Chloramphenicol

Each one of the 34 microtubes was vortexed and 50 µL was distributed in two agar plates, one containing brain–heart infusion (BHI) and the second, trypticase soy with sucrose and bacitracin (a selective medium for oral Streptococci). The agar plates were incubated at 37 °C for 24 to 48 h. In total, 96 colony-forming units (CFUs) were selected and inoculated in different culture tubes containing 5 mL of sterile BHI, and incubated for 24 h.
The Kirby–Bauer disc diffusion method determined three antibiotic resistance profiles. Tetracycline (30 µg), rifampicin (5 µg), and chloramphenicol (30 µg) discs (Oxoid, Basingstoke, UK) were applied onto the plates and incubated at 37 °C for 16 to 18 h. Resistance was considered when there was a zone of inhibition of ≤12 mm for chloramphenicol, ≤16 mm for rifampicin, and ≤14 mm for Tetracycline based on the National Committee for Clinical Laboratory Standards (NCCLS) [15 ]. Out of the 96 CFUs, 38 (39%) were resistant to at least one of the three antibiotics (Table 1).

Antibacterial activity of the selected EOs and the mixture was tested by Kirby-Bauer agar disk diffusion method following the procedures reported by Clinical and Laboratory Standards Institute (CLSI) [29 ]. Briefly, EOs and the mixture were diluted 1:10 in dimethyl sulfoxide (DMSO, Oxoid Ltd.) and one absorbent paper disk was impregnated with 10 µl of each dilution, respectively, and tested against each isolate. In this way 10 µl for each disk had 171 µg for A. triphylla, 202 µg for C. zeylanicum, 179 µg for C. citratus, 177 µg for L. cubeba, 182 µg for M. piperita, 211 µg for S. aromaticum, 101 µg (C. zeylanicum) and 105 µg (S. aromaticum) for the mixture.
A paper disk impregnated with 10 µl of DMSO was included as negative control. A commercial disk impregnated with chloramphenicol (30 µg) (Oxoid Ltd.) was used as positive control. Growth inhibition zones were calculated after incubation at 37 °C for 24 h. All tests were performed in triplicate.
The in vitro sensitivity of all Salmonella strains to chloramphenicol (30 µg) (Oxoid Ltd.) was assayed by the same method and the results were interpreted as indicated by CLSI [30 ].

Antibacterial activity of the selected EOs was tested by Kirby–Bauer agar disk diffusion method following the procedures reported by Clinical and Laboratory Standards Institute (CLSI) [19 ]. Briefly, EOs were 5% diluted in dimethyl sulfoxide (DMSO, Oxoid Ltd.), and one absorbent paper disk was impregnated with 10 µL of each dilution, respectively, and tested against each isolate.
A paper disk impregnated with 10 µL of DMSO was included as negative control. A commercial disk impregnated with chloramphenicol (30 µg) (Oxoid) was used as positive control. Growth inhibition zones were evaluated after incubation at 37 °C for 24 h. All tests were performed in triplicate.
The in vitro sensitivity of all Staphylococcus isolates to chloramphenicol (30 µg) (Oxoid) was assayed by the same method, and the results were interpreted as indicated by CLSI [20 ].

Antibiotic susceptibility testing (AST) was performed using the modified Kirby-Bauer disc diffusion method according to the clinical laboratory standard Institute (CLSI) guidelines [34 ]. The following antimicrobial drugs were employed for Gram-positive bacteria (GPB): penicillin (10 μg), trimethoprim/sulfamethoxazole (1.25/23.75 μg), clindamycin (2 μg), erythromycin (15 μg), gentamycin (10 μg), ciprofloxacin (5 μg), tetracycline (30 μg), doxycycline (30 μg) and chloramphenicol (30 μg) (Oxoid, England) and for Gram-negative bacteria (GNB): amikacin (30 μg), tobramycin (10 μg), ampicillin (10 μg), amoxicillin/clavulanate (30 μg), meropenem (10 μg), gentamycin (10 μg), ciprofloxacin (5 μg), tetracycline (30 μg), doxycycline (30 μg) and chloramphenicol (30 μg) (Oxoid, England). The sensitivity test results were interpreted according to the CLSI 2018 [34 ]. Reference strains from Ethiopian Public Health Institute, E. coli ATCC 25922 and S. aureus ATCC 25923 were used for quality control.

Antimicrobial susceptibility testing was performed on E. coli isolates according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) disc diffusion method using the following antibiotic discs, selected to represent commonly used agents for E. coli infections in human and veterinary medicine: Nalidixic acid (30 µg), Nitrofurantoin (100 µg), Piperazillin-tazobactam (36 µg), Tetracycline (30 µg), Trimethoprim (5 µg), Trimethoprim-sulfamethoxazole (25 µg), Meropenem (10 µg), Ciprofloxacin (5 µg), Ampicillin (10 µg), Cefadroxil (30 µg), Chloramphenicol (30 µg), Gentamicin (10 µg) and Mecillinam (10 µg) (Thermo Fisher Scientific Oxoid Ltd, Hants, UK). The inhibition zone diameters were interpreted according to EUCAST breakpoints⁶⁵ , or, for antibiotics with no defined clinical breakpoints (i.e. Nalidixic acid, Tetracycline and Chloramphenicol), the inhibition zone diameters were interpreted by breakpoints defined by the Normalized Resistance Interpretation method^{66 (link)}.
Phenotypic characterization to identify ESBL-producing isolates was performed using the following five antibiotic discs: Ceftazidime (10 µg), Cefotaxime (5 µg), Cefepime (30 µg), Cefoxitin (30 µg) and Amoxicillin/clavulanic acid (30/1 µg) (Thermo Fisher Scientific Oxoid Ltd, Hants, UK). Inhibition zone diameters were used to determine the phenotypes, according to EUCAST guidelines⁶⁷ .