B-196. Prophage Sequences Are more Frequently Present in Cag PAI Negative than Cag PAI Positive Isolates of Helicobacter pylori

J. B. Miller, Jr.1, K. R. Ridgway1, A. S. Gayek1, X. Liu1, K. Dondeti1, H. J. Schwab1, D. M. Theurer1, R. M. Peek, Jr2, M. H. Forsyth1;
1Coll. of William & Mary, Williamsburg, VA, 2Vanderbilt Univ. Med. Ctr./VAMC, Nashville, TN.

Background: Helicobacter pylori colonizes the human stomach and causes gastroduodenal diseases ranging from ulcer disease to cancer. A major virulence determinant of this pathogen is the cag Pathogenicity Island (cag PAI) and isolates possessing this element are more frequently associated with severe disease outcomes. We sought to identify sequences among isolates lacking this virulence determinant to define markers for isolates less likely to cause severe diseases. Method: We performed subtractive hybridization to create a plasmid library from a cag PAI negative isolate, J68. Genome subtraction was accomplished using two completely sequenced cag PAI positive strains, 26695 and J99. We screened putatively unique sequences in a two-step manner by PCR first using a battery of 36 H. pylori isolates (23 cag+, 13 cag-). Sequences yielding significantly higher genopositivity in cag- isolates were screened against a second battery of 17 isolates (8 cag+, 9 cag-). Results: Sequence analysis of this library identified six sequences absent in the genomes of the sequenced cag+ H. pylori strains. Three of these (HS8, P7, and M81) are more frequent in cag PAI negative isolates. Screening additional isolates demonstrated that P7 and M81 were present significantly more frequently (70% and 52%, respectively) among cag- than among cag+ isolates (35.5% and 13%, respectively) (p<0.05). Interestingly, the M81 sequence represents an ortholog of orf6 of H. acinonchysis prophage II. Genome walking from the M81 sequence in H. pylori J68 revealed an adjacent orf orthologous to orf7 of this prohage. Conclusion: We identified genetic sequences in H. pylori strain J68 that are more frequent among clinical isolates of lesser virulence. These genes may be adaptive in hosts infected with cag-strains of H. pylori, and may have been selected against in strains possessing the cag PAI and the accompanying increased inflammatory lifestyle. Despite the absence of prophages in the three sequenced strains of H. pylori, we demonstrate that a prophage exists in 52% of cag PAI negative strains of H. pylori, but in only 13% of cag PAI positive ones (p <0.05).