Ampicillin

The antibiotic susceptibilities of three probiotic candidates were assayed using the minimum inhibitory concentration (MIC) test strip method. Nine antibiotic strips were used for testing the bacterial strains, namely, ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin, tetracycline, and vancomycin (Liofilchem, Abruzzi, Italy). The bacteria were grown for 18 h at 37 °C in MRS medium. The cells were harvested via centrifugation at 3470× g for 5 min, washed twice with PBS (pH 7.0), and resuspended in PBS to a McFarland turbidity of 0.5. The cell suspensions were inoculated on BHI agar using swabs. The plates were dried for 15 min, and the MIC test strips were placed on the agar surface according to the manufacturer’s instructions. The plates were then incubated at 37 °C, and the results were assessed after 20 h of inoculation, according to the European Food Safety Authority (EFSA) guidelines [23 (link)].

The antibiotic resistance genes in 14 F. sanfranciscensis strains were analyzed using the CARD database (Comprehensive Antibiotic Resistance Database, http://arpcard.Mcmaster.ca, accessed on 25 December 2023). If the sequence matching degree of the resistance gene reaches 20% (e-value < 1 × 10⁻⁵), the antibiotic resistance gene is considered to exist. The Origin 2023 software was used to build a thermal map of the predicted data.
Based on the American Association for Clinical and Laboratory Standards Institute (CLSI) guidelines [34 (link)], the antibiotic resistance of the 14 F. sanfranciscensis strains was tested using the disk diffusion method. The antibiotics per disk were as follows: gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, benzathine, ampicillin, tetracycline, chloramphenicol, and mitomycin-sulfamethoxazole, purchased from Liofilchem. The strains that were classified as susceptible (S, zone diameter > 20), intermediate (IR, 15 < zone diameter < 19), or resistant (R, zone diameter ≤ 14) hinged on the diameter of the zone of inhibition around the disk [30 (link)].

The antibiotic susceptibility of B. coagulans IDCC 1201 was determined based on MIC values. In short, approximately 1–2 × 10⁸ CFU/ml of B. coagulans IDCC 1201 was spread onto each MRS agar plate. Then antibiotic (E-test) strips containing ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin, tetracycline, and vancomycin (Liofilchem, Waltham, MA, USA) were placed on the agar plates.
Cell growth inhibition was also investigated to confirm the results of the antibiotic susceptibility test. B. coagulans IDCC 1201 at 10⁶ CFU/ml and 1:1 (v/v) to each antibiotic solution at various concentrations were transferred to a 96-well plate and incubated at 37°C for 20 hr. Then, the optical density of each incubation was observed for 20 hr using a microplate reader (BioTek, Winooski, VT, USA). The cutoff values of the MICs were determined according to EFSA's technical guidelines of the EFSA on antibiotic susceptibility (EFSA, 2018 ).

A direct PCR was performed in the KEMRI-NUITM laboratory to confirm the presence of Neisseria gonorrhoeae. The direct PCR was done using the mighty amp reagents and the simpliprep machine. Specifically designed primers, forward-CGGCAGCATTCAATTTGTTAAAGC and reverse-CGCCATTTTTGTA, which were to yield a PCR product of 162 base pairs each, were used [14 ]. The thermal cycle conditions for the simpliprep machine were: 94°C, 60 sec (denaturation), 59°C, 60 secs (annealing), 72°C, 26 sec/max 429bp (1 min. kbp), (elongation), 72°C, 10 minutes (extended elongation). When the reaction was over, the samples were loaded on gel electrophoresis, stained, and viewed under ultraviolet light (UV).
The antimicrobial susceptibility testing was done by disk diffusion using 13 antimicrobials from Liofilchem Company (Ciprofloxacin (CIP,30μg), Ceftriaxone (CRO,30μg), Cefepime (FEP,25μg), Cefotaxime (CTX,30μg), Cefuroxime (CXM,30μg), Cefazolin (KZ,30μg), Ceftazidime (CAZ,30μg), Cefoxitin (FOX,30μg), Tetracycline (TE,30μg), Ampicillin (AMP,10μg), Amikacin (AK,30μg), Gentamycin (CN,30μg) and Amoxillin (AUG,30μg).The antimicrobial discs were inoculated onto a culture of Neisseria gonorrhoeae and incubated for 24–48 hours. The zone of inhibition was measured and interpreted using the CLSI 2016 guidelines.

The determination of bacterial sensitivity to selected antibiotics was performed according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The method was slightly modified to adapt to the tested strain.
Bacterial culture of Lpb. plantarum DB2, grown in MRS broth, was separated from the medium by centrifugation at 6440× g for 10 min and resuspended in sterile water, with OD values adjusted to 0.5 ± 0.05. The cell suspension was applied to MRS agar plates by uniformly swabbing a cotton swab, previously immersed in the suspension, in three directions. After inoculation, discs of the tested antibiotics (Liofilchem S.r.l., Roseto degli Abruzzi, Italy) were placed on the inoculated MRS agar. The plates were then incubated at 37 °C, and after 24 h, the appearance of inhibition zones was observed, indicating the sensitivity of the tested strain to the antibiotic.
In this study, the sensitivity of lactic acid bacteria isolate DB2 to the following antibiotics was investigated: ampicillin (10 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), clindamycin (10 µg), chloramphenicol (10 µg), streptomycin (10 µg), tetracycline (30 µg), and vancomycin (30 µg) (Liofilchem S.r.l., Roseto degli Abruzzi, Italy).