| Escherichia coli of chicken origin is an important common pathogen that seriously endangers the development of the poultry industry and can cause respiratory diseases and inflammation of other organs in chickens,affecting their performance.The irrational use of antibiotics has led to the emergence of bacterial resistance,and the formation of biofilms is one of the reasons for the emergence of resistance.The two-component system cpx AR is a typical regulatory system in chicken-derived Escherichia coli,which can regulate a variety of physiological activities of bacteria,such as susceptibility to drugs and bacterial colonisation,but the mechanisms regulating biofilm formation have not been fully elucidated.In order to investigate the relationship between the two-component system and biofilm,the following work was done in this study.1.Drug resistance profile and screening of target strains of chicken-derived Escherichia coli in HenanIn this trial,a total of 171 strains of Escherichia coli of chicken origin were isolated and identified from 195 autopsy samples of diseased and dead chickens collected in Henan during 2019-2021,with an isolation rate of 87.69%.The susceptibility of the clinical isolates was determined using the micro-broth dilution method.The test results showed that E.coli of chicken origin in Henan showed different degrees of resistance to common antibiotics,among which the resistance rates to ampicillin,ceftiofur and amoxicillin were high,ranging from 73.68% 91.89%,to the aminoglycosides kanamycin,gentamicin and amikacin from 25.14% 57.3%,between80.7% and 60.23% for the tetracyclines hygromycin and doxycycline,respectively,64.91% for florfenicol and 23.97% for ciprofloxacin.In order to obtain clinical experimental strains that meet the cpx R gene deletion among the above strains,the isolated clinical strains were screened for ampicillin and kanamycin susceptibility and biofilm forming ability,and three target strains that meet the requirements of the test were obtained.One strain E41 was randomly selected for whole-genome sequencing and the results were uploaded to NCBI.the whole-genome sequencing results were analysed in CGE,and the multilocus sequence typing was ST202,and the serotype was identified as O128,carrying multiple drug resistance genes and virulence genes.The above indicates that the drug resistance situation of Escherichia coli of chicken origin in Henan Province is more serious.2.Construction of Δcpx R and CΔcpx RIn this trial,the Red homologous recombinant gene editing technique was used to construct a cpx R gene deletion strain and a backfill strain.In this experiment,the homologous arm of the cpx R gene was amplified using the p KD4 plasmid to become the targeting fragment in the Red homologous recombination system.The targeting fragment was transferred into E41/p KD46 receptor cells by electrotransfer to obtain theΔcpx R:kan strain.The p CP20 plasmid was then used to eliminate ampicillin and kanamycin resistance in the recombinant strain,resulting in a gene deletion strainΔcpx R.The backfill strain was constructed using the overexpression plasmid p BAD/His A.A full gene fragment of cpx R was amplified in clinical strain E41,and the fragment was ligated to the p BAD/His A plasmid using an enzyme ligation process,and the resulting ligation product was transferred to DH5α receptor cells.The recombinant plasmid was verified by double digestion,and after successful verification,the plasmid was transferred to the deletion strain Δcpx R receptor cells to obtain the backfill strain CΔcpx R.In this experiment,the chicken-derived Escherichia coli two-component system cpx R gene deletion strain and backfill strain were successfully constructed,which provided a test strain to study the regulatory relationship between the twocomponent system cpx AR and biofilm.3.Study of the biological properties of E41,Δcpx R and CΔcpx RIn this experiment,to verify whether the deletion of the cpx R gene had an effect on the biological properties of the bacteria,growth curve,motility,biofilm formation,drug sensitivity and genetic stability of the deletion strain Δcpx R were measured for E41,Δcpx R and CΔcpx R.The results showed that compared to the wild strain E41,theΔcpx R deletion had no significant effect on the growth curve and biofilm formation,but the differences in motility and swimming ability of the bacteria were significant.In the drug susceptibility assay,the deletion of cpx R resulted in a 2-fold increase in the MIC of streptomycin and doxycycline for the deletion strain,and a reduction in the MIC of florfenicol to 1/2 of the original MIC.The deletion of the cpx R gene had a significant effect on bacterial drug susceptibility,while other aspects of its action were not significant and its function may be compensated for by additional factors in a compensatory manner.In the stability assay of the deletion strain,the deletion strain remained stable after 15 d of transmission.4.Detection of gene transcript levels in E41 and Δcpx R strainsIn this experiment,the transcriptomics technique was used to analyse the regulatory role of the cpx R gene on other related genes in the two-component system cpx AR.Differential and enrichment analyses of the transcriptomics results revealed 23up-regulated and 177 down-regulated genes following deletion of the cpx R gene,indicating that the cpx R gene is associated with many biological regulatory pathways.Real-time fluorescence quantitative PCR was used to validate the relevant differential genes in the flagellar system pathway,biofilm formation pathway and population sensing system,and the results showed that the differences between cpx P and lsr D genes were highly significant,and the differences between fli A,fli S,fli E and csr A genes were significant,and some genes were also not significant.In summary,the cpx R gene deletion strain was successfully constructed in the clinical strain,providing a test strain for subsequent studies,and the regulatory role of the cpx R gene on cpx P and other genes was determined,providing a theoretical reference for further studies on the regulatory relationship between the cpx AR twocomponent system and the biofilm. |
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