Antimicrobial Resistance and Virulence Genotypes of Helicobacter pylori in patients with Chronic Gastritis Luis Eduardo Bravo 1 , Mercedes Figueroa 2 , Alvaro Pazos Moncayo 2, 3 1 Department of Pathology, School of Medicine, Universidad del Valle, Cali, Colombia. 2 School of Basic Sciences, Universidad del Valle, Cali, Colombia. 3 Department of Biology, Universidad de Nariño, Pasto, Colombia . Universidad de Nariño Universidad del Valle Infection with Helicobacter pylori is the main recognized cause of gastric cancer. Colombians living in coastal Tumaco have a lower incidence of Helicobacter pylori-associated gastric cancer compared with residents of Pasto in the high Andes. A strategy for prevention of gastric cancer is chemoprevention, but the eradication of the organism with multiple antibiotic treatments is not entirely successful and is dependent in part on the resistance of the organism to the action of the antibiotics. This study aimed to detect amoxicillin and clarithromycin resistance and to determine the virulence of the infecting strain in subjects with Helicobacter pylori-associated gastritis in a southern region of Colombia. Gastric mucosa biopsy samples were collected in Tumaco, Colombia from 203 subjects undergoing endoscopy of the upper gastrointestinal tract due to dyspeptic symptoms (18-65 years old), 71.9% were women. Eight gastric mucosal biopsy samples were obtained by endoscopy; four of them for histological evaluation with Hematoxylin and eosin, and Giemsa stains; two for H. pylori isolation and in vitro antimicrobial susceptibility studies; and the other two to compare genomic variability of the isolates. H. pylori was cultured from antral biopsies and genotyped for cagA and vacA by PCR. Non-atrophic gastritis was higher en women (84,2%) than men (71,9%). In contrast, the multifocal atrophic gastritis and intestinal metaplasia were more frequent in men (10.5% vs. 5.5%) and (17.5% vs 11.8%), respectively. The prevalence of H. pylori infection was 88.7%. The infection by H. pylori was more frequent in patients with atrophic gastritis 92.9%, than in patients with non atrophic gastritis 87.8%. There were not differences by sex or gastritis type. Table 1. The 73% of the isolates had a virulent genotype, cagA (+) vacA (s1/m1), cagA (+) vacA (s1/m2), table 2. . Background Conclusions Results Methods Figure 1. Map of Colombia showing the location of the population involved in the study: Tumaco, low risk of gastric cancer. Table 1. Tumaco-Nariño-Colombia. Prevalence of H. pylori infection by histologic diagnosis and sex. Male Female Total HISTOLOGIC DIAGNOSIS n % H. pylori n % H. pylori % H. pylori Non atrophic gastritis 41 90,2 123 87,0 87,8 Multifocal Atrophic gastritis 6 83,3 8 100 92,9 Intestinal Metaplasia 10 80,0 14 100 91,7 Gastritis NOS 0,0 1 100 100 TOTAL 57 87,7 146 89,0 88,7 McFarland standards were used for standardization of inocula for susceptibility testing. The calibration curve was constructed with 26 standards, from 0.5 through 3.0 with increment of 0.1 Each standard was read a dozen times and the absorbance values were obtained at 630 nm. H. pylori inocula corresponding to McFarland turbidity standard Number 2 was selected for performing antimicrobial susceptibility testing Graph 2. Optical density of the McFarland Standard Numbers using Standard pipettes an Multichannel pipettes. cagA(+) – 183 pb ureA – 167 pb vacA m1/m2 – 406/476 pb vacA s1/s2 – 176/203 pb Graph1. H. pylori was genotyped for cagA and vacA by PCR The graph 2 shows the absorbance of the 26 McFarland standards, each standard was read a dozen times at 630 nm. The intraclass correlation coefficient was 0,97 and 0,99 for measures made with standard pipette and multichannel pipette, respectively. The optical density of the inoculum at wavelength of 630 nm was 0.24 equivalent to 2.0 Mc Farland standard with a cell density of 4.6 x10 7 CFU/ml, consistent with the findings in previous studies. The graph 1 shows the agarose gel electrophoresis of PCR products from the ureA, cagA gene and polyacrilamyde electrophoresis of the s and m alleles of vacA Table 2. Prevalence of Antibiotic Resistance by gastritis type and Helicobacter pylory genotype Variable n Prevalence of antibiotic resistance Amx-Cla Amx Cla Gastritis type Multifocal atrophic 15 20,0% 40,0% 20,0% Non atrophic 74 16,2% 31,1% 28,4% H. pylori genotype Virulent 65 12,3% 29,2% 20,0% Less virulent 24 29,2% 41,7% 41,7% Total 89 16,7% 32,2% 26,7% The table 2 shows: The 73% of the isolates consisted of virulence genotypes. Resistance to amoxicillin and clarithromycin was 32% and 27%, respectively, and 16.7% were resistant to both antibiotics. Resistance to Amoxicillin, Clarithromycin, and, to both antibiotics, was higher in the less virulence (29%, 42%, 42%) compared to the more virulence isolates (12.3%, 30%, 20% respectively). No association was observed between virulence genotypes and type of chronic gastritis. The cagA (+) vacA (s1/m1), cagA (+) vacA (s1/m2) genes were considered as virulent while the cagA (-) vacA (s2/m2) were considered less pathogenic genotypes. Minimum inhibitory concentrations (MICs) were performed following agar dilution methods. Agar dilution was performed by using twofold increments (across a range of 0.25 to 4 mg/ml) of the antimicrobial agents incorporated in Mueller-Hinton agar supplemented with 5% defibrinated sheep blood. The plates were incubated microaerophilically at 37°C for 72 h. Isolates with MICs ≥ 1 μg/ml were considered resistant to both amoxicillin and clarithromycin.. Intraclass Correlation Coefficient=0.97 Intraclass Correlation Coefficient=0.99 .1 .2 .3 .4 .5 .6 .7 .8 .9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 .5 .6 .7 .8 .9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 Standard pipette Multichannel pipette McFarland Standard Number Low-risk area MW 1 2 3 4 5 6 7 8 9 10 11 12 MW 200 170 180 ECUADOR Pacific Ocean ECUADOR Pacific Ocean • Measurement of McFarland turbidity standards with spectrophotometry is a reliable method to standardize the inoculum density for antimicrobial susceptibility testing of H. pylori strains. • Less virulence genotypes had higher resistance to both antibiotics. • The presence of antibiotic resistance in high prevalence areas of H. pylori infection in Colombia is worrisome. This study was supported by Universidad del Valle-Colombia, Universidad de Nariño-Colombia and a grant from the Departamento Administrativo de Ciencia, Tecnología e Innovación COLCIENCIAS (1106-40820549, Contrato RC. No.301-2007). The authors declare no conflict of interest. ACKNOWLEDGEMENTS