Antimicrobial Resistance and Virulence Genotypes of Helicobacter

pylori in patients with Chronic Gastritis

Luis Eduardo Bravo1, Mercedes Figueroa2, Alvaro Pazos Moncayo2, 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 x107 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

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Standard pipette Multichannel pipette

Optical density

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