Piperacillin tazobactam

Minimal inhibitory concentrations were determined according to Clinical and Laboratory Standards Institute^® (CLSI) M07-A10 (CLSI, 2015 ) in 96-well plates. Breakpoints interpretation were made according to the M100 Performance Standards for Antimicrobial susceptibility testing 28th edition (CLSI, 2019 ). Antibiotics included were amikacin (Sigma Aldrich A1774), gentamicin (Sigma Aldrich G3632), aztreonam (Sigma Aldrich PZ0038), ceftazidime (Sigma Aldrich C3809), cefepime (Sigma Aldrich PHR1763), ciprofloxacin (Sigma Aldrich 17850), levofloxacin (Sigma Aldrich 28266), doripenem (Sigma Aldrich 32138), imipenem (Sigma Aldrich I0160), meropenem (Sigma Aldrich M2574), colistin (Sigma Aldrich C4461), and piperacillin/tazobactam (Sigma Aldrich P8396/T2820). P. aeruginosa ATCC^® 27853 was used as control as according to CLSI (Supplementary Table S1).

All experiments were performed with P. aeruginosa UCBPP-PA14 (Rahme et al., 1995 (link)) and clones obtained from four antibiotic-resistant populations: CAR-10, GEN-4, PIT-1 and STR-2 (Barbosa et al., 2017 (link)). The resistant populations were previously selected for high levels of resistance against protein synthesis inhibitors from the aminoglycoside family, gentamicin (GEN; Carl Roth, Germany; Ref. HN09.1) and streptomycin (STR; Sigma-Aldrich, USA; Ref. S6501-5G), or alternatively cell-wall synthesis inhibitors from the β-lactam family, carbenicillin (CAR; Carl Roth, Germany; Ref. 6344.2) and piperacillin/tazobactam (PIT; Sigma-Aldrich, USA; Refs. P8396-1G and T2820-10MG). Resistant clones were isolated by streaking the resistant populations on LB agar plates supplemented with antibiotics and picking single colonies after an overnight growth at 37°C. Antibiotic stocks were prepared according to manufacturer instructions and frozen in aliquots for single use. Evolution experiments and resistance measurements were performed in liquid M9 minimal media supplemented with glucose (2 g/l), citrate (0.5 g/l) and casamino acids (1 g/l).

The minimum inhibitory concentrations (MICs) of antimicrobial agents were determined by using the agar dilution method¹⁶ . The tested compounds included ampicillin, aztreonam, cefotaxime, ceftazidime, cefepime, piperacillin-tazobactam, meropenem, ciprofloxacin, colistin sulfate (Sigma Chemical Company, St. Louis, MO, USA) and tigecycline (Wyeth, Puerto Rico).
According to the British Society for Antimicrobial Chemotherapy Standing Committee for Antimicrobial Susceptibility Testing (Version 14.0, 2015), the susceptible MIC breakpoint for colistin against Enterobacteriaceae is ≦2 μg/mL and it is considered resistant if MIC > 2 μg/mL. The breakpoints for tigecycline against Enterobacteriaceae are ≦1μ g/mL as susceptible and >2 μg/mL as resistant.

The E. coli isolates were considered potential carrier of ESBL enzyme when they showed resistance to cefotaxime and ceftriaxone. The MIC (minimum inhibitory concentration) of fourteen antibiotics including ceftazidime, ceftriaxone, cefotaxime, ceftizoxime, cefepime, cefixime, gentamicin, amikacin, meropenem, imipenem, ciprofloxacin, cotrimoxazole, colistin, and piperacillin/tazobactam (Sigma Chemical Co., Germany) for resistant E. coli strains was determined by the agar dilution method [3 ]. The MIC was determined on Mueller Hinton agar with twofold dilutions of antibiotics concentration (from 0.5 μg/mL to 256 μg/mL and 10 μL of microbial suspension). Microbial growth was observed and documented after 24 hours of incubation at 35°C. The result was reported according to CLSI 2011 guidelines and divided into three categories: resistant, intermediate, and susceptible. The ESBL-producing E. coli isolates were considered resistant to both cefotaxime and ceftazidime if their MIC was ≥2 μg/mL in accordance with CLSI criteria [17 ].

Salmonella isolates were serotyped via the slide agglutination assay according to the instructions provided by the manufacturer of the Salmonella antisera (Statens Serum Institute, Copenhagen, Denmark). The serovars of the isolates were determined using the Kauffmann-White scheme (Grimont and Weill, 2007 ).
Antimicrobial susceptibility testing of Salmonella isolates was performed using the agar dilution method for ampicillin, ampicillin-sulbactam, piperacillin, piperacillin-tazobactam, cefuroxime, cefotetan, ceftazidime, ceftriaxone, cefepime, aztreonam, imipenem, amikacin, gentamicin, and tobramycin (Sigma, St. Louis, MO, United States). Escherichia coli ATCC 25922 was used as a quality control strain in each run. The breakpoints for determining susceptibility or resistance to antimicrobials were determined by the guidelines of the Clinical Laboratory Standard Institute (CLSI, 2018 ).

Aztreonam, meropenem, and polymyxin B were purchased from Toronto Research Chemicals. Cefepime was purchased from Kemimac(s) Pte Ltd. Piperacillin/tazobactam and tigecycline were purchased from Sigma-Aldrich. Doripenem was obtained from Shionogi and Co. Aliquots of stock solutions of all antibiotics were prepared in sterile water and stored at −80°C. Before each experiment, the aliquots were thawed and diluted to the desired concentrations with cation-adjusted Mueller Hinton broth (Ca-MHB).