Detection And Multi-locus Sequence Typing Analysis Of Laribacter Hongkongensis Isolated From Diarrhea Patients And External Environment

Background:Laribacter hongkongensis were detected mainly in freshwater products, such as grass carp, bighead carp and tiger frogs and relevant studies to prove the existence of other hosts were in the blank so far. Lau et al (2006) collected 10 reservoirs water samples and laribacter hongkongensis were isolated from six water reservoirs in Hong Kong [8]. However, the paper that indicate laribacter hongkongensis is detected in in the water reservoir in mainland China has not yet been published.The possible source and transmission of the bacteria has not been fully elucidated.Currently the classification method of LH is genotyping, such as PFGE, ERIC-PCR, MLST and so on. Feng Jiali et al [37] used PFGE and ERIC-PCR typing LH for the LH isolated from freshwater products in Guangzhou., Woo et al [35] applied MLST to typing LH isolates from freshwater fish and human in Hong Kong. Molecular typing to study about molecular characteristics of LH is more appropriate than phenotyping because of its accuracy and stability,which can not only used for epidemiological investigations and traceable disease outbreaks, but also can be applied to analyze the relationship between LH strains from the perspective of genetic evolution. At present, China is still a lack of MLST for LH isolates and related research and analysis.Objective:1. In order to know detailed information of infectious condition of laribacter hongkongensis in diarrhea patients in Guangzhou;2. To acquire detailed information of Laribacter hongkongensis in fishery products in GuangZhou,such as freshwater fish, edible frogs and amaizon snails were sampled to detect for laribacter hongkongensis;3. To investigate the presence of L. hongkongensis in water from drinking water reservoirs in Guangzhou to known about the ecology of this bacterium in the aquatic environment;4. To further elucidate the etiology properties of LH,we carried out the following research:To analyze antibiotic resistant phenotype of LH isolates;To assess the genetical relationship of LH isolates and provide evidences for molecular epidemiology of LH by applying MLST to genotyping.Method:We collected specimens of diarrhea out patients to detect L. hongkongensis and used the self-made questionnaires to acquire general conditions,clinical features and epidemiological data from May in 2013 to November in 2014.The freshwater products including freshwater fish, edible frogs and mudsnails(Cipangopaludina cahayensis) were consumed from four markets in Guangzhou and sent to the Lab for testing LH from December in2013 to May in 2014. Water samples were collected from 6 drinking water reservoirs located in different regions of Guangzhou namely Helong, Xintang, Huangpu, Jinzhong, Hunkeng and Xinbei reservoirs. Samples were collected using aseptic principles.Biochemical tests and 16S rRNA based PCR were used to detect L.hongkongensis in diarrhea patients,frethwater products and water reservoirs samples.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 cefoperazone/ml, used to detect L.hongkongensis.The colonies like L.hongkongensis.which were oxidase,eatalase,urease,and triple sugar iron positive,would be checked by API.We also identified strains byl6S rRNA gene sequencing. The primers used for 16S rRNA gene amplification were LPW264(5’-GAGTTTGATCMTGGCTCAG-3’)and LPW265(5’-GNTACCTTGTTACGACTT-3’). The search for sequences homologous was performed by use of the BLAST program.Susceptibility test of L.hongkongensis was carried out by the disk diffusion method according to the standards by the United States CLSI/NCCLSM 100-519.The multilocus sequence typing(MLST) was performed to deseribe the bacteriological characterization of L.hongkongensis,and assess the genetical relationship. The eBURST, START2 and mega6 software were used for further analysis.Results:1. From May 2013 to November 2014,among the total 545 cases of diarrhea Patients(No.1004-1548),there was no positive strain.2. L. hongkongensis strains were isolated from the midguts and hindguts of 53 (16.36%) of 324 freshwater products, including 256 grass carps(17.97%),15 tiger frogs(46.67%) and 53 Cipangopaludina cahayensis. L. hongkongensis was not isolated from Cipangopaludina cahayensis.3. L. hongkongensis was isolated from Helong reservoir among six detected reservoirs. 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.horigkongensis detected in water reservoirs in Guangdong.The baeteria grew on MacConkey agar as nonhemolytie,gray colonies lmm in diameter after24hours 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.4.54 LH strains showed varying degrees of resistance to 19 kinds of commonly used antibiotics. Resistance rates descend as follows:cefoperazone (53/54,98.15%), cefazolin (52/54,96.30%), cephalothin (51/54,94.44%), ampicillin (44/54,81.48%), rifampicin (38/54,70.37%), erythromycin (15/54,27.78%), piperacillin (11/ 54,20.37%), tetracycline (10/54,18.52%), ceftazidime (8/54,14.81%), ciprofloxacin (7/54,12.96%), cefepime (6/54,11.11%), aztreonam (5/ 54,9.26%), streptomycin (4/54,7.41%), cefuroxime (4/54,7.41%), cotrimoxazole (3/54,5.56%), chloramphenicol (2/54,3.70%), imipenem (1/54,1.85%), amikacin (1/54,1.85%), gentamicin (0/54,0%). Various isolates were found to show resistance to cephalosporins, most of which performance the first and third-generation cephalosporin-resistantance, while show less resistance to the second, fourth generation cephalosporins. Most isolates were resistant to rifampin performance.5. Results of MLST for LH5.1 A total of 72 different STs were observed from 113 isolates of L. hongkongensis from our lab. Three STs in the three isolates from patients were observed and one ST were detected before.There were four different STs observed in the four isolates from amazonian snails.which were all new STs. forty-two STs in the fifty-one isolates from frogs and twenty-one STs in the fifty-five isolates from fish. ST101 was detected in both isolates from patient and frog.23 STs including 21 new types and ST42 and ST45 which were found before were observed in 55 LH strains isolated from fish.42 STs containing 36 new STs were detected in 51 LH isolates from frogs.ST45 was the dominant sequence type among the LH isolates from fish in Guangzhou and Jiangmen in this study,whose frequency observed is twenty-eight, followed by ST117, a total of four LH strains isolated from frogs in Guangzhou.In addition, the ST42、ST23、ST106 all appeared three times.Most LH Strains referred to ST45 were isolated from grass carp and only from bighead carp from different markets in Guangzhou and Jiangmen and detected in four seasons,suggesting no relationship among the L. hongkongensis isolates with the sampled markets.ST117 appeared in isolates from two tiger frogs and two hylarana guentheri sampled from different markets in Guangzhou.ST101 was detected in both JM-623 isolated from a diarrhea patient in Jiangmen and GZ-W59 from tiger frog in Guangzhou,demonstrating potential route of transmission. Fish source isolates, frog source isolates,snail source isolates and isolates of human origin uncrossed, suggesting that the ST-type between species may have specificity.5.2 eBURST software for further analysisEBURST software using allele number and STs for analysis, can divide all ST-types into 10 groups of clones complex. The first group (group1) including ST-type was shown in Table 3-8, and ST45 is the predicted group founder. The second group (group2) includes ST42, ST156, ST143 (ST42 to predicted group founder); the third group (group3) includes ST128, ST137; fourth group (group4) includes ST123, ST122; fifth group (groups) includes ST119, ST150; sixth group (group6) includes ST117, ST159; seventh group (group7) includes ST112, ST160; eighth group (group8) includes ST102, ST135; ninth group (group9) includes ST101, ST27; the tenth group (group10) includes ST23, ST149. ST bootstrap values reflect the credibility as the group founder (the default program n=1000), as shown in the table. The probability ST45 as the predicted group founder is 83%.5.3 START2 software for further analysis5.3.1 seven housekeeping genes composition and polymorphic sites analysisWith the analysis of seven housekeeping genes in LH strains, the differences of average G+C% content among the housekeeping gene is insignificant,and there was no DNA sequence of a strain of G+C% was significantly higher than or lower than other strains, indicating no gene horizontal transfer phenomenon from foreign bacteria or other species. The ratio of synonymous to non-synonymous substitutions (dS/dN) determines whether the gene is in selection pressure. For rho, trpE, ilvC, thiC, eno, dN/dS value< 0.05 (average:0.0221,0.0000-0.0417), indicating that the five genes were in evolutionary selection pressure.For acnB and ftsH, dN/dS value were significantly higher than the average of the other five genes and> 1, suggesting that it is under positive selection pressure.5.3.2 Analysis of seven housekeeping genes recombinationThe SSCF P value and SSUF P value of rho, thiC<0.05, and for the remaining five housekeeping genes acnB, ftsH, trpE, ilvC, eno,SSCF P value and SSUF P value>0.05, suggests the presence of gene recombination. Binary values hint the same conclusion, increasing recombinant credibility.5.3.3 Linkage disequilibrium analysis of seven housekeeping genesFish, frogs and snails source isolates alleles are all in linkage disequilibrium, showing different housekeeping genes are not mutually independent. The ISA of fish source isolates is the biggest, followed by the snail source isolates, and finally the frog source isolates. Human source isolates show linkage equilibrium, because of its limited number of strains,and reliable results should be fine.Linkage disequilibrium analysis for all isolates:Method 1 (Haubold):ISA= 0.583 (P=0.000);Method 2 (Maynard Smith):ISA=3.4978 (P=0.000). The results of the two methods consistently showed that all isolates allele are linkage disequilibrium and different LH isolates loci are not mutually independent.5.3.4 Cluster analysis of all LH isolatesGZ-SHUI (ST151 type) have closer relationship with strains isolated from fish and frog in Guangzhou, suggesting existence of a common genetic ancestry.As newly discovered source of snails in Jiangmen isolates, JM-L16 has a close genetic relationship with JM-L143 and JM-L10, and relatively far relationship with JM-L2.The study showed JM-L143 was collected from the eastern outskirts of the ponds, and the remaining three are taken from the western suburbs of rice fields.STs of human isolates HZ242, JM-623, JM-679 were ST100, ST101, ST63 respectively. ST63 was also detected in fish in Hong Kong and ST100, ST101 were new types. HZ242 had close genetic relationship with GZ-W334 and GZ-W338 isolated from fish in Guangzhou. JM-623 had the consistent sequence type with GZ-W59 isolated from frog in Guangzhou.There are some sequence types detected in Laribacter Hongkongensis isolated both in Guangzhou and Jiangmen, such as ST45, ST42, ST106, ST101. The dominant sequence type ST45 and ST42 in this study were also detected in high frequency in Hong Kong[22],which provide predictive epidemic strain.Conclusion:1. This study first reported Laribacter Hongkongensis was found in the reservoir water samples in Guangdong province, proving the existence of the bacteria in the natural water environment. Therefore, drinking reservoir water may lead to infection of Laribacter Hongkongensis.At the same time, the reservoir is common living environment for freshwater products such as freshwater fish, as a transmission environment medium, which is of great significance to the prevalence of the L.Hongkongensis2. Various isolates were found to show resistance to cephalosporins, most of which performance the first and third-generation cephalosporin-resistantance, while show less resistance to the second, fourth generation cephalosporins. Most isolates were resistant to rifampin performance.Most isolates were sensitive to imipenem, gentamycin, and Amy card magnitude.3. The MLST scheme selected seven housekeeping genes of rho, ancB, ftsH, trpE, ilvC, thiC and eno and polymorphism loci of the total number was 298.This study aimed to analyze genetic recombination, prompting that rho and thiC may exist gene recombination while the remaining five housekeeping genes of acnB, ftsH, trpE, ilvC and eno did not appear genetic recombination.The analysis of the housekeeping gene linkage disequilibrium showed that Fish, frogs and snails source isolates alleles are all in linkage disequilibrium, suggesting different housekeeping genes are not mutually independent. Human source isolates show linkage equilibrium, because of its limited number of strains,and reliable results should be fine.This study first demonstrates an MLST analysis of Laribacter hongkongensis strains of different origins isolated in mainland China including Guangzhou and Jiangmen city. A total of 63 STs(ST99-ST161) were newly appeared STs, which have not been reported before therefore complemented the MLST database. Detected for the first time in this study, GZ-SHUI (ST151 type) isolated from water reservoir in Guangzhou had a close genetic relationship with some frog and fish source isolates, indicating existence of a common genetic ancestor and transmission among reservoir water,fish and frog.As newly discovered source of snails in Jiangmen isolates, four new sequence type were observed.The same source host of LH isolates were close relatives and its relation has nothing to do with the sampling sites.HZ242 had close genetic relationship with GZ-W334 and GZ-W338 isolated from fish in Guangzhou. JM-623 had the consistent sequence type with GZ-W59 isolated from frog in Guangzhou.The results suggest human infected with LH may be related to eating uncooked fish, frogs, etc.Some sequence types detected in high frequency and in different regions, as the prediction of epidemic strains in the future, for further analysis and study on its virulence, pathogenicity and drug resistance related characteristics, provides the basis for future prevention and control of strategy formulation.