BACKGROUND
salmonella enterica serovar enteritidis  is a foodborne pathogen transmitted to humans predominately through contaminated eggs and other poultry food products  <cit> .
s. enteritidis was an infrequently reported serovar until the mid- to late 1980's when it emerged as a common cause of salmonellosis in european countries and then worldwide  <cit> .
by the 1990's s. enteritidis replaced s. enterica serovar typhimurium  as the most common serovar isolated from humans in many countries  <cit> .
in canada, s. enteritidis has been among the top three reported salmonella serovars resulting in human disease since  <dig>  accounting for between 12–21% of infections caused by salmonella  <cit> .
s. enteritidis is subtyped by a phage typing scheme implemented by ward et al.,  <cit> , and this method has identified regionally endemic s. enteritidis subtypes as well fluctuations in the predominate subtypes.
for example, s. enteritidis phage type  <dig>  was the eighth most common phage type identified in canada from 1982– <dig>  but by  <dig> pt <dig> was ranked the third most common and has subsequently remained one of the top five most prevalent phage types  <cit> .
phage typing of s. enteritidis utilizes a set of sixteen bacteriophage to generate a lytic pattern to group strains  <cit> .
typing methods based upon comparisons of whole genomic dna, plasmid dna or specific genetic determinants have also been used in place of phage typing or as supplementary techniques.
the methods most frequently used to subtype s. enteritidis include pulsed-field gel electrophoresis , plasmid profiling and restriction-hybridization based ribotyping, which have each been applied with varying degrees of success  <cit> .
a single pfge macrorestriction profile often predominated in strains of the same phage type or even amongst multiple phage types  <cit> .
alternatively, a group of strains of the same phage type can have multiple restriction patterns  <cit> .
ribotyping based upon restriction analysis of the rrna operon has utilized several schemes, such as pvuii or a double digestion with psti and sphi  <cit> .
the latter combination has been used to subtype strains of the same phage type, however, this discrimination was not always epidemiologically significant  <cit> .
plasmid profiles have indicated divergence between strains represented by the presence or absence of various low molecular weight plasmids and the  <dig> to  <dig> kbp s. enteritidis virulence plasmid  <cit> , which is similar to the  <dig> kbp pslt virulence plasmid of s. typhimurium  <cit> .
combinations of these methods have been successfully used to subtype outbreak from non-outbreak strains of s. enteritidis  <cit> .
dna microarray-based comparative genomic hybridization  and sequence-based typing  have recently been used to determine genetic relatedness between s. enteritidis strains, and there was generally a lack of diversity for strains of the same phage type  <cit> .
sbt of  <dig> clinical and poultry-related s. enteritidis strains in a scheme comprised of segments of the 16s rrna, manb, glna and pduf loci detected  <dig>   <dig>   <dig> and  <dig> polymorphic sites, respectively  <cit> .
similarly, a sbt scheme evaluating manb, mdh and fima encoded by  <dig> s. enteritidis strains revealed  <dig>   <dig> and  <dig> polymorphic sites, respectively  <cit> .
typing of the misl, spam and span loci encoding cell surface-associated proteins did not identify differences in four pt <dig> strains, but identified  <dig> polymorphic sites in pt <dig>  <cit> .
cgh analyses of a diverse collection of  <dig> s. enteritidis strains using a microarray composed of s. typhimurium, s. typhi, s. paratyphi a and s. enteritidis pt <dig> probes identified few genetic differences between these strains, including strains isolated before the emergence of s. enteritidis as a major foodborne salmonella  <cit> .
the genetic differences were predominantly at phage-encoding loci  but no significant delineation amongst s. enteritidis strains of the same phage type was observed  <cit> .
cgh analyses of pt13a and pt <dig> strains similarly identified no significant strain-to-strain variation, and these phage types were also distinguished by st64b and fels bacteriophage genetic content  <cit> .
s. enteritidis pt <dig> was identified in a large foodborne outbreak in the canadian province of ontario in  <dig>  over  <dig> cases of gastroenteritis were reported between october and december  <dig> and these were associated with the consumption of contaminated mung bean sprouts.
a single pfge profile  predominated amongst the outbreak-associated strains, and this profile was also seen in concurrent and preceding sporadic human-clinical and poultry-related isolates.
with the apparent clonality of pt <dig> isolates, it was difficult to support epidemiological links during the outbreak using pfge-based typing.
the aim of this study was to perform cgh and sbt genomic analyses in parallel with other typing methodologies to identify epidemiologically significant markers for subtyping s. enteritidis pt <dig>
RESULTS
in response to the large outbreak of s. enteritidis pt <dig>  all s. enteritidis pt <dig> strains with pfge data reported to the national microbiology laboratory were retrospectively analyzed.
this set of  <dig> strains comprised sporadic human-clinical isolates and also poultry-related agri-food isolates submitted by provincial public health laboratories in alberta, british columbia, ontario and québec.
two pfge patterns were observed:  <dig> strains had the xbai macrorestriction profile senxai.
<dig> and  <dig> strains produced the related pattern senxai.
<dig> .
pfge with blni was performed for  <dig> of these strains and all were pattern senbni.
<dig>  the inability to differentiate strains by pfge necessitated the need to attempt additional subtyping methodologies for the differentiation of both outbreak and sporadic s. enteritidis pt <dig> strains.
comparative genomic hybridization
microarray-based cgh was performed on s. enteritidis strains to identify genetic differences between strains that would potentially serve as molecular markers for subtyping s. enteritidis pt <dig> strains.
an oligonucleotide array representing all coding sequences from the sequenced genome of s. typhimurium lt <dig> and all coding sequences from sgi <dig>  <cit>  was used in two-colour cgh experiments with s. typhimurium lt <dig> genomic dna as a reference and s. enteritidis genomic dna as the test strain.
seven pt <dig> strains were analyzed, including: human isolate 05- <dig> associated with the foodborne outbreak; strain 05- <dig> isolated from mung bean sprouts presumptively identified as the causative food agent; and five other sporadic pt <dig> strains isolated from human and poultry sources that were recovered concurrently with the outbreak or previously in other regions of canada .
the cgh analyses of pt <dig> strains individually indicated the genetic content of the strains in comparison to s. typhimurium lt <dig>  and the majority of the differences corresponded to genes encoded by bacteriophages fels- <dig>  fels- <dig>  gifsy- <dig> and gifsy- <dig>  and also putative phage-related coding sequences stm2230– <dig> and stm4198– <dig> .
the oligonucleotides probes for sgi <dig> indicated that this determinant was absent in all strains, and accordingly, all examined s. enteritidis strains were sensitive to antibiotics.
the relative genetic content of s. enteritidis strains identified by microarray analysis could also be used to compare between pt <dig> strains, and few coding sequences were putatively divergent between pt <dig> strains.
dna sequencing was performed at flic and safa , and each locus was identical for all strains pt <dig> strains .
these data suggested that differences in genetic content between pt <dig> strains in the cgh dataset were partly due to technical variation rather than true biological variation.
a. plasmid profile as determined by digestion of plasmid dna with restriction enzyme bglii;  <dig> = strains did not possess large plasmid and had no dna fragments when digested with bglii
b. individual polymorphisms are in addition to insertions and deletions, in reference to pt <dig> isolate 05- <dig>
sequence-based typing
due to the lack of observable differences in genetic content between pt <dig> using cgh, additional molecular methods were attempted to differentiate these strains.
a sbt scheme targeting manb, mdh and fima  <cit>  did not identify sequence type differences amongst s. enteritidis pt <dig> .
a single nucleotide polymorphism  was observed at the manb locus for both s. enteritidis pt <dig> and pt <dig> compared to s. enteritidis pt <dig>  previously, multiple snps have been observed between s. enteritidis strains at the cyaa locus  <cit>  and additional comparative analyses of s. enteritidis phenotypic subpopulations that were descended from a common parent by whole genome mutational mapping identified polymorphisms, including small deletions, non-synonymous amino acid changes, and altered terminal codons  <cit> .
from this data set, we sequenced for each strain in our s. enteritidis panel the rata, citt, dmsa, caic and stm <dig> loci encoding membrane proteins or metabolic cellular functions, and similarly no polymorphisms were observed between pt <dig> strains.
a total of  <dig>  bp, including the flic and safa loci examined after cgh analyses, was sequenced for each strain without observing any genetic differences between the s. enteritidis pt <dig> strains.
a total of  <dig> snps were found between s. enteritidis pt <dig> and pt <dig> compared to s. enteritidis pt <dig>  and a multitude of polymorphic sites between s. typhimurium lt <dig> and s. enteritidis pt <dig> were observed .
plasmid content and plasmid rflp
the population and characteristics of plasmids harbored by salmonella has been used as a means to subtype these organisms  <cit> .
plasmid content was determined for the s. enteritidis strains, as well as s. typhimurium strain lt <dig>  and in all plasmid preparations a chromosomal dna fragment was observed .
a high molecular weight  plasmid was observed for all strains except pt <dig> strain 05- <dig> and pt <dig> strain 06- <dig>  accordingly, s. typhimurium strain lt <dig> was known to harbor the  <dig> kbp virulence plasmid pslt  <cit>  and s. enteritidis commonly harbors a ~ <dig> kbp virulence plasmid  <cit> .
strains 05- <dig> and 06- <dig> that lacked the hmw virulence plasmid had a different pfge pattern than the other s. enteritidis pt <dig> and pt <dig> strains, respectively .
the only strain from which low molecular weight  plasmids were isolated was pt <dig> strain 06- <dig>  to confirm the presence of the salmonella virulence plasmid in s. enteritidis pt <dig>  pcr screening for the spvc virulence determinant was performed.
all strains encoded spvc  except for the two s. enteritidis strains that did not posses the hmw virulence plasmid.
restriction enzyme digestion of the plasmid preparations with bglii revealed that s. enteritidis strains harboring the hmw plasmid had the same restriction fragment length polymorphism  pattern .
the s. enteritidis pt <dig> strain with lmw plasmids had a different plasmid pattern due to additional dna fragments  and the fragments in the pattern contributed by the hmw plasmid were otherwise identical to those of strains that contained only the hmw plasmid.
additionally, the rflp pattern produced by the s. typhimurium pslt differed from the s. enteritidis hmw plasmid.
rep-pcr
repetitive sequence-based pcr  methods targeting non-coding repetitive sequences are useful for bacterial subtyping and this platform has been automated and reproducibility improved through commercially-available reagents in conjunction with a microfluidics station  <cit> .
automated rep-pcr was performed on s. enteritidis pt <dig>  pt <dig> and pt <dig> strains, and a small selection of other serovars was also included to illustrate the scope of differentiation at the serovar level.
the rep-pcr amplicon patterns for all s. enteritidis pt <dig> strains clustered together and were >98% related with no significant differences .
the s. enteritidis pt <dig> and pt <dig> strains produced similar rep-pcr patterns as the pt <dig> strains  with a single amplicon being the predominant difference.
the other examined serovars were between 78–81% related to the s. enteritidis strains .
discussion
genetic homogeneity has been repeatedly observed for s. enteritidis, particularly between strains of the same phage type  <cit> .
similarly, subtyping beyond phage type utilizing methods such as ribotyping, pfge and plasmid profiling has generally only confirmed the clonal lineages discerned by phage typing without providing any further discriminatory power  <cit> .
this lack of subtyping can prevent the establishment of absolute links to contaminated agri-food sources during outbreak trace-back investigations.
during an outbreak of s. enteritidis pt <dig> in canada, the outbreak strain could not be distinguished from concurrent and geographically and temporally distinct isolates strains of s. enteritidis pt <dig>  cgh, sbt, plasmid profiling and rep-pcr were performed in an attempt to identify genetic markers suitable for subtyping s. enteritidis pt <dig>
clonally related phenotypic subpopulations of s. enteritidis have been observed and discrete allelic variation at ribosomal and metabolic loci corresponded to these pathotypes  <cit> .
in our cgh experiments, large clusters of genetic differences such as the absence of phage regions were discernible between s. enteritidis and the reference s. typhimurium for which the array was designed.
however, more subtle genetic differences such as allelic variation between pt <dig> strains could not be confirmed, which was a finding similar to previous results  <cit> .
this may have resulted from a lack of s. enteritidis specific probes.
alternatively, identifying a reliable phenotypic or pathotype difference between pt <dig> strains could aid in the identification of subtle but definitive genetic differences that impact biology within a phage type.
morales and colleagues hypothesized that genetic variation between s. enteritidis strains is better studied by performing dna sequencing for snps than through microarray-based comparative genomics  <cit> .
through sequencing, it is possible to produce de novo data for each strain instead of attempting to discern genomic content in the context of comparing hybridization data between two different strains, or possibly different serovars, as in this study .
although dna sequencing provided a higher level of detail than cgh, sequencing of the loci that were previously observed to be divergent between s. enteritidis pathotypes and other loci commonly used for sequence-based typing  did not identify any genomic differences between pt <dig> strains.
these results are similar to other published results in that discrimination between serovars was possible, but variation within a phage type was not observed  <cit> .
alternatively, some loci were significantly variable between s. enteritidis and s. typhimurium , including safa , stm <dig>  and caic .
these variable loci may prove to be useful as targets for molecular typing between salmonella serovars.
our panel of strains was selected to include the outbreak-associated strains, concurrent sporadic clinical and agri-food strains, and for comparison to unrelated strains, isolates that were previously recovered in different canadian provinces.
if the incidence of snps is less than  <dig> in  <dig>  bp it may be necessary to pursue a whole genome approach for detection of allelic variation because any one section of the genome or group of genes could be similar between clonally related pt <dig> strains relevant to human public health.
plasmid profile analyses identified s. enteritidis strains that lacked the hmw virulence plasmid or those that contained lmw plasmids, but these did not correspond to the outbreak-associated strains.
carriage of the hmw virulence plasmid were confirmed by testing for the presence of the spvc gene, which was previously observed on all s. enteritidis plasmids of ~ <dig> kbp  <cit> .
the variation in plasmid content did influence the observed xbai pfge patterns, which were otherwise identical.
additionally, rflp analyses of the plasmid preparations did not identify any further levels of genetic diversity within the plasmids, but did support that both s. enteritidis pt <dig> and pt <dig> carry a similar hmw virulence plasmid as s. enteritidis pt <dig>  furthermore, the usefulness of determining plasmid carriage as a means of subtyping was limited since the overall incidence of the hmw virulence plasmids in canada was unknown and variation of this trait may be influenced by laboratory conditions .
rep-pcr has the potential to represent genetic differences contributed throughout the genome, unlike directed sbt and plasmid profiling, and targets different genetic features than pfge.
the s. enteritidis pt <dig> strains examined in this study were not differentiated with this method and were greater than 97% related.
although there was some discrimination between the s. enteritidis pt <dig> and pt <dig> strains and the s. enteritidis pt <dig> strains , there was still greater than 95% relatedness amongst these strains.
alternatively, a select number of other serovars were examined using automated rep-pcr and this method could differentiate between serovars, which is similar to a previous rep-pcr study  <cit> .
CONCLUSIONS
s. enteritidis pt <dig> isolates in canada were observed to be essentially genetically homogeneous after microarray-based comparisons did not identify overt differences between strains and dna sequencing of eleven loci and  <dig>  bp did not identify any polymorphisms.
furthermore, pfge and plasmid rflp patterns were identical for the majority of strains, except for some changes in plasmid content that affected restriction patterns of strains not associated with a large foodborne outbreak.
rep-pcr could discriminate between serovars and phage types, but not between outbreak-related and sporadic pt <dig> isolates.
the apparent clonality of pt <dig> strains has implications for the ability to subtype this pathogen during outbreaks.

 salmonella enterica serovar enteritidis is a foodborne pathogen transmitted to humans predominately through contaminated eggs and other poultry food products. s. enteritidis was an infrequently reported serovar until the mid- to late 1980's when it emerged as a common cause of salmonellosis in european countries and then worldwide. by the 1990's s. enteritidis replaced s. enterica serovar typhimurium as the most common serovar isolated from humans in many countries. in canada, s. enteritidis has been among the top three reported salmonella serovars resulting in human disease since accounting for between 12–21% of infections caused by salmonella. s. enteritidis is subtyped by a phage typing scheme implemented by ward et al.,, and this method has identified regionally endemic s. enteritidis subtypes as well fluctuations in the predominate subtypes. for example, s. enteritidis phage type was the eighth most common phage type identified in canada from 1982– but by pt was ranked the third most common and has subsequently remained one of the top five most prevalent phage types. phage typing of s. enteritidis utilizes a set of sixteen bacteriophage to generate a lytic pattern to group strains. typing methods based upon comparisons of whole genomic dna, plasmid dna or specific genetic determinants have also been used in place of phage typing or as supplementary techniques. the methods most frequently used to subtype s. enteritidis include pulsed-field gel electrophoresis, plasmid profiling and restriction-hybridization based ribotyping, which have each been applied with varying degrees of success. a single pfge macrorestriction profile often predominated in strains of the same phage type or even amongst multiple phage types. alternatively, a group of strains of the same phage type can have multiple restriction patterns. ribotyping based upon restriction analysis of the rrna operon has utilized several schemes, such as pvuii or a double digestion with psti and sphi. the latter combination has been used to subtype strains of the same phage type, however, this discrimination was not always epidemiologically significant. plasmid profiles have indicated divergence between strains represented by the presence or absence of various low molecular weight plasmids and the to kbp s. enteritidis virulence plasmid, which is similar to the kbp pslt virulence plasmid of s. typhimurium. combinations of these methods have been successfully used to subtype outbreak from non-outbreak strains of s. enteritidis. dna microarray-based comparative genomic hybridization and sequence-based typing have recently been used to determine genetic relatedness between s. enteritidis strains, and there was generally a lack of diversity for strains of the same phage type. sbt of clinical and poultry-related s. enteritidis strains in a scheme comprised of segments of the 16s rrna, manb, glna and pduf loci detected and polymorphic sites, respectively. similarly, a sbt scheme evaluating manb, mdh and fima encoded by s. enteritidis strains revealed and polymorphic sites, respectively. typing of the misl, spam and span loci encoding cell surface-associated proteins did not identify differences in four pt strains, but identified polymorphic sites in pt. cgh analyses of a diverse collection of s. enteritidis strains using a microarray composed of s. typhimurium, s. typhi, s. paratyphi a and s. enteritidis pt probes identified few genetic differences between these strains, including strains isolated before the emergence of s. enteritidis as a major foodborne salmonella. the genetic differences were predominantly at phage-encoding loci but no significant delineation amongst s. enteritidis strains of the same phage type was observed. cgh analyses of pt13a and pt strains similarly identified no significant strain-to-strain variation, and these phage types were also distinguished by st64b and fels bacteriophage genetic content. s. enteritidis pt was identified in a large foodborne outbreak in the canadian province of ontario in over cases of gastroenteritis were reported between october and december and these were associated with the consumption of contaminated mung bean sprouts. a single pfge profile predominated amongst the outbreak-associated strains, and this profile was also seen in concurrent and preceding sporadic human-clinical and poultry-related isolates. with the apparent clonality of pt isolates, it was difficult to support epidemiological links during the outbreak using pfge-based typing. the aim of this study was to perform cgh and sbt genomic analyses in parallel with other typing methodologies to identify epidemiologically significant markers for subtyping s. enteritidis pt  in response to the large outbreak of s. enteritidis pt all s. enteritidis pt strains with pfge data reported to the national microbiology laboratory were retrospectively analyzed. this set of strains comprised sporadic human-clinical isolates and also poultry-related agri-food isolates submitted by provincial public health laboratories in alberta, british columbia, ontario and québec. two pfge patterns were observed: strains had the xbai macrorestriction profile senxai. and strains produced the related pattern senxai.. pfge with blni was performed for of these strains and all were pattern senbni. the inability to differentiate strains by pfge necessitated the need to attempt additional subtyping methodologies for the differentiation of both outbreak and sporadic s. enteritidis pt strains. comparative genomic hybridization microarray-based cgh was performed on s. enteritidis strains to identify genetic differences between strains that would potentially serve as molecular markers for subtyping s. enteritidis pt strains. an oligonucleotide array representing all coding sequences from the sequenced genome of s. typhimurium lt and all coding sequences from sgi was used in two-colour cgh experiments with s. typhimurium lt genomic dna as a reference and s. enteritidis genomic dna as the test strain. seven pt strains were analyzed, including: human isolate 05- associated with the foodborne outbreak; strain 05- isolated from mung bean sprouts presumptively identified as the causative food agent; and five other sporadic pt strains isolated from human and poultry sources that were recovered concurrently with the outbreak or previously in other regions of canada. the cgh analyses of pt strains individually indicated the genetic content of the strains in comparison to s. typhimurium lt and the majority of the differences corresponded to genes encoded by bacteriophages fels- fels- gifsy- and gifsy- and also putative phage-related coding sequences stm2230– and stm4198–. the oligonucleotides probes for sgi indicated that this determinant was absent in all strains, and accordingly, all examined s. enteritidis strains were sensitive to antibiotics. the relative genetic content of s. enteritidis strains identified by microarray analysis could also be used to compare between pt strains, and few coding sequences were putatively divergent between pt strains. dna sequencing was performed at flic and safa, and each locus was identical for all strains pt strains. these data suggested that differences in genetic content between pt strains in the cgh dataset were partly due to technical variation rather than true biological variation. a. plasmid profile as determined by digestion of plasmid dna with restriction enzyme bglii; = strains did not possess large plasmid and had no dna fragments when digested with bglii b. individual polymorphisms are in addition to insertions and deletions, in reference to pt isolate 05- sequence-based typing due to the lack of observable differences in genetic content between pt using cgh, additional molecular methods were attempted to differentiate these strains. a sbt scheme targeting manb, mdh and fima did not identify sequence type differences amongst s. enteritidis pt. a single nucleotide polymorphism was observed at the manb locus for both s. enteritidis pt and pt compared to s. enteritidis pt previously, multiple snps have been observed between s. enteritidis strains at the cyaa locus and additional comparative analyses of s. enteritidis phenotypic subpopulations that were descended from a common parent by whole genome mutational mapping identified polymorphisms, including small deletions, non-synonymous amino acid changes, and altered terminal codons. from this data set, we sequenced for each strain in our s. enteritidis panel the rata, citt, dmsa, caic and stm loci encoding membrane proteins or metabolic cellular functions, and similarly no polymorphisms were observed between pt strains. a total of bp, including the flic and safa loci examined after cgh analyses, was sequenced for each strain without observing any genetic differences between the s. enteritidis pt strains. a total of snps were found between s. enteritidis pt and pt compared to s. enteritidis pt and a multitude of polymorphic sites between s. typhimurium lt and s. enteritidis pt were observed. plasmid content and plasmid rflp the population and characteristics of plasmids harbored by salmonella has been used as a means to subtype these organisms. plasmid content was determined for the s. enteritidis strains, as well as s. typhimurium strain lt and in all plasmid preparations a chromosomal dna fragment was observed. a high molecular weight plasmid was observed for all strains except pt strain 05- and pt strain 06- accordingly, s. typhimurium strain lt was known to harbor the kbp virulence plasmid pslt and s. enteritidis commonly harbors a ~ kbp virulence plasmid. strains 05- and 06- that lacked the hmw virulence plasmid had a different pfge pattern than the other s. enteritidis pt and pt strains, respectively. the only strain from which low molecular weight plasmids were isolated was pt strain 06- to confirm the presence of the salmonella virulence plasmid in s. enteritidis pt pcr screening for the spvc virulence determinant was performed. all strains encoded spvc except for the two s. enteritidis strains that did not posses the hmw virulence plasmid. restriction enzyme digestion of the plasmid preparations with bglii revealed that s. enteritidis strains harboring the hmw plasmid had the same restriction fragment length polymorphism pattern. the s. enteritidis pt strain with lmw plasmids had a different plasmid pattern due to additional dna fragments and the fragments in the pattern contributed by the hmw plasmid were otherwise identical to those of strains that contained only the hmw plasmid. additionally, the rflp pattern produced by the s. typhimurium pslt differed from the s. enteritidis hmw plasmid. rep-pcr repetitive sequence-based pcr methods targeting non-coding repetitive sequences are useful for bacterial subtyping and this platform has been automated and reproducibility improved through commercially-available reagents in conjunction with a microfluidics station. automated rep-pcr was performed on s. enteritidis pt pt and pt strains, and a small selection of other serovars was also included to illustrate the scope of differentiation at the serovar level. the rep-pcr amplicon patterns for all s. enteritidis pt strains clustered together and were >98% related with no significant differences. the s. enteritidis pt and pt strains produced similar rep-pcr patterns as the pt strains with a single amplicon being the predominant difference. the other examined serovars were between 78–81% related to the s. enteritidis strains. discussion genetic homogeneity has been repeatedly observed for s. enteritidis, particularly between strains of the same phage type. similarly, subtyping beyond phage type utilizing methods such as ribotyping, pfge and plasmid profiling has generally only confirmed the clonal lineages discerned by phage typing without providing any further discriminatory power. this lack of subtyping can prevent the establishment of absolute links to contaminated agri-food sources during outbreak trace-back investigations. during an outbreak of s. enteritidis pt in canada, the outbreak strain could not be distinguished from concurrent and geographically and temporally distinct isolates strains of s. enteritidis pt cgh, sbt, plasmid profiling and rep-pcr were performed in an attempt to identify genetic markers suitable for subtyping s. enteritidis pt clonally related phenotypic subpopulations of s. enteritidis have been observed and discrete allelic variation at ribosomal and metabolic loci corresponded to these pathotypes. in our cgh experiments, large clusters of genetic differences such as the absence of phage regions were discernible between s. enteritidis and the reference s. typhimurium for which the array was designed. however, more subtle genetic differences such as allelic variation between pt strains could not be confirmed, which was a finding similar to previous results. this may have resulted from a lack of s. enteritidis specific probes. alternatively, identifying a reliable phenotypic or pathotype difference between pt strains could aid in the identification of subtle but definitive genetic differences that impact biology within a phage type. morales and colleagues hypothesized that genetic variation between s. enteritidis strains is better studied by performing dna sequencing for snps than through microarray-based comparative genomics. through sequencing, it is possible to produce de novo data for each strain instead of attempting to discern genomic content in the context of comparing hybridization data between two different strains, or possibly different serovars, as in this study. although dna sequencing provided a higher level of detail than cgh, sequencing of the loci that were previously observed to be divergent between s. enteritidis pathotypes and other loci commonly used for sequence-based typing did not identify any genomic differences between pt strains. these results are similar to other published results in that discrimination between serovars was possible, but variation within a phage type was not observed. alternatively, some loci were significantly variable between s. enteritidis and s. typhimurium, including safa, stm and caic. these variable loci may prove to be useful as targets for molecular typing between salmonella serovars. our panel of strains was selected to include the outbreak-associated strains, concurrent sporadic clinical and agri-food strains, and for comparison to unrelated strains, isolates that were previously recovered in different canadian provinces. if the incidence of snps is less than in bp it may be necessary to pursue a whole genome approach for detection of allelic variation because any one section of the genome or group of genes could be similar between clonally related pt strains relevant to human public health. plasmid profile analyses identified s. enteritidis strains that lacked the hmw virulence plasmid or those that contained lmw plasmids, but these did not correspond to the outbreak-associated strains. carriage of the hmw virulence plasmid were confirmed by testing for the presence of the spvc gene, which was previously observed on all s. enteritidis plasmids of ~ kbp. the variation in plasmid content did influence the observed xbai pfge patterns, which were otherwise identical. additionally, rflp analyses of the plasmid preparations did not identify any further levels of genetic diversity within the plasmids, but did support that both s. enteritidis pt and pt carry a similar hmw virulence plasmid as s. enteritidis pt furthermore, the usefulness of determining plasmid carriage as a means of subtyping was limited since the overall incidence of the hmw virulence plasmids in canada was unknown and variation of this trait may be influenced by laboratory conditions. rep-pcr has the potential to represent genetic differences contributed throughout the genome, unlike directed sbt and plasmid profiling, and targets different genetic features than pfge. the s. enteritidis pt strains examined in this study were not differentiated with this method and were greater than 97% related. although there was some discrimination between the s. enteritidis pt and pt strains and the s. enteritidis pt strains, there was still greater than 95% relatedness amongst these strains. alternatively, a select number of other serovars were examined using automated rep-pcr and this method could differentiate between serovars, which is similar to a previous rep-pcr study.  s. enteritidis pt isolates in canada were observed to be essentially genetically homogeneous after microarray-based comparisons did not identify overt differences between strains and dna sequencing of eleven loci and bp did not identify any polymorphisms. furthermore, pfge and plasmid rflp patterns were identical for the majority of strains, except for some changes in plasmid content that affected restriction patterns of strains not associated with a large foodborne outbreak. rep-pcr could discriminate between serovars and phage types, but not between outbreak-related and sporadic pt isolates. the apparent clonality of pt strains has implications for the ability to subtype this pathogen during outbreaks. 
cgh using an oligonucleotide array based upon chromosomal coding sequences of s. enterica serovar typhimurium strain lt and the salmonella genomic island successfully determined major genetic differences between s. typhimurium and s. enteritidis pt but no significant strain-to-strain differences were observed between s. enteritidis pt isolates. the aim of this study was to perform comparative genomic hybridization, dna sequence-based typing genomic analyses, plasmid analyses, and automated repetitive sequence-based pcr to identify epidemiologically significant traits capable of subtyping s. enteritidis pt salmonella enterica serovar enteritidis has emerged as a significant foodborne pathogen throughout the world and is commonly characterized by phage typing. individual loci that were identified as potentially divergent in the cgh data set were sequenced in a panel of s. enteritidis strains, and no differences were detected between the pt strains. automated rep-pcr patterns were variable between serovars, but s. enteritidis pt strains could not be differentiated. greater than base pairs of sequence for each of seven s. enteritidis pt strains were analyzed without detecting a single polymorphic site, although diversity between different phage types of s. enteritidis was observed. these data suggest that canadian s. enteritidis pt strains are highly related genetically. the ability to link strains during this outbreak was difficult due to the apparent clonality of pt isolates in canada, as there was a single dominant pulsed-field gel electrophoresis profile amongst epidemiologically linked human and food isolates as well as concurrent sporadic strains. variation in plasmid content between pt strains was observed, but macrorestriction with bglii did not identify further differences.
 salmonella enterica serovar enteritidis has emerged as a significant foodborne pathogen throughout the world and is commonly characterized by phage typing. in canada phage types and predominate and in a large foodborne pt outbreak occurred in the province of ontario. the ability to link strains during this outbreak was difficult due to the apparent clonality of pt isolates in canada, as there was a single dominant pulsed-field gel electrophoresis profile amongst epidemiologically linked human and food isolates as well as concurrent sporadic strains. the aim of this study was to perform comparative genomic hybridization, dna sequence-based typing genomic analyses, plasmid analyses, and automated repetitive sequence-based pcr to identify epidemiologically significant traits capable of subtyping s. enteritidis pt  cgh using an oligonucleotide array based upon chromosomal coding sequences of s. enterica serovar typhimurium strain lt and the salmonella genomic island successfully determined major genetic differences between s. typhimurium and s. enteritidis pt but no significant strain-to-strain differences were observed between s. enteritidis pt isolates. individual loci that were identified as potentially divergent in the cgh data set were sequenced in a panel of s. enteritidis strains, and no differences were detected between the pt strains. additional sequence-based typing was performed at the fima, mdh, manb, cyaa, citt, caic, dmsa, rata and stm loci. similarly, no diversity was observed amongst pt strains. variation in plasmid content between pt strains was observed, but macrorestriction with bglii did not identify further differences. automated rep-pcr patterns were variable between serovars, but s. enteritidis pt strains could not be differentiated.  none of the methods identified any significant variation between pt strains. greater than base pairs of sequence for each of seven s. enteritidis pt strains were analyzed without detecting a single polymorphic site, although diversity between different phage types of s. enteritidis was observed. these data suggest that canadian s. enteritidis pt strains are highly related genetically. 