Rapid And Accurate Antibiotic Susceptibility Determination Of Tet(x)and Mcr Positive E.coli And Target-Killing Mcr-positive E.coli Using RNA Biomarkers

The spread of antibiotic resistance has been an increasing global concern that seriously threatens human health and biosecurity in the 21st century.Colistin and tigecycline are the lastresort antibiotics for the treatment of infections caused by multidrug-resistant(MDR)gramnegative pathogens especially by carbapenem-resistant Enterobacteriaceae(CRE).However,the emergence of plasmid-mediated colistin resistance gene mcr-1,and tigecycline resistance genes tet(X)all over the world diminish clinical efficacies of colistin and tigecycline.The current antimicrobial susceptibility tests are mainly based on bacterial growth,which takes a long time and detects phenotype and genotype separatly.Although broad-spectrum antibiotics are effective in bacterial infection treatings,they also impair probiotics,which may cause antibiotic-related diseases.In this case,it is important to establish a rapid and robust antibiotic susceptibility determination and target-killing resistant bacteria method.In this study,we developed a rapid and robust RNA-based antibiotic susceptibility determination assay to effectively distinguish tet(X)and mcr-1-negative and-positive strains using specific RNA biomarkers in bacteria after antibiotic exposure,and established a bactericidal method that can target and kill mcr-1 positive colistinresistant bacteria based on the activation of specific gene transcription level of mcr-1.The results addressed as follows:1.Rapid and accurate antibiotic susceptibility determination of tet(X)-positive E.coli using RNA biomarkersTo identify the specific transcripts that effectively distinguish tet(X4)-positive and-negative bacteria,transcriptome profiling of the engineered tet(X4)-mediated tigecycline-resistant strain(DH5α-pUC19-tet(X4))and tet(X)-negative strain(DH5α-pUC19)was performed after 60 min tigecycline exposure(2 μg/mL).The PCA results and venn diagram suggest that tet(X4)-positive and-negative bacteria exhibit different shifts in global gene lists after tigecycline exposure at breakpoint concentration.The candidate tigecycline-specific susceptible gene list was generated by including genes with significant differential expression levels compared with the control DH5αpUC19 group,and valided in clinical isolates.25 candidate RNA biomarkers showed significant differential expression levels between the clinical tet(X4)-negative and-positive isolates after tigecycline treatment.Next,the effect of tigecycline concentration and incubation time on the expression of selected RNA biomarkers were evaluated.Global shifts of 25 selected RNA biomarkers in the tet(X4)-negative strain were observed in a very short time(5 min)and reached a peak at over 0.25 μg/mL tigecycline exposure.However,there was no significant fold change in the tet(X4)-positive tigecycline-resistant E.coli isolate.All tet(X)variants-positive groups had no remarkable response to tigecycline exposure compared with the tigecycline susceptible group.Altogether,these results suggest that the selected RNA biomarkers are also applicable for the rapid molecular AST of tet(X4)and other tet(X)variants-conferred tigecycline-resistant E.coli isolates.The accuracy verification and potential application of RBAST in clinical practice,33 clinical E.coli isolates were selected for RBAST verification.Compared with the MIC and PCR analyses,the developed RBAST correctly classified 30 of 33 isolates.The accuracy characterized by RBAST method during 3 h assay time was 90%accuracy.Collectively,these data indicate that RBAST can effectively detect tet(X)-mediated tigecycline resistance in the clinical setting.2.Rapid and accurate antibiotic susceptibility determination of MCR mediated colistinresistant E.coli using RNA biomarkersTo identify the specific transcripts that effectively distinguish mcr-1-positive and-negative bacteria,transcriptome profiling of the engineered mcr-1-mediated colistin-resistant strain(DH5αpUC19-mcr-1)and mcr-1-negative strain(DH5α-pUC19)was performed after 60 min colistin exposure(2 μg/mL)The candidate colistin-specific susceptibility gene list was generated by including genes with significantly increase or decrease(log2FC ≤-2 or≥ 2,p<0.05)in colistin susceptible groups,while no significant change(-2 ≤ Log2FC≤2)in mcr-1 positive groups after colistin exposure.There are 18 candidate mRNA biomarkers showed significantly differential expression levels between colistin-susceptible and mcr-1 positive groups after colistin treatment.When the colistin concentration reached 0.25 μg/mL,the candidate mRNA biomarkers were significantly up-regulated,while no change was found in mcr-1 positive isolate.A global shift of 18 candidate mRNA biomarkers in colistin-susceptible isolate were observed in 30 min,and reached the peak at about 60 min after colistin treatment.The accuracy verification and potential application of RBAST in clinical practice,30 clinical E.coli isolates were selected for RBAST verification and MIC and PCR analyses.The developed RBAST correctly classified 28 of 30 isolates.The accuracy characterized by RBAST method during 3 h assay time was 93%accuracy.Collectively,these data indicate that RBAST can effectively detect mcr-1-mediated colistin resistance in the clinical setting.3.Target-killing mcr-1 positive Enterobacterium based on specifically expressed mRNAA target-killing mcr-1-mediate colistin resistant Enterobacterium method is engineered based on the specific transcripts caused by the expression of mcr-1.A conjugative plasmid was contructed and a CcdB toxin protein splited with intein DnaE was chosen as bactericidal protein.With arabinose-mediated induction of CcdB-Int expression,the bacteria which have been successfully conjugated pccdB-Int plasmids died.The killing and conjugation efficiency was stable whether or not the mcr-1 exist.The candidate mcr-1-specific gene list was generated by including genes with significantly increase(log2FC≥ 2,p<0.05)in DH5α+pUC19-mcr-1 compared with DH5α+pUC19 group.The 2,000 bp upstream sequence of the candidate gene was selected as the promoter,and cloned into the upstream of CcdB-Int to construct a conjugable plasmid targeting mcr-1 positive bacteria.The candidate plasmid was transferred to EC600+pUC19-mcr-1 and control bacteria EC600+pUC19 by conjugation respectively.The ratio of recipient between EC600+pUC19-mcr-1 and EC600+pUC19 was calculated.31 candidate promoters showed targeted killing mcr-1-positive colistin resistant E.coli based on conjugation.The four candidate gene promoters with the highest efficancy also showed good targeted clearance effect on Salmonella and Klebsiella pneumoniae as well.The efficiency of target-killing mcr-1 positive bacteria was higher with the increases of culture temperature in conjugation.However,the killing efficiency began to decrease when the temperature exceeds 30℃.The killing efficiency was maintained at maximum when the conjugation pH is between 6 and 8.4.Influence of mcr-1 target gene on MCR functionTo find out the relevance of between the selected 31 genes and mcr-1,those 31 genes were knockout with suicide plasmid in E.coli EC600.We successfully constructed thirty EC600 knockout strains for further research.MIC to colistin,growth curve,competition test,mRNA expression level of mcr-1 and the level of outer membrane integrity,membrane potential and reactive oxygen species were investigated to figure out the of the phenotype of candidate gene knockout strains transformed with pUC19-mcr-1 with or without colistin treatment.The results showed that the mcr-1 transcription level of EC600 ΔintD+pUC19-mcr-1 and EC600 AyafN+pUC19-mcr-1 significantly increased compared with EC600+pUC19-mcr-1,while the competition test growth curves showed no differences.Meanwhile,the level of outer membrane integrity,membrane potential and reactive oxygen species were increased in EC600ΔintD+pUC19-mcr-1 and EC600 ΔyafN+pUC19-mcr-1 in the absence of colistin treatment.The 50%effective concentration and maximum response were also showed significant increase after colistin exposure comparer with EC600 ΔintD+pUC19-mcr-1.5.Cytotoxicity of LPS from EC600 ΔintD and EC600 ΔyafN in RAW264.7 MacrophageLipopolysaccharide(LPS)extracted from knockout strains,EC600 ΔintD+pUC19-mcr-1 and EC600 ΔyafN+pUC19-mcr-1 mentioned above,was quantitative analysed and normalized by amebocyte lysate derived blood of horseshoe crab.The mRNA expression levels and of IL-1α,IL1 β,and IL-6 and protein content in supernatant was quantified after treatment with LPS.RT-PCR results showed that the maximum response induced by LPS extracted from EC600+pUC19-mcr1,EC600 ΔintD+pUC19-mcr-1 and EC600 ΔyafN+pUC19-mcr-1 have no differences.However,the EC50 of LPS extracted from EC600 ΔintD+pUC19-mcr-1 and EC600 ΔyafN+pUC19-mcr-1 was lower than that from EC600+pUC19-mcr-1.The ELISA results showed that the level of IL1α,IL-1β and IL-6 in the supernatant were more in knockout LPS than that in WT when the stimulus concentration was 10 ng/mL.As the LPS stimulus concentration increase to 100 ng/mL,the level of.IL-1β have no significant difference between knoutout and WT strains.The EC50 of iNOS mRNA expression stimulated by LPS of EC600 ΔintD+pUC19-mcr-1 and EC600ΔyafN+pUC19-mcr-1 was significantly lower than that of EC600+pUC19-mcr-1.More NO was induced when stimulated with LPS of EC600 ΔintD+pUC19-mcr-1 and EC600 ΔyafN+pUC19mcr-1.As indicated above,LPS extracted from EC600 ΔintD+pUC19-mcr-1 and EC600ΔyafN+pUC 19-mcr-1 were significantly more toxic than LPS transferred extracted from EC600+pUC 19-mcr-1.In all,we developed a rapid and accurate RNA-based antibiotic susceptibility determination assay to effectively distinguish tet(X)and mcr-1-negative and-positive strains using specific RNA biomarkers in bacteria after antibiotic exposure with accuracy over 90%.And we successfully established a specific mcr-1 positive E.coli target-killing strategy.These results will give rise to a new concept for detecting and eliminating antibiotic-resistant infections.