Staph Infection Cholera E. Coli
J Clin Microbiol. 2006 Jul; 44(7): 2389–2397.
Identification and Characterization of Bacterial Pathogens Causing Bloodstream Infections by Dna Microarray‡
Berit E. E. Cleven
Institute for Medical Microbiology, Immunology, and Hygiene,1 Eye of Molecular Medicine Cologne, Medical Eye, University of Cologne, Goldenfelsstr. 19-21, 50935 Cologne, Federal republic of germany2
Maria Palka-Santini
Institute for Medical Microbiology, Immunology, and Hygiene,one Eye of Molecular Medicine Cologne, Medical Center, Academy of Cologne, Goldenfelsstr. xix-21, 50935 Cologne, Germany2
Jörg Gielen
Institute for Medical Microbiology, Immunology, and Hygiene,1 Center of Molecular Medicine Cologne, Medical Eye, University of Cologne, Goldenfelsstr. xix-21, 50935 Cologne, Germany2
Salima Meembor
Plant for Medical Microbiology, Immunology, and Hygiene,1 Center of Molecular Medicine Cologne, Medical Centre, University of Cologne, Goldenfelsstr. 19-21, 50935 Cologne, Federal republic of germanytwo
Martin Krönke
Institute for Medical Microbiology, Immunology, and Hygiene,1 Center of Molecular Medicine Cologne, Medical Middle, University of Cologne, Goldenfelsstr. 19-21, 50935 Cologne, Germany2
Oleg Krut
Institute for Medical Microbiology, Immunology, and Hygiene,one Centre of Molecular Medicine Cologne, Medical Center, Academy of Cologne, Goldenfelsstr. 19-21, 50935 Cologne, Germany2
Received 2005 November two; Revised 2006 Jan 11; Accepted 2006 May 1.
- Supplementary Materials
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[Supplemental material]
GUID: 78929474-CED8-4958-BC01-78A6ADCDF871
GUID: DCDFA963-9AF4-4790-BAA9-7D7283ED8FF1
Abstract
Bloodstream infections are potentially life-threatening and require rapid identification and antibiotic susceptibility testing of the causative pathogen in club to facilitate specific antimicrobial therapy. We developed a image Dna microarray for the identification and characterization of three of import bacteremia-causing species: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. The assortment consisted of 120 species-specific factor 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 almost perfect correlation betwixt phenotypic antibiotic resistance determined by conventional susceptibility testing and genotypic antibody 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 bla TEM-106 (penicillin resistance) and aacC2 (aminoglycoside resistance). Furthermore, antibiotic resistance and virulence cistron probes permitted genotypic discrimination within a species. This novel Deoxyribonucleic acid microarray demonstrates the feasibility of simultaneously identifying and characterizing bacteria in blood cultures without prior amplification of target DNA or preidentification of the pathogen.
The presence of living microorganisms in the claret of a patient is commonly indicative of a serious invasive infection requiring urgent antimicrobial therapy (xxx). The mortality associated with bloodstream infections may range from xx 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 virtually frequent etiological agents of bacteremia and fungemia in Europe (x, 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 design, is crucial for several reasons: (i) appropriate antimicrobial agents can exist selected, and thus, unnecessary treatment with ineffective antibiotics can exist avoided; (2) the prognosis of the patients can be improved; (iii) the conquering of resistance in pathogens may be decelerated; and (four) expenditure on antimicrobials and overall hospital costs tin can be reduced (2, 12).
Routine microbiological detection of bacteremia relies on enrichment of the causative pathogen using automated continuous-monitoring blood civilisation systems followed by Gram stain, subculture on agar, and subsequent biochemical identification and susceptibility testing. When the blood civilization is noted to be positive, definitive identification and antibody 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 data (e.g., virulence factors) require additional time-consuming and expensive phenotypic and genotypic tests and are not performed routinely (18).
In contempo years, numerous studies have demonstrated the value of molecular techniques in order to place and genotype leaner or fungi in blood specimens. Assays using rRNA-based oligonucleotide probes such every bit 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 let species identification just and do not provide data on antibiotic susceptibility and other strain-specific characteristics (e.1000., 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 express number of genes that tin exist 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 (unremarkably glass) in a lattice pattern. Target Deoxyribonucleic acid to exist analyzed is then labeled with a reporter molecule (eastward.g., fluorescent dye) and hybridized to the array, and specific target-probe duplexes are detected by measuring the fluorescent signals associated with each spot. In that location 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 take been applied successfully either to the detection of genes encoding resistance to β-lactam (xiv, 19, 25), erythromicin-macrolide (25, 38), tetracycline (half-dozen, 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 found a DNA-chip (microarray) using gene-specific PCR products as capture probes, which allow both the identification of bacterial species and their further label 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 civilization specimens was evaluated with clinical isolates and positive blood cultures. Nosotros demonstrate hither its loftier degree of specificity, its applicability to claret cultures, and its suitability for detecting resistance genes.
MATERIALS AND METHODS
Reference strains, clinical isolates, and culture weather.
Bacterial reference strains were obtained from the American Type Civilisation Collection (ATCC; Manassas, Va.), the Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig, Deutschland), 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 (v 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, half dozen clinical isolates), Pseudomonas aeruginosa (ATCC 27853, five clinical isolates), Klebsiella pneumoniae (three clinical isolates), Proteus mirabilisouthward (2 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 five ml of Luria-Bertani goop or tryptic soy broth (xxx g/liter; Merck) containing iii grand of yeast extract/liter. Enterococci and streptococci were grown in 10 ml of tryptic soy goop plus yeast without agitation under 5% COii. Overnight cultures were harvested afterward centrifugation at 2,560 × one thousand for 10 min. Later the supernatant was discarded, the pellet was washed in 1 ml of TE (x mM Tris-HCl [pH 7.five], 1 mM EDTA) and recovered by centrifugation at 17,900 × g for ten min. Cell pellets were used for Deoxyribonucleic acid preparation.
Blood cultures.
Positive blood cultures were used for microarray validation as they were encountered in the routine laboratory. Aerobic and anaerobic claret culture bottles (BACTEC; Becton Dickinson, Heidelberg, Germany) were inoculated with claret from patients with suspected septicemia and placed in a BACTEC 9240 blood civilisation system (Becton Dickinson), a continuous-reading, automatic, and computed blood culture system that detects the growth of microorganisms by monitoring CO2 product. Incubation was performed co-ordinate 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.ane% Triton Ten-100 and kept at room temperature for 5 min in order to disrupt human blood cells. Bacterial cells were and then harvested after centrifugation at 17,900 × g for x min, and the pellets were washed in 1 ml of TE, recovered past 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-ii organization (bioMérieux, Inc., Nürtingen, Federal republic of germany), Etest strips (AB Biodisk, Solna, Sweden) or deejay improvidence tests following the method recommended by the Clinical and Laboratory Standards Plant (9). For microarray hybridization experiments, DNA was prepared from 13 claret cultures positive for South. aureus (n = 4), Due south. epidermidis (northward = three), Due south. pneumoniae (n = 2), P. aeruginosa (n = i), E. coli (n = 2), and P. mirabilis (northward = 1). The workflow of the microarray assay is outlined in 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 atmospheric precipitation. Purified probes were deposited onto glass slides past robotic printing. (B) For hybridization assays, bacterial target Deoxyribonucleic acid 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 light amplification by stimulated emission of radiation scanner and processed past figurer assay.
DNA preparation.
Full cellular DNA was extracted and purified either by using the First-DNA All-Tissue kit (GEN-IAL GmbH, Troisdorf, Frg) post-obit the instructions of the supplier or by enzymatic lysis followed by phenol-chloroform extraction. For the latter protocol, prison cell pellets were resuspended in 500 μl of lysis buffer (20 mM Tris-HCl [pH 8.0], two mM EDTA [pH 8.0], 1.ii% Triton X-100), and lysozyme (Sigma, Taufkirchen, Federal republic of germany) was added to attain a last 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. Later incubation at 37°C for one h, cell lysates were treated with proteinase One thousand (1 mg/ml; Sigma) for 1 h at 55°C and so with RNase A (0.2 mg/ml; QIAGEN, Hilden, Germany) for ane h at 37°C. The book was increased by the addition of 200 μl of TE, and the common salt concentration was adjusted to 0.7 1000 past improver of 5 Grand NaCl. A ten% CTAB (cetyltrimethylammonium bromide) solution in 0.7 K NaCl was added to a concluding concentration of i%, followed by incubation at 65°C for 20 min in gild to release Deoxyribonucleic acid from polysaccharide Deoxyribonucleic acid complexes. Deoxyribonucleic acid was and then extracted in one case with phenol-chloroform-isoamyl booze (25:24:i) and in one case with chloroform-isoamyl alcohol (24:one) prior to precipitation with 1 book of isopropanol. Subsequently centrifugation at 17,900 × g for 30 min, Deoxyribonucleic acid pellets were washed in 70% ethanol and resuspended in l to 100 μl of TE. The concentrations, purities, and sizes of the purified Deoxyribonucleic acid preparations were determined past UV spectrophotometry (Lambda 40; Perkin-Elmer, Boston. MA) and ane% agarose gel electrophoresis.
Deoxyribonucleic acid labeling.
Total DNA from clinical isolates and blood cultures was labeled by a nonenzymatic chemic 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 equally 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 two h at 37°C co-ordinate to the recommendations by the supplier. Afterwards the book was adjusted to 200 μl with HiiO and 0.i volumes of v M NaCl were added, unbound label was removed by atmospheric precipitation with two volumes of ice-cold absolute ethanol for at least 30 min at −twenty°C. The labeled DNA was recovered by centrifugation at 17,900 × chiliad for 30 min. The pellet was washed with lxx% ethanol and resuspended in 70 μl of TE. For labeling with the Ulysis Alexa Fluor 647 kit, one μg of DNA was denatured at 95°C for 5 min, cooled on ice, and mixed with xx μl of labeling buffer and five μl of reagent, followed by incubation at 80°C for fifteen min according to the instructions of the manufacturer. Unbound dye was removed past ethanol precipitation every bit described above. The relative labeling efficiency of a reaction was evaluated by calculating the gauge ratio of bases to dye molecules (acceptable labeling ratios for nucleic acrid were ≤60). This ratio and the amount of recovered labeled DNA was adamant by measuring the absorbance of the nucleic acids at 260 nm, and the absorbance of the dye at its absorbance maximum using a Lambda twoscore UV spectrophotometer (Perkin-Elmer) and plastic disposable cuvettes for the range from 220 to 1,600 nm (UVette; Eppendorf, Hamburg, Germany).
Microarray structure.
Nosotros used cloned PCR products to generate probes for the DNA microarray. Birthday, 120 factor segments representing virulence genes, antibiotic-resistant determinants, and species-specific metabolic and structural genes from S. aureus (twoscore), Eastward. coli (31), and P. aeruginosa (49) were represented on the microarray (see Table S1 in the supplemental cloth).
S. aureus, E. coli, and P. aeruginosa genes were selected from the literature and databases and compared past Boom assay to all other sequences bachelor in the NCBI database. Primers were designed to amplify gene segments 200 to 800 bp in length devoid of credible homology with genes of other bacterial species and Homo sapiens. Cistron 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 command purposes, all gene probes were partially sequenced and verified (with the BigDye kit i.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 Deoxyribonucleic acid probe production, 120 recombinant plasmids containing S. aureus, Due east. coli, and P. aeruginosa gene segments were used for reamplification. Amplicons were purified and spotted in iv 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 degradation and spot morphology, for each batch a randomly selected microarray slide was stained past using SYBR Green Deoxyribonucleic acid dye (Molecular Probes).
Hybridization and scanning.
All experiments described in the present study stand for dual cohybridizations of 2 different target DNA samples labeled, respectively, with Cy3, Cy5, or Alexa 647 (Fig. 1). Later removal of unbound label, Cy3- and Cy5/Alexa 647-labeled DNAs were pooled and mixed with ten μg of salmon sperm Deoxyribonucleic acid and l μg of poly(A) DNA. The mixture was frozen in liquid nitrogen and lyophilized in the night. Prior to hybridization the target Dna was reconstituted in 33 μl of HiiO and 55 μl of 2× 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 four× SSC (1× SSC is 0.fifteen One thousand 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. Later 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 main wash buffer (Memorec) for 5 cycles of 1-min launder time and thirty-south soak time and in a secondary wash buffer (Memorec) for five cycles of 20-due south wash fourth dimension and xxx-southward soak time, and finally stale at 30°C with N2 (270 kPa) for iii min. Hybridized arrays were scanned with a Scan Array 5000 laser scanner (Perkin-Elmer). Light amplification by stimulated emission of radiation 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 institute and segmented in gild to select areas of recognizable signals for assay. The fluorescence intensity of each spot was measured, signal-to-local-background ratios were calculated past ImaGene, and spot morphology and deviation from the expected spot position were considered using the default ImaGene settings. The data were imported into Microsoft Admission and automatically candy. Spots with a signal-to-noise ratio of ane.2 and with at least 600 relative fluorescence units over the local groundwork 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.
RESULTS
Specificity.
In order to let the simultaneous and rapid identification of Due south. aureus, E. coli, and P. aeruginosa grown in blood civilization specimens from septicemic patients, a microarray comprising a set of 40 S. aureus, 31 Eastward. coli, and 49 P. aeruginosa factor probes 200 to 800 bp in length was developed (Table S1 in the supplemental material).
The specificity of the Dna-chip was validated kickoff with 45 well-characterized clinical isolates and reference strains of the three target species, as well equally other related bacteria and, second, with xiii claret cultures from patients with sepsis past following the workflow outlined in Fig. ane. 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 Deoxyribonucleic acid preparation as a positive internal control, hybridized with the respective probes, indicating that the labeling and hybridization had performed efficiently.
Hybridization experiments with S. aureus, East. coli, and P. aeruginosa target DNAs revealed specific hybridization with the species-specific gene probes (Fig. 2). At that place was no cross-hybridization between the three species, with the exception of the South. aureus 16S rRNA gene probe (16SSa, Fig. 2C), which also hybridized with East. coli and P. aeruginosa target Deoxyribonucleic acid.
Dna microarray analyses of 42 clinical isolates, 3 reference strains, and 13 blood cultures. Each column shows the results of an private hybridization with target Dna prepared from: S. aureus ATCC 29213 (cavalcade 1), MW2 (cavalcade 2), clinical isolates (columns 3 to vii), and positive blood cultures (columns eight 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 xix), clinical isolates (columns xx 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 (cavalcade 36), S. capitis (column 37), South. haemolyticus (cavalcade 38), S. hominis (column 39), and S. warneri (cavalcade 40). Other gram-negative species included a Proteus mirabilis positive claret 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 l), 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 (cavalcade 55), and Streptococcus pneumoniae (cavalcade 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 East. coli factor probes; (B) hybridization with P. aeruginosa gene probes; (C) hybridization with South. aureus cistron probes. Gray boxes correspond gene probes that hybridized with the corresponding target DNA; white boxes represent gene probes that showed no hybridization with the respective target DNA. Experiments performed with positive claret cultures are indicated (BC).
Identification of E. coli, P. aeruginosa, and S. aureus reference strains, clinical isolates, and blood cultures by microarray analysis corresponded past 100% with the conventional identification results (Fig. ii).
Detection and bigotry of E. coli.
All DNA samples from nine E. coli strains hybridized always with seven E. coli factor probes [envZ, fes(1) and fes(2), nfrB, yacH, yagX, and ycdS] (Fig. 2A, columns 19 to 27); in the following discussion nosotros volition refer to these genes as cadre genes. With xiv E. coli gene probes, variable hybridization was observed, including the antibiotic resistance gene probes bla TEM-106, sul, strB, and aacC2. Such a variable hybridization profile is expected for antibody resistance genes since caused resistance to antimicrobials is isolate specific. For 11 E. coli virulence factor probes (eae, eltB, escR, escT, escU, espB, hlyA, hlyB, SLTII, toxA-LTPA, and VT2vaB) no hybridization signals were detected with any of the tested East. coli isolates and blood cultures. Since these virulence genes are known to be specific for particular E. coli pathotypes (three), information technology was not surprising that they were non nowadays in the tested strains. The eae, esc, and esp genes, for case, are encoded on a chromosomal pathogenicity isle, 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 bigotry of P. aeruginosa.
Deoxyribonucleic acid samples obtained from P. aeruginosa uniformly hybridized with 32 of 49 P. aeruginosa specific factor segments, including the mexA factor probe (core genes). Variable hybridization was observed with 17 probes, assuasive for the bigotry of individual P. aeruginosa isolates (Fig. 2B, columns 12 to xviii).
Detection and discrimination of S. aureus.
Hybridization experiments performed with eleven S. aureus target DNAs revealed signals in all assays with sixteen S. aureus cistron segments (core genes) (Fig. 2C, columns 1 to 11). Variable hybridization was observed with 14 Southward. aureus cistron probes including the six antibiotic resistance gene segments aadD, aacA-aphD, blaZ, dfrA, ermA, and mecA and the virulence genes sak, ocean, sec1, and EDIN. The factor probes geh, mreA, clfB, and elkT-abcA hybridized with viii (geh), 10 (mreA and clfB), and 6 (elkT-abcA) target DNAs. However, PCR amplification of the four genes was positive for all 11 Due south. 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 non allow reliable detection of the four genes in Due south. aureus.
No hybridization was observed with ten probes, including the toxin genes seb, tst, and etb. In contrast to the customs-caused, multidrug-susceptible methicillin-resistant Due south. aureus (MRSA) strain MW2 that hybridized to mecA and blaZ only, all half dozen clinical MRSA strains showed the same multiresistant hybridization pattern, and their Deoxyribonucleic acid 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 exist located upstream and downstream, respectively, of the mecA factor in the mec chromosomal region (7, 26). Hybridization to the core gene probes permitted the identification of S. aureus, while hybridization to antibody 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 merely with the S. aureus 16S rRNA cistron probe (16SSa, Fig. 2C) and several common staphylococcal antibiotic resistance determinants (aadD, aacA-aphD, aph-A3, blaZ, true 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 South. aureus.
The Micrococcus spp. isolate showed no hybridization with the DNA-scrap (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 cistron probe (Enterococcus spp.) (Fig. 2C). Of twelve strains of seven gram-negative species (columns 41 to 52), ii 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 bla TEM-106 (β-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 factor probe.
Sensitivity.
Although the bulk 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 aforementioned isolate (Table 1) . Successful hybridization with strong fluorescent signals depends on efficiency of Dna labeling (ratio of bases per one dye molecule) and corporeality 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 iii different Deoxyribonucleic acid preparations of isolate C4242, hybridization to Pseudomonas gene probes varied from 31 to 43 probes, respectively, depending on the labeling efficiency and corporeality of DNA (Tabular array 1). The everyman number of signals was detected with 382 ng of target Deoxyribonucleic acid, 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 cistron 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.
TABLE one.
Microarray hybridization signals obtained with unlike target DNA preparations of P. aeruginosa isolates
| Isolate | DNA amt (ng) a | BDR b | No. (%) of hybridized cistron probes c |
|---|---|---|---|
| C4242 | 130* | 22 | 38 (88) |
| 382* | 75 | 31 (72) | |
| 1,350† | 48 | 43 (100) | |
| C3853 | 510* | 29 | 36 (88) |
| >2,400† | 30 | 41 (100) | |
| C3045 | 550* | 90 | 34 (89) |
| 2,950† | 41 | 38 (100) | |
| C3755 | 1,180† | 139 | 41 (95) |
| >ane,600† | twoscore | 43 (100) |
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 civilization allowed a clear and unambiguous characterization of the Due south. aureus, Due east. coli, and P. aeruginosa present in 7 of xiii tested claret specimens (Fig. 2). In accordance with the VITEK2 characterization, positive BACTEC cultures were identified by microarray hybridization as multiresistant MRSA (Fig. 2C, column viii), penicillin-resistant Due south. aureus (columns 9 and 11), and multisusceptible Southward. aureus (cavalcade 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 betwixt susceptibility testing and microarray hybridization of selected antibiotic resistance genes. (i) Due south. aureus.
For 11 Staphylococcus aureus strains and claret cultures, nosotros compared susceptibility results determined by the VITEK2 system, Etest strips, and deejay 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).
Table ii.
Correlation between phenotypic and genotypic antibody resistance for 11 S. aureus isolates and blood cultures
| Antibody | Hybridization gene probe(southward) | No. of cultures (phenotypic classification b ) | No. of isolates with antibiotic resistance gene(s) c : | |
|---|---|---|---|---|
| Present | Absent | |||
| Penicillin | mecA/blaZ | ten (R) | 10 | 0 |
| ane (S) | 0 | 1 | ||
| Oxacillin | mecA | 7 (R) | seven | 0 |
| 4 (S) | 0 | 4 | ||
| Erythromycin | ermA, ermC, | 6 (R) | half-dozen | 0 |
| or msrA | 5 (Southward) | 0 | five | |
| Tobramycin | aadD | 5 (R) | five | 0 |
| vi (Due south) | 0 | 6 | ||
| Gentamicin | aacA-aphD | 0 (R) | 0 | 0 |
| 11 (S) | 0 | 11 | ||
| Trimethoprim | dfrA | 1 (R) | 0 | ane a |
| x (Due south) | 0 | x | ||
For the Southward. aureus strains there was a 100% correlation between phenotypic resistance to penicillin and hybridization to the mecA and/or blaZ cistron (both genes confer resistance to penicillin) (see Tabular array ii). Phenotypic resistance to oxacillin correlated 100% with the hybridization of the mecA gene betwixt resistance to erythromycin and hybridization to the erythromycin resistance genes ermA, ermC, or msrSA and betwixt 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 cistron 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) East. coli and other gram-negative bacteria.
The prototype microarray harbored just four E. coli and i P. aeruginosa resistance gene probes which do not yet permit a comprehensive prediction of antibody resistance. All the same, hybridization with the Eastward. coli resistance gene probe bla TEM-106 was observed in one P. mirabilis and iv E. coli strains and correlated with phenotypic ampicillin resistance for all 5 strains (Table 3).
TABLE 3.
Correlation between ampicillin-penicillin resistance, gentamicin-tobramycin resistance, and streptomycin resistance and hybridization with the resistance cistron probes bla TEM-106, aacC2, aph-A3, and strB
| Species | Resistance phenotype a | Hybridization with: | |||
|---|---|---|---|---|---|
| bla TEM-106 b | aacC2 b | aph-A3 c | strB b | ||
| E. coli ATCC 25922 | Susceptible | - | - | - | - |
| E. coli C4821 | AMP STR | + | - | - | + |
| Due east. coli F3437 | AMP | + | - | - | - |
| Eastward. coli C3941 | AMP STR | + | - | - | + |
| E. coli F1806 d | AMP GEN TOB STR | + | + | + | + |
| E. coli C4547 | AMP (I) | - | - | - | - |
| Due east. coli C4230 | AMP | - | - | - | - |
| Due east. coli C3940 | Susceptible | - | - | - | - |
| East. coli F1642 d | STR | - | - | - | + |
| P. mirabilis C4024 | AMP STR | + | - | - | + |
| P. mirabilis C4403 | Susceptible | - | - | - | - |
| P. mirabilis F1738 d | Susceptible | - | - | - | - |
One Due east. 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 (Tabular array three). 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 iii-component efflux system responsible for intrinsic and acquired multiresistance in P. aeruginosa (β-lactams, fluoroquinolones, trimethoprim, sulfonamides, chloramphenicol, and others) (27).
DISCUSSION
Nosotros take 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 immune 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 exist causative agents of bacteremia (xxx). Our epitome microarray demonstrates the feasibility of identifying and characterizing pathogens in 1 ml of positive blood civilisation without prior amplification of the target DNA.
The microarray consisted of 120 gene segments 200 to 800 bp in length amplified from recombinant plasmids. I of import characteristic of this format is that the console of probes tin be continually extended to include sequences for additional species, variant isolates, or antibody resistance determinants as they are characterized and available. The accuracy, range, and discriminatory power of the gene-segment-based microarray can be refined past adding or removing gene probes to the console 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 past 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 volition besides permit the identification of mixed claret cultures with more than ane pathogen.
A second of import feature of this microarray format based on PCR products of extended length (100 to 3,000 bp) is that one probe per cistron is usually sufficient to produce strong signals and high specificity (35). For long probes, minor betoken mutations are likely to only slightly reduce duplex germination, which does not atomic number 82 to the loss of hybridization signals. In contrast, short oligonucleotide microarrays sometimes lack specificity and require multiple curt oligonucleotides per one gene. Volokhov et al. (38) constructed a microarray for the assay of erythromycin determinants; to increase confidence in the microarray analysis, each analyzed gene was represented by 7 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 unmarried or small point mutations, such as those responsible for resistance conferred by extended-spectrum β-lactamases (ESBLs, e.yard., TEM-, SHV-, and OXA-type ESBLs) (5). An extension of the probe console for instance, by OXA, SHV, or CMY resistance genes, will let the detection of the different β-lactamase types (19) but not discrimination within one type.
As shown for P. aeruginosa probes, the intensity of the hybridization betoken largely relies on quality and quantity of the labeled target Deoxyribonucleic acid. Under suboptimal atmospheric condition non all species-specific probes produced strong signals after hybridization with specific Deoxyribonucleic acid. PCR analyses of some S. aureus genes revealed that the genes clfB, elkT-abcA, geh, and mreA were present in all tested Due south. aureus strains, whereas non all strains produced hybridization signals with the corresponding probes. Besides the quality and quantity of the target DNA, two circumstances could explicate 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 only there is no hybridization). Second, it is impossible to predict the exact hybridization behavior of immobilized Deoxyribonucleic acid probes. Therefore, it is important to judge the collection of probes experimentally using the prototype flake 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-fleck hybridization, is essential for testing blood cultures where the bacterial diagnosis is usually uncertain. In regard to the processing time we decided to utilise a chemic labeling procedure using high-molecular-weight DNA (>12 kb). DNA fragmentation past sonication or enzymatic cleavage (restriction enzymes or DNase A) prior to the labeling reaction is difficult to control and increases the processing fourth dimension and the chance of losing DNA by an additional required precipitation step. However, large target Deoxyribonucleic acid molecules may hybridize poorly to immobilized probes due to spatial and steric constraints. Vora et al. (twoscore) reported that the sensitivity is significantly increased past using fragmented DNA (twoscore). A Deoxyribonucleic acid grooming protocol using sonication for simultaneous cell disruption and target Dna fragmentation may be the method of choice to increment the sensitivity of the microarray, in particular toward low-copy-number and/or plasmid-encoded genes which may exist underrepresented in the target Dna.
Since the focus of the present written report was to provide a proof of principle and to test the informative value of the selected capture probes, the protocols were not all the same optimized for time. However, trials with commercial Dna kits showed that DNA extraction can be performed within 1 h. Hybridizations were performed co-ordinate to standard protocols overnight. Preliminary experiments showed that this fourth dimension may be shortened to 4 h, allowing the results to be obtained inside 8 h subsequently a claret culture becomes positive.
Previous studies have shown that the detection of antibiotic resistance genes by molecular techniques has expert predictive ability for the phenotypic resistance of clinical Due south. aureus isolates (23, 36, 38). With our gene-segment-based microarray in that location was an fantabulous correlation betwixt 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 unmarried assay.
Past 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 customs-acquired MRSA. Simultaneous comprehensive resistance genotyping for oxacillin, macrolide, and aminoglycoside resistance genes (east.m., 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 effect, 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 Deoxyribonucleic acid probes directed confronting rRNA (22) were shown to provide good discrimination of fungi and gram-positive and gram-negative bacteria causing bacteremia. Notwithstanding, since these approaches are solely based on rRNA they provide no data on antibiotic resistance determinants or virulence genes of the identified strains. Another molecular arroyo 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). Even so, due to the microtiter plate format of this analysis, the number of gene probes is express and the mecA probe was the only antibiotic-resistant determinant.
On the other mitt, in that location 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 accept been express by the number of genes that can be amplified in i reaction. By combining multiplex PCR and microarrays, the microarray detection system is superior to gel electrophoretic analysis, but in this style the great potential of microarrays allowing the analysis of hundreds of genes in parallel is abandoned. Most of these systems tin can therefore be applied to preidentified pathogens just.
In contrast, the microarray presented here opens the possibility of identifying pathogens in claret cultures with concomitant further characterization in terms of antibiotic resistance and virulence without preidentification. Later on extension and further automation, the DNA-bit has the potential to provide a clinical tool for microbiological diagnostics, equally well as for epidemiological studies. It would allow clinicians to administer appropriate antibody chemotherapy in a timely fashion, improve the result of septic patients, and reduce the spread of antibiotic resistance genes.
Supplementary Material
Acknowledgments
Nosotros thank the clinical laboratory staff for providing clinical isolates and blood cultures and Ludwig Eichinger for the use of the scanning facilities.
Footnotes
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