Underrepresentation selleck inhibitor was defined when the O/E ratio value was lower than 0.5, and the Chi square value was significant (p values <0.005). Similarly, the sites were overrepresented in the sequences when the ratio O/E value was ≥2, and the Chi square value was significant (p values <0.005). In the case of WGS, we calculated Chi square only for the bacterial populations that contained more than one strain: hpEurope (26695, HPAG1, P12 and G27), and hspAmerind (V225 and Shi470), but not for hpAfrica1

with just one strain (J99). Differences in the frequency of observed and expected cognate recognition sites among H. pylori populations were examined using a pair-wise comparison test based on the medians (Wilcoxon rank sum test). For the 4 populations studied (hspWAfrica, hpEurope, hspEAsia, and hspAmerind), there were 6 possible pair-wise analyses. The p-value for the Wilcoxon rank sum test for each pair indicates the relationships among the haplotypes. Principal component analysis (PCoA) [64] was performed to detect patterns of cognate recognition profiles among strains. Non-parametric multidimensional scaling (NMDS), was used to visualize the variation

in two dimensions [65]. NMDS does not assume linearity this website of the data and does not require data transformation, which represents selleck products advantages over other classical ordination methods. The ordination algorithm for NMDS clusters groups with similarities, and based on ranked similarity distances; an iterative search for the least stress position in k-dimensions is done [65]. In vitro analysis Bacterial strains for restriction analysis Nine hspAmerind strains from Amerindian hosts (N = 9), and nine hpEurope strains from European (N = 4) and Mestizo (N = 5) hosts were used for this analysis. The 18 frozen cultures of H. pylori strains, maintained at -80°C,

were thawed and inoculated onto Brucella agar plates supplemented with 5% blood [66]. Plates were incubated at 37°C in a microaerobic atmosphere (5% CO2) in a humid chamber for 3 to 5 days [66]. H. pylori identity was confirmed by Gram staining and detection of urease and catalase activity. DNA was extracted from H. pylori cultures using the Wizard® Genomic DNA Purification Kit (Promega, MA), with the protocol MycoClean Mycoplasma Removal Kit specified by the manufacturer for gram-negative bacteria. Restriction assays Restriction endonuclease digestions were performed on the genomic DNA from 18 strains, using 16 commercially available restriction enzymes (New England BioLabs, MA) that were sensitive to methylation of the recognition sites (Additional file 1: Table S3). These enzymes were chosen because resistance to each has been reported in at least one H. pylori strain [42]. In our experiments, we controlled for the lack of restriction activity due to presence of inhibitors or high salt, by running control DNA from an H. pylori strain with a known restriction profile [18, 42].