CLSI guidelines. Bacterial colonies were isolated from a Luria-Bertani agar plate and suspended in 1X phosphate buffered saline (PBS) to a turbidity equivalent to a 0.5 McFarland standard. The bacterial suspension was then diluted 1:500 into 2X Cation Adjusted Muller-Hinton Broth (CAMHB; Fisher Scientific, United Kingdom) and vortexed to disaggregate bacterial clumps. 100 μl of the bacterial culture was transferred to the wells of a 96-well plate containing the different treatments. Treatments were prepared by serially diluting compounds 2-fold in sterile water in a 96-well plate to produce a final volume of 100 μl (total 200 μl for treatments and bacterial culture). Plates were incubated at 37°C with agitation for 18 h and the MIC was defined as the lowest concentration that caused an absence of visible growth.

E. coli CDC-0001 (BioSample accession number SAMN04014842) was used in this study. Unless otherwise stated, bacterial cultures were grown at 37°C in cation-adjusted Mueller-Hinton broth (CAMHB) (Fisher Scientific) in the presence or absence (negative controls) of taniborbactam (Venatorx Pharmaceuticals, Inc.) or cefepime hydrochloride (USP).

The minimal inhibitory concentrations (MICs) of SQQ30 peptide and TRS21 against the strains were determined according to Heydari, M., et al., 2018 [47 (link)]; 5 × 10⁵ CFU/mL of each bacteria strain was added to 95 µL of Mueller–Hinton broth (CAM-HB; Fisher scientific, Segrate, Italy) that was supplemented or not with SQQ30 (0.1–50 µM) and TRS21 (0.5–200 µM).
The positive control was represented by gentamicin for all bacterial strains.

E. coli CDC-0001 (BioSample accession number SAMN04014842) was used in this study. Unless otherwise stated, bacterial cultures were grown at 37°C in cation-adjusted Mueller-Hinton broth (CAMHB) (Fisher Scientific) in the presence or absence (negative controls) of taniborbactam (Venatorx Pharmaceuticals, Inc.) or cefepime hydrochloride (USP).

TD-SCV and revertant isolates, as defined by the phenotypic methods mentioned above, were tested for susceptibility to ciprofloxacin, clindamycin, erythromycin, oxacillin, trimethoprim-sulfamethoxazole, and vancomycin by broth dilution using brain heart infusion broth (BHI; Oxoid, Thermo Fisher Scientific) supplemented with thymidine (100 μg/ml) (23 (link), 34 (link)). S. aureus ATCC 29213 was used as a control strain; test was performed using cation-adjusted Muller-Hinton broth (CAMHB; Oxoid, Thermo Fisher Scientific). To evaluate the interference of thymidine (used to supplement the medium for susceptibility testing and the possible effect on TMP-SXT results) on bacterial growth, quality control was also performed with Enterococcus faecalis ATCC 29212 (Table 1). The double-disk diffusion method with clindamycin and erythromycin disks was performed to determine MLS_B resistance phenotypes, using MHA supplemented with thymidine (100 μg/ml). Test results were interpreted according to the CLSI standards (CLSI M100S-ED26:2016; https://clsi.org).
The mecA gene (encoding oxacillin [OXA] resistance determinant) and the ermA, ermB, ermC, msrA, and msrB genes (encoding MLS_B resistance determinants) were detected via PCR using specific primers and conditions previously described (Table 3).

The antimicrobial components ciprofloxacin (CIP, PHR1044) and benzalkonium chloride (BKC, 12060) were obtained from Sigma Aldrich (Saint Louis, MO, USA). Stock concentrations of 10 mg/mL CIP were prepared in distilled water, filtered through a 0.2 μm filter and aliquots were stored at − 20 °C. Working concentrations of CIP were based on the epidemiologic cutoff (ECOFF) value of E. coli defined by EUCAST. CIP concentrations of 0.064 mg/L (1 x ECOFF), 0.640 mg/L (10 x ECOFF) and 6.400 mg/L (100 x ECOFF) were diluted in cation-adjusted Mueller-Hinton broth with TES buffer (CAMHB, Thermo Scientific, YT3462).