Virulence factors
The presence of the genes
vacA,
cagA,
cagE and
iceA were investigated in all 155 clinical isolates positive for 16S rRNA and for
glmM. Based on the
vacA and
iceA genotypes, 138 (89%) specimens were colonized by a single
H. pylori strain. More than one strain was detected in 17 isolates and none of these patients were included in either the analysis of the relationship between clinical disease with the virulence factors or with antibiotic resistance. Since the prevalence of
H. pylori infection, in our geographic region, reaches 80%, such prevalence of multiple infection would be expected [
20].
The
vacA s and m genotype could be identified in all cases studied. Most of the isolates had the s1 signal sequence allele (62%). The prevalence of
vacA s1 was significantly higher in
H. pylori isolates from PUD patients (χ
2 = 14.3 p = 0.0002). The
vacA m1 allele was found in 41 (30%) and the m2 allele in 97 (70%) of the samples. There was no relationship between
vacA m and clinical outcome. Considering the
H. pylori isolates with only one single genotype (138), three mosaic combinations were found: s1m1 (30%), s1m2 (32%), and s2m2 (38%). Statistical analysis showed a significant association between PUD and s1m2 (χ
2 = 15.67;
p = 0.0004), and between GERD and the s2m2 genotype (χ
2 = 16.89;
p = 0.0002, Table
1).
Table 1
The vacA, iceA, cagA and cagE status of H. pylori strains and clinical outcome
vacA
| | | | |
s1 | 57 (78)* | 25 (54) | 4 (21) | 86 (62) |
s2 | 16 (22) | 21 (46) | 15 (79)** | 52 (38) |
m1 | 27 (37) | 11 (24) | 3 (16) | 41 (30) |
m2 | 46 (63) | 35 (76) | 16 (84) | 97 (70) |
s1m1 | 27 (37) | 11 (24) | 3 (16) | 41 (30) |
s1m2 | 30 (41)* | 14 (30) | 1 (5) | 45 (32) |
s2m2 | 16 (22) | 21 (46) | 15 (79)** | 52 (38) |
iceA
| | | | |
iceA1 | 12 (16) | 6 (13) | 3 (16) | 21 (15) |
iceA2 | 55 (75) | 32 (70) | 15 (79) | 102 (74) |
iceA - | 6 (9) | 8 (17) | 1 (5) | 15 (11) |
cagA
| | | | |
cagA + | 55 (75)* | 25 (54) | 9 (47) | 89 (65) |
cagA - | 18 (25) | 21 (46) | 10 (53) | 49 (35) |
cagE
| | | | |
cagE + | 60 (82) | 39 (85) | 17 (90) | 116 (84) |
cagE - | 13 (13) | 6 (15) | 2 (10) | 22 (16) |
The
iceA1 genotype was detected in 21 (15%) of the 138 isolates analyzed, while
iceA2 was found in 102 cases (74%). Fifteen isolates (11%) did not yield any PCR product for
iceA (Table
1). There was no relationship between the size of the
iceA2 amplicon and the patient's disease. In contrast to results obtained by others,
iceA2 was the most frequent genotype detected in our population [
21,
22]. In this study, the
iceA type was not associated with the clinical outcome, as previously described [
11,
21].
With regard to the
cag PAI, the
cagA gene was present in 89 (65%) of the 138
H. pylori isolates, whereas the c
agE gene was present in 116 (84%) isolates (Table
1). Both genes were absent in 11% (15/138) of the isolates and, in these cases, the
cag PAI could also be absent. There was an association between PUD and the presence of the
cagA (χ
2 = 7.954;
p = 0.0048). Our data are supported by previous reports and suggest that individuals colonized with
cagA-positive
H. pylori strains are at increased risk of developing peptic ulceration [
3,
4]. Conversely, no association was detected between
cagE genotypes and clinical outcome.
The combination of the
cagA and
vacA status was also investigated. An association was observed between the genotype,
cagA-positive
vacA s1 and PUD (χ
2 = 11.871;
p = 0.0006) and between GERD and the
cagA-negative
vacA s2 genotype (
p < 0.0001). Although our data showed a trend towards an association between the
cagA-positive,
vacA s1m2 genotype and PUD (χ
2 = 13.849;
p = 0.001), logistic regression analysis suggested that this could be explained by a higher prevalence of the
cagA and
vacA s1 alleles (OR = 5.42, 95% CI 2.6–11.3, p = 0.0006) This finding agrees with those of others reports and suggests a possible role for these factors in the pathogenesis of
H. pylori-related peptic ulceration [
3,
4]. Gastroesophageal reflux disease (GERD) results from exposure of the esophageal mucosa to acid [
23]. There is no apparent causal relationship between
H. pylori colonization and the presence of GERD or its complications, although GERD patients are infected with less virulent
H. pylori strains [
24]. Infection by
H. pylori may protect against GERD, thus these patients may have a lower frequency of
vacA s1 genotype and a higher frequency of the
vacA s2 and m2 genotypes [
4,
10,
25]. Our data support this hypothesis and logistic regression showed a negative association between the more virulent strain,
cagA-positive
vacA s1 genotype, and GERD (OR = 0.26, 95% CI 0.08–0.8, p = 0.03). Although the protective effect of infection with
cagA-positive
H. pylori strains against GERD could be explained by the lower gastric output due to the more intense gastric lesions induced by these strains [
26]. Further studies are required to determine the putative protective role of the more virulent strains.
Antimicrobial Susceptibility
H. pylori resistance to antimicrobial is of particular concern because it is a major determinant of eradication regimen failure. We tested the primary resistance to amoxicillin, clarithromycin, furazolidone, metronidazole and tetracycline in 138 H. pylori isolates.
Until recently, resistance to amoxicillin was considered to be absent or very rare, however, amoxicillin-resistant
H. pylori strains have now been identified in USA, Italy and Brazil [
16,
27,
28]. In this study, the amoxicillin resistance was found in 39% of isolates (54/138). The reason for these high rates of amoxicillin resistance remains unclear, however, since no pharmaco-epidemiological data regarding amoxicillin use in Brazil exists, it may be speculated that this drug is used in a disproportionate manner.
The increased consumption of macrolides is expected to influence the prevalence of
H. pylori resistance to clarithromycin. Resistance to clarithromycin was detected in 16% of strains (23/138). Our results agree with previous reports from Brazil, being lower those from Peru [
29] and higher than rates in Europe [
30]. Resistance to clarithromycin has a serious clinical implication, since it impairs the efficacy effect of anti-
H. pylori treatment [
31].
The occurrence of metronidazole-resistant strains may be the consequence of increased consumption of this antibiotic in the community. In this study, the resistance to metronidazol was observed in 54% (75/138) of strains. Metronidazole has been widely prescribed for other infections such as parasitic or genital infections, and the use or abuse of this inexpensive drug may contribute to the increase in metronidazole resistance, with rates ranging from 5% to 90% [
32].
Tetracyclines are a family of broad-spectrum antibiotics that have been widely used for the treatment of bacterial infections since the 1950s. Until the end of the last century, only a few cases of spontaneous tetracycline resistance in
H. pylori had been reported [
33]. During the last 3 years, however, an increase in the incidence of tetracycline resistance in
H. pylori has been reported. In this study, we found 9% (13/138) of tetracycline resistance in strains. Similar data were published in previous reports [
16,
34].
Furazolidone has been used as an alternative to overcome metronidazole resistance. In our study, resistance to furazolidone was 13% (19/138), higher than rates detected by others [
22]. The reason for the high rate of furazolidone resistance and factors leading to it are still unknown and deserve further investigation.
Thirty one strains were resistant to two agents, 19 isolates to three drugs, and 3 strains to four antimicrobials. H. pylori resistance to these antimicrobials may be partially explained by the high prevalence of this bacteria in our population. Eradication failure has been associated with several reasons, the most important being primary H. pylori resistance.
Additionally, the primary
H. pylori resistance was related to the clinical outcome, as well as to virulence factors. We were unable to detect an association between outcome and bacterial resistance, as previously reported [
35,
36]. With regard to virulence factors, no association was found when the MIC values for metronidazole, amoxicillin, clarithromycin, tetracycline, and furazolidone were compared with the different
vacA,
iceA,
cagA and
cagE genotypes, as described by several authors [
35,
37]. Thus, it seems that different virulence markers and antibiotic resistance in
H. pylori strains are not interrelated.