| In recent years,researchers have become more and more interested in Lactic acid bacteria?LAB?in combination with antibiotics to treat gastrointestinal diseases.LAB are often present in the antibiotics-containing environment,and how their adaptive evolution in such environment and whether they would cause serious safety issues have become a research focus.In this study,we analyzed the levels of antibiotic resistance of 53 Lactobacillus?L.?isolates from milk products of different geographic origins.On this basis,in order to study the adaptive evolution mechanism of LAB to antibiotics,we analyzed an antibiotics-adapted Lactobacillus plantarum P-8 strain isolated from continuous laboratory cultivation of 12 months?2400 generations?in medium containing ampicillin,clindamycin and antibiotic free,respectively.The viable counts,OD value,and antibiotic resistance were monitored during the passage,and using the Illumina Hiseq technology for whole genome resequencing of different generation strains.The main results are as follows:?1?The range of minimal inhibit concentration?MIC?values of Lactobacillus against15 antibiotics varied widely,showing varying degree of antibiotic resistance;about88.7%of the strains were resistant to one or more antibiotics,only 6 lactobacilli strains remained sensitive to all antibiotics;36 strains contained antibiotic resistance genes and antibiotic resistance genes could not be transferred between strains.?2?Compared to the control group without antibiotics,L.plantarum had significantly lower viable counts and OD value at 24h?p<0.05?along the 2400 generations of sub-cultivation with ampicillin and clindamycin.However,as the cultivation time prolonged,the viable counts and OD approached that of the control strain;the trend of MIC values change of L.plantarum P-8 in the medium with ampicillin and clindamycin was opposite.The former increased from 0.5?g/mL to 16?g/mL,and the latter remained stable.?3?We identified 4,19,and 9 single-nucleotide polymorphisms in the antibiotic free,clindamycin,and ampicillin medium,respectively.The average mutation frequencies were 7.68×10-7,1.64×10-6,and 2.52×10-6,respectively.The common mutation spectrum in three environments included CG->GC and CG->AT transversion,accounted for 37%and 32%of the total mutation,respectively.The CG->TA transition only occurred in the antibiotic environment and accounted for 15%of total mutations.Moreover,the transition and transversion ratio?Ti/Tv?in the antibiotic free medium was significantly lower than that in the antibiotic environment?p<0.05?.The above results showed that the L.plantarum cells accumulated the mutations to improve their adaptability toward an antibiotics-containing environment.?4?In the antibiotic free,clindamycin,and ampicillin medium,L.plantarum P-8encoded the same mutation site for ABC transporter,and such mutations were stable from 400 until 2400 generations.We speculated that L.plantarum P-8 strains underwent convergent evolution.The penicillin binding proteins are associated with L.plantarum P-8 antibiotic resistance.In addition,there was no mobile elements in their flanking regions.These results further demonstrated that there was no potential safety hazard for the resistant accumulation of point mutations.In summary,this study combined the methods of laboratory evolution and whole genome re-sequencing to reveal the adaptive evolutionary molecular mechanism of LAB in antibiotic environment.This study analyzed the safe use of probiotics on the level of genomics.This work has provided novel insights and basis in the safe use of probiotics. |
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