BACKGROUND
the first step in a bacterial disease is the successful establishment of a bacterial population in a host: colonization.
the conditions that determine whether a bacterial population can colonize a particular site and the density achieved are fundamental to determining the likelihood of invasive disease, transmission to other hosts and the presence of mutants resistant to antibiotics.
how these conditions are affected by prior colonization by bacteria of the same or different species has wide spread consequences for determining the sequelae of the wide-scale use of vaccines directed at specific strains or species  as well as for evaluating probiotics  <cit>  and understanding epidemiological changes in invasive bacterial diseases  <cit> .
whether bacteria can colonize or not is determined by many ecological factors including the availability of resources , host immune responses and the presence of toxins or harmful substances.
as the presence of established bacteria populations can influence all of these factors, it seems reasonable to assume that co-inhabitants often determine whether colonization can occur.
in fact co-inhabitants that are ecologically similar, should limit the colonization as the one that is better at exploiting the habitat should exclude the others through resource limitation  <cit> .
however, as a consequence of even subtle differences in resource  utilization or availability, multiple strains and species of bacteria can co-exist  <cit> .
the ability to colonize can also be influenced by interference, which includes residents populations producing harmful substances  or inducing an immune response  <cit> .
in the case of three bacterial species which colonize the human nasopharynx , epidemiological studies show that co-colonization is rarer than expected  <cit> .
these co-inhabitation patterns suggest that there may be interference or competition occurring.
in this report we apply an ecological framework to elucidate the factors contributing to the nasal colonization of neonatal rats of three bacterial species that typically colonize humans: s. pneumoniae, h. influenzae and s. aureus.
first we consider the population dynamics of each strain separately.
we provide evidence that all three species colonize the nasal passages of neonatal rats and reach an apparent steady-state density and that this level is independent of inoculum density.
to explore the effects of co-inhabitants on colonization,  <dig> hours after colonizing neonatal rats with one species we pulsed with a second inoculum of a marked strain of the same species.
the results of these pulse experiments suggest that resident s. aureus prevents co-colonization of the same strain; while for both h. influenzae and s. pneumoniae the total density is increased to allow for the co-existence of pulsed and established populations.
we repeated these experiments with the resident and invading populations being of different species and found that h. influenzae colonizes at a higher density when either s. aureus or s. pneumoniae are present and that immune-mediated competition between s. pneumoniae and h. influenzae is both site and strain specific.
RESULTS
population dynamics
all three species readily colonize the nasal passages of neonatal rats.
within  <dig> hours after one of the three species is inoculated, h. influenzae, s. aureus and s. pneumoniae reach and maintain for at least three days a constant population  in the nasal epithelium .
the population dynamics of nasal colonization did not differ in the nasal wash sample with the nasal epithelium.
the nasal epithelium, which represent the persisting colonizing bacteria  <cit> , data are shown.
the bacterial load for each of the species was not significantly different from  <dig> to  <dig> hours .
while the dynamics for both a low and high inoculum density appear to be similar, we ascertained whether bacterial load is inoculum-independent at  <dig> hours after inoculation.
for all three species the bacterial load is invariant over a wide range of inocula  , suggesting that nasal colonization rapidly reaches a steady-state that is not limited by how many bacteria are inoculated.
invasion of same species in a colonized host
to test whether nasal colonization can occur in the presence of the same species, new populations of bacteria were pulsed  into rats that were already colonized by bacteria of that species.
antibiotic markers that conferred no in vitro or in vivo fitness costs were used to distinguish the resident and pulsed populations and each experiment was repeated reversing the strains as pulsed or resident to control for any fitness differences.
as the population dynamics suggest that the bacterial load for each of these species is tightly controlled, we expected that the total density  would return to the bacterial load observed in rats without pulses.
because resident and pulsed strains of the same species utilize the same resource , co-existence of both strains is expected unless a limiting factor is available only on a first come first serve basis.
in the case of s. aureus, regardless of whether the marked strain is resident or pulsed, we find that the pulsed strain declines in density  over the course of  <dig> hours .
as the pulsed strain declines  the total bacterial load of s. aureus in the rats with the pulsed and established strain  doesn't differ from the total density of s. aureus in the rats with only the established strain .
for s. aureus the bacterial density does not exceed that observed in rats without a pulse and the resident strain has a competitive advantage.
for both s. pneumoniae and h. influenzae there is an increase in the total density in the rats with the pulse  compared to rats with only the established strain .
we saw the bacterial load increase to varying degrees, more so for h. influenzae than for s. pneumoniae, in each of four replicate experiments .
in both of these species, we observe that the pulsed and resident strains co-exist with the pulse strain becoming 25-90% of the population.
for all the species, similar pulse results were obtained in reciprocal experiments  confirming that the results were not due to fitness differences in the antibiotic marked strains.
invasion of different species in a colonized host
competition between different strains or species can be defined simply as a reduction in the density of one or both strains when both are present.
competition within the same species and particularly in the case of the same strain  is usually mediated through a limiting shared resource.
competition between species, in addition to partitioning of a shared resource, can be mediated through inhibitory agents/toxins  or predators .
previous studies suggest that production of hydrogen peroxide by s. pneumoniae may affect the densities of other species  <cit>  and that immune-mediated competition reduces s. pneumoniae density in the presence of h. influenzae  <cit> .
to evaluate the contributions of these different competitive mechanisms we performed invasion experiments  in which one species was resident and a second was introduced .
evidence for synergistic interactions between h. influenzae and s. pneumoniae or s. aureus
rather than an antagonistic interaction, we found that h. influenzae reached a higher density when invading resident populations of either s. aureus or s. pneumoniae than in the absence of these residents .
a similar increase in the bacterial density of h. influenzae was observed in vitro; when mixtures of these strains were grown in broth for  <dig> hours, h. influenzae density was 20% greater with s. pneumoniae and 19% greater with s. aureus present than when grown alone .
strain-specific, innate immune-mediated interactions between h. influenzae and s. pneumoniae
we had expected to detect immune-mediated competition between h. influenzae and s. pneumoniae, as observed in a mouse model of colonization by lysenko and colleagues  <cit> .
however, we saw no evidence of competition between h. influenzae and s. pneumoniae with the strains we initially used: tigr <dig> and eagan .
to investigate further, we tested one additional strain of s. pneumoniae, poland- <dig>  we found that this particular strain of s. pneumoniae had a reduced density in the nasal wash, but not the nasal epithelium, when invading in a neonatal rat with an established h. influenzae population .
this reduction in poland-20's population did not occur in neonatal rats which had been depleted of complement or neutrophils.
to explain why we could only observe this in one of the two strains tested and only then in the nasal wash, we hypothesized that either induction of or susceptibility to the immune response must differ in these strains and locations.
we quantified the neutrophil infiltration in the nasal epithelium by measuring the myeloperoxidase  activity at  <dig> hours after inoculation with each strain/species alone or when poland- <dig> was inoculated on an established h. influenzae population .
no difference in neutrophil infiltration is observed between rats colonized by the two different s. pneumoniae strains -20).
the neutrophil infiltration observed  <dig> hours after poland- <dig> invaded on an established h. influenzae population  was significantly higher than rats with just poland- <dig> colonizing alone.
however, neutrophil infiltration was not significantly higher than in rats with only h. influenzae.
while these results suggest that h. influenzae is primarily responsible for the neutrophil infiltration that reduces the nasal lumen populations of some strains of s. pneumoniae, s. pneumoniae may still have a role in eliciting the immune response .
we observed that the neutrophil infiltration in response to s. pneumoniae colonizing alone increases from 48- <dig> hours after inoculation, compared to the constant neutrophil presence with h. influenzae .
discussion
population dynamics
all three species that we studied  can colonize the nasal passages of neonatal rats and each reaches a bacterial load that is independent of the initial inoculum size; they increase in density when initially below this level and decline when initially above it.
this indicates that the steady-state density is tightly controlled - perhaps by a limiting resource or the host's immune response.
the total density of each of these colonizing species is relatively low and there is wide-spread variation in the densities of individuals, similar to what has been observed in colonized humans  <cit> .
invasion of same species in a colonized host
in accord with classical ecological theory of competition  <cit>  we would anticipate that established populations of one strain of a species would either prevent colonization of another strain of that species or be eliminated.
the outcome would depend on which of the strains was more fit.
in the case of genetically marked strains of the same species with equal fitness we would expect to observe co-existence.
in a series of pulse experiments with genetically marked colonizing  and resident  strains we found the situation to be more complicated than this simple interpretation.
for both h. influenzae and s. pneumoniae, the resident and the pulsed strains co-existed.
surprisingly in all replicates of these experiments, the invasion of a second population of the same species was followed by an increase in the total bacterial density.
given that the steady-state bacterial densities were independent of the initial inoculum density following a single inoculation, we had expected that the bacterial density after a second inoculation would decline to the original bacterial load.
these results might be attributable to a second inoculation leading to an expansion in the colonization area, increased immune suppression or the release of new resources - perhaps associated with an inflammatory response.
on first consideration it would seem that the results of the s. aureus pulse experiments are consistent with classical ecological theory; the established population of this species inhibited the colonization of a new strain.
moreover, as expected following the pulse by a second strain the total density of s. aureus returned to a level similar to that observed in the single inoculation experiments.
however the resident strain had the advantage no matter which marker it carried .
we interpret these results as suggesting that s. aureus is limited by a localized resource available on a 'first-come, first-serve' basis - perhaps attachment sites  <cit> .
this ecological hypothesis would account for the observations that competing strains of s. aureus were excluded from burn wounds  <cit>  and from nasal colonization in persistent human carriers  <cit> .
while these results do not exclude the possibility that variation within s. aureus strains may allow for coexistence as occasionally has been observed in humans  <cit> , they do suggest that prior nasal colonization with s. aureus can exclude similar s. aureus strains from colonizing.
invasion of different species in a colonized host
ecologically, strains of different species would be anticipated to be more divergent than those of the same species and therefore they would be expected to occupy different niches.
the results of our experiments are consistent with this interpretation as any pairwise combination of the three species can co-exist.
while we had expected some sharing of resources by these different species, we found no evidence that the presence of one species reduced the density colonizing the nasal epithelium of another species.
the only evidence for inter-specific interactions between these species observed at the nasal epithelium was in fact synergistic rather than antagonistic; h. influenzae reaches a higher density when invading resident populations of either s. aureus or s. pneumoniae than it achieves in rats not colonized by these bacteria.
since this result is also observed in vitro it seems likely due to some host-independent mechanism such as s. aureus and s. pneumoniae providing nutrients that would otherwise limit h. influenzae.
indeed, in the past h. influenzae has been identified and cultured due to the fact that it grew as satellites off of s. aureus colonies  <cit> .
to our knowledge this is the first evidence for s. aureus and s. pneumoniae increasing the density of h. influenzae during nasal colonization.
we had expected to see inter-specific antagonism not only due to resource sharing but also because of interference by toxins and harmful substances.
in fact, it has been proposed that the production of hydrogen peroxide by s. pneumoniae may affect the densities of s. aureus and h. influenzae as both are susceptible to hydrogen peroxide killing  <cit> .
however in this and a previous work that specifically addressed this issue  <cit>  we found no evidence that hydrogen peroxide produced by s. pneumoniae limits the colonizing populations of either of the two species.
this may be because the density of s. pneumoniae is too low for sufficient hydrogen peroxide production or the nasal epithelium inactivates the hydrogen peroxide produced.
taken at large, we found no ecological interaction between s. aureus and s. pneumoniae colonization that would account for the epidemiological observation that s. aureus-s. pneumoniae co-colonization is rarer than expected  <cit> .
we postulate that this epidemiological observation may be due to the bacteria preferring different hosts rather than competitive interactions within hosts  <cit> , or that competitive exclusion may only occur in immunologically mature individuals.
others have suggested that there may still be an ecological interaction based on the pneumococcal pilus  <cit>  or by induction of phage release  <cit> .
neutrophil-mediated competition
previous experiments by lysenko and colleagues in a mouse model have shown that when h. influenzae and s. pneumoniae co-colonize, s. pneumoniae's density in the nasal wash is lower than when inoculated alone due to immune-mediated competition  <cit> .
at one level, the results of our rat model experiments with h. influenzae and s. pneumoniae are consistent with their results  <cit> .
however, our results also suggest that this immune-mediated competitive interaction may only affect the colonizing s. pneumoniae population in the nasal wash  and is strain-specific.
we observed immune-mediated competition with the clinical strain of s. pneumoniae poland- <dig> but not with tigr <dig>  we hypothesize that pneumococcal strains vary either in their ability to elicit an immune response or in their suscebtibility to the immune response induced by h. influenzae.
although we can't exclude the possibility that the two strains we tested elicited different immune responses, our results suggest that there is no difference in the extent of neutrophil infiltration of the epithelium in response to colonization by either of these strains or any synergism  <cit>  between the two species.
together our results suggest that the immune response primarily elicited by h. influenzae is responsible for reducing the density of s. pneumoniae in the nasal wash and that s. pneumoniae strains may vary in their susceptibility to this innate immune response.
while we found limited evidence for immune-mediated competition, since the nasal epithelium bacterial populations of s. pneumoniae are un-altered by this innate immune response this competition may not effect the long-term carriage of s. pneumoniae in the nasal passage.
limitations
perhaps the most significant limitation and caveat associated with this study is that the neonatal rat immune system is changing during the course of these experiments, thereby restricting our ability to draw inferences about the role of the immune response and long-term colonization dynamics.
while arguably a decent model for young infants, the neonatal rats are unlikely to be an accurate model of the nasal passages of older children or adults.
another limitation of this study is that the results obtained may be strain-specific and only one or two strains for each species was tested.
the limited number of strains does not likely reflect the within species diversity in colonization strategies and this diversity should be investigated in further studies.
finally, our ability to draw inferences about the factors influencing the ecology of colonization in these neonatal rats was limited by the substantial amount of variation in densities observed in individual rats.
CONCLUSIONS
caveats and limitations aside, we believe that the application of an ecological framework to the colonization of neonatal rat model with s. aureus, s. pneumoniae and h. influenzae contributes to our understanding of the epidemiology of carriage, disease processes and the impact of vaccination on these bacteria species.
these results begin to address the mechanisms responsible for the dynamic process of nasal colonization with turnover and replacement of species, serotypes and strains in the complex community .
for example the pulse experiments results suggest that for s. pneumoniae and h. influenzae the presence  of multiple strains and serotypes would be expected in carriers as has been observed in humans  <cit> .
further, our results suggest that that h. influenzae colonization will be more successful  when preceded by either s. aureus or s. pneumoniae.
ultimately the ecology of nasal colonization informs whether vaccination  directed at one particular species will lead to the unintended consequences of increased colonization by competing  species, serotypes or strains.

 the first step in a bacterial disease is the successful establishment of a bacterial population in a host: colonization. the conditions that determine whether a bacterial population can colonize a particular site and the density achieved are fundamental to determining the likelihood of invasive disease, transmission to other hosts and the presence of mutants resistant to antibiotics. how these conditions are affected by prior colonization by bacteria of the same or different species has wide spread consequences for determining the sequelae of the wide-scale use of vaccines directed at specific strains or species as well as for evaluating probiotics and understanding epidemiological changes in invasive bacterial diseases. whether bacteria can colonize or not is determined by many ecological factors including the availability of resources, host immune responses and the presence of toxins or harmful substances. as the presence of established bacteria populations can influence all of these factors, it seems reasonable to assume that co-inhabitants often determine whether colonization can occur. in fact co-inhabitants that are ecologically similar, should limit the colonization as the one that is better at exploiting the habitat should exclude the others through resource limitation. however, as a consequence of even subtle differences in resource utilization or availability, multiple strains and species of bacteria can co-exist. the ability to colonize can also be influenced by interference, which includes residents populations producing harmful substances or inducing an immune response. in the case of three bacterial species which colonize the human nasopharynx, epidemiological studies show that co-colonization is rarer than expected. these co-inhabitation patterns suggest that there may be interference or competition occurring. in this report we apply an ecological framework to elucidate the factors contributing to the nasal colonization of neonatal rats of three bacterial species that typically colonize humans: s. pneumoniae, h. influenzae and s. aureus. first we consider the population dynamics of each strain separately. we provide evidence that all three species colonize the nasal passages of neonatal rats and reach an apparent steady-state density and that this level is independent of inoculum density. to explore the effects of co-inhabitants on colonization, hours after colonizing neonatal rats with one species we pulsed with a second inoculum of a marked strain of the same species. the results of these pulse experiments suggest that resident s. aureus prevents co-colonization of the same strain; while for both h. influenzae and s. pneumoniae the total density is increased to allow for the co-existence of pulsed and established populations. we repeated these experiments with the resident and invading populations being of different species and found that h. influenzae colonizes at a higher density when either s. aureus or s. pneumoniae are present and that immune-mediated competition between s. pneumoniae and h. influenzae is both site and strain specific.  population dynamics all three species readily colonize the nasal passages of neonatal rats. within hours after one of the three species is inoculated, h. influenzae, s. aureus and s. pneumoniae reach and maintain for at least three days a constant population in the nasal epithelium. the population dynamics of nasal colonization did not differ in the nasal wash sample with the nasal epithelium. the nasal epithelium, which represent the persisting colonizing bacteria, data are shown. the bacterial load for each of the species was not significantly different from to hours. while the dynamics for both a low and high inoculum density appear to be similar, we ascertained whether bacterial load is inoculum-independent at hours after inoculation. for all three species the bacterial load is invariant over a wide range of inocula, suggesting that nasal colonization rapidly reaches a steady-state that is not limited by how many bacteria are inoculated. invasion of same species in a colonized host to test whether nasal colonization can occur in the presence of the same species, new populations of bacteria were pulsed into rats that were already colonized by bacteria of that species. antibiotic markers that conferred no in vitro or in vivo fitness costs were used to distinguish the resident and pulsed populations and each experiment was repeated reversing the strains as pulsed or resident to control for any fitness differences. as the population dynamics suggest that the bacterial load for each of these species is tightly controlled, we expected that the total density would return to the bacterial load observed in rats without pulses. because resident and pulsed strains of the same species utilize the same resource, co-existence of both strains is expected unless a limiting factor is available only on a first come first serve basis. in the case of s. aureus, regardless of whether the marked strain is resident or pulsed, we find that the pulsed strain declines in density over the course of hours. as the pulsed strain declines the total bacterial load of s. aureus in the rats with the pulsed and established strain doesn't differ from the total density of s. aureus in the rats with only the established strain. for s. aureus the bacterial density does not exceed that observed in rats without a pulse and the resident strain has a competitive advantage. for both s. pneumoniae and h. influenzae there is an increase in the total density in the rats with the pulse compared to rats with only the established strain. we saw the bacterial load increase to varying degrees, more so for h. influenzae than for s. pneumoniae, in each of four replicate experiments. in both of these species, we observe that the pulsed and resident strains co-exist with the pulse strain becoming 25-90% of the population. for all the species, similar pulse results were obtained in reciprocal experiments confirming that the results were not due to fitness differences in the antibiotic marked strains. invasion of different species in a colonized host competition between different strains or species can be defined simply as a reduction in the density of one or both strains when both are present. competition within the same species and particularly in the case of the same strain is usually mediated through a limiting shared resource. competition between species, in addition to partitioning of a shared resource, can be mediated through inhibitory agents/toxins or predators. previous studies suggest that production of hydrogen peroxide by s. pneumoniae may affect the densities of other species and that immune-mediated competition reduces s. pneumoniae density in the presence of h. influenzae. to evaluate the contributions of these different competitive mechanisms we performed invasion experiments in which one species was resident and a second was introduced. evidence for synergistic interactions between h. influenzae and s. pneumoniae or s. aureus rather than an antagonistic interaction, we found that h. influenzae reached a higher density when invading resident populations of either s. aureus or s. pneumoniae than in the absence of these residents. a similar increase in the bacterial density of h. influenzae was observed in vitro; when mixtures of these strains were grown in broth for hours, h. influenzae density was 20% greater with s. pneumoniae and 19% greater with s. aureus present than when grown alone. strain-specific, innate immune-mediated interactions between h. influenzae and s. pneumoniae we had expected to detect immune-mediated competition between h. influenzae and s. pneumoniae, as observed in a mouse model of colonization by lysenko and colleagues. however, we saw no evidence of competition between h. influenzae and s. pneumoniae with the strains we initially used: tigr and eagan. to investigate further, we tested one additional strain of s. pneumoniae, poland- we found that this particular strain of s. pneumoniae had a reduced density in the nasal wash, but not the nasal epithelium, when invading in a neonatal rat with an established h. influenzae population. this reduction in poland-20's population did not occur in neonatal rats which had been depleted of complement or neutrophils. to explain why we could only observe this in one of the two strains tested and only then in the nasal wash, we hypothesized that either induction of or susceptibility to the immune response must differ in these strains and locations. we quantified the neutrophil infiltration in the nasal epithelium by measuring the myeloperoxidase activity at hours after inoculation with each strain/species alone or when poland- was inoculated on an established h. influenzae population. no difference in neutrophil infiltration is observed between rats colonized by the two different s. pneumoniae strains -20). the neutrophil infiltration observed hours after poland- invaded on an established h. influenzae population was significantly higher than rats with just poland- colonizing alone. however, neutrophil infiltration was not significantly higher than in rats with only h. influenzae. while these results suggest that h. influenzae is primarily responsible for the neutrophil infiltration that reduces the nasal lumen populations of some strains of s. pneumoniae, s. pneumoniae may still have a role in eliciting the immune response. we observed that the neutrophil infiltration in response to s. pneumoniae colonizing alone increases from 48- hours after inoculation, compared to the constant neutrophil presence with h. influenzae. discussion population dynamics all three species that we studied can colonize the nasal passages of neonatal rats and each reaches a bacterial load that is independent of the initial inoculum size; they increase in density when initially below this level and decline when initially above it. this indicates that the steady-state density is tightly controlled - perhaps by a limiting resource or the host's immune response. the total density of each of these colonizing species is relatively low and there is wide-spread variation in the densities of individuals, similar to what has been observed in colonized humans. invasion of same species in a colonized host in accord with classical ecological theory of competition we would anticipate that established populations of one strain of a species would either prevent colonization of another strain of that species or be eliminated. the outcome would depend on which of the strains was more fit. in the case of genetically marked strains of the same species with equal fitness we would expect to observe co-existence. in a series of pulse experiments with genetically marked colonizing and resident strains we found the situation to be more complicated than this simple interpretation. for both h. influenzae and s. pneumoniae, the resident and the pulsed strains co-existed. surprisingly in all replicates of these experiments, the invasion of a second population of the same species was followed by an increase in the total bacterial density. given that the steady-state bacterial densities were independent of the initial inoculum density following a single inoculation, we had expected that the bacterial density after a second inoculation would decline to the original bacterial load. these results might be attributable to a second inoculation leading to an expansion in the colonization area, increased immune suppression or the release of new resources - perhaps associated with an inflammatory response. on first consideration it would seem that the results of the s. aureus pulse experiments are consistent with classical ecological theory; the established population of this species inhibited the colonization of a new strain. moreover, as expected following the pulse by a second strain the total density of s. aureus returned to a level similar to that observed in the single inoculation experiments. however the resident strain had the advantage no matter which marker it carried. we interpret these results as suggesting that s. aureus is limited by a localized resource available on a'first-come, first-serve' basis - perhaps attachment sites. this ecological hypothesis would account for the observations that competing strains of s. aureus were excluded from burn wounds and from nasal colonization in persistent human carriers. while these results do not exclude the possibility that variation within s. aureus strains may allow for coexistence as occasionally has been observed in humans, they do suggest that prior nasal colonization with s. aureus can exclude similar s. aureus strains from colonizing. invasion of different species in a colonized host ecologically, strains of different species would be anticipated to be more divergent than those of the same species and therefore they would be expected to occupy different niches. the results of our experiments are consistent with this interpretation as any pairwise combination of the three species can co-exist. while we had expected some sharing of resources by these different species, we found no evidence that the presence of one species reduced the density colonizing the nasal epithelium of another species. the only evidence for inter-specific interactions between these species observed at the nasal epithelium was in fact synergistic rather than antagonistic; h. influenzae reaches a higher density when invading resident populations of either s. aureus or s. pneumoniae than it achieves in rats not colonized by these bacteria. since this result is also observed in vitro it seems likely due to some host-independent mechanism such as s. aureus and s. pneumoniae providing nutrients that would otherwise limit h. influenzae. indeed, in the past h. influenzae has been identified and cultured due to the fact that it grew as satellites off of s. aureus colonies. to our knowledge this is the first evidence for s. aureus and s. pneumoniae increasing the density of h. influenzae during nasal colonization. we had expected to see inter-specific antagonism not only due to resource sharing but also because of interference by toxins and harmful substances. in fact, it has been proposed that the production of hydrogen peroxide by s. pneumoniae may affect the densities of s. aureus and h. influenzae as both are susceptible to hydrogen peroxide killing. however in this and a previous work that specifically addressed this issue we found no evidence that hydrogen peroxide produced by s. pneumoniae limits the colonizing populations of either of the two species. this may be because the density of s. pneumoniae is too low for sufficient hydrogen peroxide production or the nasal epithelium inactivates the hydrogen peroxide produced. taken at large, we found no ecological interaction between s. aureus and s. pneumoniae colonization that would account for the epidemiological observation that s. aureus-s. pneumoniae co-colonization is rarer than expected. we postulate that this epidemiological observation may be due to the bacteria preferring different hosts rather than competitive interactions within hosts, or that competitive exclusion may only occur in immunologically mature individuals. others have suggested that there may still be an ecological interaction based on the pneumococcal pilus or by induction of phage release. neutrophil-mediated competition previous experiments by lysenko and colleagues in a mouse model have shown that when h. influenzae and s. pneumoniae co-colonize, s. pneumoniae's density in the nasal wash is lower than when inoculated alone due to immune-mediated competition. at one level, the results of our rat model experiments with h. influenzae and s. pneumoniae are consistent with their results. however, our results also suggest that this immune-mediated competitive interaction may only affect the colonizing s. pneumoniae population in the nasal wash and is strain-specific. we observed immune-mediated competition with the clinical strain of s. pneumoniae poland- but not with tigr we hypothesize that pneumococcal strains vary either in their ability to elicit an immune response or in their suscebtibility to the immune response induced by h. influenzae. although we can't exclude the possibility that the two strains we tested elicited different immune responses, our results suggest that there is no difference in the extent of neutrophil infiltration of the epithelium in response to colonization by either of these strains or any synergism between the two species. together our results suggest that the immune response primarily elicited by h. influenzae is responsible for reducing the density of s. pneumoniae in the nasal wash and that s. pneumoniae strains may vary in their susceptibility to this innate immune response. while we found limited evidence for immune-mediated competition, since the nasal epithelium bacterial populations of s. pneumoniae are un-altered by this innate immune response this competition may not effect the long-term carriage of s. pneumoniae in the nasal passage. limitations perhaps the most significant limitation and caveat associated with this study is that the neonatal rat immune system is changing during the course of these experiments, thereby restricting our ability to draw inferences about the role of the immune response and long-term colonization dynamics. while arguably a decent model for young infants, the neonatal rats are unlikely to be an accurate model of the nasal passages of older children or adults. another limitation of this study is that the results obtained may be strain-specific and only one or two strains for each species was tested. the limited number of strains does not likely reflect the within species diversity in colonization strategies and this diversity should be investigated in further studies. finally, our ability to draw inferences about the factors influencing the ecology of colonization in these neonatal rats was limited by the substantial amount of variation in densities observed in individual rats.  caveats and limitations aside, we believe that the application of an ecological framework to the colonization of neonatal rat model with s. aureus, s. pneumoniae and h. influenzae contributes to our understanding of the epidemiology of carriage, disease processes and the impact of vaccination on these bacteria species. these results begin to address the mechanisms responsible for the dynamic process of nasal colonization with turnover and replacement of species, serotypes and strains in the complex community. for example the pulse experiments results suggest that for s. pneumoniae and h. influenzae the presence of multiple strains and serotypes would be expected in carriers as has been observed in humans. further, our results suggest that that h. influenzae colonization will be more successful when preceded by either s. aureus or s. pneumoniae. ultimately the ecology of nasal colonization informs whether vaccination directed at one particular species will lead to the unintended consequences of increased colonization by competing species, serotypes or strains. 
the first step in invasive disease caused by the normally commensal bacteria streptococcus pneumoniae, staphylococcus aureus and haemophilus influenzae is their colonization of the nasal passages. when neonatal rats are colonized by any one of these species the density of bacteria in the nasal passage rapidly reaches a steady-state density that is species-specific but independent of inoculum size. when novel populations of h. influenzae and s. pneumoniae are introduced into the nasal passages of neonatal rats with established populations of the same species, residents and invaders coexisted. in the case of colonization of these species the competition is between strains of the same and different species of bacteria and the predation is mediated by the host's immune response. in mixed-species introductions, s. aureus or s. pneumoniae facilitated the invasion of another h. influenzae population; for other pairs the interaction was antagonistic and immune-mediated. for example, under some conditions h. influenzae promoted an immune response which limited the invasion of s. pneumoniae. these results suggest that multiple strains of either h. influenzae or s. pneumoniae can coexist; in contrast, s. aureus strains require a host to have no other s. aureus present to colonize. levels of colonization by h. influenzae are increased in hosts pre-colonized with either s. aureus or s. pneumoniae. nasal colonization is a dynamic process with turnover of new strains and new species. however, this was not the case for s. aureus - the established population inhibited invasion of new s. aureus populations. here, we use a neonatal rat model to explore these elements of the ecology of nasal colonization by these occasionally invasive bacteria.
 the first step in invasive disease caused by the normally commensal bacteria streptococcus pneumoniae, staphylococcus aureus and haemophilus influenzae is their colonization of the nasal passages. for any population to colonize a new habitat it is necessary for it to be able to compete with the existing organisms and evade predation. in the case of colonization of these species the competition is between strains of the same and different species of bacteria and the predation is mediated by the host's immune response. here, we use a neonatal rat model to explore these elements of the ecology of nasal colonization by these occasionally invasive bacteria.  when neonatal rats are colonized by any one of these species the density of bacteria in the nasal passage rapidly reaches a steady-state density that is species-specific but independent of inoculum size. when novel populations of h. influenzae and s. pneumoniae are introduced into the nasal passages of neonatal rats with established populations of the same species, residents and invaders coexisted. however, this was not the case for s. aureus - the established population inhibited invasion of new s. aureus populations. in mixed-species introductions, s. aureus or s. pneumoniae facilitated the invasion of another h. influenzae population; for other pairs the interaction was antagonistic and immune-mediated. for example, under some conditions h. influenzae promoted an immune response which limited the invasion of s. pneumoniae.  nasal colonization is a dynamic process with turnover of new strains and new species. these results suggest that multiple strains of either h. influenzae or s. pneumoniae can coexist; in contrast, s. aureus strains require a host to have no other s. aureus present to colonize. levels of colonization by h. influenzae are increased in hosts pre-colonized with either s. aureus or s. pneumoniae. 