Campylobacter jejuni in gastroenteritis: Detection and mechanisms of intracellular survival

Abstract

The fastidious bacterium Campylobacter jejuni has recently been identified as a leading cause of human bacillary enteritis. Delays in recognition of this important pathogen reflect inadequate isolation techniques, which cannot recover environmentally stressed viable but non-culturable forms. Therefore, a non-culture based detection systems for C. jejuni would be invaluable. Studies were undertaken to develop a PCR based detection assay for C. jejuni. Fingerprints enriched for repetitive C. jejuni chromosomal elements were generated using arbitrarily primed PCR. Dot blot screening of fingerprint products for specificity to C. jejuni identified a 496 bp product which hybridized with all C. jejuni isolates examined. No binding to other Campylobacter species or enteric genera screened was observed. The product was cloned, sequenced, and primers synthesized to three overlapping regions of the probe. A primer pair was identified which directs amplification of a 265 bp product from C. jejuni alone. Sensitivity studies demonstrated that the C. jejuni specific PCR generated product from as few as 100 lysed bacteria. The ability of C. jejuni to penetrate normally non-phagocytic host cells is believed to be a key virulence determinant. Kinetics of C. jejuni of intracellular survival have been described and indicate that the bacterium can persist and multiply within epithelial cells and macrophages in vitro. Studies by Pesci et al. demonstrate that super-oxide dismutase contributes to intra-epithelial cell survival, suggesting that bacterial factors which combat reactive oxygen species enable the organism to persist inside host cells. Experiments were conducted to determine the contribution of catalase to C. jejuni intracellular survival. The gene encoding catalase (katA) was cloned via functional complementation, sequenced, and isogenic katA mutant strains constructed. Kinetic studies of bacterial viability indicate that catalase provides resistance to hydrogen peroxide in vitro but does not have a role in intra-epithelial cell survival as growth curves for katA mutant and wild type strains generated from long term culture within HEp-2 cells are roughly identical. Catalase does however contribute to intra-macrophage survival as katA mutants were recovered from cultured peritoneal macrophages at significantly reduced numbers (p = 0.00246) relative to the wild type strain after 72 hr incubation with these cells.