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J Clin Microbiol. Oct 2005; 43(10): 5291–5294.
PMCID: PMC1248515

Multi-Virulence-Locus Sequence Typing Clarifies Epidemiology of Recent Listeriosis Outbreaks in the United States

Abstract

Multi-virulence-locus sequence typing (MVLST) was used to analyze isolates from two major listeriosis outbreaks in the United States in 1998 and 2002 that were due to consumption of contaminated hot dogs and turkey deli meat, respectively. MVLST demonstrated high epidemiological relevance and indicated that the two outbreaks were the result of one epidemic.

Listeria monocytogenes can reproduce in a wide variety of reservoirs within food processing plants and contaminates many foods, including ready-to-eat meat and poultry products. Despite implementation of Hazard Analysis and Critical Control Point systems in the meat and poultry industries, significant listeriosis outbreaks have been reported in recent years. In 1998, a multistate listeriosis outbreak due to consumption of hot dogs caused 100 illnesses and 21 deaths (1). Another multistate listeriosis outbreak in 2002 due to consumption of turkey deli products caused 53 illnesses and 11 deaths (2). Food-borne listeriosis generally has a long incubation period (7 to 60 days), which makes source tracking by classic epidemiological approaches difficult. Both DNA fragment-based and sequence-based molecular subtyping methods have been used to investigate listeriosis outbreaks. Among these methods, pulsed-field gel electrophoresis (PFGE) is currently the U.S. Centers for Disease Control and Prevention's (CDC's) “gold standard” method for epidemiological investigation of food-borne pathogens (15). Recently, multi-virulence-locus sequence typing (MVLST) was developed by Zhang et al. (18) for subtyping L. monocytogenes and provided higher discriminatory power than PFGE when analyzing genetically diverse L. monocytogenes isolates. The purpose of the present study was to use MVLST to investigate the epidemiology of the 1998 and 2002 U.S. multistate listeriosis outbreaks.

There are several basic concepts and premises of epidemiological investigation. An outbreak is defined as an acute appearance of a cluster of an illness caused by a source strain that occurs in numbers in excess of what is expected for that time and place (13). However, the same source strain may survive and spread over a long period of time and cause several outbreaks, resulting in an epidemic. A basic premise is that isolates that are part of the same chain of transmission are the descendants of the source strain and can be referred to as a clone (12). An important function of molecular subtyping is to accurately identify the clonal relationship among isolates so that it can provide early recognition of outbreaks and help identify routes of transmission. Therefore, the most important criterion for molecular subtyping is epidemiological relevance, which we define here as the ability to (i) cluster isolates that are epidemiologically associated with a particular epidemic/outbreaks and (ii) separate these isolates from those that are not epidemiologically associated with the same epidemic/outbreaks.

Ten L. monocytogenes isolates from the 1998 U.S. multistate listeriosis outbreak and 11 isolates from the 2002 U.S. multistate listeriosis outbreak were selected and obtained from the Listeria collection at the CDC. All isolates were previously identified by the CDC as being involved in these outbreaks through classic epidemiological investigations and by PFGE using two different restriction enzymes (AscI as the primary enzyme and ApaI as the secondary enzyme) (Table (Table1)1) (1, 2, 5). Bacterial strains were grown at 37°C on trypticase soy yeast extract agar. Bacterial genomic DNA was extracted using an UltraClean microbial DNA extraction kit (Mo Bio Laboratories, Solana Beach, Calif.). Intragenic regions of the six virulence genes (prfA, inlB, inlC, dal, clpP, and lisR) were targeted and sequenced as previously described by Zhang et al. (18). Multiple sequence alignments and phylogenetic analysis were performed using molecular evolutionary genetics analysis software (MEGA version 3.0) (9).

TABLE 1.
PFGE patterns, serotypes, MVLST sequence types, and ECII PCR profiles of Listeria monocytogenes isolates analyzed in this study

MVLST results are shown in Table Table1.1. All J and H isolates were compared with 28 epidemiologically unrelated L. monocytogenes isolates from Zhang et al. (18) in the same phylogenetic context (Fig. (Fig.1).1). All H and J isolates from the two recent U.S. outbreaks had identical sequences in the six virulence loci and thus were assigned to the same sequence type (Table (Table1)1) belonging to genetic lineage I (Fig. (Fig.1)1) (11, 18). Based on Wassenaar's theory that isolates that are indistinguishable by a subtyping method are clonally related (17), isolates from the 1998 and 2002 outbreaks were considered an epidemic clone (Fig. (Fig.1).1). Kathariou (7) previously assigned the 1998 outbreak isolates as belonging to epidemic clone II (ECII). Evans et al. (4) subsequently found that two L. monocytogenes serotype 4b-specific genomic regions (4bSF7 and 4bSF18) adjacent to internalin A (a virulence determinant) were not amplified by ECII PCR. In the present study, both J and H isolates were subjected to ECII PCR as described by Evans et al. (4). All J and H isolates were negative by ECII PCR (Table (Table1),1), while other 4b isolates resulted in positive amplifications (data not shown). These results confirmed our MVLST findings that J and H isolates represented a unique epidemic clone, ECII.

FIG. 1.
Neighbor-joining tree of all Listeria monocytogenes isolates based on p distance of the concatenated sequences (total of 2,641 bp) of six loci. Bootstrap values (1,000 replications) are shown at the interior branches.

In the present study, MVLST was able to identify isolates as belonging to one epidemic clone, while AscI-PFGE was able to identify and separate isolates from the two different outbreaks. In contrast, some isolates between the two outbreaks (i.e., H7596 and J1925) had the same ApaI-PFGE pattern (Table (Table1).1). In addition, many isolates within each outbreak had different ApaI-PFGE patterns (i.e., three clinical isolates, H7550, H7355, and H7557, in the 1998 outbreak). Although Tenover et al. (16) proposed that isolates with one to three band differences in PFGE profiles would probably belong to the same outbreak, this criterion has been challenged by some researchers. For example, Maslanka et al. (10) suggested that isolates with PFGE similarity values of less than 100% may not be epidemiologically related. In addition, one or two band differences in PFGE profiles are considered significant in CDC's PulseNet protocol (5) and thus may not be part of the epidemic or outbreak. Therefore, ApaI-PFGE confused the epidemiology of the two outbreaks. Also, in contrast to MVLST, neither AscI-PFGE nor ApaI-PFGE revealed a clonal relationship between isolates from the two outbreaks (Table (Table11).

In the present study, MVLST possessed excellent epidemiological relevance because it provided high discriminatory power when differentiating unrelated isolates and demonstrated the clonality of epidemic/outbreak isolates (Fig. (Fig.1).1). The reason for this is because the six genes in MVLST showed significant variation among genetically diverse isolates (18) but perfect sequence identity within all isolates from the 1998 and 2002 outbreaks. Among the six selected genes, prfA, inlB, and inlC have critical functions in intracellular survival and virulence of L. monocytogenes (8) and lisR, dal, and clpP play an important role in L. monocytogenes stress tolerance and virulence (3). Therefore, these virulence genes would be expected to be conserved among epidemic isolates. Although almost all L. monocytogenes strains harbor the above genes, only a small subpopulation of L. monocytogenes serotype 4b strains are known to cause the vast majority of listeriosis outbreaks (7). The difference between outbreak isolates and nonoutbreak isolates may be partially due to sequence variations in their virulence genes. The recombination rates of the six genes analyzed would be expected to be low, because recombination in these genes would make virulent strains less virulent and thus tend to purge them from the outbreak setting (14). This speculation is supported by a recent study which suggested that L. monocytogenes lineage I is a clonal lineage (11) with a relatively low frequency of recombination.

The choice of molecular epidemiological marker(s) is critical for maximizing epidemiological relevance. PFGE targets DNA variations at multiple endonuclease restriction sites and genomic deletions, insertions, and rearrangements that may not always correlate with epidemic properties and thus may yield data that are not epidemiologically relevant. The premise of using two enzymes for PFGE analysis is that use of one enzyme is sometimes not sufficient to differentiate epidemiologically unrelated isolates (6). In the present study, the addition of the secondary enzyme ApaI-PFGE provided additional discriminatory power but confounded the epidemiology of the two outbreaks by both separating isolates within the same outbreak and clustering isolates between the two outbreaks. In this study, MVLST clarified how MVLST and AscI-PFGE complement one another, because MVLST identified the epidemic and AscI-PFGE differentiated the two outbreaks. In addition to the selected six virulence genes, other virulence genes and genes responsible for transmission of L. monocytogenes might also be important epidemic factors and thus could be incorporated to create a multi-epidemic-locus sequence typing (MELST) strategy. We hypothesize that MELST might further improve epidemiological relevance and ultimately provide an ideal tool for investigating the epidemiology of L. monocytogenes and, potentially, other pathogens.

In the present study, MVLST indicated that the two U.S. multistate outbreaks represented an epidemic, even though classic epidemiology and PFGE did not suggest any links between the two outbreaks. Many possible vectors exist for transmission of L. monocytogenes between meat plants, including trucks, meat, pallets, and people. Further clarification of the epidemiology of epidemic clones by MVLST or MELST would allow development of intervention strategies for controlling future epidemics and outbreaks of L. monocytogenes.

Acknowledgments

This study was supported by a U.S. Department of Agriculture Special Milk Safety grant to The Pennsylvania State University.

We thank Bala Swaminathan for providing the bacterial strains and PFGE data used in this research. We also thank Natasha Brooks and John Patton for technical assistance.

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