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Journal of Bacteriology, August 2004, p. 4994-5002, Vol. 186, No. 15
0021-9193/04/$08.00+0 DOI: 10.1128/JB.186.15.4994-5002.2004
Microbial Genomics and Bioprocessing Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois 61604,1 Produce Safety and Microbiology Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Albany, California 94710,2 Animal Disease Research Unit, Agricultural Research Service, U.S. Department of Agriculture, Pullman, Washington 99164,3 Microbial Outbreaks and Special Projects Laboratory, Food Safety and Inspection Service, U.S. Department of Agriculture, Athens, Georgia 306054
Received 15 December 2003/ Accepted 23 April 2004
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ABSTRACT |
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INTRODUCTION |
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Two primary evolutionary divisions, or lineages, have been identified within L. monocytogenes on the basis of multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, ribotyping, and amplified fragment length polymorphism studies (3, 11, 24, 26). However, ribotype and virulence gene polymorphism data were used to describe the existence of a third lineage (25, 33), with some researchers suggesting that this lineage may represent a distinct taxonomic unit requiring recognition as a new species or subspecies (33, 34). Lineage-specific associations with serotypes commonly found in connection with human listeriosis (4b, 1/2b, and 1/2a) and genetic characterization of isolates from human and animal listeriosis cases have led to the suggestion that L. monocytogenes lineages differ in their pathogenic potential and host specificity (13, 33-35). However, disagreement persists about the number and composition of the major phylogenetic divisions within L. monocytogenes (2, 18, 33), the evolutionary history of lineage divergence within L. monocytogenes remains unclear, and perceived differences in virulence or host specificity need to be evaluated with respect to relative frequencies of exposure.
A solid evolutionary framework is essential for understanding the ecology and population dynamics of L. monocytogenes and for evaluating proposals regarding taxonomic revision of this important food-borne pathogen. Therefore, prfA virulence gene cluster (pVGC) sequences from 113 L. monocytogenes isolates, Listeria seeligeri, and Listeria ivanovii were used to develop a robust intraspecific phylogeny for L. monocytogenes. The pVGC is stably integrated in the same chromosomal location in these three Listeria species, and the pVGC of each species contains homologs of six virulence genes: a transcriptional regulator (prfA), two phospholipases (plcA and plcB) and a hemolysin (hly) required for lysis of host phagosomes, a metalloprotease (mpl) involved in extracellular activation of plcB, and a surface protein (actA) responsible for actin-based motility and cell-to-cell spread (31).
The primary objectives of the present study were to (i) determine the number of major phylogenetic divisions within L. monocytogenes, the genetic diversity within each of these lineages, and the distribution of serotypes across lineages; (ii) develop and use an accurate PCR-based approach for lineage identification to evaluate hypotheses of lineage-specific differences in virulence and host specificity with respect to the prevalence of individual lineages in food products; and (iii) combine analyses of phylogeny and historical demography to reconstruct the evolutionary history of lineage divergence within L. monocytogenes and to evaluate the taxonomic status of L. monocytogenes lineages within an appropriate evolutionary framework.
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MATERIALS AND METHODS |
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Phylogenetic analyses and genetic distance estimation. DNA sequences were edited and aligned by using Sequencher (version 4.1.2; Gene Codes, Ann Arbor, Mich.). In addition to the sequences generated in the present study, the pVGC sequence from L. monocytogenes strain EGD-e (GenBank accession no. AL591824) was included in the phylogenetic analyses. Prior to phylogenetic analyses, ambiguously aligned characters and nonunique pVGC haplotypes identified by using Collapse (version 1.1 [http://inbio.byu.edu/Faculty/kac/crandall_lab/programs.htm]) were removed from the data set.
Phylogenetic reconstructions were performed under both distance and maximum-parsimony frameworks. Distance analyses were performed by using the neighbor-joining algorithm and the Kimura two-parameter model of molecular evolution (15) as implemented in MEGA version 2.1 (http://www.megasoftware.net). Maximum-parsimony analyses were conducted by using the tree-bisection and reconnection method of branch swapping and the heuristic search algorithm of PAUP* version 4.0b (Sinauer Associates, Sunderland, Mass.). Relative support for individual nodes was assessed by nonparametric bootstrapping (8, 23) with 1,000 pseudoreplications of the data. For the combined pVGC data, bootstrap analyses were performed under both maximum-parsimony and distance frameworks. However, due to computational constraints, bootstrap analyses for the individual pVGC genes were performed only with the neighbor-joining algorithm. Genetic distance estimates were obtained as described for phylogenetic analyses with MEGA version 2.1, with standard errors estimated by using the bootstrap method and 1,000 pseudoreplications of the data. The significance of differences in genetic distance estimates was assessed by using one-tailed t tests and infinite degrees of freedom.
Development of an ASO-PCR multiplex for lineage identification. Three sets of primers were designed from pVGC sequences for the specific identification of isolates from each of the three L. monocytogenes lineage groups via an allele-specific oligonucleotide PCR (ASO-PCR) multiplex (Table 2). Amplifications were performed in 10-µl volumes with 0.5 µM concentrations of each primer, 2 mM MgCl2, 0.2 mM concentrations of each deoxynucleoside triphosphate, 0.5 U of AmpliTaq Polymerase (Applied Biosystems), and 100 ng of genomic DNA. Amplifications consisted of 25 cycles of 15 s at 94°C, 10 s at 56°C, and 10 s at 72°C. Amplification products were resolved on 1.5% (wt/vol) agarose gels, and scored relative to a 100-bp DNA size ladder (Invitrogen Life Technologies, Carlsbad, Calif.).
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RESULTS |
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Phylogenetic analyses of the combined pVGC data resolved three distinct L. monocytogenes lineages, with each of the lineages recovered as monophyletic groups in 100% of bootstrap replicates from both neighbor-joining and maximum-parsimony analyses (Fig. 1). Lineage designations were assigned according to the convention of Rasmussen et al. (25), by including partial hly sequences from this previous study into phylogenetic analyses of the pVGC haplotypes reported here (not shown). Based on these phylogenetic reconstructions 21, 23, and 17 unique haplotypes were identified within the pVGC data for L. monocytogenes lineages 1, 2, and 3, respectively (Fig. 1). Neighbor-joining and maximum-parsimony analyses both resolved L. monocytogenes lineages 1 and 3 as sister groups that formed a larger monophyletic group referred to here as the L1/L3 clade (Fig. 1). These lineage relationships were supported by 99 and 86% of bootstrap replicates from neighbor-joining and maximum parsimony analyses, respectively. Topological constraints that forced lineages 2 and 3 to form a single monophyletic group required nine additional steps in maximum-parsimony analyses, and constraints that forced lineages 1 and 2 into a sister-group relationship provided the worst fit to the observed data with respect to lineage relationships, requiring 15 additional steps in maximum-parsimony analyses.
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Branching patterns observed in the combined pVGC phylogeny (Fig. 1) suggest that the sampled lineage 1 haplotypes shared a single common ancestor more recently than haplotypes in the other two L. monocytogenes lineages. In order to test this hypothesis, the average genetic distance between haplotypes within each of the three lineages was determined. The average genetic distance between sampled haplotypes was significantly (P < 0.001) less for lineage 1 (0.29% ± 0.04%) than for either lineage 2 (0.73% ± 0.05%) or lineage 3 (1.17% ± 0.08%). Although this could result from biased sampling of highly related lineage 1 isolates or from differences in population substructure, these explanations are unlikely as the maximum genetic distance between lineage 2 (1.29%) or lineage 3 haplotypes (1.58%) was >2.5-fold the maximum genetic distance between lineage 1 haplotypes (0.49%). In addition, the average genetic distance between lineage 1 isolates after exclusion of the lower quartile of values (0.33% ± 0.05%) also was significantly (P < 0.001) less than the average values for the other two lineages.
Serotype distributions. Unambiguous serotype determinations were made for 93 of the 96 L. monocytogenes isolates tested, with three isolates ambiguously typed as 1/2b or 3b (Table 1). However, due to conflicts with strain history data and previously reported problems distinguishing among serotypes 4b, 4e, and 4d (22), four serotype 4e isolates were retested. Upon retesting, one isolate was confirmed as serotype 4e, two isolates were identified as serotype 4b (in agreement with strain histories), and a fourth isolate was identified as serotype 4d, confirming the previously reported difficulties in distinguishing among the 4b, 4e, and 4d serotypes. In addition to the serotype data collected here, L. monocytogenes strain EGD-e has been reported as serotype 1/2a (10), and serotype information was previously published (1) or provided with strain histories for 16 isolates from which pVGC sequence data were collected (Table 1). Serotypes were almost exclusively associated with one of the three L. monocytogenes lineages. Serotypes 4b, 1/2b, 4e and 4d were identified among lineage 1 isolates. Serotypes 1/2a, 1/2c, 3a and 3c were identified among lineage 2 isolates. However, in addition to the 4a and 4c serotypes identified among lineage 3 isolates, 10 (59%) of the 17 unique pVGC haplotypes identified within lineage 3 were from serotype 4b isolates (Fig. 1).
Lineage identification by using an ASO-PCR multiplex. An ASO-PCR multiplex was used to determine the lineage of individual L. monocytogenes isolates (Fig. 3). The accuracy of this test was evaluated by comparing the ASO-PCR multiplex results with lineage identifications based on pVGC sequence data for the 112 L. monocytogenes isolates used in the phylogenetic analyses. The ASO-PCR multiplex produced a single amplicon of the correct size for each of these isolates. In addition, no target amplicons were produced with isolates from any of the other Listeria species when the test was applied to four L. innocua, four L. ivanovii, two L. grayi, two L. welshimeri, and one L. seeligeri strain. In order to evaluate the utility of the test with a panel of isolates for which lineage identity was unknown and to estimate the frequency of the three L. monocytogenes lineages in food products, the ASO-PCR multiplex was also applied to 99 L. monocytogenes isolates from food products surveyed by the U.S. Department of Agriculture's Food Safety and Inspection Service (FSIS) (Table 3). A single target amplicon was produced for each of the 99 food isolates, with lineages 1 (47%) and 2 (51%) present at nearly equal frequencies and lineage 3 (2%) nearly absent.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Salcedo et al. (28) have suggested that the three primary divisions of L. monocytogenes are evident only from analyses of specific genes associated with virulence, and that housekeeping genes or random genetic markers are unable to distinguish more than two lineages. However, they did not include lineage 3 isolates in their analyses. In addition, all three lineages were monophyletic in analyses of mixed genome microarray data (2) and data from ribotyping and RAPD typing (18). Recognition of only two major divisions by the authors of these studies was likely due to an underestimation of diversity within lineage 3, since both included only two lineage 3 isolates, representing only one of the three known serotypes from this lineage, and no more than 2 of the 17 unique lineage 3 haplotypes reported here (Fig. 1). Therefore, the identification of three primary divisions within L. monocytogenes is not restricted to analyses of virulence associated genes but may have been hampered in some studies by inadequate sampling of variation within lineage 3.
Direct correlations between the three L. monocytogenes lineages and the most common serotypes have previously been reported, with lineage 1 containing serotypes 4b, 1/2b, 3b, and 3c; lineage 2 containing serotypes 1/2a, 1/2c, and 3a; and lineage 3 containing serotypes 4a and 4c (20). Such correlations are of interest because serotypes represent the traditional common language of L. monocytogenes subtyping, and because strains with serotypes 4b, 1/2b, and 1/2a are responsible for the vast majority of human listeriosis cases (7, 29). Similar correlations were observed in the present study, particularly with respect to serotypes 1/2a, 1/2c, and 1/2b. However, comparisons between serotype and lineage for 106 L. monocytogenes isolates for which both were unambiguously determined (Table 1 and Fig. 1) revealed for the first time that serotype 4b, which is responsible for the majority of human listeriosis cases (16) and virtually all major outbreaks of listeriosis in humans (13), is prevalent (59% of unique haplotypes) among strains from lineage 3, which is rarely associated with human listeriosis (13). These results demonstrate that serotype 4b isolates do not represent a distinct evolutionary group within L. monocytogenes and that serotype 4b cannot be used as a proxy for lineage identification.
Lineage identification, host specificity, and virulence differences. L. monocytogenes lineage-specific variation identified during the analyses of pVGC sequences was used to develop an ASO-PCR multiplex test for the specific identification of evolutionary lineage for individual L. monocytogenes isolates. This test proved to be 100% sensitive and specific in accurately assessing the lineage for 112 L. monocytogenes isolates for which lineage identity had been confirmed phylogenetically. Previously, Jinneman and Hill (14) developed a PCR-based assay for L. monocytogenes lineage identification based on sequences from the hly gene but reported that one of the lineage 3 isolates produced target amplicons indicative of both lineage 2 and lineage 3. Analyses of the hly sequences reported here indicated that by using the Jinneman and Hill test, multiple target amplicons would be produced with nine of the 17 unique lineage 3 haplotypes and that only the amplicon specific to lineage 2 isolates would be produced for one of the lineage 3 haplotypes. These predictions were confirmed by performing the Jinneman and Hill test on the isolates in Table 1. In addition to problems with specificity caused by undersampling variation in lineage 3, this test also requires at least three separate PCRs. Similar problems exist with a lineage identification test developed by Moorehead et al. (19) that is based on only 23 isolates and also requires at least three separate PCRs.
Accurate, inexpensive, and high-throughput methods for L. monocytogenes lineage identification have the potential to inform studies of the population genetics, ecology, and epidemiology of this important food-borne pathogen and can also aid in understanding the biological and regulatory significance of the evolutionary lineages that have been identified within this species. For instance, the fact that lineage 3 isolates are rarely associated with human listeriosis but are common among animal isolates led Wiedmann et al. (33), and later Jeffers et al. (13), to suggest that lineage 3 isolates show a host specificity for nonprimate mammals and limited virulence in humans. However, application of the ASO-PCR multiplex to 99 L. monocytogenes isolates surveyed by FSIS provided the first direct estimate of the prevalence of individual lineages from a broad array of food products, and indicated that lineage 3 accounts for only 2% of L. monocytogenes isolates from food (Table 3). If we assume that contaminated food is the primary cause of listeriosis in humans, the frequency of lineage 3 isolates among human sporadic cases (1%) reported by Jeffers et al. (13) is entirely consistent with the relative frequency of lineage 3 isolates in food products (Table 3). Therefore, the low frequency of association between lineage 3 isolates and human listeriosis cases likely reflects rarity of exposure and not reduced virulence for humans or specificity for nonhuman hosts. In addition, the prevalence of lineage 3 among animal isolates (37% of the animal isolates in Table 1) and the near absence among food isolates suggests that lineage 3 may be better adapted to the animal production environment than the food processing environment. Systematic comparisons of lineage-specific fitness in different environments are needed to fully evaluate this hypothesis. However, De Jesús and Whiting (6) have found that strains from lineage 3 are less likely to survive thermal inactivation than strains from the other two lineages of L. monocytogenes, indicating that lineages 1 and 2 may be better adapted to the food-processing environment than are lineage 3 isolates.
Comparison of the relative frequencies of the three L. monocytogenes lineages in food products (Table 3) and human listeriosis cases (13) suggests that lineage 1 is overrepresented and lineage 2 is underrepresented among isolates from human listeriosis cases. However, it is unclear if this reflects enhanced virulence for humans or unique ecological adaptations such as enhanced psychrotolerance and growth at refrigeration temperatures. Prevalence studies alone are insufficient to clearly demonstrate lineage-specific differences in virulence or ecological adaptations. However, the availability of complete genome sequences for L. monocytogenes lineage 1 (http://www.tigr.org) and lineage 2 (10) isolates will facilitate functional genomic studies and additional analyses of genomic variation within and between lineages that will complement comparative evaluations of virulence and comprehensive surveys of lineage prevalence in different environments. A combination of such studies will be required to fully test hypotheses regarding lineage-specific differences in virulence, host range, or ecology and to understand the genetic and evolutionary basis of such differences.
Lineage relationships and taxonomy. Previous analyses of relationships within L. monocytogenes based on shotgun DNA microarray data suggested that the single lineage 3 isolate examined in that study was distinct from a more derived group consisting of lineages 1 and 2 (36). However, these data were highly homoplasious (homoplasy index = 0.6491) in that they contained a high proportion of character state similarities that were not due to inheritance from a common ancestor, with over half of the polymorphisms distributed among polyphyletic groups (36). As noted by Zhang et al. (36), several comparative studies of Listeria genomes suggest a bias toward cell surface-related differences in genome content (4, 10, 12), indicating that different combinations of genes encoding cell surface characteristics may be favored by selection (36). This suggests that polymorphism data from such genome content studies may be inherently less reliable for use in phylogenetic reconstruction because these studies can be biased toward nonessential genes that may be lost independently in multiple evolutionary lineages or classes of genes that may be frequently involved in lateral gene transfer events due to selection.
Accurate reconstruction of the evolutionary relationships between the three L. monocytogenes lineages is essential to understanding the evolution of virulence traits and ecological adaptations within this species and is also critical in evaluating proposals to reassess the taxonomic rank of individual lineages. Both neighbor-joining and maximum-parsimony analyses of the combined pVGC data strongly support a sister-group relationship between lineages 1 and 3 (Fig. 1). The L1/L3 clade was also recovered in five of the six gene trees constructed from individual pVGC genes (Fig. 3). Lineages 1 and 2 were most closely related in the hly gene tree, which appears to reflect historical recombination between ancestors of present-day lineage 1 and lineage 2 haplotypes. However, despite the discordant hly gene tree, a sister-group relationship between lineages 1 and 2 provided the worst fit to the combined pVGC data, and the combined analyses of pVGC sequences strongly support the conclusion that lineages 1 and 3 share a common ancestor exclusive of lineage 2 (Fig. 1). This conclusion is congruent with the midpoint-rooted phylogenetic tree derived from combined analyses of ribotyping and RAPD typing data (18) and phylogenetic analyses based on mixed genome microarray data (2). In addition, the single serotype 4a isolate included in the multilocus enzyme electrophoresis study conducted by Piffaretti et al. (24) clustered with lineage 1 isolates. Although lineage association was not directly determined for this isolate, the 4a serotype appears to be specific to lineage 3 (Table 1) (20) and the other serotypes that have been identified within lineage 3 were either absent (serotype 4c) from the Piffaretti et al. (24) study or clustered with the lineage 1 group (serotype 4b), a finding consistent with the conclusion that lineages 1 and 3 are sister-groups.
Wiedmann et al. (33, 34) have suggested that lineage 3 represents a distinct taxonomic unit separate from lineages 1 and 2 and that lineage 3 should be recognized as a new species or subspecies because the small number of lineage 3 isolates examined had a distinctive ribotype fragment, a unique 16S rRNA sequence, and 70 to 76% DNA-DNA homology with the L. monocytogenes type strain from lineage 2 (27) and were predominantly serotype 4a or 4c. However, these differences were not evaluated relative to the phylogenetic history of lineage divergence within L. monocytogenes. The results of the phylogenetic analyses presented here strongly support a monophyletic L1/L3 clade exclusive of lineage 2 (Fig. 1). Therefore, recognizing lineage 3 as a new species or subspecies without equivalent recognition for lineage 1 would make L. monocytogenes paraphyletic, which is inconsistent with the modern systematic principles that taxonomy should reflect evolutionary history and taxonomic groups should comprise individuals that uniquely share a most recent common ancestor. In addition, the use of genetic or phenotypic features to circumscribe new species in the absence of an evolutionary framework for interpreting species boundaries is arbitrary and is not supported by population genetic or evolutionary theory.
The cohesion species concept proposed by Templeton (30) provides an evolutionary framework for understanding species as groups of organisms whose divergence is constrained by microevolutionary forces that maintain species as genetically and phenotypically cohesive groups. For species such as L. monocytogenes, which has a largely clonal population structure (4, 24, 25), evolutionary theory indicates that genetic drift and natural selection are the primary forces influencing species cohesion (5, 30). The results presented here, indicating a more recent coalescence for lineage 1 haplotypes than haplotypes from the other two lineages (Fig. 1), are interesting because they suggest that lineage 1 was exposed to a purge of genetic variation not observed in the other two lineages. This indicates a limitation on the extent to which these lineages are bound together by natural selection and suggests that they may represent distinct species within the framework of the cohesion species concept. Evidence from the survey of lineage prevalence in food products (Table 3) and from previous studies (6, 13), suggesting lineage-specific differences in ecological niche adaptations further support this interpretation. Given the phylogenetic relationships supported by the combined pVGC data and the fact that the L. monocytogenes type strain belongs to lineage 2 (Fig. 1), species recognition for lineage 1 would require reclassification of lineage 1 and lineage 3 isolates into two new species. However, the results of the present study should be viewed as hypothesis-generating with respect to taxonomic revision of L. monocytogenes, which will require a greater understanding of the ecology and demographic exchangeability of L. monocytogenes lineages and evaluations of the demographic history of these lineages based on genetic variation sampled from additional regions of the L. monocytogenes chromosome.
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ACKNOWLEDGMENTS |
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The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.
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FOOTNOTES |
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Supplemental material for this article may be found at http://jb.asm.org/.
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REFERENCES |
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