Cycle parameters were: initial denaturation at 92°C, 5 min; 35 cycles of denaturation at 92°C for 30s, annealing for 1 min, and extension for 1 min at 72°C; 7 min final extension; storage at 4°C. Amplification products were visualized by agarose gel electrophoresis and ethidium bromide staining. One gene pair, cj1318 and cj1336, had extensive overlapping regions of DNA sequence identity. The primers obtained could not differentiate the two genes; for the purposes of our discussion, positive results were
taken to mean that both loci were present, though this has not been unambiguously demonstrated. PCR was undertaken to detect the CJIE1 prophage and ORF11 from CJIE1. The PCR GANT61 chemical structure reaction primers and conditions have been described previously . An amplification product of approximately 750 bp signified the presence of the CJIE1 prophage while a larger amplification Blebbistatin nmr product of approximately 1700 bp indicated the presence of the ORF11 indel. A total of 496 Campylobacter spp. isolates
were tested using this PCR method. Adherence and invasion assays Assays were done according to the methods of Malik-Kale et al. , except that wells were seeded with 2 × 107 INT-407 cells the day before the assay to give a newly confluent monolayer at the time the assay began. Two strategies were used to perform the adherence and invasion assays. In the first series of experiments only two C. jejuni test isolates were assessed in each experiment along with the C. jejuni 81–176 and E. coli Top 10 control strains. This was done in order to manage the timing of steps and reduce the possibility of technical errors. Almost all of these experiments were done by a single technologist and the INT-407 cells used were between passages 65 – 120. Furthermore, a gentamicin concentration of 750
μg/ml was used to kill extracellular bacteria. A second series of experiments was done to compare the adherence and invasion of all isolates and controls in a single experiment. Fresh INT-407 cells were second obtained and used between passages 5 – 20. For these later experiments, the concentration of gentamicin was reduced to 500 μg/ml based on testing of the THZ1 solubility dmso strains used. There were no obvious differences in results using either concentration of antibiotic. Results from all assays were used to create Figure 2 and perform the statistical analyses. Similarly, results from the second series of experiments were summarized in Table 2 to show the variability between experiments and common trends when comparing isolates carrying the CJIE1 prophages versus the isolate without the prophage. Values for percent adherence (%A) and percent internalization divided by adherence (%I/A) were described previously . The value for percent adherent was obtained from by dividing the values obtained for adherent bacteria (cfu/ml) by the values obtained for input bacteria (cfu/ml) and multiplying by 100.