Research On Molecular Mechanism Of Antibiotic Resistance In Lactobacillus Casei And Lactobacillus Plantarum

Resistance of 17 Lactobacillus casei isolates and 15 Lactobacillus plantarum isolates to 11 antibiotics, and presence/absence of 20 genes implicated in antibiotic resistance in these isolates were determined. The results showed that L. plantarum isolates had the widest spectrum of MIC values for streptomycin ranging from 16 to 512 μg/mL. In particular, two isolates of L. plantarum IMAU60045 and IMAU80091 both possessed aadA and ant(6) genes implicated in resistance to streptomycin but varied in their tolerance to streptomycin as evidenced by their minimum inhibitory concentration (MIC) of 16 and 256 μg/mL respectively. Selection of high streptomycin resistance of L. plantarum IMAU60045 was performed over a 30 day period using serial passage with regular increases in streptomycin concentration to reflect the changes in resistance levels. Final MIC value of 16384 μg/mL was recorded which was 1024 fold higher than the original parental isolate. The relative quantity of gene expression (RQ) for the streptomycin resistance gene aadA and ant(6) was studied by qPCR. Expression of aadA and ant(6) genes in L. planarum isolate IMAU60045 increased, and in the first 15 days of passage, the expression of both genes increased more rapidly than in the last 15 days. The RQ value of aadA after passage was 3.35 times greater than in the original isolate and had also increased more than the expression of ant(6) after passage; RQ values for aadA and ant(6) increased by 0.702 and 0.337, respectively. Observations made in this experiment have revealed that long-term challenge with streptomycin can lead to enhanced expression of streptomycin resistance-related genes.To investigate molecular mechanisms of antibiotic resistance and adaptation in Lactobacillus, a probiotic Lactobacillus casei Zhang with systematic study was analyzed by using adaptive laboratory evolution. Mode of action of amoxicillin and gentamicin on L. casei Zhang and its resistance mechanism to the antibiotics were studied by serial passage every 24 h for 2000 generations in LSM medium containing amoxicillin or/ and gentamicin with fixed concentration. Passage in antibiotic free medium was set as control. Whole genome re-sequencing of evolved strains from different generations were performed, and antibiotic resistance, viable counts and optical density of the strains were tracked to analyze the changes of phenotype and genotype of L. casei Zhang during the passage. The results showed that the MIC for amoxicillin, gentamicin, and mixture of amoxicillin and gentamicin increased progressively and became constant at the time point of 600,1200 and 1200 generation. Passage research of L. plantarum IMAU60045 and L. casei Zhang revealed the fact that under the challenge of an antibiotic, cross-resistance to the structurally-related antibiotics of the same class developed. Associated variable degrees of increase in the MIC value for aminoglycoside and β-lactam antibiotics was detected in the corresponding antibiotic-medium evolved L. plantarum IMAU60045 and L. casei Zhang.Genetic mutations (SNPs, InDels and SV) of the three lineages of L. casei Zhang from different passaged medium were detected. The results showed that amoxicillin had great impacts on biosynthesis of cell wall, while gentamicin had influences on the cellular transcription and translation of L. casei Zhang. The morphology of the strains passaged in LSM-A presented elongate filament compared with the strain passaged in other medium. This phenomenon was mediated and influenced by genes(mrcA, pbpA, pbpB) mutation encoding high molecular penicillin-binding proteins in strains passaged in LSM-A. Meanwhile, penicillin-binding proteins have crucial role in growth and division of the cells. The passage experiment showed viable counts of L. casei Zhang passaged in LSM-A were relatively low compared with the control. On the contrary, viable counts and optical density of strains passaged in LSM-G gradually became identical with control during the evolution illustrating the different action of amoxicillin and gentamicin on L. casei Zhang which may lead to varied adaptation for L. casei Zhang when subjected to antibiotics. In our research, genes mediated antibiotic resistance related antibiotic inactive enzyme, membrane transporters, protein essential for DNA repairment and signal transduction were detected with the mutation(s). The gene mutations of L. casei Zhang made it possible to survive in the antibiotic environments by modification of active target, export of antibiotic, changing membrane permeability, inactivate and modify structure of antibiotic, mediating gene expression and balancing the energy metabolism.Study of sustained evolutionary paths of L. casei Zhang in amoxicillin or/ and gentamicin and whole genome re-sequencing analysis of its different lineages showed that no potential high level resistance phathogenic bacteria-like ’super strain’ was evoled in the passage as evidenced by their less MIC increasing of only 4 and 8 times greater than in the original strain, and no gene mutations were found in transposons or plasmid indicated that L. casei Zhang is a safe probiotic.