RFLP AND SEQUENCE-ANALYSIS OF CAMPYLOBACTER-JEJUNI AND CAMPYLOBACTER-COLI PCR PRODUCTS AMPLIFIED DIRECTLY FROM ENVIRONMENTAL-SAMPLES

Campylobacter jejuni and C. coil are difficult and laborious to isolat e and cultivate from food or environmental samples. We have developed a polymerase chain reaction (PCR) system spanning the intergenic regio ns between the flaA and flaB genes and yielding amplification products of variable sequence and slightly variable lengths, which was used fo r the typing of bacteria present in food and environmental samples. Th is procedure circumvents the necessity of cultivating bacteria and rel ies only on the isolation of the total DNA followed by a specific ampl ification step. Two typing schemes were developed The first one is bas ed on restriction fragment length polymorphisms (RFLP) obtained with t wo restriction endonucleases. The combination of digestion patterns an d amplimer lengths yielded II subtypes for the 24 strains of C. jejuni , 12 strains of C. coli, and 90 Campylobacter spp. not characterized t o the species level that were tested. The second scheme was based on d irect sequencing of the PCR amplification products. This allowed furth er 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 from Campylobacter c ells could be detected by PCR in water, surface swabs and sand and wer e typed by sequencing of the amplification products.