Detection Of Antibiotic Resistance Of Clinical Bacterial Isolates And Identification Of A Novel Tigecycline Resistance Mechanism

The clinical investigation of resistance mechanisms of multi-drug resistant bacteria has been a high priority.Infections caused by multi-drug resistant bacteria are difficult to clear and easy to recur,and the exploration of their resistance mechanisms can provide targets for the development of new antibiotics.In this study,we screened suspected novel resistance mechanism sequences from 219 clinical isolates to investigate the mechanism of novel antibiotic resistance,and the main experimental contents and results are as follows:(1)384 strains of bacteria suspected to be multi-drug resistant were isolated from hospitalderived clinical mixes.Acinetobacter baumannii and Pseudomonas aeruginosa,whose resistance mechanisms are complex and difficult to study,were excluded,and the remaining 219 isolates were tested for drug resistance phenotypes by K-B method and micro-broth dilution method.The genomes of the strains were extracted for the detection of common antibiotic resistance genes.It was found that the samples were mainly Klebsiella pneumoniae,and the resistance genes were mainly targeting β-lactams,while other types of antibiotic resistance genes were also detected.(2)Screening for resistance phenotypes that fail to detect common antibiotic resistance genes in the strains,and making them the focus of this study.Whole-genome sequencing and biomarker analysis were performed to extract annotated sequences related to their resistance phenotypes for analysis and validation.The annotated sequence KP 1-33 sul1 was targeted in the first round of screening of 13 sequenced strains,however,after gene cloning and phenotype verification,it was found that the sequence failed to confer resistance to SXT.Subsequently,nine strains that were resistant to the last line of defense antibiotics and does not harbor respective antibiotic resistance genes were selected for whole-genome sequencing to explore novel resistance mechanism,and the annotated sequences KP 1-45 tetA,KP 1-49 acrR,and KP 3-94 acrAA188 were identified as novel resistance sequences.The structural differences between TetA1-45 and TetA were found to be in the non-functional region by protein structure prediction.Therefore,KP 1-45 tetA was excluded.The insertion sequence ISKpn26 was found in the sequence KP 1-49 acrR by nr-blast sequence comparison,resulting in the loss of the transcription factor AcrR to repress the expression of the drug efflux pump protein gene acrAB,and the overexpression of acrAB directly led to the increase of drug resistance in the strain.Sequence studies of KP 3-94 acrAA188 with a point mutation revealed that the point mutation elevated the strain’s tigecycline minimum inhibitory concentration(MIC)by 1.6-fold;increased strain survival by 6-fold in tests with tigecycline stress treatment;and increased the strain’s tigecycline efflux capacity by 3-fold.These results suggest that the T188A point mutation of acrA can significantly enhance the strain’s resistance to tigecycline.In summary,clinical strains are generally antibiotic resistant and the detection rate of resistance genes is generally high.The T188A point mutation of acrA was found to significantly enhance the ability of strains to export the drugs and thus improve the resistance of strains to tigecycline in sequenced strains.