Safety Assessment Of Food-borne Lactic Acid Bacteria

Lactic acid bacteria (LAB) are closely related to our life. They are widely used as starter cultures or probiotics in yoghurt, cheese, sausage, vegetables, beverage, silages and so forth. Meanwhile, most of them are normal flora of human beings and animals, and play beneficial roles for us include alleviation of lactose intolerance, maintaining balanced intestinal flora, secreting the nutrients and improve the immunity and they have acquired a "generally recognized as safe"(GRAS) status. However, it has been demonstrated that some LAB strains, including food-borne strains, are resistance to antibiotic agents, even multidrug resistance. Some of them could also transfer the antibiotic resistance genes to other LAB or pathogens through horizontal gene transfer, and it would be detrimental to human beings. As well as this, several reports showed that LAB could exert several negative effecst, such as the possible production of undesirable metabolites. Thus, it is so important to carry out safety assessment of edible LAB. The results of this study are as follow:1. Analysis of deleterious metabolite of edible lactic acid bacteria36LAB strains were screened from different sample, including raw milk, traditional fermented milk, retailed diary and drugs. The ability of production of undesirable metabolites, including tyramine, histamine, nitroreductase and azoreductase were assessed. Some of these strains could produce tyramine or histamine, but none of them produced nitroreductase or azoreductase.2. Antibiotic resistances of edible lactic acid bacteriaThe phenotype resistances of these strains to chloramphenicol, erythromycin and tetracycline were examined by broth dilution, and the minimum inhibition concentrations (MIC) were recorded. The results showed that most strains (26/36) were resistant to one or more antibiotic agents, including26chloramphenicol resistant strains,8erythromycin resistant strains,11tetracycline resistant strains and only10strains were susceptive to all of the three agents. Fourteen strains of the antibiotic resistant LAB were multi-drug resistance strains. Then the antibiotic resistant genes were detected by PCR. two Enterococci strains were carrying erythromycin resistant genes, and one of them also had two kinds of tetracycline resistance genes, tet(M) and tet(L). Two Lactobacilli strains harboured tetracycline resistance genes too. The transferability of certain genes were analyzed by filter matings. The tetracycline resistant genes, which were carried by E. faecium KN9could not transferred to E. faecalis181,but could transferred to L. laclis subsp. cremoris MG1614by filter mating.3.Characterization of a novel mobile plasmid pKN9from E. faecium KN9In this study, we sought to investigate the genetic organization of the mobile genetic element that the carried tetracycline resistance genes in E. faecium KN9. The whole sequence (about16kb) of the element was obtained by PCR and anchored PCR amplification, using the gemnomic DNA of E. faecium KN9and L. lactis MGKN9as templates. It is so intriguing that there were two transposases that were100%identical to the corresponding gene of L. lactis MG1363. Then, we designed the primers based on the genes adjacent to transposase on the conjugative element, to define the position of transposase in donor cell. The results demonstrated that the conjugative genetic element was a13818-bp circular plasmid and we named it pKN9. Sequence analysis and sequence alignment were performed by BLAST and ORF Finder. The plasmid contained15ORFs and two among them encoded tetracycline resistande genes. This plasmid could be transferred to Lactococcus lactis in the absence of mob gene, and could directly integrate into the chromosome of the recipient. The expression levels of the tetracycline resistance genes were detected by qRT-PCR and the result showed that tet(M) gene could be induced by tetracycline and tet(L) expressed consecutively.4. Adaptive response resistance of Lb. delbrueckii JSQM1to chloramphenicolIn this study we sought to investigate whether antibiotic resistant strain will emerge under the selevtive pressure. Lb. delbrueckii JSQM1was cultured in MRS with or without sub-MIC chloramphenicol for500generations, and the MICs were determined for every100generations. The growth curve,pH variation curve were analyzed, and the fermentation properties were analyzed by HPLC. And the results showed that susceptive strains could turn to antibiotic resistant strains through mutation in the absent of certain antibiotic agent, but the resistance would disappeare in the absent of selective pressure, and the MIC of the population would decrease. The selective pressure imposed by chloramphenicol could keep the MIC of the population in the high levels. Compared with the original strain, the strains of500generations had no obvious changes in growth curve, pH variation,uptaking of glucose and production of lactic acid.