Analysis of antimicrobial susceptibility and virulence factors in Helicobacter pyloriclinical isolates

Patients

Helicobacter pylori isolates were obtained from 155 patients (86 males and 69 females; age 43.6 ± 14.9 years) living in southeastern Brazil. Eighty patients with peptic ulcer disease (PUD), 54 with non-ulcer dyspepsia (NUD) and 21 with gastroesophageal reflux disease (GERD) were included. The study was approved by the Ethics Committee of the São Francisco University and performed in accordance with the Declaration of Helsinki. Each patient signed a written informed consent prior to entering the study.

H. pyloriculture and MIC determination

H. pylori isolates were obtained by inoculating the specimens into selective media followed by incubation for 3–5 days at 37°C under microaerophilic conditions, as previously described [16]. The colonies were identified by Gram staining and by oxidase, catalase and urease production. H. pylori strains were stored at -70°C in BHI broth containing glycerol 30%. The MIC for amoxicillin, furazolidone, metronidazole, tetracycline (Sigma Chemical Co., St Louis, MO) and clarithromycin (Abbot Laboratories, North Chicago, Ill) were determined by the agar dilution method, using twofold increments (0.125 to 256 μg ml^-1) on Mueller-Hinton agar (Merck, Darmstadt, Germany) supplemented with 10% sheep blood, and incubated at 37°C under microaerophilic conditions for 72 h. All tests were performed in duplicate. Isolates were considered to be resistant when the MIC was greater than 8 μg ml^-1 for amoxicillin or metronidazole, and greater than 2 μg ml^-1 for clarithromycin, furazolidone or tetracycline [16].

Molecular Biology Methods

Genomic DNA was extracted with DNAzol™ reagent (Gibco BRL, Cincinnati, OH, USA), and the integrity of the DNA was assessed by electrophoresis in 0.8% agarose gels stained with ethidium bromide.

Polymerase chain reactions (PCR) were performed in a total volume of 50 μl containing 50 pmol of primer, 100 ng of genomic DNA, 1.0 mmol L^-1 of each of four dNTPs (Invitrogen™ Life Technologies, Alemeda, CA, USA) and 2.5 U of Taq DNA polymerase (Invitrogen™ Life Technologies). The reaction mixtures were cycled in an automated GeneAmp^® PCR System 9700 thermal cycler (PE Applied Biosystems, Foster City, CA, USA) under the following conditions: initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 1 min, annealing ranging from 45°C to 60°C for 1 min and 72°C for 1 min. The final cycle included a 7 min extension step to ensure full extension of the PCR products.

The presence of H. pylori was confirmed by PCR of the 16S rRNA [17] and glmM [18] genes. The cagA gene was analyzed using the primers D008 and R008 [19]. The cagE gene was analyzed using the primers described by Fallone et al. [10]. For analysis of the vacA m region, primers VA3-F and VA3-R were used, whereas primers VA4-F and VA4-R were used to amplify the m1 and m2 subtypes, respectively [5]. The vacA s region was analyzed using the primers, VA1-F and VA1-R [5]. For iceA genotype analysis, primers iceA1-F, iceA1-R, iceA2-F and iceA2-R were used. The primers, iceA2-F and iceA2-R, yielded a fragment of 124, 229 or 334 bp depending on the existence of repeated sequences of 105 nucleotides [3].

Statistical analysis

The association between H. pylori genotypes and clinical disease, as well as among the virulence markers and antibiotic resistance was evaluated using either the χ² test with Yates continuity correction or Fisher's exact test. Only cases containing single genotypes were included. Logistic regression analysis was used to evaulate the relationship between virulence markers and antibiotic resistance of H. pylori and clinical outcome. A logistic regression model was constructed using variables such as cagA, vacA and iceA status, and antibiotic resistance. The association of each variable with PUD or NUD or GERD (dependent variables) was tested by univariated analysis. All variables with p values of 0.25 or less were included in the full model of logistic regression and variables with p values <0.05 were remained in the model. The odds ratio (OR) and the 95% confidence interval (95% CI) were used as estimates of the risk.

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 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 cagE 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 (χ² = 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 (χ² = 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 (χ² = 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.