RFLP and Sequence Analysis ofCampylobacter jejuniandCampylobacter coliPCR Products Amplified Directly from Environmental Samples

Abstract

Campylobacter jejuniandC. coliare difficult and laborious to isolate and cultivate from food or environmental samples. We have developed a polymerase chain reaction (PCR) system spanning the intergenic regions between the flaAand flaBgenes and yielding amplification products of variable sequence and slightly variable lengths, which was used for the typing of bacteria present in food and environmental samples. This procedure circumvents the necessity of cultivating bacteria and relies only on the isolation of the total DNA followed by a specific amplification step. Two typing schemes were developed. The first one is based on restriction fragment length polymorphisms (RFLP) obtained with two restriction endonucleases. The combination of digestion patterns and amplimer lengths yielded 11 subtypes for the 24 strains ofC. jejuni, 12 strains ofC. coli, and 90Campylobacterspp. not characterized to the species level that were tested. The second scheme was based on direct sequencing of the PCR amplification products. This allowed further discrimination of strains which could not be distinguished by RFLP. Forty-six strains selected from all RFLP types could be grouped into 17 sequence types. The described approach was applied to environmental samples obtained from henhouses. DNA originating fromCampylobactercells could be detected by PCR in water, surface swabs and sand and were typed by sequencing of the amplification products.