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16 protocols using ceftazidime

1

Isolation and Antimicrobial Susceptibility of E. coli

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Isolation of E. coli was performed as follows: liquid aspirates were diluted approximately ×106 fold with sterile PBS (phosphate saline buffer). Approximately 0.05 ml volume of feces were placed into 0.5 ml of sterile PBS, vortexed to homogeneity, an aliquot was diluted approximately ×106 fold. Biopsy samples were vortexed in 0.2 ml of sterile PBS. For all samples, 0.1 ml of the resulting liquid was spread onto the Luria-Bertani agar plates. After overnight incubation on 37 °C, isolated colonies were identified with the Matrix Assisted Laser Desorbtion/Ionization (MALDI) Biotyper software (Bruker Daltonics, Germany) using the Microflex LT mass spectrometer (Bruker Daltonics, Germany). For DNA extraction, all E. coli strains were grown in the Luria-Bertani broth at 37 °C with shaking (200 RPM) overnight and collected by centrifugation. Samples and corresponding E. coli isolates are listed in Table 1.
The testing of susceptibility to ampicillin/sulbactam, ceftriaxone, cefotaxime, ceftazidime, cefepime, imipenem, meropenem, gentamicin, levofloxacin, and ciprofloxacin (all from Bio-Rad, USA) was performed by the disc-diffusion method using the Mueller-Hinton agar plates. The E. coli strain ATCC 25922 was used as a control. Current CLSI and EUCAST criteria were used for interpretation.
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2

Evaluating Antibiotic Susceptibility with EUCAST Standards

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Antibiotic susceptibility testing was determined on Müller–Hinton agar by standard disc diffusion method as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST; www.eucast.org). Seventeen antibiotics were tested, including ticarcillin, ticarcillin-clavulanic acid, piperacillin, piperacillin-tazobactam, ceftazidime, cefotaxime, cefepime, aztreonam, amikacin, tobramycin, gentamicin, ciprofloxacin, rifampicin, ertapenem, meropenem, imipenem and colistin (Bio-Rad, Marnes-la-Coquette, France). The MIC for imipenem was determined using the Etest method (bioMérieux, La Balmes les Grottes, France) and the result was interpreted according to the EUCAST breakpoint for Enterobacteriaceae (susceptible if MIC ≤ 2 mg/L)
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3

Antimicrobial Susceptibility Testing Protocol

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Antimicrobial susceptibility testing was performed on all isolates recovered from the two selective media using the disc diffusion method on Mueller–Hinton (MH) agar plates (Neogen, Lansing, Michigan) for ticarcillin (75 μg), amoxicillin/clavulanic acid (20–10 μg), cefotaxime (30 μg), ceftazidime (10 μg), temocillin (30 μg), cefoxitin (30 μg), ertapenem (10 μg), imipenem (10 μg), meropenem (10 μg), ceftazidime/avibactam (10–4 μg), aztreonam (30 μg), ciprofloxacin (5 μg), trimethoprim-sulfamethoxazole (SXT) (1.25–23.75 μg), tetracycline (30 μg), amikacin (30 μg), gentamicin (15 μg), and tobramycin (10 μg) (Bio-Rad Laboratories, Algés, Portugal), following EUCAST recommendations and breakpoint tables. Susceptibility to fosfomycin was evaluated by the disk diffusion method (50 μg) on MH agar plates supplemented with 25 μg/mL glucose-6-phosphate, according to EUCAST guidelines [24 ]. Strain E. coli ATCC 25922 was used for quality control. Multidrug resistance was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories [25 (link)].
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4

ESBL Screening in E. coli Isolates

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Escherichia coli isolates were screened for ESBL using a disc diffusion test with expanded-spectrum cephalosporins (which are hydrolysed by all TEM, SHV and CTX-M types of ESBLs) and aztreonam on Mueller-Hinton II agar (Pitout et al., 1997 (link); Pitout and Laupland, 2008 (link); Smet et al., 2008 (link); Falagas and Karageorgopoulos, 2009 (link)). The cephalosporins used were ceftazidime (30 μg), cefotaxime (30 μg), ceftriaxone (30 μg) and cefpodoxime (10 μg) (Bio-Rad, Laboratories, Hercules CA). Extended-spectrum β-lactamase phenotypic production was confirmed using the modified double-disc diffusion method or the combined-disc method (Stürenburg and Mack, 2007 (link)). cefotaxime + clavulanic acid (30 μg + 10 μg) and ceftazidime + clavulanic acid (30 μg + 10 μg) discs were used (Bio-Rad Laboratories). Extended-spectrum β-lactamase was positive when the zone diameters given by the discs with clavulanate were ≥ 5 mm larger than those without the inhibitor for at least one of the combinations. Escherichia coli ATCC 25922 (ESBL negative), E. coli ATCC 35218 (ESBL positive control), K. pneumonia ATCC 700603 (ESBL positive) and Pseudomonas aeruginosa ATCC 27853 (ESBL negative) strains were used as control strains for test performance.
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5

Antimicrobial Susceptibility Testing of P. aeruginosa

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Antimicrobial susceptibility was tested by employing disk diffusion, gradient test, and broth microdilution test according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations, 2021 [43 ]. The disks impregnated with the following antimicrobial agents were tested: imipenem (10 μg), meropenem (10 µg), ciprofloxacin (5 µg), levofloxacin (5 µg), ceftazidime (10 µg), cefepime (30 µg), amikacin (30 µg), piperacillin/tazobactam (30/6 µg), aztreonam (30 µg), ticarcillin (75 µg), ceftazidime-avibactam (10–4 µg), and ceftolozane/tazobactam (38–10 µg) (BioRad, Watford, UK). Minimum inhibitory concentrations (MICs) for colistin, imipenem, and meropenem were evaluated by ComASP Colistin (Liofilchem, Roseto, Italy) and Gradient strip test (Liofilchem, Roseto, Italy), respectively. P. aeruginosa ATCC 27853 was used as the control strain in the antibiotic susceptibility testing. MDR P. aeruginosa were defined as isolates that tested resistant to at least one antimicrobial agent of three or more different classes. Extensively drug-resistant (XDR) P. aeruginosa were defined as a subset of MDR isolates that tested non-susceptible to at least one antimicrobial agent of five different classes. P. aeruginosa was defined as pandrug-resistant (PDR) when the organism was resistant to all tested agents.
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6

Antimicrobial Susceptibility Testing Protocol

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Antimicrobial susceptibility of the clinical isolates was determined by the disk diffusion method on Mueller-Hinton II agar as recommended by the Antibiogram Committee of the French Society for Microbiology (CA-SFM: Comité de l’Antibiogramme de la Société Française de Microbiologie) [52 ]. The 14 antimicrobial drugs tested (all from Bio-Rad) were (i) beta-lactam antibiotics class: ticarcillin (TIC, 75 μg), ticarcillin-clavulanic acid (TCC, 75 / 10 μg), piperacillin (PIP, 75 μg), piperacillin-tazobactam (PPT, 75 / 10 μg) ceftazidime (CAZ, 30 μg), cefepime (FEP, 30 μg), aztreonam (ATM, 30 μg), imipenem (IPM, 10 μg) and meropenem (MEM, 10 μg), (ii) aminoglycosid antibiotics class: gentamicin (GMI, 15 μg), amikacin (AKN, 30 μg), tobramycin (TMN, 10 μg), (iii) fluoroquinolone antibiotics class: ciprofloxacin (CIP, 5 μg) and (iv) other antibiotic class: fosfomycin (FF, 50 μg + 50 μg G6P).
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7

Antimicrobial Susceptibility Testing of S. aureus

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Antimicrobial susceptibility testing of S. aureus isolates was performed using the Kirby-Bauer disk diffusion method in Muller-Hinton agar. The antimicrobials used were: penicillin (PE) 6 µg, dicloxacillin (DC) 30 µg, pefloxacin (PEF) 5 µg, cefuroxime (CXM) 30 µg, gentamicin (GE) 120 µg, cefotaxime (CTX) 30 µg, sulfamethoxazole + trimethoprim (SXT) 1.25 and 23.75 µg, tetracycline (TE) 30 µg, ampicillin (AM) 10 µg, erythromycin (E) 15 µg, ceftazidime (CAZ) 30 µg and cephalothin (CF) 30 µg (BioRad, USA).
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8

Antibiotic Susceptibility Testing Protocol

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Antibiotic susceptibility testing was performed by disk diffusion (amoxicillin/clavulanic acid, aztreonam, cefepime, cefotaxime, cefoxitin, ceftazidime, ciprofloxacin, ertapenem, gentamicin, imipenem, meropenem, piperacillin/tazobactam and trimethoprim/sulfamethoxazole; Bio-Rad, Marnes-la-Coquette, France) and minimum inhibitory concentration (MIC) by in house broth microdilution (colistin, chloramphenicol, florfenicol, flumequine and oxytetracycline) and E-test® (fosfomycin; bioMérieux, Hazelwood, MO, USA), as previously described [55 (link)].
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9

Antibiotic Susceptibility Testing Protocol

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Antibiotic susceptibility was measured with the use of the standard disc diffusion process suggested by the Clinical and Laboratory Standards Institute (CLSI) guidelines by broth microdilution and E-test (cat. nos. 537300, 501800, 533500, 501300, 501600, 506710, 513800, 526000, 523600, 525508, 522000, 503500 and 521400; AB Biodisk; BioMérieux Inc.) (17 ). A total of 14 antibiotics were tested, including ampicillin-sulbactam, trimethoprim-sulfamethoxazole, aminoglycoside antibiotic amikacin, macrolide antibiotic azithromycin, β-lactam antibiotic aztreonam, β-lactamase inhibitor tazobactam, cephalosporin antibiotics ceftazidime and cephalothin, rifampin and tigecycline, carbapenem antibiotic meropenem and colistin, and the glycopeptide antibiotics teicoplanin and vancomycin (Bio-Rad, Laboratories, Inc., Hercules, CA, USA). These antibiotics are of different classes and have different killing mechanisms on the bacteria. The E-test technique was used to determine the minimum inhibitory concentrations (MICs) of meropenem. The results were interpreted according to the CLSI guidelines from 2015 (18 ).
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10

Antimicrobial Susceptibility Testing Protocol

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Antimicrobial susceptibility was determined by the disk diffusion method, on Mueller-Hinton (MH) agar, in accordance with the guidelines of the Antibiogram Committee of the French Society for Microbiology [23 ]. The following 22 antimicrobial drugs (Bio-Rad) were tested: ampicillin (AMP, 10 μg), amoxicillin plus clavulanic acid (AMC, 30 μg), cefoxitin (FOX, 30 μg), cefotaxime (COX, 5 μg), ceftazidime (CZD, 10 μg), cefepime (FEP, 30 μg), streptomycin (SMN, 10 μg), spectinomycin (SPT, 100 μg), gentamicin (GEN, 10 μg), amikacin (AKN, 30 μg), tigecycline (TGC, 15 μg), kanamycin (KAN, 30 μg), sulfonamides (SSS, 200 μg), trimethoprim (TMP, 5 μg), trimethoprim-sulfamethoxazole (SXT, 25 μg), chloramphenicol (CHL, 30 μg), tetracycline (TET, 30 μg), nalidixic acid (NAL, 30 μg), ciprofloxacin (CIP, 5 μg), pefloxacin (PEF, 5 μg), meropenem (MEM, 10 μg), and azithromycin (AZM, 15 μg). The minimum inhibitory concentrations (MICs) of NAL and CIP were determined by E-tests (BioMérieux) on NAL-resistant isolates identified by the disk diffusion method. The recommended reference strain Escherichia coli ATCC 25922 was used as a control for antibiotic susceptibility testing. This technique was performed at the CNR-ESS.
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