Camhb

NAC stock solutions (100 mg/mL) were prepared immediately before use. NAC powder (Zambon, Bresso, Italy) was dissolved in sterile double-distilled water, pH was adjusted at 6.5–6.8 with NaOH, and the solution was filtered through a 0.22-μm membrane filter. All experiments were performed in cation-adjusted Mueller-Hinton broth (CAMHB; Becton Dickinson, Milan, Italy), starting from an appropriately concentrated medium in order to avoid broth dilution when testing high NAC concentrations.

N-acetylcysteine stock solutions (100 g/L) were prepared immediately before use, by dissolving N-acetylcysteine powder (Zambon, Bresso, Italy) in sterile double-distilled water, pH adjustment at 6.5–6.8 with NaOH, and filtering through a 0.22-μm membrane filter. All experiments were performed in CAMHB (Becton Dickinson, Milan, Italy), starting from an appropriately concentrated medium in order to avoid broth dilution when testing high N-acetylcysteine concentrations.

Columbia blood agar (COS) (bioMérieux) were used to culture the isolates. Cation-adjusted Mueller-Hinton broth (CA-MHB) (Becton, Dickinson) was used to perform the time-kill curves, and Mueller-Hinton agar (MH) plates (bioMérieux) were used for the quantification of CFU per milliliter.
The antibiotics used were meropenem, aztreonam, colistin, and amikacin (all from Sigma-Aldrich), and ceftolozane-tazobactam (Zerbaxa; MSD). The antibiotic stocks were prepared according to the Clinical and Laboratory Standards Institute (CLSI). The final concentrations used in the time-kill assays corresponded to the AUC over 24 h (Table 1) (17 (link)– (link)21 (link)).

The liquid culture media used for BMD and growth kinetics were the following: cation-adjusted Mueller–Hinton broth (CAMHB, Becton Dickinson), Brucella broth (BB, Becton Dickinson), H-medium (MERLIN), CAMHB containing 5% horse blood and 20 mg/L β-NAD (CAMHB-F, according to EUCAST SOP) [36 ], and CAMHB supplemented with 10 mL/L IsovitaleX (CAMHB-X, Thermo Fisher, Waltham, MA, USA). The in-house media were autoclaved for 15 min at 121 °C. Supplements were added when the media had cooled down; afterwards, the pH was adjusted to 7.0 ± 0.2. The solid culture media for agar dilution were prepared by adding 15 g/L agar to the culture media mentioned above.

MIC values were determined using the CLSI broth microdilution reference method with concurrent quality control (30 , 31 ). Taniborbactam was provided by Venatorx Pharmaceuticals, Inc. (Malvern, PA, USA) and tested at a fixed concentration of 4 μg/mL with cefepime (31 ). Other agents were purchased from commercial sources. Broth microdilution panels were prepared at IHMA using cation-adjusted Mueller Hinton broth (CAMHB; Becton, Dickinson) and stored at −70°C until the day of testing.
MICs were interpreted using 2021 CLSI breakpoints (31 ) with two exceptions. Meropenem-vaborbactam MICs for P. aeruginosa were interpreted using EUCAST breakpoints (≤8 μg/mL, susceptible; >8 μg/mL, resistant) (32 ). Cefepime-taniborbactam MICs against both Enterobacterales and P. aeruginosa were interpreted using provisional breakpoints of ≤16 μg/mL (susceptible) and >16 μg/mL (resistant) that were based upon published in vivo efficacy data from neutropenic murine infection models (thigh, complicated urinary tract) (33 (link), 34 (link)) and data from a safety and pharmacokinetics studies in human volunteers (35 (link), 36 (link)). These provisional breakpoints are also supported by probability of taniborbactam target attainment analyses based on simulation of human plasma exposures (J. Dowell, unpublished data).

Bacteria were cultured on Mueller-Hinton agar and tested on cation-adjusted Mueller-Hinton broth (CAMHB) (Becton Dickinson, Sparks, MD). Stock solutions of meropenem (Sandoz, Princeton, NJ) (10 to 50 mg/ml in distilled H₂O) and vaborbactam (The Medicines Company, San Diego, CA) (5 to 10 mg/ml in 90% dimethyl sulfoxide) were stored at −80°C until use.
Broth microdilution susceptibility testing was performed according to Clinical and Laboratory Standards Institute methods (40 ), using panels prepared in-house. A checkerboard assay conforming to the Moody procedures described in the Clinical Microbiology Procedures Handbook (41 ) was used to evaluate the effect of various concentrations of vaborbactam on the meropenem MIC.