Molecular Characteristics Of Acinetobacter Baumannii And Gene Expression Changes Under Tigecycline Pressure

Objective:Acinetobacter baumannii(A.baumannii)is a common nosocomial infection bacterium.It is distributed in the clinic widely and has a high detection rate.Molecular typing methods such as ERIC-PCR(enterobacterial repetitive intergenic consensus PCR),PFGE(pulsed-field gel electrophoresis),MLST(Multi locus sequence typing),etc.have been used in the investigation of epidemic events widely.The resistance of Acinetobacter baumannii has also been the focus of clinicians.Tigecycline is currently one of the limited options for clinical treatment of drug-resistant bacteria,but Acinetobacter baumannii has the risk of increasing its resistance rate.This study performed molecular typing on the Acinetobacter baumannii which isolated from a certain tertiary hospital in a certain period of time.The outbreak and clonal transmission of nosocomial infections were judged based on the typing results and the spatiotemporal distribution of the strains.This can help to further strengthen the control of nosocomial infections.At the same time,the resistance profiles of the collected strains were analyzed,and the distribution of resistance-related molecules such as AdeB,TetB,and AbaQ among the strains was detected,and the relationship between the resistance phenotype and the resistance-related molecules was analyzed.The effect of tigecycline on the resistance phenotype of A.baumannii and the expression of resistance-related molecules was further analyzed,and the resistance genes that were predominantly expressed under the pressure of tigecycline were screened.Then,we used genomic and transcriptional sequencing technology to analyze a pair of strains with increased resistance after tigecycline treatment from the same patient,and look for the cause of the change in the drug-resistant phenotype of Acinetobacter baumannii under the pressure of tigecycline,in order to provide a response strategy to slow the increase in drug resistance.Method:(1)We used ERIC-PCR as the main technical method to genotype the collected strains,combined with the spatiotemporal distribution of the strains to determine the outbreak,and clonal spread of Acinetobacter baumannii in a specific period,then,the "gold standard" PFGE was used to type 35 highly resistant strains,and compared with the ERIC-PCR typing results to verify the typing ability of ERIC-PCR in Acinetobacter baumannii in the hospital.(2)We combined PCR with agarose gel electrophoresis to detect efflux pump genes AdeB,AdeG,AdeJ,TetB,CraA,AbaQ,AbeM,MacB,AbeS and regulatory protein genes AdeR,AdeS,BaeS,and SoxR;Chi-square test was performed to compare the efflux pumps and regulatory protein carrying rates of the sensitive group strains with the non-sensitive group strains.The relationship between the efflux pump and molecular typing was analyzed.(3)According to the drug resistance spectrum of the strains,the results of molecular typing and the carrying of the efflux pump;6 strains of Acinetobacter baumannii were selected and the MIC values of each strain to tigecycline were re-measured.In vitro induction experiments were conducted in LB liquid medium with increasing tigecycline concentration gradient(experimental group),and LB pure culture of each strain was used as the self-control(control group).The MIC values of tigecycline were measured after induction and LB pure culture.The RNA of each strain was extracted,and the CT value of each efflux pump gene and regulatory protein gene was quantitatively detected by RT-qPCR after genome removal.Using self-control as a reference,the expression levels of efflux pump and regulatory protein genes of each strain were calculated after induction with tigecycline in vitro.(4)After analyzing the previous work of the collected strains and querying the patient's medical records,we selected a pair of A.baumannii which isolated from a inpatient,their resistance to tigecycline and minocycline changed from sensitive to resistance after treatment with tigecycline.They were named A.baumannii (AB711)and A.baumannii(AB721),respectively.And the drug resistance of the strains was retested.AB711 was used to perform an induction experiment in LB liquid medium with increasing tigecycline concentration gradient in vitro.After induction,the MIC value of the strain to tigecycline was recorded.The induced strain was named A.baumannii(AB712).The above three strains were subjected to genomic DNA extraction and whole genome sequencing and analysis.AB711 and AB712 were subjected to RNA extraction,transcription sequencing and analysis.Some genes were selected for reverse transcription quantitative PCR (RT-qPCR)based on the results of differentially expressed genes.The results of quantitative PCR were compared with the results of transcription sequencing.Result:(1)The 183 strains of Acinetobacter baumannii was divided into 45 clonal groups by ERIC-PCR.A,D,F,I,and J are the main subgroups,Combining the spatiotemporal distribution of the strains,no signs of outbreaks were found.In addition,molecular typing of 35 strains of Acinetobacter baumannii was performed with PFGE and ERIC-PCR.The results showed that the typing results of ERIC-PCR and PFGE for the strains were partially consistent.ERIC-PCR has certain typing ability for A.baumannii genotypes in this study.(2)The total carrying rate of AdeG is 98.36%,and the total carrying rate of regulatory protein BaeS is 96.17%.The total carrying rate of the efflux pump TetB was the lowest at 78.14%.Except for the efflux pump genes AdeG,AdeJ,and AbaQ had no significant difference in the detection rate between the drug-resistant group and the sensitive group.The other efflux pump and regulatory protein genes were detected in the drug-resistant group higher than the sensitive group.And p <0.05,the difference was statistically significant.(3)The expressions of efflux pump genes(AdeB,TetB,and AbeS)and regulatory protein gene AdeS of multidrug-resistant bacteria and extensively drug-resistant bacteria in the control group were significantly higher than those in the sensitive group,and the expression of efflux pump genes(AdeB,TetB)in extensively drug-resistant bacteria were higher than that in multidrug-resistant bacteria,the results were analyzed by t-test as p<0.01.Other resistance-related genes were expressed differently in the multidrug-resistant and extensively-resistant bacteria of the control group.Compared with the control group,the expressions of AdeB,TetB,AbaQ and AdeS in the experimental groups showed an increasing trend;the expressions of AdeB and TetB in each strain significantly increased,and P <0.01.Compared with the control group,other genes in the experimental groups,such as AbeM,MacB,AbeS,BaeS,and AdeR have different expression trends among strains.(4)After cultured in LB liquid medium with increasing tigecycline concentration gradient in vitro and then releasing the pressure from tigecycline,AB711 has undergone a two-fold increase in the MIC value of tigecycline and then reduced it to the original.This is consistent with the clinical process of AB711.Whole genome sequencing(WGS)and MLST typing showed that AB711,AB712,and AB721 belong to ST2(Pasteur)/ ST1791(Oxford).SNP-based phylogenetic tree analysis showed that AB711,AB712,and AB721 were co-origin.Under the pressure of tigecycline,functional enrichment analysis of differentially expressed genes in the transcriptome showed that genes related to the metabolic processes of benzene-containing compounds and phenol-containing compounds,translation,ribosome structure and biogenesis,amino acid transport and metabolism,and other related genes was significantly up-regulated.In addition,genes such as the efflux pump RND subunits,EmrAB,MacB,and the tetracycline operon were also up-regulated.The results of RT-qPCR and RNA-seq were basically consistent in some genes.Conclusion:(1)There were several clones of A.baumannii clinically isolated from May 2017 to March 2019 in a tertiary hospital in a certain area,but there was no outbreak of A.baumannii with specific genotypes.(2)The carrying and expression of various efflux pumps may play an important role in the drug resistance formation process of A.baumannii.(3)The high expression of TetB and AdeB may play an important role in enhancing the resistance of Acinetobacter baumannii to tigecycline.(4)Under the pressure of tigecycline,differential expression of related genes may play a major role in the change of A.baumannii's resistance to tigecycline.The specific molecular mechanism needs further study.