BACKGROUND
infection with non-typhoidal salmonella enterica is a major cause of food-borne disease in humans worldwide  <cit> .
animals and their products, particularly poultry and chicken eggs, are regarded as the main sources of this pathogen, although others, such as fresh vegetables, are also important  <cit> .
a peculiar epidemiological feature of salmonellosis is that major outbreaks and epidemics are commonly associated with a dominant serovar of s. enterica and the particular serovar involved shows temporal and geographical variation.
until the 1980s s. enterica serovar typhimurium  was the most common serovar isolated from humans worldwide.
however, in the late 1980s s. enteritidis emerged as the most common cause of human salmonellosis in europe and during the 1990s it became the most prevalent serovar in many countries worldwide  <cit> .
in uruguay, until  <dig> s. typhimurium was the most frequently isolated serovar and s. enteritidis was only isolated sporadically  <cit> .
the first significant recorded outbreak of s. enteritidis infection occurred in  <dig> and from  <dig> onwards it became the most prevalent serovar.
after  <dig> the number of isolates started to decline markedly, suggesting a post-epidemic period.
the reasons for this worldwide serovar shift are still not understood, and several hypotheses have been proposed, including the existence of a rodent reservoir for s. enteritidis, or the epidemiological change induced by vaccination of poultry against the closely related s. enterica serovar gallinarum  <cit> .
s. enteritidis is highly clonal  <cit>  so it has been difficult to discriminate genetic types by methods like multilocus sequence typing , pulsed field gel electrophoresis , random amplified polymorphism dna-pcr  or ribotyping.
dna microarray-based comparative genomic hybridization  has been used to explore genetic diversity and to search for genes involved in virulence, transmission and host specificity in several different microbial pathogens  <cit>  as well as in different serovars of s. enterica  <cit> .
in this study we have genotyped  <dig> isolates of s. enteritidis and defined a set of  <dig> isolates from before, during and after the epidemic period in uruguay, covering different sources of isolation and representing the different profiles obtained by genotyping.
to look for differences in pathogenic potential, these  <dig> isolates were assayed for their ability to invade caco- <dig> epithelial cells.
to correlate any differences in pathogenic potential with genomic variation we exploited a pan-salmonella microarray for cgh.
six other s. enteritidis isolated from distant parts of the world were included in the cgh analysis to compare the diversity seen in uruguay with that found elsewhere.
RESULTS
genotyping assays
all  <dig> s. enteritidis isolates  were subjected to rapd-pcr analysis using  <dig> different primers and were compared to s. enteritidis phage type  <dig>  strain p <dig>  the complete sequence of s. enteritidis pt <dig> p <dig> has been determined and it acts as the reference for all the analyses reported here  <cit> .
*includes eggs and other products used for human consumption.
of the s. enteritidis isolates tested in this study 96% showed the same amplification pattern as s. enteritidis pt <dig> p <dig> with all primers using rapd-pcr.
only  <dig> isolates  showed differences in the amplification pattern obtained with at least  <dig> primer.
thirty-seven isolates from different origins, periods and rapd types, were subjected to pfge after cleavage of their dna with xbai.
of these,  <dig> generated a restriction pattern identical to s. enteritidis pt <dig> p <dig>  whereas  <dig> showed subtle differences .
when both typing methods were considered together,  <dig> out of the  <dig> isolates were indistinguishable from s. enteritidis pt <dig> p <dig>  while  <dig> differed by both methods and  <dig> differed by a single typing method.
the  <dig> isolates differing by both methods included the  <dig> oldest pre-epidemic isolates ,  <dig> isolated from food  and  <dig> isolated from human blood .
overall these results revealed a high degree of genetic uniformity within s. enteritidis circulating in uruguay, with the great majority of isolates belonging to the same genetic profile as s. enteritidis pt <dig> p <dig>
next,  <dig> isolates were selected with the aim of maximizing the chances of finding divergence among the isolates.
for this, we selected isolates that span the pre-epidemic, epidemic and post-epidemic periods in uruguay and that cover any particular profile found in the rapd and/or pfge assays, and all possible sources of isolation .
the selected isolates were subjected to further phenotypic and genotypic characterization.
a- the number of primers which showed differences in rapd -pcr profiles as compared to s. enteritidis pt <dig> p <dig> is indicated.
b- expressed as percentage of identity as compared to s. enteritidis pt <dig> p125109: 96% of identity corresponds to  <dig> band of difference, 92% to  <dig> bands and 91% to  <dig> bands of difference.
c- p: number of chromosomal genes predicted as present in test isolate but absent in reference s. enteritidis pt <dig> p <dig>  a: number of chromosomal genes predicted as absent in test isolate but present in s. enteritidis pt <dig> p <dig>  numbers between brackets indicate genes from the s. enteritidis pt <dig> p <dig> virulence plasmid lost or divergent in the tested isolate .
d- results from kado and liu analysis  <cit> .
+: salmonella virulence plasmid present, -: absent.
e- results from caco- <dig> invasion assays.
-: invasiveness ≤ 30% of pt <dig>  +: > 30% of pt <dig>  all isolates marked with - showed significant differences compared to pt <dig> .
in isolate designation column, an * indicates strains previously reported  <cit> .
caco- <dig> invasion assays
in order to gain information about the pathogenic potential of s. enteritidis, the  <dig> isolates plus the s. enteritidis pt <dig> p <dig> used as reference, were assayed for their ability to invade caco- <dig> human epithelial cells .
contrary to the homogeneity observed when using the typing techniques, marked differences were observed between isolates in the cell invasion assays.
nine were impaired in their ability to invade .
these include the  <dig> oldest pre-epidemic isolates 31/ <dig> and 8/ <dig>   <dig> of  <dig> from human systemic disease, , and  <dig> from food .
one particular isolate  defective in invasiveness was also impaired for growth in lb broth .
of note,  <dig> out of these  <dig> isolates were distinct from s. enteritidis pt <dig> p <dig> when evaluated by rapd or pfge assays .
all other isolates tested were similar to s. enteritidis pt <dig> p <dig> in this invasion assay.
considering all human isolates,  <dig> out of  <dig> obtained from gastroenteritis but only  <dig> out of  <dig> from invasive disease were as invasive as s. enteritidis pt <dig> p <dig> .
overall, these results suggest that impaired invasiveness is less frequent among isolates that cause human gastroenteritis, an assumption that merit future studies with a larger panel of in vitro and in vivo phenotypical assays.
comparative genomics of s. enteritidis
these results suggest the existence of genetic determinants for the phenotypic differences that were not highlighted by the genotyping methods used.
consequently, we conducted a cgh study on the same  <dig> s. enteritidis isolates from uruguay used for the caco- <dig> invasion assays.
we also included in the cgh analysis  <dig> s. enteritidis isolates from kenya, and  <dig> isolates from the uk as external comparators.
the analysis was conducted using a pan-salmonella microarray based on the s. typhi ct <dig> genome, complemented with strain-specific genes from s. enteritidis pt <dig> p <dig>  s. typhimurium sl <dig> and dt <dig>  s. gallinarum, s. typhi ty <dig> and s. bongori .
genes specific for some of these strains were not included in previously reported s. enteritidis cgh analysis.
of  <dig> features on the microarray,  <dig> correspond to genes present in s. enteritidis pt <dig> p <dig>  and  <dig> to genes absent in s. enteritidis pt <dig> p <dig> but present in other salmonellae.
overall, the analysis produced results that extend those previously reported by others using different sets of isolates  <cit> , and confirm that there is considerable genetic homogeneity in s. enteritidis, despite geographical, temporal and source differences between the different isolates.
however, we also found a number of genomic regions and single genes that have not been described as variable among s. enteritidis field isolates.
of the  <dig> chromosomal genes from s. enteritidis pt <dig> p <dig> represented on the microarray ,  <dig> were shared by all s. enteritidis isolates tested here and are considered to be the core genome of s. enteritidis.
among these genes, only  <dig> were specific to s. enteritidis, i.e.
absent in all other sequenced salmonella strains, and they are all included in the recently annotated phage se <dig>  <cit> .
interestingly, this region was previously postulated as a region of difference between s. enteritidis and other serovars  <cit> , although more recently it was reported as absent in two s. enteritidis isolates corresponding to pt6b and pt <dig> .
considering genes that were variably present between the isolates tested,  <dig> genes known to be present on the chromosome of s. enteritidis pt <dig> p <dig> were absent, or divergent, in at least one of the s. enteritidis isolates tested .
conversely  <dig> genes were present in at least one isolate but absent from s. enteritidis pt <dig> p <dig> .
these results are summarized in figure  <dig>  these  <dig> genes together with other  <dig> genes previously described as variable present in s. enteritidis  <cit>  can be considered so far the s. enteritidis dispensable genome .
of note,  <dig> of the  <dig> regions of variability  are reported for the first time as being variable among s. enteritidis strains.
variation in plasmid genes is not included in this figure and has been treated separately .
a indicates when the reg or sing has homologous region described in other sequenced salmonella serovars .
# indicates that the genetic region was previously described as variable among s. enteritidis isolates  <cit> .
a indicates when the reg or sing has homologous region described in other sequenced salmonella serovars .
# indicates that the genetic region was previously described as variable among s. enteritidis isolates  <cit> .
detailed analysis of the genes within the dg showed that prophage-like elements constitute the major source of genetic variation distinguishing these s. enteritidis isolates.
however, this analysis also revealed some interesting differences in metabolic potential and in genes associated with restriction-modification systems .
s. enteritidis variable prophage-like regions within the dg
of the annotated prophages from s. enteritidis pt <dig> p <dig> represented on the array one kenyan and  <dig> uruguayan isolates lacked ϕse <dig> , a ~ <dig> kb phage similar to ϕst64b.
phage se <dig> is thought to be intact and a recent acquisition in s. enteritidis pt <dig> p <dig> and like ϕst64b, it carries fragments of the sope and orga genes, which have been implicated in salmonella virulence  <cit> .
two of the  <dig> uruguayan isolates that lack ϕse <dig> were isolated from human infections more than  <dig> years before the beginning of the epidemic in uruguay , whereas the other  <dig> were from food samples, one from before  and the other from the middle  of the epidemic.
similarly, porwollik and collaborators have reported that this phage  is absent in strains of s. enteritidis isolated more than  <dig> years ago and suggested that acquisition of this phage may be related to the emergence of s. enteritidis as being epidemic worldwide  <cit> .
we corroborated the presence of ϕse <dig> among the  <dig> uruguayan isolates by pcr using two set of ϕse20-specific primers that amplify fragments of sb <dig> and sb <dig> .
only isolates 31/ <dig>  8/ <dig>  53/ <dig> and 206/ <dig> were negative validating the microarray results.
we extended the pcr screening with sb <dig> primers to another  <dig> s. enteritidis isolates from the original sample set, which included  <dig> isolates from human gastroenteritis,  <dig> isolates from invasive human disease and  <dig> isolates from non-human origin .
among them we found only  <dig> other isolates that lack sb <dig>  i.e.
50/ <dig> and 211/ <dig> originating from food, 107/ <dig> from enteric disease and 209/ <dig> from invasive infection.
in summary, we found that only  <dig> out of  <dig> isolates tested from the epidemic and post-epidemic periods lack ϕse <dig>  whereas  <dig> out of  <dig> pre-epidemic isolates lack this phage.
this provides further support for the idea that the presence of ϕse <dig> is a marker for the emergence of particular isolates as epidemic strains  <cit> .
it has been proposed that s. enteritidis might be divided into two lineages based on the presence or absence of four phages, i.e.
ϕse <dig>  fels <dig> and s. typhi ct <dig> st <dig> and st <dig> phages  <cit> .
one lineage, the pt <dig> lineage, was defined as positive for ϕse <dig> and negative for fels <dig>  st <dig> and st <dig>  whereas a second lineage, the pt8-pt <dig> lineage, was defined as negative for ϕse <dig> but positive for fels <dig>  st <dig> and st <dig>  our results however, show that all uruguayan isolates tested belong to the pt <dig> lineage as defined by guard-petter  <cit> , and are negative for fels <dig>  st <dig> and st <dig> phage regions regardless of the presence or absence of ϕse <dig>  thus they do not strictly fall within the two separates groups as previously proposed  <cit> .
several prophage-related genes present on the microarray from other non-s. enteritidis serovars were found in some of the isolates.
many of them are grouped here as regions  <dig> to  <dig> .
regions  <dig> and  <dig> were only found in the kenyan s. enteritidis af <dig> isolate.
region  <dig> encodes  <dig>  genes corresponding to sequences of the s. typhi ct <dig> p2-family prophage st <dig>  <cit> .
region  <dig> harbours  <dig> genes from another p2-family prophage, ϕsope, also found in s. typhimurium and s. typhi that encodes the type iii secretion system effector protein sope important for invasion of enterocytes  <cit> .
in s. enteritidis, sope is encoded in an unrelated lambdoid phage se <dig>  <cit> , which is present in all s. enteritidis isolates tested here.
we found that the two oldest uruguayan pre-epidemic isolates  harbour  <dig> genes  that correspond to phage genes carried by s. typhimurium dt <dig> or s. typhimurium sl <dig>  or genes from ϕgifsy- <dig> of s. typhimurium lt <dig>  interestingly, regions  <dig> and 12a-b were not previously found in s. enteritidis, although this may be due to the fact that previously reported s. enteritidis cgh analysis used microarrays that lacked these regions.
both pre-epidemic isolates also carry gogb.
gogb is a ϕgifsy-1-encoded type iii secreted substrate of both spi- <dig> and spi- <dig> ttss in s. typhimurium lt <dig>  <cit> .
it has been reported that some salmonellae have gifsy- <dig> but not gogb whereas others do not have gifsy- <dig> but do have gogb, suggesting that this gene has been recently acquired by gifsy- <dig>  <cit> .
to the best of our knowledge, this is the first report of s. enteritidis harbouring this gene.
thus, we designed a pair of primers that amplifies a  <dig> bp fragment of gogb, and used them to screen for its presence among the  <dig> strains also assayed for ϕse <dig>  no other isolate was positive for gogb.
we then sequenced the pcr fragment from both pre-epidemic strains and found that the sequence has 99% of identity with s. typhimurium lt <dig> gogb.
in summary,  <dig> out of the  <dig> variable genomic regions found among s. enteritidis isolates correspond to phage-like regions, suggesting that, as in other serovars of salmonella, phages play a crucial role in the generation of genetic diversity in s. enteritidis  <cit> .
variations in the content of genes involved in metabolism
our cgh data highlighted other regions of variation.
region  <dig>  harbouring  <dig> out of  <dig> genes of the eut operon, is absent in  <dig> pre-epidemic  and  <dig> non-human epidemic  s. enteritidis isolates.
these genes encode alcohol dehydrogenase, aldehyde dehydrogenase and enzymes required for ethanolamine utilization .
s. enteritidis 32/ <dig> also lacks the pdus gene, a ferredoxin involved in propanediol utilization .
in salmonella both  <dig>  2-propanediol degradation and ethanolamine degradation require vitamin b <dig>  many enterobacteriaceae have lost the capacity to synthesize cobalamine and therefore to degrade  <dig>  2-propanediol and ethanolamine but a few genera, including salmonella and yersinia, re-acquired a  <dig> kb metabolic island encoding both the ability to synthesise cobalamine and degrade  <dig>  2-propanediol, whilst retaining the eut operon  <cit> .
although  <dig>  2-propanediol is an important source of energy for s. typhimurium and cbi mutants are significantly attenuated in their ability to grow in macrophages  <cit>  it is apparent that genes within these pathways are lost in the host-adapted s. enterica serovars including gallinarum, typhi and paratyphi a  <cit> .
region  <dig>  comprises three genes  which are absent/divergent in s. enteritidis 47/ <dig> isolated from human disease.
rpos is inducible in stationary phase, is the master regulator of the general stress response in salmonella and is required for virulence in mice  <cit> .
there are previous reports of s. typhi, s. typhimurium and s. enteritidis clinical and environmental isolates carrying mutations in rpos that result in impaired rpos functionality  <cit> .
a test of catalase activity in stationary phase is used as a method to detect rpos function  <cit> , thus we performed the test in all  <dig> isolates and found a negative result only in s. enteritidis isolate 47/ <dig>  this strongly suggest that rpos function is impaired in this isolate.
region  <dig> harbouring genes encoding nitrate reductases, cytochrome c and ferredoxin-type proteins , was also absent in  <dig> s. enteritidis  isolates from different periods of the uruguayan epidemic.
variation in s. enteritidis genomic islands
although there is a large number of genomic islands in s. enteritidis pt <dig> p <dig>  <cit>  which carry the hallmarks of having been laterally acquired, and maintain mobility functions, surprisingly our data show that most are ubiquitous in the s. enteritidis isolates tested here.
the exceptions are region  <dig>  and region  <dig>  region  <dig> is one of the largest genomic islands identified in s. enteritidis pt <dig> p <dig> , and it encodes the global transcriptional silencers h-ns  and the h-ns antagonist   <cit> .
this region was undetected using the microarray in the kenyan s. enteritidis isolate af <dig> but it is present in all other strains.
region  <dig> corresponds to the immigration control region icr in s. enteritidis pt <dig> p <dig>  <cit>  which encodes two type i restriction/modification systems.
all of these genes were not detected in the kenyan s. enteritidis isolate af <dig> and partially detected in isolate 47/ <dig>  which lacks one of the restriction enzyme subunits.
in addition to variation in genes found in large clusters in s. enteritidis pt <dig> p <dig> there was also variation in genes found as singletons .
of note is the absence of the gene ratb in s. enteritidis isolate 32/ <dig>  this gene is located within the cs <dig> genomic island in s. typhimurium, a region that is important for intestinal persistence in a mouse model  <cit> .
in s. enteritidis pt <dig> p <dig>  the genomic island is maintained but ratb is a pseudogene, as it is in the sequenced strains of the host-adapted serovars s. typhi and s. gallinarum.
variation in plasmid-encoded genes
besides chromosomal genes, the microarray incorporated genes found on salmonella virulence plasmids from serovars enteritidis, gallinarum, typhimurium and plasmids, phcm <dig> and phcm <dig>  from the multi-drug resistant s. typhi strain ct <dig>  five uruguayan isolates,  <dig> from food  and  <dig> from human disease , lack the characteristic s. enteritidis virulence plasmid.
this was confirmed by attempts to purify the plasmid .
two other uruguayan isolates, 92/ <dig> and 132/ <dig>  exhibited divergence in more than  <dig> genes and isolates 57/ <dig> and 49/ <dig> diverged in  <dig> genes found within the plasmid of s. enteritidis pt <dig> p <dig> .
included in the genes predicted as absent or divergent are the spv genes, the pef fimbrial operon as well as repa  and rsdb .
of note, isolates 92/ <dig> and 132/ <dig> also lack the few tra genes remaining in s. enteritidis pt <dig> p <dig>
despite the high degree of variability seen in these plasmids all had similar molecular weights when compared to that in s. enteritidis pt <dig> p <dig> , suggesting potential divergence in gene sequence or acquisition of novel genes.
however none of the isolates with high variation in plasmid gene content showed a positive signal for non-s. enteritidis plasmid features included in the array, suggesting that they may harbour sequence divergence or novel sequences.
in fact the only isolate showing a positive signal for non-s. enteritidis plasmid features was the kenyan s. enteritidis isolate af <dig> which harbours the complete s. enteritidis pt <dig> p <dig> virulence plasmid and shows a positive signal for  <dig> genes from the multidrug resistance plasmid, phcm <dig>  from s. typhi ct <dig>  these include genes encoding β-lactamase and streptomycin resistance.
although we cannot confirm that these are located on the plasmid there are increasing numbers of reports of drug resistance genes integrating into the virulence plasmid  <cit> .
CONCLUSIONS
the results presented here corroborate and extend previous reports demonstrating a high degree of genetic homogeneity among field isolates of s. enteritidis, irrespective of geographical, temporal and source differences.
most of the strains analysed produced highly similar profiles by rapd and pfge analysis, and those selected for further analysis showed almost indistinguishable gene content by microarray-based cgh.
the two oldest uruguayan pre-epidemic s. enteritidis isolates and a kenyan isolate  were among the most divergent.
most of the genome variation was related to prophage regions underscoring their importance as drivers for s. enteritidis evolution.
in particular half of the isolates from before the beginning of the s. enteritidis epidemic in uruguay lack ϕse <dig>  whereas absence of this phage is minimal  among s. enteritidis isolated during and after the epidemics, as detected by cgh and extended by pcr screening.
these results, together with those previously reported  <cit>  strongly suggest that this phage may have been relatively recently acquired by s. enteritidis, and that this might be related to the capacity of pt4-like strains to become prevalent.
although we are aware that the small number of pre-epidemic isolates is a limitation of this study, it is noteworthy that these are all the s. enteritidis isolates received at the national salmonella centre since the beginning of the 1970s until the end of  <dig>
the two oldest pre-epidemic isolates also carry genetic regions that were not found in s. enteritidis strains previously evaluated by cgh  <cit> , but this may be due to the fact that more genes from other serovars of salmonella are present on our microarray compared with those previously reported.
beside these, we have confirmed that  <dig> uruguayan isolates harbour gogb, a gene that has not been previously found among s. enteritidis strains.
in addition to identifying differences in the content of mobile genetic elements we were successful in identifying metabolic pathways which appear to be incomplete in some isolates.
these include those associated with the utilization of propanediol and ethanolamine as well as many genes that have previously been implicated in bacterial fitness and virulence .
we also showed that a significant number of the uruguayan s. enteritidis strains lack the salmonella virulence plasmid and others showed variation in plasmid gene content.
there was great heterogeneity in the ability of the isolates to invade caco- <dig> human epithelial cells, but our genotyping approach was insufficient to elucidate the genetic bases for these differences.
these finding are in agreement with previous reports that showed that genetically closely related s. enteritidis strains nevertheless presented important metabolic differences, and that these differences were related to the accumulation of single nucleotide polymorphism rather than with differences in gene content  <cit> .
of note, none of the genes predicted as variant among s. enteritidis in our work correspond to those described as involved in the ability to survive in the avian reproductive tract  <cit>  or in persistence in egg albumen  <cit> .
furthermore, the genetic regions related to metabolic functions found as variable in our cgh analysis do not correspond to utilization of the compounds described by morales et al.
in their comparative phenotypic analysis of s. enteritidis strains  <cit> .
a report has recently been published showing differences in genetic content among s. enteritidis isolates from prevalent phage types and the non-prevalent phage type  <dig>  <cit> .
with the exception of the plasmid-encoded genes, all other genes reported as exclusively present in the prevalent phage types, are also present in all the isolates analyzed here.
overall, our study shows that the epidemic of s. enteritidis in uruguay between  <dig> and  <dig> was caused by highly related s. enteritidis isolates, perhaps comprising a pt4-like clonal population with few whole gene differences.
to understand more clearly the link between genotype and phenotype and to differentiate between neutral variation within a population and variations associated directly with defined phenotypes, the whole genome sequences of a large number of isolates are required for association studies.
this is our future direction.

 infection with non-typhoidal salmonella enterica is a major cause of food-borne disease in humans worldwide. animals and their products, particularly poultry and chicken eggs, are regarded as the main sources of this pathogen, although others, such as fresh vegetables, are also important. a peculiar epidemiological feature of salmonellosis is that major outbreaks and epidemics are commonly associated with a dominant serovar of s. enterica and the particular serovar involved shows temporal and geographical variation. until the 1980s s. enterica serovar typhimurium was the most common serovar isolated from humans worldwide. however, in the late 1980s s. enteritidis emerged as the most common cause of human salmonellosis in europe and during the 1990s it became the most prevalent serovar in many countries worldwide. in uruguay, until s. typhimurium was the most frequently isolated serovar and s. enteritidis was only isolated sporadically. the first significant recorded outbreak of s. enteritidis infection occurred in and from onwards it became the most prevalent serovar. after the number of isolates started to decline markedly, suggesting a post-epidemic period. the reasons for this worldwide serovar shift are still not understood, and several hypotheses have been proposed, including the existence of a rodent reservoir for s. enteritidis, or the epidemiological change induced by vaccination of poultry against the closely related s. enterica serovar gallinarum. s. enteritidis is highly clonal so it has been difficult to discriminate genetic types by methods like multilocus sequence typing, pulsed field gel electrophoresis, random amplified polymorphism dna-pcr or ribotyping. dna microarray-based comparative genomic hybridization has been used to explore genetic diversity and to search for genes involved in virulence, transmission and host specificity in several different microbial pathogens as well as in different serovars of s. enterica. in this study we have genotyped isolates of s. enteritidis and defined a set of isolates from before, during and after the epidemic period in uruguay, covering different sources of isolation and representing the different profiles obtained by genotyping. to look for differences in pathogenic potential, these isolates were assayed for their ability to invade caco- epithelial cells. to correlate any differences in pathogenic potential with genomic variation we exploited a pan-salmonella microarray for cgh. six other s. enteritidis isolated from distant parts of the world were included in the cgh analysis to compare the diversity seen in uruguay with that found elsewhere.  genotyping assays all s. enteritidis isolates were subjected to rapd-pcr analysis using different primers and were compared to s. enteritidis phage type strain p the complete sequence of s. enteritidis pt p has been determined and it acts as the reference for all the analyses reported here. *includes eggs and other products used for human consumption. of the s. enteritidis isolates tested in this study 96% showed the same amplification pattern as s. enteritidis pt p with all primers using rapd-pcr. only isolates showed differences in the amplification pattern obtained with at least primer. thirty-seven isolates from different origins, periods and rapd types, were subjected to pfge after cleavage of their dna with xbai. of these, generated a restriction pattern identical to s. enteritidis pt p whereas showed subtle differences. when both typing methods were considered together, out of the isolates were indistinguishable from s. enteritidis pt p while differed by both methods and differed by a single typing method. the isolates differing by both methods included the oldest pre-epidemic isolates, isolated from food and isolated from human blood. overall these results revealed a high degree of genetic uniformity within s. enteritidis circulating in uruguay, with the great majority of isolates belonging to the same genetic profile as s. enteritidis pt p next, isolates were selected with the aim of maximizing the chances of finding divergence among the isolates. for this, we selected isolates that span the pre-epidemic, epidemic and post-epidemic periods in uruguay and that cover any particular profile found in the rapd and/or pfge assays, and all possible sources of isolation. the selected isolates were subjected to further phenotypic and genotypic characterization. a- the number of primers which showed differences in rapd -pcr profiles as compared to s. enteritidis pt p is indicated. b- expressed as percentage of identity as compared to s. enteritidis pt p125109: 96% of identity corresponds to band of difference, 92% to bands and 91% to bands of difference. c- p: number of chromosomal genes predicted as present in test isolate but absent in reference s. enteritidis pt p a: number of chromosomal genes predicted as absent in test isolate but present in s. enteritidis pt p numbers between brackets indicate genes from the s. enteritidis pt p virulence plasmid lost or divergent in the tested isolate. d- results from kado and liu analysis. +: salmonella virulence plasmid present, -: absent. e- results from caco- invasion assays. -: invasiveness ≤ 30% of pt +: > 30% of pt all isolates marked with - showed significant differences compared to pt. in isolate designation column, an * indicates strains previously reported. caco- invasion assays in order to gain information about the pathogenic potential of s. enteritidis, the isolates plus the s. enteritidis pt p used as reference, were assayed for their ability to invade caco- human epithelial cells. contrary to the homogeneity observed when using the typing techniques, marked differences were observed between isolates in the cell invasion assays. nine were impaired in their ability to invade. these include the oldest pre-epidemic isolates 31/ and 8/ of from human systemic disease,, and from food. one particular isolate defective in invasiveness was also impaired for growth in lb broth. of note, out of these isolates were distinct from s. enteritidis pt p when evaluated by rapd or pfge assays. all other isolates tested were similar to s. enteritidis pt p in this invasion assay. considering all human isolates, out of obtained from gastroenteritis but only out of from invasive disease were as invasive as s. enteritidis pt p. overall, these results suggest that impaired invasiveness is less frequent among isolates that cause human gastroenteritis, an assumption that merit future studies with a larger panel of in vitro and in vivo phenotypical assays. comparative genomics of s. enteritidis these results suggest the existence of genetic determinants for the phenotypic differences that were not highlighted by the genotyping methods used. consequently, we conducted a cgh study on the same s. enteritidis isolates from uruguay used for the caco- invasion assays. we also included in the cgh analysis s. enteritidis isolates from kenya, and isolates from the uk as external comparators. the analysis was conducted using a pan-salmonella microarray based on the s. typhi ct genome, complemented with strain-specific genes from s. enteritidis pt p s. typhimurium sl and dt s. gallinarum, s. typhi ty and s. bongori. genes specific for some of these strains were not included in previously reported s. enteritidis cgh analysis. of features on the microarray, correspond to genes present in s. enteritidis pt p and to genes absent in s. enteritidis pt p but present in other salmonellae. overall, the analysis produced results that extend those previously reported by others using different sets of isolates, and confirm that there is considerable genetic homogeneity in s. enteritidis, despite geographical, temporal and source differences between the different isolates. however, we also found a number of genomic regions and single genes that have not been described as variable among s. enteritidis field isolates. of the chromosomal genes from s. enteritidis pt p represented on the microarray, were shared by all s. enteritidis isolates tested here and are considered to be the core genome of s. enteritidis. among these genes, only were specific to s. enteritidis, i.e. absent in all other sequenced salmonella strains, and they are all included in the recently annotated phage se. interestingly, this region was previously postulated as a region of difference between s. enteritidis and other serovars, although more recently it was reported as absent in two s. enteritidis isolates corresponding to pt6b and pt. considering genes that were variably present between the isolates tested, genes known to be present on the chromosome of s. enteritidis pt p were absent, or divergent, in at least one of the s. enteritidis isolates tested. conversely genes were present in at least one isolate but absent from s. enteritidis pt p. these results are summarized in figure these genes together with other genes previously described as variable present in s. enteritidis can be considered so far the s. enteritidis dispensable genome. of note, of the regions of variability are reported for the first time as being variable among s. enteritidis strains. variation in plasmid genes is not included in this figure and has been treated separately. a indicates when the reg or sing has homologous region described in other sequenced salmonella serovars. # indicates that the genetic region was previously described as variable among s. enteritidis isolates. a indicates when the reg or sing has homologous region described in other sequenced salmonella serovars. # indicates that the genetic region was previously described as variable among s. enteritidis isolates. detailed analysis of the genes within the dg showed that prophage-like elements constitute the major source of genetic variation distinguishing these s. enteritidis isolates. however, this analysis also revealed some interesting differences in metabolic potential and in genes associated with restriction-modification systems. s. enteritidis variable prophage-like regions within the dg of the annotated prophages from s. enteritidis pt p represented on the array one kenyan and uruguayan isolates lacked ϕse, a ~ kb phage similar to ϕst64b. phage se is thought to be intact and a recent acquisition in s. enteritidis pt p and like ϕst64b, it carries fragments of the sope and orga genes, which have been implicated in salmonella virulence. two of the uruguayan isolates that lack ϕse were isolated from human infections more than years before the beginning of the epidemic in uruguay, whereas the other were from food samples, one from before and the other from the middle of the epidemic. similarly, porwollik and collaborators have reported that this phage is absent in strains of s. enteritidis isolated more than years ago and suggested that acquisition of this phage may be related to the emergence of s. enteritidis as being epidemic worldwide. we corroborated the presence of ϕse among the uruguayan isolates by pcr using two set of ϕse20-specific primers that amplify fragments of sb and sb. only isolates 31/ 8/ 53/ and 206/ were negative validating the microarray results. we extended the pcr screening with sb primers to another s. enteritidis isolates from the original sample set, which included isolates from human gastroenteritis, isolates from invasive human disease and isolates from non-human origin. among them we found only other isolates that lack sb i.e. 50/ and 211/ originating from food, 107/ from enteric disease and 209/ from invasive infection. in summary, we found that only out of isolates tested from the epidemic and post-epidemic periods lack ϕse whereas out of pre-epidemic isolates lack this phage. this provides further support for the idea that the presence of ϕse is a marker for the emergence of particular isolates as epidemic strains. it has been proposed that s. enteritidis might be divided into two lineages based on the presence or absence of four phages, i.e. ϕse fels and s. typhi ct st and st phages. one lineage, the pt lineage, was defined as positive for ϕse and negative for fels st and st whereas a second lineage, the pt8-pt lineage, was defined as negative for ϕse but positive for fels st and st our results however, show that all uruguayan isolates tested belong to the pt lineage as defined by guard-petter, and are negative for fels st and st phage regions regardless of the presence or absence of ϕse thus they do not strictly fall within the two separates groups as previously proposed. several prophage-related genes present on the microarray from other non-s. enteritidis serovars were found in some of the isolates. many of them are grouped here as regions to. regions and were only found in the kenyan s. enteritidis af isolate. region encodes genes corresponding to sequences of the s. typhi ct p2-family prophage st. region harbours genes from another p2-family prophage, ϕsope, also found in s. typhimurium and s. typhi that encodes the type iii secretion system effector protein sope important for invasion of enterocytes. in s. enteritidis, sope is encoded in an unrelated lambdoid phage se, which is present in all s. enteritidis isolates tested here. we found that the two oldest uruguayan pre-epidemic isolates harbour genes that correspond to phage genes carried by s. typhimurium dt or s. typhimurium sl or genes from ϕgifsy- of s. typhimurium lt interestingly, regions and 12a-b were not previously found in s. enteritidis, although this may be due to the fact that previously reported s. enteritidis cgh analysis used microarrays that lacked these regions. both pre-epidemic isolates also carry gogb. gogb is a ϕgifsy-1-encoded type iii secreted substrate of both spi- and spi- ttss in s. typhimurium lt. it has been reported that some salmonellae have gifsy- but not gogb whereas others do not have gifsy- but do have gogb, suggesting that this gene has been recently acquired by gifsy-. to the best of our knowledge, this is the first report of s. enteritidis harbouring this gene. thus, we designed a pair of primers that amplifies a bp fragment of gogb, and used them to screen for its presence among the strains also assayed for ϕse no other isolate was positive for gogb. we then sequenced the pcr fragment from both pre-epidemic strains and found that the sequence has 99% of identity with s. typhimurium lt gogb. in summary, out of the variable genomic regions found among s. enteritidis isolates correspond to phage-like regions, suggesting that, as in other serovars of salmonella, phages play a crucial role in the generation of genetic diversity in s. enteritidis. variations in the content of genes involved in metabolism our cgh data highlighted other regions of variation. region harbouring out of genes of the eut operon, is absent in pre-epidemic and non-human epidemic s. enteritidis isolates. these genes encode alcohol dehydrogenase, aldehyde dehydrogenase and enzymes required for ethanolamine utilization. s. enteritidis 32/ also lacks the pdus gene, a ferredoxin involved in propanediol utilization. in salmonella both 2-propanediol degradation and ethanolamine degradation require vitamin b many enterobacteriaceae have lost the capacity to synthesize cobalamine and therefore to degrade 2-propanediol and ethanolamine but a few genera, including salmonella and yersinia, re-acquired a kb metabolic island encoding both the ability to synthesise cobalamine and degrade 2-propanediol, whilst retaining the eut operon. although 2-propanediol is an important source of energy for s. typhimurium and cbi mutants are significantly attenuated in their ability to grow in macrophages it is apparent that genes within these pathways are lost in the host-adapted s. enterica serovars including gallinarum, typhi and paratyphi a. region comprises three genes which are absent/divergent in s. enteritidis 47/ isolated from human disease. rpos is inducible in stationary phase, is the master regulator of the general stress response in salmonella and is required for virulence in mice. there are previous reports of s. typhi, s. typhimurium and s. enteritidis clinical and environmental isolates carrying mutations in rpos that result in impaired rpos functionality. a test of catalase activity in stationary phase is used as a method to detect rpos function, thus we performed the test in all isolates and found a negative result only in s. enteritidis isolate 47/ this strongly suggest that rpos function is impaired in this isolate. region harbouring genes encoding nitrate reductases, cytochrome c and ferredoxin-type proteins, was also absent in s. enteritidis isolates from different periods of the uruguayan epidemic. variation in s. enteritidis genomic islands although there is a large number of genomic islands in s. enteritidis pt p which carry the hallmarks of having been laterally acquired, and maintain mobility functions, surprisingly our data show that most are ubiquitous in the s. enteritidis isolates tested here. the exceptions are region and region region is one of the largest genomic islands identified in s. enteritidis pt p, and it encodes the global transcriptional silencers h-ns and the h-ns antagonist. this region was undetected using the microarray in the kenyan s. enteritidis isolate af but it is present in all other strains. region corresponds to the immigration control region icr in s. enteritidis pt p which encodes two type i restriction/modification systems. all of these genes were not detected in the kenyan s. enteritidis isolate af and partially detected in isolate 47/ which lacks one of the restriction enzyme subunits. in addition to variation in genes found in large clusters in s. enteritidis pt p there was also variation in genes found as singletons. of note is the absence of the gene ratb in s. enteritidis isolate 32/ this gene is located within the cs genomic island in s. typhimurium, a region that is important for intestinal persistence in a mouse model. in s. enteritidis pt p the genomic island is maintained but ratb is a pseudogene, as it is in the sequenced strains of the host-adapted serovars s. typhi and s. gallinarum. variation in plasmid-encoded genes besides chromosomal genes, the microarray incorporated genes found on salmonella virulence plasmids from serovars enteritidis, gallinarum, typhimurium and plasmids, phcm and phcm from the multi-drug resistant s. typhi strain ct five uruguayan isolates, from food and from human disease, lack the characteristic s. enteritidis virulence plasmid. this was confirmed by attempts to purify the plasmid. two other uruguayan isolates, 92/ and 132/ exhibited divergence in more than genes and isolates 57/ and 49/ diverged in genes found within the plasmid of s. enteritidis pt p. included in the genes predicted as absent or divergent are the spv genes, the pef fimbrial operon as well as repa and rsdb. of note, isolates 92/ and 132/ also lack the few tra genes remaining in s. enteritidis pt p despite the high degree of variability seen in these plasmids all had similar molecular weights when compared to that in s. enteritidis pt p, suggesting potential divergence in gene sequence or acquisition of novel genes. however none of the isolates with high variation in plasmid gene content showed a positive signal for non-s. enteritidis plasmid features included in the array, suggesting that they may harbour sequence divergence or novel sequences. in fact the only isolate showing a positive signal for non-s. enteritidis plasmid features was the kenyan s. enteritidis isolate af which harbours the complete s. enteritidis pt p virulence plasmid and shows a positive signal for genes from the multidrug resistance plasmid, phcm from s. typhi ct these include genes encoding β-lactamase and streptomycin resistance. although we cannot confirm that these are located on the plasmid there are increasing numbers of reports of drug resistance genes integrating into the virulence plasmid.  the results presented here corroborate and extend previous reports demonstrating a high degree of genetic homogeneity among field isolates of s. enteritidis, irrespective of geographical, temporal and source differences. most of the strains analysed produced highly similar profiles by rapd and pfge analysis, and those selected for further analysis showed almost indistinguishable gene content by microarray-based cgh. the two oldest uruguayan pre-epidemic s. enteritidis isolates and a kenyan isolate were among the most divergent. most of the genome variation was related to prophage regions underscoring their importance as drivers for s. enteritidis evolution. in particular half of the isolates from before the beginning of the s. enteritidis epidemic in uruguay lack ϕse whereas absence of this phage is minimal among s. enteritidis isolated during and after the epidemics, as detected by cgh and extended by pcr screening. these results, together with those previously reported strongly suggest that this phage may have been relatively recently acquired by s. enteritidis, and that this might be related to the capacity of pt4-like strains to become prevalent. although we are aware that the small number of pre-epidemic isolates is a limitation of this study, it is noteworthy that these are all the s. enteritidis isolates received at the national salmonella centre since the beginning of the 1970s until the end of the two oldest pre-epidemic isolates also carry genetic regions that were not found in s. enteritidis strains previously evaluated by cgh, but this may be due to the fact that more genes from other serovars of salmonella are present on our microarray compared with those previously reported. beside these, we have confirmed that uruguayan isolates harbour gogb, a gene that has not been previously found among s. enteritidis strains. in addition to identifying differences in the content of mobile genetic elements we were successful in identifying metabolic pathways which appear to be incomplete in some isolates. these include those associated with the utilization of propanediol and ethanolamine as well as many genes that have previously been implicated in bacterial fitness and virulence. we also showed that a significant number of the uruguayan s. enteritidis strains lack the salmonella virulence plasmid and others showed variation in plasmid gene content. there was great heterogeneity in the ability of the isolates to invade caco- human epithelial cells, but our genotyping approach was insufficient to elucidate the genetic bases for these differences. these finding are in agreement with previous reports that showed that genetically closely related s. enteritidis strains nevertheless presented important metabolic differences, and that these differences were related to the accumulation of single nucleotide polymorphism rather than with differences in gene content. of note, none of the genes predicted as variant among s. enteritidis in our work correspond to those described as involved in the ability to survive in the avian reproductive tract or in persistence in egg albumen. furthermore, the genetic regions related to metabolic functions found as variable in our cgh analysis do not correspond to utilization of the compounds described by morales et al. in their comparative phenotypic analysis of s. enteritidis strains. a report has recently been published showing differences in genetic content among s. enteritidis isolates from prevalent phage types and the non-prevalent phage type. with the exception of the plasmid-encoded genes, all other genes reported as exclusively present in the prevalent phage types, are also present in all the isolates analyzed here. overall, our study shows that the epidemic of s. enteritidis in uruguay between and was caused by highly related s. enteritidis isolates, perhaps comprising a pt4-like clonal population with few whole gene differences. to understand more clearly the link between genotype and phenotype and to differentiate between neutral variation within a population and variations associated directly with defined phenotypes, the whole genome sequences of a large number of isolates are required for association studies. this is our future direction. 
overall, our results support the view that phages play a crucial role in the generation of genetic diversity in s. enteritidis and that phage se may be a key marker for the emergence of particular isolates capable of causing epidemics. our results confirm previous reports demonstrating a high degree of genetic homogeneity among s. enteritidis isolates. from these, isolates spanning different periods, genetic profiles and sources of isolation were assayed for their ability to infect human epithelial cells and subjected to comparative genomic hybridization using a salmonella pan-array and the sequenced strain s. enteritidis pt p as reference. the oldest pre-epidemic isolates lack phage se and harbour other phage encoded genes that are absent in the sequenced strain. in particular, the oldest pre-epidemic isolates carry phage-associated genetic regions not previously reported in s. enteritidis. the recent epidemic of s. enteritidis infection in uruguay has been driven by the introduction of closely related strains of phage type lineage. s. enteritidis isolates were genotyped using rapd-pcr and a selection were subjected to pfge analysis. in this study we investigate genome divergence and pathogenic potential in s. enteritidis isolated before, during and after an epidemic in uruguay. two hundred and thirty three chromosomal genes as well as the virulence plasmid were found as variable among s. enteritidis isolates. however, of the regions of variability described here are for the first time reported as being variable in s. enteritidis. besides variation in prophage, we found variation in genes involved in metabolism and bacterial fitness. ten out of the chromosomal regions that varied between different isolates correspond to phage-like regions. six other isolates from distant countries were included as external comparators.
 salmonella enterica serovar enteritidis has caused major epidemics of gastrointestinal infection in many different countries. in this study we investigate genome divergence and pathogenic potential in s. enteritidis isolated before, during and after an epidemic in uruguay.  s. enteritidis isolates were genotyped using rapd-pcr and a selection were subjected to pfge analysis. from these, isolates spanning different periods, genetic profiles and sources of isolation were assayed for their ability to infect human epithelial cells and subjected to comparative genomic hybridization using a salmonella pan-array and the sequenced strain s. enteritidis pt p as reference. six other isolates from distant countries were included as external comparators. two hundred and thirty three chromosomal genes as well as the virulence plasmid were found as variable among s. enteritidis isolates. ten out of the chromosomal regions that varied between different isolates correspond to phage-like regions. the oldest pre-epidemic isolates lack phage se and harbour other phage encoded genes that are absent in the sequenced strain. besides variation in prophage, we found variation in genes involved in metabolism and bacterial fitness. five epidemic strains lack the complete salmonella virulence plasmid. significantly, strains with indistinguishable genetic patterns still showed major differences in their ability to infect epithelial cells, indicating that the approach used was insufficient to detect the genetic basis of this differential behaviour.  the recent epidemic of s. enteritidis infection in uruguay has been driven by the introduction of closely related strains of phage type lineage. our results confirm previous reports demonstrating a high degree of genetic homogeneity among s. enteritidis isolates. however, of the regions of variability described here are for the first time reported as being variable in s. enteritidis. in particular, the oldest pre-epidemic isolates carry phage-associated genetic regions not previously reported in s. enteritidis. overall, our results support the view that phages play a crucial role in the generation of genetic diversity in s. enteritidis and that phage se may be a key marker for the emergence of particular isolates capable of causing epidemics. 