Introduction

Escherichia coli (E. coli) strains fall into four phylogenetic groups, namely A, B1, B2 and D. Group B2 and, to a lesser extent, group D, house the majority of virulent extra-intestinal E. coli, whilst groups A and B1 primarily represent commensal, low-pathogenic E. coli or enteropathogenic E. coli only for animal species [1].

Enterohemorrhagic E. coli (EHEC) represent an important pathotype of Shiga toxin-producing E. coli (STEC) group and can cause severe disease in humans ranging from diarrhoea and haemorrhagic colitis (HC) to haemolytic uremic syndrome and thrombocytopenic purpura [2, 3]. Although various EHEC serotypes like O26:H11, O91:H21, O111:H8, O113:H21, O157:H−, are frequently associated with human disease, E. coli O157:H7 is arguably the most important member of this group since this serotype is the more frequently detected during human disease outbreaks [4, 5].

Domestic ruminants like sheep, goats and particularly cattle, are considered important reservoirs for E. coli O157:H7 and play a role in the spread of the infection to man and other animals. Cattle are usually healthy carrier lacking any overt clinical symptoms related to the presence of this serotype in their gastrointestinal tract [6, 7]. In Iran, a large number of investigations about the presence of STEC have recently concerned both hospitalized humans in Tehran [8, 9] and other areas of the country [10, 11], diarrhoeic and healthy calves in Tehran [12] and slaughtered sheep in Shiraz [13]. Results of these studies reported the mostly prevalence of non-O157 Shiga toxin-producing E. coli. However, E. coli O157 was found in stool specimens of children [8] and carcasses during slaughtering of sheep [13].

In Camelidae, many serotypes of E. coli have been isolated and their pathogenic character was investigated by rabbit ileal loop assay [14] or haemolytic activity [15]. Specific survey studies have excluded the presence of STEC in camels populations, in several countries of Eastern Africa [16] and United Arab Emirates [17]. More recently, a survey of E. coli O157 in camelids from public open farm in Great Britain showed instead a low prevalence of Shiga and non-Shiga toxin-producing E. coli O157 in Llama and Alpaca species [18].

The main genetic characteristics of virulent E. coli O157:H7 is the presence of one or more Shiga toxins encoded genes as well as the Locus of enterocyte effacement (LEE), a pathogenicity island essential for attachment/effacement of bacterial cells to the intestinal epithelium and the presence of plasmid pO157 [19, 20].

Microarray technology has recently been applied for phylogenetic studies of E. coli O157:H7 [21, 22]. An oligonucleotide microarray to characterize the virulence potential of E. coli isolates was developed and validated by a collection of well-characterized reference E.coli strains [23, 24] and applied for the pathotyping of E. coli isolates from environmental and animal origin [25, 26].

This communication reports, for the first time, the isolation from dromedaries with hemorrhagic diarrhoea of two strains of E coli O157:H7 and their genetic characterization using a custom designed virulence gene microarray. The importance of DNA microarray technology in practice diagnostic is also discussed.

Materials and Methods

Strains Isolation and Identification

Faecal samples from 3 and 4 years old dromedaries, located in Gonbad-Qabus, northeast of Tehran, both with haemorrhagic diarrhoea, were collected and E. coli isolation was performed according to the protocol described by Alonso et al. [27]. The serotype identifications were done using the MAST ASSURE™ pathogenic Escherichia coli antisera kit (Mast Diagnostics, EA). Genomic DNA was extracted from isolated strains with the AccuPrep® Genomic DNA extraction kit (BIONEER, Korea) according to the manufacturer’s protocol.

DNA Labelling

Purified genomic DNA was quantified using a Nanodrop Spectrophotometer (Nanodrop Technologies, Thermo Scientific, USA) and approximately 300 ng of DNA was subjected to fluorescent labelling using the Bioprime DNA labelling system (Invitrogen Life Technologies, Burlington, Canada). Labelling efficiency and the percentage of dye incorporation was then determined by scanning the DNA sample in the Nanodrop spectrophotometer from 200 to 700 nm. Cy3 dye incorporation was calculated using a web-based percent incorporation calculator (available on web page http://www.pangloss.com/seidel/Protocols/percent_inc.html).

Virulence Oligonucleotide Microarray

The microarray version used in the present study, originally developed by Bruant et al. [24], was composed of 70-mer oligonucleotide probes targeting 264 virulence or virulence-related genes covering all known E. coli pathotypes.

Hybridizations and Data Acquisition

For each hybridization 500 ng of labelled DNA was dried under vacuum in a rotary desiccator without heating (Savant SpeedVac, ArrayIt, USA). Dried labelled DNA was resuspended in hybridization buffer (DIG Easy Hyb Buffer, Roche Diagnostics, Laval, Canada). Microarrays were pre-hybridized for at least one hour at 50 °C with a pre-heated pre-hybridization buffer containing 5× SSC, 0.1% SDS and 1.0% BSA. After pre-hybridization, the microarrays were hybridized with a solution that consisted of 25 μl of hybridization buffer, 20 μl of Bakers Yeast tRNA (10 mg/ml) (Sigma Aldrich, St. Louis, USA) and 20 μl of sonicated Salmon Sperm DNA (10 mg/ml) (Sigma Aldrich), mixed together with the labelled DNA which had previously been denatured. Microarrays were hybridized overnight at 50 °C in a SlideBooster (model SB800; Advalytix, Germany). After hybridization, stringency washes were performed with Advawash (Advalytix) using 1× SSC, 0.02% SDS preheated to 50 °C. Microarray slides were scanned with a ScanArrayLite fluorescent microarray analysis system (Perkin-Elmer, Mississauga, Canada) using with ScanArrayGx software (Perkin-Elmer, Foster City, USA). Fluorescent spot intensities were quantified with QuantArray Version 3.0 (Packard Bioscience, Boston, USA). All the microarrays were normalized using the same method. For each sub array, the mean value for each set of duplicate spotted oligonucleotides was divided by the correction factor taken from the negative controls spots. This value was then divided by the average of the empty spots to create a signal-to-noise ratio. Oligonucleotide spots with a signal-to-noise fluorescence ratio greater than the established threshold (3 in this case), were considered positive. These ratios were then converted into binary data where a value of 0 indicates a negative probe and a value of 1 a positive probe. A threshold of 3 was chosen because it best represented spot quantification. At least three arrays were hybridized to each strain and the six technical replicate points (two per array) were pooled. At least five probes of the six gene probes had to be positive before a positive score was considered.

Results

Strains Identification

The microarray data analysis of isolates confirmed the phenotypic features observed in the cultural and serotyping essays (lack of β-glucuronidase activity and delayed d-sorbitol fermentation, agglutination with O157:H7 specific monovalent antisera). The categorized virulence genes listed in Table 1 provide further support for the EHEC O157:H7 serotypes as both strains displayed positive hybridization signals for the complete locus of enterocyte effacement (LEE) gene probes (ler, eae, espA, espB, tir genes), two Shiga toxin probes (stx1 and stx2), the O157 O-antigen specific probe, various virulence plasmid (pO157) probes like katP in addition to other accessory virulence genes characterized (e.g. the type II secretion pathway gene etpD) in E. coli.

Table 1 List of main microarray positive virulence genes for E. coli O157:H7 strains isolated from two dromedaries in Teheran provincea
Full size table

Genetic Characterization

The E. coli isolates fell into two different phylogenetic groups. Strain 1 was from the D phylogroup (chuA positive and yjaA negative) whilst the strain 2 was from phylogroup B2 (chuA and yjaA positive). Although both isolates are O157 EHECs, strain 2 has approximately eight times the number of total positive virulence scores on the microarray (98 positive probe signals vs. 12, data not shown) and is just short of possessing a complete uropathogenic or meningitis-associated pathotype profile. The missing markers genes for these pathotypes are kpsM(II) or kpsM(III).

Discussion

The microarray technology applied to genetic characterization of E. coli proved suitable for the purpose of pathotyping field strains of camelid origin, confirming that the one-humped camel is a susceptible host of STEC infection, similar to other no-camel camelid species as Llama and Alpaca. The two isolates did belong to phylogenetic groups B2 and D, which have also been detected in cases of human infection. In fact, according to the classification established by Zhang et al. [22], the examined E. coli strains belong to the lineage I (presence of stx2 and nle genes), which are most commonly associated with human disease.

Comparative and epidemiological studies indicate that E. coli O157:H7 descends from the enteropathogenic E. coli O55:H7 (EPEC pathotype) by acquisition, in different evolutionary steps, of stx2 and stx1-encoding bacteriophages, plasmid pO157 and loss of their ability to ferment d-sorbitol and beta-glucoronidase activity [21, 28]. The production of Shiga toxins and ability to induce attaching and effacing lesions are considered essential in the pathogenesis of E. coli O157:H7 infection but, for inducing severe disease in humans, the possession of pO157 is required. Both strains examined in the present study have a complete set of Shiga toxin genes and the locus of enterocyte effacement genes. They also possess plasmid genes related to pO157 like the EHEC haemolysin gene (ehx) required for haemolysin synthesis, the EHEC-catalase/peroxidase gene (katP) that may help O157:H7 to colonize the host intestine in deprived oxygen conditions, the type II secretion system (etp), and the serine protease gene (espP) that influences the adherence to bovine intestinal epithelial cells and the degradation of human coagulation factor V, contributing to intestinal haemorrhages observed in HC patients [28].

No specific research has been performed in Iran on the presence of STEC strains in camels before the research described in this article. However, the STEC infections are present in Iran in humans and domestic animals, and O157:H7 serotype has been isolated in humans and sheep. The same factors (related to the STEC, to the environment and to the camel themselves) identified as possible explanations of the absence of STEC isolations (including E. coli O157:H7) in camels in northeast African countries [16], could be at work in Iran as well. However, our results can provide new evidence on the role of the camel in the human Shiga toxin-producing E. coli outbreaks.

Concerning the advantages of the use of microarrays-based technology, the recent outbreak of haemolytic uraemic syndrome in Germany and France, where a typical enteroaggregative E. coli had acquired the bacteriophage encoding stx2 gene [29], encourages the adoption of DNA microarrays-based technology as rapid method for the determination of virulence genes of E. coli isolates, before molecular sequencing. In addition, during health alert, the characterization of the virulence profiles of E. coli strains by DNA microarray technology can facilitate the activity of trace back investigations following STEC infections.