Prevalence And Antimicrobial Susceptibility Of Laribacter Hongkongensis And Enterotoxigenic Excherichia Coli Isolated From Patient With Diarrhea In Guangzhou

1 ObjectiveTo find out infectious condition and drug resistance of diarrhea causing by Enterotoxigenic Escherichia coli (ETEC) and Laribacter hongkongensis (LH) among diarrhea patients in a hospital in Guangzhou. To be aware of the detection rate of the ETEC and LH, simultaneously, with their drug resistance through the survey results in Guangzhou Haizhu district.2 Background2.1 Laribacter hongkongensisLaribacter hongkongensis is first isolated from the blood and empyema-thoracis of a patient with alcoholic liver cirrhosis in Hong Kong University by professor Woo in 2001.Through Biochemical identification and gene sequence analysis with 16SrRNA, they found that it is a new genus and species. A latest research Published in the Lancet showed that Laribacter hongkongensis may cause gastroenteritis, especially serious gastroenteritis. Consumption of freshwater fish and travel were major risk factors of the diarrhea which was studied by Microbiology department of Hong Kong University in 2004.At present, the Mainland in China of LH study is still in its infancy. They focused study on the bacteria contamination in freshwater fish and the survey on population infection is almost in a blank. So far, only four of LH isolated from 759 cases from a hospital in HangZhou by Ni Xiaoping. We have not seen other investigation about population with LH except HangZhou. Guangdong province where is adjacent to Hong Kong is the main freshwater supply base and the detection rate.of LH in freshwater fish as high as 41.7%.At present, when freshwater fish exports to Hong Kong,LH has become a pathogen must be checked to prevent the occurrence of diarrhea.However, we have not seen infection report in population in Guangdong, therefore, we are not clear the potential risk on human health caused by LH. Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea in children, adults and tourists in developing countries. According to the reports,four patients were associated with other pathogenic infections in seventeen samples of LH-positive patients, meantime, three cases were ETEC which have a dual infection with LH. The outcome told us LH and ETEC were more prone to infection dual.which increasing the patient's symptoms of diarrhea.2.2 Enterotoxigenic Escherichia coliETEC was first isolated in Calcutta in 1968 and was first isolated in Fujian by Lin Chengshui in our country. The detection rate of ETEC was focused on the years 1995-2002, the rate was range from 1.89% to 69.4%.There were only three formal epidemiological investigation in Guangdong. First,286 cases of acute diarrhea in infants and young children were tested from November 1983 to October 1984 in Guangzhou City and the rate was 16.3%;Second, The survey was performed in rural area from 1990 to 1991 with the rate was 69.4%; The third time was in 1997, a training military troop was monitored and the detection rate was 33.3%.The detection of ETEC diarrhea patients is limited to the collective unit of diarrhea outbreak in Guangdong in nearly eleven years which is also existed in other city.2.3 Antimicrobial susceptibilityAccording to the literature, the resistance rate of ETEC strains to the third-generation cephalosporin have reached 48% in Some areas of China and To ciprofloxacin and levofloxaci resistance rate as high as 66.7% and 65.7%,meantime,piperacillin and cefazolin resistance rate were similar to the third generation cephalosporins. However, high drug resistant rate of 72.4%-78.1% appeared in Penicillins, sulfonamides and tetracycline.ETEC strains had a high sensitive to Imipenem and other carbapenem. LH strains detected from Hangzhou showed different degrees of resistance to antibiotics commonly used in hospital, particularly to penicillin and third generation cephalosporins. The aim of this issue is to investigate infectious condition and drug resistance of diarrhea causing by Enterotoxigenic Escherichia coli (ETEC) and Laribacter hongkongensis (LH) among diarrhea patients in a hospital in Guangzhou, therefore,we can understand the merger rate of the two bacterial infection; infection risk factors and drug resistance spectrum and evaluate their potential risk to human health.So,we could provide some objective basis for clinical diagnosis and treatment.3 Sample collectionWe collected specimens of patients with diarrhea from intestinal disease clinic, Pediatric outpatient,gastroenterology outpatient and Emergency department. The patient's general condition, clinical features and epidemiological data were acquired from self-made questionnaire named "survey of infectious diarrhea".4 Laboratory detectionTreatment of fecal specimens: EC enriched culture medium was used for enrichment. MAC and CMAC specific culture medium were used to separate ETEC and LH. The swab was used for patients who did not have stool.Biochemical identification of LH:Suspicious positive strains of LH grew on CMAC should be Gram-negative, gray colonies of 1 mm in diameter,three sugar iron test negative and produce catalase, cytochrome oxidase after 48 h of incubation at 37℃in ambient air. API20NE is used for biochemical identification.Biochemical identification of ETEC:Suspicious positive strains of ETEC grow on MAC should be Gram-negative, pink colonies of 2-3mm in diameter,three sugar iron test positive and not produce cytochrome oxidase after 24h of incubation at 37℃in ambient air. Then, we do gene identification, the positive should be tested with API20E.Gene identification:PCR amplification of the 16SrRNA gene for LH and Enterotoxigenic gene for ETEC were performed according to previous publication.Sequence analysis:We send the PCR products to sequence analysis company if the fragment more than 100bp. Otherwise, we need to do a gene clone first. Compare sequence of PCR products in genebank using online server Blast.5 Antimicrobial susceptibility:We applied K-B disk diffusion method to antimicrobial susceptibility testing according to the standards by the United States CLSI/NCCLS M100-S19.6 Statistical Method:We establish a database using Epidata3.0 and entry the date with double people. Data analysis use software named SPSS 13.0.7 ResultsDetection conditions of LH:We did not detect LH from 646 cases in this survey.Detection conditions of ETEC:From August 2008 to October 2009,646 specimens were collected.38 patients were detected with ETEC.The incidence rate was 6%.There were fourteen cases with LT, four cases with STp,sixteen cases with STh and four cases with LT and STh. Clinical manifestations of ETEC:38 ETEC infection cases,63% patients with abdominal pain,37% with vomiting,11% with fever and 13% taking over of antibiotics. Abdominal pain in children group (age<15) and adults (age≥15) were the prominent symptoms. There were 18 cases with yellow thin watery,6 cases with mucous stool,6 cases with watery,1 case with bloody stool,4 cases with Loose stools and so on.Epidemiological features of ETEC:Epidemiological analysis of cases came from investigation form. The detection rate of Males and females showed no significant difference (χ2=0.282,P>0.05).The detection rate in less than 1 year old group is lowest and the highest detection rate in 2-year-old group.The detection peak of ETEC concentrated from February to September,the rate is from 3% to 25%,There were 2 patients with travel history,21 patients with history of eating aquatic Products and 17 cases eating out with in a weekAntimicrobial susceptibility:Resistant rates of ETEC were Ampicilin55%, Ticarcillin34%, Amoxicillin/clavulanic acid 8%, Minocycline 13%, Gentamicin 26% and Sulfamethoxazole 21%.The sensitivity rates of ETEC were Cefoperazone/sulbactam 87% and cefotaxime 89%.All strains were sensitive to Cefoxitin, ceftazidime,cefepime, Imipenem, Meropenem and Amikacin. There is only one strain resistant to ciprofloxacin.8 Conclusion:LH was found-as a new potential pathogens may cause gastroenteritis, especially one of the more serious causes of gastroenteritis. Monitoring of infection on the population is one way to assess the strain on the health risks of potentially dangerous. The survey did not detected LH because of Single-center survey. So, more research site, a larger sample size assessment of their risk of infection is necessary.Through this monitoring,we found a lower detection rate of ETEC which had the highest infection rate combined with LH and STh genotype detected in the main. Diarrhea caused by ETEC is always Watery diarrhea and has a severe clinical features. Therefore, the water samples in clinical diagnosis of patients with diarrhea should be thought of with very low detection rate of identification of the ETEC to avoid misdiagnosis and mistreatment.There were 21(55%) patients with ETEC infection.eating aquatic products and 17 (48%) eating out. This remind us that food hygiene is one of the important reasons cause ETEC diarrhea.Eating undercooked seafood may be a major risk factor for ETEC diarrhea and mixed infection with LH.Antibiotics resistance test showed that 38 strains of ETEC had a high resistance rate to penicillins, tetracycline and sulfa, meantime, they had a high susceptibility to cephalosporins. We suppose that cephalosporins could be used to treat diarrhea caused by ETEC.