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Selective Discrimination of Listeria monocytogenes Epidemic Strains by a Mixed Genome DNA Microarray Compared to Discrimination by Pulsed Field Gel Electrophoresis, Ribotyping, and Multilocus Sequence Typing

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DOI: 10.1128/JCM.42.11.5270–5276.2004

Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Selective Discrimination of

Listeria monocytogenes

Epidemic Strains by

a Mixed-Genome DNA Microarray Compared to Discrimination by

Pulsed-Field Gel Electrophoresis, Ribotyping, and

Multilocus Sequence Typing

Monica K. Borucki,

1

* So Hyun Kim,

2,3

Douglas R. Call,

2

Sandra C. Smole,

4

and Franco Pagotto

5

Animal Disease Research Unit, USDA Agricultural Research Service,

1

and College of Veterinary Medicine, Washington State

University,

2

Pullman, Washington; Seoul National University, Seoul, Korea

3

; Massachusetts State Laboratory Institute,

Massachusetts Department of Public Health, Boston, Massachusetts

4

; and Bureau of Microbial Hazards,

Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada

5

Received 13 February 2004/Returned for modification 14 April 2004/Accepted 31 July 2004

Listeria monocytogenes

can cause serious illness in humans, and subsequent epidemiological investigation

requires molecular characterization to allow the identification of specific isolates.

L. monocytogenes

is usually

characterized by serotyping and is subtyped by using pulsed-field gel electrophoresis (PFGE) or ribotyping.

DNA microarrays provide an alternative means to resolve genetic differences among isolates, and unlike PFGE

and ribotyping, microarrays can be used to identify specific genes associated with strains of interest. Twenty

strains of

L

.

monocytogenes

representing six serovars were used to generate a shotgun library, and subsequently

a 629-probe microarray was constructed by using features that included only potentially polymorphic gene

probe sequences. Fifty-two strains of

L. monocytogenes

were genotyped by using the condensed array, including

strains associated with five major listeriosis epidemics. Cluster analysis of the microarray data grouped strains

according to phylogenetic lineage and serotype. Most epidemiologically linked strains were grouped together,

and subtyping resolution was the same as that with PFGE (using AscI and ApaI) and better than that with

multilocus sequence typing (using six housekeeping genes) and ribotyping. Additionally, a majority of epidemic

strains were grouped together within phylogenetic Division I. This epidemic cluster was clearly distinct from

the two other Division I clusters, which encompassed primarily sporadic and environmental strains.

Discrimi-nant function analysis allowed identification of 22 probes from the mixed-genome array that distinguish

serotypes and subtypes, including several potential markers that were distinct for the epidemic cluster. Many

of the subtype-specific genes encode proteins that likely confer survival advantages in the environment and/or

host.

Listeria monocytogenes

is a gram-positive bacterial pathogen

that is capable of causing significant morbidity and mortality in

humans. Listeriosis is primarily a food-borne disease that has a

significant impact on specific risk groups, including pregnant

women and their fetuses, neonates, and people who are

im-munosuppressed (11).

L. monocytogenes

is capable of surviving

and replicating under a wide range of environmental

condi-tions, and this, as well as its widespread distribution, makes it

particularly hard to eradicate from food-processing plants (11).

Due to the severity of listeriosis, the United States maintains a

zero-tolerance policy regarding contamination of ready-to-eat

food products.

Although 13 serotypes of

L. monocytogenes

have been

de-scribed (25), only three serotypes (1/2a, 1/2b, and 4b) cause the

vast majority of clinical cases (26). Interestingly, although

se-rotype 1/2a is most frequently isolated from food, sese-rotype 4b

causes the majority of human epidemics (12). Thus, many have

suggested that there may be a link between serotype and

vir-ulence potential.

Numerous molecular subtyping techniques have identified

two major phylogenetic divisions within the species. Division I

consists of serotypes 1/2b, 3b, 4b, 4d, and 4e, and Division II

consists of serotypes 1/2a, 1/2c, 3a, and 3c (1–3, 5, 15, 21). A

third division, consisting of serotypes 4a and 4c and a subset of

4b strains, has also been described (8, 22, 27).

Epidemiological investigation of epidemic and sporadic

cases of listeriosis requires molecular characterization to allow

the identification of specific subtypes.

L. monocytogenes

sub-types are usually characterized by serotyping and then further

subtyped by using the current “gold standard,” pulsed-field gel

electrophoresis (PFGE) (16) or ribotyping. Multilocus

se-quence typing (MLST) has been described as a novel,

repro-ducible, and potentially discriminatory subtyping method (10,

18, 23, 24), and Revazishvili et al. (23) recently demonstrated

that MLST was able to differentiate most of the

L.

monocyto-genes

strains examined better than PFGE with AscI restriction

endonuclease digestion.

DNA subtyping with DNA microarrays may provide an

im-proved alternative to resolve genetic differences that exist

among isolates (4, 9, 28). This technique has the added

advan-tage that, unlike PFGE, ribotyping, and MLST, it can identify

specific or unique genes associated with strains of interest. For

* Corresponding author. Mailing address: USDA-ARS, 3003

ADBF, WSU, Pullman, WA 99164-6630. Phone: (509) 335-7407. Fax:

(509) 335-8328. E-mail: mborucki@vetmed.wsu.edu.

5270

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example, Call and colleagues (9) demonstrated that certain

strains of

L. monocytogenes

contained genes responsible for

repairing UV-damaged DNA, salt tolerance, and biofilm

for-mation, which would confer an advantage in certain ecological

niches such as food production environments.

In the present study, a 629-probe “condensed” microarray

was constructed using exclusively polymorphic probes.

Fifty-two strains of

L. monocytogenes

were genotyped using the

condensed array to compare the resolution of microarray

sub-typing to that of PFGE, MLST, and ribosub-typing and to identify

genetic regions that characterize subtypes.

MATERIALS AND METHODS

Bacterial strains and subtyping.Bacterial strains and sources are listed in Table 1.Listeria innocua strain ATCC 51742 was used as an outgroup for phylogenetic analysis.L. monocytogenesisolates were subtyped by using

serotyp-TABLE 1.

L. monocytogenes

strains subtyped by microarray analysis

Strain Other designation(s) Serotype Sourcea Epidemic Donorb

A050M

36046A

1/2a

Bulk milk

USDA

A061M

36582B

1/2a

Bulk milk

USDA

A070M

37952A

1/2a

Bulk milk

USDA

A259M

32490E

1/2a

Bulk milk

USDA

A437N

FDA 15c03

1/2a

Food

FDA

A501N

ILSI 33, TS4, F6854

1/2a

Food-Sp

United States, hot dog

ILSI

A502S

ILSI 34, TS14, F6900

1/2a

Human-Sp

United States, hot dog

ILSI

A503E

ILSI 35, J0161

1/2a

Human-Epi

United States, deli, 2000

ILSI

A582N

H7788

1/2a

Food

United States, hot dog

CDC

B339S

DOH 9900104

1/2b

Human-Sp

DOH

B345S

DOH 1159

1/2b

Human-Sp

DOH

B404U

NADC 2053

1/2b

Unknown

USDA

B412N

NADC 9916B

1/2b

Environmental

USDA

B430N

FDA 2492

1/2b

Food

FDA

B439N

FDA 3280

1/2b

Food

FDA

B443N

FDA 2475

1/2b

Food

FDA

B445N

FDA 2450

1/2b

Food

FDA

B507E

ILSI 39, G6003

1/2b

Food-Epi

Illinois, 1994

ILSI

B508E

ILSI 40, G6054

1/2b

Human-Epi

Illinois, 1994

ILSI

B588E

HPB1983

1/2b

Human-Epi

United Kingdom, 1989

HC

C366S

H9333

1/2c

Human

CDC

C368S

H9067

1/2c

Food

CDC

C622N

RM3000

1/2c

Soil

USDA

T590S

c

HPB1031, TS74

3b (1/2b)

Human-Sp

HC

F113V

01-3368A

4b

Bovine

USDA

F268S

33027A

4b

Bulk milk

USDA

F336S

DOH 9900094

4b

Human-Sp

DOH

F347S

DOH 1161

4b

Human-Sp

DOH

F357S

DOH 2150

4b

Human-Sp

DOH

F358S

DOH 2172

4b

Human-Sp

DOH

F405N

NADC 575

4b

Food

USDA

F428N

FDA 3365

4b

Food

FDA

F429N

FDA 3276

4b

Food

FDA

F446N

FDA 3515

4b

Food

FDA

F447N

FDA 3655

4b

Food

FDA

F469E

ILSI 1, ScottA

4b

Human-Epi

United States, Massachusetts, 1983

ILSI

F470E

ILSI 2, H7550

4b

Human-Epi

United States, hot dog, 1998

ILSI

F494E

ILSI 26, TS43, F4565

4b

Human-Epi

California, 1985

ILSI

F495E

ILSI 27, TS50, L4760

4b

Food-Epi

Halifax, Canada, 1981

ILSI

F496E

ILSI 28, TS27, L4738

4b

Human-Epi

Halifax, Canada, 1981

ILSI

F497E

ILSI 29, TS45, L3350

4b

Food-Epi

United Kingdom, 1988–1990

ILSI

F498E

ILSI 30, TS38, L3306

4b

Human-Epi

United Kingdom, 1988–1990

ILSI

F499E

ILSI 31, TS21, L4486j

4b

Food-Epi

Switzerland, 1987

ILSI

F505E

ILSI 37, J0144

4b

Food-Epi

North Carolina, 2000

ILSI

F581E

H7738

4b

Food-Epi

United States, hot dog, 1998

CDC

F583E

HPB850

4b

Human-Epi

Switzerland, 1996

HC

F584E

HPB2142

4b

Human-Epi

United States, hot dog, 1998

HC

F586E

HPB774

4b

Human-Epi

United Kingdom, 1991

HC

F589E

HPB1808

4b

Human-Epi

California, 1985

HC

F591E

HPB2262

4b

Human-Epi

Italy, 1998

HC

F592E

HPB2182

4b

Human-Epi

Canada, 1999

HC

F593E

HPB1026

4b

Human-Epi

California, 1985

HC

aSp, sporadic; Epi, epidemic.

bUSDA, U.S. Department of Agriculture Agricultural Research Service; DOH, Washington State Department of Health, Shoreline; CDC, Centers for Disease

Control and Prevention, Atlanta, Ga.; FDA, U.S. Food and Drug Administration, Bothel, Wash.; ILSI, International Life Sciences Institute.L. monocytogenesstrain collection (http://www.foodscience.cornell.edu/wiedmann/listeriadbase.htm); HC, Health Canada.

cThis isolate has serotyped as both 3b and 1/2b (25; M. K. Borucki, unpublished data).

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ing and PFGE (with AscI and ApaI restriction endonucleases) as previously described (16).

Automated ribotyping.Ribotyping was performed with the restriction enzyme EcoRI and the RiboPrinter microbial characterization system (Qualicon Inc., Wilmington, Del.), according to the manufacturer’s manual and as previously described (6, 7).

MLST.Loci were identified by searching for housekeeping genes from bothL. monocytogenes and L. innocua via GenBank (http://www.ncbi.nlm.nih.gov). These genes were mapped against theL. monocytogenesEGD genome sequence (13) to provide adequate genome coverage, and primers were chosen to amplify coding regions of 500 to 750 bp under common conditions (3 mM MgCl2and an annealing temperature of 58°C). Nucleotide sequences were obtained by PCR amplification of coding regions from the following genes:ahs,O -acetylhomo-serine sulfhydralase homolog;pstI, phosphenolpyruvate-dependent phospho-transferase enzyme I;lisK, histidine kinase homolog;lhkA, histidine kinase;dhk, dihydroxyacetone kinase; andabcZ, ABC transporter homolog Z. PCR products were purified by using QIAquick 96 PCR purification kits (Qiagen, Valencia, Calif.) and were eluted in⬃60␮l of water; 96-well plates were stored at⫺20°C. Dye terminator cycle sequencing was performed with the CEQ cycle sequencing kit (Beckman Coulter, Fullerton, Calif.) in 10-␮l reaction volumes with 10 to 20 ng of DNA. Sequencing reaction products were ethanol precipitated and dried, and samples were resuspended in 20␮l of formamide prior to separation by capillary electrophoresis with a CEQ2000XL DNA sequencer (Beckman Coulter). Sequence alignment and editing were performed with BioNumerics version 2.5 (Applied Maths, Kortrijk, Belgium). Allele sequence types were identified from 450 to 550 bp from each locus. Unweighted pair group method using arithmetic averages (UPGMA) analysis of categorical information based on the six different allele sequence types for each isolate was performed.

Microarray construction.A genomic library was constructed from 20 strains representing six serotypes (1/2a [n⫽5], 1/2b [n⫽4], 1/2c [n⫽4], 3a [n⫽1], 4b [n⫽5], and 4c [n⫽1]) and obtained from a variety of sources (human sporadic [n⫽10], epidemic [n⫽2], environmental [n⫽7], and veterinary [n⫽1]). Genomic DNA was extracted from the 20 strains by using an Easy DNA kit (Invitrogen, Carlsbad, Calif.). DNA was quantified by UV spectrophotometry, and equal amounts of genomic DNA from each strain were mixed. This pooled genomic DNA was used to construct a random shotgun library (Amplicon Ex-press, Pullman, Wash.). Briefly, 10␮g of DNA was cut with the restriction enzyme CviJI (Chimerx, Milwaukee, Wis.) or by sonification, and fragments of approximately 600 bp were gel isolated, extracted, and ligated into pUC18. Ligation products were transformed intoEscherichia coli, and 12,000 positive recombinant clones were picked and arrayed into 96-well plates. Clone inserts were amplified by PCR with M13 primers (55 pmol each), 1.5␮l of bacterial culture (template DNA), 4 U ofTaqpolymerase with 1⫻reaction buffer (Fisher, Pittsburgh, Pa.), a 0.2 mM concentration of each deoxynucleoside triphosphate (Eppendorf, Westbury, N.Y.), and 2.5 mM MgCl2in a 100-␮l reaction volume. PCR cycle conditions were 95°C for 5 min; followed by 35 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 1 min; followed by 72°C for 10 min after cycling was completed. The insert size was determined by using gel electrophoresis (1% agarose). PCR products of the correct size (500 to 1,000 bp) were purified with a Montage PCR96Cleanup kit (Millipore Corp., Bedford, Mass.) and stored at

⫺20°C until ready for printing.

PCR products were purified by sodium acetate precipitation, resuspended in 100␮l of H2O, quantified by UV spectrophotometry, and air dried. Probe DNA was then suspended in print buffer (200 mM Na2HPO4plus 0.4 M NaCl [pH 11.5]) at a final concentration of 100 ng/␮l, using a BIO-ROBOT 8000 instru-ment (Qiagen). Probes were then printed onto epoxy-coated slides (TeleChem International, Inc., Sunnyvale, Calif.) by using an Omnigrid spotter (GeneMa-chines, San Carlos, Calif.). PCR products from cloned fragments ofL. monocy-togenesribosomal and listeriolysin genes were used as positive controls, and PCR products from a mouse cDNA library were used as negative controls. After printing, the slides were UV cross-linked (120,000␮J) and stored at room temperature in the dark.

Target preparation and hybridization.Genomic DNA was extracted from target strains by using a DNeasy tissue kit (Qiagen) and quantified by using UV spectrophotometry. Target DNA (1.5␮g) was nick translated in the presence of biotin-dATP (BioNick labeling system; Invitrogen). The labeled DNA was then ethanol precipitated, resuspended in 150␮l of hybridization buffer consisting of 4⫻SSC (60 mM NaCl, 0.6 mM Na citrate [pH 7.0]) and 5⫻Denhardt’s solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), and added to the slide for overnight hybridizations at 55°C. Hybridizations and subsequent amplification steps were done in a GeneTAC hybridization station (Genomic Solutions, Ann Arbor, Mich.). Following target hybridization, the signal was amplified with a Tyramide signal amplification kit (Perkin-Elmer,

Boston, Mass.). The slides were washed twice at 23°C for 30 s with TNT buffer (100 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% Tween 20). Subsequent wash steps used two washes (30 s each) in TNT buffer, and all subsequent manipula-tions occurred at approximately 23°C. Streptavidin conjugated to horseradish peroxidase (1:100 in hybridization buffer) was incubated on the slide for 30 min, followed by washing and incubation with 10% equine serum (Sigma-Aldrich) in 2⫻SSC for 30 min. Biotinyl tyramide (1:50 in amplification buffer [tyramide signal amplification biotin system]) was then incubated on each slide for 10 min, followed by washing and a 30-min incubation with 2␮g of streptavidin per ml conjugated to Alexa Fluor 546 (Molecular Probes, Eugene, Oreg.) in 1⫻ SSC–5⫻Denhardt’s solution. The slides were given a final wash, followed by drying and imaging with a ScanArray 4000XL laser scanner (Packard BioChip Technologies, Downers Grove, Ill.).

Signal analysis. Quantarray software (Packard Biochip Technologies) was used to quantify signal intensity. The final output included median intensity values, and data were normalized by dividing the median signal intensity by the median signal intensity of the ribosomal positive control. Data were managed by using MS Excel (Microsoft Corp., Redmond, Wash.) spreadsheets.

Data analysis.Our analysis was limited to only those probes that were bimo-dally distributed such that both positive hybridizations (high signal) and negative hybridizations (low signal) were clearly identified. The selection process was based on a previously published algorithm (14). Briefly, for each probe, intensity values were assigned to either a “low” or a “high” cluster. After intensity values for all hybridization experiments were assigned to these two clusters, cluster averages and standard deviations were calculated. If cluster averages were dif-ferent by greater than three standard deviations, the probe was considered bimodal. Ninety bimodal probes were selected for analysis by this technique. An additional 30 bimodal probes were selected for analysis as described previously (4).

For dendrogram construction, probes with normalized intensity readings of less than 0.2 were assigned a score of 1, probes with normalized intensity read-ings of greater than 0.2 but less than 0.4 were scored as 2 (and treated as ambiguous data in the phylogenetic analysis), and probes with normalized in-tensity readings of greater than 0.4 were scored as 3. A matrix was constructed and processed with PAUP (version 4.0b8a; Sinauer Associates, Inc., Sunderland, Mass.). UPGMA and Treeview (20) were used to construct a dendrogram that summarized genetic relationships between samples. Stepwise discriminant func-tion analysis (DFA) (NCSS 2001 statistical software; NCSS, Kaysville, Utah) was used to identify probes characteristic of divisions and subtypes. Data were also examined by using a spreadsheet (Microsoft Excel) to identify probes that con-sistently discriminated between various dendrogram clusters.

Sequence analysis.Probes of interest were retrieved from the clone library and sequenced by using two-pass automated sequencing, and data were analyzed by using DNASTAR (DNASTAR, Madison, Wis.). Nucleotide sequences were compared to existing nucleotide and protein sequences present in the GenBank database by using BLASTn and BLASTx searches. Seven of these probe se-quences were selected to identify how sequence divergence was reflected by signal intensity on the microarray. PCR primers were designed to amplify a 500-to 600-bp region of the corresponding sequences from 15L. monocytogenes

isolates representing the two primary phylogenetic divisions. The resulting PCR products were sequenced, and percent sequence similarity was calculated.

Nucleotide sequence accession numbers.The DNA sequences of the MLST loci have been deposited in GenBank under accession numbers AY622010 through AY622039 (abcZ), AY622040 through AY622069 (ahs), AY622070 through AY622099 (dhk), AY622100 through AY622129 (lhkA), AY622130 through AY622159 (lisK), and AY622160 through AY622189 (ptsI).

RESULTS

A shotgun library was constructed by mixing equal molar

amounts of genomic DNAs from 20 strains (six serotypes) of

L.

monocytogenes

(4, 9). A 2,000-probe screening microarray was

constructed from the clone library and screened for

polymor-phic probes by hybridizing the genomic DNAs from 80 strains

of

L. monocytogenes

to the array. These strains were obtained

from diverse sources (human epidemic, human sporadic,

envi-ronmental, and veterinary) and included seven serotypes (1/2a,

1/2b, 1/2c, 3a, 4a, 4b, and 4e). The 685 probes identified as

polymorphic were sequenced, and nucleotide sequences were

compared to identify replicate probes. Six hundred twenty-nine

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probes were identified as unique, and the closest protein match

for each probe was identified by using BLASTx searches

against GenBank. The probes were then used to construct a

condensed array consisting entirely of polymorphic and

char-acterized probes.

Fifty-two

L. monocytogenes

strains were hybridized to the

condensed array, and subsequent data analysis identified 130

bimodally distributed probes. Data analyses were limited to

these probes to maximize the likelihood of identifying

subtype-or division-specific DNA sequences.

Phylogenetic divisions and subgroups.

Comparative

mi-croarray analysis grouped strains according to previously

de-scribed phylogenetic divisions, and all strains were grouped by

serotype (Fig. 1). Division I (D1) consisted of two main

sub-groups (D1a and D1b). Interestingly, the D1b subgroup,

con-sisting of human sporadic and environmental serotype 1/2b

strains, clustered more closely to Division II (D2) strains than

to D1a strains. However, DFA and subsequent sequence

anal-ysis were unable to identify probes with sequences unique to

D2 and D1b. Indeed, sequence analysis of 13 of the 14 probes

revealed that the majority of sequence differences occurred

between the major divisions (D1 and D2). Three probes that

differentiated between serovars 1 and 4 (probes 55 and 205) or

between serotypes (probe 1083) were identified, and it is likely

that serovar-specific probes may have influenced the

topolog-ical position of the D1b subcluster.

To allow serotype or source clusters to be easily visualized

on the dendrogram (Fig. 1), strains were coded by serotype (A

([1/2a], B [1/2b], C [1/2c], F [4b], or T [3b]), three-digit lab

strain identification number, and source (E [epidemic], S

[spo-radic], N [environmental or food], M [bulk milk], or V

[veter-inary]). Interestingly, most strains within serotype 4b grouped

according to source as well as serotype, with a majority of

serotype 4b epidemic strains forming a monophyletic group

within D1a.

Stepwise DFA was used to identify 22 probes that differed

among divisions and subclusters. Thirteen of these probes were

further investigated by PCR and sequence analysis (Table 2).

Sequence data revealed that five of the probes were division

specific, four were subcluster specific, and four were serovar or

serotype specific.

Reproducibility and resolution.

To verify that assay

repli-cates yielded similar results, genomic DNAs from isolates

B339S and B345S were extracted, purified, labeled, and

hy-bridized in two separate experiments. As expected, the

repli-cates for both strains clustered together (Fig. 1).

Resolution of the condensed array was compared to that of

the current gold standard, PFGE with AscI and ApaI

restric-tion endonuclease digesrestric-tion (16), by characterizing a panel of

28 strains by using both techniques. Resolution was similar for

the two techniques, with both microarray analysis and PFGE

dividing the 28 strains into 10 distinct subtypes (Fig. 1).

Addi-tionally, nine epidemiologically unrelated strains were grouped

into four subtypes by using ribotyping and MLST with six

housekeeping genes (Table 3). These strains separated into

five distinct groups when characterized by microarray analysis

and PFGE (with AscI and ApaI).

A panel of 10 isolates associated with four different

epidem-ics were subtyped by using the condensed array. Most (9 of 10)

epidemiologically linked isolates grouped together on the

den-drogram (Fig. 1).

DISCUSSION

Microarray analysis grouped strains by phylogenetic

divi-sions and serotype. However, a subcluster of Division I, D1b,

grouped more closely to D2 than to D1a. This subcluster

con-sisted of serotype 1/2b strains from human sporadic and

envi-ronmental sources. This grouping was consistent even when

data were analyzed using three different cluster algorithms

(UPGMA, neighbor joining, and Ward’s minimum variance),

using different intensity range scores (i.e., with

0.15 scored as

1), or simply using normalized intensity data to produce the

dendrogram (Ward’s minimum variance). Two probes (probes

55 and 891) identified by DFA as differentiating D1b from D1a

were sequenced and found to be serovar specific

(differenti-ated serovar 1 from serovar 4) (Table 2). Therefore, it likely

that a combination of probe differences makes D1b appear

more similar to D2 than to D1a.

D1 strains were separated into four main subclusters, with

D1a containing three subclusters and D1b consisting of a single

1/2b subcluster. One of the subclusters within D1a included 15

of the 17 serotype 4b strains associated with epidemics (Fig. 1).

DFA was used to identify three probes that are most useful in

defining this subcluster, and further analysis of these probes is

under way.

Strains epidemiologically linked to particular epidemics

were included in the microarray analysis to determine whether

microarray subtyping did indeed group these strains together.

Isolates obtained from patients and implicated foods from the

1981 Halifax epidemic (F495E and F496E), the 1994 Illinois

epidemic (B507E and B508B), and the 1998 multistate

demic (F470E, F581E, and F584E) grouped according to

epi-demic (Fig. 1). Two of the three strains associated with the

1988 to 1990 United Kingdom epidemic also grouped together.

Investigation of the later outbreak identified pa

ˆte

´ as the likely

source of an observed upsurge in listeriosis cases; however, no

samples of pa

ˆte

´ eaten by patients with listeriosis were available

for subtyping (19). Interestingly, the two strains from this

out-break that did cluster together were both obtained from

pa-tients, whereas strain F497E, a strain also associated with this

epidemic but in a separate cluster, was a food isolate.

Strain A503E, a serotype 1/2a isolate that caused a

multi-state deli meat-associated epidemic in 2000, clustered with

three other 1/2a strains (Fig. 1). Two of these strains are

particularly interesting, because one (A501N) was isolated

from the same food-processing plant in 1988 as A503E and

another (A502S) was from a human sporadic case associated

with A501N (17).

The resolutions of four different subtyping methods were

compared using a subset of strains (Fig. 1; Table 3).

Microar-ray analysis and PFGE subtyping showed the highest

resolu-tion, MLST had moderate subtyping resoluresolu-tion, and ribotyping

had the lowest resolution. The microarray analysis subtyping

resolution was similar to that of PFGE with two enzymes, the

current gold standard for molecular subtyping of

L.

monocy-togenes

strains (16). Nevertheless, occasionally the two

tech-niques placed strains in different groups (Fig. 1; Table 3). This

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[image:5.585.71.509.81.629.2]

FIG. 1. UPGMA representation of genetic relationships between 52

L. monocytogenes

isolates and one

L. innocua

isolate (INN) based on

hybridization data derived from 130 bimodally distributed microarray probes. Phylogenetic divisions are indicated as D1a and D1b (Division I) and

D2 (Division II). Isolates B339S and B345S (in boldface) were tested for processing and analysis reproducibility in two separate experiments.

Isolates with the same AscI and ApaI PFGE restriction patterns are shown in the same color. Nine isolates were subtyped by PFGE, MLST, and

ribotyping and are labeled with a diamond symbol. Ten isolates from the following four different epidemics were tested for subtype grouping: the

1981 Halifax epidemic (HA) (isolates F495E and F496E), the 1994 Illinois epidemic (IL) (isolates B507E and B508B), the 1998 multistate

frankfurter-associated epidemic (HD) (isolates F470E, F581E, and F584E), and the 1988 to 1990 United Kingdom epidemic (UK) (isolates F497E,

F498E, and F586E).

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is not surprising, because the two techniques sample the

ge-nome differently.

Microarray analysis and subsequent DFA processing of data

resulted in the identification of 22 subtype-specific probes.

Thirteen of these probes were further analyzed by PCR and

sequence analysis (Table 2). Sequence analysis indicated that the

microarray hybridization was capable of detecting approximately

10% sequence divergence between strains. These data agree with

the microarray sensitivity threshold reported previously (9),

al-though microarray sensitivity is obviously dependent on

hybrid-ization conditions, sequence content, and signal analysis.

The 22 probes identified as important for division and

sub-type definition included seven probes with sequence similarity

to cell wall-associated proteins (probes 119, 205, 265, 321, 553,

657, 891, and 951). Three of these were serovar or serotype

specific (Table 2). Five probes had sequence similarity to

pro-teins important for survival in the environment or host (probes

57, 837, 1133, 1229, and 1263), and four probes were similar in

sequence to virulence-associated proteins (probes 55, 875, 887,

and 1117).

[image:6.585.46.541.80.301.2]

In conclusion, these data indicate that microarray analysis

has a resolution similar to that of PFGE and better than those

of MLST with housekeeping genes and ribotyping. Microarray

analysis accurately clustered epidemiologically linked strains.

TABLE 2. Protein similarity and sequence analysis of

L. monocytogenes

subtype- specific probes

Probe Protein similaritya Putative function Probe specificity

1117

bvr

locus

Virulence gene regulation

Division

b

951

Internalin-like (LPXTG motif)

Cell surface

Division

c

887

lisRK

Stress tolerance, virulence

Division

c

875

ORFA (LPXTG motif),

L. innocua

Division

b

657

TagD

Cell wall synthesis

Division

b

523

ABC transporter, permease

Molecule transport

Division

c

203

Imo0737

Division

b

199

Imo1434

Division

c

119

Internalin-like (LPXTG motif)

Cell surface

Division

b

57

Multidrug efflux transporter

Molecule transport

Division

c

1133

UV damage repair protein

Stress response

Division and subcluster

c,d

837

Exinuclease ABC

Stress response

Division and subcluster

c

321

Internalin-like (LPXTG motif)

Cell surface

Division and subcluster

c

141

Imo1965

Division and subcluster

c

1263

RNase PH

Temperature response

Serovar

b

1229

Two-component sensor protein KdpD

Osmoregulation

Serovar

b

1083

Helicase

Replicative DNA helicase, DnaC

Serotype

891

Autolysin

Cell wall lysis

Serovar

c

553

Glycosyltransferase

Cell wall synthesis

Serovar

b

265

MurZ, Rho

Cell wall synthesis, transcription factor

Serovar

b

205

Rhamnose synthetase

Cell wall synthesis

Serovar

c

55

Amidase 4b protein

Cell adherence

Serovar

c

aBLASTx searches showed that all probes had the greatest sequence similarity toL. monocytogenesproteins unless noted otherwise. bSpecificity as determined by microarray analysis.

cSpecificity as determined by microarray and sequence analyses. dSequence data specific for both division and subclusters.

TABLE 3.

L. monocytogenes

strains of different origin subtyped by five different methods

Strain Previous

designationa Yr Origin Serotype

MLST

typeb Ribotype

PFGE Microarray resultc

AscI ApaI

F583E

HPB850

1996

Switzerland

4b

222422

DUP-1038

A

A

d

F589E

HPB1808

1985

California

4b

222422

DUP-1038

A

a

F586E

HPB774

1991

United Kingdom

4b(x)

632222

DUP-1042

B

b

F593E

HPB1026

1985

California

4b

632222

DUP-1042

B

b

F591E

HPB2262

1998

Italy

4b

632222

DUP-1042

C

B

e

T590S

HPB1031

NA

f

United States

3b (1/2b)

333223

DUP-1042

D

c

B588E

HPB1983

1998

Canada

1/2b

333223

DUP-1042

D

C

e

F584E

HPB2142

1998

United States

4b

225226

DUP-1044

E

d

F592E

HPB2182

1999

Canada

4b

225226

DUP-1044

E

D

e

aF. Pagotto et al., unpublished data.

bGene and number of alleles:abcZ, 6;ahs, 6; dhk, 5;lhkA, 6;lisK, 7;ptsI, 8. cMicroarray analysis able () or not able () to further differentiate isolates. dOne band missing.

eOne extra band. fNA, not available.

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[image:6.585.42.544.531.675.2]
(7)

Most epidemic-related strains formed a monophyletic cluster

within Division I. Additionally, microarray analysis allowed

identification of 22 probes that simultaneously distinguish

di-visions, serotypes, and subtypes.

ACKNOWLEDGMENTS

Funding was provided by the USDA Agricultural Research Service

(grant CWU 5348-32000-017-00D) and the Agricultural Animal

Health Program (College of Veterinary Medicine, Washington State

University).

We gratefully acknowledge the excellent technical assistance

pro-vided by James Reynolds, Kevin Tyler, Edith Orozco, Dave Tibbals,

and Melissa Krug.

L. monocytogenes

isolates were kindly provided by

Lewis Graves (Centers for Disease Control and Prevention), Jinxin Hu

(Washington State Department of Health), Karen Jinneman (U.S.

Food and Drug Administration), Lisa Gorski (USDA Agricultural

Research Service), and Martin Wiedmann (Cornell University).

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Figure

TABLE 1. L. monocytogenes strains subtyped by microarray analysis
FIG. 1. UPGMA representation of genetic relationships between 52 L. monocytogeneshybridization data derived from 130 bimodally distributed microarray probes
TABLE 3. L. monocytogenes strains of different origin subtyped by five different methods

References

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