The human isolate from the 1988 listeriosis case (F6900) and a human (J0161) and a food (J2818) isolate from the 2000 multi-state listeriosis outbreak in the US (Table 1) were sequenced using the 454 GS20 Genome Sequencer (454 Life Sciences, Branford, CT), which uses the "sequencing by synthesis" technology 29. Chromosomal sequences ranged from 2.96 Mb to 2. 97 Mb, the sequencing depth ranged from 24 to 29.15-fold. The sequenced genomes captured between 93.79 and 96.08% of the EGD-e reference genome (Table 2).

In addition to the chromosomal sequences, a plasmid sequence was found in the 2000 human isolate (J0161). This plasmid represented two non-overlapping contigs of 69,915 bp and 12,763 bp. These two plasmid contigs showed a very high level of nucleotide identity (>99%) with pLM80, an approx. 80 kb plasmid previously reported in the serotype 4b (lineage I) strain H7858 28. The two plasmid contigs included all 95 ORFs previously identified in pLM80 28. Genes found in the plasmid contigs included at least 11 genes annotated as transposon genes in pLM80 and two genes associated with cadmium resistance. The observation that this plasmid was only sequenced in the 2000 human isolate (J0161) suggests either a loss of the plasmid in the other isolates (during propagation in the food processing plant, the food or the human host for the 1988 isolate or during isolation) or an acquisition of this plasmid in the 2000 human isolate.

To identify genomic differences between the human and food isolate from the 1988 case as well as a food and human isolate from the 2000 outbreak, we aligned the 4 genomes using TBA (Threaded-Blockset Aligner) 30 and refined this alignment using MUSCLE 31. The refined alignment of the four genomes had a total length of 2,922,773 bp (including gaps) (Figure 1). The aligned sequences for each genome ranged from 2,921,243 to 2,921,655 (Table 2). As between 1.2% and 1.7% of the genome sequences for each isolate could not be included in the alignment, it is possible that the four isolates contain some differences in addition to those detailed below. Regions not included in the alignment most likely represent regions not sequenced in one or more genomes, deletions/insertions, or genome fragments replaced by a non-homologous sequence (i.e., within the comK prophage). Specifically, while the length of the comK prophage sequence ranged from 38,876 to 42,001 bases in the four isolates (Table 2), the comK prophage alignment (including gaps) was only 28,886 bp, indicating extensive diversification including presence of non-homologous prophage sequences in the different isolates (as described in detail below). This difference represents 25% of the non-aligned sequences in the whole genome alignment, although this prophage represents less than 2% of the genome size.

The final alignment was divided in three distinct non-overlapping alignments representing (i) the comK prophage (28,886 bp), (ii) the tRNA Thr-4 prophage (34,703 bp), and (iii) the backbone sequence (2,859,184 bp) representing the rest of the chromosomal sequence. Identification of polymorphisms and recombination were carried out separately for each alignment. The comK prophage alignment had 1,274 polymorphic sites that differentiated the two 1988 isolates from the two 2000 isolates; at all of these sites, the isolates from the same year were identical to each other. In contrast, the tRNA Thr-4 prophage alignment had one confirmed polymorphic site, which differentiated the 1988 isolates from the 2000 isolates; this site represents a nonsynonymous transversion (Table 3). Surprisingly, besides the polymorphisms found in the comK prophage, the polymorphism in the tRNA Thr-4 prophage represents the only SNP that differentiates the 1988 isolates from the 2000 isolates.

Among the 11 confirmed polymorphisms in the backbone sequence (excluding the comK and Thr-4 prophage) of the J0161 subculture sequenced at Broad, eight were specific to J2818 (2000 food isolate), one was specific to F6900 (1988 human isolate), and one was specific to J0161 (2000 human isolate) (Table 3). In addition, one polymorphism (SNP #3; Table 3) was present in the J0161 subculture sequenced at Broad, but was not found in the J0161 subculture used for re-sequencing at Cornell (see "Methods"). Among the eight polymorphisms specific to the 2000 food isolate (J2818, Table 3), three fall in intergenic regions and five fall in coding sequences, including one representing a synonymous change and four representing nonsynonymous changes (Table 3). The 2000 human and food isolates thus differ from each other in four nonsynonymous sites, including one substitution (SNP 10; Table 3) in addB, which encodes a protein that is part of the enzyme AddAB 3233. AddAB is analogous to E. coli RecBCD, which is responsible for processing double-stranded breaks in the chromosome 3334.

As the 2000 human and food isolates differed by 9 SNPs, we identified additional human isolates associated with the 2000 outbreak to validate these SNPs; unfortunately no additional food isolates associated with this outbreak were available for SNP validation. Nine isolates with ribotype DUP-1053A (representing the ribotype of the 1988 and 2000 food and human isolates) obtained between August and November of 2000 from human clinical cases in Ohio, Michigan and New York were found to be slow rhamnose-fermenters. These isolates were also found, by PFGE using ApaI and AscI for restriction of DNA, to be indistinguishable from each other and from the 1988 and 2000 human and food isolates sequenced. As these results strongly suggested that these nine isolates were from humans that were infected as part of the 2000 outbreak, we screened these isolates for the 12 confirmed polymorphisms identified among the genome sequences for the human and food isolates from the 1988 case and the 2000 outbreak as well as for the presence of two plasmid-specific sequences. All 9 isolates were positive by PCR for both plasmid sequences, indicating that the plasmid found in J0161 is present in most other human isolates associated with this outbreak. None of these nine human isolates had any of the polymorphisms specific to F6900 (1988 human isolate) or J2818 (2000 food isolate).

Furthermore, none of the nine isolates shared the one SNP specific to the subculture of the 2000 human isolate (J0161) that was sequenced at Broad (SNP 3; Table 3), but that was not found in the J0161 subculture that was used for re-sequencing at Cornell, supporting the hypothesis that this polymorphism arose during laboratory passage of the J0161 subculture sequenced at Broad. Nevertheless, all nine human isolates showed (i) nt sequences identical to J0161 (the sequenced 2000 human isolate) at the eight sites that carry unique SNPs in the 2000 food isolate (J2818), (ii) the SNP unique to the 2000 human isolate J0161 (SNP 2; Table 3) as well as (iii) the tRNA Thr-4 prophage SNP (SNP 12; Table 3) that differentiates the 1988 isolates from the 2000 isolates. SNP 2 thus does not represent a random mutation unique to the sequenced human isolate from the 2000 outbreak (e.g., mutation that arose during passage or infection), but was found in all human clinical isolates surveyed from this outbreak.

While it is unlikely that any of the few nonsynonymous substitutions observed would affect the fitness of L. monocytogenes strains tested here, we performed preliminary experiments to test whether the nonsynonymous sites differentiating the 2000 human and food isolates (may affect the ability of the 2000 human and food isolates to multiply inside macrophage cells. We specifically surmised that the nonconservative Trp → Cys change in addB (i.e., SNP 10, Table 3) could alter the function of the enzyme AddAB 3233, which is analogous to E. coli RecBCD 3334. As RecBCD deficient E. coli mutants have been shown to be more susceptible to oxidative DNA damage 35; we hypothesized that the 2000 food and human isolates may differ in their ability to survive inside activated macrophages (i.e., J774 cells stimulated with LPS) where they may be exposed to oxidative stress. We used FSL R2-499 (a subculture of J0161 confirmed as having SNP 10 and thus encoding the same addB allelic variant as J0161) and the 2000 food isolate J2818 for these experiments. As no apparent difference in intracellular growth of these two strains was observed when the strains were initially characterized in separate intracellular growth assays (Additional file 1), we performed triplicate competition experiments by infecting J774 cells with both strains simultaneously; competition assays like this typically provide for more sensitive means to identify whether one strain has a competitive advantage over another strain in a given environment 3637. In the competitive intracellular growth assay the relative recovery of both isolates after 9 h of intracellular growth did not differ significantly (P > 0.05; chi-square test); overall, 16 and 14 colonies, respectively, with the J2818 and the J0161 addB allelic type were recovered (3/10, 7/10, and 6/10 isolates carried the J2818 addB allelic type in replicates 1, 2, and 3, respectively). While these data suggest that the mutations that differentiate the two 2000 isolates do not affect the intracellular growth capabilities, of these isolates, in activated J774 macrophage cells, future phenotypic and animal experiments would be needed to further test whether the 2000 human and food isolate genotypes differ in their fitness. As no data on the effect of an L. monocytogenes addB null mutation are available, it is unclear which physiological functions may be affected by nonsynonymous mutations in this gene. Future experiments thus should involve characterization of an addB null mutant before conducting additional phenotypic and animal experiments to determine the effect of the observed nonsynonymous mutation in addB and/or other nonsynonymous mutations differentiating the 2000 human and food isolates.

Overall, we only found a single synonymous nucleotide difference (SNP 11; Table 3) between the 2000 food isolate (J2818) and the 1988 isolates and no synonymous nucleotide difference between the 2000 human isolate (J0161) and the 1988 isolates. In order to determine whether this low number of synonymous changes is consistent with previously reported mutation rates for bacteria, we calculated the estimated number of generations between the 1988 isolates and the 2000 food isolates (J2818) using previously reported mutation rates 2338, including 3.30 – 4.50 × 10^-10 per bp per generation for experimental Myxococcus xanthus populations 3 and 1.44 × 10^-10 per bp per generation for experimental E. coli populations 2. Ochman et al. 38 estimated a mutation rate of 4.50 × 10^-9 per bp per year (approximately 2.25 × 10^-11 per bp per generation assuming ~200 generations per year) for natural populations of E. coli and 8.20 × 10^-9 per bp per year (approximately 2.05 × 10^-10 per bp per generation assuming 40 generations per year) for natural populations of Buchnera. The number of generations was calculated as the (number of synonymous substitutions, i.e., 1])/[(number of synonymous sites, i.e., 5.65 × 10⁵) × (mutation rate)]. Using the two extremes of the suggested mutation rates (2.25 × 10^-11 and 4.50 × 10^-10 per bp per generation) we estimated that between 3,933 and 78,666 generations separated the 1988 isolates from the 2000 isolates. Assuming a 12 year time span between the two sets of isolates, these mutation rates translate into 328 and 6,556 generations per year (0.90 – 17.96 generations per day), yielding a generation time ranging from 1.34 to 26.67 hours. The generation time estimate (1.34 h) based on the low mutation rate (2.25 × 10^-11 mutations per bp per generation) proposed for natural populations of E. coli 38 is only about 2-fold longer than the estimated generation time for L. monocytogenes in rich media broth at 30°C (0.7 h) 39.

Other L. monocytogenes generation times reported were 14.2 h (in rich-media at 4°C 39) and 111.3 h (for L. monocytogenes in soil exposed to temperatures fluctuating between -29°C to 12°C 40). We thus propose that a mutation rate closer to 4.50 × 10^-10 (as estimated from an experimental population of M. xanthus 3) is more likely to represent the mutation rate for L. monocytogenes, as a generation time of approx 24 – 28 h seems plausible for L. monocytogenes in natural environments. Under this mutation rate, generation time, and assuming a Poisson distribution, the probability of observing zero or one synonymous substitution is roughly the same (~36.8%), which is consistent with the observation that the 2000 food isolate (J2818) differed from the 1988 isolates by one synonymous change, while the 2000 human isolate (J0161) showed no synonymous changes as compared to the 1988 isolates. In conclusion, the low rate of mutations observed for the isolates studied here is thus clearly within the range expected based on previously reported mutation rates for other bacteria.

As the comK prophage in the 2000 isolates showed considerable divergence from the comK prophage in the 1988 isolates (1,274 nt differences between the 1988 and 2000 prophage sequences), further analyses were performed on the comK prophage sequences. Initial BLAST searches against (i) the F6854 genome, (ii) genome sequences deposited in GenBank 41, and (iii) genome sequences in the L. monocytogenes database at the Broad Institute 42 found that the following prophages showed the highest similarity to the 2000 isolate prophage sequence: (i) the comK prophage sequence in L. monocytogenes FSL J1-194 (BLAST score of 2.23 × 10⁴ bits), (ii) the comK prophage in F6854 (the 1988 human isolate; 1.88 × 10⁴ bits), (ii) the phage A500 (1.03 × 10⁴ bits) (GenBank accession DQ003637), (iii) the comK prophage in EGD-e (1.01 × 10⁴ bits) 43, and (iv) the phage A118 (8.82 × 10³ bits) 44. Additionally, BLAST searches of the individual genes in the comK prophage against these same databases, showed that 35 of the 65 genes in the 2000 isolates showed the best match with genes in the FSL J1-194 prophage (Additional file 2); 14 and 11 genes in the 2000 isolates showed the best match with genes from the phage A118 and the EGD-e comK prophage, respectively. While these data suggest that diversification of the comK prophage in the 2000 isolates does not represent a prophage replacement with a sequenced prophage, replacement could have occurred with a prophage not represented in the available nucleotide sequence databases.

To determine whether recombination events could have contributed to the diversification of the comK prophage, recombination analyses were performed on an alignment of the four comK prophage sequences for the 1988 and the 2000 isolates and the prophage most closely related to the comK prophage in the 2000 isolates (i.e., the comK prophage in isolate FSL J1-194). Sawyer's test provided evidence for recombination among the five prophage sequences in the alignment (i.e., inner fragments, P < 0.0001) as well as for recombination with sequences not included in this alignment (i.e., outer fragments, P < 0.0001). Overall, five inner recombination events involving the comK prophage in the two 2000 isolates and FSL J1-194 were initially identified. Visual analysis of the prophage sequences suggests at least three independent recombination events in the two 2000 isolates, relative to the two 1988 isolates; for these three recombinant regions (marked as R1, R2, and R3 in Figure 2) the prophage sequences in two 2000 isolates were more similar to FSL J1-194 than to the prophage sequences in the two 1988 isolates (Figure 2). Analyses of nt identities between the 1988 and 2000 isolates and FSL J1-194 for all individual ORFs in the comK prophage (Additional file 2), also supported that extensive recombination occurred in this prophage. While 15 genes showed 100% identity between the 1988 and 2000 prophage genomes (e.g. LMOf6854_2341.9; Additional file 2), 24 other genes showed identities < 95% between the 1988 and 2000 prophage genomes and 100% identity between the 2000 isolates and FSL J1-194 (e.g. LMOf6854_2352.2; Additional file 2), suggesting introduction of these genes into the ancestor of the 2000 isolates from a phage or prophage sequence closely related to that found in FSL J1-194. Eleven prophage genes in the two 2000 isolates showed < 98% identity to the corresponding prophage sequence in the 1988 isolates and a best match with a sequence other than that of FSL J1-194 (e.g. LMOf6854_2371; Additional file 2), suggesting introduction from a distinct phage or prophage genotype.

For 18 genes present in the comK prophage of the 1988 isolates (i.e., F6854 and F6900), no homologues were identified in the genomes of the 2000 isolates; 15 of these genes appear to have been replaced in the 2000 isolates by non-homologous genes (Additional file 2). These deletions and replacements in the 2000 isolates are likely consequences of recombination events that integrated or replaced large sequences, as supported by the observation that some observed gene deletions or replacements were located in large recombinant fragments identified by Sawyer's test. For example, the eight adjacent genes LMOf6854_2352.2 to LMOf6854_2360.1 found in the 1988 isolates appear to have been replaced by a block of seven genes in the 2000 isolates (Sawyer's tests identified a recombinant fragment, which corresponds to this replacement event). Two of these eight genes present in the 1988 isolates (LMOf6854_2352.6 and LMOf6854_2352.7) show no homology to the genes present in the 2000 prophage (Additional file 2).

While a few singletons (i.e., single base differences found in only one isolate) were observed in the genome backbone of the two 1988 and the two 2000 isolates, the only polymorphisms that differentiate the 1988 isolates from the 2000 isolates fall within the two prophages inserted into comK and tRNA-Thr-4. While the tRNA-Thr-4 prophage region showed only one SNP that differentiated the 1988 and 2000 isolates, the comK prophage region differs considerably between the two 1988 and the two 2000 isolates. Our data suggest that multiple recombination events lead to diversification of the comK prophage, although the occurrence of a single prophage replacement event cannot be excluded. Recombination events seem to have replaced a number of phage genes (including several genes that were annotated as encoding proteins with essential roles in the lytic cycle) with allelic variants or non-homologous genes. For example the gene encoding the major capsid in the 1988 isolates (LMOf6854_2352.7) was replaced with a non-homologous major capsid gene (LMPG_02241.2) in the 2000 isolates. Mechanistically, the observed recombination events in the comK prophage most likely occurred through infection with one or more phages in one or more cells of the 1988 genotype, followed by subsequent recombination or phage replacement. In addition to diversification of the integrated prophage genomes observed here, it has been well documented that phages can be vectors for horizontal transfer of genes located in the genome backbone 45 and that phages are capable of generalized transduction in L. monocytogenes 46. While we did not find any evidence for horizontal gene transfer outside the prophage region in the isolates studied here, horizontal gene transfer in genes in the backbone genome has been well documented in L. monocytogenes 474849. While very little is known about the frequency of phage insertion and phage transduction in bacteria in natural environments, Hodgson 46 reported that rates of phage transduction of Listeria under laboratory conditions ranged from about 1 to 300 transductants per 10⁷ PFU. In conclusion, while short term evolution of L. monocytogenes in natural environments involves limited diversification of the genomic backbone, phage-mediated genetic changes appear to be major mechanisms for diversification and evolution of L. monocytogenes during short evolutionary time frames. This is particularly interesting as there is no evidence for phage-encoded virulence genes in Listeria, unlike for a number of other pathogens where phage mediated transfer and diversification of virulence genes appear to represent an important mechanism for diversification.

Our data are consistent with the overall idea that bacteriophages play multiple important roles in the evolution of bacterial populations in natural environments, including (i) providing selective pressure on bacterial populations (e.g., 505152) and (ii) providing a mechanism for rapid horizontal exchange of genetic material (e.g., 53). Contributions of bacteriophages to genetic diversification also have been well characterized in different pathogenic and non-pathogenic bacterial species. For example, the V. cholerae CTX genetic element represents the genome of a lysogenic bacteriophage, which carries the genes for cholera toxin 53. This lysogenic phage can be induced in toxigenic strains and the resulting phage particle appears to be able to transduce non-toxigenic V. cholerae 54. Similarly, in different E. coli pathotypes, shiga-toxin genes as well as genes encoding other virulence factors are located on different prophages 55. Diversification of prophages integrated in the chromosome of bacteria also appears to occur rapidly and commonly and contributes to bacterial genome evolution. For example, in enterohemorrhagic E. coli considerable diversification of prophages carrying virulence determinants has been observed, likely indicating independent infections of host bacteria by different bacteriophages carrying these virulence determinants 55. Similarly, considerable diversity has been observed in the genome of the Vibrio CTX prophage (e.g., 56). The importance of bacteriophages and lysogenic prophages in the evolution of gram-positive pathogens has also been documented (e.g., 57). For example, the rapid emergence of serotype M3 group A Streptococcus (GAS) has been associated with the acquisition of a prophage that contained a unique combination of virulence genes, which was probably generated through several recombination events 58. Moreover, most of the genetic diversity among GAS strains seems to be phage-related 5960. Prophages and diversification of prophage-associated genes thus appears to play an important role in the evolution of various bacterial species.

In addition to the diversification in the comK prophage, we also found that the four isolates differed in their plasmid content; a plasmid sequence was only found in the 2000 human isolate, as well as in nine additional human isolates linked to this outbreak. While our findings suggest that plasmid loss or acquisition events occurred, it can not be determined whether these changes occurred during L. monocytogenes survival in the processing plant, during passage of isolates, or during human infection. Rapid diversification of L. monocytogenes due to plasmid loss or acquisition has previously been reported and isolates from the same outbreak have been shown to differ in their plasmid profiles 21. However, it is important to note that the media commonly used to isolate L. monocytogenes (particularly media used for isolation from food samples) contain acriflavine, a plasmid curing agent 6162. Therefore, it is possible that the isolates for which no plasmid sequence was obtained lost this plasmid during isolation or during laboratory passage.

Our data showed that the human and food isolates from a sporadic case in 1988 and the human and food isolates from an outbreak in 2000, which were linked to the same food processing facility, had genome sequences that showed no major inversions, deletions and insertions, although minor changes like these cannot be ruled out since the genome sequences have not been fully finished and assembled. While the four isolates showed a total of 12 confirmed SNPs, all 11 SNPs that occurred in the genome backbone represented singletons, i.e., they were found in only one of the four isolates. The only mutations that differentiated the 1988 isolates from the 2000 isolates were (i) one single SNP in the tRNA-Thr-4-prophage and (ii) a large number of SNPs and deletions in the comK prophage, which most likely represent multiple recombination events. This high level of overall genome conservation between the human and food isolates from the 1988 listeriosis case and the 2000 outbreak strongly supports that these two incidences were caused by the same L. monocytogenes strain, which is likely to have persisted over at least 12 years in the processing plant that produced the food linked to both the 1988 case and the 2000 outbreak. Persistence is also supported by the observation that the PFGE type represented by the persistent strain seems rare (it was only found once among 495 food isolates characterized by PFGE 22). Re-introduction from the immediate environment surrounding the plant is a possible, but unlikely, alternative to persistence of the specific strain in the processing plant, particularly as ready-to-eat meat processing plants will have controls in place to minimize introduction of microorganisms. Persistence in turkey flocks providing raw materials for this plant is even less likely as the heat treatment used for production of frankfurters and deli meats inactivates L. monocytogenes 63. Similarly, re-introduction through a persistently infected worker is highly unlikely as people rarely shed L. monocytogenes 6465. Importantly, even in the unlikely scenario that the unique strain linked to listeriosis cases in 1998 and 2000 was reintroduced, rather than having persisted in the plant, the isolates characterized here would still represent a set of closely related isolates with a parent-descendant relationship, which have multiplied in a non-controlled environment over about 12 years. Thus, the alternative scenarios outlined above do not affect the main conclusions of our study, including that phage mediated mechanisms are critical for short term diversification of L. monocytogenes.

Overall, our data demonstrate the utility of using full genome sequencing for follow-up investigations to epidemiological and source tracking studies. In particular, full genome sequencing clearly allowed for differentiation of the L. monocytogenes isolates from the 1988 case and the isolates associated with the 2000 outbreak, even though these isolates were previously found to be identical by other subtyping methods, including PFGE 26. Genome sequencing not only represents the most sensitive subtyping approach conceivable, but also provides for specific identification of differences between isolates, which can be used to assess whether differences between two isolates are likely to have occurred within a short time frame or not. This assessment is critical for source tracking as genomic diversification that can lead to different genomic restriction profiles can clearly occur over short time periods (e.g., through diversification of prophages, loss of plasmids). Rapid diversification in certain hot spots (e.g., prophages) represents a challenge as the current gold standard method for bacterial subtyping is a genomic restriction profiling approach (i.e., PFGE 66), which means that closely related isolates may have different PFGE types (as seen in some outbreaks (e.g., 2167). Importantly though, the standard restriction enzymes used for L. monocytogenes PFGE analyses (i.e., ApaI and AscI 68) only interrogate <500 nt for SNPs; based on the genome sequence for F6854, there are 39 restriction sites for ApaI, which recognizes a 6 bp motif (yielding 234 bp of DNA interrogated by this enzyme), and 15 restriction sites for AscI, which recognizes a 8 bp motif (yielding 120 bp of DNA interrogated by this enzyme). Thus, genetically distinct isolates may share the same PFGE pattern. For example, Nightingale et al. 69 found that L. monocytogenes isolates with the same PFGE pattern may differ by presence/absence of premature stop codons in a number of genes, including the virulence gene inlB. In contrast to PFGE, our sequence analyses used >2.9 Mb of aligned sequences for subtype discrimination, thus providing for vastly improved detection of substitutions. While we cannot exclude that the isolates characterized show differences in their genomes in addition to those reported here (as the genomes have not been closed and include gaps), this approach clearly provides for a comprehensive sampling of the genomic diversity in the characterized isolates. Future use of full genome sequencing for epidemiological investigations of bacterial infectious disease outbreaks thus seems feasible assuming anticipated continued decreases in the cost of DNA sequencing 70 and availability of computational tools that allow for rapid and reliable genome assembly and comparisons.

Analysis of the single nucleotide polymorphism in the genome backbone of the isolates from both the 1988 listeriosis case and the 2000 listeriosis outbreak also provided us with the ability to probe the evolution of L. monocytogenes isolates during environmental persistence and during human infection. Based on our data, we hypothesize that the ancestor of F6854 and F6900 (1988 isolates) contaminated the plant in or before 1988. The single SNP differentiating the 1988 human isolate from the 1988 food isolate is consistent with a very close ancestral relationship between these two isolates; the unique SNP in the human isolate could have occurred in the ancestor of the human isolate (F6900) during multiplication in either the plant, the contaminated food, or the infected human or during isolation and propagation of the isolate. The ancestor of F6854 remained in the plant and persisted until 2000. Between 1988 and 2000, the comK prophage of that strain was replaced (through one or several recombination events) by the prophage found in the 2000 isolates. Moreover, one mutation that differentiates the 1988 isolates from the 2000 isolates occurred in the tRNA prophage (possibly through a substitution or due to a recombination event). After the comK prophage replacement and the tRNA mutation, one mutation in J0161 (the 2000 human isolate) and eight mutations in J2818 (the 2000 food isolate) occurred and differentiated these two isolates from each other and from the 1988 isolates. The substitution that is unique to the 2000 human isolate must have occurred during growth prior to infection and isolation as it was found in the sequenced human isolate (J0161) as well as in an additional 9 human isolates (see Figure 3). On the other hand, the substitutions in the 2000 food isolate could have occurred during multiplication in the plant, the contaminated food, or during isolation and laboratory propagation. The observation that the 2000 food isolate carries eight unique SNPs, which clearly separated this isolate from the other three isolates, while carrying a comK prophage region identical to the 2000 human isolate, suggests that these mutations occurred after the two 2000 isolates diverged from the 1988 strain. The possibility that these SNPs arose during laboratory passages is supported by our observation that two subcultures of the 2000 human isolates differed by one SNP as well as observations that two subcultures of Bacillus anthracis strain Porton differed by a number of SNPs 71.

The primary isolates characterized here were a human and a food isolate from a 1988 sporadic listeriosis case in the US 24 (isolates F6900 and F6854; Table 1) as well as a human and a food isolate from a 2000 listeriosis outbreak in the US 25 (isolates J0161 and J2818; Table 1). Isolates F6900, J0161 and J2818 used for preparation of DNA for genome sequencing were directly sent from the US Centers for Disease Control and Prevention (CDC) to Anza Therapeutics, Inc. for genomic DNA isolation. Subcultures used for re-sequencing (to confirm selected polymorphisms) and for tissue culture were independently provided to Cornell from the CDC. At the CDC, isolates were stored at -70°C in sheep's blood (isolate F6900) or in trypticase soy broth with 20% glycerol (J0161 and J2818). At Cornell, all isolates were stored at -80°C in brain heart infusion broth with 15% glycerol.

F6854, the food isolate from the 1988 listeriosis case (Table 1) was isolated from direct plating of the food sample on lithium chloride – phenylethanol- moxalactam (LPM) agar. While the specific details of the isolation procedures for J2818, the 2000 food isolate, were not available, standard procedures for isolation of L. monocytogenes from meat products in 2000 would involve primary enrichment in University of Vermont (UVM) broth and secondary enrichment in Fraser Broth (FB) or Buffered Listeria Enrichment Broth (BLEB), followed by plating on Modified Oxford (MOX)(MLG 8.06 and MLG 8A.03, the protocols are available at 72); all of these enrichment media as well as MOX include acriflavine, a potential DNA modifying and plasmid curing agent 6162.

Standard protocols for the isolation of L. monocytogenes from human patients involve collection of blood or cerebrospinal fluid (CSF) with a syringe or directly into blood culture bottles. The blood culture enrichment is sub-cultured onto appropriate plating media (e.g. trypticase soy agar supplemented with 5% defibrinated sheep's blood) at regular intervals. While human isolate F6900 (Table 1) was isolated via blood culture by this protocol (Swaminathan and Graves, unpublished), the procedure used for isolation of human isolate J0161 is not available. Unfortunately, no specific data on the number of passages for each isolate are available.

High molecular weight genomic DNA was isolated from overnight cultures grown in buffered yeast media. Bacteria were lysed using lysin from phage 10403 46 that had been cloned and purified as described 73. Genomic DNA was isolated using QIAGEN genomic-tip 100/G columns per manufacturer's recommendations.

Genome sequences for isolates J0161 (human isolate, 2000 listeriosis outbreak) 2526, J2818 (food isolate, 2000 listeriosis outbreak) 2526, F6900 (human isolate, 1998 listeriosis case) 24, and isolate FSL J1-194 (unrelated human isolate from a sporadic case 42 were determined at the Broad Institute using the 454 technology 29. 454 data were assembled using the 454 Newbler assembler, version v1.0.52.60 74. Assembly quality metrics for each of the four genome assemblies are described in Table 2. In all cases, the assembly covers ≥93% of the finished reference and the N50 contig size is at least 148 kb. N50 contig size is a length-weighted median representing the size contig in which the typical base is found.

Whole genome shotgun end sequencing of plasmids with 4 to 10 kb inserts and fosmids with 40 kb inserts was performed by ABI BigDye chemistry and detected on ABI 3730 machines as described previously 75.

The draft genomes of J0161 (version 1), J2818 (version 2), F6900 (version 2) (NCBI accession nos. AARW00000000.1, AARX00000000.1, and AARU00000000.1), sequenced as described above, as well as the unfinished sequence of F6854 (AADQ00000000.1), obtained from the Comprehensive Microbial Resource 76 were used for all analyses. In addition to the chromosome sequences for these four isolates; we also analyzed the plasmid sequence found in the 2000 human isolate (J0161). The genome sequence of F6854 represented a pseudomolecule in which gaps between the contigs were closed with random sequences using the fully sequenced genome of EGD-e as a reference; all details on the construction of this pseudomolecule were described by 28. The synthetic sequences introduced to close gaps were deleted from F6854 after the initial genome alignment using TBA (as detailed below) and prior to further manipulations and analyses.

The genome sequence for the lineage I strain FSL J1-194 (version 2) (NCBI accession no. AARJ00000000.1), also determined at the Broad Institute, was used for recombination analyses; like the other genomes used here, the genome of FSL J1-194 has not been fully sequenced and closed.

The package TBA (Threaded-Blockset Aligner) 30 was used to align the five genomes (i.e., the 1998 and 2000 human and food isolates as well as FSL J1-194) with the package's default settings. TBA runs a series of blastz alignments (i.e., pairwise sequence alignment) between the genome sequences for each isolate. These alignments are subsequently used to build multiple alignment blocks; each block contains the sequences for the isolates included in a pairwise alignment that had been created in the first step. If, for example, J0161 did not have a specific fragment "A" or if the fragment had not been sequenced in this isolate, J0161 did not appear in the block for fragment "A". Because the genome sequence of four of the five isolates is represented in multiple contigs (Table 1), several blocks were created; each block could not contain more than one contig from a given isolate. The blocks were filtered to keep only those blocks that contained sequences from all isolates analyzed. The F6854 sequence was used as a reference to order blocks such that the first block started at the start of the F6854 sequence and the last block ended at the end of the F6854 sequence. After completion of the alignment, 62 ambiguous sites in the F6854 genome sequence (e.g., R, S, W, etc.) were manually changed to the consensus sequence of the other isolates at the same position (at all of these sites isolates J0161, J2818, and F6900 had the same nt; thus, these manipulations would not underestimate the number of true differences between the 1988 and 2000 isolates or between the food and human isolates). The alignment blocks were subsequently edited using custom perl scripts (developed by the authors) and BioEdit version 7.0.5.2 77 to create one multiple alignment of the sequences in which the blocks were ordered according to the F6854 chromosomal sequence. This alignment was further refined using the program MUSCLE version 3.6 31 using a gap open penalty of -75.0, a gap extension penalty of -1.0, and the default settings for all other parameters.

The final alignment was separated into three sub-alignments, including alignments for (i) the prophage inserted into comK; (ii) the prophage inserted into tRNA-Thr-4; and (iii) the genome backbone sequence without the two prophage sequences. These alignments were initially analyzed using DnaSP version 4.0 78 to identify polymorphic sites in all three alignments. A perl script was developed to identify positions with gaps in the alignment and to identify which sequences had the gaps.

The number of synonymous sites in the genome of F6854 (i.e., 5.65 × 10⁵ synonymous sites) was calculated as previously described 3 using all annotated protein coding sequences with the exception of the ORFs present within the two prophages.

The coding sequences for all genes annotated in the comK prophage in the 2000 isolates (J2818 and J0161) were used as queries in blastn searches against (i) the genomic sequence of F6854 (1988 food isolate); (ii) the Listeria database at the Broad Institute website 42; and (iii) GenBank 41. For those comK prophage genes present in the 2000 isolates that did not have homologous sequences in the 1988 human and food isolates (F6854 and F6900) in the initial blastn search, the absence of a homologue was confirmed by inspection of the corresponding region of the prophage in F6854 (1988 food isolate).

Recombination analyses were performed using Sawyer's test implemented in GENECONV 79; GENECONV default settings were used for the analyses with the exception that sites with gaps were not considered and mismatches were allowed within the recombinant fragment (gscale = 3).

All 43 polymorphic sites in the backbone alignment and the single polymorphic site in the tRNA Thr-4 prophage were initially checked by comparison with pre-existing Sanger trace files available from NCBI for isolates J0161, J2818 and F6854. If Sanger trace files were not available, polymorphisms were confirmed by re-sequencing; for this purpose, PCR primers were designed, using the F6854 genome as reference, to amplify the regions flanking the polymorphisms (Additional file 3). A total of 33 pairs of primers were designed since in a few cases multiple polymorphic sites could be amplified in a single fragment. PCR products were purified using Exonuclease I (0.5 U/μl) and Shrimp alkaline phosphatase (0.05 U/μl) (USB, NEB) and sequenced, using standard Sanger fluorescent sequencing methods, at the Core Laboratories Center at Cornell University, USA. Among the PCRs performed for sequencing, five amplified more than one fragment (three amplified two fragments and two amplified three fragments), suggesting that those regions represented repetitive regions in the chromosome. Blastn searches of the target sequences that were used to design these five primer sets against the genomes of F6854 and EGD-e confirmed that the fragments amplified by these primers represented almost identical repetitive sequences and DNA sequencing showed that the polymorphic sites initially identified in these regions were due to misalignments of repetitive sequences. Overall, 11 polymorphisms were confirmed by re-sequencing; importantly, the one SNP in the genome backbone initially identified as differentiating the two 1988 and the two 2000 isolates was not confirmed. Interestingly, one polymorphism in the 2000 food isolate (J0161; SNP #3; Table 3) was not confirmed by re-sequencing, but was confirmed in the trace files available in NCBI, indicating that this polymorphism most likely occurred during laboratory passage of the J0161 subculture sequenced at the Broad Institute (as the sequence at this site in the J0161 subculture at Cornell matched the other 2000 isolate and the two 1988 isolates); we considered this polymorphism as a confirmed polymorphism as it was present in the actual isolate sequenced. In summary, thus, among all 44 polymorphisms identified initially, 12 were confirmed. Among the 32 polymorphism not confirmed, eight represented sequencing errors in F6854 (which was sequenced with the Sanger method) and one represented a sequencing error in J2818; 12 of the polymorphisms that were not confirmed represent problems in the number of bases within homopolymeric tracts and misplacement of the gap in the alignment, and 11 represented misalignments of repetitive regions (Additional file 4).

We also chose genome alignment gaps (representing possible indels) that (i) differentiated the two 1988 isolates from the two 2000 isolates (26 gaps) or (ii) differentiated the two human isolates from the two food isolates (34 gaps) for verification. Gaps were initially verified using Sanger trace files available for isolates J0161, J2818 and F6854 and sequencing by synthesis technologies data files. For a total of six gaps that could not be verified either due to ambiguous sequence data or lack of Sanger sequencing data, verification was performed using re-sequencing; PCR primer design and sequencing were performed as described above. One additional gap was present in a sequence representing a multicopy gene (i.e., a 23S rRNA sequence); no validation of this gap was attempted.

Verification of the 59 gaps by comparison with pre-existing trace files available in NCBI or by re-sequencing showed that all of these gaps represented alignment artifacts or artifacts due to the sequencing by synthesis technology. Thus, none of the short indels that were initially identified as differentiating the two 1998 from the two 2000 isolates or the two human from the two food isolates could be confirmed and these initially identified indels appear to represent sequencing errors, consistent with previous reports that the sequencing by synthesis strategy often incorrectly characterizes homopolymeric tracts 3.

In order to identify additional human isolates linked to the 2000 outbreak, the Pathogen Tracker 80 database was used to identify human isolates that shared the same ribotype as the outbreak strain (DUP-1053A) and were isolated during the time frame of the outbreak (May to November of 2000) in some of the states (New York, Michigan and Ohio) that were included in the outbreak. Selected isolates identified through this approach were tested for rhamnose fermentation as well as by PFGE to identify specific isolates that matched the outbreak strain. Rhamnose fermentation was tested as previously described 81 with the exception that only one tube without a mineral oil overlay was used. PFGE with the restriction enzymes ApaI and AscI was performed using the US CDC standard PulseNet protocol 68. Nine isolates matching the rhamnose fermentation characteristics and PFGE profile of the 1988 and 2000 isolates were selected to validate the polymorphisms described in Table 3 using PCR amplification and subsequent sequencing (see Additional file 3 for primers) of the regions containing these polymorphisms. All 9 of these isolates were also screened for the presence of two plasmid-specific sequences using PCR (see Additional file 3 for primers).

Unfortunately neither additional food nor additional human isolates linked to the 1988 case were available for analyses. Furthermore, no additional food or plant environmental isolates from 2000 or the period between 1998 and 2000 were available for analyses.

L. monocytogenes strains were grown in BHI at 37°C with shaking at 220 RPM. Log phase (OD₆₀₀ = 0.4) L. monocytogenes strains in BHI were diluted 1:100 and grown at 37°C to early stationary phase (defined as 3 h after the OD₆₀₀ reached 1.0). Aliquots of the stationary phase cultures were flash frozen in liquid nitrogen and cultures were enumerated after thawing. The murine macrophage cell line J774A.1 (ATCC TIB-67) was maintained using Dulbecco's minimal essential medium (DMEM) with Earle's salts and 1% sodium pyruvate (Gibco; Gaithersburg, MD) containing 10% fetal bovine serum (Gibco), 1.5 g/L sodium bicarbonate (Gibco), and 100 μg/ml each penicillin G and streptomycin (J774 medium).

Intracellular growth assays were performed as described by Conte et al., 82 with minor modifications. Briefly, 48 h prior to the assay, J774 cells were seeded into 24 well plates (Corning) at a density of 2 × 10⁵ cells/well in J774 medium without antibiotics. Twenty-four hours after seeding, the medium was replaced with J774 medium supplemented with 0.1 μg/ml E. coli O55:B5 LPS (Sigma). Thirty minutes prior to infection, the medium was replaced with fresh J774 medium without antibiotics. For infection, approximately 1 × 10⁶ CFU L. monocytogenes were added to each well, resulting in an MOI of approximately 1. At 30 minutes post infection, the monolayers were washed once with PBS to remove any unassociated L. monocytogenes, and the medium was replaced with J774 media containing 50 μg/ml gentamycin to kill any extracellular L. monocytogenes. Infected J774 cells in different wells were washed 3 times with PBS and lysed with ice-cold distilled water after at 1, 5, 7, or 9 h post infection. Intracellular L. monocytogenes were enumerated by plating the appropriate dilutions of the J774 lysate on BHI.

Intracellular growth assays were initially performed separately with the two isolates to be tested (i.e., J0161 and J2818), but were also performed in a competition experiment format 3637, where activated J774 cells were infected with both L. monocytogenes isolates (mixed at a 1:1 ratio). To assess the frequency of each strain after intracellular growth in the competition experiment, ten colonies recovered after 9 h of intracellular growth were selected from each of the three replicates for PCR amplification and sequencing of the region containing SNP 10 (Table 3) using primers RHO105-2327842F and RHO106-2327842R (Additional file 3).