Ceftazidime caz

All strains used in this study are derived from E. coli K12 W3110. Individual outer membrane porin deleted (W3110ΔompF and W3110ΔompC) and porinless (W3110 ΔompF ΔompC) strains were gift from M. G. P. Page. W3110ΔFC was transformed with the empty pBAD24 vector, or recombinant derivatives containing K. aerogenes omp35 or omp36, E. cloacae ompE35 or ompE36, or K. pneumoniae ompK35 or ompK36. All strains were cultured in Mueller Hinton II Broth or Luria Broth supplemented with ampicillin (100 µg/ml), and protein expression was induced with 0.02% l-arabinose (Sigma) for 2 h at 37 °C. Carbapenems including ertapenem (ETP) and meropenem (MEM), were purchased from Sequoia Research Products Ltd. (United Kingdom); cephalosporins including ceftazidime (CAZ), cefotaxime (CTX), and cefepime (FEP); and penicillins including piperacillin (PIP) and ticarcillin (TIC) were from Sigma. Anhydrous pentane, hexadecane and hexane was purchased from Carl Roth GmbH, Co. 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phospho-l-serine (POPS) were purchased from Avanti Polar Lipids (Alabama, USA). All solutions were prepared with 18.2 MΩ cm Millipore-grade water unless otherwise noted.

Antimicrobial susceptibility profiles for each strain are listed in Table 1. Minimal inhibitory concentrations (MIC) were first determined by conventional BMD testing following CLSI guidelines for medium, inoculum, and incubation temperature [47 ]. BMD susceptibility testing panels were prepared with Cation-Adjusted Mueller Hinton Broth (CAMHB) in house. β-lactam antibiotics selected for this study were amoxicillin-clavulanic acid (AMC) (Toku-E, Bellingham, WA and USP, Frederick, MD), ceftazidime (CAZ) (Sigma Aldrich, St. Louis, MO) and imipenem (IPM) (Toku-E, Bellingham, WA). Two-fold antibiotic concentrations were tested ranging from 0.06/0.03–128/64 μg/ml AMC, 0.06–128 μg/ml CAZ, and 0.03–64 μg/ml IPM. B. pseudomallei strains were classified as resistant (R), intermediate (I), or susceptible (S) based on interpretive criteria outlined by CLSI [47 ]. MICs were recorded after 16 to 20 h of incubation at 35 °C ambient air, with the exception of MSHR1655 (43 h). Incubation was extended due to insufficient growth in the control well of the BMD panel.

Fecal swabs or stool samples (200 mg) were initially suspended in 2 ml of sterile 0.9% saline. Next, an aliquot of each sample were plated onto four sets of MacConkey media (Merck, Germany): (i) control plate without antibiotics, (ii) biplate agar containing cefotaxime (CTX) (Sigma, USA) and ceftazidime (CAZ) (Sigma, USA) at 1 mg/L concentration to screen for ESBL- and/or pAmpC-producing isolates [17 (link)], (iii) medium containing ciprofloxacin (CIP) (Sigma, USA) at 1 mg/L to screen for FQ-resistant isolates [18 (link)], and (iv) medium supplemented with 1 mg/L ertapenem (ERT) (Sigma, USA) to screen for carbapenem-resistant strains [25 , 26 (link)]. All of the plates were incubated overnight at 37 °C.

Susceptibility assays were performed using the broth microdilution (BMD) method and cation-adjusted Mueller-Hinton II broth (Becton, Dickinson and Company, Sparks, MD, USA), following Clinical and Laboratory Standards Institute guidelines (55 ), or Etest, following the manufacturer’s (AB bioMérieux, Marcy l’Etoile, France) guidelines. Ceftazidime (CAZ) was purchased from Sigma-Aldrich, and imipenem (IMP) and meropenem (MEM) were bought from the United States Pharmacopeia (Rockville, MD). Avibactam was purchased from Advanced ChemBlocks (Burlingame, CA, USA).

A total of 25g of each vegetable were obtained with a sterile scalpel and placed in bags containing 225ml of buffered peptone water (BD, Franklin Lakes, NJ, United States). Samples were homogenized in a stomacher (IUL Instruments, Spain) for 1minute and then incubated at 37°C for 18–24h. The same microbiological procedure was followed for water samples after removing the full gauzes from the cassettes and placed them in peptone water. After incubation, two plates with MacConkey agar medium (BD) were inoculated with 100μl from each stomacher bag; one plate was supplemented with 2μg/ml of ceftazidime (CAZ; Sigma, Germany) and the other with 2μg/ml of ciprofloxacin (CIP; Sigma, Germany). In all experiments, K. pneumoniae SCL 2346 (CAZ MIC >16μg/ml and CIP MIC >2μg/ml) and E. coli ATCC 25922 were used as resistant and susceptible controls, respectively. All plates were incubated at 37°C for 18–24h. Colonies were selected according to morphology, using a magnifying glass. This selection was made by classifying the colonies according to standard patterns: colony shape, color (pigmentation), texture, and edge shape, as described previously (Higuera-Llantén et al., 2018 (link)). Distinct morphotypes phenotypically consistent with Enterobacterales were further identified by MALDI-TOF (Bruker Daltonics, Germany).