Trimethoprim sulfamethoxazole

The antibiotic susceptibility profiles for the VT-MRSA isolates were determined using the MIC technique via the microdilution method according to the CLSI 2013 [32 ], as described in the previous section. MIC tests were performed for ampicillin (Sigma-Aldrich), ceftazidime (Pfizer; México DF, México), ceftriaxone (Roche; Mexico DF, México), ciprofloxacin (Bayer; Levenkusen Westfalia, Germany), erythromycin (MP Biomedicals; Solon, OH, USA), kanamycin (Sigma-Aldrich), meropenem (Astra Zeneca; México DF, Mexico), rifampicin (MP Biomedicals), gentamicin (MP Biomedicals), and trimethoprim-sulfamethoxazole (Sigma-Aldrich).

NMR spectra were measured with a JEOL Eclipse 500 FT-NMR spectrometer (¹H, 500 MHz; ¹³C 125 MHz). Column chromatography was carried on silica-gel (Kieselgel 60, 70–230 mesh, Merck, Germany), a thin layer chromatography (TLC) on pre-coated Silica-gel F254 (0.25 mm, Merck) and Sephadex LH-20 (25–100 µM, Sigma, U.S.A). Mueller-Hinton broth (MHB) and Mueller-Hinton agar (MHA) (Difco Laboratories, Baltimore, MD, USA). Ampicillin, amoxicillin/clavulanic acid, chloramphenicol, cephalothin, sulfisoxazole, nalidixic acid, norfloxacin, streptomycin, trimethoprim/sulfamethoxazole, ticarcillin, ciprofloxacin, N,N′-Dicyclohexylcarbodiimide, Sodium azide, Tris, Triton X-100 and solvents were purchased from Sigma Aldrich (St. Louis, USA).

Standard amikacin, ciprofloxacin, doxycycline, ertapenem, gentamicin, imipenem (U.S. Pharmacopeia, Rockville, MD, USA), ampicillin, cephalothin, cefuroxime, ceftriaxone, ceftazidime, colistin sulfate, doripenem, meropenem, trimethoprim/sulfamethoxazole (Sigma-Aldrich, St. Louis, MO, USA), fosfomycin (Ercros, Barcelona, Spain), and tigecycline (Pfizer, New York, NY, USA) were used for antimicrobial susceptibility testing. The minimum inhibitory concentration (MIC) determinations and susceptibility interpretation criteria followed the Clinical Laboratory and Standard Institute (CLSI) and Federal Drug Administration (FDA) standards [11 ,12 (link)]. The MICs of the drugs, except tigecycline and colistin, were measured by agar dilution in Mueller–Hinton agar (Oxoid, Basingstoke, UK) according to CLSI recommendations.¹¹ For fosfomycin susceptibility, glucose-6-phosphate (25 mg/mL) was added to the agar plate. tigecycline and colistin MICs were determined by microdilutions in freshly prepared cation-adjusted Mueller–Hinton broth (CAMHB). E. coli ATCC 25922 was used as the control strain [13 (link)]. The MICs of other agents were determined using the custom-designed panels for Gram-negative bacilli (Sensititre, Thermo Fisher Scientific, Oakwood Village, OH, USA).

The following drugs were tested: amoxicillin-clavulanate (Sigma, lot 100M08228v; USP, lot J0G109), azithromycin (USP, lot G; Sigma, lot E446421/1v), cefdinir (Sigma, lot 117K1392), cefpodoxime (USP, lot HDKD09), clarithromycin (USP, lot GIG324), doxycycline (Sigma, lot BCBF9827V), penicillin (Sigma, lot BCBF3866V), solithromycin (Cempra, lot EKS11646), and trimethoprim-sulfamethoxazole (Sigma, lots 000M4110V and BCBF0534V). The drugs were dissolved and diluted for testing in accordance with the recommendations of Clinical and Laboratory Standards Institute (CLSI) document M100-S25 (12 ).

The antimicrobials ampicillin, ceftriaxone, ceftazidime, gentamicin, tetracycline, ciprofloxacin, nalidixic acid, chloramphenicol, trimethoprim-sulfamethoxazole, ertapenem, and colistin were obtained from Sigma-Aldrich (St. Louis, MO, USA). MICs of the antimicrobial agents were determined by the agar-dilution technique, except the broth-microdilution technique was used for colistin. The Clinical and Laboratory Standards Institute criteria were used for interpreting the MICs³⁹ .

Disk diffusion method was performed using commercial antibiotic disks and Muller–Hinton agar, according to the CLSI recommendations [14 ]. Minimum inhibitory concentration (MIC) was determined for four antibiotics, i.e., ceftriaxone, ciprofloxacin, nitrofurantoin, and trimethoprim–sulfamethoxazole (Sigma–Aldrich), by using the 96-well broth microdilution (CLSI, 2018) method. Stock antibiotic solutions were prepared accordingly with CLSI and conserved at −20 °C. The broth microdilution testing domain was of 4 to 1 µg/mL for ciprofloxacin, 4 to 1 µg/mL for ceftriaxone, 150 to 18 µg/mL for nitrofurantoin, and 100 to 25 µg/mL for trimethoprim–sulfamethoxazole. All panels were incubated at 37 °C and read after 18–24 h. The lowest antibiotic concentration that completely inhibited the visible microbial growth and was confirmed by the spectrophotometer reading was recorded as the MIC value.

MICs were determined using the broth microdilution method, following the CSLI guidelines [48 ]. The tests were conducted in ISO-Sensitest Broth (Oxoid, Hamsphire, UK), according to recommendations of the International Standard ISO 20776-1. Standard antibiotics were also included in the assays in order to compare their activity and efficacy. In particular, ciprofloxacin, ampicillin, vancomycin, trimethoprim-sulfamethoxazole (Sigma Aldrich, Madrid, Spain), and colistin (MPBiomedicals, Eschwege, Germany) were investigated.
Briefly, broth microdilution was performed in polystyrene 96-well round bottomed plates in a final volume of 100 μL. Serial two-fold dilutions of the antimicrobial ranging from 128 mg/L to 0.03 mg/L were used. In addition, wells with bacteria alone and with growth media alone were included as positive and negative controls, respectively. As for the inoculum, after an overnight incubation in agar plates, bacterial colonies were resuspended in 0.9% NaCl to reach a 0.5 McFarland (equivalent to 1.5 × 10⁸ CFU/mL) and diluted to obtain a final concentration in the well of 5 × 10⁵ CFU/mL. Finally, the plates were incubated for 18 h at 37 °C. MIC values were expressed in mg/L. We performed three technical and three biological replicates for each agent.