Tetracycline

Because the disc diffusion method only allows the analysis of the impact of a single concentration of an antibiotic, in the second stage of the experiment, the E-test strips with exponentially decreasing antibiotic concentrations were applied for all of the antibiotics that were previously used in the disc diffusion test.
The cultures with the E-tests were exposed and incubated in the same way as described for the disc diffusion method. The E-tests containing cefoxitin, amoxicillin, amoxicillin/clavulanic acid, erythromycin, clindamycin, tetracycline, ciprofloxacin, gentamicin, and teicoplanin were obtained from Liofilchem (Roseto degli Abruzzi, Italy). The analyses were performed on M–H agar in accordance with the E-test manufacturer’s recommendations.
The location of the Petri dishes in the RMF reactor chamber and the location of antibiotic discs and E-tests in Petri dishes are presented in Figure 7.

A total of 18 antibiotics were used in the susceptibility test, by disk diffusion, with amoxicillin, amoxicillin and clavulanic acid, cefoxitin, cefotaxime, ceftazidime, ceftiofur, cefepime, ceftaroline, aztreonam, meropenem, ciprofloxacin, gentamicin, sulfamethoxazole-trimethoprim, tigecycline, tetracycline, fosfomycin, chloramphenicol, and nitrofurantoin (Liofilchem, Roseto degli Abruzzi, Italy). The interpretation was performed according to CLSI [45 ]. tigecycline resistance was interpreted according to EUCAST [46 ]. E. coli ATCC 25922 was used as quality control. In accordance with EUCAST standards, the minimal inhibitory concentration (MIC) for colistin was determined by the broth microdilution method [46 ]. MDR profiles were determined according to standard criteria [26 (link)]. ESBL detection was performed using the double-disk synergy test (DDST) [45 ].

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 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)].

Eleven carbapenem resistance genes (bla_IMP, bla_VIM, bla_NDM, bla_SPM, bla_AIM, bla_DIM, bla_GIM, bla_SIM, bla_KPC, bla_BIC, and bla_OXA–48) (Poirel et al., 2011 (link)) were screened using PCR detection from the presumptive carbapenemase-producing bacteria. The amplicons were sequenced (Macrogen) and identified using NCBI BLAST¹. For the screened carbapenemase-producing bacteria, MDR to 16 antibiotics was determined using Kirby-Bauer disk diffusion, and resistance to colistin was determined using broth dilution methods. For MDR, the following antibiotic disks were used: ampicillin-sulbactam (10/10 μg), cefotaxime (30 μg), ceftazidime (30 μg), chloramphenicol (30 μg) ciprofloxacin (5 μg), colistin (2 mg/L), doripenem (10 μg), fosfomycin (200 μg), gentamicin (10 μg), levofloxacin (5 μg), meropenem (10 μg), netilmicin (10 μg), piperacillin (100 μg), tetracycline (30 μg), tobramycin (10 μg), and trimethoprim-sulfamethoxazole (1.25/23.75 μg) (Liofilchem, Roseto degli Abruzzi, Italy). Resistance to the antibiotics was determined according to the Clinical and Laboratory Standards Institute (CLSI) guideline (Clinical Laboratory Standars and Institue, 2016 ). Subsequently, MICs of 16 antibiotics for E. coli strain N7 were evaluated using the broth dilution method (Hasselmann, 2003 (link)).

The Kirby Bauer disk diffusion method was used with Muller Hinton agar (Oxoid Ltd. Basingstoke, Hampshire, England) to determine antimicrobial susceptibility patterns of the isolates and CLSI M100 2020 was used to interpret the results [30 ].
The following antimicrobial discs were used: ampicillin (10μl), amoxicillin/clavulanic acid (20/10μl), piperacillin/tazobactam (100/10μl), cefazolin (30μl), cefuroxime (30μl), ceftazidime (30μl) obtained from Hardy Diagnostics, Santa Maria, CA, USA. Ceftriaxone (30μl), cefotaxime (30μl), cefepime (30μl), imipenem (10μl), meropenem (10μl), amikacin (30μl), gentamicin (10μl), and tobramycin(10μl) were obtained from OXOID LTD., Basingstoke, Hampshire, England. Nalidixic acid(30μl), ciprofloxacin(5μl), trimethoprim/sulfamethoxazole (1.25/23.75μl), nitrofurantoin(300μl), and tetracycline(30μl) from Liofilchem, Roseto degli Abruzzi, Italy.