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Journal of Clinical Microbiology, April 2008, p. 1435-1450, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.02207-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Multiple-Locus Variable-Number Tandem-Repeat Analysis as a Tool for Subtyping Listeria monocytogenes Strains{triangledown}

Katharine E. Volpe Sperry,1* Sophia Kathariou,2 Justin S. Edwards,1 and Leslie A. Wolf1

North Carolina State Laboratory of Public Health, Raleigh, North Carolina,1 North Carolina State University, Raleigh, North Carolina2

Received 14 November 2007/ Returned for modification 18 January 2008/ Accepted 28 January 2008


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ABSTRACT
 
Listeria monocytogenes, like many other food-borne bacteria, has certain strains that are commonly linked to outbreaks. Due to the relatively low numbers of affected individuals, outbreaks of L. monocytogenes can be difficult to detect. The current technique of molecular subtyping in PulseNet laboratories to identify genetically similar strains is pulsed-field gel electrophoresis (PFGE). While PFGE is state-of-the-art, interlaboratory comparisons are difficult because the results are highly susceptible to discrepancies due to even minor variations in experimental conditions and the subjectivity of band marking. This research was aimed at the development of a multiple-locus variable-number tandem-repeat analysis (MLVA) that can be implemented in PulseNet laboratories to replace or complement existing protocols. MLVA has proven to be a rapid and highly discriminatory tool for subtyping many bacteria. In this study, a novel MLVA method for L. monocytogenes strains was developed utilizing eight loci multiplexed into two PCRs. The PCR products were separated by capillary gel electrophoresis for high throughput and accurate sizing, and the fragment sizes were analyzed and clustered based on the number of repeats. When tested against a panel of 193 epidemiologically linked and nonlinked isolates, this MLVA for L. monocytogenes strains demonstrates strong epidemiological concordance. Since MLVA is a high-throughput screening method that is fairly inexpensive, easy to perform, rapid, and reliable, it is well suited to interlaboratory comparisons during epidemiological investigations of food-borne illness.


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INTRODUCTION
 
Listeria monocytogenes, the causative agent of listeriosis, is a dangerous food-borne pathogen. Listeriosis causes meningitis, encephalitis, and septicemia, primarily in the elderly or in immunocompromised patients. It is most severe, however, in pregnant women and neonates due to its ability to cross the placenta and infect the fetus, causing congenital defects, stillbirth, and miscarriage. L. monocytogenes is most commonly acquired through consumption of contaminated foods such as unpasteurized or incompletely pasteurized cheeses and ready-to-eat foods, especially deli-type meats, due to its ability to grow at 4°C and to contaminate the food-processing environment. Given the severity of L. monocytogenes infections and potentially tragic outcomes, improving the detection of outbreaks and the discriminatory power of molecular subtyping methods is clearly a priority for food safety initiatives (7, 48). Public health laboratory scientists and epidemiologists play a critical role in this initiative by subtyping food-borne bacteria and performing outbreak investigations.

Bacterial subtyping is used to determine the relatedness among different isolates as part of an epidemiologic investigation. PulseNet, the international molecular subtyping network for food-borne bacteria developed and managed by the Centers for Disease Control and Prevention (CDC), utilizes pulsed-field gel electrophoresis (PFGE) as one key method for early detection of strains linked to potential outbreaks. This subtyping method compares DNA fragment patterns generated by macrorestriction digests of total genomic DNA that are separated by electrophoresis. The resulting banding patterns are compared to determine similarity. When clusters of isolates with similar PFGE profiles are detected, public health laboratories share these data with the epidemiologists, who then perform food history investigations to track the source of the organism. PulseNet PFGE protocols have been standardized and disseminated to public health laboratories by the CDC (32). These protocols must be strictly followed to ensure that the results are comparable from laboratory to laboratory.

The CDC developed an international database so that PulseNet laboratories can submit normalized PFGE patterns, thus enabling interlaboratory comparisons. PulseNet laboratories use the patterns submitted to the database to detect clusters of cases, to identify increases in the occurrence of a specific subtype, and to identify outliers to an outbreak. Subtyping by PulseNet laboratories in concert with epidemiological investigations has in many cases identified and/or confirmed the source of an outbreak (50). At the end of 2006, the L. monocytogenes database consisted of a total of 7,753 gel images (tiff files) including 920 unique AscI patterns and 1,262 unique ApaI patterns. Since the inception of the L. monocytogenes database, 77 clusters have been identified including 17 outbreaks linked to likely sources (Steven G. Stroika, PulseNet National Database Team, CDC, Atlanta, GA, personal communication).

L. monocytogenes has many serotypes, although serotypes 1/2a, 1/2b, and 4b are implicated in most cases of human disease (20). Serotype 4b is responsible for the majority of outbreaks and has been shown to be highly clonal (11, 17). Among the outbreak strains of serotype 4b, two strain subtypes continually reemerge. These are known as epidemic clone I (ECI) and epidemic clone II (ECII). These clones have been responsible for many outbreaks within North America and Europe (23, 34, 54). Due to the clonal nature of L. monocytogenes, novel subtyping methods are required to accurately discriminate among these common strain types. The current method, PFGE, is very labor-intensive and somewhat subjective. PFGE also relies on computer-based band marking, which can be inaccurate and requires manual interpretation by trained personnel to identify clusters. In practice, even minor deviations in experimental conditions can produce pronounced differences in patterns. Significant differences in patterns can also be attributed to the presence of mobile genetic elements. Other methods developed to subtype L. monocytogenes strains include multilocus sequence typing (15, 44, 56) and suspension microarray analysis based on specific genes or single-nucleotide polymorphisms (8, 9, 21). These methods tend to be technically demanding and expensive although the data are portable and nonsubjective. Since these methods are based on DNA sequences, they are genetically relevant.

Multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) is a proven, rapid, and highly discriminatory subtyping method for agents such as Bacillus anthracis, Francisella tularensis, and Escherichia coli (25, 28, 35, 36). This type of analysis has been successful because bacteria have highly variable repeated elements throughout their genomes. VNTRs are short segments of DNA that have hypervariable copy numbers. It is thought that the variation in copy number is due to slipped-strand mispairing during DNA polymerase mediated duplications or possibly due to recombination (51). Despite mutations that may occur within the tandem repeat, the unit length remains relatively constant while the copy number varies. The tandem repeats are in stable regions of the genome, and they are not likely to be associated with mobile genetic elements, such as plasmids. The difference in copy numbers at specific loci is used to measure relatedness of strains in this subtyping scheme. To date, only limited information is available on MLVA applications with L. monocytogenes. In a recently published study, six loci were employed to subtype 45 isolates. Most of the isolates included were serotype 1/2a; strains of serotypes 1/2b and 4b were not sufficiently represented in this study (40). The current research was aimed at the development of MLVA for L. monocytogenes strains that can be implemented in PulseNet laboratories to replace or complement existing protocols.


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MATERIALS AND METHODS
 
Bacterial strains and nucleic acid extraction. A total of 193 L. monocytogenes isolates were acquired from the culture collections at the CDC (Atlanta, GA), North Carolina State University (Raleigh, NC), North Carolina State Laboratory of Public Health ([NCSLPH] Raleigh, NC) (clinical specimens from 2001 to 2006), and the American Type Culture Collection (Manassas, VA) (Table 1). Many of the strains included have been described previously in the World Health Organization (WHO) international multicenter L. monocytogenes subtyping study as well as other publications (6, 10, 27).


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  		TABLE 1. L. monocytogenes isolate information

Each isolate was streaked for isolation and grown on 5% sheep blood agar (BBL blood agar base [infusion agar]; BD, Franklin Lakes, NJ) at 35°C overnight. Cultures were preserved using a Microbank cryo-preservation system (Pro-Lab Diagnostic, Austin, TX), per the manufacturer's directions, and stored at –70°C. Multiple methods for nucleic acid preparation were used. A loopful (using a sterile calibrated 1-µl inoculating loop) of pure bacterial growth was used for all methods. The MLVA protocol utilized the "boil prep" method (28, 35). Briefly, the bacteria were suspended in 100 µl of sterile nuclease-free H2O (Amresco, Solon, OH); the suspension was boiled at 95 to 100°C for 10 min and immediately chilled on ice to aid in cell lysis. Cell suspensions were centrifuged at 8,000 x g for 10 min to separate cellular debris. The clarified supernatant was used in the PCR. During initial development and for nucleic acid sequencing, DNA was extracted using a DNeasy Tissue kit (Qiagen, Valencia, CA) or MagNA Pure LC DNA Isolation III kit (Roche, Indianapolis, IN) per the manufacturer's directions for gram-positive organisms. Extracted DNA was stored at –20°C.

PFGE. All PFGE was performed using PulseNet standardized procedures with Asc1 and Apa1 restriction enzymes (www.cdc.gov/pulsenet/protocols.htm) (32). Many of the PFGE patterns were downloaded from the PulseNet international database. The PFGE patterns that could not be acquired from the PulseNet database were analyzed by NCSLPH's PulseNet laboratory. Analysis was performed using BioNumerics (Applied Maths, Austin, TX) cluster analysis. The average from experiments was used to cluster the similarity matrix determined by each single enzyme analysis (AscI and ApaI) using the Dice coefficient with a 1.5 tolerance and the unweighted pair group method with arithmetic mean (UPGMA).

Genome analysis and primer design. The two fully sequenced L. monocytogenes genomes, EGDe (accession number AL591824) (29) and F2365 (AE017262) (41), were used for analysis of tandem repeats. The two genomes were initially scanned individually using the Tandem Repeat Finder program (http://tandem.bu.edu/trf/trf.html) (4). The Genomes, Polymorphism and Minisatellites strain comparison page in the Microorganisms Tandem Repeat Database(http://minisatellites.u-psud.fr/) was then used to scan and compare both genomes (18, 19, 52). Primers were designed utilizing Primer3 software (http://frodo.wi.mit.edu/) (45). Each primer was designed in the flanking region of the tandem repeat to produce a fragment size no larger than 600 bp. Primers were synthesized by Proligo (Boulder, CO) and Integrated DNA Technologies (Coralville, IA). For fragment analysis, the forward primers were labeled with one of three WellRed dye-labeled phosphoramidites (D2, D3, and D4) and purified by high-performance liquid chromatography.

PCR amplification and fragment analysis. Initial analysis of each locus was performed utilizing QuantiTect Sybr Green PCR (Qiagen, Valencia, CA) per the manufacturer's instructions on an iQ iCycler (Bio-Rad, Hercules, CA) with 0.3 µM unlabeled primer and 2 µl of DNA in 25-µl reaction mixtures; the PCR program consisted of 35 cycles, annealing at 50°C, and melt curve analysis. Standard PCR was performed per the manufacturer's directions using HotStar Taq polymerase (Qiagen, Valencia, CA), 0.3 µM of each primer, and 2 µl of DNA in 25-µl reaction mixtures; the PCR program consisted of 35 cycles and annealing at 50°C. Initial multiplexed PCR used a Multiplex PCR kit (Qiagen, Valencia, CA) per the manufacturer's instructions with 0.3 µM of each labeled primer (Table 2) and 2 µl of DNA in 25-µl reaction mixtures; the PCR program consisted of 35 cycles and annealing at 50°C. The finalized protocol consisted of two multiplexed PCRs (R1 and R2), each with four primer sets (concentrations are given in Table 2) using 1.5 U of Platinum Taq DNA polymerase, 2 mM MgCl2, 0.2 mM of the deoxynucleoside triphosphates, 1x PCR buffer, PCR-grade water (Invitrogen, Carlsbad, CA), and 1 µl of DNA in 10-µl reaction mixtures. The cycling conditions used were as follows: 95°C for 5 min and 35 cycles of 94°C for 20 s, 50°C for 20 s, and 72°C for 20 s, followed by one cycle of 72°C for 5 min and an indefinite hold at 4°C on an MJF Tetrad (Bio-Rad, Hercules, CA) (28).


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  		TABLE 2. MLVA Primers for L. monocytogenes

Fragments were sized by combining 1 µl of a 1:60 dilution of the PCR product with 20 µl of deionized formamide (sample loading solution) and 0.08 µl of DNA size standard 600 (Beckman Coulter, Fullerton, CA) (9, 28). Fragment analysis was performed on a Beckman Coulter CEQ 8000 genetic analyzer (Beckman Coulter, Fullerton, CA) using the Frag-Test method, and fragments were analyzed with default fragment analysis parameters edited to reflect the DNA size standard 600 and quartic model (28). Estimated fragment size, peak height, and dye for each isolate were exported in comma-delimited format (.cvs) and imported in BioNumerics (Applied Maths, Austin, TX). Customized scripts were developed by Applied Maths (Austin, TX) and are available at www.applied-maths.com. These scripts are used to import the fragment sizes (VNTRimport_v3) and to calculate copy numbers (VNTRcalc). Null alleles were coded as negative. UPGMA cluster analysis of copy number was performed with a categorical multistate coefficient.

Sequence verification. The loci and flanking regions were amplified with HotStar Taq Polymerase (Qiagen, Valencia, CA) as described above. The PCR products were purified using the QIAquick PCR Purification kit (Qiagen, Valencia, CA). Cycle sequencing was performed per the manufacturer's directions with the CEQ DTCS (dye terminator cycle sequencing) Quick Start kit (Beckman Coulter, Fullerton, CA) using 0.2 µM primer and 25 to 100 fmol of template DNA. Unincorporated dye terminators were removed with the DyeEx 2.0 spin kit (Qiagen, Valencia, CA) using an additional wash step of 300 µl of sterile nuclease-free sequence grade water (Amresco, Solon, OH) prior to the application of the sample or by using Clean Seq (Agencourt Biosciences, Beverly, MA) per the manufacturer's instructions. All samples were run on the CEQ 8000 genetic analyzer (Beckman Coulter, Fullerton, CA) using the LFR-1 method and default sequence analysis parameters.

Stability and reproducibility determinations. Two L. monocytogenes strains, EGDe and Li2 (ATCC, Manassas, VA), were chosen for the stability study. Each was passaged on sheep blood agar plates 45 times, approximately 3 times per week. Samples were boiled as described previously at each passage. Ten evenly spaced isolates were tested by MLVA to determine the stability of each locus.

Reproducibility of the assay was determined by testing 100 isolates via the two multiplexed PCRs. This analysis was performed in triplicate on three different runs by two technicians.


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RESULTS AND DISCUSSION
 
Identification of suitable loci. A total of 75 tandem repeats were identified within the two fully sequenced genomes of L. monocytogenes (EGDe and F2365). Forty-three of these repeated elements that were under 600 bp in total length and had a unit length of 3 to 21 bp (Table 3) were analyzed. Initial screening of the loci was performed in a pilot study with a panel of 10 isolates consisting of two L. monocytogenes isolates, representing each of the following serotypes: 1/2a, 1/2b, 1/2c, 3b, and 4b. This pilot study of all loci was performed using Sybr green PCR with unlabeled primers for rapid and inexpensive preliminary screening. To ensure accurate amplification, primers where redesigned as needed.


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  		TABLE 3. All loci examined

The Genomes, Polymorphism and Minisatellites website allowed us to compare the tandem repeats found within the two fully sequenced genomes. Some of the tandem repeats found individually in each of the genomes were, in fact, the same or overlapping. This was seen with Lm-8, Lm-9, and Lm-36; Lm-6, Lm-7, Lm-23, and Lm-35; and Lm-26 and Lm-37. The 37 remaining loci were tested on an expanded panel of 79 isolates (Table 1) with labeled primers using capillary electrophoresis fragment analysis to determine subtyping ability. The tandem repeats that did not produce accurate and reproducible results within the serotypes causing the majority of human illness (1/2a, 1/2b, and 4b) were eliminated from the study (Table 3). Lm-5, Lm-29, and Lm-34 were eliminated for their failure to amplify serotypes 1/2b, 3b, and 4b (Table 3).

In order to verify that the size determined by fragment analysis was indeed due to a change in repeat number and not to other genetic events, such as insertions and deletions, sequence analysis of both DNA strands was performed. For each locus, two isolates of each different allele size identified were sequenced. When necessary, primers were redesigned to provide more consistent sequencing results (data not shown). Sequence analysis showed that Lm-1, Lm-4, Lm-20, Lm-25, Lm-27, and Lm-40 had variability in the flanking regions that could not be avoided even with primer redesign. The variability included insertions and deletions that produced a change in fragment size that was not due to variations in repeat number. Since the sequence variability is not related to the tandem repeat, these loci were eliminated from this study. Loci that had very low diversity or did not affect subtyping ability were also eliminated (Table 3). Thus, a total of eight loci (Lm-2, Lm-3, Lm-8, Lm-10, Lm-11, Lm-15, Lm-23, and Lm-32) remained in this study.

Two of these loci, Lm-3 and Lm-10, were identified independently by Murphy et al. (LM-TR-1 and LM-TR-4) (40). In our study we also independently identified the other four loci described previously (40). These four loci were not chosen for inclusion in our L. monocytogenes MLVA for several reasons. Lm-5 (LM-TR-6) did not amplify serotypes 1/2b, 3b, and 4b, while Lm-26 (LM-TR-2) was found to have very low diversity. Genomic analysis showed that the other two loci (LM-TR-3 and LM-TR-5) overlapped. These tandem repeats were equivalent to our Lm-4, which was eliminated from the study due to sequence variability in the flanking region, possibly due to the overlapping repeat and not to a difference in copy number within the tandem repeat.

Assay development. Eight of the remaining tandem repeats provided adequate diversity and were thoroughly evaluated with all 193 isolates (Table 1) for their ability to subtype these strains into epidemiologically significant clusters. Partial repeats were seen in Lm-10 and Lm-23. In all cases, these resulted in half of a repeat. For the purpose of analysis in BioNumerics, the allele size was changed from 12 to 6 and 6 to 3, respectively, to account for these half-repeats. In some instances no amplification was observed at a particular locus. Lack of amplification (a null allele) could be due to mutation at the primer site resulting in no PCR product. Since a null allele is different from a locus having zero repeats, it is denoted as –2 by BioNumerics (Table 4). Null alleles were observed in Lm-3, Lm-11, and Lm-32.


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  		TABLE 4. L. monocytogenes VNTR Loci

A multiplexed PCR protocol was developed for these eight loci consisting of two reactions with four loci in each (Fig. 1). This assay was made to be concordant with the protocols previously developed for PulseNet laboratories to subtype E. coli and Salmonella enterica serotype Typhimurium (9, 28). To determine subtyping capabilities, the complete panel of 193 isolates consisting of both known outbreak and sporadic strains and including isolates from each of the serotypes of interest was analyzed (Table 1). The Simpson's index of diversity (49) for the eight tandem repeats ranged from 33.8% to 84.5% (Table 4). Based on the complete panel of isolates and the 54 unique MLVA profiles they produced, the calculated Simpson's index of diversity for the assay is 94%.


Figure 1
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  		FIG. 1. Listeria monocytogenes MLVA. Two multiplex PCRs including four loci each were analyzed on the CEQ 8000 (Beckman Coulter). Red peaks, size standard 600; black peaks, Lm-8, Lm-15 and Lm-23; green peaks, Lm-10, Lm-11, and Lm-32; blue peaks, Lm-2 and Lm-3. Multiplex PCR R1 is shown in the top panel, and R2 is shown in the bottom panel. nt, nucleotide.

The stability of each locus was evaluated to determine the effect of laboratory passage. All fragment sizes varied by less than ±1 bp (0.02 bp to 0.88 bp), indicating that these loci are stable during routine laboratory manipulation. The multiplexed MLVA was shown to be reproducible by determining the copy number for each locus on a panel of 100 isolates. Although the fragment sizes showed slight variation (less than ±1 bp), the copy number was determined to be 100% reproducible (data not shown).

Comparative effectiveness of MLVA and select other methods. Copy numbers as determined by BioNumerics of all 193 isolates are shown in Table 1. A panel of 123 isolates (Table 1) was analyzed by this multiplexed MLVA and compared to PFGE (AscI and ApaI). Cluster analysis of these isolates reveals that MLVA efficiently separates isolates of genomic division 1 (lineage II) (serotypes 1/2a and 1/2c) from those of genomic division 2 (lineage I) (serotypes 1/2b, 3b, 4b, and a single isolate of 4d) (Fig. 2). Only a single clinical isolate of serotype 4c was available and was not included in the cluster analysis; however, the MLVA type is indicated in Table 1. The clear differentiation between the major genomic divisions (lineages) was in agreement with similar findings from numerous other subtyping approaches (for a review, see references 8, 10, 17, 44, 55, and 56).


Figure 2
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  		FIG. 2. Cluster analysis of 123 L. monocytogenes isolates based on MLVA type using the categorical coefficient and UPGMA. Genomic divisions are denoted as follows: •, genomic division 1 (serotypes 1/2a and 1/2c); {blacksquare}, genomic division 2 (serotypes 1/2b and 3b); {blacklozenge}, genomic division 2 (serotype 4b). The EC group is indicated if known.

Comparisons of MLVA and PFGE techniques can be quite difficult since they evaluate different types of genetic events. While PFGE relies on changes in the restriction enzyme site, MLVA relies on copy number changes of tandem repeats. Both are successful in grouping closely related and differentiating unrelated L. monocytogenes strains. PFGE and MLVA comparisons were performed by separating the isolates into groups based on serotype.

Although the MLVA and PFGE techniques clustered this diverse set of isolates differently, the results were very similar. Seven unique profiles were produced for 32 1/2a and 1/2c isolates examined by MLVA (Fig. 3A), while PFGE (AscI and ApaI) (Fig. 3B) produced 11 unique profiles based on cluster analysis in BioNumerics. These different profiles were in some cases due to one- or two-band differences. For instance, several strains (Fig. 3B, filled circles) exhibited a one-band difference in the ApaI pattern. The strains with this profile included isolate J0161 from the 2001 multistate outbreak as well as food, clinical, and environmental isolates implicated in listeriosis cases from 1988 to 1990. Epidemiological studies have revealed that these isolates are associated with the same food processing plant and likely represent long-term contamination of that facility with the same strain (42). Lastly, this cluster also includes two clinical isolates from North Carolina (NC2002-327 and NC2004-454, isolated in 2002 and 2004, respectively) which were indistinguishable by PFGE, suggesting that this strain type continues to circulate in food.


Figure 3
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  		FIG. 3. Cluster analysis of 32 L. monocytogenes serotype 1/2a and 1/2c isolates. Symbols next to isolate names indicate similar isolates in MLVA and PFGE dendrograms. If isolates have indistinguishable patterns, then they are listed with one representative pattern. (A) Results of MLVA typing using the categorical coefficient and UPGMA. (B) Results of two-enzyme PFGE analysis (AscI and ApaI). The average from experiments was used to cluster the similarity matrix determined by single-enzyme analysis using the Dice coefficient with a 1.5 tolerance and UPGMA cluster.

Seven unique MLVA profiles and 12 unique PFGE (AscI and ApaI) profiles were detected among the 31 isolates of serotypes 1/2b and 3b, based on nine epidemiological groups and three unlinked isolates (Fig. 4). As described above, several PFGE profiles differed by only one to two bands. The isolates from the gastroenteritis outbreak in Italy (Table 1) were found to have identical MLVA and PFGE profiles. This MLVA profile was also found in three other isolates from the United States and the United Kingdom (Fig. 4, filled squares). The ApaI patterns for these isolates were also identical; however, differences were seen in the AscI pattern for the United Kingdom isolate. The chocolate milk outbreak isolates (Table 1; Fig. 4, indicated by an arrow) were also identical by both PFGE and MLVA.


Figure 4
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  		FIG. 4. Cluster analysis of 31 L. monocytogenes serotype 1/2b and 3b isolates. Symbols next to isolate names indicate similar isolates in MLVA and PFGE dendrograms. If isolates have indistinguishable patterns, then they are listed with one representative pattern. (A) Results of MLVA typing using the categorical coefficient and UPGMA. (B) Results of two-enzyme PFGE analysis (AscI and ApaI). The average from experiments was used to cluster the similarity matrix determined by single-enzyme analysis using the Dice coefficient with a 1.5 tolerance and UPGMA cluster.

Comparisons of 60 serotype 4b isolates showed the most significant differences between MLVA and PFGE (AscI and ApaI) (Fig. 5). Nine unique MLVA profiles and 26 unique PFGE (AscI and ApaI) profiles were produced based on cluster analysis of these isolates. The MLVA clearly separated the sporadic isolates from outbreak isolates. The MLVA was also able to group isolates by epidemic clone groups. Analyses based on unique genomic markers, gene cassettes, and single nucleotide polymorphisms have also found similarities between the strains associated with the epidemic clone groups (21, 23, 33, 34, 54-56). In efforts to increase the subtyping ability of this MLVA for L. monocytogenes, six loci (Lm-38 to Lm-43) were designed specifically for serotype 4b. Two of these loci had very low diversity, and one had variability in the flanking region. The remaining three loci did not affect the subtyping ability, clustering the 4b isolates identically as the panel of eight loci used in the multiplexed assay (Table 3).


Figure 5
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  		FIG. 5. Cluster analysis of 60 L. monocytogenes serotype 4b isolates. Symbols next to isolate names indicate similar isolates in MLVA and PFGE dendrograms. If isolates have indistinguishable patterns, then they are listed with one representative pattern. EC groups are indicated as follows: {blacksquare}, ECI; •, ECII. (A) Results of MLVA typing using the categorical coefficient and UPGMA. (B) Results of two-enzyme PFGE analysis (AscI and ApaI). The average from experiments was used to cluster the similarity matrix determined by single-enzyme analysis using the Dice coefficient with a 1.5 tolerance and UPGMA cluster.

Comparisons were made between the serotype 4b MLVA results and previously published work with multilocus genotyping (MLGT) (21) and multivirulence locus sequence typing (MVLST) (56). The ECI isolates grouped together in three main clusters, differing by only one locus, using MLVA (Fig. 5A, filled squares). The epidemic groups (EG) from the WHO multicenter study (6) are used here simply to denote epidemiological information (Table 1). EG-9, -13, -15, -16, -18, and -21 and EG-12 and -17 were identical by MLVA. PFGE clustering of EG-9, -15, -16, -17, and -18 showed identical AscI patterns and closely related ApaI patterns. EG-12 and EG-16 were identical, except for isolate G4021, which matched EG-9. PFGE did not cluster all isolates from EG-13 together nor did it cluster all isolates from EG-21 together (Fig. 5B, filled squares). MVLST clustered EG-12, -13, and -16 as identical to one another (56). MLGT clustered EG-12, -13, and -16 as similar although some differences were seen between the outbreaks (21). ECII isolates were not represented in the WHO multicenter study (6) since the ECII strain type was not identified until the multistate hotdog outbreak of 1998 to 1999 (13, 14, 23). ECII isolates grouped together into four clusters differing by one locus using MLVA (Fig. 5A, filled circles). As described previously, PFGE of these isolates with AscI produced two distinct patterns and PFGE with ApaI produced four closely related patterns (Fig. 5, filled circles) (31). These clusters correlated well with those identified by MLVA. The ECII isolates also clustered as identical by MVLST (15). In future studies, MLVA results could be more thoroughly compared to those of MVLST and MLGT by including additional isolates and serotypes.

In conclusion, this study details the development of an MLVA method for L. monocytogenes that consists of two multiplexed PCRs. The loci were selected based on their ability to subtype primarily serotypes 1/2a, 1/2b, and 4b. All of the loci selected have relatively small repeat units of 6 bp to 15 bp. The MLVA is a high-throughput, rapid assay with much improved data portability compared to PFGE. The most notable advantage of the MLVA is the lack of subjectivity. While PFGE relies on stringent adherence to subjective band-marking procedures, MLVA generates exact fragment sizes that are directly imported into BioNumerics. This allows the data transfer and interlaboratory comparison to be seamless. An added benefit for PulseNet laboratories is that different food-borne organisms (e.g., E. coli, S. enterica serotype Typhimurium, and L. monocytogenes) can be analyzed simultaneously using capillary gel electrophoresis. These advantages make MLVA an ideal choice for the next generation of food-borne subtyping tests in public health laboratories.


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ACKNOWLEDGMENTS
 
This study was part of the PulseNet USA initiative for the development of the next generation of subtyping methods.

This work was supported by Grant Cooperative Agreement Number U60-CCU303019 from the CDC and APHL. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of APHL or CDC.

We gratefully acknowledge Denise Griffin, Shadia Barghothi, Debra Springer, and Memory Dalton, NCSLPH PulseNet Team, for PFGE data and isolate preparation; Robin Siletzky, North Carolina State University, for her technical expertise, strain preparation, strain typing, and assistance in accumulating epidemiological information; Lewis Graves, CDC, for providing isolates and epidemiological and serotype information as well as scientific discussion; PulseNet, National Database Team, for PFGE data; Shari Shea, APHL, for assistance with administration of the contract; Bala Swaminathan and Eija Trees, CDC, for scientific discussion; North Carolina Department of Agriculture and Consumer Services for submission of isolates; and Shermalyn Greene and Rachel Gast, NCSLPH, for their scientific discussion and critical review of the manuscript.


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FOOTNOTES
 
* Corresponding author. Mailing address: North Carolina State Laboratory of Public Health, 306 N Wilmington St., Raleigh, NC 27601. Phone: (919) 807-8816. Fax: (919) 733-8695. E-mail: katesperry{at}gmail.com Back

{triangledown} Published ahead of print on 6 February 2008. Back


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Journal of Clinical Microbiology, April 2008, p. 1435-1450, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.02207-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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