Amoxicillin clavulanic acid

After PCR validation, the E. coli suspensions were subjected to antimicrobial susceptibility testing on Müller–Hinton agar using the Kirby–Bauer disk diffusion method, in accordance with the Clinical and Laboratory Standards Institute guidelines, using E. coli ATCC 25922 as an internal quality control. The antimicrobial disks used for the test included ampicillin (10 μg/disk), amoxicillin/clavulanic acid (20/10 μg/disk), ceftriaxone (30 μg/disk), meropenem (10 μg/disk), aztreonam (30 μg/disk), gentamicin (10 μg/disk), trimethoprim/sulfamethoxazole (1.25/23.75 μg/disk), amikacin (30 μg/disk), and ciprofloxacin (CIP) (5 μg/disk) (Oxoid Ltd., Hampshire, United Kingdom).

The antimicrobial susceptibility was evaluated according to Clinical and Laboratory Standarts Institude (2012 ) by using disc diffusion method. Briefly, isolates were cultured aerobically in 10 mL Mueller-Hinton (MH) broth (Merck, Darmstadt, Germany) at 37°C for 24 h. Overnight cultures, grown on MH broth (OD adjusted to 0.5 MacFarland unit), were swabbed evenly with sterile non-toxic cotton swab on MH agar plates and left to dry for 2 to 4 min. Then, antimicrobial sensitivity discs were placed on the culture by using a disk dispenser and incubated at 37°C for 24 h. The tested antimicrobials were amoxicillin/clavulanic acid (AMC, 30 μg), amoxycillin (AML, 30 μg), ceftazidime (CAZ, 30 μg), cephazolin (KZ, 30 μg), ciprofloxacin (CIP, 5 μg), erythromycin (E, 15 μg), chloramphenicol (C, 30 μg), ampicillin (AMP, 10 μg), penicillin (P, 10 μg), streptomycin (S, 10 μg), tetracycline (TE, 30 μg), kanamycin (K, 30 μg), gentamicin (CN, 10 μg), vancomycin (VA, 30 μg), nalidixic acid (NA, 30 μg), and suphamethoxazole/trimethoprim (SXT, 25 μg) (Oxoid, Hamphire, United Kingdom). The multiple antibiotic resistance (MAR) index was calculated as “a/b,” where “a” the number of antibiotics for a particular isolate was resistant and “b” the total number of antibiotics tested (Krumperman, 1983 (link)).

Antimicrobial susceptibility profiles of the isolates were determined using the disk diffusion method according to the standard procedures of the Clinical and Laboratory Standards Institute (CLSI, 2013 ). Escherichia coli (ATCC25922) (Microbiologics, USA) was used as the quality control strain. The following 17 antibiotic discs (Oxoid) were used: amikacin, amoxicillin+clavulanic acid, ampicillin+sulbactam, azithromycin, cefepime, cefixime, cefotaxime, cefoxitin, cefperazone, ceftazidime, ceftriaxone, chloramphenicol, gentamycin, imipenem, levofloxacin, meropenem, and trimethoprim-sulphamethexazole. MDR was defined according to the guidelines of the European Society of Clinical Microbiology and Infectious Diseases (Magiorakos et al., 2012 (link)).

Disk diffusion assay was performed to assess the antibiotic resistance/susceptibility pattern of bacterial isolates. Antimicrobial susceptibility testing was carried out on Muller-Hinton agar (Oxoid, England) using the single disc diffusion technique against tetracycline (30 μg), penicilin G (10 μg), erythromycin (15 μg), gentamicin (10 μg), ciprofloxacin (5 μg), norfloxacillin (10 μg), trimethoprim-sulphamethoxazole (25 μg), nitrofurantonin (300 µg), doxycycline (30 µg), ceftriaxone (30 μg), ampicillin (10 μg) and amoxicillin clavulanic acid (10 μg) (all Oxoid, England). Selection of these antibiotics was based on the frequently used in the country for the treatment of otitis media. Results were reported as sensitive, intermediate and resistance according to Clinical Laboratory Standards Institute (CLSI 2015 ) guide lines. An isolate was defined as being multidrug resistant if it is resistant to three or more of the antimicrobial agents tested and based on the antimicrobial categories as stated by Magiorakos et al (2012 (link)).

Antimicrobial susceptibility testing of E. coli isolates were performed by the Kirby-Bauer disk diffusion assay on Mueller-Hinton agar medium (Oxoid, Basingstoke, England) as recommended by Clinical Laboratory Standard Institute (CLSI, 2011), against 15 antimicrobial agents from different categories including: amikacin (30 μg), amoxicillin (10 μg), amoxicillin-clavulanic acid (30 μg), ceftazidime (30 μg), ceftriaxone (30 μg) cefuroxime (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamicin (10 μg), nalidixic acid (30 μg), nitrofurantoin (50 μg), ofloxacin (5 μg), tetracycline (30 μg), tobramicin (10 μg) and trimethoprim- sulfamethoxazole (25 μg) (Oxoid, Basingstoke, England). E. coli isolate was considered non-susceptible to an antimicrobial agent when it tested resistant, intermediate or non-susceptible when using clinical breakpoints as interpretive criteria, provided by the CLSI, (2011). MDR patterns of E. coli isolates were defined as non-susceptibility to at least one agent in three or more antimicrobial categories (Magiorakos et al., 2012 ).

Salmonella isolates were evaluated for antimicrobial resistance using the Kirby–Bauer disk diffusion method according to the Clinical and Laboratory Standards Institute guidelines (Clinical and Laboratory Standards Institute [Clsi], 2018 ). Susceptibility to the following 22 antibiotics was tested: ampicillin, amoxicillin-clavulanic acid, cefazolin, cefoxitin, ceftriaxone, ceftazidime, cefotaxime, ceftiofur, cefepime, aztreonam, imipenem, gentamicin, kanamycin, amikacin, streptomycin, tetracycline, ciprofloxacin, enrofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, chloramphenicol, and florfenicol (Oxoid, Basingstoke, United Kingdom).