The Detection And Characterization Of Laribacter Hongkongensis In Diarrhea Patients And Freshwater Products In Jiangmen

1 BackgroundL.hongkongensis was first isolated from the blood and empyema of a cirrhotic patient by Professor Yuen in microbiology Department, HongKong University in 2001. They found it was a new genus and species of bacteria through biochemical identification and gene sequence analysis with 16S rRNA. A late research published in the Lancet showed that L.hongkongensis may cause gastroenteritis, especially serious gastroenteritis,and consumption of freshwater fish and travel were major risk factors for diarrhea. At present, The study in Mainland China is still basic. They focused on the bacteria contamination in freshwater fish, but the survey in population is almost in the blank. So far, only 13 strains of L.hongkongensis isolated from 2428 cases from a hospital in HangZhou by Ni Xiaoping. We have not seen other investigation in population with L.hongkongensis. Guangdong Province, where is adjacent to HongKong, supply freshwater products to it, and the detection rate of L.hongkongensis in freshwater fish as high as 41.7%.At present,when freshwater fish are exported to Hong Kong, L.hongkongensis must be checked to prevent the occurrence of diarrhea. However, we have not seen the report of infectious case in population in Guangdong province. Therefore, we do not know clearly about the potential risk on human health cansed by L.hongkongensis. So we carry out this study in Jiangmen.2 Objective2.1 To know the detailed information of infectious condition of L.-hongkongensis in diarrhea patients and freshwater products in Jiangmen city2.2 To describe the bacteriological chalacterization of L.hongkongensis, assess the genetical relationship of isolates, and provide evidences for molecular epidemiology of L.hongkongensis.3 MethodsWe collected fecal specimens of diarrhea outpatients and freshwater products to detect L.hongkongensis, and used the self-made questionnaires to acquire the patients, general condition, clinical features and epidemiological data.Biochemical tests and 16S rRNA based PCR were used to detect L.hongkongensis in diarrhea patients and freshwater products. We put the fecal specimens of diarrhea outpatients in Cary-blair agar immediately then took it to laboratory in 24 hours,then the cefoperazone MacConkey agar(CMA),which was modified from MacConkey agar by adding 16ug of cefoperazone/ml and lmg of dextrose/ml,to used to detect L.hongkongensis.The colonies like L.hongkongensis,which were oxidase, catalase, urease, and triple sugar iron positive,would be checked by API20NE. We also identified strains by 16S rRNA gene sequencing. The primers used for 16S rRNA gene amplification were LPW264 (5'-GAGTTTGATCMTGGCTCAG-3') and LPW265 (5'-GNTACCTTGTTACGACTT-3').The 50-μl PCR mixture contained 2μl of 100μmol/liter of each deoxynucleoside triphosphate,1μl of 50 pmol of each primer,5μl of 10×buffer (which contained 25 mmol/liter MgC12),2 U Taq polymerase, and 2μl of template DNA. The PCR temperature program was controlled at 94℃for 3 min, followed by 94℃for 1 min and then 55℃for 1 min and 72℃for 2 min (for 35 cycles), with a final extension at 72℃for 5 min. Ten microliters of each amplified product was electrophoresed in a 1.0%(wt/vol) agarose gel with a molecular marker (DNA marker DL2000; The search for sequences homologous was performed by use of the BLAST program.Susceptibility test of L.hongkongensis from people to 18 antimicrobial agents was carried out by the disk diffusion method according to the standards by the United States CLSI/NCCLSM100-519.The pulsed field gel electrophoresis (PFGE) was performed to describe the bacteriological chalacterization of L.hongkongensis, and assess the genetical relationship. The un-weighted pair-group mean arithmctic (UPGNA) of BioNumeries v4.0 software was used for cluster analysis.4 Results4.1 The detection form populationWe have collected 804 fecal specimens form diarrhea outpatients in the different hospitals in eight months in the Jiangmen.There were two fecal specimens positive for L.hongkongensis. There two strains, isolated in October, which were tested by biochemistry and gene sequence of 16S rRNA with BLAST.As results,they showed 99% of homology with L.hongkongensis reported in GeneBank.This was the first report of L.hongkongensis infectious cases of community-acquired diarrhea patients in population in Guangdong.The bacteria grew on MacConkey agar as nonhemolytic, gray colonies 1 mm in diameter after 24 hours of incubation at 37℃in ambient air. They were CAT, OX, URE, ADH, NO3, MAN, GNT,and CAP positive. They were TRP, ESC, GEL, ADI, MLT, CIT, PAC negative or don't assimilate any sugar. JM1 was sensitive to FEP, IPM, SXT, ATM, CN, AK, CIP, C and TE, but resistant to KF, CXM, CFP, KZ, CAZ, DA and MTZ,and was intermediate to PRL and AMP. JM2 was same to JM1 except resistanting to PRL and AMP,and being intermediate to TE and MTZ.4.2 The detection form freshwater products.We collected 425 samples of freshwater products totally. The L. hongkongensis were isolated from 25 (5.9%)of specimens, including 19 (30.1%) of the 63 grass carps,3(1.13%)of the 265 Amazonian snails,l of 5 wide Chinese tiger frogs(20.0%), 1 of 2 giant spiny frogs, and zero from 15 small fish,10 anodontas,23 white corbiculas,42 river snails and 18 wuhang fish.4.3 The PFGE results for Laribacter hongkongensisThe PFGE condition used for L.hongkongensis were as:OD 5.0, cell disruption for 8h, SPe I 20U cutS for 6h, switch time of 2.16 to 63.80s,120°field angle,6V/cm of electric field intersity, electrophoresis 20h.We used the same reagents, instruments and parameters to perform PFGE,27 strains were cut by SPe I into 10 to 14 bands ranging from 30Kb to 700Kb. Through analysis by Bionumerics V4.0 soft,we grouped the 27 strains, whose SID were between 46.6% to 100%, into 23 clusters(the strains with the same SID were considered as one cluster).The isolates got the SID score 100%, between Y16 and Y25,Y28 and Y29,Y35and Y39,W2 and W8。5 Conclusion:were detected from two diarrhea outpatients in Jiangmen,which implied L.hongkongensis may cause diarrhea although the detection rate was not high. We also found the L.hongkongensis in many freshwater products,and the results of PFGE showed there were strains getting SID score 100%, these indicated L.hongkongensis had aggregation of species and regions.