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Department of Food Science, 412 Stocking Hall, Cornell University, Ithaca, NY 14853, USA
Correspondence
Angela Roberts
ajr29{at}cornell.edu
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ABSTRACT |
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Present address: Gorton's, 128 Rogers St, Gloucester, MA 01930, USA.
Present address: Department of Biology, Centro de Investigação de Recursos Naturais, University of Azores, Portugal.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Listeriosis symptoms in humans can include encephalitis, meningitis, septicaemia and abortion (Low & Donachie, 1997). Epidemiological, population genetics and evolutionary studies of L. monocytogenes using various subtyping methods have been critical to improving our understanding of how L. monocytogenes is transmitted from animals or the environment through foods to humans. Commonly used subtyping methods for L. monocytogenes include serotyping, multilocus enzyme electrophoresis, and DNA-based methods such as pulsed-field gel electrophoresis (PFGE), ribotyping, PCR-restriction length polymorphism (PCR-RFLP) analysis and multilocus sequence typing (Wiedmann, 2002a, b). Results from subtyping studies utilizing many of these methods have established that L. monocytogenes can be divided into at least three distinct evolutionary lineages. Two phylogenetic lineages were identified initially by multilocus enzyme electrophoresis (Piffaretti et al., 1989), and the existence of these lineages was subsequently confirmed by partial DNA sequencing of a virulence gene (Rasmussen et al., 1991), PFGE (Brosch et al., 1994) and ribotyping (Graves et al., 1994). Rasmussen et al. (1995) provided the first evidence of a third L. monocytogenes phylogenetic lineage, lineage III, based on analyses of partial DNA sequences for flaA, iap and hly. Ribotyping and PCR-RFLP analyses of L. monocytogenes virulence genes, as well as comparative genomics and DNA sequencing studies, further confirmed the existence of at least three phylogenetic lineages in L. monocytogenes (Doumith et al., 2004; Ward et al., 2004; Wiedmann et al., 1997). Interestingly, these three L. monocytogenes lineages appear to correlate well with serotypes: lineage I isolates represent serotypes 1/2b, 3b, 3c and 4b, while lineage II isolates represent serotypes 1/2a, 1/2c and 3a (Nadon et al., 2001). While lineage III isolates initially were reported to represent serotypes 4a and 4c (Nadon et al., 2001), recent characterization of a larger number of lineage III isolates indicated that a number of isolates in this lineage are serotype 4b (Nightingale et al., 2005; Ward et al., 2004).
L. monocytogenes isolates belonging to lineage III are rare and have several unique genetic and phenotypic characteristics. Of the more than 1800 L. monocytogenes isolates in our strain collection that have been subtyped, for example, only 52, or less than 3 %, belong to lineage III (http://www.pathogentracker.net). Lineage III isolates often have unique ribotypes (e.g. DUP-1061, DUP-1059) (Jeffers et al., 2001), hly PCR-RFLP types and serotypes (e.g. 4a, 4c) (Nadon et al., 2001; Wiedmann et al., 1997). Interestingly, lineage III strains are more prevalent among isolates from animals with clinical listeriosis than among human clinical isolates. For example, 10·5 % of the 76 animal isolates characterized by Jeffers et al. (2001) were lineage III, while human lineage III prevalence has been consistently less than 2·5 % (Jeffers, 1998; Jeffers et al., 2001). While these findings led some to hypothesize that lineage III isolates may have reduced human pathogenic potential (Jeffers, 1998; Jeffers et al., 2001; Wiedmann et al., 1997), more recent data reporting an exceptionally low prevalence (<1·0 %) of lineage III among food isolates (Gray et al., 2004; Ward et al., 2004) suggest that the uncommon occurrence of human listeriosis cases due to lineage III strains may be explained by the rarity of foodborne exposure to these strains.
The goal of this study was to gain a more comprehensive knowledge of the phenotypic and genotypic characteristics of L. monocytogenes lineage III strains to facilitate a better understanding of the ecology and transmission of these strains. To achieve this goal, we assembled the largest set of lineage III isolates characterized to date (46 isolates) to (i) determine the phylogenetic relationship among lineage III isolates, (ii) compare actA, hly and lmaA genotypes and rhamnose fermentation phenotypes of lineage III isolates, and (iii) characterize the pathogenic potential of lineage III isolates using a cell culture plaque assay.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Forty-six of those isolates were chosen for inclusion in this study (Table 1); six isolates were eliminated because their sources of isolation (e.g. isolation from two animals on the same farm), ribotypes, actA types and hly types suggested that they were clones of other isolates already included in our isolate set. Our final lineage III isolate set characterized here included isolates from human clinical cases (n=20), animals with and without signs of listeriosis (n=13), foods (n=6), as well as seven isolates of unknown origin. All stock cultures were stored at –80 °C in 15 % (v/v) glycerol and grown and, unless otherwise noted, maintained in brain heart infusion (BHI) agar or broth (Difco/Becton Dickinson).
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). The Riboprinter generated DuPont IDs (e.g. DUP-1061) for the majority of the isolates analysed. If the Riboprinter was unable to assign a DuPont ID (i.e. for a new pattern with <0·85 similarity to existing patterns in the DuPont database), we assigned a unique type designation based on the ‘Ribogroup’ that had been assigned by the instrument (e.g. ribogroup 116-110-S-2). All DuPont IDs were confirmed by visual inspection. If an assigned DuPont ID included more than one distinct ribotype pattern (e.g. patterns differing by a single weak band) then each pattern was designated with an additional letter (e.g. DUP-1059A and DUP-1059B). Ribotype patterns for isolates in this study are available for comparison through Pathogen Tracker (www.pathogentracker.net).
Virulence gene allele characterization.
Virulence gene alleles for actA and hly were determined by PCR-RFLP as described previously (Wiedmann et al., 1997). Detection of the putative virulence gene lmaA was carried out by PCR using primers lmaA-F (5'-TTC TGC TGG TGC TAC AGG TG-3'and lmaA-R (5'-CCA ACA AGG TCT AAC TGT AAA CCG-3'), which amplify an approximately 420 bp fragment of the lmaA ORF (Schaferkordt & Chakraborty, 1997). Each reaction contained 1 unit Taq polymerase (Promega), 1·5 mM MgCl2, 1x PCR buffer, 50 µM of each dNTP and 0·5 µM of each primer. Thermocycling conditions for the lmaA PCR were 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s.
actA and sigB sequencing.
actA and sigB PCR amplification and sequencing was performed using standard methods as described by Nightingale et al. (2005). For actA, an approximately 600 bp region at the 3' end of this gene (nt 1275 to 1920), previously identified as being the most discriminatory region within actA (Cai et al., 2002), was amplified and sequenced. For sigB, an 840 bp DNA fragment (nt –30 to 810) containing the complete sigB ORF was amplified and sequenced. DNA sequencing was performed at Cornell University's Bioresource Center or by Macrogen (Seoul, Korea). All DNA sequences are available online through Pathogen Tracker (www.pathogentracker.net).
Descriptive analysis of sequence data.
Descriptive analyses were performed as described by Nightingale et al. (2005). Analysis was based on a 561 nt actA alignment and a 573 nt sigB alignment (representing 29·2 % and 73·5 % of the respective ORFs). actA and sigB sequences for lineage I and II isolates reported by Nightingale et al. (2005) were used for comparison purposes. Analysis was performed for alignments containing all isolates studied here (n=163) as well as separately for alignments that were stratified by lineage (I, II and III) and subgroups within lineage III (i.e, IIIA, IIIB and IIIC). Briefly, nucleotide diversity (
, mean pairwise nucleotide difference per site; and k, mean pairwise nucleotide difference per sequence), number of polymorphic sites, number of mutations, number of alleles, Tajima's D test for neutrality, number of synonymous mutations (S), number of nonsynonymous mutations (N), and dN/dS ratio averaged over all isolates were calculated for each gene using DnaSP version 3.99 (Rozas et al., 2003). Alleles, defined by a unique combination of polymorphisms within an individual gene, and sequence types, defined by a unique combination of alleles, were also assigned using DnaSP.
Phylogenetic analysis.
Phylogenetic analyses were performed essentially as described by Nightingale et al. (2005). Briefly, MODELTEST (Posada & Crandall, 1998) was used to optimize parameters to infer maximum-likelihood phylogenetic trees in PAUP* (Swofford, 1997). Due to the large size of our dataset (n=163, lineages I, II and III), a single isolate was selected to represent each unique sequence type (n=67) to infer phylogenetic trees. Maximum-likelihood trees were generated for each individual gene in PAUP*. Heuristic searches were performed using equal weights for all sites and the tree-bisection-reconnection branch-swapping algorithm was employed.
Plaque assay.
We used a plaque assay previously described by Sun et al. (1990) to determine the ability of selected lineage III isolates to spread from cell to cell as represented by plaque size. Briefly, duplicate wells of mouse fibroblast L2 cells (a gift from D. A. Portnoy, Department of Molecular and Cell Biology, University of California, Berkeley) were infected with 1x105 c.f.u. and 3x104 c.f.u. of L. monocytogenes. After a 1 h incubation at 37 °C, the monolayers were washed and overlaid with Dulbecco's Modified Eagle's Medium (DMEM) containing 10 µg gentamicin ml–1 and 1·4 % agar. Following 3 days of incubation, a second overlay of DMEM containing 6 % Neutral Red solution and 1·4 % agar was added. Plaques were counted and their sizes determined using the SigmaScan Pro 5.0 software (Statistical Solutions) to calculate plaque area. Plaque size and number were normalized by expressing them as a percentage of the internal control strain, 10403S.
Rhamnose fermentation.
We tested the ability of our isolates to ferment rhamnose by inoculating Oxidative Fermentative (OF) Test Medium (prepared as described by Atlas, 1993) containing 0·55 mM rhamnose with a stab of cells taken from individual colonies on agar plates. Two tubes per isolate were stabbed; one was overlaid with mineral oil, and both were incubated at 37 °C for 48 h. Observations were taken at 24 and 48 h, and a reaction was scored as positive if the media turned yellow in both tubes, indicating the fermentative formation of acid from rhamnose.
Statistical analysis.
The chi-squared test was used to test for significant associations between lineage III subgroup and isolate source, lmaA genotype, hly PCR-RFLP type, actA type and rhamnose fermentation phenotype. A one-way analysis of variance (ANOVA) was used to compare mean plaque sizes of isolates classified to different lineage III subgroups (i.e., IIIA, IIIB and IIIC). A two-sided one-sample t-test was used to determine if the mean plaque size for all lineage III isolates was significantly different from the plaque size for the internal control strain, 10403S. Statistical significance was declared at P
0·05 and all analyses were performed in Statistix 7 (Analytical Software).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) and 46 lineage III isolates were used to infer the molecular phylogeny of isolates previously classified as lineage III based on EcoRI ribotype and hly PCR-RFLP analyses. sigB and actA were chosen for sequencing because they are located on different regions of the L. monocytogenes chromosome (721·2 kb apart), and because they have different functions: actA is a virulence gene while sigB is a general stress-response gene. Moreover, previous work showed that actA and sigB are highly polymorphic and discriminatory, but show limited indication of horizontal gene transfer, making them suitable for reconstructing the evolutionary history of a group of L. monocytogenes isolates (Cai et al., 2002). A total of 57 unique actA alleles and 30 unique sigB alleles, resulting in 67 unique sequence types (STs) were differentiated among all 163 isolates (I, II and III). Among the 46 lineage III isolates, a total of 30 actA and 18 sigB alleles, resulting in 32 STs, were differentiated (Table 2). One lineage III food isolate (FSL J2-076) contained a point mutation in sigB leading to a premature stop codon at codon 51; at the first nucleotide position in this codon (nt 151) a ‘T’ substitution, which resulted in a TAA stop codon instead of the CAA codon found in all other isolates, was identified.
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). Subgroup IIIA includes 29 isolates, representing 18 STs and 10 EcoRI ribotypes. Subgroup IIIB includes 10 isolates, 9 STs and 3 ribotypes, while subgroup IIIC includes 7 isolates, 5 STs and 3 ribotypes. EcoRI ribotypes, STs, and actA and sigB alleles were unique for each lineage III subgroup; i.e. no EcoRI ribotype, ST or allelic type was represented in more than one lineage III subgroup, further supporting isolate classification into these subgroups.
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). actA showed a considerably higher level of nonsynonymous mutations as compared to sigB sequences (Table 2). Most of the genetic diversity within L. monocytogenes appears to be attributable to the presence of divergent evolutionary lineages within this bacterial species. When sequence data were analysed after stratification by lineage (i.e. lineage I, II, IIIA, IIIB and IIIC), the genetic diversity observed within each of these subpopulations was considerably reduced (Table 2). While lineage III isolates showed the greatest level of genetic diversity, as these isolates contained the majority of actA and sigB polymorphisms, the number of polymorphisms observed within lineage IIIA, IIIB and IIIC subgroups for both actA and sigB was much lower, suggesting a barrier for genetic exchange between these lineage III subgroups (Table 2). Lineage IIIA appears to be the most genetically diverse subpopulation within lineage III, as determined by the elevated measures of nucleotide diversity (i.e. and k values; Table 2).
Tajima's D test not only tests the hypothesis that sequences have evolved according the neutral theory but also encompasses information about a given population's demographics and thus can be used to make inferences about an organism's population structure (Nielsen, 2001; Simonsen et al., 1995). Our data indicate that actA and sigB have evolved under neutrality, as none of the Tajima's D test statistic values, based on all isolates, were significantly different from the expected value of zero. In a previous study, we showed that large positive Tajima's D test statistics, indicating a subdivided population structure for L. monocytogenes, became much smaller or negative when analyses were performed for lineage I and II isolates separately (Nightingale et al., 2005). Similarly, the large positive Tajima's D values observed for both actA and sigB based on alignments containing all isolates studied here (i.e. lineages I, II and III), which were reduced upon stratification of the full dataset by lineage, supports the concept that L. monocytogenes has a subdivided population structure. Similarly, for the most part, Tajima's D values observed for all lineage III isolates became smaller or negative when lineage III isolates were divided into the subgroups that were resolved in actA and sigB phylogenetic trees (IIIA, IIIB and IIIC), providing additional evidence that lineage III also represents a subdivided population.
Genetic and phenotypic characteristics of lineage III subgroups
Based on preliminary evidence that at least some lineage III strains lack the ability to ferment rhamnose (Wiedmann et al., 1997), we assayed all 46 lineage III isolates for rhamnose fermentation. There was an exclusive and significant association (P<0·001) between lineage III subgroup and ability to ferment rhamnose. Specifically, all lineage IIIA isolates fermented rhamnose, while all lineage IIIB and IIIC isolates were rhamnose fermentation negative (Table 1).
Since preliminary evidence also indicated that lineage III isolates often had unique genotypic characteristics (Jeffers et al., 2001; Wiedmann et al., 1997), we further characterized all lineage III isolates for actA type (type 3 or 4, indicating absence or presence of a proline-rich repeat, respectively), hly PCR-RFLP type, and the presence or absence of the virulence-associated gene lmaA (Table 1). There was an overall significant association (P=0·034) between lineage III subgroup and actA type. Specifically, actA type 4 was significantly overrepresented among lineage IIIA isolates (79 % of IIIA isolates carried actA type 4), while actA types 3 and 4 appeared to be equally prevalent among lineage IIIB and IIIC isolates. Overall, lineage III isolates represented eight different hly PCR-RFLP types; however, there was no significant association between lineage III subgroup and hly type. In general, lineage IIIA had the most hly type diversity, with all eight hly PCR-RFLP types represented, while lineages IIIB and IIIC contained only three types each (1c, 1d, 4b and 1, 4a, 4b, respectively). Subgroups IIIA, IIIB and IIIC contained predominantly hly PCR-RFLP type 4b (28 %, 80 % and 57 %, respectively). Interestingly, 19 lineage III isolates did not yield a PCR product for lmaA, indicating the absence of this putative virulence gene (Schaferkordt & Chakraborty, 1997) in some lineage III isolates. While the majority (88 %) of IIIB and IIIC isolates carried lmaA, only 58 % of lineage IIIA isolates were confirmed to carry this gene, and this difference was statistically significant (P=0·002). Serotypes were available for 31 of the 46 lineage III isolates characterized and included serotypes 4a (n=16), 4b (n=7) and 4c (n=8). There were no associations between lineage III subgroup and serotype, and all three subgroups contained at least one isolate representing each serotype.
Distribution of lineage III isolates among different sources
Of the 46 lineage III isolates characterized, 20 were obtained from human clinical cases, 13 from animals both with and without clinical listeriosis, 6 from foods, and 7 from unknown sources. Human clinical isolates were found in all lineage III subgroups and there were no significant associations between lineage III subgroup and source of isolation. There were, furthermore, no significant associations between isolate source (human, animal or food) and actA type, hly type, lmaA type or rhamnose fermentation phenotype.
Cell culture characterization of lineage III isolates
Since isolates in all three lineage III subgroups were obtained from human listeriosis cases, selected isolates from these three subgroups were characterized using a cell culture plaque assay to assess their ability to spread from cell to cell. Plaque size for the 14 isolates characterized ranged from 53 % to 114 %, with an overall mean plaque size of 88±21 % (mean±SD) which represents a marginally significantly smaller size than the lineage II internal control strain, 10403S (P=0·05). There was no significant difference in the mean plaque size of isolates representing the three lineage III subgroups; specifically, lineage IIIA isolates had a mean plaque size of 100±23 % (n=4), lineage IIIB isolates had a mean plaque size of 84±22 % (n=7), while lineage IIIC isolates had a mean plaque size of 80±8 % (n=3). In contrast, Wiedmann et al. (1997) reported a mean plaque size for lineage III isolates (n=5) that was significantly larger than the 10403S control (126·2±6·8 %). In that study, however, one small plaque-forming isolate (L99, plaque size 53·5±7·9 %) was excluded from analysis based on its previously reported virulence-attenuated phenotype (Chakraborty et al., 1994). When isolate L99 is included, however, the mean lineage III plaque size is 114±30 %, which is not significantly different from the mean plaque size for the lineage III isolates characterized here.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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L. monocytogenes isolates previously designated lineage III represent a subdivided population that can be partitioned into at least three genetically and phylogentically distinct subgroups
Using phylogenetic analysis of sigB and actA sequences for 46 isolates, we found that L. monocytogenes isolates identified as lineage III based on EcoRI ribotyping and hly PCR-RFLP analysis represent three distinct subgroups, which we have termed IIIA, IIIB and IIIC. The relevance of these three lineage III subgroups is further supported by the fact that EcoRI ribotypes are unique to lineage III subgroups; i.e. a given EcoRI ribotype does not occur in more than one subgroup, similar to the observation that EcoRI ribotypes as well as ribotype subset are unique to lineages I and II (Jeffers, 1998; Jeffers et al., 2001; Wiedmann et al., 1997). Additionally, we showed that actA and sigB alleles and thus sequence types are exclusive to these lineage III subgroups. The absence of shared alleles has been proposed as a criterion to resolve subpopulations within a set of intraspecific data (Lan & Reeves, 2001) and our data strongly suggest that the three subgroups identified within lineage III do, in fact, represent genetically distinct populations. Interestingly, classification of lineage III isolates into multiple distinct subgroups is also consistent with previous studies, which used phylogenetic analysis of DNA sequences for one or more L. monocytogenes gene(s) to classify and characterize L. monocytogenes isolates; most of these studies showed that lineage III isolates were highly diverse and often formed multiple distinct clusters in phylogenetic trees (Meinersmann et al., 2004; Moorhead et al., 2003; Ward et al., 2004). For example, the sigB phylogeny for 8 lineage III (including 6 isolates used here) and 15 other L. monocytogenes isolates reported by Moorhead et al. (2003) also showed separation of lineage III isolates into subgroups, including completely separate clustering of the IIIC isolate FSL W1-110. Similarly, lineage III isolates also grouped in multiple separate clusters in the addB and truB phylogenic trees for 5 lineage III and 30 other L. monocytogenes isolates reported by Meinersmann et al. (2004), even though the IIIC isolate FSL W1-110 generally clustered with the two IIIA isolates. However, these previous studies were limited by the small number of lineage III isolates characterized, and the larger lineage III isolate collection used in the current study allowed the lineage III subgroups, particularly subgroup IIIC, to be defined. We conclude that there is abundant and convincing evidence that isolates previously grouped into L. monocytogenes lineage III represent at least three distinct populations. While we refer to the lineage III subgroups as IIIA, IIIB and IIIC to be consistent with previous designation of these isolates as lineage III and reflecting that serotypes 4a, 4b and 4c are found among all three subgroups, phylogenetic analyses indicate that these three subgroups may represent separate evolutionary lineages.
While lineage III isolates are most prevalent among animal isolates, they are occasionally isolated from human cases and appear to have the ability to cause human disease
Interestingly, we found that all three lineage III subgroups identified here included isolates from human clinical cases, indicating that lineage III strains have the ability to cause human disease. Our finding that all lineage III isolates tested were able to form plaques in a cell culture cytopathogenicity assay indicates that lineage III strains possess the virulence genes necessary to spread from cell to cell, demonstrating the pathogenic potential of this L. monocytogenes population. These data indicate that lineage III may not show attenuated human virulence as previously proposed (Jeffers et al., 2001; Wiedmann et al., 1997). While over 40 % of the lineage III isolates characterized here came from human clinical cases of listeriosis, it is important to point out that human clinical isolates are overrepresented in our strain collection used to assemble the lineage III isolates set for this study. In general, lineage III isolates are more common among animal listeriosis cases (representing about 10 % of animal clinical isolates: Jeffers et al., 2001) than among human clinical isolates or food isolates, representing 2·2 % and 0·4 % of isolates in these two categories, respectively (Gray et al., 2004). Consistent with the results reported by Ward et al. (2004), these data thus suggest that rarity of human exposure to lineage III isolates through consumption of contaminated foods, rather than reduced virulence, may account for the fact that lineage III isolates cause few human listeriosis cases and have never been linked to a human listeriosis outbreak. We hypothesize that the genetic and phenotypic characteristics that are unique to lineage III, both those characteristics that have been already identified and those that remain to be discovered, make lineage III strains, on average, less well suited to survive the stressful conditions associated with food and food-production environments, explaining the rare human foodborne exposure to lineage III strains. Supporting this hypothesis, a study on thermal inactivation D-values and growth characteristics of lineage I, II and III isolates (De Jesus & Whiting, 2003) previously found that lineage III isolates had the longest mean lag duration time at 7 °C (a temperature typical of refrigerated food storage) and the lowest mean thermal inactivation D-value, suggesting that lineage III isolates are, on average, less resistant to heat and cold stress than isolates belonging to lineages I and II. There were no differences in lag duration times between the lineages at 37 °C, which supports our assertion that lineage III strains have the potential to grow normally and be fully virulent once consumed by a mammalian host. Djordjevic et al. (2002) also found that lineage III, along with lineage II, has a decreased ability to form biofilms on PVC microtitre plates. Since biofilm formation is likely to contribute to the ability of L. monocytogenes to persist in different environments and to subsequently be introduced into the human food chain, a reduced ability to form biofilms may also contribute to a lower prevalence of lineage III isolates in foods. Interestingly, we also found here that one subgroup IIIA isolate carried a premature stop codon in sigB, which encodes the stress-responsive alternative sigma factor 
B (Becker et al., 1998). While this mutation was only found in a single isolate sequenced, occurrence of this mutation may indicate that at least some lineage III strains experience limited selection to maintain functional stress-response systems in their natural reservoirs, consistent with their potentially reduced stress resistance. While the observations outlined above provide initial evidence that lineage III strains are characterized by reduced environmental survival capability while maintaining the ability to cause mammalian disease, future research on genetic and phenotypic characteristics of the different lineage III subgroups and their ecology and epidemiology is needed to better understand the evolution and transmission of these L. monocytogenes strains.
Lineage III isolates have unusual phenotypic characteristics that may complicate their identification
Our data confirm and extend previous initial evidence (Wiedmann et al., 1997) that lineage III isolates have unique phenotypic and genetic characteristics that may complicate their detection using various standard assays for the identification of L. monocytogenes. While previous studies noted that some lineage III isolates are unable to ferment rhamnose, rhamnose fermentation is frequently included as a differential biochemical test in rapid identification kits such as API Listeria (BioMerieux) and Micro-ID (Organon Technika) used to identify pathogenic L. monocytogenes (Bille et al., 1992; Sado et al., 1998). Since all subgroup IIIB and IIIC isolates are rhamnose-negative, identification of these L. monocytogenes isolates based on biochemical patterns could be ambiguous. In addition, the lack of lmaA PCR amplification in some lineage III isolates combined with the absence of a number of other L. monocytogenes-specific genes, including several confirmed and putative internalin genes (e.g. as determined by DNA array analyses; Doumith et al., 2004; Y. Jia and others, unpublished data) indicates that PCR assays targeting certain L. monocytogenes-specific genes may also fail to detect some or all L. monocytogenes lineage III strains. Our results indicate the importance of including L. monocytogenes isolates representing all three lineage III subgroups when validating assays for the specific detection of L. monocytogenes, particularly considering that all three lineage III subgroups have been found to cause human listeriosis cases. To this end, the lineage III isolates characterized here are available to other researchers and commercial entities.
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ACKNOWLEDGEMENTS |
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Received 8 September 2005;
revised 28 November 2005;
accepted 28 November 2005.
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