| In this study,the object was Lactobacillus plantarum ATCC 14917,and the whole genome resequencing technology was used to study its resistance during the evolution of ?-lactam antibiotics,and analysis the changes of phenotype and probiotic characteristics in order to obtain probiotics with low fitness cost and high antibiotic resistance,and provide important strain resources for functional food research and development.The first part is about that the study on the resistance of L.plantarum to ?-lactam antibiotics.Macroscopic broth dilution method was used to study the antibiotic resistance(minimum inhibitory concentration,MIC)during the evolution of L.plantarum.The results showed that the MIC values of the original L.plantarum strains on cephalexin,penicillin,amoxicillin and ampicillin were respectively at 32 ?g/mL,1?g/mL,0.5 ?g/mL and 0.5 ?g/mL,in addition to being resistant to cephalexin,the original L.plantarum strains were sensitive to penicillin,amoxicillin and ampicillin.The laboratory evolutionary method was used to study the adaptive evolution of L.plantarum in the environment of ?-lactam antibiotics.When the antibiotic resistance reached the maximum,the MIC values of L.plantarum to cephalexin,penicillin,amoxicillin and ampicillin were respectively increased to 8192 ?g/mL,16?g/mL,16?g/mL,and 64?g/mL,and maintained stable antibiotic resistance,and the antibiotic resistance was increased by 256 times,16 times,32 times,and 128 times than the original strain.The four stable and highly resistant strains constructed were continued to evolve in antibiotic-free medium to study the resistance stability of the strains.The results showed that the resistance of L.plantarum to cephalexin was reduced from 8192?g/mL to 4096 ?g/mL,the resistance to penicillin remained stable,the resistance to amoxicillin was reduced from 16 ?g/mL to 1 ?g/mL,and the resistance to ampicillin was reduced to 4 ?g/mL.The antibiotic resistance was still higher than that of the original strain,indicating that the resistance of L.plantarum is an irreversible process.The second part is about that phenotypic and probiotic characteristics of L.plantarum during evolution.The results showed that for highly resistant strains and resistant reverting strains,the morphology of the colonies showed a smooth milky white bulge,the metabolism pattern of 49 carbohydrates was basically the same,and the tolerance to different concentrations of salt was maintained.Regarding cell morphology,the cell morphology of continuous cultured strains in different environments varies.In addition,in terms of the biomass of the strain,the overall biomass of the strain that evolved after deselection pressure was higher than that of the strain that evolved in the antibiotic environment.As for the probiotic characteristics of the strains,the highly resistant strains have reduced their adaptability due to the high resistance,so the acidity tolerance,bile salt tolerance and adhesion characteristics of the strains were all lower than those evolved under deselective pressure.The third part is about that whole genome resequencing and bioinformatics analysis of L.plantarum.Genome-wide resequencing of strains that have evolved to stable resistance in different environments focuses on nonsynonymous mutations related to antibiotic resistance,phenotype and probiotic characteristics.The distribution of mutation products related to the antibiotic resistance of the strain is:hypothetical protein,LPXTG-motif cell wall anchoring domain protein and acetyl transferase GNAT family were detected in strains resistant to cephalexin,penicillin,amoxicillin and ampicillin,one or more of these products can still be detected in the antibiotic-resistant reverting strains.TetR family and serine D-Ala-D-Ala carboxypeptidases were also detected in strains resistant to penicillin,amoxicillin and ampicillin,and the products were restored when antibiotics were removed.At the same time,a new mutant product serine-type D-Ala-D-Ala carboxypeptidase was added to the cephalexin-resistant reverting strain.Major factor superfamily proteins were detected in cephalexin,amoxicillin and ampicillin-resistant strains and penicillin-resistant reverting stain,and disappeared in ampicillin-resistant restorers.A unique product ABC transporter was detected in the penicillin-resistant reverting strain,and the universal stress family protein was detected in the amoxicillin-resistant reverting strain,while the gene mutations of Ser/Thr phosphatase family protein,LrgA family protein,and LrgB-like protein were existed in ampicilin-resistant restorer strain.The distribution of products related to the phenotype and probiotic characteristics of the strains is:hypothetical protein and tRNA dimethyl allyltransferase were detected in four highly resistant L.plantarum and resistant reverting strains,and a new mutant product YibE/F-like protein was detected in resistant reverting strains.The putative foldase protein PrsA 2 was present in the cephalexin-resistant and ampicillin-resistant revertins,and the diguanylate cyclase(GGDEF)domain protein was only present in the penicillin-resistant reverting strains.Phosphoglycerate mutase family protein was present in cephalexin-resistant and ampicillin-resistant reverting strains,sugar transport protein was present in cephalexin-resistant and penicillin-resistant reverting strains,and bile acid 7-alpha dehydratase was present in the amoxicillin-resistant and ampicillin-resistant reverting strains.In addition,there were two unique gene mutation products,phosphoenolpyruvate-dependent sugar phosphotransferase system,EIIA 2 and PTS system sucrose-specific IIBC component in ampicillin-resistant reverting strains.After analysis of orthologous clusters and metabolic pathways,these gene-annotated products mainly affect important functions such as cell processes and signal transduction,information storage and processing,and metabolism of strains,and it also played an important role in acquiring resistance and maintaining certain phenotypes and probiotic characteristics.to L.plantarum during adaptive evolution.Mobility testing of antibiotic-resistant genes confirmed that antibiotic-resistant genes in highly antibiotic-resistant strains are not located on movable elements,indicating that the resistance of L.plantarum to ?-lactam antibiotics does not constitute a hidden danger. |
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