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Antimicrobial Agents and Chemotherapy, October 2003, p. 3290-3295, Vol. 47, No. 10
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.10.3290-3295.2003
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 Department of Pathobiology, University of Washington, Seattle, Washington 981952

Received 24 April 2003/ Returned for modification 6 June 2003/ Accepted 2 July 2003


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
We developed and tested a glass-based microarray suitable for detecting multiple tetracycline (tet) resistance genes. Microarray probes for 17 tet genes, the ß-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 65°C, and detected with Tyramide Signal Amplification, Alexa Fluor 546, and a microarray scanner. Using a detection threshold of 3x 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.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
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 resistance genes are identified, this information can be used to investigate the diversity and the spatial and temporal distribution of resistance genes within and between different host reservoirs, populations, and environments (1, 14). When identifying specific resistance genes, researchers have used a variety of methods, including DNA-DNA hybridization on membranes 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 substrate (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 of ca. 550-bp PCR products. This microarray functions with a high degree of specificity and is suitable for detection of multiple tetracycline resistance genes from multiple genera of bacteria.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
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).


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

 

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  		TABLE 2. Field isolates and other test strains used in this study

 
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 (InforMax 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 µl) were composed of 1x 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 95°C followed by 30 cycles of 96°C for 30 s, 60°C for 30 s, and 72°C for 30 s and a final 10-min extension time at 72°C. Each PCR product was verified by gel electrophoresis and ethanol precipitated. Products were resuspended 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 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/µl in print buffer (100 mM Na2HPO4, 200 mM NaCl, 0.01% sodium dodecyl sulfate [SDS]; pH ~11), heat denatured (95°C, 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 chemistry and served as orientation points for image processing. After being spotted, slides were baked for 1 h at 130°C 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 µg) was nick translated according to the manufacturer's 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 precipitated and resuspended in 75 µl of hybridization buffer, composed of 4x SSC (0.6 M NaCl plus 0.6 M Na citrate) and 5x Denhardt's 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 (95°C for 3 min), briefly centrifuged, and held on ice until applied to the slide. Blocking buffer was aspirated and immediately replaced with 35 µl of denatured target per well. Slides were then placed into a humidified, conical tube (50 ml) and submerged into a water bath overnight at 65°C.

After incubation, the labeled target was aspirated and could be stored at -20°C for replicate hybridizations. Slides were washed with a mixture of 1x SSC and 0.2% SDS at hybridization temperature (4 min) followed by 0.1x SSC-0.2% SDS at room temperature (4 min) and 0.1x 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 2x 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 1x SSC-5x Denhardt's 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 (Applied 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 confirmed 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
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
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 positive 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).



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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.

 
Threshold for detection. Our positive control hybridizations yielded some minor cross-hybridization with other probes—most commonly involving tet(A) (Fig. 1). Therefore, we selected an arbitrary threshold for positive detection based on three standard deviations (3x 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 3x SD 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 3x 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 information where more than one tet gene may be present.



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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 3x SD for all tet probes.

 
Sample hybridizations. We tested 48 isolates on the microarray: 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 absence 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 applicability 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 detection 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 process more efficient, and because PCR is not required to generate targets, the array is limited only by the physical constraints 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 requiring 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 ß-lactams (3). Longer probes, such as the PCR products employed 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.

This project received financial assistance from the Morris Animal Foundation (Englewood, Colo.) and the Agricultural Animal Health Program (Washington State University, Pullman).


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Veterinary Microbiology and Pathology, Washington State University, 402 Bustad Hall, Pullman, WA 99164-7040. Phone: (509) 335-6313. Fax: (509) 335-8529. E-mail: drcall{at}wsu.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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Antimicrobial Agents and Chemotherapy, October 2003, p. 3290-3295, Vol. 47, No. 10
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.10.3290-3295.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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