M ller hinton agar

C. jejuni NCTC11168 was used in all experiments and was grown under microaerophilic conditions (4% O₂, 10% CO₂, 86% N₂) at 42°C on Müller-Hinton agar (Oxoid) plates supplemented with vancomycin (10 µg/ml) and trimethoprim (5 µg/ml). The initial inoculum for the experiments was prepared by taking the bacteria from frozen glycerol stocks stored at −80°C and then restreaking them onto a second plate, which was then scraped into Müller-Hinton broth (Oxoid) to resuspend the cells.
To apply a population bottleneck, the required number of colonies was picked with a loop (for smaller bottleneck sizes) or estimated and then scraped off the plate (for larger bottleneck sizes) before being resuspended in Müller-Hinton broth and then serially diluted onto agar plates. Individual colonies were picked from the inoculum (60 colonies) and the final population (30 colonies) after five successive bottlenecks. The repeat numbers, expression states, and phasotypes for 28 phase-variable loci of these colonies were determined with the 28-locus PV assay (29 (link)).

Due to logistic constraints, a third of the study isolates (20/68, 29 %) were randomly selected for phenotypic susceptibility testing by minimum inhibitory concentrations (MICs). MICs were performed by the agar dilution method [50 (link)], according to the European Committee on Antimicrobial Susceptibility Testing v9.0 (EUCAST, 2019) guidelines. Stock solutions of 1000 mg l⁻¹ were initially prepared, from which the working solutions were made. For each antibiotic, duplicate twofold serial dilutions (from 32 mg l⁻¹ to 0.03 mg l⁻¹) were done in molten Müller–Hinton agar (Oxoid, Basingstoke, UK). The results were interpreted according to EUCAST breakpoint tables (http://www.eucast.org). Where EUCAST cut-off values were not available, the recommended cut-off values from the Clinical Laboratory Standards Institute (https://www.clsi.org) were used.

A set of 300 isolated colonies of E. coli were randomly selected for antibiotic resistance profiling against clinically relevant antibiotics. These included 100 from untreated sludge from each WWTP (sample points 1 and 4) and 100 from after MAD at WWTP two (sample point 5). All antibiotic testing was carried out according to CLSI disk diffusion guidelines (Clinical Laboratory Standards Institute, 2017). Selected isolates were first re-streaked from MLGA onto nutrient agar and incubated for 24 hours at 37°C. The isolate was then resuspended in phosphate buffered saline (PBS) to an OD₆₀₀ of 0.5 using a MacFarland standard, plated onto Müller-Hinton agar (Oxoid) and incubated at 35°C for 18–20 hours. Zones of inhibition were measured to the nearest millimetre.
The antibiotics used were ciprofloxacin (5 μg), gentamicin (10 μg), meropenem, (10 μg), ampicillin (10 μg) (used to test for amoxicillin resistance (Clinical Laboratory Standards Institute, 2017)), trimethoprim (5 μg), and four different third generation cephalosporins—ceftazidime (30 μg), cefoperazone (75 μg), ceftiofur (30 μg) and cefpodoxine (30 μg) (all antibiotics were from Oxoid., Basingstoke, UK).

Antimicrobial susceptibility testing was performed using VITEK^®2 automated system (biomérieux, Marcy-l’Etoile, France). In addition, Etests^® (biomérieux, Marcy-l’Etoile, France) for ceftazidime were performed on Müller-Hinton-Agar (Oxoid, Schwerte, Germany) to determine the minimal inhibitory concentration (MIC). All MICs were interpreted according to the 2023 European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints version 13.1 (EUCAST, 2023 ).

A total of 32 GAS isolates, including 27 erythromycin-resistant [minimum inhibitory concentration (MIC) ≥ 1 μg/mL] and 5 erythromycin-susceptible strains isolated throughout Italy from children with pharyngitis (Varaldo et al., 1999 (link)), were examined. All strains had previously been characterized (Facinelli et al., 2001 (link); Spinaci et al., 2004 (link), 2006 (link)) in terms of erythromycin resistance phenotype/genotype [erm(B)/cMLS (n = 6); erm(B)/iMLS (n = 5); erm(TR)/iMLS (n = 6); mef(A)/M (n = 10)]; emm type (12 different emm types); the presence of the prtF1 gene, and cell invasiveness. Each of the 32 strains is a clone identified among Italian GAS isolates.
Blood agar base (BAB) supplemented with 5% sheep blood, Müller-Hinton agar (MHA) supplemented with 5% sheep blood, Müller-Hinton cation-adjusted broth (CAMHB) supplemented with 3% laked sheep blood, brain heart infusion (BHI) agar and broth, Todd-Hewitt broth (THB) and Tryptone Soya Broth (TSB), all from Oxoid (Basingstoke, UK) were used throughout the study. Isolates were maintained in glycerol at –70°C and subcultured twice on BAB before testing.

Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa ATCC 27853, Pseudomonas putida and Pseudomonas aeruginosa polyresistant bacteria were used in the experiments. The bacteria are available from our own strain collection at the Department of Medical Microbiology and Immunology, Medical School, University of Pécs, Pécs, Hungary. The strains were plated on Müller-Hinton agar (Oxoid Ltd., Basingstoke, UK) and 5% sheep blood agar media and incubated for at least 1 day at 25 °C, 37 °C and 42 °C. Fatty acid composition of bacterial strains was determined following 1, 3 and 5 days of the incubation. The antimicrobial sensitivity of strains was detected using a filter paper disc method. We used the product of Oxoid Ltd., Basingstoke, UK and we fully adhered to protocol suggested by Oxoid firm. The Pseudomonas aeruginosa ATCC 27853 was sensitive to ciprofloxacin, ceftazidime and imipenem—cilastatin, the Pseudomonas aeruginosa polyresistant was resistent to the same antimicrobial drugs.

Synthesized compounds were prepared for Antibacterial Activity by dissolving in Dimethyl Sulfoxide (DMSO) and then diluted in sterile distilled water to obtain a final concentration of 100 mg/mL. To check the effect of solvent on the growth of microorganisms, DMSO (100 µL) was used as negative control [48 (link)]. The antibacterial activity of different compounds was evaluated by agar well diffusion method. Briefly, 100 μL of 0.5 MacFarland bacterial suspensions (1.5 × 10⁸ CFU/mL) was spread on Müller-Hinton agar (Oxoid, Basingstoke, UK). Wells were made on the agar plates using sterile cork borer, then 100 μL of each prepared compound was introduced into appropriately marked wells. The antibacterial activity was evaluated by measuring the diameter of the inhibition zone for each tested organism compared with the negative and positive control after an incubation period of 24 h at 37 °C. DMSO (100 µL) was used as a negative control, and an antibiotic disc of Ciprofloxacin (5 μg) was used as a positive control [48 (link),49 ].