BACKGROUND
ensuring the high microbiological quality of environmental water used as a source of recreational or drinking water is an important worldwide problem  <cit> .
poor microbiological quality of water results from contamination by microorganisms of human or animal fecal origin, and leads to the risk of gastro-enteritis in humans.
such contamination is caused by fecal bacteria from  point source pollution, e.g., treated effluents from wastewater treatments plants  which primarily treat wastewater of human origin, or  nonpoint source pollution consisting of inputs of microorganisms of mainly animal origin, via run-off or leaching from pasture or manured soils  <cit> .
the world health organization and, more recently, european guidelines , use escherichia coli as the bacterial indicator species for fecal contamination of water.
epidemiological studies have been used to determine threshold values for concentrations of e. coli in water above which there is a risk of gastro-enteritis  <cit> .
e. coli is a commensal bacterium of the gastro-intestinal tract of humans and vertebrate animals  <cit> .
to survive in an aqueous environment it must resist environmental stressors   <cit>  and avoid predation by protozoa  <cit> .
some authors have suggested that some of these e. coli strains might then persist by becoming naturalized in fresh water and soil  <cit> .
the aquatic environment can thus be considered a secondary habitat, where some authors have even shown the possible growth of e. coli  <cit> .
the diversity of e. coli populations in their secondary habitats has been studied by analyzing the sequences of the gene uida  <cit> , palindromic repetitive sequences  <cit> , ribotypes  <cit> , and profiles of antibiotic resistance  <cit> .
using these methods, the dynamics of e. coli populations have been investigated and, in some cases, it has been possible to discriminate between the human or animal origin of the contamination.
the structure of an e. coli population is characterized by four main phylo-groups   <cit> .
strains of the phylo-groups a and b <dig> are mainly found as commensals in humans and vertebrate animals, with the a phylo-group strains being predominant in humans and the b <dig> strains in animals  <cit> .
extraintestinal infections are mainly caused by the strains of the phylo-groups b <dig> and d  <cit> .
although strains of the b <dig> and d phylo-groups are typically less abundant as commensals, the distribution of the four phylo-groups can vary according to diet or climate  <cit> .
it also has been suggested that some strains could be host-specific, such as b <dig> strains exhibiting the hly  gene, found only in animals, and b <dig> o <dig> o-type strains, found only in humans  <cit> .
the objective of this study was to investigate the effects of various hydrological conditions on the structure of the e. coli population collected from stream water in a small rural watershed in northern france .
land use in the watershed is almost entirely agricultural with a low population density.
results show that an increase of fecal contamination was accompanied by a change in the distribution of phylo-groups in the e. coli population, represented by a change in the ratio of a to b <dig> phylo-groups.
e. coli b <dig> isolates were the dominant phylo-group isolated in the water.
among e. coli b <dig> isolates, some epidemiological types  seem to be specific to water that is only slightly contaminated.
RESULTS
e. coli population structure in creek water in relation to hydrological conditions and watershed land use
e. coli were enumerated and the population structure analyzed by phylo-grouping in three sets of samples collected under different hydrological and agricultural land-use conditions .
in this study, the e. coli population structure in creek water is analyzed from a single sample integrating all the daily samples.
the origin  of specific strains was investigated, in addition to the phylo-grouping, by hly gene detection in the e. coli b <dig> isolates and o <dig> typing of e. coli b <dig> isolates, as well as by studying the antibiotic resistance pattern.
statistical analyses  were performed in order to compare hydrological conditions .
a  <dig> septic tanks  were located between  <dig> to  <dig> m from the creek.
one malfunctioning septic tank  was located  <dig> m from the sampling point.
b nephelometric turbidity unit
c suspended sediment concentration
during the dry period , when cattle were being grazed, but when there was no runoff or leaching, the water was slightly contaminated by e. coli  .
the structure of the e. coli population was significantly different from the structures analyzed from the other sample collection periods , with a majority of e. coli b <dig> isolates  .
this structure argues for contamination by e. coli b <dig> isolates that are better adapted to the aquatic environment  <cit> , rather than for residual bovine fecal contamination, as the isolates were devoid of the hly gene and sensitive to all antibiotics  <cit> .
resistanceb
n: numbers of isolates
a e. coli isolates resistant to one or more antibiotics
b chl: chloramphenicol; tet: tetracyclin; str: streptomycin
nd: not detected
c hly gene detection by pcr method
d serotype o <dig> detection by pcr method
it was during the wet period , when there was no grazing, but when there was a malfunctioning septic system , that the lowest value of e. coli  was measured in the water.
the e. coli population was characterized by a high proportion of phylo-group a isolates  , followed by e. coli b <dig> isolates without the hly gene .
none of the e. coli was resistant to the seven antibiotics tested .
this e. coli population is probably due to an input of solely human origin, as the structure corresponds to that already described for human commensal e. coli in france  <cit> .
the rainfall event that occurred during the dry period  resulted in runoff from the pastures and leaching of soils.
the density of the e. coli in the stream water  was two orders of magnitude higher than that measured for the two other periods .
during this rain event, an input of e. coli from cattle contamination  was added to that from human contamination .
the structure of the e. coli population was characterized by two main phylo-groups, b <dig>  and a .
some e. coli b <dig> isolates with the hly gene, presumably of animal origin were detected   <cit> .
more than 60% of these isolates were resistant to at least one of the three antibiotics used in veterinary medicine   <cit>  , suggesting an animal origin.
thus, it appears that both hydrological conditions and current land use in the watershed might affect the structure of the e. coli a and b <dig> populations in the stream.
in contrast, the hydrological and land-use conditions did not exert a significant influence on the phylo-groups b <dig> and d, which were the least abundant phylo-groups recovered from the water .
no human-specific b <dig> o <dig> o-type strain was isolated during any sampling conditions, which is consistent with the low frequency of these strains in the e. coli population  <cit> .
changes in e. coli population structure during a rain event
in order to better understand the effect of a rain event on the structure of an e. coli population, we selected three out of the twenty-four hourly samples.
our selection represented three key moments  showing how the turbidity and e. coli density evolved.
it would not have been possible to observe this evolution using just a sample that integrated all the daily samples.
the rain event consisted of  <dig> mm of rain that fell during a wet period, during which there were  <dig> cattle being grazed in the watershed  .
five hours before rainfall began, the level of e. coli contamination was low , and the small number of isolates did not permit analysis of the structure of the e. coli population .
during the rain event, the turbidity increased, as did the number of e. coli, consistent with previous work demonstrating a correlation between bacteria and particles  <cit> .
six hours after the rainfall event the e. coli density reached a value of  <dig>   <dig> cfu/ <dig> ml, at which point the structure of the e. coli population was characterized by a majority of e. coli phylo-group a , with 63% being resistant to at least one antibiotic , suggesting fecal contamination of human origin resulting from leaching of soils and from surface runoff .
this structure was significantly different from that observed in the less contaminated water analyzed  <dig> hours after the rainfall event .
at that time the e. coli density had decreased to  <dig>   <dig> cfu/ <dig> ml , and e. coli b <dig> isolates  were the predominant e. coli phylo-group.
these isolates are mainly hly positive  with 31% resistant to at least one antibiotic , suggesting that there had been an input on the soils of e. coli of bovine origin that was then introduced into the water through run-off and/or leaching.
resistancec
resistancec
resistancec
amx/tic/chl/sxt/str
amx/tic/sxt/str
chl
amx/tic/chl
nd: not detected
n: numbers of isolates
a timing in relation to rainfall
b e. coli isolates resistant to one or more antibiotics
c amx: amoxicillin; tic: ticarcillin chl: chloramphenicol; tet: tetracyclin; str: streptomycin; sxt:trimethoprim + sulfamethoxazole
d hly gene detection by pcr method
e serotype o <dig> detection by pcr method
we cannot exclude an input from wild animals , although wild e. coli strains are usually not resistant to antibiotics  <cit> .
these results indicate that during the rain event, an increase in microbial contamination was accompanied by a modification of the structure of the e. coli population, resulting in a high ratio of phylo-groups a/b <dig>  in contrast, in the water collected  <dig> h after the rain event, and only slightly contaminated by e. coli, the majority of e. coli isolates belonged to the b <dig> phylo-group.
diversity of e. coli b <dig> strains isolated from the creek water
as e. coli b <dig> was the dominant phylo-group isolated in water from the bébec, accounting for between 15% to 87% of the e. coli population , we investigated further the diversity of e. coli b <dig> isolates by  sequencing the uida gene  and comparing the sequences obtained with those in the mlst pasteur database in order to find the uida allele,  detecting the presence of hly and determining molecularly the o-type,  studying the antibiotic resistance profile.
a total of  <dig> epidemiological types  were identified among the  <dig> e. coli b <dig> isolated from the water  and the proportion of each ets differed for each sampling event .
aamx: amoxicillin; chl: chloramphenicol; tet: tetracyclin; all of epidemiological types of e. coli b <dig> strains were non-resistant to tic: ticarcillin; sxt: trimethoprim + sulfamethoxazole; str: streptomycin; and cip: ciprofloxacin.
bnt: not o <dig>  o <dig>  o <dig>  o <dig>  o <dig>  o45b, o <dig>  o <dig>  o <dig>  o <dig>  o <dig>  o <dig>  o <dig>  or o <dig>
the binary coding stands for presence  or absence  of hly gene amplification, and resistance  or non-resistance  to antibiotics.
in the most contaminated water , the diversity of e. coli b <dig> strains  was higher  than in less contaminated water  .
at the peak of the turbidity, e. coli density reached a value of  <dig>   <dig> cfu/ <dig> ml, the diversity of e. coli b <dig> strains was higher  than the diversity observed when turbidity and e. coli density decreased  .
among the  <dig> ets, strains of the group et <dig>  were present in all samples, regardless of the hydrological condition or the current land use in the watershed.
however, they made up a greater proportion of the strains under non-storm conditions: during the dry period ,  <dig> et <dig> / <dig> e. coli b <dig> were present, and during the wet period   <dig> et <dig> / <dig> e. coli b <dig> were present .
in contrast, other ets were present only under certain hydrological conditions and/or land-use conditions.
et <dig>  and et <dig>  were present only during the dry period.
et <dig>  was present after the rain event only when the turbidity decreased after the peak had been reached .
these results indicate that specific e. coli b <dig> ets are more abundant in water that is only slightly contaminated, suggesting better survival of these ets.
these results strengthen the hypothesis of walk et al.,  <cit> , that some strains of e. coli b <dig> phylo-group are persistent in water and might correspond to strains with an adaptive advantage in water.
however, it must be pointed out that in this work, the e. coli a <dig> isolates , without any amplification of the genes chua, yjaa and the fragment tspe <dig> c <dig>  could correspond to the new clades of escherichia recently described which appear to be environmentally adapted  <cit> .
CONCLUSIONS
in environmental water, the occurrence of e. coli, a bacterial indicator of fecal contamination, is related to both the use of the watershed by livestock and humans combined and the hydrological conditions  <cit> .
in this study, focused on a small rural watershed composed of pasture and human occupation, we showed that both the number and the structure of the population of e. coli were modified by hydrological conditions and use of the watershed.
in this watershed, following rainfall, an increase of fecal contamination was accompanied by a modification of the distribution of phylo-groups in the e. coli population, represented by change in the ratio of a to b <dig> phylo-groups.
e. coli b <dig> strains were the dominant phylo-group isolated in the water.
among e. coli b <dig> isolates, some ets seem to be specific to water that is only slightly contaminated, suggesting different survival abilities among e. coli b <dig> strains.
the results from this study do not question the choice of e. coli as a bacterial indicator of microbial quality of water dce 2006/7/ce .
they rather indicate that the structure of an e. coli population in water is not stable, but depends on the hydrological conditions, on current use of the watershed land, and on both the origin and intensity of the contamination by fecal bacteria.

 ensuring the high microbiological quality of environmental water used as a source of recreational or drinking water is an important worldwide problem. poor microbiological quality of water results from contamination by microorganisms of human or animal fecal origin, and leads to the risk of gastro-enteritis in humans. such contamination is caused by fecal bacteria from point source pollution, e.g., treated effluents from wastewater treatments plants which primarily treat wastewater of human origin, or nonpoint source pollution consisting of inputs of microorganisms of mainly animal origin, via run-off or leaching from pasture or manured soils. the world health organization and, more recently, european guidelines, use escherichia coli as the bacterial indicator species for fecal contamination of water. epidemiological studies have been used to determine threshold values for concentrations of e. coli in water above which there is a risk of gastro-enteritis. e. coli is a commensal bacterium of the gastro-intestinal tract of humans and vertebrate animals. to survive in an aqueous environment it must resist environmental stressors and avoid predation by protozoa. some authors have suggested that some of these e. coli strains might then persist by becoming naturalized in fresh water and soil. the aquatic environment can thus be considered a secondary habitat, where some authors have even shown the possible growth of e. coli. the diversity of e. coli populations in their secondary habitats has been studied by analyzing the sequences of the gene uida, palindromic repetitive sequences, ribotypes, and profiles of antibiotic resistance. using these methods, the dynamics of e. coli populations have been investigated and, in some cases, it has been possible to discriminate between the human or animal origin of the contamination. the structure of an e. coli population is characterized by four main phylo-groups. strains of the phylo-groups a and b are mainly found as commensals in humans and vertebrate animals, with the a phylo-group strains being predominant in humans and the b strains in animals. extraintestinal infections are mainly caused by the strains of the phylo-groups b and d. although strains of the b and d phylo-groups are typically less abundant as commensals, the distribution of the four phylo-groups can vary according to diet or climate. it also has been suggested that some strains could be host-specific, such as b strains exhibiting the hly gene, found only in animals, and b o o-type strains, found only in humans. the objective of this study was to investigate the effects of various hydrological conditions on the structure of the e. coli population collected from stream water in a small rural watershed in northern france. land use in the watershed is almost entirely agricultural with a low population density. results show that an increase of fecal contamination was accompanied by a change in the distribution of phylo-groups in the e. coli population, represented by a change in the ratio of a to b phylo-groups. e. coli b isolates were the dominant phylo-group isolated in the water. among e. coli b isolates, some epidemiological types seem to be specific to water that is only slightly contaminated.  e. coli population structure in creek water in relation to hydrological conditions and watershed land use e. coli were enumerated and the population structure analyzed by phylo-grouping in three sets of samples collected under different hydrological and agricultural land-use conditions. in this study, the e. coli population structure in creek water is analyzed from a single sample integrating all the daily samples. the origin of specific strains was investigated, in addition to the phylo-grouping, by hly gene detection in the e. coli b isolates and o typing of e. coli b isolates, as well as by studying the antibiotic resistance pattern. statistical analyses were performed in order to compare hydrological conditions. a septic tanks were located between to m from the creek. one malfunctioning septic tank was located m from the sampling point. b nephelometric turbidity unit c suspended sediment concentration during the dry period, when cattle were being grazed, but when there was no runoff or leaching, the water was slightly contaminated by e. coli. the structure of the e. coli population was significantly different from the structures analyzed from the other sample collection periods, with a majority of e. coli b isolates. this structure argues for contamination by e. coli b isolates that are better adapted to the aquatic environment, rather than for residual bovine fecal contamination, as the isolates were devoid of the hly gene and sensitive to all antibiotics. resistanceb n: numbers of isolates a e. coli isolates resistant to one or more antibiotics b chl: chloramphenicol; tet: tetracyclin; str: streptomycin nd: not detected c hly gene detection by pcr method d serotype o detection by pcr method it was during the wet period, when there was no grazing, but when there was a malfunctioning septic system, that the lowest value of e. coli was measured in the water. the e. coli population was characterized by a high proportion of phylo-group a isolates, followed by e. coli b isolates without the hly gene. none of the e. coli was resistant to the seven antibiotics tested. this e. coli population is probably due to an input of solely human origin, as the structure corresponds to that already described for human commensal e. coli in france. the rainfall event that occurred during the dry period resulted in runoff from the pastures and leaching of soils. the density of the e. coli in the stream water was two orders of magnitude higher than that measured for the two other periods. during this rain event, an input of e. coli from cattle contamination was added to that from human contamination. the structure of the e. coli population was characterized by two main phylo-groups, b and a. some e. coli b isolates with the hly gene, presumably of animal origin were detected. more than 60% of these isolates were resistant to at least one of the three antibiotics used in veterinary medicine, suggesting an animal origin. thus, it appears that both hydrological conditions and current land use in the watershed might affect the structure of the e. coli a and b populations in the stream. in contrast, the hydrological and land-use conditions did not exert a significant influence on the phylo-groups b and d, which were the least abundant phylo-groups recovered from the water. no human-specific b o o-type strain was isolated during any sampling conditions, which is consistent with the low frequency of these strains in the e. coli population. changes in e. coli population structure during a rain event in order to better understand the effect of a rain event on the structure of an e. coli population, we selected three out of the twenty-four hourly samples. our selection represented three key moments showing how the turbidity and e. coli density evolved. it would not have been possible to observe this evolution using just a sample that integrated all the daily samples. the rain event consisted of mm of rain that fell during a wet period, during which there were cattle being grazed in the watershed. five hours before rainfall began, the level of e. coli contamination was low, and the small number of isolates did not permit analysis of the structure of the e. coli population. during the rain event, the turbidity increased, as did the number of e. coli, consistent with previous work demonstrating a correlation between bacteria and particles. six hours after the rainfall event the e. coli density reached a value of cfu/ ml, at which point the structure of the e. coli population was characterized by a majority of e. coli phylo-group a, with 63% being resistant to at least one antibiotic, suggesting fecal contamination of human origin resulting from leaching of soils and from surface runoff. this structure was significantly different from that observed in the less contaminated water analyzed hours after the rainfall event. at that time the e. coli density had decreased to cfu/ ml, and e. coli b isolates were the predominant e. coli phylo-group. these isolates are mainly hly positive with 31% resistant to at least one antibiotic, suggesting that there had been an input on the soils of e. coli of bovine origin that was then introduced into the water through run-off and/or leaching. resistancec resistancec resistancec amx/tic/chl/sxt/str amx/tic/sxt/str chl amx/tic/chl nd: not detected n: numbers of isolates a timing in relation to rainfall b e. coli isolates resistant to one or more antibiotics c amx: amoxicillin; tic: ticarcillin chl: chloramphenicol; tet: tetracyclin; str: streptomycin; sxt:trimethoprim + sulfamethoxazole d hly gene detection by pcr method e serotype o detection by pcr method we cannot exclude an input from wild animals, although wild e. coli strains are usually not resistant to antibiotics. these results indicate that during the rain event, an increase in microbial contamination was accompanied by a modification of the structure of the e. coli population, resulting in a high ratio of phylo-groups a/b in contrast, in the water collected h after the rain event, and only slightly contaminated by e. coli, the majority of e. coli isolates belonged to the b phylo-group. diversity of e. coli b strains isolated from the creek water as e. coli b was the dominant phylo-group isolated in water from the bébec, accounting for between 15% to 87% of the e. coli population, we investigated further the diversity of e. coli b isolates by sequencing the uida gene and comparing the sequences obtained with those in the mlst pasteur database in order to find the uida allele, detecting the presence of hly and determining molecularly the o-type, studying the antibiotic resistance profile. a total of epidemiological types were identified among the e. coli b isolated from the water and the proportion of each ets differed for each sampling event. aamx: amoxicillin; chl: chloramphenicol; tet: tetracyclin; all of epidemiological types of e. coli b strains were non-resistant to tic: ticarcillin; sxt: trimethoprim + sulfamethoxazole; str: streptomycin; and cip: ciprofloxacin. bnt: not o o o o o o45b, o o o o o o o or o the binary coding stands for presence or absence of hly gene amplification, and resistance or non-resistance to antibiotics. in the most contaminated water, the diversity of e. coli b strains was higher than in less contaminated water. at the peak of the turbidity, e. coli density reached a value of cfu/ ml, the diversity of e. coli b strains was higher than the diversity observed when turbidity and e. coli density decreased. among the ets, strains of the group et were present in all samples, regardless of the hydrological condition or the current land use in the watershed. however, they made up a greater proportion of the strains under non-storm conditions: during the dry period, et / e. coli b were present, and during the wet period et / e. coli b were present. in contrast, other ets were present only under certain hydrological conditions and/or land-use conditions. et and et were present only during the dry period. et was present after the rain event only when the turbidity decreased after the peak had been reached. these results indicate that specific e. coli b ets are more abundant in water that is only slightly contaminated, suggesting better survival of these ets. these results strengthen the hypothesis of walk et al.,, that some strains of e. coli b phylo-group are persistent in water and might correspond to strains with an adaptive advantage in water. however, it must be pointed out that in this work, the e. coli a isolates, without any amplification of the genes chua, yjaa and the fragment tspe c could correspond to the new clades of escherichia recently described which appear to be environmentally adapted.  in environmental water, the occurrence of e. coli, a bacterial indicator of fecal contamination, is related to both the use of the watershed by livestock and humans combined and the hydrological conditions. in this study, focused on a small rural watershed composed of pasture and human occupation, we showed that both the number and the structure of the population of e. coli were modified by hydrological conditions and use of the watershed. in this watershed, following rainfall, an increase of fecal contamination was accompanied by a modification of the distribution of phylo-groups in the e. coli population, represented by change in the ratio of a to b phylo-groups. e. coli b strains were the dominant phylo-group isolated in the water. among e. coli b isolates, some ets seem to be specific to water that is only slightly contaminated, suggesting different survival abilities among e. coli b strains. the results from this study do not question the choice of e. coli as a bacterial indicator of microbial quality of water dce 2006/7/ce. they rather indicate that the structure of an e. coli population in water is not stable, but depends on the hydrological conditions, on current use of the watershed land, and on both the origin and intensity of the contamination by fecal bacteria. 
escherichia coli is a commensal bacterium of the gastro-intestinal tract of human and vertebrate animals, although the aquatic environment could be a secondary habitat. the aim of this study was to investigate the effect of hydrological conditions on the structure of the e. coli population in the water of a creek on a small rural watershed in france composed of pasture and with human occupation. during the wet period and the rainfall events, contamination from human sources was predominantly characterized by strains of the a phylo-group, whereas contamination by cattle mainly involved b phylo-group strains resistant to antibiotics and exhibiting hly. isolates of the b phylo-group devoid of hly and sensitive to antibiotics were particularly abundant during the dry period. it became apparent, after studying the distribution in the four main e. coli phylo-groups, the presence of the hly gene and the antibiotic resistance pattern, that the e. coli population structure was modified not only by the hydrological conditions, but also by how the watershed was used. the structure of an e. coli population in water is not stable, but depends on the hydrological conditions and on current use of the land on the watershed.
 escherichia coli is a commensal bacterium of the gastro-intestinal tract of human and vertebrate animals, although the aquatic environment could be a secondary habitat. the aim of this study was to investigate the effect of hydrological conditions on the structure of the e. coli population in the water of a creek on a small rural watershed in france composed of pasture and with human occupation.  it became apparent, after studying the distribution in the four main e. coli phylo-groups, the presence of the hly gene and the antibiotic resistance pattern, that the e. coli population structure was modified not only by the hydrological conditions, but also by how the watershed was used. isolates of the b phylo-group devoid of hly and sensitive to antibiotics were particularly abundant during the dry period. during the wet period and the rainfall events, contamination from human sources was predominantly characterized by strains of the a phylo-group, whereas contamination by cattle mainly involved b phylo-group strains resistant to antibiotics and exhibiting hly. as e. coli b was the main phylo-group isolated in water, the diversity of e. coli b isolates was further investigated by studying uida alleles, the presence of hly, the o-type, and antibiotic resistance. among the forty epidemiolgical types identified, five e. coli b ets were more abundant in slightly contaminated water.  the structure of an e. coli population in water is not stable, but depends on the hydrological conditions and on current use of the land on the watershed. in our study it was the ratio of a to b phylo-groups that changed. however, a set of b phylo-group isolates seems to be persistent in water, strengthening the hypothesis that they may correspond to specifically adapted strains. 