Susceptibility testing was performed by Kirby Bauer disk diffusion method using 22 antibiotics: amikacin, ampicillin, cefpodoxime, ceftazidime, ceftriaxone, cefuroxime, ciprofloxacin, ertapenem, imipenem, nalidixic acid, nitrofurantoin, tetracycline, trimethoprim/sulfamethoxazole (Becton Dickinson, NJ), ampicillin-sulbactam, aztreonam, cefazoline, cefepime, cefotaxime, ceftiofur, chloramphenicol, kanamycin, and streptomycin (Oxoid), according to the Clinical and Laboratory Standards Institute, CLSI.18 ESCs and ciprofloxacin-resistant isolates were selected and cryopreserved for further analysis. Multiresistance was defined as the resistance to three or more structural classes of antibiotics19 (link) and M100 from CLSI (30th edition, 2020) was used for interpretation and antimicrobial class classification (Glossary I).20 Isolates showing an intermediate resistant pattern were included in the resistant group for the analysis of antibiotic susceptibility but excluded from the minimal inhibitory concentration (MIC) assays. MICs were performed manually using the microdilution method on isolates that showed resistance to cefotaxime, ciprofloxacin, and nalidixic acid as recommended by CLSI.21 Concentrations from 0.125 (μg/mL) to 256 (μg/mL) were evaluated for each antibiotic.
Ertapenem
Manufactured by BD
Sourced in Germany, Spain, United States
Ertapenem is a broad-spectrum antibiotic medication used in the treatment of various bacterial infections. It belongs to the class of carbapenems, a group of powerful antibiotics effective against a wide range of gram-positive and gram-negative bacteria. Ertapenem is administered intravenously or intramuscularly and is designed to inhibit bacterial cell wall synthesis, thereby preventing the growth and replication of infectious bacteria.
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Lab products found in correlation
Imipenem, by BD (3 mentions)
Ciprofloxacin, by BD (3 mentions)
Trimethoprim sulfamethoxazole, by BD (3 mentions)
Meropenem, by BD (3 mentions)
Ampicillin, by BD (2 mentions)
Ceftazidime, by BD (2 mentions)
Tetracycline, by BD (2 mentions)
Cefpodoxime, by BD (2 mentions)
Ceftriaxone, by BD (2 mentions)
Amikacin, by BD (2 mentions)
Minocycline, by BD (2 mentions)
Gentamicin, by BD (2 mentions)
Kanamycin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Ceftiofur, by Thermo Fisher Scientific (1 mentions)
Nalidixic acid, by BD (1 mentions)
Cefuroxime, by BD (1 mentions)
Nitrofurantoin, by BD (1 mentions)
Aztreonam, by Thermo Fisher Scientific (1 mentions)
Ampicillin sulbactam, by Thermo Fisher Scientific (1 mentions)
5 protocols using ertapenem
1
Antibiotic Susceptibility Testing Protocol
2
Antimicrobial Susceptibility Testing Protocol
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During the antimicrobial susceptibility testing step, the following were used: the diffusion method on Mueller– Hinton Agar (Becton Dickinson, Frankfurt, Germany) and imipenem (10 µg) and meropenem (10 µg) discs plus ertapenem (10 µg) for K. pneumoniae only (Becton Dickinson, Germany). The results of the antimicrobial susceptibility tests were interpreted according to the European Committee on Antimicrobial Susceptibility Testing recommendations (EUCAST, Breakpoint tables for bacteria, Clinical breakpoints—bacteria v. 12.0) [22 ]. P. aeruginosa was purchased from American Type Culture Collection (ATCC 27853) and E. coli ATCC 25922 strains served as a susceptibility testing quality control.
3
Conjugation of Ertapenem-Resistant Plasmids
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Conjugation experiments were carried out in Luria-Bertani broth (LB, Oxoid) with sodium azide-resistant Escherichia coli J53 and Acinetobacter baylyi (MIC of rifampicin 16 mg/L) as the recipients. Transconjugants were selected by plating onto LB agar plates (Oxoid) supplemented with 100 mg/L of sodium azide and 0.125 mg/L of ertapenem (Sigma, Madrid, Spain) using E. coli J53 as recipient, and 8 mg/L of rifampicin (Sigma, Madrid, Spain) and ertapenem, respectively, using A. baylyi as recipient.
Plasmid DNA was extracted by the Kieser method, electroporated into E. coli DH10B and plated on MacConkey agar (Becton Dickinson, Madrid, Spain) supplemented with ertapenem at 0.125 mg/L. 11 (link)
Plasmid DNA was extracted by the Kieser method, electroporated into E. coli DH10B and plated on MacConkey agar (Becton Dickinson, Madrid, Spain) supplemented with ertapenem at 0.125 mg/L. 11 (link)
4
Antibiotic Susceptibility Testing Protocol
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The following CLSI recommended quality control (QC) strains from the American Type Culture Collection (ATCC, Manassas, VA) were used: Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 25923 (26 ). Kirby-Bauer disk diffusion with amikacin, ampicillin, ampicillin/sulbactam, aztreonam, cefazolin, cefepime, cefotetan, ceftazidime, ceftazidime/avibactam (Hardy Diagnostics), ceftriaxone, ciprofloxacin, doxycycline, ertapenem, gentamicin, imipenem, levofloxacin, meropenem, minocycline, piperacillin/tazobactam, tobramycin, and trimethoprim/sulfamethoxazole (all disks were from BD unless otherwise indicated) was performed and interpreted in accordance with CLSI guidelines (26 ). Gradient diffusion with imipenem/relebactam (bioMérieux, Durhman, NC) and meropenem/vaborbactam (Liofilchem, Waltham, MA) was performed and interpreted in accordance with package inserts and CLSI guidelines (26 ). Mueller-Hinton (MH) II agar (BD) was used for both AST methods.
5
Antibiotic Susceptibility Testing Protocol
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Antimicrobial susceptibility testing was done by the Kirby-Bauer standard disk diffusion method [11] according to CLSI guidelines [12] for different antimicrobial agents like: ceftazidime (30 µg), cefotaxime (30 µg), cefpodoxime (10 µg), ceftriaxone (30 µg), cefepime (30 µg), aztreonam (30 µg), ampicillin (10 µg), piperacillin (100 µg), cefoxitin (30 µg), gentamicin (120 µg), amikacin (30 µg), ciprofloxacin (5 µg), tetracycline (30 µg), minocycline (30 µg), chloramphenicol (30 µg), trimethoprim/sulfamethoxazole (1.25 µg/23.75 µg), colistin (10 µg), ertapenem (10 µg) and meropenem (10 µg) (BD Diagnostics, Franklin Lakes, NJ, USA).
The MIC values (mg/L) of cefotaxime, ertapenem, meropenem, amikacin, gentamicin and tigecycline were determined using Etest method (AB Biodisk, Solna, Sweden) and were interpreted according to CLSI guidelines as modified in 2013. The clinical breakpoints for meropenem were as follows: susceptible (S) ≤1.0 mg/L, intermediate (I) 2.0–3.0 mg/L, and resistant (R) ≥4.0 mg/L. The same for ertapenem were as follows: S ≤0.5 mg/L, I: 1.0 mg/L, R ≥2 mg/L. MIC50 and MIC90 of meropenem were calculated as the MIC at which 50% and 90% of the isolates were inhibited.
The MIC values (mg/L) of cefotaxime, ertapenem, meropenem, amikacin, gentamicin and tigecycline were determined using Etest method (AB Biodisk, Solna, Sweden) and were interpreted according to CLSI guidelines as modified in 2013. The clinical breakpoints for meropenem were as follows: susceptible (S) ≤1.0 mg/L, intermediate (I) 2.0–3.0 mg/L, and resistant (R) ≥4.0 mg/L. The same for ertapenem were as follows: S ≤0.5 mg/L, I: 1.0 mg/L, R ≥2 mg/L. MIC50 and MIC90 of meropenem were calculated as the MIC at which 50% and 90% of the isolates were inhibited.
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