Antibiotic Susceptibility Of Potentially Probiotic Lactobacillus Strains

Lactobacillus bacteria, as probiotics, are widely used in food, feed, medical and health related fields. In recent years, ever-growing studies have shown that antibiotics, due to abuse in the treatment of diseases, may lead bacterial strains (or species) to have high tolerance to antibiotics. Once these probiotics which carry transferable resistance factors are used in food industry, perhaps some hazards related to food safety take place. We isolated 34 Lactobacillus strains from various food environments including sourdough, fermented sausages, sauerkraut, dairy products, and host intestinal tracts. The objectives of this study were to: (1) assay the susceptibility of 34 lactobacilli strains to antibiotics; (2) detect plasmids of the strains that have higher resistance to antibiotics; (3) investigate the growth performances of the plasmid-containing strains under simulated gastrointestinal conditions; and (4) analyze the relationship between the plasmid profiles and resistance patterns of the strains by plasmid elimination. The present study also establishes a safety evaluation system for probiotic Lactobacillus strains according to the molecular mechanisms of antibiotic-resistant lactobacilli strains. The following are main results.1. The sensitivities of the 34 lactobacilli strains to various antibiotics were tested. Among them, 100% of lactobacilli bacteria were resistant to bacitracin, polymyxin B, kanamycin, and nalidixic acid. 82.4% (28/34) of these strains were tolerant to vancomycin, 91.2% (31/34) to streptomycin, and 88.2% (30/34) to gentamicin, respectively. It means that these drug resistances might be widespread at least among the tested lactobacilli strains, although the antibiotic-resistant distribution was species-specific. Moreover, 38.2% (13/34) of these strains tolerated penicillin,17.6% (6/34) Cephalothin, 41.2% (14/34) ampicillin, 8.8% (3/34) tetracycline and chloramphenicol, and 29.4% (10/34) rifampicin, respectively. The susceptibility of the 34 lactobacilli strains to these antibiotics were high, depending on species tested.2. 16 antibiotic-resistant strains were selected to extract plasmid, in which 6 strains were proved to contain plasmid. They were Lactobacillus pentosaceus strains CH8, Lp-A and Lp-4, Lactobacillus helveticus LLB, Lactobacillus acidophilus LL, and Lactobacillus plantarum L11, respectively. Additionally, the simulation of gastrointestinal environments showed that plasmid-containing strains CH8, Lp-A, LLB, LL, and L11 survived better in harsh stresses at 37°C. Potentially, these plasmid-containing strains that are able to survived to challenge the gastrointestinal stresses might transfer their antibiotic-resistant genes to other intestinal micro-flora, having a harm to the host.3. Plasmid elimination showed that strain CH8 contained chloramphenicol andβ-lactam resistance genes on its plasmids. PCR confirmed that strain CH8 carried 2 plasmids with a size of 10kb. Their sequences were proved to haveβ-lactam resistance gene blr. Moreover, these plasmids carrying blr were transformed into Escherichia coli HB101, a highly suscept strain to chloramphenicol. It was seen that strain HB101 grew very well on 4μg/mL chloramphenicol-containing ager medium at 37°C for 48h.In conclusions, our study formed a 3-step procedure to assay the safety of potentially probiotic lactobacilli strains. The procedure included: firstly, the detections of antibiotic susceptibility and antibiotic-resistant plasmids; secondly, the investigation of the survival of lactobacilli strains carrying plasmids and resistances in gastrointestinal circumstances; thirdly, the growth observation of susceptive E.coli transformant containing antibiotic-resistant plasmids on specific antibiotic media.