After the 35th cycle, the extension step was prolonged for 10 min in order to complete synthesis of all strands after which the samples were kept at 4°C until analysis. A negative control lacking of the DNA template was included in each experiment. The H. pylori strains used as RXDX-101 research buy positive controls in the PCR tests included H. pylori ATCC 43504 and H. pylori ATCC 49503. Detection of PCR products was performed

by gel electrophoresis. Samples (5 μL) of final PCR products were loaded onto 1.5% agarose gel and subjected to electrophoresis in 1X TAE (0.04 mol/L Tris–acetate, 0.001 mol/L EDTA) buffer for 60–90 min at 100 V. The gels were stained with ethidium bromide and photographed under UV light trans-illumination. A 100-bp DNA ladder (BioLab New England, Celbio, Milan, Italy) was included on each gel as a molecular size standard. Susceptibility testing The minimum inhibitory concentration (MIC) was assayed by the standard agar dilution method according to the AZD5363 guidelines of the Clinical and Laboratory Standards Institute (CLSI) [29] using CB. Twofold

serial dilutions of the compound tested ranging from 0.016 μg/mL to 1.024 μg/mL were used. Frozen stock cultures were thawed and subcultured on CB and grown for 3 days under microaerophilic conditions. Bacterial growth was taken from the plates, resuspended in BB and grown under shaking (125 rpm) at 37°C for 24 h. H. MEK inhibitor pylori cultures in the exponential phase of growth were diluted with BB to contain about 5 × 107 CFU/mL by adjusting the turbidity of the suspension to match the MacFarland no. 1 standard. Ten-microliter

aliquots of the suspension were inoculated on CB containing twofold serial dilutions of the compound tested. Compound-free click here CB media were included in each experiment to confirm the viability of the inoculum and to observe the growth of any contaminants. CB incorporating twofold serial dilutions of the solvent dimethil sulfoxide was included as a growth control to ensure that the viability of the H. pylori strains was not affected by the dimethil sulfoxide used to dissolve the compound. All plates were incubated at 37°C in a microaerophilic atmosphere and examined after 3 days. For quality control, H. pylori ATCC strains 43504 and 49503 were tested in each run. Amoxicillin (Sigma Aldrich S.r.l., Italy), and clarithromycin (Abbott S.p.A., Italy), were used as control compounds for comparative analyses. According to CLSI breakpoints, the resistance breakpoints were 0.5 μg/mL for amoxicillin and 1 μg/mL for clarithromycin [29]. The MIC was considered the lowest concentration at which the compound inhibited the development of visible bacterial growth on the agar plates. All MIC determinations were performed in duplicate for each strain. Results To type the H. pylori strains isolated from the patients examined in this study, we amplified by PCR different alleles of the genes of the two major virulence factors of this bacteria, cagA and vacA.