The pellet was re-suspended in 200 μl of PBS, 25 μl of the H. pylori cells were mixed with 15 μl of the plasmid at a final concentration of 30 ng/μl. The mix was plated on Brucella agar supplemented with 5% sheep blood (BAB) and incubated as described above. After 24 h, the colonies were collected with a sterile swab Fostamatinib cost and diluted

in series from 10-1 to 10-6 in 900 μl Brucella broth (BB). The first four dilutions were spotted on selective media: BAB + Str [20 μg/mL], or Km [10 μg/mL], depending of the phenotype to be selected. The two last dilutions were inoculated onto non-selective BAB plates. After 5 days of incubation, colony-forming units (CFU) were counted on both the selective and non-selective plates, and transformation efficiency was calculated by comparing CFU numbers on the two types of media. CFU counts used for this analysis this website were over a range of 30 – 300, to maximize statistical accuracy [67]. Differences in the rates of transformation were

compared using the t-test, and the variance among strains was determined using the F-test. Horizontal DNA transfer during co-culture To evaluate the ability of H. pylori hspAmerind or hpEurope strains to obtain DNA from each other, the co-culture assay was performed as previously described [32]. The strains and plasmids used for these experiments are listed in Table 3. In summary, in addition to the single plasmid strains explained above, we produced double-resistant hspAmerind and hpEurope strains by transforming the single resistant strains described above with an additional suicide plasmid, pAD1-Cat [32]. This suicide plasmid, which carries a ureAB fragment from H. pylori strain 60190 with a central exogenous cat cassette (1127 bp), gets incorporated into the genomic ureA locus, creating chloramphenicol resistant (CmR) strains [32]. To determine the rates of DNA transformation from Baricitinib a donor hspAmerind strain to a recipient hpEurope strain, a single plasmid hspAmerind

strain (99–33 or 99–35) with resistance to antibiotic “”X”" (used as a donor) and a double plasmid hpEurope strain (08–97 or 08–100) with resistance to antibiotics “”Y/Z”" (used as recipient), were co-cultured; transformants were selected by double or triple antibiotic resistance: “”X/Y”" or “”X/Y/Z”", respectively. To investigate the rates of transformation from a donor hpEurope strain to a recipient hspAmerind strain, we performed the same experiment but with the reverse phenotype, i.e. donor = hpEurope with single resistance “”X”"; recipient = hspAmerind with double resistance “”Y/Z”", and transformants with double or triple antibiotic resistance: X/Y”" or “”X/Y/Z”", were evaluated.