BACKGROUND
in the developing world, every child under 5 years of age experiences approximately three episodes per year of diarrhea  <cit> .
although more than  <dig> viral, bacterial, and parasitic causes of diarrhea have been identified to date, only a few etiological agents cause the vast majority of diarrheal diseases in children in the developing world.
these include rotavirus, diarrheagenic escherichia coli, campylobacter jejuni, shigella spp., non-typhoidal salmonella, giardia lamblia, cryptosporidium spp.
and entamoeba histolytica <cit> .
unfortunately, a large proportion of cases of diarrheal disease are of unknown etiology.
there are many reasons for this problem, including fragility of causative agents, exacting growth requirements, and lack of recognition of some organisms as enteric pathogens.
here, we used the previously described strategy of 16s rrna gene polymerase chain reaction  and sequencing technology  <cit>  to analyze quantitatively the densities of different bacterial species in fecal samples of patients with diarrhea of unknown etiology at different times relative to hospital admission, and analyzed the features of the dominant species.
methods
study design
children with diarrhea without antibiotic treatment who were admitted to the children’s hospital, shanxi province, china from august  <dig> to  <dig>   <dig> were screened for enteric pathogens, including shigella, salmonella, enterotoxigenic e. coli, enteroinvasive e. coli , enteropathogenic e. coli , shiga-toxin-producing e. coli, enteroaggregative adherence e. coli , and common diarrhea viruses, including group a rotavirus, human calicivirus , enteric adenovirus  and human astrovirus .
the targeted virulence genes of enteric bacterial pathogens included heat-labile , heat-stable  enterotoxins, shiga-like toxin , bundle forming pili , enteric attaching and effacing locus , eaec specific probe, and the genes encoding invasive plasmid antigens   <cit> .
the group a rotaviruses were detected by commercially available enzyme-linked immunosorbent assay  according to the manufacturer’s instructions .
total viral dna and rna were extracted from fecal specimens prepared in phosphate-buffered saline at 10% using the qiaamp minelute virus spin kit  according to the manufacturer’s recommendations.
hucv, enteric adv and hastv were detected by pcr as described previously  <cit> .
g. lamblia and ent.
histolytica were detected using direct microscopy with a saline preparation of the specimen.
the clinical history and physiological findings of each patient were documented on standardized case report forms.
fecal samples from five healthy and five hospitalized children at the same location but with no apparent diarrhea were analyzed as controls.
libraries of the 16s rrna gene were constructed for each fecal sample, with a minimum size of  <dig> analyzable sequences  <cit> .
analyzing dominant fecal bacterial species by 16s rrna gene sequence technology
all fecal samples were collected in triplicate; one for timely isolation and detection of the enteric pathogens; one stored at −20°c for 16s rrna sequence analysis; and one stored in 20% glycerol at −80°c for isolation of the putative pathogens suggested by the 16s rrna gene analysis.
the dna was extracted from a 200-mg fecal sample, which was measured and adjusted to 100 ng/μl of each sample for pcr.
the universal eubacterial primers 27 f-519r  were used to amplify a 500-bp region of the 16s rrna gene.
lataq polymerase  was used for pcr under the following conditions: 95°c for 5 min, followed by 20 cycles of: 95°c for 30 s, 52°c for 30 s, and 72°c for 1 min; and a final elongation step at 72°c for 10 min.
the pcr products were extracted from sliced gels and cloned into the pgemr-t easy vector system .
they were then transformed into competent e. coli jm <dig>  a total of  <dig> white clones for each fecal sample were randomly selected for enrichment.
the purified plasmid dna was used for sequence analysis.
to verify the repeatability, we repeated the 16s rrna gene analysis of feces at admission for nine children with diarrhea of unknown etiology.
the 16s rrna gene sequences were analyzed for chimeric constructs using the chimera check program within the ribosomal database project.
species-level identification was performed using a 16s rrna gene sequence similarity of ≥99% compared with the prototype strain sequence in the genbank.
identification at the genus level was defined as a 16s rrna gene sequence similarity of ≥97% with that of the prototype strain sequence in the genbank, and the sequences were listed by genus.
the sequences matched attributable to either e. coli or shigella sp.
were listed as e. coli/shigella sp.
isolation of suggested fecal-dominant streptococcus
strains of streptococcus were isolated from fecal samples using kf streptococcus agar, and identified using the microscan walkaway si  <dig> system.
the full length of the 16s rrna gene sequence was obtained for confirmation of identification.
pulsed-field gel electrophoresis was performed according to the protocol for streptococcus suis <cit> .
the dna was digested with  <dig> u smai .
a dendrogram of isolates was drawn using bionumerics software .
clustering of patterns was performed using the unweighted pair group with arithmetic averaging .
genome sequencing and analysis of streptococcus lutetiensis
the genome of s. lutetiensis  <dig> isolated from patient  <dig> was sequenced using a combination of  <dig> sequencings with a roche  <dig> flx and paired end sequencing derived from the puc <dig> library using an abi  <dig> automated dna analyzer .
the genome was predicted using glimmer software  <cit> .
all putative open reading frames  were annotated using non-redundant nucleotides and proteins in the ncbi, swissport and kegg databases.
blastn and artemis comparison tool  were used for the pair alignment.
orthologous gene clusters were searched for using the orthomcl pipeline.
we clustered these orthologous genes according to their presence or absence in different genome sequences among streptococcus spp., and then a phylogenic tree was constructed using the neighbor-joining method.
genome islands were defined as having abnormal gc content with at least five continuous genes.
the homologous genes within each island were compared with the references using blastn with an e-value cutoff at 1×10– <dig>
nucleotide sequence accession numbers
the genbank accession numbers reported in this study are cp <dig> for the genome sequence of s. lutetiensis strain 033; and jn <dig> and jn <dig> for the 16s rrna gene sequences of s. gallolyticus subsp.
pasteurianus strains  <dig> and  <dig>  respectively.
ethics statement
feces samples were acquired with the written informed consent from the parents of the children with diarrhea and normal children.
this study was reviewed and approved by the ethics committee of the national institute for communicable disease control and prevention, china cdc, according to the medical research regulations of the ministry of health, china .
RESULTS
detection of enteric pathogens in feces of children with diarrhea
from august  <dig> to  <dig>   <dig>  fecal samples were obtained from  <dig> children with diarrhea admitted to the children’s hospital, shanxi province, china .
thirty-two of  <dig> children with diarrhea yielded negative culture for common enteric bacterial pathogens, such as salmonella, vibrio or diarrheagenic e. coli.
shigella sonnei was isolated from one patient .
the 16s rrna gene sequences of fecal samples were also negative for salmonella, vibrio or yersinia spp.
eleven children with diarrhea were diagnosed with shigella or diarrheagenic e. coli, including two with eaec, one with epec, and eight with eiec/shigella, according to virulence gene detection results .
these  <dig> children belonged to a group of  <dig> who had the 16s rrna gene sequence of e. coli/shigella sp.
the 16s rrna gene sequence of bacteroides fragilis was detected in five children with diarrhea, but its virulence gene heat-labile protein toxin was not detected.
twelve of  <dig> children with diarrhea were positive for the clostridium 16s rrna gene sequence, but the virulence gene toxin a or b of clostridium difficile was not detected.
three samples were positive for group a rotavirus by elisa and none tested positive for hucv, adv and hastv .
dominant fecal bacteria in children with diarrhea of unknown etiology
we divided the  <dig> children with diarrhea into three groups based on the etiological diagnosis.
group a included  <dig> children who were infected with diarrheagenic e. coli or shigella species and rotaviruses.
group b included  <dig> children with diarrhea of unknown etiology with only one fecal sample collected at admission.
group c included nine children with diarrhea of unknown etiology from whom three fecal samples were collected, including one at admission, one during recovery, and one after recovery .
the 16s rrna gene sequencing data revealed that  <dig> of  <dig> children with diarrhea of unknown etiology had streptococcus as the dominant fecal bacterial genus at admission.
among the remaining eight children, escherichia , klebsiella , enterococcus  or ruminococcus  was the most dominant bacterial genus .
we analyzed fecal samples from five healthy and five hospitalized children at the same location but with no apparent diarrhea as controls.
none of the genera escherichia, enterococcus, klebsiella, ruminococcus and streptococcus was dominant within the control fecal samples taken from five healthy children.
none of five hospitalized children at the same location but with no apparent diarrhea had streptococcus as the dominant genus, although one of them had the percent of streptococcus to  <dig> % in fecal microbiota.
there were no species in the streptococcus bovis group in the  <dig> children in the control group .
in nine children with diarrhea of unknown etiology in group c, eight had streptococcus as the most dominant fecal bacterial genus at admission, one with s. lutetiensis, two with s. gallolyticus subsp.
pasteurianus, two with streptococcus salivarius, and three with streptococcus sp.
.
we divided these nine children in group c into two, according to the most dominant fecal bacterial species at admission.
group c <dig> included one child whose most dominant species was e. coli.
the percentage of e. coli in the fecal microflora of patient  <dig>  was increased from  <dig> % at admission to  <dig> % during treatment, and then dropped to  <dig> % after recovery , based on  <dig> analyzed 16 s rrna gene sequences.
* +: 6–10/ high power field  ++: >10/hpf.
**: fecal samples collected at patient discharge from hospital.
group c <dig> included eight children with diarrhea, who were further divided into three subgroups, based on the most dominant fecal bacterial species at admission.
group c2a included two children who had s. salivarius as the most dominant fecal bacterial species.
group c2b included three children who had streptococcus sp.
as the most dominant species.
group c2c included three children who had s. bovis group as the most dominant species .
for patient  <dig>  in group c2a, the percentage of s. salivarius in the fecal microflora was reduced from  <dig> % at admission to  <dig> % during recovery , based on  <dig> sequences analyzed.
patient  <dig>  had the percentage of s. salivarius in the fecal microflora of  <dig> % at admission, which increased to  <dig> % during recovery and then to  <dig> % after recovery .
group c2b had streptococcus sp.
as the dominant fecal species at admission.
for patient  <dig> , the percentage of streptococcus sp.
in fecal microflora was reduced from  <dig> % to  <dig> % during recovery , and then to  <dig> % after recovery  , based on  <dig> 16s rrna gene sequences analyzed.
for patient  <dig> , the percentage of streptococcus sp.
in fecal microflora was reduced from  <dig> % at admission to  <dig> % during recovery  and then to  <dig> % after recovery  , based on  <dig> 16s rrna gene sequences analyzed.
for patient  <dig> , the percentage of streptococcus sp.
in fecal microflora was reduced from  <dig> % at admission to  <dig> % during recovery  and then to zero after recovery  , based on  <dig> 16s rrna gene sequences analyzed.
all three patients in group c2c had s. bovis group as their most dominant fecal bacterial species at admission.
for patient  <dig> , the percentage of s. bovis group in fecal microflora was reduced from  <dig> % at admission to zero during recovery  .
it was not detected in feces sampled at discharge from the hospital, after 5 days of treatment.
for patient  <dig> , the percentage of s. bovis group in fecal microflora was reduced from  <dig> % at admission to zero during recovery  .
it was not detected in feces sampled at discharge from hospital, after 5 days of treatment.
for patient  <dig> , the percentage of s. bovis group in fecal microflora was reduced from  <dig> % at admission to zero after 4 days of treatment .
it was not detected in the feces sampled at discharge from hospital, after 9 days of treatment.
isolation and identification of the s. bovis group from feces
we attempted to culture the dominant bacterial species as identified by the 16s rrna gene analysis from the feces of all nine patients in group c .
four patients  had negative cultures even on non-selective blood agar; possibly because antibiotics had been given before the hospital consultation.
patient  <dig> had seven isolates belonging to the s. bovis group in the feces samples collected at admission, patient  <dig> had  <dig>  and patient  <dig> had  <dig>  according to the results of the microscan walkaway si  <dig> system, all isolates of the s. bovis group were identified as biotype ii .
we then amplified, cloned, and sequenced the major portion of the 16s rrna gene from each isolate.
the strains isolated from patient  <dig> were identified as s. lutetiensis and those from patients  <dig> and  <dig> were s. gallolyticus subsp.
pasteurianus.
a dendrogram comparing representative 16s rrna gene sequences of the isolated s. bovis group strains with other streptococcus species mapped our isolates within the s. bovis group .
chromosomal dna from the  <dig> strains of the s. bovis group from the three patients were digested with restriction enzyme smai and analyzed using pulsed-field gel electrophoresis .
strains from each patient  were found to have unique restriction patterns.
genome sequence and comparison of the s. bovis group with s. lutetiensisstrain 033
we sequenced the entire genome of the s. lutetiensis strain  <dig> and compared it withits close relatives, s. gallolyticus subsp.
pasteurianus and s. gallolyticus subsp.
gallolyticus  <cit> .
to the best of our knowledge, this is the first time the genome of s. lutetiensis has been completely sequenced.
the genome of strain  <dig> contained  <dig> ,547 bp with a gc content of  <dig> %.
it had  <dig> trnas and  <dig> rrnas .
fifty-five tandem repeated regions were identified in the genome with the highest number of tandem repeats duplicated  <dig> times .
a total of  <dig>  orfs >300 bp  were identified.
of these,  <dig> % matched clusters of orthologous groups  in the database with e-values <1×10– <dig> .
twenty genomic islands  in the genome of s. lutetiensis  <dig> were identified.
of these, five were antibiotic-resistance islands and two were putative pathogenicity islands .
notably, gi- <dig> was found to contain four glycosyl transferase genes, four pilin-related genes, and > <dig> transposase genes or putative transposase genes that have been reported to be associated with virulence in streptococcus pneumoniae,neisseriaceae, and others  <cit> .
gi- <dig> encodes a colonization-associated adhesion factor previously described in s. suis <cit> .
gi- <dig> encodes the capsule polysaccharide  genes that are associated with the virulence of pathogenic streptococci; for example, s. pneumoniae and s. suis   <cit> .
five gis were unique to s. lutetiensis and have not been identified in other species of this genus.
two were phage related, one encoded a cellobiose phosphorylase-like protein, one encoded an atpase, and one had an unknown function.
we found the hemolytic toxin cylz in s. lutetiensis that activates the neutrophil signaling pathways in the brain endothelium and contributes to the development of meningitis identified in s. agalactiae <cit> .
the gene for sortase , also identified in the genome of s. lutetiensis, was found to be associated with adhesion to epithelial cells and with colonization of pathogenic streptococci  <cit>  .
together with the biochemical tests, the results of 16s rrna gene sequencing with  <dig> complete genomes for streptococci, two draft genomes , and four segment 16s rrna genes  in the s. bovis group were selected for an evolutionary study.
the reference strain of s. lutetiensis  was found to be the nearest strain to the s. lutetiensis genome sequence in our study, showing the same 16s rrna gene sequences.
compared with the nearest species s. infantarius subsp.
infantarius baa- <dig> and eu <dig>  strain  <dig> had two and three nucleotide differences in the 16s rrna genes, respectively.
an entire genome comparative analysis was performed on the four completed genomes of s. gallolyticus subsp.
gallolyticus baa- <dig>  s. gallolyticus subsp.
gallolyticus atcc <dig>  and s. gallolyticus subsp.
pasterurianus atcc <dig> in the s. bovis group.
the s. lutetiensis sequenced genome in our study was found to be phylogenetically related to the genome of s. gallolyticus subsp.
pasterurianus atcc43144; and  <dig> % of the genes were found in the homologous genes in atcc <dig>   <cit> .
although large-scale genome rearrangements, inversions and deletions were observed, the four genomes displayed the same collinear structure .
we found  <dig> % of the genes of s. gallolyticus subsp.
pasterurianus and  <dig> % of the genes of s. gallolyticus subsp.
gallolyticus were not present in s. lutetiensis, suggesting that the genome of s. lutetiensis strain  <dig> was similar to that of s. gallolyticus subsp.
pasterurianus .
discussion
selective media are routinely used to isolate particular pathogens from mixtures of bacterial species from the feces of patients with diarrhea.
however, they cannot be used to isolate putative bacterial agents of diarrhea of unknown etiology.
the important feature of the direct sequencing of the 16s rrna gene in the fecal samples is the ability to identify most of the existing bacterial species  <cit> .
using this technique, we analyzed the dynamics of the fecal bacteria flora in nine patients with diarrhea of unknown etiology.
we examined three fecal samples per patient, at admission, during recovery, and after recovery.
we speculated that the putative causative enteric pathogens were dominant in the intestine when infection was established.
the number of causative pathogens in the intestine may decrease during treatment and after recovery.
eight of nine patients  who provided all three specimens with unknown etiology at admission had as the dominant streptococcus genus in their fecal samples.
there is a report of a child who developed hemolytic uremic syndrome with group a beta hemolytic streptococcus-positive diarrhea  <cit> .
streptococci are also numerous in the fecal microflora of patients with irritable bowel syndrome patients  <cit> .
so, the role of streptococci in the fecal microflora of children with diarrhea deserved further research.
three patients from group c <dig> had streptococcus as the dominant genus, and all showed a reduced the percentage of streptococcus sp.
in fecal microflora of during and after recovery.
two patients had s. salivarius as the dominant species with one showing a reduced the percentage of streptococcus sp.
in fecal microflora during and after recovery.
the other patient showed an increase.
three patients had the s. bovis group as the dominant species, and all showed a reduced the percentage of s. bovis group in fecal microflora during and after recovery.
this observation suggests that the association of the s. bovis group with diarrhea is worthy of further investigation.
s. bovis is divided into three biotypes, i , ii/ <dig> , and ii/ <dig> , based upon mannitol fermentation and β-glucuronidase activities.
s. gallolyticus subsp.
gallolyticus is known to be associated with endocarditis and colon carcinoma.
s. infantarius, s. lutetiensis and s. gallolyticus subsp.
pasteurianus are associated with non-colonic cancer and meningitis.
children with signs of gastrointestinal disturbance at presentation associated with s. bovis were also reported  <cit> .
the dominant species from the nine patients of group c were cultured and four showed that they were negative.
thirty-six strains of the s. bovis group were isolated from three patients, and pfge analysis showed that they had their own unique restriction pattern, indicating that the strains within individual patients were identical.
the isolates were identified as s. lutetiensis and s. gallolyticus subsp.
pasteurianus.
we determined and analyzed the full genome sequence of the s. lutetiensis strain isolated from a child with diarrhea.
two previously recognized pathogenicity islands were identified in the genome.
gi- <dig> was found to encode a cps gene cluster involved in the pathogenicity of s. suis <cit> .
gi- <dig> was found to encode glycosyl transferase, the virulence factor in s. pneumoniae <cit> .
eight additional virulence factors were identified in the s. bovis group.
these included the putative hemolytic toxin cylz and the sortase gene associated with adhesion and colonization  <cit> .
CONCLUSIONS
we studied the dynamics of the fecal microbial community in children with diarrhea of unknown etiology and found for the first time that strains of the s. bovis group were among the predominant bacteria in some of the patients at admission, and showed a reduction in numbers during treatment and recovery.
in addition, we report the first genome sequence of a s. lutetiensis isolate, identifying putative pathogenic islands and virulence genes.
however, it was hard to detect all the infectious agents and there were many non-infectious factors that may cause diarrhea; therefore, additional studies are needed to clarify the potential contribution of these bacteria to diarrhea in children.
competing interests
the authors declare that they have no competing interests.
authors’ contribution
dj, ll, zl, hj, cy and jx conceived and designed the experiments.dj, sl,yxu and xb performed the experiments.cc, zz, pd, hw, yxiong, hz and lw carried out the molecular genetic studies and participated in the sequence alignment.hs contributed reagents and materials.
dj, mg and jx wrote the manuscript.
all authors read and approved the final manuscript.

 in the developing world, every child under 5 years of age experiences approximately three episodes per year of diarrhea. although more than viral, bacterial, and parasitic causes of diarrhea have been identified to date, only a few etiological agents cause the vast majority of diarrheal diseases in children in the developing world. these include rotavirus, diarrheagenic escherichia coli, campylobacter jejuni, shigella spp., non-typhoidal salmonella, giardia lamblia, cryptosporidium spp. and entamoeba histolytica. unfortunately, a large proportion of cases of diarrheal disease are of unknown etiology. there are many reasons for this problem, including fragility of causative agents, exacting growth requirements, and lack of recognition of some organisms as enteric pathogens. here, we used the previously described strategy of 16s rrna gene polymerase chain reaction and sequencing technology to analyze quantitatively the densities of different bacterial species in fecal samples of patients with diarrhea of unknown etiology at different times relative to hospital admission, and analyzed the features of the dominant species. methods study design children with diarrhea without antibiotic treatment who were admitted to the children’s hospital, shanxi province, china from august to were screened for enteric pathogens, including shigella, salmonella, enterotoxigenic e. coli, enteroinvasive e. coli, enteropathogenic e. coli, shiga-toxin-producing e. coli, enteroaggregative adherence e. coli, and common diarrhea viruses, including group a rotavirus, human calicivirus, enteric adenovirus and human astrovirus. the targeted virulence genes of enteric bacterial pathogens included heat-labile, heat-stable enterotoxins, shiga-like toxin, bundle forming pili, enteric attaching and effacing locus, eaec specific probe, and the genes encoding invasive plasmid antigens. the group a rotaviruses were detected by commercially available enzyme-linked immunosorbent assay according to the manufacturer’s instructions. total viral dna and rna were extracted from fecal specimens prepared in phosphate-buffered saline at 10% using the qiaamp minelute virus spin kit according to the manufacturer’s recommendations. hucv, enteric adv and hastv were detected by pcr as described previously. g. lamblia and ent. histolytica were detected using direct microscopy with a saline preparation of the specimen. the clinical history and physiological findings of each patient were documented on standardized case report forms. fecal samples from five healthy and five hospitalized children at the same location but with no apparent diarrhea were analyzed as controls. libraries of the 16s rrna gene were constructed for each fecal sample, with a minimum size of analyzable sequences. analyzing dominant fecal bacterial species by 16s rrna gene sequence technology all fecal samples were collected in triplicate; one for timely isolation and detection of the enteric pathogens; one stored at −20°c for 16s rrna sequence analysis; and one stored in 20% glycerol at −80°c for isolation of the putative pathogens suggested by the 16s rrna gene analysis. the dna was extracted from a 200-mg fecal sample, which was measured and adjusted to 100 ng/μl of each sample for pcr. the universal eubacterial primers 27 f-519r were used to amplify a 500-bp region of the 16s rrna gene. lataq polymerase was used for pcr under the following conditions: 95°c for 5 min, followed by 20 cycles of: 95°c for 30 s, 52°c for 30 s, and 72°c for 1 min; and a final elongation step at 72°c for 10 min. the pcr products were extracted from sliced gels and cloned into the pgemr-t easy vector system. they were then transformed into competent e. coli jm a total of white clones for each fecal sample were randomly selected for enrichment. the purified plasmid dna was used for sequence analysis. to verify the repeatability, we repeated the 16s rrna gene analysis of feces at admission for nine children with diarrhea of unknown etiology. the 16s rrna gene sequences were analyzed for chimeric constructs using the chimera check program within the ribosomal database project. species-level identification was performed using a 16s rrna gene sequence similarity of ≥99% compared with the prototype strain sequence in the genbank. identification at the genus level was defined as a 16s rrna gene sequence similarity of ≥97% with that of the prototype strain sequence in the genbank, and the sequences were listed by genus. the sequences matched attributable to either e. coli or shigella sp. were listed as e. coli/shigella sp. isolation of suggested fecal-dominant streptococcus strains of streptococcus were isolated from fecal samples using kf streptococcus agar, and identified using the microscan walkaway si system. the full length of the 16s rrna gene sequence was obtained for confirmation of identification. pulsed-field gel electrophoresis was performed according to the protocol for streptococcus suis. the dna was digested with u smai. a dendrogram of isolates was drawn using bionumerics software. clustering of patterns was performed using the unweighted pair group with arithmetic averaging. genome sequencing and analysis of streptococcus lutetiensis the genome of s. lutetiensis isolated from patient was sequenced using a combination of sequencings with a roche flx and paired end sequencing derived from the puc library using an abi automated dna analyzer. the genome was predicted using glimmer software. all putative open reading frames were annotated using non-redundant nucleotides and proteins in the ncbi, swissport and kegg databases. blastn and artemis comparison tool were used for the pair alignment. orthologous gene clusters were searched for using the orthomcl pipeline. we clustered these orthologous genes according to their presence or absence in different genome sequences among streptococcus spp., and then a phylogenic tree was constructed using the neighbor-joining method. genome islands were defined as having abnormal gc content with at least five continuous genes. the homologous genes within each island were compared with the references using blastn with an e-value cutoff at 1×10– nucleotide sequence accession numbers the genbank accession numbers reported in this study are cp for the genome sequence of s. lutetiensis strain 033; and jn and jn for the 16s rrna gene sequences of s. gallolyticus subsp. pasteurianus strains and respectively. ethics statement feces samples were acquired with the written informed consent from the parents of the children with diarrhea and normal children. this study was reviewed and approved by the ethics committee of the national institute for communicable disease control and prevention, china cdc, according to the medical research regulations of the ministry of health, china.  detection of enteric pathogens in feces of children with diarrhea from august to fecal samples were obtained from children with diarrhea admitted to the children’s hospital, shanxi province, china. thirty-two of children with diarrhea yielded negative culture for common enteric bacterial pathogens, such as salmonella, vibrio or diarrheagenic e. coli. shigella sonnei was isolated from one patient. the 16s rrna gene sequences of fecal samples were also negative for salmonella, vibrio or yersinia spp. eleven children with diarrhea were diagnosed with shigella or diarrheagenic e. coli, including two with eaec, one with epec, and eight with eiec/shigella, according to virulence gene detection results. these children belonged to a group of who had the 16s rrna gene sequence of e. coli/shigella sp. the 16s rrna gene sequence of bacteroides fragilis was detected in five children with diarrhea, but its virulence gene heat-labile protein toxin was not detected. twelve of children with diarrhea were positive for the clostridium 16s rrna gene sequence, but the virulence gene toxin a or b of clostridium difficile was not detected. three samples were positive for group a rotavirus by elisa and none tested positive for hucv, adv and hastv. dominant fecal bacteria in children with diarrhea of unknown etiology we divided the children with diarrhea into three groups based on the etiological diagnosis. group a included children who were infected with diarrheagenic e. coli or shigella species and rotaviruses. group b included children with diarrhea of unknown etiology with only one fecal sample collected at admission. group c included nine children with diarrhea of unknown etiology from whom three fecal samples were collected, including one at admission, one during recovery, and one after recovery. the 16s rrna gene sequencing data revealed that of children with diarrhea of unknown etiology had streptococcus as the dominant fecal bacterial genus at admission. among the remaining eight children, escherichia, klebsiella, enterococcus or ruminococcus was the most dominant bacterial genus. we analyzed fecal samples from five healthy and five hospitalized children at the same location but with no apparent diarrhea as controls. none of the genera escherichia, enterococcus, klebsiella, ruminococcus and streptococcus was dominant within the control fecal samples taken from five healthy children. none of five hospitalized children at the same location but with no apparent diarrhea had streptococcus as the dominant genus, although one of them had the percent of streptococcus to % in fecal microbiota. there were no species in the streptococcus bovis group in the children in the control group. in nine children with diarrhea of unknown etiology in group c, eight had streptococcus as the most dominant fecal bacterial genus at admission, one with s. lutetiensis, two with s. gallolyticus subsp. pasteurianus, two with streptococcus salivarius, and three with streptococcus sp.. we divided these nine children in group c into two, according to the most dominant fecal bacterial species at admission. group c included one child whose most dominant species was e. coli. the percentage of e. coli in the fecal microflora of patient was increased from % at admission to % during treatment, and then dropped to % after recovery, based on analyzed 16 s rrna gene sequences. * +: 6–10/ high power field ++: >10/hpf. **: fecal samples collected at patient discharge from hospital. group c included eight children with diarrhea, who were further divided into three subgroups, based on the most dominant fecal bacterial species at admission. group c2a included two children who had s. salivarius as the most dominant fecal bacterial species. group c2b included three children who had streptococcus sp. as the most dominant species. group c2c included three children who had s. bovis group as the most dominant species. for patient in group c2a, the percentage of s. salivarius in the fecal microflora was reduced from % at admission to % during recovery, based on sequences analyzed. patient had the percentage of s. salivarius in the fecal microflora of % at admission, which increased to % during recovery and then to % after recovery. group c2b had streptococcus sp. as the dominant fecal species at admission. for patient, the percentage of streptococcus sp. in fecal microflora was reduced from % to % during recovery, and then to % after recovery, based on 16s rrna gene sequences analyzed. for patient, the percentage of streptococcus sp. in fecal microflora was reduced from % at admission to % during recovery and then to % after recovery, based on 16s rrna gene sequences analyzed. for patient, the percentage of streptococcus sp. in fecal microflora was reduced from % at admission to % during recovery and then to zero after recovery, based on 16s rrna gene sequences analyzed. all three patients in group c2c had s. bovis group as their most dominant fecal bacterial species at admission. for patient, the percentage of s. bovis group in fecal microflora was reduced from % at admission to zero during recovery. it was not detected in feces sampled at discharge from the hospital, after 5 days of treatment. for patient, the percentage of s. bovis group in fecal microflora was reduced from % at admission to zero during recovery. it was not detected in feces sampled at discharge from hospital, after 5 days of treatment. for patient, the percentage of s. bovis group in fecal microflora was reduced from % at admission to zero after 4 days of treatment. it was not detected in the feces sampled at discharge from hospital, after 9 days of treatment. isolation and identification of the s. bovis group from feces we attempted to culture the dominant bacterial species as identified by the 16s rrna gene analysis from the feces of all nine patients in group c. four patients had negative cultures even on non-selective blood agar; possibly because antibiotics had been given before the hospital consultation. patient had seven isolates belonging to the s. bovis group in the feces samples collected at admission, patient had and patient had according to the results of the microscan walkaway si system, all isolates of the s. bovis group were identified as biotype ii. we then amplified, cloned, and sequenced the major portion of the 16s rrna gene from each isolate. the strains isolated from patient were identified as s. lutetiensis and those from patients and were s. gallolyticus subsp. pasteurianus. a dendrogram comparing representative 16s rrna gene sequences of the isolated s. bovis group strains with other streptococcus species mapped our isolates within the s. bovis group. chromosomal dna from the strains of the s. bovis group from the three patients were digested with restriction enzyme smai and analyzed using pulsed-field gel electrophoresis. strains from each patient were found to have unique restriction patterns. genome sequence and comparison of the s. bovis group with s. lutetiensisstrain 033 we sequenced the entire genome of the s. lutetiensis strain and compared it withits close relatives, s. gallolyticus subsp. pasteurianus and s. gallolyticus subsp. gallolyticus. to the best of our knowledge, this is the first time the genome of s. lutetiensis has been completely sequenced. the genome of strain contained,547 bp with a gc content of %. it had trnas and rrnas. fifty-five tandem repeated regions were identified in the genome with the highest number of tandem repeats duplicated times. a total of orfs >300 bp were identified. of these, % matched clusters of orthologous groups in the database with e-values <1×10–. twenty genomic islands in the genome of s. lutetiensis were identified. of these, five were antibiotic-resistance islands and two were putative pathogenicity islands. notably, gi- was found to contain four glycosyl transferase genes, four pilin-related genes, and > transposase genes or putative transposase genes that have been reported to be associated with virulence in streptococcus pneumoniae,neisseriaceae, and others. gi- encodes a colonization-associated adhesion factor previously described in s. suis. gi- encodes the capsule polysaccharide genes that are associated with the virulence of pathogenic streptococci; for example, s. pneumoniae and s. suis. five gis were unique to s. lutetiensis and have not been identified in other species of this genus. two were phage related, one encoded a cellobiose phosphorylase-like protein, one encoded an atpase, and one had an unknown function. we found the hemolytic toxin cylz in s. lutetiensis that activates the neutrophil signaling pathways in the brain endothelium and contributes to the development of meningitis identified in s. agalactiae. the gene for sortase, also identified in the genome of s. lutetiensis, was found to be associated with adhesion to epithelial cells and with colonization of pathogenic streptococci. together with the biochemical tests, the results of 16s rrna gene sequencing with complete genomes for streptococci, two draft genomes, and four segment 16s rrna genes in the s. bovis group were selected for an evolutionary study. the reference strain of s. lutetiensis was found to be the nearest strain to the s. lutetiensis genome sequence in our study, showing the same 16s rrna gene sequences. compared with the nearest species s. infantarius subsp. infantarius baa- and eu strain had two and three nucleotide differences in the 16s rrna genes, respectively. an entire genome comparative analysis was performed on the four completed genomes of s. gallolyticus subsp. gallolyticus baa- s. gallolyticus subsp. gallolyticus atcc and s. gallolyticus subsp. pasterurianus atcc in the s. bovis group. the s. lutetiensis sequenced genome in our study was found to be phylogenetically related to the genome of s. gallolyticus subsp. pasterurianus atcc43144; and % of the genes were found in the homologous genes in atcc. although large-scale genome rearrangements, inversions and deletions were observed, the four genomes displayed the same collinear structure. we found % of the genes of s. gallolyticus subsp. pasterurianus and % of the genes of s. gallolyticus subsp. gallolyticus were not present in s. lutetiensis, suggesting that the genome of s. lutetiensis strain was similar to that of s. gallolyticus subsp. pasterurianus. discussion selective media are routinely used to isolate particular pathogens from mixtures of bacterial species from the feces of patients with diarrhea. however, they cannot be used to isolate putative bacterial agents of diarrhea of unknown etiology. the important feature of the direct sequencing of the 16s rrna gene in the fecal samples is the ability to identify most of the existing bacterial species. using this technique, we analyzed the dynamics of the fecal bacteria flora in nine patients with diarrhea of unknown etiology. we examined three fecal samples per patient, at admission, during recovery, and after recovery. we speculated that the putative causative enteric pathogens were dominant in the intestine when infection was established. the number of causative pathogens in the intestine may decrease during treatment and after recovery. eight of nine patients who provided all three specimens with unknown etiology at admission had as the dominant streptococcus genus in their fecal samples. there is a report of a child who developed hemolytic uremic syndrome with group a beta hemolytic streptococcus-positive diarrhea. streptococci are also numerous in the fecal microflora of patients with irritable bowel syndrome patients. so, the role of streptococci in the fecal microflora of children with diarrhea deserved further research. three patients from group c had streptococcus as the dominant genus, and all showed a reduced the percentage of streptococcus sp. in fecal microflora of during and after recovery. two patients had s. salivarius as the dominant species with one showing a reduced the percentage of streptococcus sp. in fecal microflora during and after recovery. the other patient showed an increase. three patients had the s. bovis group as the dominant species, and all showed a reduced the percentage of s. bovis group in fecal microflora during and after recovery. this observation suggests that the association of the s. bovis group with diarrhea is worthy of further investigation. s. bovis is divided into three biotypes, i, ii/, and ii/, based upon mannitol fermentation and β-glucuronidase activities. s. gallolyticus subsp. gallolyticus is known to be associated with endocarditis and colon carcinoma. s. infantarius, s. lutetiensis and s. gallolyticus subsp. pasteurianus are associated with non-colonic cancer and meningitis. children with signs of gastrointestinal disturbance at presentation associated with s. bovis were also reported. the dominant species from the nine patients of group c were cultured and four showed that they were negative. thirty-six strains of the s. bovis group were isolated from three patients, and pfge analysis showed that they had their own unique restriction pattern, indicating that the strains within individual patients were identical. the isolates were identified as s. lutetiensis and s. gallolyticus subsp. pasteurianus. we determined and analyzed the full genome sequence of the s. lutetiensis strain isolated from a child with diarrhea. two previously recognized pathogenicity islands were identified in the genome. gi- was found to encode a cps gene cluster involved in the pathogenicity of s. suis. gi- was found to encode glycosyl transferase, the virulence factor in s. pneumoniae. eight additional virulence factors were identified in the s. bovis group. these included the putative hemolytic toxin cylz and the sortase gene associated with adhesion and colonization.  we studied the dynamics of the fecal microbial community in children with diarrhea of unknown etiology and found for the first time that strains of the s. bovis group were among the predominant bacteria in some of the patients at admission, and showed a reduction in numbers during treatment and recovery. in addition, we report the first genome sequence of a s. lutetiensis isolate, identifying putative pathogenic islands and virulence genes. however, it was hard to detect all the infectious agents and there were many non-infectious factors that may cause diarrhea; therefore, additional studies are needed to clarify the potential contribution of these bacteria to diarrhea in children. competing interests the authors declare that they have no competing interests. authors’ contribution dj, ll, zl, hj, cy and jx conceived and designed the experiments.dj, sl,yxu and xb performed the experiments.cc, zz, pd, hw, yxiong, hz and lw carried out the molecular genetic studies and participated in the sequence alignment.hs contributed reagents and materials. dj, mg and jx wrote the manuscript. all authors read and approved the final manuscript. 
the sequences of the 16s rrna genes extracted from fecal samples provide insights into the dynamics of fecal microflora. eight of nine children whom three consecutive fecal samples were collected had streptococcus as the dominant fecal bacterial genus, including three in the streptococcus bovis group and three streptococcus sp., which was reduced during and after recovery. we studied children with diarrhea who were admitted to the children’s hospital in shanxi province during eleven of children with diarrhea of unknown etiology had streptococcus as the most dominant fecal bacterial genus at admission. we sequenced the genome of s. lutetiensis and identified five antibiotic islands, two pathogenicity islands, and five unique genomic islands. the identified virulence genes included hemolytic toxin cylz of streptococcus agalactiae and sortase associated with colonization of pathogenic streptococci. we isolated strains that were possibly from the s. bovis group from feces sampled at admission, which were then identified as streptococcus lutetiensis from one child and streptococcus gallolyticus subsp. microbial communities16s rrna gene analysisstreptococcus lutetiensisgenome analysispathogenic island pasteurianus from children with diarrhea of unknown etiology, and found pathogenic islands and virulence genes in the genome of s. lutetiensis. we identified s. lutetiensis and s. gallolyticus subsp.
 the sequences of the 16s rrna genes extracted from fecal samples provide insights into the dynamics of fecal microflora. this potentially gives valuable etiological information for patients whose conditions have been ascribed to unknown pathogens, which cannot be accomplished using routine culture methods. we studied children with diarrhea who were admitted to the children’s hospital in shanxi province during  nineteen of children with diarrhea could not be etiologically diagnosed by routine culture and polymerase chain reaction methods. eleven of children with diarrhea of unknown etiology had streptococcus as the most dominant fecal bacterial genus at admission. eight of nine children whom three consecutive fecal samples were collected had streptococcus as the dominant fecal bacterial genus, including three in the streptococcus bovis group and three streptococcus sp., which was reduced during and after recovery. we isolated strains that were possibly from the s. bovis group from feces sampled at admission, which were then identified as streptococcus lutetiensis from one child and streptococcus gallolyticus subsp. pasteurianus from two children. we sequenced the genome of s. lutetiensis and identified five antibiotic islands, two pathogenicity islands, and five unique genomic islands. the identified virulence genes included hemolytic toxin cylz of streptococcus agalactiae and sortase associated with colonization of pathogenic streptococci.  we identified s. lutetiensis and s. gallolyticus subsp. pasteurianus from children with diarrhea of unknown etiology, and found pathogenic islands and virulence genes in the genome of s. lutetiensis. microbial communities16s rrna gene analysisstreptococcus lutetiensisgenome analysispathogenic island 