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Journal of Clinical Microbiology, July 2006, p. 2389-2397, Vol. 44, No. 7
0095-1137/06/$08.00+0     doi:10.1128/JCM.02291-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of Bacterial Pathogens Causing Bloodstream Infections by DNA Microarray{ddagger}

Berit E. E. Cleven,1,{dagger} Maria Palka-Santini,1,{dagger} Jörg Gielen,1 Salima Meembor,1 Martin Krönke,1,2 and Oleg Krut1*

Institute for Medical Microbiology, Immunology, and Hygiene,1 Center of Molecular Medicine Cologne, Medical Center, University of Cologne, Goldenfelsstr. 19-21, 50935 Cologne, Germany2

Received 2 November 2005/ Returned for modification 11 January 2006/ Accepted 1 May 2006


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ABSTRACT
 
Bloodstream infections are potentially life-threatening and require rapid identification and antibiotic susceptibility testing of the causative pathogen in order to facilitate specific antimicrobial therapy. We developed a prototype DNA microarray for the identification and characterization of three important bacteremia-causing species: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The array consisted of 120 species-specific gene probes 200 to 800 bp in length that were amplified from recombinant plasmids. These probes represented genes encoding housekeeping proteins, virulence factors, and antibiotic resistance determinants. Evaluation with 42 clinical isolates, 3 reference strains, and 13 positive blood cultures revealed that the DNA microarray was highly specific in identifying S. aureus, E. coli, and P. aeruginosa strains and in discriminating them from closely related gram-positive and gram-negative bacterial strains also known to be etiological agents of bacteremia. We found a nearly perfect correlation between phenotypic antibiotic resistance determined by conventional susceptibility testing and genotypic antibiotic resistance by hybridization to the S. aureus resistance gene probes mecA (oxacillin-methicillin resistance), aacA-aphD (gentamicin resistance), ermA (erythromycin resistance), and blaZ (penicillin resistance) and the E. coli resistance gene probes blaTEM-106 (penicillin resistance) and aacC2 (aminoglycoside resistance). Furthermore, antibiotic resistance and virulence gene probes permitted genotypic discrimination within a species. This novel DNA microarray demonstrates the feasibility of simultaneously identifying and characterizing bacteria in blood cultures without prior amplification of target DNA or preidentification of the pathogen.


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INTRODUCTION
 
The presence of living microorganisms in the blood of a patient is usually indicative of a serious invasive infection requiring urgent antimicrobial therapy (30). The mortality associated with bloodstream infections may range from 20 to 50% and depends on several factors, including the pathogen and host (30). Many septic episodes are nosocomial and may be due to microorganisms with increased antimicrobial resistance. Staphylococcus aureus, Escherichia coli, coagulase-negative staphylococci (CoNS), Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus spp., Streptococcus spp., Candida albicans, and Enterobacter cloacae are the most frequent etiological agents of bacteremia and fungemia in Europe (10, 20, 29) and the United States (4, 30). Rapid and reliable detection of bloodstream infections, including characterization of the pathogen to the species level and determination of its antibiotic susceptibility pattern, is crucial for several reasons: (i) appropriate antimicrobial agents can be selected, and thus, unnecessary treatment with ineffective antibiotics can be avoided; (ii) the prognosis of the patients can be improved; (iii) the acquisition of resistance in pathogens may be decelerated; and (iv) expenditure on antimicrobials and overall hospital costs can be reduced (2, 12).

Routine microbiological detection of bacteremia relies on enrichment of the causative pathogen using automated continuous-monitoring blood culture systems followed by Gram stain, subculture on agar, and subsequent biochemical identification and susceptibility testing. When the blood culture is noted to be positive, definitive identification and antibiotic susceptibilities are usually not available earlier than 24 to 72 h. In general, automated identification systems type pathogens to the species level; additional strain-specific information (e.g., virulence factors) require additional time-consuming and expensive phenotypic and genotypic tests and are not performed routinely (18).

In recent years, numerous studies have demonstrated the value of molecular techniques in order to identify and genotype bacteria or fungi in blood specimens. Assays using rRNA-based oligonucleotide probes such as fluorescence in situ hybridization (16, 17, 24) or microarrays (1, 22) have been shown to allow rapid species identification in blood cultures. However, methods solely based on rRNA probes allow species identification only and do not provide information on antibiotic susceptibility and other strain-specific characteristics (e.g., virulence genes). For the molecular detection of antibiotic resistance in staphylococci, several multiplex PCR-based assays have been described (23, 34, 36). The major drawback of multiplex PCR is the limited number of genes that can be analyzed in one reaction and that a preidentification to the species level is required.

A promising genotyping method that allows the simultaneous identification of a wide variety of genes is provided by the DNA microarray technology (43). DNA probes specific to selected genes are spotted on a solid substrate (usually glass) in a lattice pattern. Target DNA to be analyzed is then labeled with a reporter molecule (e.g., fluorescent dye) and hybridized to the array, and specific target-probe duplexes are detected by measuring the fluorescent signals associated with each spot. There are two types of DNA microarrays: one is the oligonucleotide-based array and the other is the PCR product-based array (43). DNA microarrays of both formats have been applied successfully either to the detection of genes encoding resistance to ß-lactam (14, 19, 25), erythromicin-macrolide (25, 38), tetracycline (6, 25), and gentamicin-aminoglycoside (25) antibiotics or to the analysis of virulence factors (3, 11, 39, 42).

The aim of the present study was to establish a DNA-chip (microarray) using gene-specific PCR products as capture probes, which allow both the identification of bacterial species and their further characterization in regard to antibiotic resistance and virulence. The practicability and specificity of the DNA microarray for the identification and characterization of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa grown in blood culture specimens was evaluated with clinical isolates and positive blood cultures. We demonstrate here its high degree of specificity, its applicability to blood cultures, and its suitability for detecting resistance genes.


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MATERIALS AND METHODS
 
Reference strains, clinical isolates, and culture conditions. Bacterial reference strains were obtained from the American Type Culture Collection (ATCC; Manassas, Va.), the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Germany), or the Network on Antimicrobial Resistance in Staphylococcus aureus (Herndon, Va.). Clinical isolates were obtained from our routine microbiology laboratory. The following bacteria were used for evaluation of the specificity of the microarray: Staphylococcus aureus (ATCC 29213, NRS123 alias MW2, five clinical isolates), Staphylococcus epidermidis (five clinical isolates), Staphylococcus capitis (clinical isolate), Staphylococcus haemolyticus (clinical isolate), Staphylococcus hominis (clinical isolate), Staphylococcus warneri (clinical isolate), Staphylococcus auricularis (clinical isolate), Micrococcus spp. (clinical isolate), Escherichia coli (ATCC 25922, six clinical isolates), Pseudomonas aeruginosa (ATCC 27853, five clinical isolates), Klebsiella pneumoniae (three clinical isolates), Proteus mirabilis (two clinical isolates), Serratia marcescens (two clinical isolates), Enterobacter cloacae (clinical isolate), Enterobacter aerogenes (clinical isolate), Acinetobacter baumannii (clinical isolate), Stenotrophomonas maltophilia (clinical isolate), Enterococcus spp. (clinical isolate), Enterococcus faecalis (clinical isolate), and Streptococcus pneumoniae (clinical isolate).

Bacterial strains and clinical isolates were grown overnight at 37°C with constant shaking in 5 ml of Luria-Bertani broth or tryptic soy broth (30 g/liter; Merck) containing 3 g of yeast extract/liter. Enterococci and streptococci were grown in 10 ml of tryptic soy broth plus yeast without agitation under 5% CO2. Overnight cultures were harvested after centrifugation at 2,560 x g for 10 min. After the supernatant was discarded, the pellet was washed in 1 ml of TE (10 mM Tris-HCl [pH 7.5], 1 mM EDTA) and recovered by centrifugation at 17,900 x g for 10 min. Cell pellets were used for DNA preparation.

Blood cultures. Positive blood cultures were used for microarray validation as they were encountered in the routine laboratory. Aerobic and anaerobic blood culture bottles (BACTEC; Becton Dickinson, Heidelberg, Germany) were inoculated with blood from patients with suspected septicemia and placed in a BACTEC 9240 blood culture system (Becton Dickinson), a continuous-reading, automated, and computed blood culture system that detects the growth of microorganisms by monitoring CO2 production. Incubation was performed according to the manufacturer's recommendations. Bottles with a positive growth index were removed from the incubator, and aliquots of 1 ml of the blood culture suspensions were taken aseptically with a needle syringe. One 1-ml aliquot of the blood culture suspensions was mixed with 1 ml of 0.1% Triton X-100 and kept at room temperature for 5 min in order to disrupt human blood cells. Bacterial cells were then harvested after centrifugation at 17,900 x g for 10 min, and the pellets were washed in 1 ml of TE, recovered by centrifugation, and used for DNA preparation. A second 1 ml-aliquot was examined by Gram stain and subcultured on agar plates. The organisms grown on agar plates were characterized and tested for susceptibility using a VITEK-2 system (bioMérieux, Inc., Nürtingen, Germany), Etest strips (AB Biodisk, Solna, Sweden) or disk diffusion tests following the method recommended by the Clinical and Laboratory Standards Institute (9). For microarray hybridization experiments, DNA was prepared from 13 blood cultures positive for S. aureus (n = 4), S. epidermidis (n = 3), S. pneumoniae (n = 2), P. aeruginosa (n = 1), E. coli (n = 2), and P. mirabilis (n = 1). The workflow of the microarray assay is outlined in Fig. 1.


Figure 1
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  		FIG. 1. Workflow for hybridization assay used with the prototype microarray. (A) For DNA-chip construction, capture probes were produced by PCR amplification of plasmid-cloned gene segments, followed by ethanol precipitation. Purified probes were deposited onto glass slides by robotic printing. (B) For hybridization assays, bacterial target DNA was extracted from positive blood cultures, clinical isolates, or reference strains and then labeled with fluorescent dyes and hybridized to the spotted DNA capture probes. Images of fluorescent, hybridized probes were acquired by using a laser scanner and processed by computer analysis.

DNA preparation. Total cellular DNA was extracted and purified either by using the First-DNA All-Tissue kit (GEN-IAL GmbH, Troisdorf, Germany) following the instructions of the supplier or by enzymatic lysis followed by phenol-chloroform extraction. For the latter protocol, cell pellets were resuspended in 500 µl of lysis buffer (20 mM Tris-HCl [pH 8.0], 2 mM EDTA [pH 8.0], 1.2% Triton X-100), and lysozyme (Sigma, Taufkirchen, Germany) was added to reach a final concentration of 0.8 mg/ml. In addition, lysostaphin (Sigma) was added to a final concentration of 0.2 mg/ml to promote staphylococcal lysis or mutanolysin (0.5 U/µl; Sigma) was added to lyse streptococci and enterococci. After incubation at 37°C for 1 h, cell lysates were treated with proteinase K (1 mg/ml; Sigma) for 1 h at 55°C and then with RNase A (0.2 mg/ml; QIAGEN, Hilden, Germany) for 1 h at 37°C. The volume was increased by the addition of 200 µl of TE, and the salt concentration was adjusted to 0.7 M by addition of 5 M NaCl. A 10% CTAB (cetyltrimethylammonium bromide) solution in 0.7 M NaCl was added to a final concentration of 1%, followed by incubation at 65°C for 20 min in order to release DNA from polysaccharide DNA complexes. DNA was then extracted once with phenol-chloroform-isoamyl alcohol (25:24:1) and once with chloroform-isoamyl alcohol (24:1) prior to precipitation with 1 volume of isopropanol. After centrifugation at 17,900 x g for 30 min, DNA pellets were washed in 70% ethanol and resuspended in 50 to 100 µl of TE. The concentrations, purities, and sizes of the purified DNA preparations were determined by UV spectrophotometry (Lambda 40; Perkin-Elmer, Boston. MA) and 1% agarose gel electrophoresis.

DNA labeling. Total DNA from clinical isolates and blood cultures was labeled by a nonenzymatic chemical labeling method using the Label-It Cy3/Cy5 kits (Mirus, Madison, WI) or the ULYSIS Alexa Fluor 647 nucleic acid labeling kit (Molecular Probes, Eugene, OR). Prior to labeling, PCR products amplified from three selected recombinant plasmids (1 µl each; 30 ng/µl) were added to each reaction to serve as internal positive controls. For labeling with the Label-It Cy3/Cy5 kit, 5 µg of high-molecular-weight DNA (>12 kb) was mixed with 7.5 µl of reagent in a total volume of 50 µl, followed by incubation for 2 h at 37°C according to the recommendations by the supplier. After the volume was adjusted to 200 µl with H2O and 0.1 volumes of 5 M NaCl were added, unbound label was removed by precipitation with 2 volumes of ice-cold absolute ethanol for at least 30 min at –20°C. The labeled DNA was recovered by centrifugation at 17,900 x g for 30 min. The pellet was washed with 70% ethanol and resuspended in 70 µl of TE. For labeling with the Ulysis Alexa Fluor 647 kit, 1 µg of DNA was denatured at 95°C for 5 min, cooled on ice, and mixed with 20 µl of labeling buffer and 5 µl of reagent, followed by incubation at 80°C for 15 min according to the instructions of the manufacturer. Unbound dye was removed by ethanol precipitation as described above. The relative labeling efficiency of a reaction was evaluated by calculating the approximate ratio of bases to dye molecules (acceptable labeling ratios for nucleic acid were ≤60). This ratio and the amount of recovered labeled DNA was determined by measuring the absorbance of the nucleic acids at 260 nm, and the absorbance of the dye at its absorbance maximum using a Lambda 40 UV spectrophotometer (Perkin-Elmer) and plastic disposable cuvettes for the range from 220 to 1,600 nm (UVette; Eppendorf, Hamburg, Germany).

Microarray construction. We used cloned PCR products to generate probes for the DNA microarray. Altogether, 120 gene segments representing virulence genes, antibiotic-resistant determinants, and species-specific metabolic and structural genes from S. aureus (40), E. coli (31), and P. aeruginosa (49) were represented on the microarray (see Table S1 in the supplemental material).

S. aureus, E. coli, and P. aeruginosa genes were selected from the literature and databases and compared by BLAST analysis to all other sequences available in the NCBI database. Primers were designed to amplify gene segments 200 to 800 bp in length devoid of apparent homology with genes of other bacterial species and Homo sapiens. Gene segments were amplified by using puReTaq Ready-To-Go PCR beads (Amersham Biosciences, Freiburg, Germany) and cloned into the pDrive cloning vector (QIAGEN) according to the recommendations of the suppliers and transformed into competent Escherichia coli (XL1-Blue) cells using the calcium chloride protocol (31).

For quality control purposes, all gene probes were partially sequenced and verified (with the BigDye kit 1.1 and a 377 DNA sequencer; Applied Biosystems, Foster City, CA). All sequences obtained were identical or nearly identical to those obtained from the database. For DNA probe production, 120 recombinant plasmids containing S. aureus, E. coli, and P. aeruginosa gene segments were used for reamplification. Amplicons were purified and spotted in four replicates per slide (Memorec, Cologne, Germany) (Fig. 1). Prior to spotting, the DNA concentrations were normalized to ensure the deposition of equal DNA amounts. To verify probe deposition and spot morphology, for each batch a randomly selected microarray slide was stained by using SYBR Green DNA dye (Molecular Probes).

Hybridization and scanning. All experiments described in the present study represent dual cohybridizations of two different target DNA samples labeled, respectively, with Cy3, Cy5, or Alexa 647 (Fig. 1). After removal of unbound label, Cy3- and Cy5/Alexa 647-labeled DNAs were pooled and mixed with 10 µg of salmon sperm DNA and 50 µg of poly(A) DNA. The mixture was frozen in liquid nitrogen and lyophilized in the dark. Prior to hybridization the target DNA was reconstituted in 33 µl of H2O and 55 µl of 2x hybridization solution (Memorec, Cologne, Germany), chemically denatured with 11 µl of denaturation buffer D1 (Mirus), and neutralized with 11 µl of buffer N1 (Mirus) according to the instructions of the supplier. Hybridization was automatically performed with a TECAN hybridization station (HS400; TECAN, Salzburg, Austria). The arrays were prewashed at 60°C for 1 min with 0.2% sodium dodecyl sulfate and 4x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and prehybridized in 120 µl of denatured prehybridization buffer (Memorec) for 30 min at 60°C with mild agitation. After injection of 110 µl of labeled DNA, hybridization was performed at 60°C for 18 h with mild agitation. The arrays were washed at 50°C in a primary wash buffer (Memorec) for five cycles of 1-min wash time and 30-s soak time and in a secondary wash buffer (Memorec) for five cycles of 20-s wash time and 30-s soak time, and finally dried at 30°C with N2 (270 kPa) for 3 min. Hybridized arrays were scanned with a Scan Array 5000 laser scanner (Perkin-Elmer). Laser light of wavelengths at 532 and 635 nm were used to excite Cy3 dye and Cy5/Alexa 647 dye, respectively. Fluorescent images were analyzed by using ImaGene software (BioDiscovery, El Segundo, CA). Spots were found and segmented in order to select areas of recognizable signals for analysis. The fluorescence intensity of each spot was measured, signal-to-local-background ratios were calculated by ImaGene, and spot morphology and deviation from the expected spot position were considered using the default ImaGene settings. The data were imported into Microsoft Access and automatically processed. Spots with a signal-to-noise ratio of 1.2 and with at least 600 relative fluorescence units over the local background in all three replicas were considered positive. Cutoff values for these parameters were empirically determined in pilot experiments and used to tag spots either as positive or negative.


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RESULTS
 
Specificity. In order to allow the simultaneous and rapid identification of S. aureus, E. coli, and P. aeruginosa grown in blood culture specimens from septicemic patients, a microarray comprising a set of 40 S. aureus, 31 E. coli, and 49 P. aeruginosa gene probes 200 to 800 bp in length was developed (Table S1 in the supplemental material).

The specificity of the DNA-chip was validated first with 45 well-characterized clinical isolates and reference strains of the three target species, as well as other related bacteria and, second, with 13 blood cultures from patients with sepsis by following the workflow outlined in Fig. 1. Positive blood cultures were processed as they were encountered in the routine laboratory. Hybridization results were compared to conventional identification results obtained by routine diagnostics.

In all assays, three PCR-amplified DNA segments, which had been added to each DNA preparation as a positive internal control, hybridized with the corresponding probes, indicating that the labeling and hybridization had performed efficiently.

Hybridization experiments with S. aureus, E. coli, and P. aeruginosa target DNAs revealed specific hybridization with the species-specific gene probes (Fig. 2). There was no cross-hybridization between the three species, with the exception of the S. aureus 16S rRNA gene probe (16SSa, Fig. 2C), which also hybridized with E. coli and P. aeruginosa target DNA.


Figure 2
Figure 2
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  		FIG.2. DNA microarray analyses of 42 clinical isolates, 3 reference strains, and 13 blood cultures. Each column shows the results of an individual hybridization with target DNA prepared from: S. aureus ATCC 29213 (column 1), MW2 (column 2), clinical isolates (columns 3 to 7), and positive blood cultures (columns 8 to 11); P. aeruginosa ATCC 27853 (column 12), clinical isolates (columns 13 to 17), and positive blood cultures (column 18); E. coli ATCC 25922 (column 19), clinical isolates (columns 20 to 25), and positive blood cultures (columns 26 and 27); S. epidermidis clinical isolates (columns 28 to 32) and blood cultures (columns 33 to 35); and clinical isolates of CoNS S. auricularis (column 36), S. capitis (column 37), S. haemolyticus (column 38), S. hominis (column 39), and S. warneri (column 40). Other gram-negative species included a Proteus mirabilis positive blood culture (column 41), clinical isolates of Proteus mirabilis (columns 42 and 43), Serratia marcescens (columns 44 and 45), Klebsiella pneumonia (columns 46 to 48), Stenotrophomonas maltophilia (column 49), Acinetobacter baumannii (column 50), Enterobacter cloacae (column 51), and Enterobacter aerogenes (column 52). Other gram-positive species included clinical isolates of Micrococcus spp. (column 53), Enterococcus spp. (column 54), Enterococcus faecalis (column 55), and Streptococcus pneumoniae (column 56) and two positive blood cultures of S. pneumoniae (columns 57 and 58). (A) Hybridization of DNA prepared from bacterial isolates, reference strains, and blood cultures with E. coli gene probes; (B) hybridization with P. aeruginosa gene probes; (C) hybridization with S. aureus gene probes. Gray boxes represent gene probes that hybridized with the respective target DNA; white boxes represent gene probes that showed no hybridization with the respective target DNA. Experiments performed with positive blood cultures are indicated (BC).

Identification of E. coli, P. aeruginosa, and S. aureus reference strains, clinical isolates, and blood cultures by microarray analysis corresponded by 100% with the conventional identification results (Fig. 2).

Detection and discrimination of E. coli. All DNA samples from nine E. coli strains hybridized always with seven E. coli gene probes [envZ, fes(1) and fes(2), nfrB, yacH, yagX, and ycdS] (Fig. 2A, columns 19 to 27); in the following discussion we will refer to these genes as core genes. With 14 E. coli gene probes, variable hybridization was observed, including the antibiotic resistance gene probes blaTEM-106, sul, strB, and aacC2. Such a variable hybridization profile is expected for antibiotic resistance genes since acquired resistance to antimicrobials is isolate specific. For 11 E. coli virulence gene probes (eae, eltB, escR, escT, escU, espB, hlyA, hlyB, SLTII, toxA-LTPA, and VT2vaB) no hybridization signals were detected with any of the tested E. coli isolates and blood cultures. Since these virulence genes are known to be specific for particular E. coli pathotypes (3), it was not surprising that they were not present in the tested strains. The eae, esc, and esp genes, for example, are encoded on a chromosomal pathogenicity island, which is typical for enteropathogenic E. coli exhibiting the unique virulence mechanism known as attaching and effacing (13). The alpha-hemolysin (hly) operon is encoded on a large plasmid of enterohemorrhagic E. coli strains (32).

Detection and discrimination of P. aeruginosa. DNA samples obtained from P. aeruginosa uniformly hybridized with 32 of 49 P. aeruginosa specific gene segments, including the mexA gene probe (core genes). Variable hybridization was observed with 17 probes, allowing for the discrimination of individual P. aeruginosa isolates (Fig. 2B, columns 12 to 18).

Detection and discrimination of S. aureus. Hybridization experiments performed with 11 S. aureus target DNAs revealed signals in all assays with 16 S. aureus gene segments (core genes) (Fig. 2C, columns 1 to 11). Variable hybridization was observed with 14 S. aureus gene probes including the six antibiotic resistance gene segments aadD, aacA-aphD, blaZ, dfrA, ermA, and mecA and the virulence genes sak, sea, sec1, and EDIN. The gene probes geh, mreA, clfB, and elkT-abcA hybridized with 8 (geh), 10 (mreA and clfB), and 6 (elkT-abcA) target DNAs. However, PCR amplification of the four genes was positive for all 11 S. aureus target DNAs (results not shown), suggesting that the four genes were present in all of the strains investigated and that these gene probes did not allow reliable detection of the four genes in S. aureus.

No hybridization was observed with 10 probes, including the toxin genes seb, tst, and etb. In contrast to the community-acquired, multidrug-susceptible methicillin-resistant S. aureus (MRSA) strain MW2 that hybridized to mecA and blaZ only, all six clinical MRSA strains showed the same multiresistant hybridization pattern, and their DNA hybridized to the ermA (erythromycin resistance), mecA (oxacillin resistance), and aadD (tobramycin resistance) genes. As for the majority of multiresistant MRSA strains, the ermA and aadD genes were shown to be located upstream and downstream, respectively, of the mecA gene in the mec chromosomal region (7, 26). Hybridization to the core gene probes permitted the identification of S. aureus, while hybridization to antibiotic resistance gene probes allowed for the discrimination of strains.

Discrimination of E. coli, P. aeruginosa, and S. aureus from related bacterial species. Cohybridization experiments performed with related bacterial species confirmed the high specificity of the DNA-chip (Fig. 2). For S. epidermidis and all other CoNS, cross-hybridization was observed only with the S. aureus 16S rRNA gene probe (16SSa, Fig. 2C) and several common staphylococcal antibiotic resistance determinants (aadD, aacA-aphD, aph-A3, blaZ, cat, dfrA, ermA, ermC, mdrSA, and mecA) (Fig. 2C, columns 28 to 36). There was no cross-hybridization with other metabolic or virulence genes of S. aureus.

The Micrococcus spp. isolate showed no hybridization with the DNA-chip (column 53). Streptococci (columns 56 to 58) and enterococci (columns 54 and 55) showed hybridization with the staphylococcal 16S RNA gene probe and once with the staphylococcal aph-A3 aminoglycoside resistance gene probe (Enterococcus spp.) (Fig. 2C). Of twelve strains of seven gram-negative species (columns 41 to 52), two hybridized with the S. aureus 16S rRNA gene probe (Klebsiella pneumoniae and Proteus mirabilis, Fig. 2C, columns 41 and 47), and one clinical isolate of Proteus mirabilis hybridized with the E. coli resistance genes blaTEM-106 (ß-lactam resistance), sul (sulfonamide resistance), and strB (streptomycin resistance) (Fig. 2A, column 42). Serratia, Stenotrophomonas, Acinetobacter, and Enterobacter species showed no cross-hybridization with any gene probe.

Sensitivity. Although the majority of P. aeruginosa probes allowed unambiguous identification, some probes showed variable hybridization patterns when microarray hybridization was performed with different target DNA samples prepared from the same isolate (Table 1) . Successful hybridization with strong fluorescent signals depends on efficiency of DNA labeling (ratio of bases per one dye molecule) and amount of labeled DNA. For the different target DNA preparations of four clinical isolates, variable hybridization was observed with 14 gene probes (uvrDII, vsmI, pa1069, rhlR, rhlA, rhlB, 1046, pyocinS, pyocinS1im, plcR, plcN, PHZb, rbf303, and pIIAp2). For example, for three different DNA preparations of isolate C4242, hybridization to Pseudomonas gene probes varied from 31 to 43 probes, respectively, depending on the labeling efficiency and amount of DNA (Table 1). The lowest number of signals was detected with 382 ng of target DNA, which, however, showed a high base-to-dye ratio (BDR) of 75. Overall, our results suggest that various amounts of DNA and BDRs influenced the hybridization results of few gene probes. However, irrespective of the varying quality and quantity of the labeled target DNA, 35 of the 49 P. aeruginosa gene probes showed robust hybridization results in all performed experiments.


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  		TABLE 1. Microarray hybridization signals obtained with different target DNA preparations of P. aeruginosa isolates

Detection and characterization of pathogens in blood cultures. Although DNA prepared from blood cultures comprises a mixture of human and bacterial DNA, the resulting hybridization signals obtained with DNA from 1 ml of positive blood culture allowed a clear and unambiguous characterization of the S. aureus, E. coli, and P. aeruginosa present in 7 of 13 tested blood specimens (Fig. 2). In accordance with the VITEK2 characterization, positive BACTEC cultures were identified by microarray hybridization as multiresistant MRSA (Fig. 2C, column 8), penicillin-resistant S. aureus (columns 9 and 11), and multisusceptible S. aureus (column 10), E. coli (Fig. 2A, columns 26 and 27), and P. aeruginosa (Fig. 2B, column 18) and discriminated from oxacillin-resistant Staphylococcus epidermidis (columns 33 to 35), Proteus mirabilis (column 43), and Streptococcus pneumoniae (columns 57 and 58).

Correlation between susceptibility testing and microarray hybridization of selected antibiotic resistance genes. (i) S. aureus. For 11 Staphylococcus aureus strains and blood cultures, we compared susceptibility results determined by the VITEK2 system, Etest strips, and disk diffusion tests with the results of the microarray hybridization assay for the simultaneous detection of antibiotic resistance genes (Table 2) . The presence or absence of resistance genes as indicated by microarray hybridization was confirmed by PCR with gene-specific primers (results not shown).


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  		TABLE 2. Correlation between phenotypic and genotypic antibiotic resistance for 11 S. aureus isolates and blood cultures

For the S. aureus strains there was a 100% correlation between phenotypic resistance to penicillin and hybridization to the mecA and/or blaZ gene (both genes confer resistance to penicillin) (see Table 2). Phenotypic resistance to oxacillin correlated 100% with the hybridization of the mecA gene between resistance to erythromycin and hybridization to the erythromycin resistance genes ermA, ermC, or msrSA and between resistance to tobramycin and hybridization to the aadD gene. Furthermore, they all showed 100% correlation between phenotypic susceptibility to gentamicin and no hybridization to the resistance genes aacA-aphD. Notably, the dfrA gene of the trimethoprim-resistant strain MW2 (MIC of 1 µg/ml) was not detected by microarray hybridization, whereas PCR amplification revealed the presence of the dfrA gene.

(ii) E. coli and other gram-negative bacteria. The prototype microarray harbored only four E. coli and one P. aeruginosa resistance gene probes which do not yet allow a comprehensive prediction of antibiotic resistance. Nevertheless, hybridization with the E. coli resistance gene probe blaTEM-106 was observed in one P. mirabilis and four E. coli strains and correlated with phenotypic ampicillin resistance for all five strains (Table 3).


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  		TABLE 3. Correlation between ampicillin-penicillin resistance, gentamicin-tobramycin resistance, and streptomycin resistance and hybridization with the resistance gene probes blaTEM-106, aacC2, aph-A3, and strB

One E. coli blood culture showed also resistance to tobramycin and gentamicin. This phenotypic resistance correlated with the hybridization of the aacC2 gene probe for aminoglycoside resistance and the S. aureus aph-A3 probe for tobramycin-kanamycin resistance (Table 3). For one P. mirabilis and four E. coli strains, phenotypic resistance to streptomycin correlated with hybridization to the strB probe (Table 3).

All P. aeruginosa strains hybridized with the mexA gene probe (Fig. 2) and showed phenotypic resistance to tetracycline, trimethoprim-sulfamehoxazole, penicillin (ampicillin and mezlocillin), and cephalosporin (cefazolin, cefixime, and cefuroxime). The mexA-mexB-oprM operon is a determinant for a three-component efflux system responsible for intrinsic and acquired multiresistance in P. aeruginosa (ß-lactams, fluoroquinolones, trimethoprim, sulfonamides, chloramphenicol, and others) (27).


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DISCUSSION
 
We have established a novel, highly specific DNA microarray for the identification and characterization of S. aureus, E. coli, and P. aeruginosa in blood cultures. Our DNA-chip allowed simultaneous species identification and detection of important virulence and antibiotic resistance genes in a single assay. It was successful in identifying all 27 E. coli, P. aeruginosa, and S. aureus strains and in discriminating them from 21 closely related gram-positive and gram-negative bacterial strains which are also known to be causative agents of bacteremia (30). Our prototype microarray demonstrates the feasibility of identifying and characterizing pathogens in 1 ml of positive blood culture without prior amplification of the target DNA.

The microarray consisted of 120 gene segments 200 to 800 bp in length amplified from recombinant plasmids. One important feature of this format is that the panel of probes can be continually extended to include sequences for additional species, variant isolates, or antibiotic resistance determinants as they are characterized and available. The accuracy, range, and discriminatory power of the gene-segment-based microarray can be refined by adding or removing gene probes to the panel without significantly increasing the complexity or costs. In this pilot study, three important species causing bacteremia were selected to provide a proof of principle. The range of organisms that can be identified is easily expanded by increasing the number of gene probes in the array. For example, the addition of a few probes specific for S. epidermidis and other CoNS will allow for the species identification of CoNS. Furthermore, due to a specific hybridization pattern for each species, it will also allow the identification of mixed blood cultures with more than one pathogen.

A second important feature of this microarray format based on PCR products of extended length (100 to 3,000 bp) is that one probe per gene is usually sufficient to produce strong signals and high specificity (35). For long probes, minor point mutations are likely to only slightly reduce duplex formation, which does not lead to the loss of hybridization signals. In contrast, short oligonucleotide microarrays sometimes lack specificity and require multiple short oligonucleotides per one gene. Volokhov et al. (38) constructed a microarray for the analysis of erythromycin determinants; to increase confidence in the microarray analysis, each analyzed gene was represented by seven oligoprobes. However, for routine diagnostics this leads to an undesired increase in complexity.

A limitation of the long probes as used here is the reduced sensitivity to single or minor point mutations, such as those responsible for resistance conferred by extended-spectrum ß-lactamases (ESBLs, e.g., TEM-, SHV-, and OXA-type ESBLs) (5). An extension of the probe panel for example, by OXA, SHV, or CMY resistance genes, will allow the detection of the different ß-lactamase types (19) but not discrimination within one type.

As shown for P. aeruginosa probes, the intensity of the hybridization signal largely relies on quality and quantity of the labeled target DNA. Under suboptimal conditions not all species-specific probes produced strong signals after hybridization with specific DNA. PCR analyses of some S. aureus genes revealed that the genes clfB, elkT-abcA, geh, and mreA were present in all tested S. aureus strains, whereas not all strains produced hybridization signals with the corresponding probes. Besides the quality and quantity of the target DNA, two circumstances could explain the discrepancy. First, extensive sequence variations between a specific probe and the corresponding gene present in a certain strain may produce false negatives (i.e., the gene is present but there is no hybridization). Second, it is impossible to predict the exact hybridization behavior of immobilized DNA probes. Therefore, it is important to judge the collection of probes experimentally using the prototype chip and to select only probes that produce robust results.

The use of a single protocol for all bacterial species, comprising all steps of DNA preparation and DNA-chip hybridization, is essential for testing blood cultures where the bacterial diagnosis is usually uncertain. In regard to the processing time we decided to use a chemical labeling procedure using high-molecular-weight DNA (>12 kb). DNA fragmentation by sonication or enzymatic cleavage (restriction enzymes or DNase A) prior to the labeling reaction is difficult to control and increases the processing time and the chance of losing DNA by an additional required precipitation step. However, large target DNA molecules may hybridize poorly to immobilized probes due to spatial and steric constraints. Vora et al. (40) reported that the sensitivity is significantly increased by using fragmented DNA (40). A DNA preparation protocol using sonication for simultaneous cell disruption and target DNA fragmentation may be the method of choice to increase the sensitivity of the microarray, in particular toward low-copy-number and/or plasmid-encoded genes which may be underrepresented in the target DNA.

Since the focus of the present study was to provide a proof of principle and to test the informative value of the selected capture probes, the protocols were not yet optimized for time. However, trials with commercial DNA kits showed that DNA extraction can be performed within 1 h. Hybridizations were performed according to standard protocols overnight. Preliminary experiments showed that this time may be shortened to 4 h, allowing the results to be obtained within 8 h after a blood culture becomes positive.

Previous studies have shown that the detection of antibiotic resistance genes by molecular techniques has good predictive power for the phenotypic resistance of clinical S. aureus isolates (23, 36, 38). With our gene-segment-based microarray there was an excellent correlation between genotypic detection of antibiotic resistance determinants and phenotypic detection using conventional susceptibility testing. The detection of the resistance genes mecA, blaZ, ermA, ermC, msrSA, aadD, and aacA-aphD by microarray hybridization allowed for the reliable prediction of oxacillin, penicillin, erythromycin, tobramycin, and gentamicin resistance in a single assay.

By microarray hybridization it was possible to discriminate multidrug-resistant MRSA, resistant to methicillin-penicillin and other antibiotics, and MRSA resistant to methicillin-penicillin only, which is frequently encountered as community-acquired MRSA. Simultaneous comprehensive resistance genotyping for oxacillin, macrolide, and aminoglycoside resistance genes (e.g., mecA, aadD, aacA-aphD, ermA, ermB, ermC, and msrSA) by microarray hybridization allows the rapid identification of multiresistant MRSA or macrolide- or aminoglycoside-susceptible MRSA and, in consequence, permits other therapeutic options and may reduce reliance on vancomycin (26, 28).

Recently, a 23S rRNA gene oligonucleotide microarray (1) and a macroarray (96-well format) of DNA probes directed against rRNA (22) were shown to provide good discrimination of fungi and gram-positive and gram-negative bacteria causing bacteremia. However, since these approaches are solely based on rRNA they provide no information on antibiotic resistance determinants or virulence genes of the identified strains. Another molecular approach applying the commercial Hyplex BloodScreen multiplex PCR-enzyme-linked immunosorbent assay system to positive blood cultures was shown to be well suited for the direct and specific identification of the most common pathogenic bacteria and the direct detection of the mecA gene of S. aureus (41). However, due to the microtiter plate format of this assay, the number of gene probes is limited and the mecA probe was the only antibiotic-resistant determinant.

On the other hand, there are numerous studies on the detection of virulence genes and/or antibiotic resistance genes based on multiplex PCR (15, 21, 23, 33, 36, 37) or assays combining multiplex PCR and microarray detection systems (8, 38, 39). However, all assays using multiplex PCR have been limited by the number of genes that can be amplified in one reaction. By combining multiplex PCR and microarrays, the microarray detection system is superior to gel electrophoretic analysis, but in this way the great potential of microarrays allowing the analysis of hundreds of genes in parallel is abandoned. Most of these systems can therefore be applied to preidentified pathogens only.

In contrast, the microarray presented here opens the possibility of identifying pathogens in blood cultures with concomitant further characterization in terms of antibiotic resistance and virulence without preidentification. After extension and further automation, the DNA-chip has the potential to provide a clinical tool for microbiological diagnostics, as well as for epidemiological studies. It would allow clinicians to administer appropriate antibiotic chemotherapy in a timely fashion, improve the outcome of septic patients, and reduce the spread of antibiotic resistance genes.


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ACKNOWLEDGMENTS
 
We thank the clinical laboratory staff for providing clinical isolates and blood cultures and Ludwig Eichinger for the use of the scanning facilities.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute for Medical Microbiology, Immunology and Hygiene, Medical Center, University of Cologne, Goldenfelsstr. 19-21, 50935 Cologne, Germany. Phone: 49-221-47832103. Fax: 49-221-47832134. E-mail: oleg.krut{at}uni-loeln.de. Back

{ddagger} Supplemental material for this article may be found at http://jcm.asm.org/. Back

{dagger} B.E.E.C. and M.P.-S. contributed equally to this study. Back


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Journal of Clinical Microbiology, July 2006, p. 2389-2397, Vol. 44, No. 7
0095-1137/06/$08.00+0     doi:10.1128/JCM.02291-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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