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Applied and Environmental Microbiology, June 2007, p. 4082-4088, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.01820-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

DNA Microarray-Based Identification of Serogroups and Virulence Gene Patterns of Escherichia coli Isolates Associated with Porcine Postweaning Diarrhea and Edema Disease ^,

Weiqing Han,^1,2,3 Bin Liu,^1,2,3 Boyang Cao,^1,2,3 Lothar Beutin,⁴ Ulrike Krüger,⁴ Hongbo Liu,^1,2,3 Yayue Li,^1,2,3 Yanqun Liu,^1,2,3 Lu Feng,^1,2,3 and Lei Wang^1,2,3*

TEDA School of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin 300457, China,¹ Tianjin Key Laboratory of Microbial Functional Genomics, 23 Hongda Street, TEDA, Tianjin 300457, China,² Tianjin Research Center for Functional Genomics and Biochip, 23 Hongda Street, TEDA, Tianjin 300457, China,³ National Reference Laboratory for Escherichia coli, Federal Institute for Risk Assessment (BfR), Diedersdorfer Weg 1, D-12277 Berlin, Germany⁴

Received 2 August 2006/ Accepted 13 April 2007

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 
Escherichia coli strains causing postweaning diarrhea (PWD) and edema disease (ED) in pigs are limited to a number of serogroups, with O8, O45, O138, O139, O141, O147, O149, and O157 being the most commonly reported worldwide. In this study, a DNA microarray based on the O-antigen-specific genes of all 8 E. coli serogroups, as well as 11 genes encoding adhesion factors and exotoxins associated with PWD and ED, was developed for the identification of related serogroups and virulence gene patterns. The microarray method was tested against 186 E. coli and Shigella O-serogroup reference strains, 13 E. coli reference strains for virulence markers, 43 E. coli clinical isolates, and 12 strains of other bacterial species and shown to be highly specific with reproducible results. The detection sensitivity was 0.1 ng of genomic DNA or 10³ CFU per 0.3 g of porcine feces in mock samples. Seventeen porcine feces samples from local hoggeries were examined using the microarray, and the result for one sample was verified by the conventional serotyping methods. This microarray can be readily used to screen for the presence of PWD- and ED-associated E. coli in porcine feces samples.

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 
Postweaning diarrhea (PWD) and edema disease (ED) are two of the most prevalent porcine diseases worldwide. They account for substantial economical losses and are major causes of death in weaned pigs (3, 14). PWD is primarily caused by enterotoxigenic Escherichia coli (ETEC), while ED is caused by verocytotoxigenic E. coli. Virulence factors associated with PWD and ED include adhesion factors (F4, F5, F6, F18, F41, and intimin) and exotoxins (STa, STb, LT, Stx2e, and EAST1) (2, 12, 13, 23). Pathogenic strains colonize the small intestine through different types of adhesion factors and generate one or more exotoxins responsible for the diseases (12, 16). PCR-based methods have been developed to detect these virulence factor genes in porcine E. coli (13, 23, 24).

E. coli causing PWD and ED in pigs is limited to a few O serogroups, including O8, O45, O138, O139, O141, O147, O149, and O157, which are most commonly reported worldwide (12, 14, 16, 22). For example, these eight serogroups accounted for 81.2% of all isolates examined and 88.9% of typeable isolates in Denmark (12). Conventional O serotyping of E. coli strains in clinical specimens and environmental samples is laborious and time-consuming. The presence of capsules, capsular-like fimbriae, and rough lipopolysaccharide complicates the O serotyping of porcine-pathogenic E. coli strains and prompted us to develop a molecular typing method.

The O antigen, which consists of repeats of an oligosaccharide unit (O unit), is part of the lipopolysaccharide in the outer membrane of gram-negative bacteria and contributes major antigenic variability to the cell surface. There are 186 O-antigen forms recognized for E. coli (including Shigella) (11). Genes involved in biosynthesis of O antigen are normally clustered on the chromosome between two housekeeping genes, galF and gnd, in E. coli. Some O-antigen genes, including those encoding glycosyltransferases, O-unit flippase (Wzx), and O-antigen polymerase (Wzy), are often specific for different O antigens (36) and can be used as targets in molecular typing. PCR assays based on O-antigen-specific genes of E. coli O45, O138, O139, and O157 have been developed by us and others (6, 35, 36).

Conventional PCR is used to amplify a single target gene but the method is laborious for detection of multiple serogroup-specific genes and virulence factor genes. Multiplex PCR can amplify multiple targets in a single reaction, but a clear length differentiation among PCR products is required, which leads to a challenge for primer design. Recently DNA microarrays have been applied to microbial detection and community analysis (4, 19, 26, 27, 31, 34, 38). The approach involves the immobilization of numerous oligonucleotide DNA probes on a solid support to which fluorescence-labeled amplified target DNA is hybridized, which was shown to be rapid, reliable, and sensitive (5, 20, 25, 37). In this study, the O-antigen gene clusters of E. coli O141, O147, and O149 were sequenced and analyzed. These data were combined with published data from other O-antigen gene clusters to develop a genotyping microarray. The microarray also included genes encoding adhesion factors and exotoxins of porcine E. coli strains. The microarray was examined for its specificity and sensitivity and applied to 17 porcine feces samples.

All strains were inoculated into Luria-Bertani medium and incubated overnight at 37°C. Genomic DNA was prepared as previously described (1). O-antigen gene clusters from E. coli O141, O147, and O149 reference strains were sequenced using the protocol described previously (10), and sequences of 15,601 bp (12 orf genes), 10,252 bp (8 orf genes), and 8,729 bp (9 orf genes) were obtained between galF and gnd, respectively (see Fig. S1 in the supplemental material). All orf genes were assigned functions based on amino acid identities of their products to proteins of known functions and named accordingly (see Tables S1, S2, and S3 in the supplemental material). Sequences of E. coli O8, O45, O138, O139, and O157 O-antigen gene clusters were retrieved from GenBank.

The wzy gene was employed as the target gene for typing of E. coli O45, O138, O139, O141, O147, O149, and O157. Genes from the O-antigen gene cluster of E. coli O8 share high-level identity (from 88.4% to 96.3%) to those from the Klebsiella pneumoniae O5 wb gene cluster (21), which is also responsible for the synthesis of O antigen. The specific region of the glycosyltransferase gene orf469, which is the most heterogeneous gene, was used for typing E. coli O8. E. coli O149 and Shigella boydii type 1 have identical O antigens (8), and their O-antigen gene clusters were found to be nearly identical (99.8% to 100% identity for each gene set). The O-antigen structures of E. coli O147 and Shigella flexneri 6 are similar, with the only difference being the presence or absence of an O acetyl group on the side chain (7, 15), and their O-antigen gene clusters are expected to share high-level DNA identity. The invasion plasmid antigen gene (ipaH) has been shown to be unique to Shigella and enteroinvasive E. coli (EIEC) and can be used for the detection of Shigella from non-EIEC E. coli (17, 30, 33). None of the E. coli strains belonging to serogroups O147 and O149 are recognized as EIEC. Therefore, the ipaH gene was used as the target gene to differentiate E. coli O147 from S. flexneri 6 and E. coli O149 from S. boydii type 1. Genes encoding adhesion factors (F4, F5, F6, F18, F41, and intimin) and exotoxins (STa, STb, LT, Stx2e, and EAST1) were selected as target genes to reveal the virulence gene patterns of PWD- and ED-associated serogroups, and their sequences were retrieved from GenBank.

A total of 21 pairs of primers were designed based on each of the target genes described above, and they were divided into two groups to generate target DNAs by multiplex PCR (Table 1). Group 1 targets the O-antigen-specific genes and the ipaH gene, and group 2 targets the virulence genes. In addition, both groups contain the primer pair for amplifying the 16S rRNA gene as the internal positive control. The primer concentrations were optimized based on the final intensities of hybridization signals, which can be analyzed by the interpretation software developed in-house. An attempt to use the same concentration (0.2 µM) for all of the primers failed due to the detection of negative or weak fluorescence signals for some of the genes. Therefore, different primer concentrations (0.1 µM to 0.6 µM) were tested, and the best combinations are listed in Table 1. Multiplex PCR was performed with 50 to 100 ng of template DNA in a final volume of 50 µl containing the following: 1x PCR buffer; 2.5 mM MgCl₂; 400 µM (each) dATP, dCTP, dGTP, and dTTP; and 2.5 U Taq DNA polymerase. The PCR cycle was performed with the initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 1.5 min, concluding with a final elongation at 72°C for 5 min. The agarose gel images for the multiplex PCR are shown in Fig. S2 in the supplemental material. PCR products were then purified with the Microcon centrifugal filter devices kit (Millipore Corporation). To label PCR products, only reverse primers were used in a PCR, and 0.3 µl of 25 nM Cy3-dUTP was included. Ten microliters of the purified amplification products generated from the first-round multiplex PCR was added as the template. All labeled DNA was purified with the Microcon centrifugal filter devices kit and stored at –20°C in the dark until use.

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TABLE 1. Primers and their concentrations in multiplex PCRa

Probes based on each of the target genes were designed using OligoArray 2.0 (28) (Table 2), synthesized with a 5' amidocyanogen modifier, and added with a poly(T) spacer consisting of 15 thymine nucleotides. Probes (1 µg/µl in 50% dimethyl sulfoxide) were spotted onto an aldehyde group modified glass slide (CEL Corporation) to make a microarray, and three replicates were spotted for each probe to eliminate any possible physical defects in the glass slide. A probe based on the conserved region of the 16S rRNA gene was used as a positive control (Table 2). A probe containing 40 poly(T) oligonucleotides labeled with Cy3 at the 3' end was used as the positional reference and printing control (see Fig. S3 in the supplemental material). For each target gene, one to four probes were used, and the layout of the array is shown in Fig. S3 in the supplemental material. To carry out the microarray assay, 10 µl of the labeled target DNA was mixed with 10 µl of preheated (50°C) hybridization buffer (25% formamide, 0.1% sodium dodecyl sulfate, 6x SSPE [1x SSPE is 0.18 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA {pH 7.7}]) and hybridized with the array at 50°C for 15 h. The hybridized microarray was scanned with a laser beam of 532 nm using the 4100A biochip scanner (Axon Corporation) with the following parameters: photomultiplier tube gain of 600 and pixel size of 5 µm. Two files were generated, one for the images saved as .tif files and the other for the signal intensity, saved as .gpr. The signal-to-noise ratio was calculated for each spot using Bactarray Analyzer 1.0, developed in-house, with the threshold set at 3.0. A positive detection result was reported when all the probes of the given target gene generated hybridization signals above the signal-to-noise-ratio threshold.

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TABLE 2. Oligonucleotide probes used in this study

The specificity of the DNA microarray was tested using 186 E. coli and Shigella O-serogroup reference strains, which represent all of the 186 O serogroups recognized, 13 E. coli reference strains for virulence markers, and 12 strains of other bacterial species (see Table S4 in the supplemental material). Multiple strains of different sources representing each of the target serogroups and virulence genes were used to test the microarray, and the numbers of strains are 6, 3, 9, 9, 6, 3, 7, and 9 for O8, O45, O138, O139, O141, O147, O149, and O157, respectively, and 10, 3, 2, 21, 3, 10, 19, 21, 11, 16, and 15 for virulence factors F4, F5, F6, F18, F41, intimin, STa, STb, LT, Stx2e, and EAST1, respectively. The sources of these strains are given in Table S4 in the supplemental material. All of the strains, belonging to the eight serogroups or carrying virulence markers, consistently hybridized to their corresponding probes, indicating that the primers/probes can well represent the corresponding serogroups or virulence genes. The hybridization patterns observed for 15 reference strains are shown in (Fig. 1). All Shigella strains tested in this study yielded signals with ipaH gene-targeted probes, while all E. coli strains tested negative. None of the E. coli strains belonging to other serogroups and strains of other bacterial species, which are likely to be present in the small intestine, bound to the serogroup-specific probes on the microarray.

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FIG. 1. Hybridization patterns for different E. coli strains. (1) E. coli O8. (2) E. coli O45. (3) E. coli O138. (4) E. coli O139. (5) E. coli O141. (6) E. coli O147. (7) E. coli O149. (8) E. coli O157. (9) E. coli strain harboring virulence factors STap, STb, and F6. (10) E. coli strain harboring virulence factors STap, F5, and F41. (11) E. coli strain harboring virulence factors LT, EAST1, STap, STb, and F4. (12) E. coli strain harboring virulence factors Stx2e and F18. (13) E. coli strain harboring virulence factor intimin. (14) S. flexneri 6. (15) S. boydii type 1.

A double-blind test was carried out with 43 clinical isolates (see Table S4 in the supplemental material) which have been characterized for their virulence genes by conventional PCR (see Table S5 in the supplemental material) and for their O and H serotypes with specific antisera at the Federal Institute for Risk Assessment (BfR) in Berlin, Germany. A complete agreement with the results of conventional methods was obtained, and the hybridization patterns for representative clinical isolates are shown in Fig. S4 in the supplemental material. These results showed that the microarray assay was specific and reliable.

The microarray was applied for examination of 17 porcine feces samples (0.3 g) obtained from asymptomatic adult pigs from 4 local hoggeries. After 10 h of enrichment in LB medium at 37°C, 16 out of the 17 samples gave positive signals to at least 1 serogroup, and only sample 17 gave a negative signal (see Table S6 in the supplemental material). In order to confirm the results, the culture of sample 15, which gave positive signals for O8, O45, and O147 strains, was selected and plated to isolate single colonies. A total of 126 single bacterial colonies were picked and screened by PCR using O8-, O45-, and O147-specific primers, respectively. One each of O8- and O45-positive colonies were found when 30 colonies were screened, and their identities were confirmed by using the Biolog Microstation system (Biolog, Inc.) and the conventional O serotyping methods with commercial antisera (Institute of Veterinary Drug Control, Beijing, China) (data not shown). We did not detect any O147 strains after all 126 colonies were tested, and it is possible that the corresponding colonies were missed, which is a common problem for conventional antiserum serotyping. This indicates that the microarray is more sensitive than conventional serotyping methods. The O8 and O45 isolates were further screened for their virulence gene patterns using the same microarray, and the O45 strain was found to harbor an exotoxin EAST1-encoding gene. It has been reported that EAST1 contributes to the virulence of bacterial strains; however, the significance of this gene as a pathogenicity factor is not conclusive (32). The results for these 17 porcine feces samples showed that the microarray could identify PWD- and ED-related E. coli strains in the presence of other microorganisms and is highly specific.

To test the sensitivity of the microarray, serial dilution of genomic DNA (0.01 to 100 ng) was done for each of the eight target reference strains. The DNA dilutions were mixed with 500 ng of background DNA extracted from sample 17, which was not found to contain any target serogroup strains by microarray analysis, using the QIAamp DNA stool minikit (QIAGEN Inc.), and used as templates for multiplex PCR. Positive signals could be obtained from as little as 0.1 ng of genomic DNA. For porcine feces mock samples, serial dilutions from each of the pure cultures of the eight strains were mixed with approximately 0.3 g of porcine feces from sample 17. The spiked samples were cultured in 30 ml of LB medium at 37°C for 6 h, and 50 µl of genomic DNA was isolated with the QIAamp DNA stool minikit, out of which 3 µl was used as the template in the microarray assay. Cells from all eight strains were detected at levels as low as 10³ CFU per 0.3 g of porcine feces.

It has been widely accepted that serotyping can be used as an epidemiological marker for pathogenicity of E. coli (2, 29). Conventionally, antigenic analysis of different serogroups in E. coli is performed by carrying out laborious agglutination reactions using antisera raised against the O reference strains. In addition, cross-reactions among different serogroups often occur, giving equivocal results; O serotyping fails if O-rough strains (which do not produce an O antigen as a result of mutations in one or more of the multiple genes controlling O-antigen synthesis and polymerization) are present, which was found not infrequently with isolates from PWD and ED (35). PCR-based tests amplifying specific genes in the E. coli O-antigen gene clusters have been found to be serogroup specific (6, 35). In this study, through sequencing of three E. coli O-antigen gene clusters which have not been identified before, we obtained specific genes for all of the eight major E. coli serogroups associated with PWD and ED.

Nevertheless, serotyping only indirectly identifies porcine-pathogenic strains. Therefore, 11 virulence factor genes associated with PWD and ED were also targeted in our microarray to give further indications of possible pathogenicity of the targeted serogroups. E. coli pathogenicity is a complex, multifactorial mechanism involving a large number of virulence factors which vary according to the pathotype, and a single component might not be sufficient to transform an E. coli strain into a pathogenic one but could only play a role in combination with other virulence determinants. The inclusion of virulence gene probes in the microarray is useful for virulence assessment of E. coli strains. Because several virulence traits are located on transferable genetic elements (9), it is likely that new pathogenic serogroups may emerge. The distribution and frequencies of pathogenic serogroups can vary considerably from one geographic region to another and over time in a given region. Characterization for the presence of virulence factor genes will detect all of the potential pathogenic strains, even if they appear in serogroups other than the currently recognized pathogenic ones.

In this study, the microarray assay was applied to 254 strains, including 186 E. coli and Shigella O serogroup reference strains, 13 E. coli reference strains for virulence markers, 43 E. coli clinical isolates, and 12 strains of other bacterial species. The microarray was also applied to 17 porcine feces samples. All results showed that the microarray assay is specific and reliable. The applicable samples of the microarray fall into two categories: bacterial isolates and porcine feces. For bacterial isolates, the microarray can be utilized as a specific and reliable alternative to the conventional O serotyping and PCR-based detection of virulence genes to characterize both serogroups and virulence gene patterns simultaneously.

When the microarray assay is directly applied to porcine feces samples, it can be used as an efficient preliminary screening method for surveillance of pig farms or for an epidemiological investigation in a certain region. The first step is to detect the presence of potential pathogenic O serogroups directly with porcine feces samples using the microarray; once the pathogenic serogroups are found, further plating can be carried out to isolate the targeted serogroup colonies; finally, the isolated strains can then be examined by the same microarray for the identification of virulence gene patterns. Although the microarray can be applied to detect both pathogenic serogroups and virulence genes directly with porcine feces samples at the same time, the serogroup-specific and virulence genes might be from different host strains.

The diseases of PWD and ED commonly occur in piglets within the first 14 days after weaning (12). Adult pigs carrying the PWD- and ED-associated serogroup strains with related virulence genes may not show any symptoms but can serve as an infection source for piglets, leading to an outbreak of PWD and ED in a large pig farm due to rapid transmission of pathogens, so preliminary screening for the presence of PWD- and ED-related E. coli strains using this microarray is important when starting to breed or buy piglets. This is the first DNA microarray for comprehensive detection of serogroups and pathotypes of E. coli associated with PWD and ED and is promising as a new diagnostic tool for investigations of sporadic infections and outbreaks and for surveillance of serogroup/pathotype distribution of these strains in different geographic locations.

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 
The DNA sequences of the E. coli O141, O147, and O149 O-antigen gene clusters have been deposited in GenBank under the accession numbers DQ868765, DQ868766, and DQ868764.

 
This work was supported by the NSFC Key Program (30530010) and funds from the Science and Technology Committee of Tianjin City (05YFGZG04700) to L.W. and L.F.

 
* Corresponding author. Mailing address: TEDA School of Biological Sciences and Biotechnology, Nankai University, 23 Hongda Street, TEDA, Tianjin 300457, China. Phone: 86-22-66229588. Fax: 86-22-66229596. E-mail: wanglei{at}nankai.edu.cn

Published ahead of print on 20 April 2007.

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

Top ABSTRACT INTRODUCTION Nucleotide sequence accession... REFERENCES

 

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Applied and Environmental Microbiology, June 2007, p. 4082-4088, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.01820-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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