BACKGROUND
the gastrointestinal  tract of humans is colonized by escherichia coli within about  <dig> hours of birth  <cit> .
this facultative anaerobe is then stably maintained as a relatively minor, but critical, component of the large intestine microflora with a cell density approximately  <dig> times lower than the predominant bacterial genera, such as bacteriodes, clostridia, and anaerobic streptococci.
e. coli adheres to, and primarily subsists on, the mucin layer that coats the epithelial cells of the large intestine.
a dominant, resident strain will normally persist in the gi tract for periods of months to years, until it is eventually replaced by one of the many transient strains continually passing through the intestinal lumen.
the basis for these periodic shifts is not known and has recently become the focus of a large body of research  <cit> .
in part, this increased interest in the dynamics of e. coli strains is due to dysbiosis, or microbial imbalances of the normal human microflora of the gi tract.
this common outcome of antibiotic therapies is now considered to be a contributing factor to many chronic and degenerative diseases such as irritable bowel syndrome and rheumatoid arthritis  <cit> .
attempts to re-establish a healthy microbial flora, alleviate gi disorders, and control pathogenic e. coli in humans and animals through the administration of probiotics, are limited by our ability to choose and properly administer the most appropriate strains  <cit> .
understanding strain dynamics of e. coli in the gi tract may provide a more sound approach to both probiotic strain choice and methods of administration  <cit> .
one powerful predictor of the ability of a strain of e. coli to competitively exclude or displace other strains is the production of one or more of a large family of narrow spectrum antimicrobials, the bacteriocins.
theoretical studies have shown that bacteriocin production enhances the invasion and establishment success of the producing strains  <cit> .
in vivo studies further demonstrate that bacteriocin production improves the establishment success of its producing strain  <cit> .
similar results were obtained when mice harboring bacteriocin-sensitive strains were co-caged with mice harboring bacteriocin-producing strains.
within a relatively short period  the sensitive strains had been displaced by the bacteriocin-producing strains  <cit> .
e. coli are prolific producers of their own species-specific bacteriocins, known as colicins, which were first identified over  <dig> years ago  <cit> , and given the name colicin to identify the producing species.
the frequency of colicin production varies among e. coli populations depending on the host species diet  <cit> , the relatedness of the e. coli strains present  <cit> , and the habitat quality  <cit> .
however, on average, forty percent of the strains in any population are likely to produce one or more colicins  <cit> .
over thirty colicins have been characterized to date, all of which are plasmid-encoded, high molecular weight proteins that are induced in times of stress  <cit> .
upon release of colicins from the producing cell, the toxins kill their targets primarily by membrane permeabilization or nucleic acid degradation  <cit> .
genes encoding colicin functions are found in clusters that include a toxin-encoding gene; an immunity gene, encoding a protein conferring self-specific protection to the cell against its own colicin; and, frequently, a lysis gene, encoding a protein involved in colicin release via lysis or pseudo-lysis of the producing cell  <cit> .
it has recently been suggested that bacteriocin production is a critical factor in determining the establishment success of probiotic bacteria in humans and animals  <cit> .
to investigate this hypothesis, we introduced e. coli strains differing only in the carriage and identity of bacteriocin-encoding plasmids into the gi tract of mice.
the importance of bacteriocin production in colonization and persistence of their e. coli hosts in the mouse intestine was elucidated over time providing a rare and novel glimpse into the impact of bacteriocins on the establishment of enteric bacteria in the mouse gi tract.
RESULTS
this study was designed to examine the colonization and persistence of colicinogenic e. coli strains in the mouse gi tract following a single administration.
to this end, six isogenic strains of e. coli bzb <dig> were created differing in only two characters:  the ability to produce a colicin ; and  the identity of the colicin produced .
mice treated with streptomycin to eradicate their resident enterobacterial flora were inoculated with streptomycin resistant bacteriocin producing  strains that were then monitored for  <dig> days by weekly sampling of mouse pellets.
the persistence and population density of colicin producers in the mouse gi tract
a statistically significant difference in strain persistence was observed over the course of the experiment  =  <dig> , p <  <dig> ).
a second repeated-measure anova, which excluded the colicin-free control strain, revealed significant difference in persistence times among the colicin strains  =  <dig> , p <  <dig> ).
these results indicate that the significant differences in strain abundance observed over time is not solely due to the rapid disappearance of the colicin free control strain and presumably reflect differences between the more slowly declining strains  and the highly persistent strains .
one week after the initial strain introduction into the mouse gi tract, no significant differences in density were observed between the different colicin-producing strains  =  <dig> , p =  <dig> ; no significant contrasts).
a simple one-way anova indicated no such differences at the end of the experiment either  =  <dig> , p =  <dig> ).
however, the orthogonal contrasts analysis indicated a significant difference in the density of the control strain versus all other colicinogenic strains  =  <dig> , p =  <dig> ).
the doubling time of colicin producers isolated from the mouse gi tract
an average strain generation time was determined from five colonies isolated from each colicin treatment at days  <dig> and  <dig> .
an increase in doubling time was observed for all strains, ranging from 6–33% relative to day  <dig>  =  <dig> , p <  <dig> ).
however, the degree of increase varied among strains, as indicated by a significant interaction term  =  <dig> , p =  <dig> ), with the non-colicin producing strain experiencing the greatest increase in generation time .
1growth rate is expressed in generations/h.
discussion
the abundance and diversity of bacteriocin production in microbial populations point to the fundamental role these potent toxins serve in mediating strain dynamics in microbial systems.
indeed, most species of bacteria have been shown to possess bacteriocins  <cit>  and levels of production within a species can be as high as 95%.
for example, nearly 40% of the e. coli isolated from fecal samples of animals and humans were shown to be colicinogenic  <cit> , while greater than 95% of the pseudomonas aeruginosa isolated from environmental and clinical sources are bacteriocin producers  <cit> .
numerous in silico and in vitro studies have shown that colicinogenic e. coli rapidly out-compete their colicin sensitive counterparts, due to the lethality of colicin production  <cit> .
in the present study, the average increase in the generation time of producer strains was lower then that monitored for the colicin free cells .
similar to other e. coli strains established in streptomycin-treated mice  <cit> , we observed an increase of ~30% in doubling time, while the colicinogenic strains increase in generation time was more moderate .
in contrast, in an in vivo study of bacteriocins employing the same mouse model as described here, did not detect an increased persistence of colicinogenic enteric bacteria  <cit> .
however, in that study persistence was monitored for only  <dig> days.
our data suggest that over a longer period of time,  <dig> days in the present study, the benefit of colicinogenicity becomes more apparent , with colicin producers maintaining significantly higher densities than their non-colicin producing counterparts.
the colicin-based advantage observed in the present in vivo study reflects a similar advantage to colicin production as has been detected in prior in silico and in vitro studies  <cit> .
our results are even more promising with respect to the advantage gained from colicin production when the sampling method employed here is considered, as fecal-based sampling will generally underestimate the actual density of the strain in the gi tract  <cit> .
there is one further colicin-based in vitro study, which employed the same mouse model described here, but which differed significantly in experimental design.
in this latter study the focus was on the interaction  between colicinogenic and non-colicinogenic strains, while the current study focuses on the ability of colicinogenicity to enhance strain maintenance  <cit> .
this prior colicin competition study revealed that colicin production enhances strain persistence when mice equilibrated with colicin producing strains are co-caged with mice equilibrated with colicin sensitive strains  <cit> .
thus, although the intent of the two studies is quite different, both reveal that colicinogenicity has a significant and positive effect on the ability of a strain to be maintained in the gi tract of a streptomycin-treated mouse.
many studies in humans and livestock have shown that probiotic bacteria have the ability to re-establish an indigenous microflora after perturbations of the normal intestinal flora  <cit> .
probiotic bacteria provide this health benefit in many ways and the production of toxins, in particular bacteriocins, was proposed as a leading candidate in this process  <cit> .
e. coli strain nissle  <dig>  a producer of microcins h <dig> and m  <cit> , is a well characterized probiont in humans and livestock  <cit> .
this strain was found to be effective in treating chronic inflammatory bowel disease  <cit>  and in inhibiting the adhesion of enteric pathogens to the gi epithelial cells of infants  <cit> .
e. coli strain h <dig> inhibits the invasion of the enetric pathogen shigella flexneri in germ-free mice, probably due to the production of microcin c <dig>  <cit> , colicins e <dig> and ib, as well as aerobin and an unidentified phage  <cit> .
in order for a probiotic strain to exert its beneficial effect in the gi tract, it is essential for the cells to become established.
feeding trials with a variety of probiotic strains have shown that the strains often disappear from the gi tract within weeks of administration  <cit> .
several studies have examined methods to increase strain persistence using prebiotics  <cit> ; synbiotic dietary supplements  <cit> ; and addition of uptake systems.
this latter mechanism involves inserting the listerial betaine uptake system, betl  <cit> , into the probiotic strains such as bifidobacterium breve strain ucc <dig>  <cit>  and lactobacillus salivarius strain ucc <dig>  <cit> .
the present study suggests that production of a bacteriocin may serve a similar beneficial function.
CONCLUSIONS
we have shown that bacteriocin-producing strains of e. coli, but not their bacteriocin-free counterparts, were recovered from the feces of mice over extended periods of sampling following a single administration of the strains.
these results suggest that colicinogenicity is beneficial in increasing e. coli persistence in the mouse gi tract.

 the gastrointestinal tract of humans is colonized by escherichia coli within about hours of birth. this facultative anaerobe is then stably maintained as a relatively minor, but critical, component of the large intestine microflora with a cell density approximately times lower than the predominant bacterial genera, such as bacteriodes, clostridia, and anaerobic streptococci. e. coli adheres to, and primarily subsists on, the mucin layer that coats the epithelial cells of the large intestine. a dominant, resident strain will normally persist in the gi tract for periods of months to years, until it is eventually replaced by one of the many transient strains continually passing through the intestinal lumen. the basis for these periodic shifts is not known and has recently become the focus of a large body of research. in part, this increased interest in the dynamics of e. coli strains is due to dysbiosis, or microbial imbalances of the normal human microflora of the gi tract. this common outcome of antibiotic therapies is now considered to be a contributing factor to many chronic and degenerative diseases such as irritable bowel syndrome and rheumatoid arthritis. attempts to re-establish a healthy microbial flora, alleviate gi disorders, and control pathogenic e. coli in humans and animals through the administration of probiotics, are limited by our ability to choose and properly administer the most appropriate strains. understanding strain dynamics of e. coli in the gi tract may provide a more sound approach to both probiotic strain choice and methods of administration. one powerful predictor of the ability of a strain of e. coli to competitively exclude or displace other strains is the production of one or more of a large family of narrow spectrum antimicrobials, the bacteriocins. theoretical studies have shown that bacteriocin production enhances the invasion and establishment success of the producing strains. in vivo studies further demonstrate that bacteriocin production improves the establishment success of its producing strain. similar results were obtained when mice harboring bacteriocin-sensitive strains were co-caged with mice harboring bacteriocin-producing strains. within a relatively short period the sensitive strains had been displaced by the bacteriocin-producing strains. e. coli are prolific producers of their own species-specific bacteriocins, known as colicins, which were first identified over years ago, and given the name colicin to identify the producing species. the frequency of colicin production varies among e. coli populations depending on the host species diet, the relatedness of the e. coli strains present, and the habitat quality. however, on average, forty percent of the strains in any population are likely to produce one or more colicins. over thirty colicins have been characterized to date, all of which are plasmid-encoded, high molecular weight proteins that are induced in times of stress. upon release of colicins from the producing cell, the toxins kill their targets primarily by membrane permeabilization or nucleic acid degradation. genes encoding colicin functions are found in clusters that include a toxin-encoding gene; an immunity gene, encoding a protein conferring self-specific protection to the cell against its own colicin; and, frequently, a lysis gene, encoding a protein involved in colicin release via lysis or pseudo-lysis of the producing cell. it has recently been suggested that bacteriocin production is a critical factor in determining the establishment success of probiotic bacteria in humans and animals. to investigate this hypothesis, we introduced e. coli strains differing only in the carriage and identity of bacteriocin-encoding plasmids into the gi tract of mice. the importance of bacteriocin production in colonization and persistence of their e. coli hosts in the mouse intestine was elucidated over time providing a rare and novel glimpse into the impact of bacteriocins on the establishment of enteric bacteria in the mouse gi tract.  this study was designed to examine the colonization and persistence of colicinogenic e. coli strains in the mouse gi tract following a single administration. to this end, six isogenic strains of e. coli bzb were created differing in only two characters: the ability to produce a colicin; and the identity of the colicin produced. mice treated with streptomycin to eradicate their resident enterobacterial flora were inoculated with streptomycin resistant bacteriocin producing strains that were then monitored for days by weekly sampling of mouse pellets. the persistence and population density of colicin producers in the mouse gi tract a statistically significant difference in strain persistence was observed over the course of the experiment =, p < ). a second repeated-measure anova, which excluded the colicin-free control strain, revealed significant difference in persistence times among the colicin strains =, p < ). these results indicate that the significant differences in strain abundance observed over time is not solely due to the rapid disappearance of the colicin free control strain and presumably reflect differences between the more slowly declining strains and the highly persistent strains. one week after the initial strain introduction into the mouse gi tract, no significant differences in density were observed between the different colicin-producing strains =, p =; no significant contrasts). a simple one-way anova indicated no such differences at the end of the experiment either =, p = ). however, the orthogonal contrasts analysis indicated a significant difference in the density of the control strain versus all other colicinogenic strains =, p = ). the doubling time of colicin producers isolated from the mouse gi tract an average strain generation time was determined from five colonies isolated from each colicin treatment at days and. an increase in doubling time was observed for all strains, ranging from 6–33% relative to day =, p < ). however, the degree of increase varied among strains, as indicated by a significant interaction term =, p = ), with the non-colicin producing strain experiencing the greatest increase in generation time. 1growth rate is expressed in generations/h. discussion the abundance and diversity of bacteriocin production in microbial populations point to the fundamental role these potent toxins serve in mediating strain dynamics in microbial systems. indeed, most species of bacteria have been shown to possess bacteriocins and levels of production within a species can be as high as 95%. for example, nearly 40% of the e. coli isolated from fecal samples of animals and humans were shown to be colicinogenic, while greater than 95% of the pseudomonas aeruginosa isolated from environmental and clinical sources are bacteriocin producers. numerous in silico and in vitro studies have shown that colicinogenic e. coli rapidly out-compete their colicin sensitive counterparts, due to the lethality of colicin production. in the present study, the average increase in the generation time of producer strains was lower then that monitored for the colicin free cells. similar to other e. coli strains established in streptomycin-treated mice, we observed an increase of ~30% in doubling time, while the colicinogenic strains increase in generation time was more moderate. in contrast, in an in vivo study of bacteriocins employing the same mouse model as described here, did not detect an increased persistence of colicinogenic enteric bacteria. however, in that study persistence was monitored for only days. our data suggest that over a longer period of time, days in the present study, the benefit of colicinogenicity becomes more apparent, with colicin producers maintaining significantly higher densities than their non-colicin producing counterparts. the colicin-based advantage observed in the present in vivo study reflects a similar advantage to colicin production as has been detected in prior in silico and in vitro studies. our results are even more promising with respect to the advantage gained from colicin production when the sampling method employed here is considered, as fecal-based sampling will generally underestimate the actual density of the strain in the gi tract. there is one further colicin-based in vitro study, which employed the same mouse model described here, but which differed significantly in experimental design. in this latter study the focus was on the interaction between colicinogenic and non-colicinogenic strains, while the current study focuses on the ability of colicinogenicity to enhance strain maintenance. this prior colicin competition study revealed that colicin production enhances strain persistence when mice equilibrated with colicin producing strains are co-caged with mice equilibrated with colicin sensitive strains. thus, although the intent of the two studies is quite different, both reveal that colicinogenicity has a significant and positive effect on the ability of a strain to be maintained in the gi tract of a streptomycin-treated mouse. many studies in humans and livestock have shown that probiotic bacteria have the ability to re-establish an indigenous microflora after perturbations of the normal intestinal flora. probiotic bacteria provide this health benefit in many ways and the production of toxins, in particular bacteriocins, was proposed as a leading candidate in this process. e. coli strain nissle a producer of microcins h and m, is a well characterized probiont in humans and livestock. this strain was found to be effective in treating chronic inflammatory bowel disease and in inhibiting the adhesion of enteric pathogens to the gi epithelial cells of infants. e. coli strain h inhibits the invasion of the enetric pathogen shigella flexneri in germ-free mice, probably due to the production of microcin c, colicins e and ib, as well as aerobin and an unidentified phage. in order for a probiotic strain to exert its beneficial effect in the gi tract, it is essential for the cells to become established. feeding trials with a variety of probiotic strains have shown that the strains often disappear from the gi tract within weeks of administration. several studies have examined methods to increase strain persistence using prebiotics; synbiotic dietary supplements; and addition of uptake systems. this latter mechanism involves inserting the listerial betaine uptake system, betl, into the probiotic strains such as bifidobacterium breve strain ucc and lactobacillus salivarius strain ucc. the present study suggests that production of a bacteriocin may serve a similar beneficial function.  we have shown that bacteriocin-producing strains of e. coli, but not their bacteriocin-free counterparts, were recovered from the feces of mice over extended periods of sampling following a single administration of the strains. these results suggest that colicinogenicity is beneficial in increasing e. coli persistence in the mouse gi tract. 
the strains' persistence, population density, and doubling time were monitored over a period of days. early in the experiment only minor differences in population density between the various colicin-producing and the non-producing control strains were detected. in an attempt to evaluate the importance of bacteriocin production for escherichia coli strain residence in the gastrointestinal tract, a murine model experimental evolution study was undertaken. the data presented here support prior claims that bacteriocin production may play a significant role in the colonization of e. coli in the gastrointestinal tract. further, this study suggests that the ability to produce bacteriocins may prove to be a critical factor in determining the success of establishing probiotic e. coli in the gastrointestinal tract of humans and animals. six colicin-producing, yet otherwise isogenic, e. coli strains were administered and established in the large intestine of streptomycin-treated mice. the ability of a bacterial strain to competitively exclude or displace other strains can be attributed to the production of narrow spectrum antimicrobials, the bacteriocins.
 the ability of a bacterial strain to competitively exclude or displace other strains can be attributed to the production of narrow spectrum antimicrobials, the bacteriocins. in an attempt to evaluate the importance of bacteriocin production for escherichia coli strain residence in the gastrointestinal tract, a murine model experimental evolution study was undertaken.  six colicin-producing, yet otherwise isogenic, e. coli strains were administered and established in the large intestine of streptomycin-treated mice. the strains' persistence, population density, and doubling time were monitored over a period of days. early in the experiment only minor differences in population density between the various colicin-producing and the non-producing control strains were detected. however, over time, the density of the control strains plummeted, while that of the colicin-producing strains remained significantly higher =; p < ).  the data presented here support prior claims that bacteriocin production may play a significant role in the colonization of e. coli in the gastrointestinal tract. further, this study suggests that the ability to produce bacteriocins may prove to be a critical factor in determining the success of establishing probiotic e. coli in the gastrointestinal tract of humans and animals. 