ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2003, p. 32903295 Vol. 47, No.

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0066-4804/03/$08.000 DOI: 10.1128/AAC.47.10.32903295.2003
Copyright 2003, American Society for Microbiology. All Rights Reserved.

Identifying Antimicrobial Resistance Genes with DNA Microarrays

Douglas R. Call,1* Marlene K. Bakko,1 Melissa J. Krug,1 and Marilyn C. Roberts2
Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University,
Pullman, Washington 99164,1 and Department of Pathobiology, University of Washington,
Seattle, Washington 981952
Received 24 April 2003/Returned for modification 6 June 2003/Accepted 2 July 2003

We developed and tested a glass-based microarray suitable for detecting multiple tetracycline (tet) resistance
genes. Microarray probes for 17 tet genes, the -lactamase blaTEM-1 gene, and a 16S ribosomal DNA gene
(Escherichia coli) were generated from known controls by PCR. The resulting products (ca. 550 bp) were
applied as spots onto epoxy-silane-derivatized, Teflon-masked slides by using a robotic spotter. DNA was
extracted from test strains, biotinylated, hybridized overnight to individual microarrays at 65C, and detected

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with Tyramide Signal Amplification, Alexa Fluor 546, and a microarray scanner. Using a detection threshold
of 3 the standard deviation, we correctly identified tet genes carried by 39 test strains. Nine additional strains
were not known to harbor any genes represented on the microarray, and these strains were negative for all 17
tet probes as expected. We verified that R741a, which was originally thought to carry a novel tet gene, tet(I),
actually harbored a tet(G) gene. Microarray technology has the potential for screening a large number of
different antibiotic resistance genes by the relatively low-cost methods outlined in this paper.

Antimicrobial resistance testing is an invaluable method for
characterizing clinical and environmental bacteria, although a
resistance phenotype provides only limited information about
the identity of the actual resistance genes harbored by the
microbes (15). In contrast, when the specific antibiotic resis-
tance genes are identified, this information can be used to
investigate the diversity and the spatial and temporal distribu-
tion of resistance genes within and between different host res-
ervoirs, populations, and environments (1, 14). When identi-
fying specific resistance genes, researchers have used a variety
of methods, including DNA-DNA hybridization on mem-
branes and gene-specific PCR assays (10, 14). PCR assays are
relatively rapid and can be used with a variety of sample types,
such as individual bacterial colonies or environmental samples
(4, 5, 9). Multiplex PCR assays have been devised for detection
of a large number of genes (12), and this approach could be
used for different classes of antibiotic resistance genes. For
example, it should be possible to devise multiplex PCRs to
detect the 35 genetically diverse tetracycline resistance genes
(8).
DNA microarrays offer an alternative method for screening
for the presence of a wide diversity of genes. In this format,
probes specific to each gene are deposited onto a solid sub-
strate (usually glass) in a lattice pattern. DNA is then labeled
and hybridized to the array, and specific target-probe duplexes
are detected with a reporter molecule (21). In this study, we
investigated the feasibility of using DNA microarrays as a tool
to screen bacterial isolates for tetracycline resistance genes.
Our initial efforts involved short oligonucleotide probes (25-
mer), but we found this array design to be insensitive to low-
copy-number genes (data not shown). Herein we demonstrate
a more conventional format in which microarray probes consist
* Corresponding author. Mailing address: Department of Veteri-
nary Microbiology and Pathology, Washington State University, 402
Bustad Hall, Pullman, WA 99164-7040. Phone: (509) 335-6313. Fax:
(509) 335-8529. E-mail: drcall@wsu.edu.
of ca. 550-bp PCR products. This microarray functions with a
high degree of specificity and is suitable for detection of mul-
tiple tetracycline resistance genes from multiple genera of bac-
teria.
MATERIALS AND METHODS
Controls and test isolates. Seventeen cloned tet genes were used as PCR
templates and hybridization controls for the microarray (Table 1). A diverse
group of test isolates (n 48), including 12 different genera, were assembled
from previous studies (10, 14) and from bovine sources (Table 2). We included
Escherichia coli transconjugants that harbored tet genes from other sources,
including isolate 33, which harbors plasmid R714a from a Providencia species
(13). All isolates either harbored known tet genes or required MICs that were
consistent with resistance based on National Committee for Clinical Laboratory
Standards breakpoints for E. coli (17, 18).
Microarray probes. We used PCR to generate probes for the microarray.
Cloned DNA from 17 of 35 recognized tet genes was extracted by using a DNeasy
kit (Qiagen, Valencia, Calif.) (Table 1) (http://faculty.washington.edu/marilynr/).
Primers for each gene were identified with Primer3 software (20) with the goal
of producing products ca. 550 bp in length (Table 1). This probe size was selected
based on previous success by similar procedures for microarray hybridizations
(6). Additional PCR products were used as controls, including the 16S ribosomal
DNA (rDNA) gene (rrn from E. coli) and the blaTEM-1 gene (from pBR322). All
predicted PCR products were compared by using Vector NTI software (Infor-
Max Inc., Bethesda, Md.) to verify that probe sequences were unique (85%
sequence identity between predicted probe sequences). Oligonucleotides were
synthesized by Invitrogen Corp. (Carlsbad, Calif.) with no modifications and with
basic desalting after synthesis. PCR mixtures (100 l) were composed of 1
reaction buffer (Fisher Scientific, Pittsburgh, Pa.), 200 nM each deoxynucleoside
triphosphate (dNTP), 2 mM MgCl2, 4 U of Taq polymerase, 400 nM each primer,
and ca. 40 ng of template DNA. The thermal cycler conditions included 2 min of
denaturation at 95C followed by 30 cycles of 96C for 30 s, 60C for 30 s, and
72C for 30 s and a final 10-min extension time at 72C. Each PCR product was
verified by gel electrophoresis and ethanol precipitated. Products were resus-
pended in sterile water and quantified by spectrophotometry.
Slide preparation. Multiple microarrays were printed on glass slides so that
independent microarrays were contained within each of 12 wells defined by a
Teflon-masked surface (Erie Scientific, Portsmouth, N.H.); the hydrophobic
nature of the masking permitted independent samples to be hybridized within
each well. Slides were derivatized with an epoxy-silane monolayer as described
previously (7). Briefly, slides were sonicated for 2 min in a detergent solution
(Contrad 70 detergent; Fisher Scientific), rinsed with deionized water, and dried
by compressed air. Slides were then soaked for 1 h in 3 N HCl, rinsed, dried, and

3290
VOL. 47, 2003 TETRACYCLINE RESISTANCE MICROARRAY 3291

TABLE 1. Primers and predicted amplification products used as microarray probes

GenBank
Gene Primersa Locationc Size (bp)d
accession no.b

tet(A) TTGGCATTCTGCATTCACTC X75761 1822 494

GTATAGCTTGCCGGAAGTCG 2316

tet(B) CAGTGCTGTTGTTGTCATTAA V00611 275 571

GCTTGGAATACTGAGTGTAA 846

tet(C) ATATCGTCCATTCCGACAGC Y19114 92 502

CTGACTGGGTTGAAGGCTCT 594

tet(D) GCAAACCATTACGGCATTCT X65876 1638 546

GATAAGCTGCGCGGTAAAAA 2184

tet(E) TATTAACGGGCTGGCATTTC L06940 518 544

AGCTGTCAGGTGGGTCAAAC 1062

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tet(G) GCTCGGTGGTATCTCTGCTC AF133140 1209 550
CAAAGCCCCTTGCTTGTTAC 1759

tet(H) TCAACCACACTTTGGATGCT U00792 986 550

CCCATTTTTGTGCCAATTTC 1536

tet(L) ACTGGGTGAACACAGCCTTT X60828 322 548

CAGGAATGACAGCACGCTAA 870

tet(M) ACACGCCAGGACATATGGAT M85225 740 536

ATTTCCGCAAAGTTCAGACG 1276

tet(O) GCGGTAATTATGGGAAACGA M18896 735 550

TTTCCCGCTGTTCAGATTTC 1285

tet(S) CGCTACATTTGCGAGACTCA L09756 1267 555

GAATGCCACTACCCAAAGGA 1822

tet(W) GGGAAATTGTTCGGACAGAC AJ222769 1090 549

AACGGATACCATCCCTGACA 1639

tet(Z) GATGAGATGGGGAAGGTTCA AF121000 15666 544

CTTGTTGGTAACCCGGAAGA 16210

tet(30) CCGTCATGCAATTTGTGTTC AF090987 1281 550

TAGAGCACCCAGATCGTTCC 1831

tetA(P) TTGGGGGAGTTTTAACAGGA L20800 1292 564

TTCCAAAAATTCCAAACCAA 1856

tetB(P) TGGGGCAAATTTCAACAAAG L20800 2710 537

TCAACAACTCCCCCATTTTC 3247

otr(B) CAAGAAACTGGCGATCGTG AF061335 969 545

GGAACCAGGTCATGACGAAC 1514

blaTEM-1 CCAATGCTTAATCAGTGAGG L09137 1629 858

ATGAGTATTCAACATTTCCG 2487

16S rDNA AGAGTTTGATCMTGGCTCAG AE000452 8 528

ATTACCGCGGCTGCTGG 536
a
Primers designed for this study. Primers for the blaTEM-1 and 16S rDNA genes were adopted from references 2 and 16, respectively.
b
GenBank accession number for the reference sequence used to design the microarray probes.
c
Primer location within the reference sequence.
d
Predicted size of the microarray probe.

then incubated in a 2% solution of 3-glycidoxypropyltrimethoxysilane (Sigma
Aldrich, St. Louis, Mo.) in high-performance liquid chromatography HPLC-
grade methanol. After 15 min, slides were rinsed in 100% methanol and dried by
compressed air.
Microarray construction. PCR products were diluted to 75 ng/l in print
buffer (100 mM Na2HPO4, 200 mM NaCl, 0.01% sodium dodecyl sulfate [SDS];
pH 11), heat denatured (95C, 5 min) with a thermal cycler, and allowed to cool
to room temperature. Each probe was then deposited at a fixed location within
each masked well by a robotic spotter (BioRobotics Microgrid II; Woburn,
Mass.). Each probe was printed as four replicate spots within each array, and
every array included an arbitrary oligonucleotide probe (25-mer) conjugated with
biotin. These biotin pseudoprobes served as positive controls for the detection
3292 CALL ET AL. ANTIMICROB. AGENTS CHEMOTHER.

TABLE 2. Field isolates and other test strains used in this study
Isolate no.a Species Geneb Intensityc Thresholdd

1 Acinetobacter sp. None 11,130

2 Acinetobacter sp. None 1,113
3 Acinetobacter woffii None 259
4 Escherichia coli None 4,549
5 Escherichia coli None 15,589
6 Pantoea sp. None 17,606
7 Providencia rettgeri None 12,467
9 Pseudomonas putida None 23,694
10 Ralstonia pickettii None 15,160
11 Aeromonas hydrophila tet(A) 4,895 4,035
12 Aeromonas hydrophila tet(A) 33,819 26,808
13 Citrobacter freundii tet(A) 65,535 51,741
14 Citrobacter freundii tet(A) 62,044 48,931
EC3e Escherichia coli tet(A),(B) 65,535 73,099
EC4 Escherichia coli tet(A) 26,433 21,129
EC5 Escherichia coli tet(A) 65,535 52,592

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EC6 Escherichia coli tet(A) 26,002 20,713
EC8 Escherichia coli tet(A) 49,000 38,695
EC9 Escherichia coli tet(A) 14,199 11,304
15 Escherichia coli tet(A) 61,251 55,416
SSuTA1 Escherichia coli tet(A) 48,778 38,454
SSuTA2 Escherichia coli tet(A) 38,426 30,303
SSuTCH1 Escherichia coli tet(A) 33,717 26,769
SSuTCH2 Escherichia coli tet(A) 36,390 28,889
SSuTCH3 Escherichia coli tet(A) 9,964 8,073
17 Pseudomonas fluorescens tet(A) 48,640 39,580
18 Pseudomonas fluorescens tet(A) 61,988 51,568
19 Pseudomonas fluorescens tet(A) 29,881 24,043
20 Pseudomonas fluorescens tet(A) 51,838 41,179
22 Pseudomonas sp. tet(A) 59,082 46,700
24 Brevundimonas vesicularis tet(B) 60,669 51,384
25 Enterobacter sakazakii tet(B) 59,673 50,585
SSuT1 Escherichia coli tet(B) 24,672 20,814
SSuT2 Escherichia coli tet(B) 34,982 29,552
SSuT3 Escherichia coli tet(B) 52,593 44,385
SSuTA3 Escherichia coli tet(B) 61,319 48,373
26 Serratia liquifaciens tet(B) 64,873 57,964
27 Citrobacter freundii tet(D) 12,974 10,353
28 Citrobacter freundii tet(D) 8,499 6,872
29 Aeromonas hydrophila tet(E) 65,535 60,134
30 Aeromonas hydrophila tet(E) 23,357 18,635
31 Aeromonas hydrophila tet(E) 63,840 50,459
32 Aeromonas hydrophila tet(E) 39,391 31,303
33f Escherichia coli tet(G) 32,777 25,887
34 Acinetobacter radioresistens tet(H) 42,763 37,004
EC2 Escherichia coli tet(H) 65,535 51,935
35 Moraxella sp. tet(H) 60,039 53,763
EC1 Escherichia coli tet(Z) 65,535 52,207
a
Isolate reference number. Both EC2 and EC3 harbored cloned genes and do not represent field isolates.
b
Gene detected by the DNA microarray. Gene identity was confirmed by previous work (14) or by PCR with gene-specific primers (strains designated with an SSuT
prefix).
c
Median pixel intensity of the microarray probe.
d
Detection threshold based on the average median pixel intensity for tet probes plus 3 SD.
e
EC3 was clearly positive for both tet(A) and tet(B).
f
Isolate 33 harbored plasmid R714a from a strain of Providencia (13).

chemistry and served as orientation points for image processing. After being
spotted, slides were baked for 1 h at 130C under vacuum (22 Hg) and stored at
room temperature while protected from light.
Hybridization and detection. Target DNA was prepared by growing a test
isolate in Luria-Bertani (LB) broth or on an LB plate. Cells were pelleted, and
DNA was extracted with a DNeasy kit (Qiagen) followed by quantification by
spectrophotometry. Target DNA (1 g) was nick translated according to the
manufacturers instructions (biotin-dATP; BioNick kit; Invitrogen Corp.), except
reaction mixtures were incubated for 2 h. The labeled DNA was used directly
with hybridization buffer for the 48 test strains, whereas 1:1,000 dilutions were
prepared from pure plasmid extractions containing cloned positive control genes
(Table 1). To more closely simulate test samples, positive control clones were
also prepared with a DNeasy kit extraction so that both genomic DNA and
plasmid DNA were present in the labeling reaction mixture, and a 1:5 dilution
was used in the subsequent hybridizations. Labeled DNA was ethanol precipi-
tated and resuspended in 75 l of hybridization buffer, composed of 4 SSC (0.6
M NaCl plus 0.6 M Na citrate) and 5 Denhardts solution (0.001% Ficoll,
0.001% polyvinylpyrrolidone, 0.001% bovine serum albumin). In preparation for
hybridization, slides were incubated with TNB (100 mM Tris-HCl [pH 7.5], 0.15
mM NaCl) with 0.5% blocking reagent (biotin Tyramide Signal Amplification
[TSA] kit; Perkin-Elmer Life Sciences, Boston, Mass.) for 30 min at room
temperature. Labeled DNA was heat denatured (95C for 3 min), briefly cen-
trifuged, and held on ice until applied to the slide. Blocking buffer was aspirated
and immediately replaced with 35 l of denatured target per well. Slides were
VOL. 47, 2003 TETRACYCLINE RESISTANCE MICROARRAY 3293

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FIG. 1. Hybridization results illustrating specificity of tet gene probes. Probes were printed in quadruplicate in two columns. From top to bottom
of each array, the left column included tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(L), tet(M), and tet(O). The right column included
tetP(A), tetP(B), tet(S), tet(W), tet(Z), otr(B), tet(30), 16S rDNA, blaTEM-1, and a biotin marker.

then placed into a humidified, conical tube (50 ml) and submerged into a water
bath overnight at 65C.
After incubation, the labeled target was aspirated and could be stored at
20C for replicate hybridizations. Slides were washed with a mixture of 1 SSC
and 0.2% SDS at hybridization temperature (4 min) followed by 0.1 SSC0.2%
SDS at room temperature (4 min) and 0.1 SSC at room temperature (4 min)
(11). Stringent washes were followed by three 1-min washes in TNT buffer (0.1
M Tris-HCl [pH 7.5], 0.15 M NaCl, 0.05% Tween 20). A streptavidin-horseradish
peroxidase conjugate (TSA kit) was diluted 1:100 in TNB and incubated in each
well for 30 min, followed by three 1-min washes in TNT. Ten percent fetal equine
serum in 2 SSC was incubated in each well for 30 min to provide a protein
surface for tyramide binding followed by three 1-min washes in TNT buffer.
Biotinyl tyramide (diluted 1:50 in amplification diluent) was incubated in each
well for 10 min. After biotinyl tyramide had been washed from the slide, 2-mg/ml
streptavidin conjugated to Alexa Fluor 546 (Molecular Probes, Eugene, Oreg.)
was added in 1 SSC5 Denhardts solution, and the slides were incubated for
1 h before being given three 1-min washes in TNT followed by drying by high-
speed centrifugation. Images were captured with an arrayWoRxe scanner (Ap-
plied Precision, Issaquah, Wash.), and image quantification was accomplished
with softWoRx software (Applied Precision). Median pixel values are reported
as signal intensity (averaged for replicate probes). Positive detection was con-
firmed by either PCR (primers from Table 1) or by DNA-DNA hybridization (10,
14). Resistant strains that failed to hybridize to any tet probes were hybridized a
second time to confirm the finding.
RESULTS AND DISCUSSION
Microarray validation. In all cases, there was a one-to-one
correspondence between a control gene and its respective
probe, and many hybridizations with cloned genes were posi-
tive for the blaTEM-1 probe (Fig. 1). The latter result reflected
the presence of the ampicillin resistance gene harbored by
many of the cloning vectors. The tetA(P) and tetB(P) genes
were contained on the same plasmid, so both probes were
expected to be visible for this hybridization. We subsequently
nick translated individual PCR products and demonstrated
that both probes were specific for their respective targets (data
not shown).
Threshold for detection. Our positive control hybridizations
yielded some minor cross-hybridization with other probes
most commonly involving tet(A) (Fig. 1). Therefore, we se-
lected an arbitrary threshold for positive detection based on
three standard deviations (3 SD) above the average signal
intensity for all tet probes on the array (excluding 16S rDNA,
blaTEM-1, and biotin spots). For example, the results for 3 SD
3294 CALL ET AL. ANTIMICROB. AGENTS CHEMOTHER.

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FIG. 2. Example of quantified signal intensity for positive control hybridizations tet(A) (upper panel) and tet(S) (lower panel). The horizontal
line represents a detection threshold equivalent to the average median intensity plus 3 SD for all tet probes.
VOL. 47, 2003
for the tet(A) and tet(S) hybridizations (Fig. 1) were 54,532 and
55,003, which yielded very clear detection thresholds for these
hybridizations (Fig. 2). We adopted this procedure for scoring
subsequent hybridizations. If more than one tet probe had a
high signal intensity, then a 3 SD detection threshold may
not be appropriate. For example, the threshold for the tetA(P)
and tetB(P) hybridization (Fig. 1) was 73,754, which exceeds
the maximum possible signal intensity, 65,535. Therefore, each
hybridization needs to be examined to avoid loss of informa-
tion where more than one tet gene may be present.
Sample hybridizations. We tested 48 isolates on the mi-
croarray: 38 isolates were identified as having a single tet gene,
and 1 isolate (EC3) harbored two tet genes, tet(A) and tet(B).
The nine remaining strains did not hybridize to any of the tet
genes present on the array (Table 2). In the latter case, all nine
field isolates were positive for the 16S rDNA probe, indicating
that failure to detect a gene was not due to failed labeling and
hybridization procedures, but presumably was due to the ab-
sence of a corresponding probe. Both positive and negative
hybridization results for 38 isolates were described previously
by using DNA-DNA hybridization for the 17 tet genes (10, 14).
Positive detections for the remaining nine isolates (Table 2)
were confirmed with gene-specific primers (this study). The E.
coli isolate harboring plasmid R741a (isolate 33) was shown to
have tet(G), and this was confirmed by sequence analysis (data
not shown).
Assay design. For the proposed technique to have applica-
bility in a high-throughput setting, it would clearly require
shorter incubation times and automation. In this study, we
used overnight incubations before proceeding with TSA detec-
tion simply as a matter of convenience. We also tested the
feasibility of reducing the hybridization incubation to 3 or 6 h
by using tet(B) as a test target. Probe signal was not saturated
at 3 h (intensity, 54,266), but saturation was evident by 6 h
(intensity, 65,535), indicating that a truncated hybridization
procedure is feasible.
Our prototype microarray demonstrates the feasibility of
developing relatively high-throughput, planar microarrays for
identification of resistance genes. These probe fragments can
be cloned into a common vector to make the production pro-
cess more efficient, and because PCR is not required to gen-
erate targets, the array is limited only by the physical con-
straints of the slide. For example, we have designed a new
masked formatting that accommodates over 400 probes per
well. If no masking is used, then it is not unusual to incorporate
over 10,000 probes on a single slide. Nevertheless, microarrays
designed from PCR products have the disadvantage of requir-
ing positive control constructs for the PCR template. These
long probes are less sensitive to minor point mutations, such as
those responsible for protection against extended-spectrum
-lactams (3). Longer probes, such as the PCR products em-
ployed here, are clearly more sensitive for target detection
(22), but a compromise may be achieved by using long (ca., 70-
to 100-mer) oligonucleotide probes or peptide nucleic acids
(19).
ACKNOWLEDGMENTS
We are very grateful to Stacey LaFrentz for technical assistance.
TETRACYCLINE RESISTANCE MICROARRAY 3295
This project received financial assistance from the Morris Animal
Foundation (Englewood, Colo.) and the Agricultural Animal Health
Program (Washington State University, Pullman).
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