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Brief Communication |
Correspondence: 1Corresponding Author: Chitrita DebRoy, Gastroenteric Disease Center, Department of Veterinary and Biomedical Sciences, The Pennsylvania State University, 104 Wiley Lab, Wiley Lane, University Park, PA 16802
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Abstract |
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Key Words: Animals • detection • E. coli • Enterohemorrhagic • PCR • petting zoo • Shigatoxin-producing
During 2004 to 2005, 3 outbreaks of Escherichia coli infections occurred among agricultural fair, festival, and petting zoo visitors in North Carolina, Florida, and Arizona.3 A total of 173 cases, including 22 cases of hemolytic uremic syndrome, were reported in these 3 outbreaks. Although no fatalities occurred, illness primarily affected children who visited petting zoos at these events.3 The E. coli O157 outbreak in petting zoos in Florida has been linked to goats, sheep, and cows.3 Genetic fingerprinting of the bacteria reflected an identical strain of E. coli O157:H7, was the causative agent for most of the outbreaks.3 Escherichia coli O157:H7, referred to as enterohemorrhagic E. coli (EHEC), represents a subgroup of Shigatoxin-producing E. coli (STEC) that has been shown to cause bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans, and to pose considerable threat for human health. There were 11 published outbreaks of EHEC reported between 1966 through 2000.2 Other recent outbreaks of E. coli O157:H7 associated with farm visits occurred in Pennsylvania, Washington,5 Canada,7 and North Wales.13 The morbidity and the mortality associated with outbreaks of gastrointestinal illnesses caused by STEC has highlighted the threat they pose to public health. Therefore, monitoring the presence of E. coli in animals will assure prompt diagnosis and identify the source of infection that may assist in risk management. Conventional methods to detect bacteria in fecal samples include detection and identification of E. coli O157:H7 by propagation in sorbitol MacConkey agar culture, followed by serologic and biochemical tests,9 which are cumbersome, time consuming, and not very sensitive, because it is unable to detect non-O157 STEC.11,14 Recently, it has been shown that enzyme-linked immunoabsorbent assays may be more sensitive for detecting STEC strains than conventional culture methods.11 The advent of nucleic-acid based assay systems, e.g., polymerase chain reaction (PCR) has led to the emergence of improved, rapid, and reliable methods of microbial identification and surveillance.
The direct detection of pathogenic bacteria in fecal swabs is a challenging task, hampered by the presence of PCR-inhibitory substances, such as polysaccharides, bilirubin, and bile salts, that are frequently associated with feces. Furthermore, the presence of high numbers of indigenous microflora4,8 also makes it harder to detect the pathogenic strains. There, therefore, is a need to develop a sample preparation strategy that can effectively restore the pathogenic bacteria or the target DNA in the fecal sample. Although there are several reports on the detection and the identification of E. coli O157 from feces,4,6,8,10,12 no standard screening protocol has been described to detect STEC strains in fecal swabs of animals by using PCR methods. Animals carry both nonpathogenic and pathogenic strains of E. coli. While the pathogenic strains do not pose a threat to the health of the animal, they can be extremely harmful to humans, especially to children and immunocompromised individuals. The proximity to animals such as cows, goats, and sheep, coupled with poor hygiene and sanitation can contribute to an outbreak of gastrointestinal illness. The objective of the present study was to develop a PCR assay to screen for STEC in fecal swabs of animals that is sensitive, specific, rapid, and reliable. The method has been applied successfully for detecting STEC in petting zoo animals for risk management.
Rectal swabs from animals (n = 104), placed in BBLTM CultureSwab collection and transport media,a were obtained from a petting zoo. By using sterile scissors, the cotton part of the swab was cut and dropped into a test tube that contained 5 ml of Tryptic Soy Broth (TSB) media. The bacteria in the swab were grown overnight in an incubator shaker at 37°C. The samples (500 µl) were centrifuged in a microcentrifuge at 12,000 x g for 1 minute, and DNA was extracted from the cell pellet by using an Aqua-pure Genomic DNA kitb and the manufacturer's protocol. DNA lysis solution (300 µl) was added to the bacterial pellet and mixed well. The solutions were then incubated at 80°C for 5 minutes. Ribonuclease (1.5 µL) provided in the kit was added to each tube, mixed, and incubated further at 37°C for 45 minutes. A protein precipitation solution (100 µl) supplied by the manufacturer was then added. The solution was mixed and centrifuged at 13,000 x g for 3 minutes. The DNA in the supernatant was precipitated with 300 µl of isopropanol. The DNA was recovered by centrifuging the tubes at 13,000 x g for 1 minute and was washed once with 300 µl 70% ethanol. The DNA was resuspended in sterile double-distilled water (50 µl) and was incubated at 65°C for 30 minutes and stored at –20°C until use. For determining the sensitivity of the PCR assay, chicken feces (1 g) was added to 10 ml of sterile phosphate buffered saline solution, and the mixture was vortexed until all large fecal particles were homogenized. The fecal mixture (90 µl) was pipetted into sterile 2-ml microcentrifuge tubes and inoculated with 10 µl E. coli O157:H7 (ATCC strain 43895) at concentrations ranging from 0 to 106 CFU. Sterile swabs were dipped into each of the serially diluted inoculum to allow all liquids to absorb completely in the swabs. The swabs were then cut aseptically into 5-ml TSB media to simulate the condition of samples from petting zoo. The sample tubes were incubated at 37°C overnight, with aeration. DNA was extracted from the cell pellet as described above.
For amplification of E. coli genomic DNA, the primers for wecA gene, as listed in Table 1, were used. The gene is essential for enterobacterial common antigen and has been found to be specific for detecting E. coli.1 Two sets of multiplex PCR were conducted, one for detecting Shiga toxins 1 and 2 (stx1, stx2) and eae and a second multiplex for detecting the rfbE gene of O157 and the fliC gene for H7. The primersc used for both the multiplex assays are listed in Table 1. Reaction contents for each PCR (11-µl total reaction volume) consisted of 3 µl of template DNA, 0.5 µM of primers, 0.18 mM concentration of each of the 4 deoxynucleotide phosphates (dNTPs), 2 mM MgCl2 (for E. coli genomic DNA amplification), and 4 mM MgCl2 (for multiplex reactions), 0.4 IU of Taq DNA polymerase,d 50 mM Tris (pH 8.3), 250 µg/ml Bovine Serum Albumin (BSA), 2% sucrose, and 0.1 mM Cresol Red. The PCR was performed in a RapidCyclere by following a specific Rapid-Cycle DNA amplification protocol15 used for this particular thermocycler. The reactions consisted of 30 cycles of template denaturation at 94°C for 0 s for all reactions. Primer annealing and extension times are indicated in Table 2. The amplification products were subjected to electrophoresis in 1% agarose gels at 200 V for 30 minutes for all assays. The gels were stained with ethidium bromide and were visualized under ultraviolet light. Positive samples were identified based on the presence of bands of the expected sizes compared with results with a positive control strain (E. coli ATCC 43895).
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a. Transport Media, Becton Dickinson, NJ.
b. Aqua-pure Genomic DNA kit, Bio-Rad Laboratories, CA.
c. Primers, Integrated DNA Technologies Inc., Coralville, IA.
d. Taq DNA polymerase, PGC Scientific, Gaithersburg, MD.
e. RapidCycler, Idaho Technologies Inc., Salt Lake City, UT.
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