Sensititre nephelometer

The MIC of the parental isolates was determined using Broth Microdilution in Mueller-Hinton broth. Bacterial strains were isolated on Mueller-Hinton agar plates and three colonies were resuspended in 0.9% NaCl to MacFarland 0.5 (determined using a Sensititre Nephelometer, Thermo Fisher Scientific) and diluted 1:10,000 in Mueller-Hinton broth (to ~10⁴ cells/mL). Aliquots of 50 μL were mixed with 50 μL of medium containing serially-diluted antibiotics in 96-well round-bottomed plates. MICs were determined after incubation at 37 °C for 18 h. The MIC of the isolates with variable copy number (see “Characterization of isolates with variable initial copy number”) was measured by Etest strips (BioMerieux) for tobramycin, tetracycline, and cefotaxime. Overnight cultures of the bacterial isolates were diluted 1:20 in PBS (8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na₂HPO₄, and 0.24 g/L KH₂PO₄) and the cell suspensions were evenly spread onto agar plates using sterile cotton swabs. The Etest strips were applied and the plates were read after 24 h of incubation at 37 °C. For piperacillin-tazobactam, the MIC was determined using Broth Microdilution as previously described, using an initial culture inoculum of 10⁶ cells/mL.

Whole genome sequencing of the bacterial isolates was performed as previously reported^{25 (link),27 (link)}. Briefly, an aliquot of the bacterial culture in tryptic soy broth was diluted with 0.85% sterile saline solution to the desired inoculum density of 1 × 10⁶ CFU per ml using Thermo Scientific™ Sensititre™ Nephelometer and the chilled culture tubes were submitted for DNA extraction and sequencing. DNA extraction was performed using the Qiagen DNeasy Blood & Tissue kit (Lucigen, WI, USA) according to the manufacturer’s protocol as was previously described^{20 (link)}. Sequencing libraries were prepared using the Nextera XT library preparation kit (Illumina Inc., CA, USA). Sequencing was performed on the Illumina MiSeq platform (Illumina Inc., CA, USA) using the v2 reagent kit (2 × 250 nt paired-end chemistry), which yielded 250-bp paired-end reads.

MICs for
susceptibility testing and MIC₉₀ assays were determined
according to CLSI standards for liquid MIC (M07) using the direct
colony suspension method for inoculum preparation. Appropriate inoculation
suspension density was assessed using a Sensititre Nephelometer (Thermo
Fisher Scientific) with a Sensititre 0.5 McFarland Standard. MBC and
time-kill assays were conducted according to CLSI guidelines (M26).
MIC in the presence of serum was done using a 50% (final concentration)
pooled, heat-inactivated (56 °C, 1 h), sterile-filtered (Filtropur
S plus 0.2, Sarstedt) human serum.

Bacterial growth experiments were performed in two biological replicates with two technical replicates for each strain on Bioscreen C (Thermo Labsystems, Helsinki, Finland) for 24 h at 37 °C. Briefly, 250 μL of MHB-II was inoculated with cells from LB agar plates to a final cell density of approximately 10⁶ CFU/mL, using a Sensititre™ Nephelometer (Thermo Fisher Scientific, Roskilde, Denmark) with a 0.5 McFarland standard (1–2 × 10⁸ CFU/mL). The cultures were then grown without antibiotic or supplemented with either 128 mg/L CTX, 0.5 mg/L GEN, or a combination of CTX and GEN of 1 + 0.25 mg/L. The OD₆₀₀ was measured every 20 min with continuous shaking, and the growth curves were obtained using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA).

The NMIC/ID-4 panel of the BD Phoenix system was employed to analyze a total of 1022 bacterial isolates. Initially, a McFarland 0.5 stock solution was prepared in 4.5 mL of Phoenix ID broth [17 (link)] using a Sensi Titre™ nephelometer (Thermo Fisher Scientific, Waltham, MA, USA). The resulting bacterial ID solution was then placed in the designated ID field on the Phoenix panel. Subsequently, 50 µL of the microbial solution was added to each chemical reactivity well on the panel. Any excess suspension was absorbed by the cushion located on the underside of the panels. The panel was sealed with a rubberized coating, and the display code was read before being directly inserted into the Phoenix machine.
For the cultivated bacterial isolates, the ID results obtained from the Phoenix system were compared with those of the traditional API system, which served as the reference standard. Accurate identification rates were subsequently determined at both the genus and species levels based on these comparisons.

The bacterial cells used for antibacterial activity and phenotypic characterization were prepared as follows. Initially, bacterial colonies were re-streaked on LB agar plates with or without antibiotics (kanamycin or ampicillin) and resuspended in nuclease-free water (NFW) to 0.5 McFarland turbidity using a Sensititre™ Nephelometer (Thermo Fisher Scientific, Waltham, MA, USA). Individual cells were inoculated into Sensititre™ Cation-adjusted Mueller–Hinton broth (Thermo Fisher Scientific, Waltham, MA, USA) at a 1000-fold dilution and were grown further at 37 °C for 16 h without shaking [43 (link)].