Epidemiological Study,Pathogenicity And Genome Sequence Analysis Of Laribacter Hongkongensis

Objectives:1.To investigate the prevalence of Laribacter hongkongensis(LH)in different hosts,we collected the stool samples from the hospital outpatients with diarrhea in Guangzhou,the patients with liver disease,the healthy population,the wild type Rattus norveum and the freshwater products.2.To further study the pathogenesis of LH,nude mice were used as animal models to explore the pathogenicity of different host-derived strains.3.In order to understand the genome similarity and difference of LH from different host sources,genome characterization and polymorphism were analyzed by whole genome sequencing.Method:1.Specimen collection:collected the stool samples from the hospital outpatients with diarrhea in Guangzhou,the patients with liver disease,the healthy population,the wild type Rattus norveum and the freshwater products.2.Identification and isolation of LH strain:biochemical screening and PCR amplification of specific fragments for confirmed.3.The resistance phenotype of LH strain was determined by disk diffusion method.4.MLST was used to determine the allele spectrum and ST number of LH strain.Gene analysis and cluster analysis were carried out using eBURST and START2 analysis software.5.The nude mice were divided into 7 groups:diarrhea isolate(HLHK9 and LH679),hepatopathy patient isolate(LHNF250),Rattus norvegicus isolate(LHR251),grass carp isolate(LHY19),tiger frog isolate(LHW73),and normal saline.6.Different host-derived LH,including LHNF250,LHR251,LHY19,and LHW73 were sequenced by second-generation sequencing technology Illumina HiSeq2000.Results:1.LH prevalence:One LH strain was isolated from stool samples of 600 patients with liver disease,168 cases of diarrhea patients and 150 cases of healthy people(500 copies)were not detected;the positive rate was 1.05%in wild-type Rattus norvema stool specimen,32.4%and 71.4%in the contents of swab of grass carp and tiger frog,respectively.2.LH strains showed varying degrees of resistance to 19 kinds of commonly used antibiotics.The highest total resistance rate was cefalotin(100%),followed by cefazolin(96.7%),rifampicin(93.3%),and cefoperazone(93.3%).LH strains showed to be sensitive for cefepime,imipenem,aztreonam,gentamicin and amikacin.4.MLST classification:LHNF250,LHR251 and LHR398 isolates were divided into ST-162,ST-163 and ST-164,respectively.Genealogy and cluster analysis showed that ST-162,ST-163 and ST-164 were dispersed,and the relationship between one brown rat-derived isolate was close to that of human isolates.5.LH-infected nude mice:All nude mice in the six experimental groups did not develop diarrhea,and the pathological sections showed that the degree of lesion(degeneration,inflammatory cell infiltration and intestinal mucosal atrophy)of colonic tissue varied from high to low was:LH679,HLHK9 and LHW73>LHR251>LHR251>LHYL and LHNF250.The degree of liver lesion(hepatic sinusoid dilatation,stem cell edema and lymphocyte infiltration)from high to low was:LH679>HLHK9 and LHW73>LHR251>LHY19 and LHNF250.6.The genomic size of LHNF250,LHH251,LH19 and LHW73 were 3,072,799bp,3,063,288bp,3,265,250bp,3,205,168bp respectively.The predicted genes were 2,880,2,879,3,113 and 3,031 respectively.The genomic average GC 62.35%,62.40%,62.53%and 62.48%respectively.There were 65,63,62 and 64 tRNAs,respectively.Conclusion1.Isolation from the stool of a cirrhosis patient suggested that cirrhosis was a potential risk factor for LH infection.LH strains were isolated from Rattus norvegicus,which suggests that Rattus norvegicus may be a potential host of LH,other than humans and freshwater organisms;the positive rate of LH in freshwater fish and tiger frogs is consistent with previously reported in the literatures.The above findings help us to understand the distribution of LH in nature.2.All LH isolates are resistant to at least one type of antibiotic.Most of the strains showed resistance to rifampicin and the first or the third generations of cephalosporin antibiotics,while the second and the fourth generation cephalosporins showed lower resistance.3.Liver disease patient and Rattus norvegicus isolates were new ST-type.Cluster analysis suggested that murine-derived strains had more close genetic relationship with diarrhea patients,but the linkages between them were to be further studied.4.According to the pathological changes of colonic tissue and liver tissue,the virulence of the strains may be different.The pathogenicity of LH from different host sources was discussed,and the basic experimental data of LH were improved.5.The genome size,GC content and genomic components of four strains of LH sequencing strains were consistent with the genome of different host strains,and the genome of multiple resistant strains carried genes related to their resistance.