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
.3.4
.5 .6 .7 .8 .9 1
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1.9 2
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2.9 3 .5 .6 .7 .8 .9 1
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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