Searching for antibiotic-susceptible bioprotective lactic acid bacteria to control dangerous biological agents in artisanal cheese

Abstract

Minas artisanal cheese (MAC) samples (n = 59) from 16 municipalities across five traditional MAC-producing regions in Brazil were used to prospect antibiotic-susceptible protective lactic acid bacteria (LAB) against three pathogenic bacteria found in the MAC. From 291 LAB isolates, 84 genetically diverse strains were selected via rep-PCR. MALDI-TOF identification revealed multiple species, predominantly Enterococcus faecalis (n = 37), Enterococcus faecium (n = 21), Lactiplantibacillus plantarum (n = 5) and Lacticaseibacillus rhamnosus (n = 3). The antagonistic activity of these strains was evaluated against Enterococcus faecalis ATCC 29212, Listeria monocytogenes ATCC 5779, and Escherichia coli O157:H7 using spot-on-lawn assays. Several strains showed strong inhibitory effects against E. coli and L. monocytogenes, with halo/colony ratios reaching 4.86 and 4.47, respectively. No antimicrobial peptide producing strain was observed. Antibiotic susceptibility was tested against nine antibiotics, and five strains were susceptible to all antibiotics, while 53 strains were susceptible to 5–8 antibiotics. However, five strains were resistant to all antibiotics, showing the highest resistance to gentamicin (66.7%), cotrimoxazole (58.3%), and streptomycin (57.2%). Resistance genes (aacA-aphD, ermA/B, tetM/O/K/L/S, blaZ, and vanA/B) were screened, and 40 strains harbored at least one gene. Taken together, these results revealed three antibiotic-susceptible bioprotective lactobacilli (L. rhamnosus 52, L. plantarum 177, and L. plantarum 272G) as superior strains, whose efficacy in eliminating E. coli O157 and Listeria monocytogenes in the milk matrix between 7- and 21-days post-inoculation was confirmed. These findings confirm the potential of these autochthonous lactobacilli to improve the safety of dairy, paving the way for their applications in product development in future projects.