| Objective1 To understand the the incidence of diarrhea caused by Laribacter hongkongensis, we collected stool samples in two hospitals in Guangzhou for LH detection. To understand the ecological characteristics of LH, we exquisite frogs and snails in the wild reservoir to detect LH.2 To further explore the mechanism of multidrug resistance of LH, expression levels of LH integrase genes in the biofilm and liquid phase were compared.3 To better understand the pathogenic mechanism of LH, animal experiments had been done.Methods1 The detection of LH in patients with diarrhea in a hospital and community in Guangzhou and fresh water animals surroundings.1.1 samples collection and data acquisition:May 2009 to March 2009 we collected stool specimens of gastroenteritis patients with diarrhea who were more than 14 years old in hospital infection department, gastroenterology clinic and emergency department. Patients' characteristics, clinical manifestations and epidemiological items were obtained through "survey of infectious diarrhea". Wildlife specimens were captured in the reservoirs near the hospital.1.2 Laboratory testing:Specimens collected from patients with diarrhea and fresh water animals were corresponding inoculated in EC broth and LH selective medium then cultured at 37℃for 24h. After biochemical identification (including Gram staining, double-enzyme test, urea base, triple sugar iron culture base and API20NE biochemical system) and the gene identification positive strains were screened. For the fragments more than 100bp were delivered directly to the Shanghai sequencing companies to sequence the LH-positive strains.1.3 Statistical Methods:set a database with epidata3.0, double entry the data.2 The expression of classⅠintegrase gene in state of biological membrane and liquid phase2.1 LH in biological membrane and liquid phase were culturedShake culture of the integrase-positive strains of the bacterium LH overnight, take one part of it as liquid bacteria stored at -20℃; the other part was inoculated to be biofilm with the carrier of ordinary coverslip, cultured for 12 days under the temperature of 37℃, exchange the medium everyday. Then wash the biofilm with saline, stain and scrap the biofilm on the carrier with a cell scraper. The biofilm bacteria collected was then scattered,washed and stored at -20℃for use.2.2 Preparation of cDNAAccording to Omega's small amount of total RNA extraction kit instructions, total RNA was extracted from 13 LH biofilm and liquid bacteria culture; After measuration RNA was transcript into cDNA with M-MLV reverse transcriptase of Promega Corporation.2.3 Comparison of the level of integration of gene expression in state of biofilm and liquid,Take the cDNA above as templates, amplify the fragment by RT-PCR with total reaction volume of 20μL, which include lOμL SYBR Green Mix, each primer 1μL (2mM), cDNA template 6μL. Reaction conditions was 94℃30s; 95℃15s,58℃45s,72℃30s,80℃5s,40 cycles, extension 20s. They reacted for 5 cycles before joining the internal parameters of each primer, 1μL to continue.; analised by 1.5% agarose gel electrophoresis and then the gray scale scanning.3 Study of pathogenicity through animal experiments3.1 OD-CFU shaped curvePrepare the bacterial suspension, diluted and measured their OD values. Regard OD values and viable amount of variables separately as X and Y, draw the correlation curve. The results showed that when the wavelength was 580-600nm, OD value of culture medium can more accurately reflect the amount of live LH. So LH was measured at the appropriate wavelength to get OD-CFU curve equation.3.2 LH virulence testTo the original laboratory strain isolated in Jiangmen as experimental strains, the normal level mice were randomly divided into two major groups (experimental and control, non-pathogenic E. coli TG1 was control). Each large group was further divided into five groups (five/group). With the method of intraperitoneal injection animals were gave different mumber levels of LH, continuously attacked for 7 days. Observed their clinical performance, conditions of death and survival, then calculated the median lethal dose by karber method.3.3 Three-day-old neonatal rabbits infected by LH Three-day-old rabbits were randomly divided into 4 groups,3 groups were orally inoculated with different doses of fresh bacterial suspension and the control group was orally inoculated with sterile saline. Experimental animals were then sacrificed for gavage, anatomy and tissue biopsy.Results1 Detection of LH in diarrhea patients in hospitals and freshwater in GuangzhouFrom May 2009 to October 2009, among the total 238 cases of diarrhea patients, 127 male,111 female, there was no positive strain. Freshwater species were 144 cases, including 120 snails, accounting for 83.3%; fushou spiral of 73 (50.69% of the total number of samples), the local spiral of 47 (32.64%).24 frogs, accounting for 16.7%; jinxian frog 10 (6.94%), toad 14 (9.72%).13 LH positive strains were found.2 in Fushou snails, the positive rate was 2.74%; no positive detection in local spirals; 3 in jinxian frogs, the positive rate was 30%; 8 in toads, positive rate was 57.14%.2 the expression level of classⅠintegrase gene in the biological membrane and liquid state in LHThe fragments and internal reference primers of 26 samples were amplified by RT-PCR. The classⅠintegrase mRNA were present in all the Products. All fragments of classⅠintegrase band and the internal reference bands were clearly visible. Through gray scale scanning, integrase gene expression in state of biofilm was about 3 times higher than that in liquid. ClassⅠbiofilm culture group integrase gene expression was 2.545±0.674, liquid culture group integrase classⅠexpression was 0.9736±0.7549, paired t test analysis was used, the difference was significance (P<0.05), proved that bacteria in the biofilm state of integrase gene expression of typeⅠliquid culture bacteria different from the ordinary classⅠunder the integrase gene expression. 3 Study of pathogenicity of LH by animal experimentsAccording to the results of preliminary experiments, the selected wavelength of 600nm was used to obtain the OD-CFU curve equation, which is y=5×108x-3×107; The median lethal dose was calculated and LH's LD50=1.504×109. The animal experiments showed that LH was not isolated from the hard stools and rectal swabs; dissection found no abnormal heart and lung, stomach swelling, covered with yellow mucus in the small intestine, large intestine will be caking and yellow, abdominal cavity was normal; blood culture results were negative; intestinal tissue cultures were isolated from LH; biopsy revealed Oral bacterial content of 3×1015cfu/ml group had inflammation of the small intestine, large intestine shows immune cells; in which the small intestine 7 Inflammation is not heavy, vascular congestion of the expansion, should appear on the neutrophils, but not observed, the large intestine with mild congestion. Oral bacterial content of 3×1013cfu/ml group revealed mild inflammation of the small intestine biopsy, suspected of degeneration, hyperemia was not obvious; colon degeneration, cell edema.ConclusionsLH as a newly discovered potential pathogens, which may lead to more severe gastroenteritis gastroenteritis, the monitoring of human infection is important in assessment of potential danger. None LH positive strains were found among 238 cases of diarrhea this time, so the multi-center and a larger sample size is necessary. In this study, the average detection rate of wild frogs and edible frogs had no significant differences. The host of LH was not limited to freshwater fish but also other fresh products which help us to understand their distribution in nature more fully. Since LH has multiple drug resistance, the study of its resistance mechanism caused by integrase is necessary; this study showed that mRNA of integration in the biofilm state is 3 times than that in liquid. As well as, the pathogenicity of LH were explored by animal experiments. |
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