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Detection of Laribacter Hongkongensis in Patients with Diarrhea and Freshwater Animals and the Bacterial Characterization

Author: ZhangOu
Tutor: HuJing；ChenQing
School: Southern Medical University,
Course: Epidemiology and Biostatistics,
Keywords: Diarrhea Hong Kong seagull -shaped bacteria Biofilm Integrase Animal experiments Virulence
CLC: R446.5
Type: Master's thesis
Year: 2011
Downloads: 18
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Abstract

Research purposes to collect stool specimens of diarrheal patients in two hospitals in Guangzhou for the Guangzhou area by the Hong Kong seagull-shaped bacteria due to the incidence of diarrhea, LH detection. In order to understand the ecological characteristics of Hong Kong seagull-shaped bacteria, designated collecting the Hetang frogs and snails sample LH detection explore LH reservoir bacteria in freshwater animals in the wild. 2 to further explore the LH multiple drug resistance mechanism, by comparing the LH integrase gene expression levels in the biofilm and the liquid state, in-depth study on the the LH integration of the sub. 3 is an in-depth understanding of the Hong Kong seagull-shaped bacteria pathogenic mechanisms Preliminary animal experiments pathogenic. The research methods Guangzhou hospitals and community clinics diarrhea hongkongensis-shaped bacteria detected in the patients and the surrounding waters wild frogs and snails research. 1.1 specimen collection and data acquisition: from May 2009 to October 2009 to collect hospital infection disease outpatient, the Gastroenterology outpatient and emergency department gastroenteritis diarrhea stool specimens of patients over 14 years old. The general condition of the patient, the clinical manifestations and epidemiological data obtained by homemade infectious diarrhea questionnaire. Reservoirs of wildlife specimens in the service area of ??the hospital to receive specimens captured. 1.2 laboratory testing: the diarrhea patient specimens collected wildlife specimens after corresponding treatment vaccination LH selective medium and EC broth at 37 ℃ for 24h, biochemical identification (including Gram stain, dual-enzyme test, urea and the the trisaccharide iron medium well API20NE of biochemical identification strips) and gene identification screening the LH positive strains. Identified, according to the sequencing results ultimately LH positive strains were sequenced amplified fragments above 100bp product directly sent to the British completed. 1.3 Statistical methods: epidata3.0 the establishment of a database, the data double entry. Hong Kong seagull-shaped bacteria class Ⅰ integrase gene expression levels in the state of biological membrane and liquid phase detection the 2.1 LH bio-film and liquid phase were cultured overnight shaking culture integrase enzyme positive LH strains of bacilli part as liquid state bacteria saved at -20 ℃, the other part is seeded in six-well plates containing medium, biofilm carrier for ordinary coverslip at 37 ° C for static culture 12d medium was changed every day for board. 12d with saline flush carrier coverslip, staining, cell scraping scraping the biofilm on the carrier. Collect the biofilm status bacteria break up and wash, and stored at -20 ℃. 2.2 Preparation of integrated operation 13 LH biofilm bacteria and liquid bacterial culture were extracted total RNA enzyme-positive strains cDNA Omega total RNA miniprep kit instructions, the entire ice; Promega Corporation M-measured concentration MLV reverse transcriptase cDNA synthesis manual operation, the operation of total RNA of reverse transcriptase become cDNA. 2.3 Comparison of the biofilm and the liquid state under integration of the level of gene expression in order to extract into the biofilm and the liquid state of the cDNA as a template, RT-PCR amplification, and the total reaction volume of 20μL, wherein 10μL SYBR Green Mix, each primer 1μL (2mM), cDNA template 6 μL reaction conditions of 94 ° C denaturation 30s; 95 ° C 15s, 58 ° C 45s, for 30 seconds at 72 ° C, 80 ° C 5s, 40 cycles, extension 20S. After five cycles, the addition of the internal reference of each primer, 1μL continue. 1.5% agarose gel electrophoresis analysis of observed and gray-scale scanning, through statistical processing differences. 3 Preliminary through animal studies seagull-shaped bacteria pathogenic study 3.1 Hong Kong seagull-shaped bacteria OD-CFU curve plotting logarithmic phase bacterial suspensions were prepared by enrichment after continuous delivery times dilution, selected the appropriate wavelength measured gradient of OD value, averaged, and the viable count. OD value and viable volume as a variable, to draw the correlation curve. The results show that the wavelength in the 580-600nm, the culture liquid OD value can be more accurately reflect the amount of LH in viable, so LH BACTERIA OD value was measured in a suitable wavelength and OD-CFU curve equation. The 3.2 LH virulence experimental LH original strains isolated in our laboratory for the experimental strains, 50 ordinary level Kunming mice were randomly divided into two groups (experimental bacteria and control bacteria, to control the use of non-pathogenic E. coli TG1). Each group was randomly divided into five groups (5 / group), intraperitoneal injection to the animals in each group were inoculated with different gradient broth continuous tapping bacteria 7d, daily observation of clinical performance, according to the death and survival record calculated the median lethal dose of LH karber law. 3.3 three-day-old suckling rabbits infected Hong Kong seagull-shaped bacteria the experiment eight 3-day-old suckling rabbits were randomly divided into four groups, three groups were orally inoculated Hong Kong seagull-shaped bacteria of different doses of fresh bacterial suspension, orally inoculated with sterile saline control group. Gavage fasting (suckling rabbits and females separately) 4h, every time intragastric 1ml / 2 times a day for 10 days. Daily blind species, and clinical observation. Gavage were killed after the end of the experimental animals, anatomy, bacterial culture and biopsy of tissue to do. Results in a hospital in Guangzhou diarrhea patients and freshwater animals digestive tract LH detection: from May 2009 to October 2009, stool specimens collected 238 cases of diarrhea patients, including 127 cases of male and female 111 cases, not isolated to positive strains. 144 total catch of wild freshwater animals: snails 120, accounting for 83.3%; including apple snails 73 (50.69% of the total number of specimens), local snails 47 (32.64%). 24 frogs, accounting for 16.7%; golden frog 10 (6.94%), toad 14 (9.72%). A total of 13 samples isolated LH: apple snails in samples isolated LH, the positive rate was 2.74%; the local snails not positive; 3 golden frog isolated LH, the positive rate was 30%; isolated toad 8, the positive rate was 57.14%. 2 hongkongensis shaped bacteria class Ⅰ integrase gene in biological film and a liquid phase state under expression levels were 26 samples RT-PCR, and amplified simultaneously internal reference primers. The product was purified by gel electrophoresis analysis of the liquid culture bacteria and biofilm bacteria have class Ⅰ integrase mRNA. The sample class Ⅰ integrase strip and internal reference bands are clearly visible. Grayscale scanning biofilm bacteria Ⅰ integrase gene expression average is about 3 times higher than the liquid bacterial culture The biofilm culture group Ⅰ integron gene expression levels to 2.545 ± 0.674 liquid culture group Ⅰ integrase expression as 0.9736 ± .7549 Finally, paired t-test analysis of the relative gray scan results, the two groups, the difference was statistically significance (P lt; 0.05), to prove the state of the biofilm bacteria Ⅰ integrase gene expression different from the ordinary liquid bacterial culture Ⅰ integrase gene expression. Animal studies Preliminary seagull-shaped bacteria pathogenic study based on the results of preliminary experiments, selected the OD value the measured wavelength 600nnm LH broth, and LH OD-CFU curve equation y = 5 × 10gx-3 × 107; through on virulence experiment calculation LH median lethal dose LD50 = 1.504 × 109. The three-day-old suckling rabbits infected Hong Kong seagull-shaped bacteria experiments showed that blind species of hard stools and rectal swabs were not isolated LH; anatomy found no abnormal heart and lung, stomach swelling, small intestine covered with yellow mucus, large intestine of caking and yellow will no abnormal abdominal cavity; blood culture results were negative; intestinal tissue cultures were isolated LH; pathological Oral bacterial amount for a 3 × 1015cfu/ml group of small intestine inflammation, large intestine visible immune cells; No. 7, in which the small intestine inflammation is not heavy, vascular congestion expansion should appear neutrophils, but did not observe, colon mild hyperemia. Oral bacterial amount small of 3 × 1014cfu/ml group submucosal edema, visible lesions; colorectal cell edema. Oral bacterial amount of 3 × 1013cfu/ml group small intestinal biopsy revealed mild inflammation suspected degeneration, congestion is not obvious; colon vacuolar degeneration, cell edema. Research conclusion LH as a new discovery of potential pathogens, may cause gastroenteritis especially the more severe gastroenteritis pathogens, monitoring of the population infected is to assess one of the potentially dangerous bacteria on population health risks ; the survey of 238 cases of diarrhea in the Guangzhou two medical units charged crowd failed to isolate LH, multi-center, as well as a larger sample size of their assessment of the risk of infection is necessary. The experiment average detection rate of wild frogs and edible frog difference was not statistically significant, the detection of apple snails (2.74%) of the positive rate for the first time reported. Visible, Hong Kong seagull-shaped bacteria of the insurance bacteria host is not confined to freshwater fish and other freshwater products and wildlife research allows us a more comprehensive understanding of their distribution in nature, so as to achieve effective prevention. Since LH with multiple drug resistance, its study of the mechanisms of resistance integrase is necessary; studies LH the integrase gene mRNA expression in bacterial biofilms state is three times the liquid phase. From one point of view, the results of this study explain integron-positive strains of multi-drug resistance problem, and further explore the molecular characteristics to be. Through animal experiments, the study also LH pathogenic preliminary exploration perfect LH basis of experimental data, and the animal experiments, and laid a foundation for the following research.

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