BACKGROUND
equine gut microbiota is poorly characterised and studies are currently underway to increase understanding of how defined microbial communities are able to interfere with different bacterial aetiologies of diarrhoea in horses.
recently, high-throughput amplicon sequencing analysis has been introduced to investigate the intestinal microbiota of healthy horses and horses with colitis .
however, the equine faecal microbiome is still largely unknown, as are interspecies interactions and their contribution to animal health  <cit> .
in horses, diarrhoea and colitis have been associated with a number of different pathogenic agents, including clostridium difficile, clostridium perfringens, salmonella spp and escherichia coli .
clostridium difficile is amongst the most important agents of diarrhoea and serious colitis in horses  <cit> .
while both adult horses and foals can suffer c. difficile enteric disease, it seems that foals are more likely to be colonised by the bacterium  <cit> .
a diagnosis of c. difficile infection  requires clinical suspicion as well as detection of pre-formed c. difficile toxins tcda and/or tcdb  in non-enriched specimens.
other possible causes of acute colitis must also be ruled out  <cit> .
however, in most cases of diarrhoea, the aetiology remains unclear and the prevalence of c. difficile colonisation in hospitalised horses has rarely been addressed  <cit> .
as in humans, the major risk factors for the development of cdi are antibiotic treatment and hospitalisation  <cit> .
however, some cases of infection have been also reported in horses without previous exposure to these risk factors, including in foals at  <dig> to 5 days of age  <cit> .
furthermore, it has been reported that up to 7 % of horses carry spores of c. difficile without showing any signs of diarrhoea  <cit> .
the first objective of this study was to provide further information on the nature of the bacterial community present in horses developing diarrhoea, including possible alterations in the microbiota profiles as a result of antibiotic treatment, through comparison with faeces from horses without diarrhoea.
this study also aimed to examine, by culture of horse faeces at admission to an equine clinic, the carriage rate of c. difficile.
isolates obtained were characterised in terms of pcr-ribotype, toxigenic activity and antibiotic resistance.
further metagenetic analyses were performed to compare the faecal microbiota of c. difficile in colonised and non-colonised horses.
RESULTS
bacterial community present in horses with and without diarrhoea
a group of  <dig> horses with diarrhoea at the moment of sampling were compared with  <dig> non-diarrhoeic horses via metagenetic analysis.
all of the animals  tested negative for c. difficile by faeces culture .
pyrosequencing yielded between  <dig>  and  <dig>  reads per sample .
the microbiota composition for each horse is presented at phylum level , genus level  and species level .
the more abundant bacterial families found for both groups were lachnospiraceae , ruminococcaceae , verrucomicrobiaceae , and prevotellaceae .
bacterial diversity of the gut microbiota in diarrhoea was lower than in non-diarrhoeic horses .
this effect was observed both in terms of species richness and in the population evenness .
principal coordinate analysis  of the diarrhoeic and non-diarrhoeic horses did show the sample distribution along the  <dig> main axes  .
analysis of molecular variance  revealed a significant difference between the variance of both groups taken as a single group and the variance of each group :  <dig>  and  <dig>  among and within groups respectively; amova test statistic fs:  <dig> ; p-value:  <dig> ).
unifrac weighted analysis further showed that both groups share different population structure .
thus, the microbiota structure of each group was showed statistically different from each other.
the relative abundance of each genus in both groups were compared in order to identify the populations responsible for this difference , leading to statistical differences for actinobacillus, porphyromonas, rc <dig> gut group, roseburia and a taxonomically undefined population belonging to the ruminococcaceae  .table  <dig> detailed information on twenty c. difficile negative horses studied via high-throughput amplicons sequencing analysis with and without diarrhoea
nsaids nonsteroidal anti-inflamatory dugs
pen penicillin, gen gentamicin, xnl ceftiofur, sxt trimethoprim/sulfamethoxazole, lz metronidazole
f-m flunixin meglumine
1time before antibiotic administration in days 2antibiotic treatment in progress at the time of sampling
fig.
<dig> microbiota faecal composition at phylum level for horses with and without diarrhoea.
bar chart detailing the relative abundance of the  <dig> core phylotypes common to the two groups of horses
fig.
<dig> microbiota faecal composition at genus level  for horses with and without diarrhoea.
samples  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and 29: horses with diarrhoea.
samples  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and 30: horses without diarrhoea.
the unclassified populations correspond to defined groups of the genus level for which a taxonomical classification assignation to the genus cannot be attributed.
these populations are therefore labelled with the first defined superior hierarchical taxonomic level followed by “_unclassified” to prevent confusion
fig.
<dig> bacterial biodiversity, bacterial richness and bacterial evenness in c. difficile negative horses with and without diarrhoea.
box plot of richness, evenness and diversity values showed that the microbiota structure of each group  is statistically different from each other.
whiskers represent minimum and maximum value.
bottom and top of the box are the first and the third quartile.
the median is shown as a band inside the box
fig.
<dig> bacterial genus whose relative abundance was statistically different between the  <dig> groups.
result of a white test .
box plot showing mean relative sequence abundance of actinobacillus, porphyromonas, rc <dig>  roseburia and ruminococcaceae_unclassified in horses with and without diarrhea.
the error bar indicates the diversity between samples in terms of proportions of sequences
clostridium difficile prevalence in horses at admission and strain characterisation
during the three-month study period, the total number of horses admitted at the clinic for either emergencies or consultation was  <dig>  with  <dig> hospitalisations.
a total of  <dig> samples were collected from the  <dig> hospitalised horses enrolled in the study.
two horses were sampled on two different times because they suffered a diarrhoeal episode during hospitalisation.
however, these horses did not test positive for c. difficile on any of the sample days.
the overall prevalence of c. difficile in the faecal microbiota of hospitalised horses at admission was  <dig>  % .
all horses testing positive were adult animals aged between four and 16 years old.
altogether,  <dig> of the total of  <dig> horses studied  presented gastrointestinal problems at admission, but c. difficile was isolated from only three of these animals .
in these three horses a nasogastric tube had been passed before the faecal collection.
however, none of them had previously received an antibiotic treatment.
nineteen horses  had diarrhoea at admission but all tested negative for the bacterium .
the remaining  <dig> horses sampled  were not affected by gastrointestinal disorders.
in this group, c. difficile was detected in two horses.
the clinical diagnoses in these two infected horses were multiple wounds and wound with tendon injury, respectively.
both had received a similar antibiotic treatment  and non-steroidal anti-inflammatory drugs .
age  and the gender  of the two horses were also the same .
in terms of antimicrobial therapy,  <dig> out of  <dig> horses studied  had previously received antimicrobial therapy , but most  tested negative for c. difficile.
among them, the most common drug used was ceftiofur, which was administered to  <dig>  horses.table  <dig> detailed information on c. difficile positive horses, including molecular characterization and antibiotic resistance of the isolates
tcda tcdb
cdta cdtb
tcdc
a
gyra
mut mutation
ce cytotoxicity assay using confluent monolayer mrc- <dig> cells
gen gentamicin, pen penicillin, cc clindamycin, te tetracycline, va vancomycin, mxf moxifloxacin, ra rifampicin, lz metronidazole, e erythromycin, xnl ceftiofur
iintermediate antimicrobial resistance
apresence of deletions in the regulator gene tcdc
bpresence of mutation in the gyra gene associated with moxifloxacin resistance
ccolic secondary to a gaseous distension of the caecum and a retraction of the colon
dcolic secondary to a pelvic flexure impaction
etime before antibiotic administration in days fantibiotic treatment in progress at the time of sampling
four of the equine isolates were positive for tcda, tcdb and binary toxin cdt genes while only one was non-toxinogenic.
the presence of tcdb was confirmed by cytotoxicity assay using confluent monolayer mrc- <dig> cells.
none of the isolates presented an  <dig>  39-base pair deletion or a deletion at  <dig> of the tcdc gene.
five different pcr-ribotypes were detected.
only one strain had a ribotype profile associated with the reference cardiff collection number .
the remaining isolates were identified as ucl <dig>  ucl <dig>  ucl23f and ucl <dig> .
pcr-ribotypes ucl <dig>   <dig> and ucl23f were isolated from three animals with gastrointestinal problems while pcr-ribotypes ucl <dig> and ucl <dig> were recovered from the two horses with wounds .
only the non-toxigenic strain pcr-ribotype ucl <dig> showed resistance to metronidazole  = 40 μg/ml, average of two essays) and erythromycin.
for clindamycin, only one isolate  was susceptible, while all others were fully resistant.
intermediate resistance for penicillin was observed in all of the isolates tested.
the isolate pcr-ribotype ucl <dig> also exhibited intermediate resistance to tetracycline, while all the rest were susceptible.
there was no vancomycin, moxifloxacin or rifampicin resistance detected, but all the strains were resistant to ceftiofur .
microbiota composition for c. difficile positive and negative horses
stool samples from all horses testing positive for c. difficile  were studied in order to obtain further information about the microbiota composition of the colonised subjects.
as during the entire study period  only five animals were positive for the bacterium, we could only use five c. difficile-negative horses as control group, but with similar clinical history .
in both c. difficile colonised and non-colonised horses, the dominant taxa were lachnospiraceae , bacteroidales  and ruminococcaceae .
in the group of c. difficile positive horses, only one animal  presented a predominance of bacteroides  and akkermansia .
the same bacteroides genus was found in another c. difficile positive sample  at a level of  <dig>  % .
only  <dig> distinct otus  were identified, representing 25-40 % of the relative abundance of the microbial taxa in the faecal samples .fig.
<dig> microbiota faecal composition at genus level  of c. difficile culture-positive and -negative horses.
samples hf_ <dig>   <dig>   <dig>   <dig> and 10: c. difficile positive horses detected by faeces culture.
samples hf_ <dig>   <dig>   <dig>   <dig> and 08: c. difficile negative horses detected by faeces culture
however, bacterial biodiversity, bacterial richness and bacterial evenness were not statistically different between c. difficile colonised and non-colonised horses .
ordination analysis with pcoa supported the grouping of most of the individuals into one group , which confirmed by non significant results from amova   <dig>  and  <dig>  among and within groups respectively; test statistic for amova   <dig> ; p-value:  <dig> ) and unifrac analysis of sample clustering .
indeed, white test abundance population comparison between groups performed at the different taxonomical levels identified two taxa  for which the relative abundance was statistically different .
microbiota composition relation with gastro-intestinal disorder diagnostics
we grouped the microbiota profiling data from the  <dig> samples from diarrhoeic and non diarrhoeic samples with the  <dig> c. difficile positive and negative samples together and assigned them to diagnosis categories: colic , enteritis , enterocolitis , diarrhoea  and others .
we used this clustering in order to identify genus whose abundance could be related to one particular category.
statistical abundance comparison with anova <dig> between colic, diarrhoea and other categories highlight a higher abundance of escherichia and streptococcus genera in colic group compared to the others and higher abundance of akkermansia, fusobacterium, porphyromonas and xylanibacter genera in the diarrhoea group .
enteritis and enterocolitis group, having only one sample could not be included in statistical abundance comparison but each sample was dominated by  <dig> defined genus: bacteroides and parabacteroides in the enteritis sample and porphyromonas and fusobacterium enterocolitis sample .
discussion
high-throughput amplicon sequencing analysis is one of the methods of choice in the study of complex gut microbiota ecosystems  <cit> .
however, most of the studies reported bacteria populations at the phylum and class level while the genus and species level were explored only in a few recent studies  <cit> .
higher taxonomic resolutions  may reveal more differences in population structure than phylum or class level  <cit>  and provide the degree of precision necessary for clinical diagnosis  <cit> .
as previously demonstrated, selecting the accurate region of 16s ribosomal dna  gene to sequence is essential in determining the utility of microbial genomics for species-level assignments  <cit> .
in the present study we report genus and species labelling based on v1-v <dig> region.
to determine whether the presence of diarrhoea was related to changes in the composition of the faecal microbiota of horses, a strict screening process was carried out among all the samples obtained over three months, in order that the two groups  were as similar as possible and therefore comparable.
as observed in a previous study investigating the microbiota in the equine large intestine via 16s ribosomal dna sequencing  <cit> , a great part of the sequences  were not identical  to sequence entries present in silva database .
even among the sequences identical to known entries, the species name was seldom taxonomically defined.
these findings underline the lack of knowledge regarding a good part of the horse gut microbiota stressing the need for further research on fundamental microbiology either on taxonomic as well on the functional level.
interestingly, akkermansia was found in 90 % of horses studied, with a relative abundance ranging between  <dig>  % and  <dig>  %.
this bacterium is an appealing candidate to become a human probiotic, selected based on established mechanisms of preventative treatment of obesity and diabetes .
only one previous study  <cit>  describes the genus akkermansia in the equine intestinal microbiota in this study we reported the presence of akkermansia muciniphila and akkermansia eu <dig> in the faecal microbiota of horses with and without diarrhoea.
akkermansia eu <dig> population identified in this study is 100 % identical to the akkermansia eu <dig> genbank entry .
it represents a potential new akkermansia species as it shares only 90 % of nucleotide identity with akkermansia muciniphila.
the relevance of this finding deserves further investigation.
overall, the composition of the microbiota of all horses studied was dominated by the same taxa as previously described  <cit> .
however, the differences observed in the cumulative mean relative abundance among individuals in these dominant bacterial groups may be linked to recent dietary history  <cit>  or to antimicrobial therapy  <cit> .
diarrhoea has been associated with changes in the faecal microbiota composition of humans but alterations in the equine gut microbiota has been rarely addressed  <cit> .
in the present study, actinobacillus  and porphyromonas taxa  were detected more abundantly in horses with diarrhoea.
these results contrast with a previous study of commensal bacteria in acute diarrhoea in children, where porphyromonas species were in the lowest proportions during acute diarrhoea compared with levels during periods of normal gastrointestinal health  <cit> .
fusobacteria were found in horses with diarrhoea but not in any of the horses with normal faeces.
in a previous study, fusobacterium spp.
was also found in higher percentages in horses with colitis, which could be a consequence of overgrowth due to bacterial dysbiosis or an aetiological agent of disease  <cit> .
in human beings, fusobacteria have been associated with colorectal carcinomas and adenomas  <cit> .
however, there are no previous studies describing this bacterium as an equine enteropathogen  <cit> .
after clustering of the different feces samples into more defined diagnosis categories, genus escherichia and streptococcus were more abundant in horses with diarrhoea without any other symptoms.
a more refined analysis revealed that the species involved were escherichia coli and streptococcus equinus.
if the first is long known to be associated with diarrhoea, the second is the most common streptococcus found in horse feces.
in the colic group, fusobacterium and porphyromonas genera were found in higher amount compared to other groups but were composed of yet unknown species.
as mentioned above, little is known regarding the involvement of these bacteria in gastro-intestinal disorders.
finally, two horses suffered from enteritis and enterocolitis and their 16s profiling revealed a domination  of genus bacteroides in the enteritis case and genus porphyromonas in the enterocolitis case.
the bacteroides population was mainly represented by bacteroides heparinolyticus.
this species, originally characterised as an agent of periodontitis in human  <cit> , is phylogenetically related to bacteroides fragilis which is well-known enterotoxinogenic bacteria involved in human infections  <cit> .
however, there is still no evidence of b. heparinolyticus involvement in gut disorder in horse or in human and it is thus unclear whether its abundance is related to the symptomatology.
we are aware that the limited size of the analysed cohort reduces the strength and the scope of our results.
larger cohort studies will be needed to improve our knowledge on diarrhoea impact on horse microbiota.
in the present study, the carriage of c. difficile at the time of admission was examined.
the overall prevalence found was  <dig>  % .
none of the positive animals had diarrhoea at the moment of sampling which may suggest that c. difficile colonisation in these horses was transient in most cases.
in the literature, there is only one previous study that investigated the presence of c. difficile in horses at admission to a veterinary teaching hospital  <cit> .
our results correlate with the findings of this study, which reported a prevalence of  <dig>  % .
in another previous study conducted at the same belgian equine clinic, we observed a c. difficile colonisation rate of  <dig>  % .
however, in that study, animals were not only sampled at the time of admission but also tracked during their hospitalisation, which could explain the higher prevalence found  <cit> .
from the five c. difficile positive horses found, three of them presented gastrointestinal problems  with a nasogastric tube passed before sampling.
nasogastric tube placement has been previously identified as a risk factor for c. difficile infection  <cit> .
two other horses without enteric perturbations were also colonised by c. difficile.
both had suffered wounds and were treated with antibiotics.
previous studies reported intestinal flora perturbations and antibiotic exposure as the most significant risk factors for c. difficile proliferation in horses  <cit> .
there were five different pcr-ribotypes detected among the five c. difficile positive animals, which suggests that a wide variety of c. difficile strains circulate in horses, as previously reported  <cit> , including pcr-ribotype  <dig>  all the isolates were resistant to ceftiofur and four out of five were also resistant to clindamycin, which agrees with the findings of previous studies  <cit> .
only one isolate was resistant to metronidazole and erythromycin.
surprisingly, this strain  was the only non-toxigenic isolate.
a high degree of resistance to antimicrobials  in non-toxigenic strains has been reported previously  <cit>  but the role in disease development or prevention is still unknown  <cit> .
we were unable to identify c. difficile by pyrosequencing analysis in the stool samples with positive cultures.
in humans, it is considered that feces harbour up to  <dig> bacteria per gram  <cit> .
thus, our sampling of thousands of sequences limits our detection ability to populations above  <dig> bacteria per gram.
the horses testing positive by culture did not have any clinical signs of c. difficile disease and the isolate was obtained only after three days of enrichment.
while the results of high-throughput amplicon sequencing analysis are limited by the small number of animals positive for c. difficile, and by the fact that none of the animals suffered cdi, the findings for each colonised horse should not be dismissed as they provide a first insight, albeit limited, about the impact of c. difficile colonisation in the horses’ gut microbiota, which merits further investigation.
CONCLUSIONS
metagenomic analysis is a promising tool to identify correlations between changes in the gut microbiota and intestinal diseases.
the abundance of actinobacillus, porphyromonas and fusobacteria in the faecal microbiota of diarrhoeic horses deserves special attention, as it can be a result of microbiome dysbiosis, and therefore a cause of intestinal disease, or in the case of fusobacteria, may act as equine enteric pathogen.
furthermore, the high proportion of akkermansia in all of the horses studied and its role in the intestine merits further investigation.
clostridium difficile colonisation seems to be transient in all of the horses studied without overgrowth to trigger infection.
for a great variety of bacterial species the currently available systems are not able to confidently assign taxonomy, which shows how complex and still unknown the equine microbiome is.

 equine gut microbiota is poorly characterised and studies are currently underway to increase understanding of how defined microbial communities are able to interfere with different bacterial aetiologies of diarrhoea in horses. recently, high-throughput amplicon sequencing analysis has been introduced to investigate the intestinal microbiota of healthy horses and horses with colitis. however, the equine faecal microbiome is still largely unknown, as are interspecies interactions and their contribution to animal health. in horses, diarrhoea and colitis have been associated with a number of different pathogenic agents, including clostridium difficile, clostridium perfringens, salmonella spp and escherichia coli. clostridium difficile is amongst the most important agents of diarrhoea and serious colitis in horses. while both adult horses and foals can suffer c. difficile enteric disease, it seems that foals are more likely to be colonised by the bacterium. a diagnosis of c. difficile infection requires clinical suspicion as well as detection of pre-formed c. difficile toxins tcda and/or tcdb in non-enriched specimens. other possible causes of acute colitis must also be ruled out. however, in most cases of diarrhoea, the aetiology remains unclear and the prevalence of c. difficile colonisation in hospitalised horses has rarely been addressed. as in humans, the major risk factors for the development of cdi are antibiotic treatment and hospitalisation. however, some cases of infection have been also reported in horses without previous exposure to these risk factors, including in foals at to 5 days of age. furthermore, it has been reported that up to 7 % of horses carry spores of c. difficile without showing any signs of diarrhoea. the first objective of this study was to provide further information on the nature of the bacterial community present in horses developing diarrhoea, including possible alterations in the microbiota profiles as a result of antibiotic treatment, through comparison with faeces from horses without diarrhoea. this study also aimed to examine, by culture of horse faeces at admission to an equine clinic, the carriage rate of c. difficile. isolates obtained were characterised in terms of pcr-ribotype, toxigenic activity and antibiotic resistance. further metagenetic analyses were performed to compare the faecal microbiota of c. difficile in colonised and non-colonised horses.  bacterial community present in horses with and without diarrhoea a group of horses with diarrhoea at the moment of sampling were compared with non-diarrhoeic horses via metagenetic analysis. all of the animals tested negative for c. difficile by faeces culture. pyrosequencing yielded between and reads per sample. the microbiota composition for each horse is presented at phylum level, genus level and species level. the more abundant bacterial families found for both groups were lachnospiraceae, ruminococcaceae, verrucomicrobiaceae, and prevotellaceae. bacterial diversity of the gut microbiota in diarrhoea was lower than in non-diarrhoeic horses. this effect was observed both in terms of species richness and in the population evenness. principal coordinate analysis of the diarrhoeic and non-diarrhoeic horses did show the sample distribution along the main axes. analysis of molecular variance revealed a significant difference between the variance of both groups taken as a single group and the variance of each group: and among and within groups respectively; amova test statistic fs:; p-value: ). unifrac weighted analysis further showed that both groups share different population structure. thus, the microbiota structure of each group was showed statistically different from each other. the relative abundance of each genus in both groups were compared in order to identify the populations responsible for this difference, leading to statistical differences for actinobacillus, porphyromonas, rc gut group, roseburia and a taxonomically undefined population belonging to the ruminococcaceae.table detailed information on twenty c. difficile negative horses studied via high-throughput amplicons sequencing analysis with and without diarrhoea nsaids nonsteroidal anti-inflamatory dugs pen penicillin, gen gentamicin, xnl ceftiofur, sxt trimethoprim/sulfamethoxazole, lz metronidazole f-m flunixin meglumine 1time before antibiotic administration in days 2antibiotic treatment in progress at the time of sampling fig. microbiota faecal composition at phylum level for horses with and without diarrhoea. bar chart detailing the relative abundance of the core phylotypes common to the two groups of horses fig. microbiota faecal composition at genus level for horses with and without diarrhoea. samples and 29: horses with diarrhoea. samples and 30: horses without diarrhoea. the unclassified populations correspond to defined groups of the genus level for which a taxonomical classification assignation to the genus cannot be attributed. these populations are therefore labelled with the first defined superior hierarchical taxonomic level followed by “_unclassified” to prevent confusion fig. bacterial biodiversity, bacterial richness and bacterial evenness in c. difficile negative horses with and without diarrhoea. box plot of richness, evenness and diversity values showed that the microbiota structure of each group is statistically different from each other. whiskers represent minimum and maximum value. bottom and top of the box are the first and the third quartile. the median is shown as a band inside the box fig. bacterial genus whose relative abundance was statistically different between the groups. result of a white test. box plot showing mean relative sequence abundance of actinobacillus, porphyromonas, rc roseburia and ruminococcaceae_unclassified in horses with and without diarrhea. the error bar indicates the diversity between samples in terms of proportions of sequences clostridium difficile prevalence in horses at admission and strain characterisation during the three-month study period, the total number of horses admitted at the clinic for either emergencies or consultation was with hospitalisations. a total of samples were collected from the hospitalised horses enrolled in the study. two horses were sampled on two different times because they suffered a diarrhoeal episode during hospitalisation. however, these horses did not test positive for c. difficile on any of the sample days. the overall prevalence of c. difficile in the faecal microbiota of hospitalised horses at admission was  %. all horses testing positive were adult animals aged between four and 16 years old. altogether, of the total of horses studied presented gastrointestinal problems at admission, but c. difficile was isolated from only three of these animals. in these three horses a nasogastric tube had been passed before the faecal collection. however, none of them had previously received an antibiotic treatment. nineteen horses had diarrhoea at admission but all tested negative for the bacterium. the remaining horses sampled were not affected by gastrointestinal disorders. in this group, c. difficile was detected in two horses. the clinical diagnoses in these two infected horses were multiple wounds and wound with tendon injury, respectively. both had received a similar antibiotic treatment and non-steroidal anti-inflammatory drugs. age and the gender of the two horses were also the same. in terms of antimicrobial therapy, out of horses studied had previously received antimicrobial therapy, but most tested negative for c. difficile. among them, the most common drug used was ceftiofur, which was administered to horses.table detailed information on c. difficile positive horses, including molecular characterization and antibiotic resistance of the isolates tcda tcdb cdta cdtb tcdc a gyra mut mutation ce cytotoxicity assay using confluent monolayer mrc- cells gen gentamicin, pen penicillin, cc clindamycin, te tetracycline, va vancomycin, mxf moxifloxacin, ra rifampicin, lz metronidazole, e erythromycin, xnl ceftiofur iintermediate antimicrobial resistance apresence of deletions in the regulator gene tcdc bpresence of mutation in the gyra gene associated with moxifloxacin resistance ccolic secondary to a gaseous distension of the caecum and a retraction of the colon dcolic secondary to a pelvic flexure impaction etime before antibiotic administration in days fantibiotic treatment in progress at the time of sampling four of the equine isolates were positive for tcda, tcdb and binary toxin cdt genes while only one was non-toxinogenic. the presence of tcdb was confirmed by cytotoxicity assay using confluent monolayer mrc- cells. none of the isolates presented an 39-base pair deletion or a deletion at of the tcdc gene. five different pcr-ribotypes were detected. only one strain had a ribotype profile associated with the reference cardiff collection number. the remaining isolates were identified as ucl ucl ucl23f and ucl. pcr-ribotypes ucl and ucl23f were isolated from three animals with gastrointestinal problems while pcr-ribotypes ucl and ucl were recovered from the two horses with wounds. only the non-toxigenic strain pcr-ribotype ucl showed resistance to metronidazole  = 40 μg/ml, average of two essays) and erythromycin. for clindamycin, only one isolate was susceptible, while all others were fully resistant. intermediate resistance for penicillin was observed in all of the isolates tested. the isolate pcr-ribotype ucl also exhibited intermediate resistance to tetracycline, while all the rest were susceptible. there was no vancomycin, moxifloxacin or rifampicin resistance detected, but all the strains were resistant to ceftiofur. microbiota composition for c. difficile positive and negative horses stool samples from all horses testing positive for c. difficile were studied in order to obtain further information about the microbiota composition of the colonised subjects. as during the entire study period only five animals were positive for the bacterium, we could only use five c. difficile-negative horses as control group, but with similar clinical history. in both c. difficile colonised and non-colonised horses, the dominant taxa were lachnospiraceae, bacteroidales and ruminococcaceae. in the group of c. difficile positive horses, only one animal presented a predominance of bacteroides and akkermansia. the same bacteroides genus was found in another c. difficile positive sample at a level of  %. only distinct otus were identified, representing 25-40 % of the relative abundance of the microbial taxa in the faecal samples.fig. microbiota faecal composition at genus level of c. difficile culture-positive and -negative horses. samples hf_ and 10: c. difficile positive horses detected by faeces culture. samples hf_ and 08: c. difficile negative horses detected by faeces culture however, bacterial biodiversity, bacterial richness and bacterial evenness were not statistically different between c. difficile colonised and non-colonised horses. ordination analysis with pcoa supported the grouping of most of the individuals into one group, which confirmed by non significant results from amova and among and within groups respectively; test statistic for amova; p-value: ) and unifrac analysis of sample clustering. indeed, white test abundance population comparison between groups performed at the different taxonomical levels identified two taxa for which the relative abundance was statistically different. microbiota composition relation with gastro-intestinal disorder diagnostics we grouped the microbiota profiling data from the samples from diarrhoeic and non diarrhoeic samples with the c. difficile positive and negative samples together and assigned them to diagnosis categories: colic, enteritis, enterocolitis, diarrhoea and others. we used this clustering in order to identify genus whose abundance could be related to one particular category. statistical abundance comparison with anova between colic, diarrhoea and other categories highlight a higher abundance of escherichia and streptococcus genera in colic group compared to the others and higher abundance of akkermansia, fusobacterium, porphyromonas and xylanibacter genera in the diarrhoea group. enteritis and enterocolitis group, having only one sample could not be included in statistical abundance comparison but each sample was dominated by defined genus: bacteroides and parabacteroides in the enteritis sample and porphyromonas and fusobacterium enterocolitis sample. discussion high-throughput amplicon sequencing analysis is one of the methods of choice in the study of complex gut microbiota ecosystems. however, most of the studies reported bacteria populations at the phylum and class level while the genus and species level were explored only in a few recent studies. higher taxonomic resolutions may reveal more differences in population structure than phylum or class level and provide the degree of precision necessary for clinical diagnosis. as previously demonstrated, selecting the accurate region of 16s ribosomal dna gene to sequence is essential in determining the utility of microbial genomics for species-level assignments. in the present study we report genus and species labelling based on v1-v region. to determine whether the presence of diarrhoea was related to changes in the composition of the faecal microbiota of horses, a strict screening process was carried out among all the samples obtained over three months, in order that the two groups were as similar as possible and therefore comparable. as observed in a previous study investigating the microbiota in the equine large intestine via 16s ribosomal dna sequencing, a great part of the sequences were not identical to sequence entries present in silva database. even among the sequences identical to known entries, the species name was seldom taxonomically defined. these findings underline the lack of knowledge regarding a good part of the horse gut microbiota stressing the need for further research on fundamental microbiology either on taxonomic as well on the functional level. interestingly, akkermansia was found in 90 % of horses studied, with a relative abundance ranging between  % and  %. this bacterium is an appealing candidate to become a human probiotic, selected based on established mechanisms of preventative treatment of obesity and diabetes. only one previous study describes the genus akkermansia in the equine intestinal microbiota in this study we reported the presence of akkermansia muciniphila and akkermansia eu in the faecal microbiota of horses with and without diarrhoea. akkermansia eu population identified in this study is 100 % identical to the akkermansia eu genbank entry. it represents a potential new akkermansia species as it shares only 90 % of nucleotide identity with akkermansia muciniphila. the relevance of this finding deserves further investigation. overall, the composition of the microbiota of all horses studied was dominated by the same taxa as previously described. however, the differences observed in the cumulative mean relative abundance among individuals in these dominant bacterial groups may be linked to recent dietary history or to antimicrobial therapy. diarrhoea has been associated with changes in the faecal microbiota composition of humans but alterations in the equine gut microbiota has been rarely addressed. in the present study, actinobacillus and porphyromonas taxa were detected more abundantly in horses with diarrhoea. these results contrast with a previous study of commensal bacteria in acute diarrhoea in children, where porphyromonas species were in the lowest proportions during acute diarrhoea compared with levels during periods of normal gastrointestinal health. fusobacteria were found in horses with diarrhoea but not in any of the horses with normal faeces. in a previous study, fusobacterium spp. was also found in higher percentages in horses with colitis, which could be a consequence of overgrowth due to bacterial dysbiosis or an aetiological agent of disease. in human beings, fusobacteria have been associated with colorectal carcinomas and adenomas. however, there are no previous studies describing this bacterium as an equine enteropathogen. after clustering of the different feces samples into more defined diagnosis categories, genus escherichia and streptococcus were more abundant in horses with diarrhoea without any other symptoms. a more refined analysis revealed that the species involved were escherichia coli and streptococcus equinus. if the first is long known to be associated with diarrhoea, the second is the most common streptococcus found in horse feces. in the colic group, fusobacterium and porphyromonas genera were found in higher amount compared to other groups but were composed of yet unknown species. as mentioned above, little is known regarding the involvement of these bacteria in gastro-intestinal disorders. finally, two horses suffered from enteritis and enterocolitis and their 16s profiling revealed a domination of genus bacteroides in the enteritis case and genus porphyromonas in the enterocolitis case. the bacteroides population was mainly represented by bacteroides heparinolyticus. this species, originally characterised as an agent of periodontitis in human, is phylogenetically related to bacteroides fragilis which is well-known enterotoxinogenic bacteria involved in human infections. however, there is still no evidence of b. heparinolyticus involvement in gut disorder in horse or in human and it is thus unclear whether its abundance is related to the symptomatology. we are aware that the limited size of the analysed cohort reduces the strength and the scope of our results. larger cohort studies will be needed to improve our knowledge on diarrhoea impact on horse microbiota. in the present study, the carriage of c. difficile at the time of admission was examined. the overall prevalence found was  %. none of the positive animals had diarrhoea at the moment of sampling which may suggest that c. difficile colonisation in these horses was transient in most cases. in the literature, there is only one previous study that investigated the presence of c. difficile in horses at admission to a veterinary teaching hospital. our results correlate with the findings of this study, which reported a prevalence of  %. in another previous study conducted at the same belgian equine clinic, we observed a c. difficile colonisation rate of  %. however, in that study, animals were not only sampled at the time of admission but also tracked during their hospitalisation, which could explain the higher prevalence found. from the five c. difficile positive horses found, three of them presented gastrointestinal problems with a nasogastric tube passed before sampling. nasogastric tube placement has been previously identified as a risk factor for c. difficile infection. two other horses without enteric perturbations were also colonised by c. difficile. both had suffered wounds and were treated with antibiotics. previous studies reported intestinal flora perturbations and antibiotic exposure as the most significant risk factors for c. difficile proliferation in horses. there were five different pcr-ribotypes detected among the five c. difficile positive animals, which suggests that a wide variety of c. difficile strains circulate in horses, as previously reported, including pcr-ribotype all the isolates were resistant to ceftiofur and four out of five were also resistant to clindamycin, which agrees with the findings of previous studies. only one isolate was resistant to metronidazole and erythromycin. surprisingly, this strain was the only non-toxigenic isolate. a high degree of resistance to antimicrobials in non-toxigenic strains has been reported previously but the role in disease development or prevention is still unknown. we were unable to identify c. difficile by pyrosequencing analysis in the stool samples with positive cultures. in humans, it is considered that feces harbour up to bacteria per gram. thus, our sampling of thousands of sequences limits our detection ability to populations above bacteria per gram. the horses testing positive by culture did not have any clinical signs of c. difficile disease and the isolate was obtained only after three days of enrichment. while the results of high-throughput amplicon sequencing analysis are limited by the small number of animals positive for c. difficile, and by the fact that none of the animals suffered cdi, the findings for each colonised horse should not be dismissed as they provide a first insight, albeit limited, about the impact of c. difficile colonisation in the horses’ gut microbiota, which merits further investigation.  metagenomic analysis is a promising tool to identify correlations between changes in the gut microbiota and intestinal diseases. the abundance of actinobacillus, porphyromonas and fusobacteria in the faecal microbiota of diarrhoeic horses deserves special attention, as it can be a result of microbiome dysbiosis, and therefore a cause of intestinal disease, or in the case of fusobacteria, may act as equine enteric pathogen. furthermore, the high proportion of akkermansia in all of the horses studied and its role in the intestine merits further investigation. clostridium difficile colonisation seems to be transient in all of the horses studied without overgrowth to trigger infection. for a great variety of bacterial species the currently available systems are not able to confidently assign taxonomy, which shows how complex and still unknown the equine microbiome is. 
however, none of the positive animals had diarrhoea at the moment of sampling. the abundance of some taxa in the faecal microbiota of diarrhoeic horses can be a result of microbiome dysbiosis, and therefore a cause of intestinal disease, or some of these taxa may act as equine enteric pathogens. the overall prevalence of c. difficile in the total samples of hospitalised horses at admission was  %, with five different pcr-ribotypes identified, including pcr-ribotype two colonised horses displayed a decreased bacterial species richness compared to the remaining subjects studied, which shared the same bacteroides genus. clostridium difficile has been identified as an important cause of diarrhoea in horses. clostridium difficile colonisation seems to be transient in all of the horses studied, without overgrowth to trigger infection. the prevalence of c. difficile in hospitalised horses at the time of admission is also reported. the equine faecal microbiota is very complex and remains largely unknown, while interspecies interactions have an important contribution to animal health. bacterial diversity of the gut microbiota in diarrhoea is lower than that in non-diarrhoeic horses in terms of species richness and in population evenness. a large proportion of the sequences were unclassified, showing the complexity of horses’ faecal microbiota. statistical differences for actinobacillus, porphyromonas, rc group, roseburia and ruminococcaceae were revealed.
 the equine faecal microbiota is very complex and remains largely unknown, while interspecies interactions have an important contribution to animal health. clostridium difficile has been identified as an important cause of diarrhoea in horses. this study provides further information on the nature of the bacterial communities present in horses developing an episode of diarrhoea. the prevalence of c. difficile in hospitalised horses at the time of admission is also reported.  bacterial diversity of the gut microbiota in diarrhoea is lower than that in non-diarrhoeic horses in terms of species richness and in population evenness. statistical differences for actinobacillus, porphyromonas, rc group, roseburia and ruminococcaceae were revealed. fusobacteria was found in horses with diarrhoea but not in any of the horses with non-diarrheic faeces. in contrast, akkermansia was among the three predominant taxa in all of the horses studied. the overall prevalence of c. difficile in the total samples of hospitalised horses at admission was  %, with five different pcr-ribotypes identified, including pcr-ribotype two colonised horses displayed a decreased bacterial species richness compared to the remaining subjects studied, which shared the same bacteroides genus. however, none of the positive animals had diarrhoea at the moment of sampling.  the abundance of some taxa in the faecal microbiota of diarrhoeic horses can be a result of microbiome dysbiosis, and therefore a cause of intestinal disease, or some of these taxa may act as equine enteric pathogens. clostridium difficile colonisation seems to be transient in all of the horses studied, without overgrowth to trigger infection. a large proportion of the sequences were unclassified, showing the complexity of horses’ faecal microbiota. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2015 