Chloramphenicol

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)].

Samples were prepared following the normalized method ISO 6887-2 [26 ]. For each biscuit, 100 g was crushed in aseptic conditions, and 25 g was taken and transferred into a sterile flask containing 225 mL of sterile saline (NaCl 0.85%). The mixture was homogenized and left at room temperature (25 ± 2°C) for 30 min. Then, serial dilutions were made (10⁻¹ to 10⁻⁶). The total aerobic mesophilic flora was enumerated on Plate Count Agar (PCA, LiofilChem, Italy) following the pour plate method ISO 4833-1 [27 ]. Yeast and mould enumeration was performed on Sabouraud agar supplemented with chloramphenicol (LiofilChem, Italy), using the method ISO 21527-1 [28 ]. The method ISO 6888-2 [29 ] was used to enumerate Staphylococcus spp. while total coliforms and E. coli were enumerated following the method ISO4832 [30 ]. Following incubation, colonies forming units appearing in the Petri dishes were counted. The microbial counts obtained from triplicate experiments were summarized as mean loads, and the final results were expressed as colonies forming units per gram of biscuits.

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)).

A 100 mL aliquot of melted ice was filtered and the membrane placed on Sabouraud dextrose agar containing chloramphenicol (0.5 g/L) (Liofilchem, Roseto degli Abruzzi, Italy). After 8 days of incubation at 28 °C, yeast colonies were identified using a semi-automated sugar assimilation system (API ID 32C, Biomerieux, Marcy l’Etoile, France), while filamentous fungi were identified by evaluating their macroscopic and microscopic morphological characteristics according to the methods described elsewhere [18 ].

Iodine (≥99.0%), copper iodide (CuI) and Mueller Hinton Broth (MHB) were obtained from Sigma Aldrich (Gillingham, UK). 1,4,7,10-Tetraoxacyclododecan (12-crown-4) was received from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Disposable sterilized Petri dishes with Mueller Hinton II agar and McFarland standard sets were bought from Liofilchem Diagnostici (Roseto degli Abruzzi (TE), Italy). The bacterial strains E. coli WDCM 00013 Vitroids, P. aeruginosa WDCM 00026 Vitroids, K. pneumoniae WDCM00097 Vitroids, C. albicans WDCM 00054 Vitroids, and Bacillus subtilis WDCM0003 Vitroids were received from Sigma-Aldrich Chemical Co. S. pneumoniae ATCC 49619, S. aureus ATCC 25923, E. faecalis ATCC 29212, S. pyogenes ATCC 19615, P. mirabilis ATCC 29906 were purchased from Liofilchem. Gentamicin (9125, 30 µg/disc), cefotaxime (9017, 30 µg/disc), chloramphenicol (9128, 10 µg/disc), streptomycin (SD031, 10 µg/disc), and nystatin (9078, 100 IU/disc) were obtained from Liofilchem. Methanol (analytical grade) was received from EMSURE (Merck KGaA, Darmstadt, Germany). Acetonitrile (analytical grade) was purchased from MTEDIA (TEDIA Company, Fairfield, OH, USA). Sterile filter paper discs with a diameter of 6 mm were bought from Himedia (Mumbai, India). Ultrapure water was utilized and all reagents were of analytical grade and used as received.

Methylene blue (MB) photosensitizer in powder form was purchased from VWR International BVBA (Leuven, Belgium). In addition, Mueller-Hinton agar (MHA), nutrient broth, and nutrient agar powder were purchased from Oxoid Ltd. (Basingstoke, Hants, UK). The following classes of meropenem, gentamicin, fosfomycin, colistin, chloramphenicol, doxycycline, cefazolin, and nitrofurantoin antibiotic discs were purchased from Liofilchem® (S.r.I. Roseto, Italy). All other chemicals and reagents were obtained from Sigma-Aldrich (St. Louis, USA).