Rifampicin

A modification of the standard disk diffusion method (rAST) was evaluated for the detection of antimicrobial resistance. The rAST followed CLSI standards in all aspects, except for the inoculum preparation. CLSI guideline recommends preparing the inoculum for AST with a direct suspension of isolated colonies selected from 18–24 h agar plate incubation [6 ]. In this study, we used colonies selected from a rapid culture grown (4–6 h agar plate incubation). The inoculum was prepared from the rapid culture grown on a 150 mm Mueller–Hinton Agar (bioMérieux, France), and then, disks were applied and plates incubated at 35°C ± 1° and incubated for 18 h. The following antimicrobial agents were evaluated for GN: amikacin 30 μg, amoxicillin-clavulanate 20/10 μg, ampicillin 10 μg, ampicillin-sulbactam 10/10 μg, cefepime 30 μg, ceftazidime 30 μg, cefuroxime 30 μg, ciprofloxacin 5 μg, gentamicin 10 μg, meropenem 10 μg, piperacillin-tazobactam 100/10 μg, and trimethoprim-sulfamethoxazole 23.75/1.25 μg (Oxoid®, Thermo-Fisher, USA). For GP, cefoxitin 30 μg, clarithromycin 15 μg, clindamycin 2 μg, doxycycline 30 μg, erythromycin 15 μg, gentamicin 10 μg, levofloxacin 5 μg, rifampicin 5 μg, and trimethoprim-sulfamethoxazole 23.75/1.25 μg (Oxoid®) were evaluated. The inhibition zones were analyzed after 18–24 h, and the results were interpreted by CLSI proposed breakpoints [6 ].

Antibiotic susceptibility testing was performed by disk diffusion method following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The tested antimicrobial agents were: cefoxitin (30 μg), gentamicin (10 μg), tetracycline (30 μg), trimethoprim-sulfamethoxazole (1.25/23.75 μg), erythromycin (15 μg), clindamycin (2 μg), ciprofloxacin (5 μg), mupirocin (200 μg), rifampicin (5 μg), fusidic acid (10 μg) and penicillin (1 μg) (all Oxoid, Basingstoke, UK). Isolates were classified as susceptible or resistant based on S. aureus epidemiological cut-off values issued by the EUCAST. erythromycin-induced clindamycin resistance was detected by Disk approximation test (D-test). The reference strain S. aureus ATCC 29213 was used as internal quality control. The nitrocefine test was completed using beta-lactamase identification sticks (Oxoid, Basingstoke, UK). The sizes of inhibition zone diameters were independently read by at least three operators and then averaged to obtain the final inhibition zone diameters (in mm).

Antibiotic resistance phenotypes were determined by agar diffusion method [20 ].The bacterial inoculum was prepared by bacterial dilution in sterile MilliQ water. This inoculum was seeded in plates with Muller Hinton agar (Oxoid, Basingstoke, England) and after placed the antibiotics disks. The tested antibiotics were as follows: the beta-lactams ticarcillin (75 μg), ticarcillin/clavulanic acid (75/10 μg), ceftazidime (10 μg), cefepime (30 μg), imipenem (10 μg), andmeropenem (10 μg); the aminoglycosides streptomycin (10 μg) and gentamicin (10 μg); tobramycin (10 μg) andamikacin (30 μg); the quinolones levofloxacin (5 μg), nalidixic acid (30 μg), and ciprofloxacin (10 μg); the polymyxincolistin sulfate (10 μg); the sulfonamide sulfamethoxazole (25 μg) andsulfamethoxazole/trimethoprim (23.75/1.25 μg); fosfomycin (50 μg); rifampicin (5 μg); and tetracycline (30 μg) (Oxoid, Basingstoke, England). The strains Escherichia coli ATCC 25922 and Pseudomonas aeruginosa DSM 1117 (ATCC 27853) were included as quality controls. Phenotypes were classified as resistant, intermediary or susceptible according to the manufacturer’s instructions.

The antibiotic susceptibility of the selected Bacillus strains was tested using a disk diffusion method according to Clinical and Laboratory Standard Institute (CLSI) performance standards for antimicrobial susceptibility testing [22 (link)]. Eleven kinds of antibiotics (Oxoid, Basingstoke, UK) were used: Ampicillin (AMP, 10 µg), Chloramphenicol (C, 30 µg), Ciprofloxacin (CIP, 5 µg), Erythromycin (E, 15 µg), Gentamycin (CN, 10 µg), Kanamycin (K, 30 µg), Tetracycline (TE, 30 µg), Teicoplanin (TEC, 30 µg), Vancomycin (VA, 30 µg), Rifampicin (RD, 30 µg), and Streptomycin (S, 10 µg). Bacillus cultures, adjusted to approximately 1 × 10⁸ CFU/mL using the 0.5 McFarland standard, were spread onto nutrient agar plates. Antibiotic discs were loaded onto the agar. The diameter of the inhibition zone for each antibiotic was detected after incubation at 37 °C for 24 h.

Bacillus cereus ATCC14579, Escherichia coli ATCC25922, Pseudomonas aeruginosa ATCC27853, and Staphylococcus aureus ATCC25923 (ATCC, USA) were used as pathogenic representatives for antibacterial screening in this work. The NSS inoculum of each standard bacterium with approximately 1.5 × 10⁸ cells/mL was swabbed on NA. Five mg of each prepared crude extract was impregnated into sterile 6 mm diameter filter paper discs (Whatman, UK) before being placed onto the swabbed NB. The discs containing ampicillin (10 μg/disc), ceftriaxone (30 μg/disc), chloramphenicol (30 μg/disc), and rifampicin (5 μg/disc) (Oxoid, UK) were used as positive control for Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus, respectively. After incubation for overnight, the antibacterial activity of each crude extract was determined by measuring the IZD in mm.