BACKGROUND
listeria monocytogenes is a gram-positive, facultative intracellular bacterial pathogen that can cause severe disease in humans, other mammals and birds  <cit> .
human listeriosis is relatively rare despite our likely frequent encounters with l. monocytogenes, which is ubiquitously present in the environment , farm and rural environments, and urban environments  <cit> .
the vast majority of human listeriosis is foodborne and the most commonly implicated vehicles are ready-to-eat food products such as meat, dairy, seafood, and fresh produce that are contaminated with l. monocytogenes during processing  <cit> .
l. monocytogenes can be introduced into food processing facilities and food products due to cross-contamination with environmental sources or from the feces of food production animals.
l. monocytogenes can persist within food processing environments for long periods of time, due in part to its ability to grow at wide-ranging temperatures and ph  and the ability to form biofilms promoting adherence to food processing surfaces  <cit> .
the persistence of a single subtype of l. monocytogenes in processing facilities or on equipment has been reported from several months to more than  <dig> years  <cit> .
given the widespread occurrence of l. monocytogenes, subtyping of clinical and food isolates is required to establish epidemiologic links during routine surveillance, outbreak investigations, and for source tracking.
there are  <dig> known serotypes of l. monocytogenes but the vast majority of human disease cases are caused by strains belonging to serotypes 4b, 1/2a, and 1/2b, severely limiting the utility of this subtyping method for differentiating l. monocytogenes  <cit> .
large clonal outbreaks owing to contaminated food sources such as coleslaw, milk, cheese, hot dogs and deli meats, have been predominately caused by serotypes 4b and 1/2a strains  <cit> .
accordingly, additional subtyping methods are required to better characterize outbreak isolates.
several molecular subtyping methods have been developed and applied to l. monocytogenes, including pulsed-field gel electrophoresis , ribotyping, multilocus variable-number tandem repeat analysis , and sequence-based subtyping  <cit> .
pfge has been adopted by pulsenet as the internationally standardized method for molecular subtyping of l.monocytogenes and has been essential in the detection and investigation of listeriosis outbreaks in canada and worldwide  <cit> .
dna sequencing has enabled analyses of l. monocytogenes genomes and furthered the understanding of this pathogen's biology and phylogeny.
comparative analyses of genome sequences have elucidated the genetic differences between l. monocytogenes serotypes, the acquisition and evolution of virulence and pathogenic traits among listeria spp., and the genetic basis underlying the unique survival and growth characteristics of l. monocytogenes  <cit> .
genome sequencing has also permitted the development of multi-locus sequence typing  protocols, which in combination with other typing methods, have validated three evolutionary lineages for l. monocytogenes  <cit> .
the lineages reflect the serotype distribution, with serotypes 1/2b, 3b and 4b segregating to lineage i and serotypes 1/2a and 1/2c segregating to lineage ii.
risk assessments based upon genetic lineages have suggested that l. monocytogenes subtypes may have unique ecological niches and may also differ in their pathogenic potential or host adaptations  <cit> .
the majority of available bacterial genome sequences have been generated using the sanger chain termination sequencing chemistries.
although this technology was instrumental in the emergence of the field of genomics, it is time and resource intensive  <cit> .
several post-sanger sequencing technologies  have since been developed that enable extremely rapid whole-genome sequencing and a broader application of comparative genomics  <cit> .
the high-throughput pyrosequencing method commercialized as the  <dig> or gs flx™ platform has been applied for rapidly determining the genome sequences of bacterial isolates.
l. monocytogenes serotype 1/2a caused a nationwide outbreak of listeriosis associated with ready-to-eat meat products in canada during the summer of  <dig>  the outbreak resulted in  <dig> deaths and at least  <dig> illnesses.
whereas the majority of outbreak-associated isolates  had an indistinguishable asci pfge pattern, one clinical isolate from ontario and several isolates from the affected food production facility exhibited an asci pfge pattern that differed by a single restriction enzyme fragment compared to the primary outbreak strain.
we report herein the first real-time application of whole-genome sequencing during an active listeriosis outbreak investigation.
high-throughput pyrosequencing was applied to characterize two outbreak-associated isolates of l. monocytogenes in order to obtain a thorough genetic characterization of the outbreak strains and to determine the genetic basis for the single asci restriction fragment change.
RESULTS
primary outbreak isolate harbors unique plasmid plm <dig> and prophage ϕlmc1
public health responses to foodborne outbreaks require bacterial subtyping data to definitively link affected individuals with contaminated food sources and other suspected patients.
the genetic relatedness between clinical and food isolates is routinely determined using methods such as pfge, however, with the latest generation of dna sequencing technologies it is theoretically possible to now determine the complete genetic blueprint of bacterial isolates within the scope of an outbreak.
in this study, draft genome sequences suitable for comparative analysis between outbreak isolates and to reference genomes were available within three days.
these data allowed for discovery of novel prophage and genomic islands, and the subsequent sequence closure and the confirmation of sequence variants allowed for annotation of polymorphisms and other traits associated with micro-diversity.
clinical isolate 08- <dig> was selected as the reference outbreak strain  and clinical isolate 08- <dig> differed only in the asci restriction pattern .
high-throughput pyrosequencing was performed on both isolates and closed circular chromosomal sequences were obtained for each isolate .
a  <dig> kbp contig present in the 08- <dig> sequence assembly but absent in 08- <dig> was identified as a circular plasmid and designated plm <dig> .
comparison of the chromosomal sequences for isolates 08- <dig> and 08- <dig> with publicly available listeria spp.
genomes  and with select loci used for mlst  indicated that these isolates were members of evolutionary lineage ii and clonal complex  <dig> .
a.
'rte', ready to eat; 'csf', cerebrospinal fluid
b.
'+', amplicon of expected size detected using pcr using oligonucleotides described in additional file 6; '-', no amplicon detected
c. 'fs', frameshift resulting in a truncated coding sequence encoding butyrate kinase; 'wt', wild type.
confirmed by sanger-based dna sequencing of amplicons generated by high fidelity pcr.
d. 'wt', wild type; '- <dig> bp', in-frame deletion of  <dig> codons.
confirmed by sanger-based dna sequencing of amplicons generated by pcr.
e. number of snp's relative to the hypothetical last common ancestor presented in fig.
<dig>  sanger-based dna sequence confirmation of snps was completed after pcr amplification using oligonucleotides described in additional file  <dig>
the asci restriction patterns of 08- <dig> and 08- <dig> were indistinguishable, except for a single band shift of approximately  <dig> kbp .
accordingly, comparative sequence alignment revealed a  <dig> kbp contiguous region integrated adjacent to  the trna-ser gene that was unique to 08- <dig> and encoding several putative bacteriophage-related coding sequences  .
blast analyses indicated that this prophage was unique among sequenced listeria genomes, although it was comprised of coding sequences similar to determinants from several known phages associated with gram-positive microbes, including l. monocytogenes phages a <dig>  a <dig> and b <dig> .
the presence of prophage ϕlmc <dig> accounted for the asci restriction pattern difference between isolates 08- <dig> and 08- <dig>
the extrachromosomal circular plasmid  harbored in isolate 08- <dig> encoded putative determinants for replication, partitioning, heavy metal resistance and dna translocation .
many of these features were homologous to sequences encoded by plasmids from l. innocua , l. monocytogenes serotype 4b isolate h <dig>  and bacillus anthracis , including a replication and regulation control center .
two regions of plm <dig> were related to heavy metal resistance, including a transposon-associated cada that exhibited high sequence identity to pli <dig>  a second, distinct region unique to plm <dig> was comprised of cada  and cadc genes, with the latter encoding a cadmium-efflux arsr-family regulatory accessory protein  homolog.
resistance to the heavy metal cadmium has been recently discovered to be associated with resistance to quaternary ammonium compounds , which may contribute to l. monocytogenes persistence in food production facilities  <cit> .
notably, several coding sequences exhibited sequence similarity to type iv secretion systems , including dna processing , dna coupling  and membrane channel formation  .
additional predicted membrane-associated proteins were present within this region, and all coding sequences were oriented in the same direction , which is consistent with the operon-style organization of most known t4ss.
varying degrees of similarity between pli <dig>  plm <dig> and pxo <dig> plasmids have already been established  <cit> , and our data further support a chimeric nature for listeria plasmids.
plm <dig> and ϕlmc <dig> exhibit non-homogeneous distribution within a wider panel of outbreak-associated isolates
during the outbreak, serotype 1/2a l. monocytogenes isolates were recovered from clinical samples, contaminated food products and the food processing environment that shared the two asci pfge patterns observed for the sequenced isolates .
to determine the distribution of ϕlmc <dig> and plm <dig> amongst outbreak-associated isolates, pcr-based screening was conducted by targeting ϕlmc <dig> loci encoding a putative phage terminase large subunit and phage tail tape measure protein, and plm <dig> loci encoding vird <dig> and fic .
all isolates with asci pattern lmaci.
<dig> lacked the ϕlmc <dig> determinants by pcr, confirming that this novel sequence insertion accounts for the restriction enzyme pattern shift relative to pfge pattern lmaci.
<dig>  bacteriophage insertions have previously been recognized as contributing to macromolecular genetic diversity between related outbreak-associated l. monocytogenes strains  <cit> .
the pcr screening assays for plasmid determinants confirmed that plm <dig> was absent in isolate 08- <dig>  and this plasmid was also absent in other outbreak-related isolates , including one clinical isolate .
the sequence of plm <dig> revealed an absence of asci restriction sites and presence of a single apai restriction site, but this element did not contribute to the pfge patterns resulting from these enzymes.
in contrast, plasmid plm <dig>  had been reported as visible in apai pfge digests  <cit> .
just as a variable presence of plasmid plm <dig> was observed in this canadian outbreak, plasmid plm <dig> was sporadically present in isolates during the 1998- <dig> multi-state serotype 4b frankfurter usa outbreak  <cit> .
lack of stable vertical transmission of plm <dig> during the outbreak or laboratory culture  might account for plasmid absence.
alternatively, plm <dig> may have been carried only in a subset of l. monocytogenes strains that subsequently caused the outbreak.
presence of determinants related to bacterial conjugation  further confounds any speculation on the transmission of this plasmid.
whole genome comparisons enable construction of an evolutionary model of the outbreak isolates
the genomes of 08- <dig> and 08- <dig> were collinear, and with the exception of the prophage and plasmid unique to 08- <dig>  all genetic diversity was accounted for by  <dig> short indels and  <dig> confirmed snps.
the two indels included a single c/t base pair insertion in 08- <dig> that introduced a frameshift truncation into coding sequence lm5578_ <dig> .
secondly, there was a  <dig> bp  in-frame deletion in the gltx gene  of 08- <dig> encoding a putative glutamyl-trna synthetase.
dna sequencing confirmed that this gltx deletion was present in all examined lmaci.
<dig> isolates but that a wild type allele was present in all isolates typed as lmaci.
<dig> .
alternatively, a wild type buk gene was only observed in isolate 08- <dig> and a truncated butyrate kinase was encoded in all other isolates, independent of pfge pattern .
directed pcr and dna sequencing of the snp sites within the panel of clinical, food and environmental isolates was also performed .
of the  <dig> snps,  <dig> were only present in isolate 08- <dig>  the singular remaining non-coding snp was observed in each of lmaci.
<dig> isolates  but was absent in all of the lmaci.
<dig> isolates, including 08- <dig>
the  <dig> snps observed in the genome of clinical isolate 08- <dig> included  <dig> non-synonymous,  <dig> synonymous and  <dig> intergenic mutations .
such a predominance of non-synonymous changes suggests that isolate 08- <dig> was under strong positive selection for functional divergence rather than purifying selection .
the non-synonymous mutations occurred in nine cog categories, including four changes related to transcription, but no mutations occurred in cogs related to dna repair, recombination or replication .
only four non-synonymous changes represented functionally analogous amino acid changes  and none were in exported proteins carrying signal peptides.
to place this finding into context, listeria species have been reported as having the strongest purifying selection  of all the prokaryotes following comparison of multiple completed genome sequences  <cit> .
furthermore, genome sequencing of four serotype 1/2a lineage ii isolates from an endemic clone contaminating a single food production facility from  <dig> to  <dig> revealed only  <dig> total snps outside of the bacteriophage determinants, and a maximum of  <dig> snps were observed between any two given strains  <cit> .
this low number of snps in an endemic clone spanning over  <dig> years suggested that non-controlled natural populations of l. monocytogenes are stable and exhibit limited genetic micro-diversity  <cit> .
if a similar mutation rate occurred in the serotype 1/2a l. monocytogenes isolates recovered during this  <dig> canadian outbreak, passage of several decades would be expected in order to attain  <dig> total snps.
we are assuming that 08- <dig> shares a very recent ancestor as 08- <dig> and thus speculate that 08- <dig> was subjected to pressures resulting in elevated mutation rates and an abundance of non-synonymous changes.
snp coordinates are in relation to the genome of 08- <dig> and the nucleotide composition refers to the positive dna strand unless the snp lies within a coding sequence on the negative  strand, and in these instances are marked in brackets.
we propose a model to describe the chromosomal evolution of strains involved in this nation-wide outbreak using the distribution and segregation of genetic traits such as snps, indels and prophage .
this model assumes that the last common ancestor  of all strains encoded wild type buk and gltx loci, ϕlmc <dig> was absent, and the wild type state of all snp positions was represented by the sequences in 08- <dig> with the exception of intergenic snp at  <dig>  isolate 08- <dig> is predicted to be a direct descendent of the lca, as it is the only isolate encoding a wild type buk gene.
the remainder of the lmaci.
<dig> isolates differed from the lca by only the single frameshift in buk, generating ancestor  <dig> .
from ancestor  <dig>  acquisition of three independent traits  occurred, resulting in a third lineage defined by the lmaci.
<dig> pfge pattern.
in light of the high numbers of non-synonymous snps in 08- <dig> and macromolecular genetic changes such as bacteriophage integration in the lineage represented by 08- <dig>  we speculate that three distinct, but highly related strains were circulating in this outbreak .
the diversifying snps in 08- <dig> may have occurred in response to adverse environmental conditions prior to infection or host-specific conditions during infection, and ϕlmc <dig> likely arose by acquisition from other natural populations in the affected production facility.
genome sequence alignments reveal that the outbreak isolates belong to evolutionary lineage ii - clonal complex 8
phylogenetic comparison to listeria genome sequences representing each of the three l. monocytogenes evolutionary lineages, l. welshimeri and l. innocua indicated that 08- <dig> and 08- <dig> are both lineage ii strains .
the most closely related genome was strain egde, a serotype 1/2a lab-passaged animal isolate from  <dig>  <cit> .
for detection of large-scale genomic rearrangements, a dot plot comparison of 08- <dig> versus the egde genome sequence was performed .
this revealed that the common chromosomal sequences were collinear, with the exception of a large  <dig> kbp inverted segment around the putative origin of replication  .
symmetric inversions around the origin of replication have previously been observed between other bacterial genome sequences  <cit> .
in contrast to only  <dig> snps being observed between 08- <dig> and 08- <dig>   <dig> high quality snps and  <dig> estimated indels were revealed in a comparison of 08- <dig> and egde .
these included  <dig> synonymous snps,  <dig> non-synonymous snps and  <dig> indels occurring within  <dig> predicted coding sequences, with  <dig> to  <dig> non-synonymous snps and/or indels occurring per gene.
moreover, two large contiguous regions present in both 08- <dig> and 08- <dig> represent probable prophages  that are absent in egde .
ϕlmc <dig> was highly related to phage b <dig>  and ϕlmc <dig> was inserted at comk and related to listeria phage a <dig>
comparative analysis of the regions used for multi-locus sequence typing  indicated that isolates 08- <dig> and 08- <dig> are more closely related to lineage ii clonal complex  <dig>  than to egde .
the mlst determinants used by ragon et.
al.
<cit>  were extracted in silico from the genome sequences and 08- <dig> was identical to a previously characterized cc <dig>  sequence type  <dig>  strain .
the extracted sequences for 08- <dig> were also identical to st <dig> except for one snp within the abcz locus not currently listed in the institute pasteur's l. monocytogenes mlst database.
the sole previous member of st <dig> was identified as a clinical serotype 1/2a isolate derived from a cns infection in new zealand in  <dig>  <cit> .
phylogenetic trees based on mlst and whole genome sequences were relatively congruent when considering evolutionary lineages, however genome sequences provide a more precise placement relative to other examined isolates.
the pyrosequencing data also provided the opportunity to analyze the entire genome for micro and macro-diversity, including the full repertoire of phylogenetically relevant loci.
virulence-associated determinants of the outbreak isolates informs lineage- and strain-specific virulence potential
the surface protein internalin, encoded by the inla locus, is a principal virulence determinant promoting mammalian host cell invasion via interaction with e-cadherin on epithelial cells  <cit> .
truncation variants of inla have been associated with decreased pathogenicity and significantly reduced invasiveness for human intestinal epithelial cells  <cit> .
using nomenclature assigned by ragon et al.
<cit> , isolates 08- <dig> and 08- <dig> both encode a full length inla allele type  <dig> that has previously been observed in isolates related to human illness  <cit> .
this singular determinant however cannot account for differences in pathogenicity since foodborne isolates have been observed with intact inla loci  <cit>  and inla allele  <dig> is also encoded by environmental isolates  <cit> .
l. monocytogenes does encode multiple additional internalin and internalin-like coding sequences, although the function of each paralog is currently unknown  <cit> .
isolates 08- <dig> and 08- <dig> both encode inlb, inlc, inlc <dig>  inld, inle, inlf, inlg, inli, inlj and  <dig> other leucine-rich internalin-like coding sequences .
the presence of an intact inla locus and this compilation of other internalin-like loci may account in part for the pathogenicity of the serotype 1/2a outbreak strains sequenced in this study, but this requires further examination.
the listeria pathogenicity island  <dig>  encodes six significant virulence-associated loci: prfa , plca, plcb , hly , mpl  and acta .
previous phylogenetic analysis of this region from food, clinical, animal and environmental isolates indicated that this gene cluster was genetically diverse and clustered based on the three l. monocytogenes evolutionary lineages  <cit> .
blastn comparative analyses of this ~ <dig> kbp region from 08- <dig> and 08- <dig> identified a very high sequence identity with other lineage ii isolates, including  <dig> % identity to the edge reference genome; ≥ 98% identity to all  <dig> lineage ii isolates characterized by ward et al.
<cit> , and ≥ 98% nucleotide identity to all  <dig> lineage ii isolates characterized by orsi et al.
<cit> .
in comparison, all lipi- <dig> regions encoded by evolutionary lineage i and iii strains in these previous studies were ≤ 95% identical to our lineage ii strains.
prfa regulates additional virulence determinants encoded elsewhere on the chromosome, such as bsh encoding a bile salt hydrolase that promotes survival within the gut  <cit>  and uhpt encoding for a hexose phosphate permease for utilization of host carbon sources  <cit> .
these latter two determinants were also encoded within 08- <dig> and 08- <dig>  the presence of these determinants along with lipi- <dig> is well conserved across l. monocytogenes strains independent of evolutionary lineage, so are unlikely to enhance virulence potential unless the allelic diversity observed between lineages results in a phenotypic change.
in general, the contribution of lineage-specific genome differences to virulence and pathogenicity of l. monocytogenes are not well understood.
for example, recent in silico analyses have indicated that while there are lineage ii-specific genomic regions not present in lineage i, their role in virulence is not clear  <cit> .
furthermore, the presence of the listeria pathogenicity island lipi- <dig> operon encoding listeriolysin s has been associated with evolutionary lineage i strains causing foodborne outbreaks  <cit> .
this region was absent in the genomes of both 08- <dig> and 08- <dig>
outbreak isolates harbor a putative mobile genetic island encoding translocation functions
a large  <dig>  kbp contiguous region  was present in both sequenced genomes yet absent in all publicly available listeria genome sequences to date, including egde .
the bordering coding sequences lm5578_ <dig> and lm5578_ <dig> were each 98% identical to contiguous egde coding sequences lmo <dig> and lmo <dig>  respectively, implying that the  <dig> kbp operon-like structured region represents a genomic insertion within the ancestral chromosome of these isolates.
accordingly, putative serine recombinases are encoded in this region  and  <dig> bp imperfect inverted repeats are present at the borders in the intergenic regions between loci 1849/ <dig> and 1903/ <dig> , indicating that this is a horizontally acquired genetic island, hereafter designated listeria genomic island  <dig> .
coding sequences within lgi <dig> exhibited sequence homology and were similarly organized as contiguous regions present within several environmental firmicutes, including clostridium kluyveri, c. bolteae and desulfitobacterium hafniense.
these lgi1-like regions also appeared to be horizontally-acquired, based on skewed g/c content relative to neighboring sequences  <cit> .
the genetic organization and predicted functions of several lgi <dig> loci resembled the proposed b. anthracis pxo <dig> plasmid-encoded secretion system  <cit> , including several coding sequences that resembled putative type ii and type iv secretion systems  .
prototypical members of these systems are involved in pilus biogenesis and translocation of dna-protein complexes or virulence effectors, respectively.
canonical t4ss genes predicted within lgi <dig> include virb <dig>  vird <dig>  and virb <dig>  which encode atpases involved in substrate recruitment to the transfer complex and substrate translocation, and virb <dig> and virb <dig> subunit genes, which form the core membrane-spanning transfer complex .
genes that likely contribute to pilus-like surface appendages were also detected , and the presence of a dnag gene encoding a putative primase neighboring the vird <dig> coupling protein homolog suggests that this genetic island may be mobilizable .
besides pxo <dig>  type iv secretion-like systems  features have also been described in gram-positive plasmids such as b. anthracis plasmid pxo <dig>  <cit>  and the pheromone-inducible conjugative plasmid pcf <dig> of enterococcus faecalis  <cit> .
gram-positive t4sls also encode a putative peptidoglycan hydrolase and adhesin, both of which are predicted functions in lgi <dig> .
lm5788_ <dig> encodes a c-terminal nlpc/p <dig> domain, and is predicted to be involved in assembly of the core substrate transfer complex as a virb1-like peptidoglycan hydrolase.
a p <dig> domain is also present in the autolytic virulence determinant designated listeria invasion associated protein   <cit> .
lm5578_ <dig> is predicted to encode a sel1-like repeat  family protein that contains helical domains mediating protein-protein interactions  <cit> .
although protein adhesins are often used in gram-positive bacteria to mediate conjugative attachment to target cells, slr proteins also are involved for eukaryotic cell entry by legionella pneumophilia  <cit> .
whether this t4sls is involved in translocation of pathogenicity effector molecule  is tempting to speculate, but further study is required.
lgi <dig> also encoded a homolog  to the multidrug efflux proton:drug antiporter emre implicated in resistance to toxic cationic hydrophobic compounds such as quaternary ammonium compounds and tetracycline  <cit> .
this gene was flanked by a marr-family transcriptional regulator loci  and a putative dna-directed rna polymerase sigma- <dig> subunit  encoding a specialized ecf  family sigma factor, which is part of the bacterial stress response regulon  <cit> .
a two component signal transduction system  and restriction modification components  also were present.
cumulatively, lgi <dig> is unique compared to all currently sequenced l. monocytogenes but the contribution to pathogenicity or environmental persistence is unconfirmed.
CONCLUSIONS
high-throughput dna sequencing rapidly provided the complete genetic content of two l. monocytogenes outbreak isolates.
within three days of project commencement, draft genome sequences were available that were suitable to begin comparative analyses such as genome alignments, preliminary annotation of coding sequences, and identification of traits associated with macro-diversity and micro-diversity.
this allowed us to determine evolutionary lineages and unequivocally define the full breadth of genetic variation between two subtype variants identified by the internationally pulsenet standardized pfge typing method.
whole genome sequencing therefore enabled robust real-time characterization of virulence determinants and genetic diversity  within a natural l. monocytogenes population.
these novel markers were then applied for a rapid assessment of the genetic relatedness of additional clinical, food, and environmental isolates recovered during the outbreak.
the distribution of the snp, indel and prophage traits indicated that three distinct but highly related strains were likely involved.
further characterization of the snps indicated that clinical isolate 08- <dig> was likely subjected to positive selective pressures resulting in a higher frequency of non-synonymous mutation than would normally be expected for l. monocytogenes.
selective pressure for adaptive change could have resulted from host specific conditions during infection or adverse food storage conditions, but with only a single available isolate representative of this snp genotype it is not possible to identify the micro-diversification timeline.
previous comparative genomic studies of outbreak-associated l. monocytogenes have found several strain and serotype-specific coding sequences between serotype 4b and 1/2a strains, but amongst these there were few relevant genetic traits suggestive of virulence potential  <cit> .
one notable exception was variation in the complement of internalin-like coding determinants  <cit> , and consistent with this finding, a large composition of internalin-like determinants was observed in the current study.
in addition, there were two large-scale genetic insertions unique to our serotype 1/2a strains.
prophage ϕlmc <dig> was composed of genes related to previously characterized phage determinants, but on the whole, represented a novel listeria phage.
a ~ <dig> kbp genetic island  unique amongst all other currently sequenced l. monocytogenes isolates was also present in 08- <dig>  encoding putative translocation, resistance, and regulatory determinants.
while l. monocytogenes serotype 1/2a can be frequently isolated from food processing environments, the majority of invasive listeriosis outbreaks to date have resulted from lineage i serotype 4b strains  <cit> .
lineage ii serotype 1/2a strains have more frequently been associated with outbreaks of listerial gastroenteritis  <cit> .
our current study demonstrates that lineage ii strains can also cause large outbreaks of severe invasive disease and is consistent with a global trend towards serotype 1/2a predominance.
whole genome sequencing allowed us to detect within these outbreak isolates a repertoire of genetic determinants involved in diversification and microevolution.
these features may have a role in virulence and pathogenicity, as well as survival within food processing environments and in foods.
genomic studies can therefore facilitate a greater understanding of the lineage-specific and strain-specific features of l. monocytogenes and how they contribute to this pathogen's ecology and virulence.
this knowledge may ultimately lead to the development of methods to better assess the risks posed by individual l. monocytogenes strains.
this study also provides a proof-of-concept that the latest generation dna sequencing platforms have a place in real-time public health responses to bacterial pathogens.
whole genome sequencing may not be ready to be applied routinely as a subtyping method, but public health laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole genome sequences in the context of molecular epidemiology.
most of the current subtyping methods such as pfge and mlst capture only a small proportion of the true genetic content, so it is still difficult to interpret robust data sets such as whole genomes when all previous characterizations have been comparatively limited in detail.
it is foreseeable that the burgeoning capacity for whole genome sequencing will soon provide a cost effective alternative to the current subtyping methods after a reconciliation has been made between the true nature of subtypes, the diversity revealed by classical methods and the micro- and macro-diversity readily identified by next-generation sequencing technologies.
as more genomes from clinical isolates are completed and compared , it will become increasingly feasible to apply genome sequencing for responses to bacterial outbreaks.

 listeria monocytogenes is a gram-positive, facultative intracellular bacterial pathogen that can cause severe disease in humans, other mammals and birds. human listeriosis is relatively rare despite our likely frequent encounters with l. monocytogenes, which is ubiquitously present in the environment, farm and rural environments, and urban environments. the vast majority of human listeriosis is foodborne and the most commonly implicated vehicles are ready-to-eat food products such as meat, dairy, seafood, and fresh produce that are contaminated with l. monocytogenes during processing. l. monocytogenes can be introduced into food processing facilities and food products due to cross-contamination with environmental sources or from the feces of food production animals. l. monocytogenes can persist within food processing environments for long periods of time, due in part to its ability to grow at wide-ranging temperatures and ph and the ability to form biofilms promoting adherence to food processing surfaces. the persistence of a single subtype of l. monocytogenes in processing facilities or on equipment has been reported from several months to more than years. given the widespread occurrence of l. monocytogenes, subtyping of clinical and food isolates is required to establish epidemiologic links during routine surveillance, outbreak investigations, and for source tracking. there are known serotypes of l. monocytogenes but the vast majority of human disease cases are caused by strains belonging to serotypes 4b, 1/2a, and 1/2b, severely limiting the utility of this subtyping method for differentiating l. monocytogenes. large clonal outbreaks owing to contaminated food sources such as coleslaw, milk, cheese, hot dogs and deli meats, have been predominately caused by serotypes 4b and 1/2a strains. accordingly, additional subtyping methods are required to better characterize outbreak isolates. several molecular subtyping methods have been developed and applied to l. monocytogenes, including pulsed-field gel electrophoresis, ribotyping, multilocus variable-number tandem repeat analysis, and sequence-based subtyping. pfge has been adopted by pulsenet as the internationally standardized method for molecular subtyping of l.monocytogenes and has been essential in the detection and investigation of listeriosis outbreaks in canada and worldwide. dna sequencing has enabled analyses of l. monocytogenes genomes and furthered the understanding of this pathogen's biology and phylogeny. comparative analyses of genome sequences have elucidated the genetic differences between l. monocytogenes serotypes, the acquisition and evolution of virulence and pathogenic traits among listeria spp., and the genetic basis underlying the unique survival and growth characteristics of l. monocytogenes. genome sequencing has also permitted the development of multi-locus sequence typing protocols, which in combination with other typing methods, have validated three evolutionary lineages for l. monocytogenes. the lineages reflect the serotype distribution, with serotypes 1/2b, 3b and 4b segregating to lineage i and serotypes 1/2a and 1/2c segregating to lineage ii. risk assessments based upon genetic lineages have suggested that l. monocytogenes subtypes may have unique ecological niches and may also differ in their pathogenic potential or host adaptations. the majority of available bacterial genome sequences have been generated using the sanger chain termination sequencing chemistries. although this technology was instrumental in the emergence of the field of genomics, it is time and resource intensive. several post-sanger sequencing technologies have since been developed that enable extremely rapid whole-genome sequencing and a broader application of comparative genomics. the high-throughput pyrosequencing method commercialized as the or gs flx™ platform has been applied for rapidly determining the genome sequences of bacterial isolates. l. monocytogenes serotype 1/2a caused a nationwide outbreak of listeriosis associated with ready-to-eat meat products in canada during the summer of the outbreak resulted in deaths and at least illnesses. whereas the majority of outbreak-associated isolates had an indistinguishable asci pfge pattern, one clinical isolate from ontario and several isolates from the affected food production facility exhibited an asci pfge pattern that differed by a single restriction enzyme fragment compared to the primary outbreak strain. we report herein the first real-time application of whole-genome sequencing during an active listeriosis outbreak investigation. high-throughput pyrosequencing was applied to characterize two outbreak-associated isolates of l. monocytogenes in order to obtain a thorough genetic characterization of the outbreak strains and to determine the genetic basis for the single asci restriction fragment change.  primary outbreak isolate harbors unique plasmid plm and prophage ϕlmc1 public health responses to foodborne outbreaks require bacterial subtyping data to definitively link affected individuals with contaminated food sources and other suspected patients. the genetic relatedness between clinical and food isolates is routinely determined using methods such as pfge, however, with the latest generation of dna sequencing technologies it is theoretically possible to now determine the complete genetic blueprint of bacterial isolates within the scope of an outbreak. in this study, draft genome sequences suitable for comparative analysis between outbreak isolates and to reference genomes were available within three days. these data allowed for discovery of novel prophage and genomic islands, and the subsequent sequence closure and the confirmation of sequence variants allowed for annotation of polymorphisms and other traits associated with micro-diversity. clinical isolate 08- was selected as the reference outbreak strain and clinical isolate 08- differed only in the asci restriction pattern. high-throughput pyrosequencing was performed on both isolates and closed circular chromosomal sequences were obtained for each isolate. a kbp contig present in the 08- sequence assembly but absent in 08- was identified as a circular plasmid and designated plm. comparison of the chromosomal sequences for isolates 08- and 08- with publicly available listeria spp. genomes and with select loci used for mlst indicated that these isolates were members of evolutionary lineage ii and clonal complex. a.'rte', ready to eat;'csf', cerebrospinal fluid b.'+', amplicon of expected size detected using pcr using oligonucleotides described in additional file 6;'-', no amplicon detected c.'fs', frameshift resulting in a truncated coding sequence encoding butyrate kinase;'wt', wild type. confirmed by sanger-based dna sequencing of amplicons generated by high fidelity pcr. d.'wt', wild type;'- bp', in-frame deletion of codons. confirmed by sanger-based dna sequencing of amplicons generated by pcr. e. number of snp's relative to the hypothetical last common ancestor presented in fig. sanger-based dna sequence confirmation of snps was completed after pcr amplification using oligonucleotides described in additional file the asci restriction patterns of 08- and 08- were indistinguishable, except for a single band shift of approximately kbp. accordingly, comparative sequence alignment revealed a kbp contiguous region integrated adjacent to the trna-ser gene that was unique to 08- and encoding several putative bacteriophage-related coding sequences. blast analyses indicated that this prophage was unique among sequenced listeria genomes, although it was comprised of coding sequences similar to determinants from several known phages associated with gram-positive microbes, including l. monocytogenes phages a a and b. the presence of prophage ϕlmc accounted for the asci restriction pattern difference between isolates 08- and 08- the extrachromosomal circular plasmid harbored in isolate 08- encoded putative determinants for replication, partitioning, heavy metal resistance and dna translocation. many of these features were homologous to sequences encoded by plasmids from l. innocua, l. monocytogenes serotype 4b isolate h and bacillus anthracis, including a replication and regulation control center. two regions of plm were related to heavy metal resistance, including a transposon-associated cada that exhibited high sequence identity to pli a second, distinct region unique to plm was comprised of cada and cadc genes, with the latter encoding a cadmium-efflux arsr-family regulatory accessory protein homolog. resistance to the heavy metal cadmium has been recently discovered to be associated with resistance to quaternary ammonium compounds, which may contribute to l. monocytogenes persistence in food production facilities. notably, several coding sequences exhibited sequence similarity to type iv secretion systems, including dna processing, dna coupling and membrane channel formation. additional predicted membrane-associated proteins were present within this region, and all coding sequences were oriented in the same direction, which is consistent with the operon-style organization of most known t4ss. varying degrees of similarity between pli plm and pxo plasmids have already been established, and our data further support a chimeric nature for listeria plasmids. plm and ϕlmc exhibit non-homogeneous distribution within a wider panel of outbreak-associated isolates during the outbreak, serotype 1/2a l. monocytogenes isolates were recovered from clinical samples, contaminated food products and the food processing environment that shared the two asci pfge patterns observed for the sequenced isolates. to determine the distribution of ϕlmc and plm amongst outbreak-associated isolates, pcr-based screening was conducted by targeting ϕlmc loci encoding a putative phage terminase large subunit and phage tail tape measure protein, and plm loci encoding vird and fic. all isolates with asci pattern lmaci. lacked the ϕlmc determinants by pcr, confirming that this novel sequence insertion accounts for the restriction enzyme pattern shift relative to pfge pattern lmaci. bacteriophage insertions have previously been recognized as contributing to macromolecular genetic diversity between related outbreak-associated l. monocytogenes strains. the pcr screening assays for plasmid determinants confirmed that plm was absent in isolate 08- and this plasmid was also absent in other outbreak-related isolates, including one clinical isolate. the sequence of plm revealed an absence of asci restriction sites and presence of a single apai restriction site, but this element did not contribute to the pfge patterns resulting from these enzymes. in contrast, plasmid plm had been reported as visible in apai pfge digests. just as a variable presence of plasmid plm was observed in this canadian outbreak, plasmid plm was sporadically present in isolates during the 1998- multi-state serotype 4b frankfurter usa outbreak. lack of stable vertical transmission of plm during the outbreak or laboratory culture might account for plasmid absence. alternatively, plm may have been carried only in a subset of l. monocytogenes strains that subsequently caused the outbreak. presence of determinants related to bacterial conjugation further confounds any speculation on the transmission of this plasmid. whole genome comparisons enable construction of an evolutionary model of the outbreak isolates the genomes of 08- and 08- were collinear, and with the exception of the prophage and plasmid unique to 08- all genetic diversity was accounted for by short indels and confirmed snps. the two indels included a single c/t base pair insertion in 08- that introduced a frameshift truncation into coding sequence lm5578_. secondly, there was a bp in-frame deletion in the gltx gene of 08- encoding a putative glutamyl-trna synthetase. dna sequencing confirmed that this gltx deletion was present in all examined lmaci. isolates but that a wild type allele was present in all isolates typed as lmaci.. alternatively, a wild type buk gene was only observed in isolate 08- and a truncated butyrate kinase was encoded in all other isolates, independent of pfge pattern. directed pcr and dna sequencing of the snp sites within the panel of clinical, food and environmental isolates was also performed. of the snps, were only present in isolate 08- the singular remaining non-coding snp was observed in each of lmaci. isolates but was absent in all of the lmaci. isolates, including 08- the snps observed in the genome of clinical isolate 08- included non-synonymous, synonymous and intergenic mutations. such a predominance of non-synonymous changes suggests that isolate 08- was under strong positive selection for functional divergence rather than purifying selection. the non-synonymous mutations occurred in nine cog categories, including four changes related to transcription, but no mutations occurred in cogs related to dna repair, recombination or replication. only four non-synonymous changes represented functionally analogous amino acid changes and none were in exported proteins carrying signal peptides. to place this finding into context, listeria species have been reported as having the strongest purifying selection of all the prokaryotes following comparison of multiple completed genome sequences. furthermore, genome sequencing of four serotype 1/2a lineage ii isolates from an endemic clone contaminating a single food production facility from to revealed only total snps outside of the bacteriophage determinants, and a maximum of snps were observed between any two given strains. this low number of snps in an endemic clone spanning over years suggested that non-controlled natural populations of l. monocytogenes are stable and exhibit limited genetic micro-diversity. if a similar mutation rate occurred in the serotype 1/2a l. monocytogenes isolates recovered during this canadian outbreak, passage of several decades would be expected in order to attain total snps. we are assuming that 08- shares a very recent ancestor as 08- and thus speculate that 08- was subjected to pressures resulting in elevated mutation rates and an abundance of non-synonymous changes. snp coordinates are in relation to the genome of 08- and the nucleotide composition refers to the positive dna strand unless the snp lies within a coding sequence on the negative strand, and in these instances are marked in brackets. we propose a model to describe the chromosomal evolution of strains involved in this nation-wide outbreak using the distribution and segregation of genetic traits such as snps, indels and prophage. this model assumes that the last common ancestor of all strains encoded wild type buk and gltx loci, ϕlmc was absent, and the wild type state of all snp positions was represented by the sequences in 08- with the exception of intergenic snp at isolate 08- is predicted to be a direct descendent of the lca, as it is the only isolate encoding a wild type buk gene. the remainder of the lmaci. isolates differed from the lca by only the single frameshift in buk, generating ancestor. from ancestor acquisition of three independent traits occurred, resulting in a third lineage defined by the lmaci. pfge pattern. in light of the high numbers of non-synonymous snps in 08- and macromolecular genetic changes such as bacteriophage integration in the lineage represented by 08- we speculate that three distinct, but highly related strains were circulating in this outbreak. the diversifying snps in 08- may have occurred in response to adverse environmental conditions prior to infection or host-specific conditions during infection, and ϕlmc likely arose by acquisition from other natural populations in the affected production facility. genome sequence alignments reveal that the outbreak isolates belong to evolutionary lineage ii - clonal complex 8 phylogenetic comparison to listeria genome sequences representing each of the three l. monocytogenes evolutionary lineages, l. welshimeri and l. innocua indicated that 08- and 08- are both lineage ii strains. the most closely related genome was strain egde, a serotype 1/2a lab-passaged animal isolate from. for detection of large-scale genomic rearrangements, a dot plot comparison of 08- versus the egde genome sequence was performed. this revealed that the common chromosomal sequences were collinear, with the exception of a large kbp inverted segment around the putative origin of replication. symmetric inversions around the origin of replication have previously been observed between other bacterial genome sequences. in contrast to only snps being observed between 08- and 08- high quality snps and estimated indels were revealed in a comparison of 08- and egde. these included synonymous snps, non-synonymous snps and indels occurring within predicted coding sequences, with to non-synonymous snps and/or indels occurring per gene. moreover, two large contiguous regions present in both 08- and 08- represent probable prophages that are absent in egde. ϕlmc was highly related to phage b and ϕlmc was inserted at comk and related to listeria phage a comparative analysis of the regions used for multi-locus sequence typing indicated that isolates 08- and 08- are more closely related to lineage ii clonal complex than to egde. the mlst determinants used by ragon et. al. were extracted in silico from the genome sequences and 08- was identical to a previously characterized cc sequence type strain. the extracted sequences for 08- were also identical to st except for one snp within the abcz locus not currently listed in the institute pasteur's l. monocytogenes mlst database. the sole previous member of st was identified as a clinical serotype 1/2a isolate derived from a cns infection in new zealand in. phylogenetic trees based on mlst and whole genome sequences were relatively congruent when considering evolutionary lineages, however genome sequences provide a more precise placement relative to other examined isolates. the pyrosequencing data also provided the opportunity to analyze the entire genome for micro and macro-diversity, including the full repertoire of phylogenetically relevant loci. virulence-associated determinants of the outbreak isolates informs lineage- and strain-specific virulence potential the surface protein internalin, encoded by the inla locus, is a principal virulence determinant promoting mammalian host cell invasion via interaction with e-cadherin on epithelial cells. truncation variants of inla have been associated with decreased pathogenicity and significantly reduced invasiveness for human intestinal epithelial cells. using nomenclature assigned by ragon et al., isolates 08- and 08- both encode a full length inla allele type that has previously been observed in isolates related to human illness. this singular determinant however cannot account for differences in pathogenicity since foodborne isolates have been observed with intact inla loci and inla allele is also encoded by environmental isolates. l. monocytogenes does encode multiple additional internalin and internalin-like coding sequences, although the function of each paralog is currently unknown. isolates 08- and 08- both encode inlb, inlc, inlc inld, inle, inlf, inlg, inli, inlj and other leucine-rich internalin-like coding sequences. the presence of an intact inla locus and this compilation of other internalin-like loci may account in part for the pathogenicity of the serotype 1/2a outbreak strains sequenced in this study, but this requires further examination. the listeria pathogenicity island encodes six significant virulence-associated loci: prfa, plca, plcb, hly, mpl and acta. previous phylogenetic analysis of this region from food, clinical, animal and environmental isolates indicated that this gene cluster was genetically diverse and clustered based on the three l. monocytogenes evolutionary lineages. blastn comparative analyses of this ~ kbp region from 08- and 08- identified a very high sequence identity with other lineage ii isolates, including % identity to the edge reference genome; ≥ 98% identity to all lineage ii isolates characterized by ward et al., and ≥ 98% nucleotide identity to all lineage ii isolates characterized by orsi et al.. in comparison, all lipi- regions encoded by evolutionary lineage i and iii strains in these previous studies were ≤ 95% identical to our lineage ii strains. prfa regulates additional virulence determinants encoded elsewhere on the chromosome, such as bsh encoding a bile salt hydrolase that promotes survival within the gut and uhpt encoding for a hexose phosphate permease for utilization of host carbon sources. these latter two determinants were also encoded within 08- and 08- the presence of these determinants along with lipi- is well conserved across l. monocytogenes strains independent of evolutionary lineage, so are unlikely to enhance virulence potential unless the allelic diversity observed between lineages results in a phenotypic change. in general, the contribution of lineage-specific genome differences to virulence and pathogenicity of l. monocytogenes are not well understood. for example, recent in silico analyses have indicated that while there are lineage ii-specific genomic regions not present in lineage i, their role in virulence is not clear. furthermore, the presence of the listeria pathogenicity island lipi- operon encoding listeriolysin s has been associated with evolutionary lineage i strains causing foodborne outbreaks. this region was absent in the genomes of both 08- and 08- outbreak isolates harbor a putative mobile genetic island encoding translocation functions a large kbp contiguous region was present in both sequenced genomes yet absent in all publicly available listeria genome sequences to date, including egde. the bordering coding sequences lm5578_ and lm5578_ were each 98% identical to contiguous egde coding sequences lmo and lmo respectively, implying that the kbp operon-like structured region represents a genomic insertion within the ancestral chromosome of these isolates. accordingly, putative serine recombinases are encoded in this region and bp imperfect inverted repeats are present at the borders in the intergenic regions between loci 1849/ and 1903/, indicating that this is a horizontally acquired genetic island, hereafter designated listeria genomic island. coding sequences within lgi exhibited sequence homology and were similarly organized as contiguous regions present within several environmental firmicutes, including clostridium kluyveri, c. bolteae and desulfitobacterium hafniense. these lgi1-like regions also appeared to be horizontally-acquired, based on skewed g/c content relative to neighboring sequences. the genetic organization and predicted functions of several lgi loci resembled the proposed b. anthracis pxo plasmid-encoded secretion system, including several coding sequences that resembled putative type ii and type iv secretion systems. prototypical members of these systems are involved in pilus biogenesis and translocation of dna-protein complexes or virulence effectors, respectively. canonical t4ss genes predicted within lgi include virb vird and virb which encode atpases involved in substrate recruitment to the transfer complex and substrate translocation, and virb and virb subunit genes, which form the core membrane-spanning transfer complex. genes that likely contribute to pilus-like surface appendages were also detected, and the presence of a dnag gene encoding a putative primase neighboring the vird coupling protein homolog suggests that this genetic island may be mobilizable. besides pxo type iv secretion-like systems features have also been described in gram-positive plasmids such as b. anthracis plasmid pxo and the pheromone-inducible conjugative plasmid pcf of enterococcus faecalis. gram-positive t4sls also encode a putative peptidoglycan hydrolase and adhesin, both of which are predicted functions in lgi. lm5788_ encodes a c-terminal nlpc/p domain, and is predicted to be involved in assembly of the core substrate transfer complex as a virb1-like peptidoglycan hydrolase. a p domain is also present in the autolytic virulence determinant designated listeria invasion associated protein. lm5578_ is predicted to encode a sel1-like repeat family protein that contains helical domains mediating protein-protein interactions. although protein adhesins are often used in gram-positive bacteria to mediate conjugative attachment to target cells, slr proteins also are involved for eukaryotic cell entry by legionella pneumophilia. whether this t4sls is involved in translocation of pathogenicity effector molecule is tempting to speculate, but further study is required. lgi also encoded a homolog to the multidrug efflux proton:drug antiporter emre implicated in resistance to toxic cationic hydrophobic compounds such as quaternary ammonium compounds and tetracycline. this gene was flanked by a marr-family transcriptional regulator loci and a putative dna-directed rna polymerase sigma- subunit encoding a specialized ecf family sigma factor, which is part of the bacterial stress response regulon. a two component signal transduction system and restriction modification components also were present. cumulatively, lgi is unique compared to all currently sequenced l. monocytogenes but the contribution to pathogenicity or environmental persistence is unconfirmed.  high-throughput dna sequencing rapidly provided the complete genetic content of two l. monocytogenes outbreak isolates. within three days of project commencement, draft genome sequences were available that were suitable to begin comparative analyses such as genome alignments, preliminary annotation of coding sequences, and identification of traits associated with macro-diversity and micro-diversity. this allowed us to determine evolutionary lineages and unequivocally define the full breadth of genetic variation between two subtype variants identified by the internationally pulsenet standardized pfge typing method. whole genome sequencing therefore enabled robust real-time characterization of virulence determinants and genetic diversity within a natural l. monocytogenes population. these novel markers were then applied for a rapid assessment of the genetic relatedness of additional clinical, food, and environmental isolates recovered during the outbreak. the distribution of the snp, indel and prophage traits indicated that three distinct but highly related strains were likely involved. further characterization of the snps indicated that clinical isolate 08- was likely subjected to positive selective pressures resulting in a higher frequency of non-synonymous mutation than would normally be expected for l. monocytogenes. selective pressure for adaptive change could have resulted from host specific conditions during infection or adverse food storage conditions, but with only a single available isolate representative of this snp genotype it is not possible to identify the micro-diversification timeline. previous comparative genomic studies of outbreak-associated l. monocytogenes have found several strain and serotype-specific coding sequences between serotype 4b and 1/2a strains, but amongst these there were few relevant genetic traits suggestive of virulence potential. one notable exception was variation in the complement of internalin-like coding determinants, and consistent with this finding, a large composition of internalin-like determinants was observed in the current study. in addition, there were two large-scale genetic insertions unique to our serotype 1/2a strains. prophage ϕlmc was composed of genes related to previously characterized phage determinants, but on the whole, represented a novel listeria phage. a ~ kbp genetic island unique amongst all other currently sequenced l. monocytogenes isolates was also present in 08- encoding putative translocation, resistance, and regulatory determinants. while l. monocytogenes serotype 1/2a can be frequently isolated from food processing environments, the majority of invasive listeriosis outbreaks to date have resulted from lineage i serotype 4b strains. lineage ii serotype 1/2a strains have more frequently been associated with outbreaks of listerial gastroenteritis. our current study demonstrates that lineage ii strains can also cause large outbreaks of severe invasive disease and is consistent with a global trend towards serotype 1/2a predominance. whole genome sequencing allowed us to detect within these outbreak isolates a repertoire of genetic determinants involved in diversification and microevolution. these features may have a role in virulence and pathogenicity, as well as survival within food processing environments and in foods. genomic studies can therefore facilitate a greater understanding of the lineage-specific and strain-specific features of l. monocytogenes and how they contribute to this pathogen's ecology and virulence. this knowledge may ultimately lead to the development of methods to better assess the risks posed by individual l. monocytogenes strains. this study also provides a proof-of-concept that the latest generation dna sequencing platforms have a place in real-time public health responses to bacterial pathogens. whole genome sequencing may not be ready to be applied routinely as a subtyping method, but public health laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole genome sequences in the context of molecular epidemiology. most of the current subtyping methods such as pfge and mlst capture only a small proportion of the true genetic content, so it is still difficult to interpret robust data sets such as whole genomes when all previous characterizations have been comparatively limited in detail. it is foreseeable that the burgeoning capacity for whole genome sequencing will soon provide a cost effective alternative to the current subtyping methods after a reconciliation has been made between the true nature of subtypes, the diversity revealed by classical methods and the micro- and macro-diversity readily identified by next-generation sequencing technologies. as more genomes from clinical isolates are completed and compared, it will become increasingly feasible to apply genome sequencing for responses to bacterial outbreaks. 
a large, multi-province outbreak of listeriosis associated with ready-to-eat meat products contaminated with listeria monocytogenes serotype 1/2a occurred in canada in subtyping of outbreak-associated isolates using pulsed-field gel electrophoresis revealed two similar but distinct asci pfge patterns. the chromosomes were collinear, but differences included single nucleotide polymorphisms and three indels, including a kbp prophage that accounted for the observed difference in asci pfge patterns. high-throughput pyrosequencing of two l. monocytogenes isolates was used to rapidly provide the genome sequence of the primary outbreak strain and to investigate the extent of genetic diversity associated with a change of a single restriction enzyme fragment during pfge. the distribution of these traits was assessed within further clinical, environmental and food isolates associated with the outbreak, and this comparison indicated that three distinct, but highly related strains may have been involved in this nationwide outbreak. notably, these two isolates were found to harbor a kbp putative mobile genomic island encoding translocation and efflux functions that has not been observed in other listeria genomes. this study confirms that the latest generation of dna sequencing technologies can be applied during high priority public health events, and laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole genome sequences in the context of molecular epidemiology. high-throughput genome sequencing provided a more detailed real-time assessment of genetic traits characteristic of the outbreak strains than could be achieved with routine subtyping methods.
 a large, multi-province outbreak of listeriosis associated with ready-to-eat meat products contaminated with listeria monocytogenes serotype 1/2a occurred in canada in subtyping of outbreak-associated isolates using pulsed-field gel electrophoresis revealed two similar but distinct asci pfge patterns. high-throughput pyrosequencing of two l. monocytogenes isolates was used to rapidly provide the genome sequence of the primary outbreak strain and to investigate the extent of genetic diversity associated with a change of a single restriction enzyme fragment during pfge.  the chromosomes were collinear, but differences included single nucleotide polymorphisms and three indels, including a kbp prophage that accounted for the observed difference in asci pfge patterns. the distribution of these traits was assessed within further clinical, environmental and food isolates associated with the outbreak, and this comparison indicated that three distinct, but highly related strains may have been involved in this nationwide outbreak. notably, these two isolates were found to harbor a kbp putative mobile genomic island encoding translocation and efflux functions that has not been observed in other listeria genomes.  high-throughput genome sequencing provided a more detailed real-time assessment of genetic traits characteristic of the outbreak strains than could be achieved with routine subtyping methods. this study confirms that the latest generation of dna sequencing technologies can be applied during high priority public health events, and laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole genome sequences in the context of molecular epidemiology. 