| Proteus vulgaris(P.vulgaris)strains are common opportunistic pathogens,both environmentally and clinically,usually causing gastrointestinal infection,urinary tract cross infection and other diseases under given conditions.Antibiotics such as ciprofloxacin are widely used in the treatment of infection caused by species P.vulgaris,but also lead to the emergence of a large number of drug-resistant bacteria,which seriously affects the clinical treatment effect and endangers human life and health.A strain of genus Proteus with two endogenous plasmids was isolated from the intestines of Penaeus vannamei in our laboratory in the early stage,which was identified as species P.vulgaris and named as P3 M in this study.P3 M was comprehensively identified by whole-genome sequencing(WGS)and a series of experiments to elucidate its mechanism of ciprofloxacin and polymyxin resistance as a multidrug resistant bacterium.The specific research contents of this study are shown as follows:1.WGS was used to identify the P3 M genome,including the number and function of genes,GC content,non-coding RNA(nc RNA),repeat sequences and other detailed information.All the functional genes were divided into 22 groups and six metabolic pathways according to COG database and KEGG database,respectively.218 antibiotic resistance genes(ARGs)belonging to 36 categories of antibiotics were found on the P3 M genome,and P3 M were obviously resistant to 16 common antibiotics by the determination of minimum inhibitory concentration(MIC),indicating that P3 M was a foodborne multidrug resistant bacterium.The checkerboard MIC test showed that the combination of tetracycline and ciprofloxacin could significantly inhibit the growth of P3 M and play a synergistic bactericidal effect.Further bioinformatics analysis showed that there were two kinds of two-component systems(TCS)Bae S-Bae R and Cpx ACpx R,and several kinds of mobile genetic elements(MGEs),including plasmids,sitespecific recombinases and direct repeats on the P3 M genome.In this study,we identified the genomic and resistance characteristics of foodborne multidrug-resistant P.vulgaris strain P3 M,which will lay a foundation for further understanding the transmission and transfer of antibiotic resistance of species P.vulgaris in the environment.2.There are two endogenous plasmids in P3 M,p3M-2A and p3M-2B,the size of which is 2,656 bp and 5,903 bp,respectively.p3M-2B carries a quinolone resistance gene qnr D,while p3M-2A does not carry any resistance gene.qnr D-carrying plasmids can be divided into 2.7 kb and 4.3 kb according to their size,but p3M-2B does not meet this rule.Plasmid elimination and qRT-PCR experiments showed that p3M-2A could significantly promote the expression of qnr D on p3M-2B plasmid,and ORF1 played a major regulatory role in this process.Linear plasmid alignment analysis showed that ORF1-4 on p3M-2B was the most conserved sequence necessary for the function of qnr D-carrying plasmids,while ORF5 might be an exogenous DNA sequence obtained by homologous recombination of direct repeats DR-C during the long-term evolution process.In this study,we proposed the possible formation process model of larger qnr D-carrying plasmids such as p3M-2B,which provided a new idea for the horizontal transfer of antibiotic resistance genes.3.In this study,nc RNA Csi R(ciprofloxacin stress-induced nc RNA)involved in the regulation of ciprofloxacin resistance in P3 M was identified under the treatment of ciprofloxacin.The cell survival experiment showed that the survival rate of the Csi Rdeficient strain was significantly higher than that of the wild-type strain P3 M,indicating that Csi R played a negative regulatory role.Through further target prediction,qRT-PCR,microscale thermophoresis(MST)and other experiments,we identified the target gene emr B of Csi R participated in the regulation of ciprofloxacin resistance,and proved that the interaction between Csi R and emr B m RNA 5’ UTR region affected the translation process of emr B.In addition,through sequence alignment,we found that the interaction sites between Csi R and emr B m RNA were highly conserved in other sequenced P.vulgaris strains,suggesting that this regulatory mechanism may be ubiquitous in this species.In this study,we revealed the important regulatory role of nc RNA Csi R in the regulation of ciprofloxacin resistance in P.vulgaris species,providing a theoretical basis for further clarification of the mechanism of multidrug resistance in Proteus genera.4.In this study,nc RNA Psi R(polymyxin B stress-induced nc RNA)that can specifically respond to polymyxin stress signal was identified through WGS and qRTPCR.The survival rate of the mutant strain Δpsi R under polymyxin treatment condition was significantly lower than that of the wild-type strain P3 M,indicating that this nc RNA was involved in the regulation of polymyxin resistance of the strain.Through target prediction,qRT-PCR and MST experiments,pgs A,the target gene of Psi R involved in the regulation of polymyxin resistance was identified.Later,we specified the regulatory mechanism of Psi R through experiments such as half-life measurement,that is,the interaction between Psi R and pgs A m RNA unfolded the stem-loop structure in front of the binding site of pgs A m RNA,thus improving the stability of pgs A at the post-transcriptional level and enhancing the polymyxin resistance of P3 M.In this study,we revealed the important role of nc RNA in the regulation of antibiotic resistance and its highly conserved evolution in Proteus genera,laying a foundation for further understanding the mechanism of polymyxin resistance in Proteus in the future.In conclusion,this research reveals the multidrug resistance characteristics and evolution characteristics of Proteus vulgaris strain P3 M,and the plasmids and nc RNAs involved in the regulation of ciprofloxacin and polymyxin resistance were also systematically identified.At the same time,this study also clarified the mechanism of nc RNAs involved in the resistance regulation process and their important guiding significance for the study of the generation and transmission of drug resistance in other bacteria within the same species and genus,laying a foundation for future research in related fields. |
PDF Full Text Request