Temporal And Spatial Distribution Of Antibiotic Resistance Genes In Liver Infected With Campylobacter Jejuni

Campylobacter jejuni(C.jejuni)is an important food-borne pathogen and symbiotic bacteria that is widely prevalent in the world.It mainly causes gastroenteritis,endocarditis and sepsis in humans and animals,and can cause immune injury diseases(reactive arthritis,hepatitis and Guillain-Barre syndrome(GBS).Antibiotic resistance genes(ARGs)can be captured by human normal flora,conditional pathogens and pathogens through migration of resistance elements.Human infection carries the pathogen of ARGs,which may be difficult to treat.It is very important to study multi-drug resistant of Campylobacter jejuni.Dong Jun,a member of the research group,had previously studied the epidemiology of Campylobacter jejuni in poultry in central China.The results showed that Campylobacter jejuni strains(ST464CCs)had the strongest ability to form biofilm,and the overall resistance to various antibiotics was high.However,it is unclear whether multi-drug resistant Campylobacter jejuni causes antibiotic resistance genes(ARGs)to be transmitted to other bacteria in the body during chronic infection after infection.In this study,we hypothesized that ARGs can be transmitted to bacteria in the liver and change the structure of microbial community in the process of chronic liver infection by multi-drug resistant of Campylobacter jejuni.The design experiment was as follows:80 Kunming mice were randomly divided into four groups:A,B,C and D.They were inoculated with 6×10~9 CFU/ml,3×10~9 CFU/ml,1.5×10~9 CFU/ml bacterial liquid and Bush broth respectively.The experiment of mice infection lasted for eight weeks.By observing clinical symptoms,counting clinical scores,calculating visceral index,pathological changes,pathological sections,pathological scores and other correlation analysis and pathogenicity data,we can effectively evaluate the autoimmune pathological injury model and explore the characteristic changes of liver injury;detect the types of ARGs by microbial qPCR DNA chip;construct the clone libraries of intI1,tnpA and 16S rRNA,and conclude.Quantitative detection of ARGs by qPCR was carried out to explore the temporal and spatial distribution characteristics of ARGs in the liver.The microbial communities in liver samples were analyzed by 16S rRNA nested PCR amplification and high-pass sequencing techniques,and the migration patterns of ARGs in the organs and the distribution characteristics of ARGs were revealed.The main results are as follows:(1)Establishment of pathological model of autoimmune injury caused by Campylobacter jejuni infection.According to the best modeling conditions obtained from the experiment,the rats in each group were observed at 8:00 a.m.and 6:00 p.m.from Monday to Sunday.The results showed that Kunming mice in group A showed clinical symptoms such as decreased activity,shortness of breath,slight tremor,weak hind limbs movement and disordered abdominal fur after infection with medium concentration of bacterial solution in group B,which were lower than those in group C.The clinical symptoms of bacterial contamination solution were obvious,and the clinical symptoms score could reach 4-5 points.There was no obvious abnormal activity in the low dose group,and the clinical symptom score was 0.At 8:00 a.m.every Tuesday,Kunming mice in each concentration group were dissected.The main pathological changes were mesentery and small intestine effusion.Compared with the control group,the liver and spleen were markedly enlarged and inflamed,which was consistent with the clinical symptoms of infection.Histopathological sections showed that the central veins of the liver were arranged irregularly,there were lesions in the hepatocytes,and there were slight lesions in the cecum and mucosa.The lamina propria showed hyperemia.The results showed that Campylobacter jejuni ST464 inoculated the Kunming rats with different doses,which caused significant changes in the organ index at different time points,especially the spleen and liver showed obvious characteristic changes,and the characteristics of the small intestine were slightly obvious;The heart,lungs,kidneys,etc.did not show obvious characteristic changes,which is likely to have a great relationship with the functional role of various organs.Kunming mice can be caused a diffuse chronic infection of the liver via the model of abdominal infection.(2)Distribution characteristics of antibiotic resistance genes in liver of Campylobacter jejuni at different concentrations and at different time points.Under laboratory conditions,kunming mice were challenged according to high concentration,low concentration of three concentrations of bacteria liquid and Bush broth(control group)and samples were taken for eight consecutive weeks.A total of 10 drug resistance genes were detected by microbial qPCR DNA chip,which belonged to?-lactam,macrolide and fluoroquinolone,accounting for 73.97%,23.25%and 2.77%,respectively.From the Sperman correlation analysis,the intI1/tnpA abundance showed a positive significant relationship,p<0.05.The intI1 gene and tnpA play a key role in the capture of ARGs and their horizontal transfer during the propagation of ARGs.The resistance gene ermB(macrolides antibiotics)showed a positively significant relationship with the liver organ index(Liver index),p<0.05.The ermB gene has a significant effect on the pathological changes of the liver during the chronic infection process.This is a positively significant relationship between Acidobacteria and SHV(Beta-lactam).Through Adonis analysis,R~2=0.49531 indicates that the group explained the difference in the difference group;P<0.02,indicating that the test has high credibility,and most of the similar samples between the microorganisms Community structure similarity is relatively high.The high-throughput sequencing results of 16S rRNA Miseq showed that in three groups of high,medium and low concentrations of multi-drug resistant C.jejuni infection,Proteobacteria,Acidobacteria and Firmicutes are the most important microflora in the microbial community on Phylum level,and their abundances are 38.73%,33.23%,19.7%,accounting for 91.66%of the total microbes.Obviously;Rhodococcus,Achromobacter and Staphylococcus have been shown to be a common dominant genus on genus level,with the exception of dominant species,other species have certain differences.In the high-concentration group,multi-drug resistant of Campylobacter jejuni will reduce the diversity and abundance of microorganisms in the process of chronic infection,which may promote the growth and reproduction of resistant dominant bacteria to a certain extent in the microbial community.In summary,in this study,the liver of multi-drug resistant of C.jejuni ST464infection in the chronic process can be detected three major classes of antibiotics(10kinds of ARGs)in liver based on the distribution of spatial and temporal,which preliminary indicates the important impacts between the liver index,microbial community and ARGs.The influence of ARGs on the bacteria and microbial community structure transmitted to the liver provides more possible data support for controlling the spread of ARGs in the future.