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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,
2,3Douglas R. Call,
2Sandra C. Smole,
4and Franco Pagotto
5Animal Disease Research Unit, USDA Agricultural Research Service,
1and College of Veterinary Medicine, Washington State
University,
2Pullman, 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
5Received 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
cHPB1031, 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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[image:2.585.46.539.79.576.2]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⬃60l 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 20l 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, 10g 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), 1.5l 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 100l 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 (120,000J) 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 (1.5g) was nick translated in the presence of biotin-dATP (BioNick labeling system; Invitrogen). The labeled DNA was then ethanol precipitated, resuspended in 150l 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 2g 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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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
b951
Internalin-like (LPXTG motif)
Cell surface
Division
c887
lisRK
Stress tolerance, virulence
Division
c875
ORFA (LPXTG motif),
L. innocua
Division
b657
TagD
Cell wall synthesis
Division
b523
ABC transporter, permease
Molecule transport
Division
c203
Imo0737
Division
b199
Imo1434
Division
c119
Internalin-like (LPXTG motif)
Cell surface
Division
b57
Multidrug efflux transporter
Molecule transport
Division
c1133
UV damage repair protein
Stress response
Division and subcluster
c,d837
Exinuclease ABC
Stress response
Division and subcluster
c321
Internalin-like (LPXTG motif)
Cell surface
Division and subcluster
c141
Imo1965
Division and subcluster
c1263
RNase PH
Temperature response
Serovar
b1229
Two-component sensor protein KdpD
Osmoregulation
Serovar
b1083
Helicase
Replicative DNA helicase, DnaC
Serotype
891
Autolysin
Cell wall lysis
Serovar
c553
Glycosyltransferase
Cell wall synthesis
Serovar
b265
MurZ, Rho
Cell wall synthesis, transcription factor
Serovar
b205
Rhamnose synthetase
Cell wall synthesis
Serovar
c55
Amidase 4b protein
Cell adherence
Serovar
caBLASTx 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
fUnited 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.
on May 15, 2020 by guest
http://jcm.asm.org/
[image:6.585.42.544.531.675.2]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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