Mueller hinton agar (mha)

Bacterial identification was performed using the API 20E system (bioMérieux, Marcy-l’Etoile, France). Antibiograms for 32 antibiotics (amoxicillin, amoxicillin/clavulanic acid, aztreonam, ceftazidime, cefalotine, cefmandole, cefotaxime, cefepime, cefoxitine, imipenem, meropenem, Moxalactam, pipéracilline, ticarcilline, ticarcilline/acide clavulanique, piperacilline/tazobactam, fosfomycine, colistine, rifampicine, cotrimxazole, ciprofloxacine, pefloxacine; norfloxacine, nalidixic acid, tetracycline, tigecycline, chloramphenicol, kanamycine, amikacine, netilmicine, tobramycine, gentamicine) were determined by the disc diffusion method on Mueller-Hinton agar (BioRad, Marnes-La-Coquette, France) and the susceptibility breakpoints were determined and interpreted as recommended by the Clinical and Laboratory Standards Institute [17 ]. All plates were incubated at 37 °C for 18 h. Minimum inhibitory concentrations (MICs) of given ß-lactams were determined using the Etest technique (bioMérieux, Marcy l’Etoile, France) for the following β-lactam antibiotics: amoxicillin ± clavulanic acid, cefoxitin, cefotaxime, ceftazidime, aztreonam, cefepime and imipenem. Confirmation of ESBL producers was performed by double disc synergy testing between ticarcillin/clavulanate and aztreonam and/or ceftazidime and/or cefepime [18 (link)].

Antimicrobial susceptibilities were determined by disk diffusion on Mueller–Hinton agar (Bio-Rad, Marnes-La-Coquette, France) and the minimum inhibitory concentrations (MIC) of colistin and doxycycline were determined using the microbroth dilution method. E. coli strain ATCC25922 and P. aeruginosa strain ATCC27853 were used as reference control strains, and the aforementioned tests were performed following the Clinical and Laboratory Standards Institute (CLSI) guidelines [36 ]. Susceptibility was interpreted according to the criteria of the CLSI (M100-S29) [37 ], except for susceptibility to tigecycline, which was interpreted following the FDA criteria (susceptible: ≤2 mg/L; resistant: ≥8 mg/L) for Enterobacteriaceae.

Antibiotic susceptibility testing (AST) was performed for all isolates by disk diffusion method on Mueller-Hinton agar (Bio-Rad^®, Marnes-la-Coquette, France) according to the criteria of the European Committee on Antimicrobial Susceptibility Testing¹. Antibiotic disks were purchased from Bio-Rad^®. Consensus recommendations (Magiorakos et al., 2012 (link)) were used to evaluate the proportion of Multi Drug-Resistant (MDR, not susceptible to at least three antimicrobial groups) and extensively Drug Resistant (XDR, not susceptible to at least six antimicrobial groups) isolates, considering the following six antimicrobial groups: penicillin (ticarcillin, ticarcillin-clavulanate, piperacillin, piperacillin-tazobactam), cephalosporins (ceftazidime, cefepime), monobactams (aztreonam), carbapenems (imipenem, meropenem), aminoglycosides (gentamicin, tobramycin, amikacin), and fluoroquinolones (ciprofloxacin, levofloxacin). The other isolates – resistant to none, one or two classes of antipseudomonal agents – were defined as non-MDR. The P. aeruginosa strain ATCC 27853 was used as a quality control strain in each experiment.
Of note in a parallel study (Cottalorda et al., unpublished), the antimicrobial resistance profiles, identified for isolates collected from the first urine sample, were also explored for these seven patients.

For antimicrobial tests: Mueller Hinton agar and potato dextrose agar (PDA) were purchased from Bio-Rad (Bio-Rad, France). For the wounds healing evaluation: Balance rocking, scissors, razor blade, knife blade, pliers, cotton, syringe, beaker, spatula, “Cicaflora^®”, physiological serum, HCl (2%) and ether were used too.

Antimicrobial susceptibility assay of the bacterial strains was performed using agar disc diffusion assay method recommended by the Clinical and Laboratory Standards Institute [9 ]. Commercially available antimicrobial discs (Mast Diagnostics, U.K) of erythromycin (15 µg), gentamicin (10 µg), sulfamethoxazole-trimethoprim (25 µg), tetracycline (30 µg), ciprofloxacin (5 µg), azithromycin (15 µg), ampicillin (10 µg), ceftriaxone (30 µg), cefixime (5 µg), and mecillinam (10 µg) were placed on Mueller–Hinton agar (Bio-Rad) seeded with bacteria cultured in their early log phase, as previously described CLSI [9 ]. Following incubation, the plates were examined, and the inhibitory zone diameters for individual antimicrobial agents were measured and recorded as susceptible and resistant based on the breakpoints for respective antibiotic susceptibility of CLSI (CLSI 2010). Organisms resistant to 3 or more classes of antibiotics are designated as MDR. Zone diameter, breakpoints (mm): erythromycin (15 μg) S-I-R: >26, 23–25, <2; gentamicin (10 μg): >15, 13–14, <12; sulfamethoxazole trimethoprim (25 μg): >26, 23–25, <22; tetracycline (30 μg): >26, 23–25, <22; ciprofloxacin (5 μg): >21, 15–20, <14; ampicillin (10 µg): >17,14–16, <13; ceftriaxone (30 μg): >16, 13–15, <12; cefixime (5 μg): >19, 16–18, <15; azithromycin (15 μg): >18, 14–17, <13; mecillinam (10 μg): >15, 12–14, <11.

Each rat was treated with bronchoalveolar lavage and then was sacrificed in a CO₂ chamber and its lungs were aseptically removed and homogenized in 10 mL of normal saline. The homogenates were centrifuged at 2500×g for 10 min, and the pellets were suspended in 2.5 mL of normal saline. The counts of K. pneumonia organisms were obtained after plating serial dilutions of lung homogenates on Mueller–Hinton agar (Bio-Rad, CA, USA). The colonies were counted after 24 h of incubation at 37 °C. The lowest level of detection was 100 CFU/lung, and the rats were considered to be microbiologically cured if they were found to have counts below this level (Vasseur et al., 2014 (link)).

Antibiotic susceptibility was performed by the disc diffusion method on Mueller Hinton agar (Bio-Rad, Marne la Coquette, France) according to the recommendations of the Antibiogram Committee of the French Society for Microbiology (Comité de l’Antibiogramme de la Société Française de Microbiologie) [9 ]. The following antibiotic discs (drug concentration in μg) were tested: amoxicillin (25 μg), amoxicillin/clavulanic acid (20/10 μg), ampicillin (10 μg), imipenem (10 μg), cefotaxime (30 μg), ceftriaxone (30 μg), ciprofloxacin (5 μg), norfloxacin (5 μg), amikacin (30 μg), gentamicin (15 μg) and trimethoprim/ sulfamethoxazole (1.25/23.75 μg), all from Bio-Rad (Bio-Rad, Marne la Coquette, France).
ESBL phenotypes were detected by double-disk synergy according to the method described by Jarlier et al. [10 (link)]. Disks of cefotaxime and ceftriaxone were placed 20 mm from an amoxicillin/clavulanate disk. Enhancement of the inhibition zone of the third-generation cephalosporin toward the amoxicillin/clavulanate disk indicated the possible presence of an ESBL. Escherichia coli ATCC 25922 was used as control.