BACKGROUND
laribacter hongkongensis is a newly discovered, facultative anaerobic, gram-negative, motile, sea gull-shaped rod that belongs to the neisseriaceae family of β-proteobacteria.
it was first recovered from the blood and empyema thoracis of a patient with alcoholic liver cirrhosis in hong kong in  <dig>  <cit> .
since the patient's underlying disease and the presence of ascites suggested that the gastrointestinal tract may be a possible source of infection, l. hongkongensis was intensively sought in human fecal specimens.
during a period of two months, the bacterium was recovered from the stool of three patients with community-acquired gastroenteritis on charcoal cefoperazone deoxycholate agar.
a similar finding was observed in three other patients in switzerland  <cit> .
subsequently, in a multi-centered prospective study using a newly developed selective medium  <cit> , the bacterium was shown to be associated with community-acquired gastroenteritis and traveler's diarrhea  <cit> .
l. hongkongensis is likely to be globally distributed, as travel histories from patients suggested that it is present in at least four continents, including asia, europe, africa and central america  <cit> .
recently, l. hongkongensis has also been reported from another coastal province in mainland china  <cit> .
in a recent review, l. hongkongensis, together with enterotoxigenic bacteroides fragilis and klebsiella oxytoca, were included as newly appreciated agents associated with acute diarrhea  <cit> .
although the causative role of l. hongkongensis in gastroenteritis is yet to be established  <cit> , these data provide strong evidence that the bacterium is a potential diarrheal pathogen that warrants further investigations.
l. hongkongensis has been found in the intestines of healthy freshwater fish but not other studied animals that are commonly used for cooking in hong kong  <cit> .
the bacterium was recovered from the guts of 24% of  <dig> freshwater fish studied, with the highest recovery rates from grass carp  and bighead carp  and during spring and summer  <cit> .
moreover, l. hongkongensis has also been recovered from drinking water reservoirs in hong kong  <cit> .
the presence of a heterogeneous population of l. hongkongensis by pulsed-field gel electrophoresis  among isolates from freshwater fish  <cit>  and the association of l. hongkongensis gastroenteritis with fish consumption  <cit>  suggested that freshwater fish is likely the major reservoir of the bacterium and the source of human infections.
a highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of l. hongkongensis.
previously, we have used pfge for typing l. hongkongensis  <cit> .
however, due to experimental variations, pfge patterns are difficult to compare among different laboratories.
as multi-locus sequence typing  is well known to be highly reproducible and discriminative for bacteria, we developed such a typing system for l. hongkongensis using the sequence information of the l. hongkongensis complete genome sequence project.
in this article, we report the development of an mlst scheme for l. hongkongensis using  <dig> isolates from humans and fish.
methods
l. hongkongensis isolates
a total of  <dig> l. hongkongensis isolates, including  <dig> isolates recovered from humans and  <dig> isolates from fish, were used in this study .
l. hongkongensis isolate hku <dig> was recovered from the blood culture and empyema pus of a patient with bacteremic empyema thoracis  <cit> .
the other  <dig> isolates from humans  were recovered from the stool of patients with community-acquired gastroenteritis  <cit> .
isolates hlhk 2– <dig> were recovered from patients in switzerland while the rest were from patients in hong kong.
the  <dig> isolates from fish were recovered from the guts of freshwater fish sampled from retail food markets in hong kong  <cit> .
these included  <dig> isolates  recovered from grass carp ,  <dig> isolates  from bighead carp,  <dig> isolates  from mud carp  and three isolates  from large-mouth bass.
the identification of all l. hongkongensis isolates were confirmed phenotypically by standard conventional biochemical methods and genotypically by 16s rrna gene sequencing  <cit> .
dna extraction
bacterial dna extraction was modified from our previous published protocol  <cit> .
briefly,  <dig> μl of naoh  was added to  <dig> μl of bacterial cells suspended in distilled water and the mixture was incubated at 60°c for  <dig> min, followed by addition of  <dig> μl of tris-hcl , achieving a final ph of  <dig> .
the resultant mixture was diluted 100× and  <dig>  μl of the diluted extract was used for pcr.
pcr amplification and sequencing
extracted dna from the  <dig> isolates of l. hongkongensis was used as the template for amplification of seven housekeeping genes , using primers listed in table  <dig>  the seven housekeeping genes were chosen because either the gene itself or other genes in the same metabolic pathway has been used in mlst schemes of other bacteria.
the sequences of the seven genes were obtained from our on-going l. hongkongensis complete genome sequence project .
the pcr mixture  contained denatured l. hongkongensis dna, pcr buffer ,  <dig> mm mgcl <dig>   <dig> μm of each deoxynucleoside triphosphates and  <dig>  u ampli taq gold dna polymerase .
for rho, trpe, ilvc, thic and eno, the sample was amplified in  <dig> cycles of 94°c for  <dig> min, 55°c for  <dig>  min and 72°c for  <dig> min, and with a final extension at 72°c for  <dig> min in an automated thermal cycler .
for acnb and ftsh, the sample was amplified using a reannealing temperature of 60°c.
twenty microliters of each amplified product was electrophoresed in 2%  agarose gel, with a molecular size marker .
electrophoresis in tris-borate-edta buffer was performed at  <dig> volts for  <dig> min.
the gel was stained with ethidium bromide  for  <dig> min, rinsed and photographed under ultraviolet light illumination.
the pcr product was gel-purified using the qiaquick pcr purification kit .
both strands of the pcr product were sequenced using bigdye terminator cycle sequencing kit version  <dig>  with an abi prism  <dig> dna analyzer according to manufacturers' instructions  and the pcr primers.
bioedit version  <dig> .
<dig>  was used for reading the sequences and aligning the forward and backward reads  <cit> .
allele and sequence type assignment
the nucleotide sequences of the seven gene loci used for mlst in all the l. hongkongensis isolates were aligned and compared with those of isolate hlhk <dig> using clustal w multiple alignment  <cit>  implemented in bioedit version  <dig> .
<dig>   <cit> .
an arbitrary number was assigned to each distinct allele at a locus.
the numbered alleles at each locus were combined in order to establish the sequence type  for each isolate.
each st was numbered in the order of identification .
the data have been deposited in our laribacter hongkongensis complete genome sequence and mlst database http://mlstdb.hku.hk:14206/mlst_index.html
sequence analysis
the proportions of nucleotide alterations that led to a change in the amino acid sequence  and the proportions of nucleotide alterations that did not lead to a change in the amino acid sequence  were calculated with start <dig> http://pubmlst.org/software/analysis/ <cit> .
phylogenetic analysis was performed using clonalframe algorithm with the software package clonalframe version  <dig> , using  <dig>  burn-in cycles and  <dig>  further iterations  <cit> .
over  <dig> trees were generated from which a 75% majority-rule consensus tree was derived with mega version  <dig>   <cit> .
sts were grouped into lineages with eburst  <cit> .
the members of an eburst lineage were defined as groups of two or more independent isolates where each had identical alleles at six or more loci with at least one other member of the group.
the linkage disequilibrium between alleles at the seven gene loci was measured using the standardized index of association  with lian  <dig>  http://pubmlst.org/analysis/ <cit> .
split decomposition analysis was performed using the splitstree program   <cit> .
sawyer's test analysis for intragenic recombination was performed with start <dig> http://pubmlst.org/software/analysis/ <cit> .
gene tree congruence analysis was performed using the shimodaira-hasegawa  test  <cit>  as implemented in paup  <dig> b <dig> using the rell method and  <dig> bootstrap replicates  <cit> .
ninety-seven sts were selected and used in the sh test.
maximum-likelihood trees for each mlst gene of the  <dig> sts were inferred under a general time-reversible model, with an estimated gamma distribution, using phyml v <dig>   <cit> .
RESULTS
variation at the seven mlst loci
single bands of the expected sizes were observed for each gene locus amplified using the specific primers.
among the  <dig> bp of the seven loci, a total of  <dig> polymorphic sites were observed in the  <dig> isolates of l. hongkongensis.
two hundred and sixty-five and  <dig> polymorphic sites were observed in the  <dig> isolates from humans and  <dig> isolates from fish respectively.
no insertion, deletion or premature termination was observed in any of the polymorphic sites.
allelic profiles were assigned to the  <dig> isolates of l. hongkongensis .
the alleles defined for the mlst system were based on sequence lengths of between  <dig> bp  and  <dig> bp .
the median number of alleles at each locus was  <dig> .
the dn/ds ratio for the seven gene loci are shown in table  <dig>  all seven genes showed very low dn/ds ratios of <  <dig>  , indicating that no strong positive selective pressure is present.
asscf, sum of the squares of condensed fragments
bp-value indicating statistically significant  evidence for recombination are marked with asterisks
cmcf, maximum condensed fragment
relatedness of l. hongkongensis isolates
a total of  <dig> different sts were assigned to the  <dig> l. hongkongensis isolates, with  <dig> of the  <dig> sts identified only once .
thirty-eight different sts were observed in the  <dig> isolates from humans and  <dig> different sts observed in the  <dig> isolates from fish, with  <dig> sts in  <dig> grass carp,  <dig> sts in  <dig> bighead carps,  <dig> sts in the  <dig> mud carps and two sts in three large mouth bass.
three sts  were found in both isolates from humans and fish.
the most common st  was identified nine times, followed by st- <dig>  and st- <dig> .
the overall discriminatory power for the  <dig> isolates was  <dig> , that for the isolates from  <dig> humans was  <dig>  and for the isolates from fish was  <dig> .
clonalframe was used to construct a dendrogram using the concatenated nucleotide sequences of the seven gene loci of the  <dig> isolates .
eburst grouped the isolates into  <dig> lineages, with  <dig> sts in group  <dig>   <dig> sts in group  <dig>  seven sts in group  <dig>  three sts in groups 4– <dig> and two sts in groups 7– <dig>  whereas  <dig> sts did not belong to any of the  <dig> groups .
these  <dig> singleton sts were isolated from  <dig> patients and  <dig> fish .
all these  <dig> groups were also observed as clusters in the dendrogram .
groups  <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> contained only isolates from fish, group  <dig> contained  <dig> isolates from fish and two isolates from humans, group  <dig> contained three isolates from fish and one isolate from human, group  <dig> contained one isolate from fish and two isolates from humans, and groups  <dig>   <dig> and  <dig> contained only isolates from human.
isa measurement showed significant linkage disequilibrium in both isolates from humans and fish.
the isa for the isolates from humans and fish were  <dig>   and  <dig>  , indicating that the isolates from fish were more clonal than the isolates from humans.
only one interconnected network  was detected in the split graphs .
the p-value  of sum of the squares of condensed fragments in sawyer's test showed evidence of intragenic recombination in the rho, acnb and thic loci, but the p-value  of maximum condensed fragment in these gene loci did not show evidence of intragenic recombination .
congruence analysis showed that all the pairwise comparisons of the  <dig> mlst loci were incongruent, indicating that recombination played a substantial role in the evolution of l. hongkongensis.
.
ap values  represent differences in likelihood score between the maximum likelihood topology of each locus
no relationships were observed among the l. hongkongensis isolates with respect to their years of isolation; the locations of the hospitals, age and sex of the patients and the presence of plasmids in the isolates from patients  <cit> ; nor to the species of the fish and the locations of the markets where the fish were purchased.
discussion
a highly discriminative mlst scheme was developed for l. hongkongensis.
seven housekeeping genes with very low dn/ds ratios of the range of  <dig>  –  <dig> , similar to the housekeeping genes in other mlst schemes, were employed to produce a highly discriminative mlst scheme, with discriminatory power of  <dig> , comparable to the mlst schemes of other pathogenic bacteria, for molecular typing of l. hongkongensis.
when the same l. hongkongensis isolate was subcultured  <dig> times, no difference was observed between the sequences of the seven gene loci in the original isolate and the one after  <dig> subcultures .
therefore, these seven loci are discriminative enough for typing, but not evolving too rapidly to an extent that will mask genetic relatedness, as in the case of helicobacter pylori, another urease positive, s-shaped and motile alimentary tract microbe  <cit> .
the l. hongkongensis isolates recovered from fish were clustered.
in our previous study on ecoepidemiology of l. hongkongensis using pfge of spei digested chromosomal dna, it was found that the isolates from fish and humans were clustered separately  <cit> .
analysis of the sequences of the seven gene loci using both dendrogram and eburst groups revealed a similar phenomenon to the previous ecoepidemiology study, although the clustering pattern of the isolates in the present study was different from that in the previous one .
eburst group analysis showed that six of the  <dig> groups consisted exclusively of isolates from fish, whereas three of the  <dig> groups consisted exclusively of isolates from humans .
all these  <dig> eburst groups were also found in clusters in the dendrogram , although isa measurement showed that the isolates from fish were probably more clonal than the isolates from humans.
all these results of clustering of isolates from fish and humans into different groups observed in both the previous pfge and the present mlst studies suggested that some clones of l. hongkongensis could be more virulent than others.
although the isolates from fish appeared more clonal than the isolates from humans, a heterogeneous population of l. hongkongensis existed in the same ecosystem.
sts recovered from the same species of fish or the same fish market did not cluster together.
over 80% of freshwater fish consumed in hong kong are imported from fish farms in mainland china, whereas the remaining 20% are locally reared in fish farms in rural areas of hong kong.
since the same species of freshwater fish in a particular market is usually obtained from the same fish farm and multiple sts were present in l. hongkongensis isolates recovered from the same species purchased from the same market, it implied that multiple clones of l. hongkongensis probably existed in the same aquaculture farm in mainland china or hong kong.
CONCLUSIONS
seven housekeeping genes with very low dn/ds ratios were employed to produce a highly discriminative mlst scheme for molecular typing of l. hongkongensis.
abbreviations
bp: base pairs; dn: non-synonymous substitution; ds: synonymous substitution; dna: deoxyribonucleic acid; isa: standardized index of association; m: molar; min: minutes; ml: milliliter; mlst: multilocus sequence typing; pcr: polymerase chain reaction; pfge: pulsed-field gel electrophoresis; st: sequence type; μl: microliter.
authors' contributions
pcyw conceived the study and drafted the manuscript.
pcyw, jllt, skpl and kyy participated in the design of the study.
pcyw and jllt supervised the study.
pcyw, jllt and aklt analyzed the data.
ht constructed the database and website.
kmc, ekyl, jkhc, sslm, dmwt and lmwc carried out the pcr and sequencing experiments.
skpl and kyy corrected the manuscript.
all authors read and approved the final manuscript.
supplementary material
additional file 1
characteristics of l. hongkongensis isolates used in the present study.
the tabulated data describe the background epidemiological and mlst characteristics of the  <dig> l. hongkongensis isolates in this study.
click here for file
additional file 2
eburst groups of l. hongkongensis isolates.
the tabulated data provide the detailed compositions of each eburst group of l. hongkongensis isolates.
click here for file
acknowledgements
this work was partly supported by the research fund for the control of infectious diseases of the health, welfare and food bureau of the hong kong sar government and research grant council grant, university development fund, outstanding young researcher award, hku special research achievement award and the croucher senior medical research fellowship, the university of hong kong.

 laribacter hongkongensis is a newly discovered, facultative anaerobic, gram-negative, motile, sea gull-shaped rod that belongs to the neisseriaceae family of β-proteobacteria. it was first recovered from the blood and empyema thoracis of a patient with alcoholic liver cirrhosis in hong kong in. since the patient's underlying disease and the presence of ascites suggested that the gastrointestinal tract may be a possible source of infection, l. hongkongensis was intensively sought in human fecal specimens. during a period of two months, the bacterium was recovered from the stool of three patients with community-acquired gastroenteritis on charcoal cefoperazone deoxycholate agar. a similar finding was observed in three other patients in switzerland. subsequently, in a multi-centered prospective study using a newly developed selective medium, the bacterium was shown to be associated with community-acquired gastroenteritis and traveler's diarrhea. l. hongkongensis is likely to be globally distributed, as travel histories from patients suggested that it is present in at least four continents, including asia, europe, africa and central america. recently, l. hongkongensis has also been reported from another coastal province in mainland china. in a recent review, l. hongkongensis, together with enterotoxigenic bacteroides fragilis and klebsiella oxytoca, were included as newly appreciated agents associated with acute diarrhea. although the causative role of l. hongkongensis in gastroenteritis is yet to be established, these data provide strong evidence that the bacterium is a potential diarrheal pathogen that warrants further investigations. l. hongkongensis has been found in the intestines of healthy freshwater fish but not other studied animals that are commonly used for cooking in hong kong. the bacterium was recovered from the guts of 24% of freshwater fish studied, with the highest recovery rates from grass carp and bighead carp and during spring and summer. moreover, l. hongkongensis has also been recovered from drinking water reservoirs in hong kong. the presence of a heterogeneous population of l. hongkongensis by pulsed-field gel electrophoresis among isolates from freshwater fish and the association of l. hongkongensis gastroenteritis with fish consumption suggested that freshwater fish is likely the major reservoir of the bacterium and the source of human infections. a highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of l. hongkongensis. previously, we have used pfge for typing l. hongkongensis. however, due to experimental variations, pfge patterns are difficult to compare among different laboratories. as multi-locus sequence typing is well known to be highly reproducible and discriminative for bacteria, we developed such a typing system for l. hongkongensis using the sequence information of the l. hongkongensis complete genome sequence project. in this article, we report the development of an mlst scheme for l. hongkongensis using isolates from humans and fish. methods l. hongkongensis isolates a total of l. hongkongensis isolates, including isolates recovered from humans and isolates from fish, were used in this study. l. hongkongensis isolate hku was recovered from the blood culture and empyema pus of a patient with bacteremic empyema thoracis. the other isolates from humans were recovered from the stool of patients with community-acquired gastroenteritis. isolates hlhk 2– were recovered from patients in switzerland while the rest were from patients in hong kong. the isolates from fish were recovered from the guts of freshwater fish sampled from retail food markets in hong kong. these included isolates recovered from grass carp, isolates from bighead carp, isolates from mud carp and three isolates from large-mouth bass. the identification of all l. hongkongensis isolates were confirmed phenotypically by standard conventional biochemical methods and genotypically by 16s rrna gene sequencing. dna extraction bacterial dna extraction was modified from our previous published protocol. briefly, μl of naoh was added to μl of bacterial cells suspended in distilled water and the mixture was incubated at 60°c for min, followed by addition of μl of tris-hcl, achieving a final ph of. the resultant mixture was diluted 100× and μl of the diluted extract was used for pcr. pcr amplification and sequencing extracted dna from the isolates of l. hongkongensis was used as the template for amplification of seven housekeeping genes, using primers listed in table the seven housekeeping genes were chosen because either the gene itself or other genes in the same metabolic pathway has been used in mlst schemes of other bacteria. the sequences of the seven genes were obtained from our on-going l. hongkongensis complete genome sequence project. the pcr mixture contained denatured l. hongkongensis dna, pcr buffer, mm mgcl μm of each deoxynucleoside triphosphates and u ampli taq gold dna polymerase. for rho, trpe, ilvc, thic and eno, the sample was amplified in cycles of 94°c for min, 55°c for min and 72°c for min, and with a final extension at 72°c for min in an automated thermal cycler. for acnb and ftsh, the sample was amplified using a reannealing temperature of 60°c. twenty microliters of each amplified product was electrophoresed in 2% agarose gel, with a molecular size marker. electrophoresis in tris-borate-edta buffer was performed at volts for min. the gel was stained with ethidium bromide for min, rinsed and photographed under ultraviolet light illumination. the pcr product was gel-purified using the qiaquick pcr purification kit. both strands of the pcr product were sequenced using bigdye terminator cycle sequencing kit version with an abi prism dna analyzer according to manufacturers' instructions and the pcr primers. bioedit version. was used for reading the sequences and aligning the forward and backward reads. allele and sequence type assignment the nucleotide sequences of the seven gene loci used for mlst in all the l. hongkongensis isolates were aligned and compared with those of isolate hlhk using clustal w multiple alignment implemented in bioedit version.. an arbitrary number was assigned to each distinct allele at a locus. the numbered alleles at each locus were combined in order to establish the sequence type for each isolate. each st was numbered in the order of identification. the data have been deposited in our laribacter hongkongensis complete genome sequence and mlst database http://mlstdb.hku.hk:14206/mlst_index.html sequence analysis the proportions of nucleotide alterations that led to a change in the amino acid sequence and the proportions of nucleotide alterations that did not lead to a change in the amino acid sequence were calculated with start http://pubmlst.org/software/analysis/. phylogenetic analysis was performed using clonalframe algorithm with the software package clonalframe version, using burn-in cycles and further iterations. over trees were generated from which a 75% majority-rule consensus tree was derived with mega version. sts were grouped into lineages with eburst. the members of an eburst lineage were defined as groups of two or more independent isolates where each had identical alleles at six or more loci with at least one other member of the group. the linkage disequilibrium between alleles at the seven gene loci was measured using the standardized index of association with lian http://pubmlst.org/analysis/. split decomposition analysis was performed using the splitstree program. sawyer's test analysis for intragenic recombination was performed with start http://pubmlst.org/software/analysis/. gene tree congruence analysis was performed using the shimodaira-hasegawa test as implemented in paup b using the rell method and bootstrap replicates. ninety-seven sts were selected and used in the sh test. maximum-likelihood trees for each mlst gene of the sts were inferred under a general time-reversible model, with an estimated gamma distribution, using phyml v.  variation at the seven mlst loci single bands of the expected sizes were observed for each gene locus amplified using the specific primers. among the bp of the seven loci, a total of polymorphic sites were observed in the isolates of l. hongkongensis. two hundred and sixty-five and polymorphic sites were observed in the isolates from humans and isolates from fish respectively. no insertion, deletion or premature termination was observed in any of the polymorphic sites. allelic profiles were assigned to the isolates of l. hongkongensis. the alleles defined for the mlst system were based on sequence lengths of between bp and bp. the median number of alleles at each locus was. the dn/ds ratio for the seven gene loci are shown in table all seven genes showed very low dn/ds ratios of <, indicating that no strong positive selective pressure is present. asscf, sum of the squares of condensed fragments bp-value indicating statistically significant evidence for recombination are marked with asterisks cmcf, maximum condensed fragment relatedness of l. hongkongensis isolates a total of different sts were assigned to the l. hongkongensis isolates, with of the sts identified only once. thirty-eight different sts were observed in the isolates from humans and different sts observed in the isolates from fish, with sts in grass carp, sts in bighead carps, sts in the mud carps and two sts in three large mouth bass. three sts were found in both isolates from humans and fish. the most common st was identified nine times, followed by st- and st-. the overall discriminatory power for the isolates was, that for the isolates from humans was and for the isolates from fish was. clonalframe was used to construct a dendrogram using the concatenated nucleotide sequences of the seven gene loci of the isolates. eburst grouped the isolates into lineages, with sts in group sts in group seven sts in group three sts in groups 4– and two sts in groups 7– whereas sts did not belong to any of the groups. these singleton sts were isolated from patients and fish. all these groups were also observed as clusters in the dendrogram. groups and contained only isolates from fish, group contained isolates from fish and two isolates from humans, group contained three isolates from fish and one isolate from human, group contained one isolate from fish and two isolates from humans, and groups and contained only isolates from human. isa measurement showed significant linkage disequilibrium in both isolates from humans and fish. the isa for the isolates from humans and fish were and, indicating that the isolates from fish were more clonal than the isolates from humans. only one interconnected network was detected in the split graphs. the p-value of sum of the squares of condensed fragments in sawyer's test showed evidence of intragenic recombination in the rho, acnb and thic loci, but the p-value of maximum condensed fragment in these gene loci did not show evidence of intragenic recombination. congruence analysis showed that all the pairwise comparisons of the mlst loci were incongruent, indicating that recombination played a substantial role in the evolution of l. hongkongensis.. ap values represent differences in likelihood score between the maximum likelihood topology of each locus no relationships were observed among the l. hongkongensis isolates with respect to their years of isolation; the locations of the hospitals, age and sex of the patients and the presence of plasmids in the isolates from patients; nor to the species of the fish and the locations of the markets where the fish were purchased. discussion a highly discriminative mlst scheme was developed for l. hongkongensis. seven housekeeping genes with very low dn/ds ratios of the range of –, similar to the housekeeping genes in other mlst schemes, were employed to produce a highly discriminative mlst scheme, with discriminatory power of, comparable to the mlst schemes of other pathogenic bacteria, for molecular typing of l. hongkongensis. when the same l. hongkongensis isolate was subcultured times, no difference was observed between the sequences of the seven gene loci in the original isolate and the one after subcultures. therefore, these seven loci are discriminative enough for typing, but not evolving too rapidly to an extent that will mask genetic relatedness, as in the case of helicobacter pylori, another urease positive, s-shaped and motile alimentary tract microbe. the l. hongkongensis isolates recovered from fish were clustered. in our previous study on ecoepidemiology of l. hongkongensis using pfge of spei digested chromosomal dna, it was found that the isolates from fish and humans were clustered separately. analysis of the sequences of the seven gene loci using both dendrogram and eburst groups revealed a similar phenomenon to the previous ecoepidemiology study, although the clustering pattern of the isolates in the present study was different from that in the previous one. eburst group analysis showed that six of the groups consisted exclusively of isolates from fish, whereas three of the groups consisted exclusively of isolates from humans. all these eburst groups were also found in clusters in the dendrogram, although isa measurement showed that the isolates from fish were probably more clonal than the isolates from humans. all these results of clustering of isolates from fish and humans into different groups observed in both the previous pfge and the present mlst studies suggested that some clones of l. hongkongensis could be more virulent than others. although the isolates from fish appeared more clonal than the isolates from humans, a heterogeneous population of l. hongkongensis existed in the same ecosystem. sts recovered from the same species of fish or the same fish market did not cluster together. over 80% of freshwater fish consumed in hong kong are imported from fish farms in mainland china, whereas the remaining 20% are locally reared in fish farms in rural areas of hong kong. since the same species of freshwater fish in a particular market is usually obtained from the same fish farm and multiple sts were present in l. hongkongensis isolates recovered from the same species purchased from the same market, it implied that multiple clones of l. hongkongensis probably existed in the same aquaculture farm in mainland china or hong kong.  seven housekeeping genes with very low dn/ds ratios were employed to produce a highly discriminative mlst scheme for molecular typing of l. hongkongensis. abbreviations bp: base pairs; dn: non-synonymous substitution; ds: synonymous substitution; dna: deoxyribonucleic acid; isa: standardized index of association; m: molar; min: minutes; ml: milliliter; mlst: multilocus sequence typing; pcr: polymerase chain reaction; pfge: pulsed-field gel electrophoresis; st: sequence type; μl: microliter. authors' contributions pcyw conceived the study and drafted the manuscript. pcyw, jllt, skpl and kyy participated in the design of the study. pcyw and jllt supervised the study. pcyw, jllt and aklt analyzed the data. ht constructed the database and website. kmc, ekyl, jkhc, sslm, dmwt and lmwc carried out the pcr and sequencing experiments. skpl and kyy corrected the manuscript. all authors read and approved the final manuscript. supplementary material additional file 1 characteristics of l. hongkongensis isolates used in the present study. the tabulated data describe the background epidemiological and mlst characteristics of the l. hongkongensis isolates in this study. click here for file additional file 2 eburst groups of l. hongkongensis isolates. the tabulated data provide the detailed compositions of each eburst group of l. hongkongensis isolates. click here for file acknowledgements this work was partly supported by the research fund for the control of infectious diseases of the health, welfare and food bureau of the hong kong sar government and research grant council grant, university development fund, outstanding young researcher award, hku special research achievement award and the croucher senior medical research fellowship, the university of hong kong.
laribacter hongkongensis is a newly discovered, facultative anaerobic, gram-negative, motile, sea gull-shaped rod associated with freshwater fish borne gastroenteritis and traveler's diarrhea. congruence analysis showed that all the pairwise comparisons of the mlst loci were incongruent, indicating that recombination played a substantial role in the evolution of l. hongkongensis. all seven genes showed very low dn/ds ratios of <, indicating that no strong positive selective pressure is present. standardized index of association measurement showed significant linkage disequilibrium in isolates from both humans and fish. the p-value of sum of the squares of condensed fragments in sawyer's test showed evidence of intragenic recombination in the rho, acnb and thic loci, but the p-value of maximum condensed fragment in these gene loci did not show evidence of intragenic recombination. a highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of l. hongkongensis. in this study, a multilocus sequence typing system was developed for l. hongkongensis. the system was used to characterize l. hongkongensis isolates, including from humans and from fish. a website for l. hongkongensis mlst was set up and can be accessed at http://mlstdb.hku.hk:14206/mlst_index.html. a highly reproducible and discriminative mlst system was developed for l. hongkongensis. the overall discriminatory power was. the median number of alleles at each locus was.
 laribacter hongkongensis is a newly discovered, facultative anaerobic, gram-negative, motile, sea gull-shaped rod associated with freshwater fish borne gastroenteritis and traveler's diarrhea. a highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of l. hongkongensis. in this study, a multilocus sequence typing system was developed for l. hongkongensis. the system was used to characterize l. hongkongensis isolates, including from humans and from fish.  fragments of seven housekeeping genes were amplified and sequenced. among the bp of the seven loci, polymorphic sites were observed. the median number of alleles at each locus was. all seven genes showed very low dn/ds ratios of <, indicating that no strong positive selective pressure is present. a total of different sequence types were assigned to the isolates, with sts identified only once. the overall discriminatory power was. eburst grouped the isolates into lineages, with six groups containing only isolates from fish and three groups only isolates from humans. standardized index of association measurement showed significant linkage disequilibrium in isolates from both humans and fish. the isa for the isolates from humans and fish were and, indicating the isolates from fish were more clonal than the isolates from humans. only one interconnected network was detected in the split graphs. the p-value of sum of the squares of condensed fragments in sawyer's test showed evidence of intragenic recombination in the rho, acnb and thic loci, but the p-value of maximum condensed fragment in these gene loci did not show evidence of intragenic recombination. congruence analysis showed that all the pairwise comparisons of the mlst loci were incongruent, indicating that recombination played a substantial role in the evolution of l. hongkongensis. a website for l. hongkongensis mlst was set up and can be accessed at http://mlstdb.hku.hk:14206/mlst_index.html.  a highly reproducible and discriminative mlst system was developed for l. hongkongensis. 