Response of intestinal epithelial cells to Trichuris suis excretory-secretory products and the influence on Campylobacter jejuni invasion under in vitro conditions.

We previously developed a swine animal model in which natural host resistance to Campylobacter jejuni is altered by experimental infection with low numbers of the nematode Trichuris suis. Pigs naturally colonized with C. jejuni experience colitis because of the invasion of the bacterium approximately 21 days after exposure to T. suis. To better understand the mechanism of T. suis-dependent C. jejuni colitis, we evaluated the effects of T. suis excretory-secretory products (ESPs) on intestinal epithelial cells (IECs) and the influence of ESP on C. jejuni invasion in IECs under in vitro conditions. Viability assays revealed a dose-dependent cytotoxic response in ESP-treated IECs, particularly IPEC-1 and INT407 cells. Transepithelial electrical resistance dropped significantly in IPEC-1 cells treated on apical and basolateral surfaces, but not in those treated only on apical surfaces. Using the gentamicin-killing assay, reduced numbers of intracellular C. jejuni were recovered from IECs treated with ESP at 1 mg protein/ml concentration. This observation can be at least partially explained by a novel antibacterial activity in ESP. Contrary to our hypothesis, ESP at subtoxic concentrations did not enhance invasion. In addition to mechanical damage from worms, these results suggest that soluble products released by T. suis contribute to IEC damage at the site of worm attachment.

Trichuris suis: detection of antibacterial activity in excretory-secretory products from adults.

Antibacterial activity was detected in excretory-secretory products (ESP) of adult Trichuris suis cultured in vitro in serum-free media. Gram-negative bacteria (Campylobacter jejuni, Campylobacter coli, and Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) were sensitive to ESP. Susceptibility was dependent on the concentration of ESP but not on the inoculum size. Preliminary assessment of the mode of action suggests a bacteriocidal mechanism. This antibacterial activity was heat stable and resistant to digestion with pronase E and trypsin. Based on ultrafiltration experiments, the activity is less than 10,000 MW. This excreted/secreted antibacterial activity from T. suis is likely a component of a humoral defense system for this helminth.

Identification of ciprofloxacin-resistant Campylobacter jejuni by use of a fluorogenic PCR assay.

Fluoroquinolones are one class of antimicrobial agents commonly used to treat severe Campylobacter jejuni infection. C. jejuni strains resistant to high levels of the fluoroquinolone ciprofloxacin (MIC >/=16 microg/ml) have been predominantly characterized with a C-->T transition in codon 86 of gyrA. The gyrA gene encodes one subunit of DNA gyrase, which is a primary target for fluoroquinolone antibiotics. This study establishes a rapid PCR-based TaqMan method for identifying ciprofloxacin-resistant C. jejuni strains that carry the C-->T transition in codon 86 of gyrA. The assay uses real-time detection, eliminating the need for gel electrophoresis. Optimization of the assay parameters using purified Campylobacter DNA resulted in the ability to detect femtogram levels of DNA. The method should be useful for monitoring the development of ciprofloxacin resistance in C. jejuni. Compiled nucleotide sequence data on the quinolone resistance-determining region of gyrA in Campylobacter indicate that sequence comparison of this region is a useful method for tentative identification of Campylobacter isolates at the species level.