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Applied and Environmental Microbiology, September 2007, p. 5692-5697, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.00419-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Pathotyping Escherichia coli by Using Miniaturized DNA Microarrays{triangledown} ,{dagger}

Muna F. Anjum,1* Muriel Mafura,1 Peter Slickers,2 Karin Ballmer,3 Peter Kuhnert,3 Martin J. Woodward,1 and Ralf Ehricht2

Department of Food and Environmental Safety, Veterinary Laboratories Agency-Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom,1 CLONDIAG Chip Technologies GmbH, Jena, Germany,2 Institute of Veterinary Bacteriology, Vetsuisse Faculty of the University of Bern, Bern, Switzerland3

Received 22 February 2007/ Accepted 4 July 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 Design and validation of...
 Pathotyping clinical isolates.
 Conclusion.
 REFERENCES
 
The detection of virulence determinants harbored by pathogenic Escherichia coli is important for establishing the pathotype responsible for infection. A sensitive and specific miniaturized virulence microarray containing 60 oligonucleotide probes was developed. It detected six E. coli pathotypes and will be suitable in the future for high-throughput use.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
Design and validation of...
 Pathotyping clinical isolates.
 Conclusion.
 REFERENCES
 
Pathogenic Escherichia coli strains constitute a significant public health problem worldwide (12). In contrast to their nonpathogenic counterparts, these strains have acquired specific virulence attributes that allow them to cause a spectrum of human and animal illnesses (10, 15). Numerous methods exist for the detection of pathogenic E. coli, including geno- and phenotypic marker assays for the detection of virulence genes and their products (7, 17, 21, 23). These methods have the common drawback of screening a relatively small number of determinants simultaneously. DNA microarrays offer a viable alternative due to their ability to screen multiple markers simultaneously.

The aim of this work was to develop a simple high-throughput system based in a microtube (details are available from CLONDIAG, Jena, Germany) (13, 20) for pathotyping E. coli isolates sent to clinical diagnostic laboratories.


   Design and validation of miniaturized virulence arrays.
Top
 ABSTRACT
 INTRODUCTION
 Design and validation of...
Pathotyping clinical isolates.
 Conclusion.
 REFERENCES
 
A miniaturized E. coli oligonucleotide virulence array was designed containing 39 virulence, 7 bacteriocin, and 15 control (rrl and gad) gene probes (Table 1). Eighteen genes were specific to a particular E. coli pathotype, 13 were common between 2 or more pathotypes, and 7 were unassigned. The design of probes/primers and the specificity were tested as previously described (1, 13).


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  		TABLE 1. Probes and primers used in the miniaturized microarraya

 
Control strains were used to validate each probe present on the array (Table 1). PCR amplification and sequencing, using primers given in Appendix 1 of the supplemental material, verified the presence of the probes in control strains. The sequenced genes showed between 92 and 100% sequence identity to the respective target gene and showed 100% sequence identity to the probe and primer regions (data not shown).

Genomic DNA was extracted from cells grown aerobically overnight at 37°C in LB broth, using a DNeasy tissue kit (catalog no. 69504; QIAGEN). One microgram of genomic DNA from each strain was used as a template in a multiplex linear amplification and labeling reaction with the set of 60 primers (Table 1), as previously described (1). The amplified products were added to ArrayTubes for hybridizations performed according to the method of Ballmer et al. (1, 13).

The sequenced strains EDL933, CFT073, and E2348/69 were used to estimate assay sensitivity to ensure strong signal intensity with minimal nonspecific cross-hybridization. Optimization included varying the concentrations of genomic DNA used for labeling (2 to 0.05 µg), the primers present in the linear multiplex mix (0.135 to 0.810 µM), and the poly-horseradish peroxidase-streptavidin conjugate used for detecting hybridization (50 to 400 pg/µl). The minimal concentration of genomic DNA found to reliably detect all expected genes was 1.0 µg, while a concentration of 0.135 µM per primer in the stock solution was sufficient for the detection of target DNA (Fig. 1). The optimal concentration of poly-horseradish peroxidase-streptavidin conjugate was found to be 200 pg/µl; concentrations above or below this value resulted in high background or no detectable reaction at all (data not shown).


Figure 1
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  		FIG. 1. Optimization of the genomic concentration used in this study. The optimal concentration of genomic DNA from EDL933 used for the detection of genes on the virulence oligonucleotide miniaturized microarray chip was assessed using (a) 2 µg, (b) 1 µg, (c) 0.5 µg, (d) 0.1 µg, and (e) 0.05 µg of DNA. The six biotinylated marker spots (C) are visible in all arrays. A concentration of 0.135 µM per primer in the stock solution and 200 pg/µl of poly-horseradish peroxidase-streptavidin conjugate were used for these assays.

 
The spot signal intensity was derived by calculating the quantitative staining value with IconoClust software (version 2; CLONDIAG). The data were normalized using the signal intensity of the gad probe, and the normalized signal intensity for genes within positive and negative control strains was used to differentiate between present (signal intensity value above 0.4) and absent (signal intensity value below 0.3) genes. Genes with signal intensity values between 0.3 and 0.4 were considered ambiguous. Two replicate hybridizations were performed for each control strain, and the 95% confidence interval of error across replicate hybridizations was 1.6 to 3% (see Appendix 2 in the supplemental material).

The specificity of each probe was estimated by comparing array data with PCR and sequenced data from control strains. In all cases, the virulence gene(s) known to be present within positive control strains was clearly identified by array, while two negative control strains, including the sequenced strain MG1655, showed the presence of only 23S rRNA and gad genes (see Appendix 2 in the supplemental material). For many positive control strains, additional virulence genes were detected (Table 2). Furthermore, PCR amplification in all control strains of five randomly chosen genes (eae, astA, ehx or hlyA, iss, and mcmA), showed 100% correlation between array and PCR data, indicating the probes to be highly specific with minimum cross-reactions (data not shown).


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  		TABLE 2. Virulence determinants detected within each positive control strain

 

   Pathotyping clinical isolates.
Top
 ABSTRACT
 INTRODUCTION
 Design and validation of...
 Pathotyping clinical isolates.
Conclusion.
 REFERENCES
 
A panel of 63 E. coli human and animal clinical isolates were pathotyped using the virulence miniaturized microarray (see Appendix 3 in the supplemental material). For five strains, two hybridization reactions were performed and the 95% confidence interval of error between replicates was 0.9 to 5.0%. Only one hybridization reaction was performed for the remaining test strains.

Fifty-five of the isolates hybridized to more than one virulence determinant and were readily designated within a recognized pathotype, mostly matching the clinical diagnosis where available. Five isolates that harbored only the iss gene and/or microcins and three isolates that hybridized to only control genes could not be pathotyped. These isolates may harbor virulence genes not present on our array. Several isolates with novel combinations of genes were detected and included two shigatoxigenic E. coli strains, one with senB, iss, cma, cba, and mchBCF genes and another with astA, cdtB, and cnf genes. The most commonly detected gene was iss, which was present in half the strains tested. Other genes which were detected in at least 10 or more isolates included eae, ehx, astA, iroN, mchF, mchB, mchC, f17A (three variants combined), f17G, mcmA, cba, cma, and prfB/papB. Genes virF, pet, hlyE, fasA, and cfa were not detected in any test isolate (see Appendix 3 in the supplemental material).


   Conclusion.
Top
 ABSTRACT
 INTRODUCTION
 Design and validation of...
 Pathotyping clinical isolates.
 Conclusion.
REFERENCES
 
Several E. coli virulence arrays for genotyping have been described previously (2-5, 9, 11, 18). These arrays use mostly a glass slide printed with oligonucleotide probes or PCR products for target genes and fluorescent Cy dyes to label DNA used for hybridization. This system is time consuming, with expensive reagents and requires a skilled technician. In contrast, the microtube-based array system used in this study has a short assay time due to an amplification step and inexpensive reagents and requires low technical skills, making it amenable for use in clinical diagnostic laboratories. In the future, the routine use of virulence microarrays in such laboratories will not only allow rapid detection and designation of the pathotypes of strains sent to diagnostic laboratories but also enable emergent strains harboring novel virulence combinations to be detected before such strains spread to become a health problem.


   ACKNOWLEDGMENTS
 
We are grateful to the Enteric Reference Laboratory at VLA for the provision of E. coli strains and in particular to Katherine Sprigings and Louise Finch. We thank Elke Müller and Jana Sachtschal for their assistance.

This project was funded through the VLA seedcorn fund.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Food and Environmental Safety, Veterinary Laboratories Agency-Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom. Phone: 44 (0)1932 359476. Fax: 44 (0)1932 357268. E-mail: m.anjum{at}vla.defra.gsi.gov.uk Back

{triangledown} Published ahead of print on 13 July 2007. Back

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


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 INTRODUCTION
 Design and validation of...
 Pathotyping clinical isolates.
 Conclusion.
 REFERENCES
 

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Applied and Environmental Microbiology, September 2007, p. 5692-5697, Vol. 73, No. 17
0099-2240/07/$08.00+0     doi:10.1128/AEM.00419-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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