Ceftriaxone

For zoliflodacin, agar dilution was performed, according to Clinical and Laboratory Standards Institute (CLSI) guidelines (M07-A9 and M100-S24; www.clsi.org), on GCVIT agar plates (3.6% Difco GC Medium Base agar (BD, Diagnostics, Sparks, MD, United States) supplemented with 1% IsoVitalex (BD, Diagnostics, Sparks, MD, United States). Additionally, a microbroth dilution method for zoliflodacin (in triplicate) was performed in the medium used in the HFIM, that is, modified fastidious broth (mFB), as previously described (Foerster et al., 2020 (link)). Etest was used for MIC testing of ceftriaxone, cefixime, ciprofloxacin, and azithromycin, in accordance with the manufacturer’s instructions (bioMérieux, Marcy-l’Etoile, France).

The antibiotic susceptibility profile of the isolates was identified using the standard disk diffusion method on Mueller–Hinton agar (bioMérieux, Marcy l’Etoile, France). Sixteen different antibiotics were tested, including amoxicillin (20 μg), amoxicillin-clavulanic acid (20/10 μg), piperacillin-tazobactam (30/6 μg), cephalotin (30 μg), ceftriaxone (30 μg), cefepime (30 μg), ertapenem (10 μg), imipenem (10 μg), amikacin (30 μg), gentamicin (10 μg), ciprofloxacin (5 μg), Fosfomycin (200 μg), nitrofurantoin (100 μg), tobramycin (10 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), and colistin (10 μg) (bioMérieux, Marcy l’Etoile, France). The minimal inhibitory concentration (MIC) of colistin, ertapenem, and imipenem was identified using the microdilution and the E-test methods, respectively. Each strain was considered to be resistant to colistin, ertapenem, and imipenem if their MICs were greater than 2 mg/L, 1 mg/L, and 8 mg/L, respectively. The results were interpreted according to the European Microbial Medical Sensitivity Committee (EUCAST) 2017 (http://www.Sfmicrobiology.Org/Userfiles/Files/Files/CASFM/CASF%20V2_0_MAI2017.PDF, accessed on 25 September 2020).

To confirm the transfer of resistance phenotype, antibiotic susceptibility patterns of the donor, recipients, and transconjugants were determined after their growth on Mueller–Hinton (MH, Difco) agar by disk diffusion method in accordance with Clinical and Laboratory Standards Institute [CLSI] (2014) using commercially available disks (Becton Dickinson Company, Sparks, MD) namely, ampicillin (AMP), cefuroxime (CXM), ceftriaxone (CRO), cefotaxime (CTX), cefotaxime/clavulanic acid (CTX-CLA), ceftazidime (CAZ), ceftazidime/clavulanic acid (CAZ-CLA), chloramphenicol (CHL), nalidixic acid (NA), ciprofloxacin (CIP), ofloxacin (OFX), norfloxacin (NOR), imipenem (IPM), streptomycin (STR), azithromycin (AZM) tetracycline (TET), trimethoprim/sulfamethoxazole (SXT). To measure the increase of resistance in the transconjugants, MICs of antibiotics (ceftriaxone, imipenem, tetracycline, and sulfamethoxazole/trimethoprim) were determined by E test (AB bioMérieux, Solna, Sweden) in comparison with the wild NDM-positive Salmonella isolate.

Frozen isolates were re-cultured and serotyped based on the White-Kaufman-Le Minor standard method [12 ]. Confirmed S. Typhi isolates were screened for antibiotic susceptibility using the Kirby Bauer disc diffusion method and results were interpreted based on the 2017 CLSI guidelines [13 ]. The following antibiotics were used; ciprofloxacin (5 μg), ceftriaxone (30 μg), chloramphenicol (30 μg), nalidixic acid (30 μg), tetracycline (30 μg) and ampicillin (10 μg) (Oxoid, United Kingdom). Minimum inhibitory concentration (MIC; mg/L) for ceftriaxone, ciprofloxacin, and azithromycin were done using the E-test (bioMérieux, Marcy l’Étoile, France). Escherichia coli ATCC 25922 was used as a quality control. Multi-drug resistance (MDR) was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories [14 (link)].

For determination of zoliflodacin MICs (mg/L), agar dilution on GCVIT agar plates (Foerster et al., 2019 (link)) and microbroth dilution [in triplicates in the HFIM medium, i.e., modified Fastidious Broth (mFB)] were performed, as previously described (Jacobsson et al., 2021 (link)). Etest was used to determine MICs (mg/L) of ceftriaxone, cefixime, ciprofloxacin, and azithromycin, in accordance with the manufacturer’s instructions (bioMérieux, Marcy-l’Etoile, France).