APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 0099-2240/99/$04.0010 Apr. 1999, p. 1636–1643 Vol. 65, No. 4 Copyright © 1999, American Society for Microbiology. All Rights Reserved. Detection of Small Numbers of Campylobacter jejuni and Campylobacter coli Cells in Environmental Water, Sewage, and Food Samples by a Seminested PCR Assay ASTRID S. WAAGE, 1 * TRAUTE VARDUND, 1 VIDAR LUND, 2 AND GEORG KAPPERUD 1,3 Department of Bacteriology 1 and Department of Environmental Medicine, 2 National Institute of Public Health, 0403 Oslo, and Department of Pharmacology, Microbiology, and Food Hygiene, Norwegian College of Veterinary Medicine, 0033 Oslo, 3 Norway Received 11 March 1998/Accepted 20 January 1999 A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples. Water and sewage samples were fil- tered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were pre- pared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campy- lobacter flagellin genes, flaA and flaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8,700 heterotrophic organisms per ml and 10,000 CFU of coliform bac- teria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes nec- essary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as <3 CFU per g of food could be detected with samples subjected to overnight enrichment, while variable results were obtained for samples analyzed without prior enrichment. This rapid and sensitive nested PCR assay provides a useful tool for specific detection of C. jejuni or C. coli in drinking water, as well as environmental water, sewage, and food samples containing high levels of background organisms. Campylobacter jejuni was isolated from human stools for the first time in 1972 (11). Since then, the development of selective stool culture media has revealed that thermophilic Campylo- bacter species, particularly C. jejuni and Campylobacter coli, are common causes of diarrhea in most parts of the world (2). It is estimated that in the United States, Campylobacter strains cause more than two million cases of diarrhea annually, which is similar to the number of cases of Salmonella enteritis (49). The natural habitats of most Campylobacter species are the intestines of birds and other warm-blooded animals, including seagulls and several other wild birds (22). In most cases the host is a carrier that does not exhibit symptoms, but it may have acquired immunity through an earlier Campylobacter infection (41). Campylobacter cells may enter the environment, including drinking water, through the feces of animals, birds, or infected humans. These organisms are not able to grow but may survive in the environment for several weeks at temperatures around 4°C (8, 9). Different kinds of poultry, especially broiler chick- ens, are some of the most important sources of Campylobacter infection in humans, and the water supply has been shown to be a prominent factor in colonization of chickens on broiler farms (23, 42). In addition, contaminated drinking water has been the cause of several large outbreaks of campylobacter enteritis (9, 51). Eleven of 57 reported outbreaks of Campy- lobacter infection in the United States between 1978 and 1986 were waterborne (53), and all of these outbreaks were related to drinking unboiled surface water, contamination of ground- water with surface water, inadequate disinfection, or contam- ination by avian wildlife feces. The infective dose of Campylobacter cells is very small; it has been estimated that ca. 500 cells of C. jejuni can cause human illness (7, 44). This means that even very small numbers of Campylobacter cells in water or food may be a potential health hazard. Moreover, the presence of Campylobacter cells is not correlated with the level of microorganisms that are indicators of fecal contamination of water (10). Thus, sensitive methods are needed to detect Campylobacter cells in environmental and drinking water sources. There are several problems concerning detection of Campy- lobacter cells in water, including the small numbers and slow growth rates of the organisms. The traditional methods cur- rently used are time-consuming and laborious, requiring pro- longed incubation, selective enrichment to reduce the growth of the background flora, and biochemical identification. Campylobacter cells may also enter a viable but nonculturable state due to starvation and physical stress, which may explain the failure of the culture techniques to isolate the organisms from contaminated water samples implicated in outbreaks of infection (21, 45). The PCR is an extensively used genetic approach for detect- ing infectious agents (13, 50), and a number of PCR assays for detecting Campylobacter cells have been developed during the past few years. These assays have been used to detect Campy- lobacter cells in poultry (12, 15–17, 29, 31, 61), feces (28, 37, 39, 43, 57), dairy products (1, 12, 20, 60), sewage (27), and water (18, 20, 26, 38). In this paper, we describe a nonselective enrichment proce- dure followed by a rapid and simple DNA preparation step and a seminested PCR assay for detection of C. jejuni and C. coli in water and food samples. We found that the seminested PCR assay, which was described previously by Wegmu ¨ller et al. (60), specifically amplifies the intragenic flaA-flaB sequence from C. jejuni and C. coli. The sensitivity of the procedure was * Corresponding author. Mailing address: Department of Bacteriol- ogy, National Institute of Public Health, P. B. 4404, Torshov, N-0403 Oslo, Norway. Phone: 47 22 04 22 00. Fax: 47 22 04 25 18. E-mail: as [email protected]. 1636 Downloaded from https://journals.asm.org/journal/aem on 13 August 2023 by 2402:800:62f0:6afe:60c3:27bd:6bc:a7ea.
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