Research On The Resistance Of Lactobacillus Plantarum To Aminoglycoside Antibiotics

Lactobacillus plantarum,as a common probiotic in the food and medical industry,have a lot of food value and medicinal value.However,the drug resistance of most lactic acid bacteria has potential transferability,which will have negative effects on the health of the body,so it is necessary to evaluate the safety of probiotic to drug resistance before application.In view of this,L.plantarum ATCC14917 were selected to evolve in aminoglycoside antibiotics to screen the strains with high resistance.The phenotypic characteristics of high resistance L.plantarum ATCC14917 in evolution process were studied,the resistance mechanism and the transferability of resistance genes of L.plantarum ATCC14917 were explored combined with the whole genome sequencing at the molecular level,which preliminarily evaluated the safety of L.plantarum ATCC 14917 to aminoglycoside anti-biotics.Firstly,the resistance of L.plantarum ATCC14917 to aminoglycoside antibiotics was studied.Resistance test showed that the minimum inhibitory concentration(MIC)of original L.plantarum ATCC14917 against streptomycin,neomycin,gentamicin and kanamycin were 16 ?g/mL,32?g/mL,16 ?g/mL and 64 ?g/mL,respectively,showing L.plantarum ATCC14917 was resistant to streptomycin,and sensitive to neomycin,gentamicin and kanamycin.In order to further study the safety of aminoglycoside antibiotic resistance in L.plantarum ATCC14917,the high-level resistant strains were constructed by using laboratory evolution approach.At the end of evolution,the MIC of L.plantarum ATCC14917 to streptomycin,neomycin,gentamicin and kanamycin had reached 131,072 pg/mL,256 ?g/mL,512 ?g/mL and 1024?g/mL which was 8192,64,128 and 64-fold higher than that in the original isolates.The high resistant L.plantarum ATCC14917 isolate was then passaged in antibiotic-free medium to determine the stabiliity of resistance.The MIC of the L.plantarum ATCC14917 to streptomycin,neomycin,gentamicin and kanamycin decreased to 4096 ?g/mL,32 ?g/mL,64 ?g/mL and 128 pg/mL after 30 days passage but still showed high resistance against the cut-off values,which indicated that the acquisition of drug resistance was an irreversible process.Secondly,the phenotypic stability ofL.plantarum ATCC 14917 in the evolution was investigated.During laboratory evolution,the cell morphology,colony morphology,living counts,bacterial liquid density and fermentation activity of L.plantarum ATCC 14917 strains grown in antibiotics and free-antibiotics medium were studied.The results showed that the high drug resistance and continuous passage did not change the characteristics mentioned above,indicating L.plantarum ATCC14917 had good phenotypic stability.Thirdly,whole genomic analysis of L.plantarum ATCC 14917 was performed.Whole-genome sequencing of strains under different passages condition were used to investigate the mechanism of L.plantarum ATCC14917 to aminoglycoside antibiotics and adaptation in the presence and absence of selection pressure.CLC Genomics Workbench was used to detect gene mutations associated with drug resistance during the passage process.Six non-synonymous mutation genes,including 2 SNPs,1 MNV and 3 SVs,were verified by Sanger sequencing in highly resistant L.plantarum ATCC14917.Thereinto,ribosomal protein S12 detected at the 385923 position in GL379762 and drug resistance MFS transporter detected at the 355220-355227 position in the GL379762 of L.plantarum ATCC 14917 were found in the strains that evolved in four antibiotics,which indicated that ribosomal protein S12 and drug resistance MFS transporter were vital substances leading to the high resistance of L.plantarum ATCC 14917 to aminoglycoside antibiotics.In addition,Ser/Thr phosphatase family protein detected at the 120032-120045 position in the GL379767 in streptomycin,neomycin and gentamicin resistant strains and IrgA protein detected at 404368-404377 position in the GL379764 in streptomycin and kanamycin resistant strain,resulted in the enhancement of Lplantarum ATCC 14917 to corresponding antibiotics.After the selective pressure of antibiotics was removed,only ribosomal protein.S12 was detected in strains resistant to four antibiotics.Furthermore,two hypothetical protein detected at 18853-18854 position in GL379768 and detected at 259982 position in GL379766 were found respectively in streptomycin and gentamic in resistant strains,which may be related to the resistance response of Lactobacillus plantarum to streptomycin and gentamicin.The analysis of the mobility of the detected mutations showed that there were no aminoglycoside antibiotic resistance-related genes in L.plantarum ATCC14917 located on the mobile element,which demonstrated that the resistance of L.plantarum ATCC 14917 to aminoglycoside antibiotics was safety.