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)
Ertapenem
Manufactured by Bio-Rad
Sourced in France
Ertapenem is a broad-spectrum antibiotic medication used to treat a variety of bacterial infections. It is a member of the carbapenem class of antibiotics, which are effective against both Gram-positive and Gram-negative bacteria. Ertapenem is primarily used to treat complicated intra-abdominal infections, complicated skin and skin structure infections, and community-acquired pneumonia.
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Lab products found in correlation
Gentamicin, by Bio-Rad (6 mentions)
Ciprofloxacin, by Bio-Rad (6 mentions)
Imipenem, by Bio-Rad (6 mentions)
Amikacin, by Bio-Rad (5 mentions)
Piperacillin tazobactam, by Bio-Rad (4 mentions)
Cefotaxime, by Bio-Rad (4 mentions)
Cefepime, by Bio-Rad (4 mentions)
Amoxicillin clavulanic acid, by Bio-Rad (4 mentions)
Aztreonam, by Bio-Rad (3 mentions)
Ceftazidime, by Bio-Rad (3 mentions)
Meropenem, by Bio-Rad (3 mentions)
Cefoxitin, by Bio-Rad (3 mentions)
Trimethoprim sulfamethoxazole, by Bio-Rad (3 mentions)
Ticarcillin, by Bio-Rad (2 mentions)
Piperacillin, by Bio-Rad (2 mentions)
Tobramycin, by Bio-Rad (2 mentions)
Rifampicin, by Bio-Rad (2 mentions)
Ticarcillin clavulanic acid, by Bio-Rad (2 mentions)
Colistin, by Bio-Rad (2 mentions)
Streptomycin, by Bio-Rad (2 mentions)
8 protocols using ertapenem
1
Evaluating Antibiotic Susceptibility with EUCAST Standards
2
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)].
3
Antibiotic Resistance Profiling of GNB Isolates
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All GNB isolates which were non-naturally colistin-resistant and grown on the LBJMR medium were subjected to an AST according to the current (DD) test method (Kirby-Bauer procedure). The minimal inhibition concentration (MIC) was confirmed by CLSI and EUCAST guidelines [28 ]. AST was performed with a definite turbidity bacterial suspension in NaCl (0.5 McFarland; 1.5 × 108 cells/mL). Antibiogram test included the following sixteen antibiotics: amoxicillin (AMX), amoxicillin-clavulanic acid (AMC), cefepime (FEP), piperacillin/tazobactam (TPZ), cefalotin (KF), ceftriaxone (CRO), ertapenem (ETP), imipenem (IMP), fosfomycin (FF), nitrofurantoin (F), trimethoprim-sulfamethoxazole (SXT), amikacin (AK), ciprofloxacin (CIP), doxycycline (DO), colistin (CT), and gentamicin (GT) (Bio-Rad, Marne-la-Coquette, France). Hierarchical clustering of the antibiotic resistance phenotype was performed using Multi-Experiment Viewer (MeV 4.9.0).
Strains with a narrow diameter zone of inhibition (ZOI) less than 15 mm were picked out to confirm the minimal inhibition concentration value using other complementary tests, namely the E-tests method (BioMérieux) and UMIC test (Biocentric Bandol, France) [29 (link)]. Furthermore, strains were considered to be multidrug-resistant (MDR) if bacteria were resistant to more than three different classes of antibiotics.
Strains with a narrow diameter zone of inhibition (ZOI) less than 15 mm were picked out to confirm the minimal inhibition concentration value using other complementary tests, namely the E-tests method (BioMérieux) and UMIC test (Biocentric Bandol, France) [29 (link)]. Furthermore, strains were considered to be multidrug-resistant (MDR) if bacteria were resistant to more than three different classes of antibiotics.
4
Phenotypic and Genotypic Characterization of E. coli
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Isolates were identified to species level using API20E kits (bioMérieux, Marcy l’Étoile, France) and 16S rRNA sequencing. All identified E. coli isolates were tested for resistance to amikacin, ciprofloxacin, gentamicin, tazobactam, trimethoprim/sulfamethoxazole and ertapenem (Biorad, Hercules, CA) by the disk agar diffusion technique. The zone diameters of each drug were interpreted using the criteria published by the CLSI [49 ].Isolates, which showed intermediate resistance, or resistance to ertapenem were further tested for confirmation by MIC determination using Etest strips for ertapenem, meropenem, and imipenem. All these isolates (n = 27) were further characterized using WGS.
5
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)].
6
Antibiotic Resistance Profiling of Bacterial Strains
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Antibiotic susceptibility profiles were determined for every wild-type and transconjugant strain by the disc diffusion method, following the EUCAST guidelines (www.eucast.org ; Supplementary Data 2 ). We used the following antimicrobials agents: imipenem (10 µg), ertapenem (10 µg), amoxicillin-clavulanic acid (20/10 µg), rifampicin (30 µg), streptomycin (300 µg), chloramphenicol (30 µg), and amikacin (30 µg; BioRad, CA, USA). pOXA-48-carrying and pOXA-48-free strains were pre-cultured in Müller-Hinton (MH) broth at 37 °C in 15 ml test tubes with continuous shaking (250 r.p.m.), and disc diffusion antibiograms were performed on MH agar plates (BBL, Becton Dickinson, MD, USA).
7
Antibiotic Susceptibility Testing Protocol
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Antibiotic susceptibility was determined by disc diffusion on Mueller–Hinton agar, in accordance with the guidelines of the Antibiogram Committee of the French Society for Microbiology (CA-SFM & EUCAST, 2014 ). The following 32 antimicrobial drugs (Bio-Rad) were tested: ampicillin, ticarcillin, piperacillin, piperacillin/tazobactam, cefamandole, cefoperazone, cefoxitin, cefotaxime, amoxicillin/clavulanic acid, ticarcillin/clavulanic acid, imipenem, meropenem, ertapenem, cefepime, ceftazidime, streptomycin, spectinomycin, kanamycin, amikacin, gentamicin, netilmicin, tigecycline, isepamicin, nalidixic acid, pefloxacin, ciprofloxacin, sulfonamides, trimethoprim, sulfamethoxazole/trimethoprim, chloramphenicol, tetracycline and azithromycin. Minimal inhibitory concentration (MIC) values for nalidixic acid, ciprofloxacin and azithromycin were determined by Etests (bioMérieux). E. coli CIP 76.24 (ATCC 25922) was used as a control.
8
Antibiotic Susceptibility Testing Protocol
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Growth medium was LB Lennox (LBL) and all other chemicals, unless stated otherwise, were purchased from Sigma Chemical Co. Ampicillin stock solution 100 mg/ml in Double Distilled Water (DDW) was kept as single use aliquots at −20 °C. ertapenem (MERCK) stock solution (20 mg/ml in DDW) was kept at −20 °C. Kanamycin stock solution (30 mg/ml in DDW) was kept at −20 °C.
Antibiotic content in disks: Ampicillin 10 μg (Bio-rad) and Kanamycin 7.5 μg (Bio-rad), ertapenem 0.25 μg, cefazolin 7.5 μg (OXOID), ciprofloxacin 0.1 μg, imipenem 2.5 μg (OXOID).
Glucose (D-glucose) was purchased from JT Baker.
Mueller-Hinton (MH) plates and MH + 5% sheep blood (MH-BD) agar plates were purchased from Novamed.
Antibiotic content in disks: Ampicillin 10 μg (Bio-rad) and Kanamycin 7.5 μg (Bio-rad), ertapenem 0.25 μg, cefazolin 7.5 μg (OXOID), ciprofloxacin 0.1 μg, imipenem 2.5 μg (OXOID).
Glucose (D-glucose) was purchased from JT Baker.
Mueller-Hinton (MH) plates and MH + 5% sheep blood (MH-BD) agar plates were purchased from Novamed.
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