Prevalence of clarithromycin resistance in Helicobacter pylori in Santiago, Chile, estimated by real-time PCR directly from gastric mucosa

Helicobacter pylori is a gram-negative bacillus that colonizes the gastric mucosa. The infection is associated with both gastric and extragastric diseases such as peptic ulcer disease, gastric atrophy, gastric adenocarcinoma, gastric MALT (mucosa-associated lymphoid tissue) lymphoma, and iron deficiency anemia. The evidence strongly suggests that H.pylori eradication is beneficial in treatment or prevention of those diseases [1]. One treatment for H.pylori eradication is the triple-therapy that comprises clarithromycin, amoxicillin, and proton pump inhibitor (PPI), nevertheless, triple-therapy is not recommended in regions with high clarithomycin resistance rate [1]. Global claritrhomycin-resistance rates in adults are contrasted among various countries, from 1% in The Netherlands to 37.6% in Italy [2]. In light of this observation, the last Maastricht V/Florence consensus on management of H.pylori [1] recommends that in high-clarithromycin resistance regions (> 15% resistance prevalence), quadruple therapy is necessary. If dual clarithromycin and metronidazole resistance is > 15%, bismuth-containing quadruple therapy is the recommended first-line treatment.

Clarithromycin belongs to the class of macrolide antibiotics. The mechanism of action is the inhibition of protein synthesis of bacteria by binding to the 23S ribosomal subunit (23S rRNA). Mutations in the peptidyltransferase region encoded in domain V of 23S rRNA are responsible for macrolide resistance [3]. Three mutations in the 23S rRNA gene account for nearly all clarithromycin-resistant (ClaR) strains: A2143G, A2142G and A2142C to a small extent [3]. Standard method (antibiogram), after culture or molecular tests, can be used to detect H.pylori and clarithromycin resistance directly from the gastric biopsy [1]. Epidemiological studies evaluating regional clarithromycin resistance have been conducted based on detection of A2142G and A2143G in DNA isolated from gastric biopsies [4, 5]. Commercial tests detecting both mutations are available [3], performing at a sensitivity rate of > 94% and a specificity rate of > 98.5% compared to the culture susceptibility test. Recently, Tamayo et al. [6] found a high concordance between Etest for clarithromycin and 23S rRNA mutations. To date, only Garrido and Toledo [7] have evaluated primary resistance of H.pylori to clarithromycin in clinical isolates from Santiago, Chile. They found primary resistance in 10 out of 50 isolates by agar dilution. Resistant isolates harbor A2142G or A2143G mutations.

Metronidazole is a nitroimidazole synthesized as an inactive antibiotic prodrug. The bactericidal effect of the drug is mainly explained by the cytotoxic effect of molecules resulting from a reduction of metronidazole intracellularly in facultative anaerobic bacteria. Reduction of metronidazole in H.pylori is mediated mainly by a nonessential oxygen-insensitive NADPH nitroreductase encoded by the rdxA gene. NADPH-flavin-oxidoreductase (FrxA) is also involved in the reduction of metronidazole [3]. Various authors found that metronidazole resistance arises from inactivating mutations in rdxA found only in resistant isolates, and missense mutations have also been involved in metronidazole resistance [8]. On the other hand, the role of inactivating mutations in the frxA gene is controversial since truncating mutations have been found in sensitive isolates, and some studies report that frxA mutations alone may not be enough to confer resistance [8]. Vallejos et al. [9] studied resistance to clarithromycin and metronidazole in 50 isolates obtained from patients in Santiago, Chile. Forty-five and twenty percent respectively were resistant, resulting in 13.2% of the prevalence of dual clarithromycin and metronidazole resistance.

A total of 191 subjects were included in this study, 124 were female (64.9%) and 67 male (35.1%). The median age was 46 years (range 19 years to 82 years). None of them was treated for H.pylori eradication. Ninety-three (93) out of 191 DNA samples obtained from gastric mucosa were H.pylori-positive (48.7%). Clarithomycin resistance was detected using a 5’exonuclease assay in 29 samples (31.2% [95%CI 22.0–41.6%]). Of them, 9 harbored 2142G (31.0%) and 20 (69.0%) harbored 2143G. All ClaR samples were confirmed by Sanger sequencing. Both Sanger sequencing and 5’exonuclease allowed us to detect heteroresistance, i.e., the coexistence of resistant and susceptible bacterial strains in the same patient [12]. Sixteen (16) out of 93 H.pylori subjects were homoresistant to clarithromycin (17.2%). In addition, we detected heteroresistance in 13 out of 93 patients (14.0%).

The rdxA gene was sequenced in 28 ClaR samples. One ClaR sample was not available at the time of analysis. A nonsense substitution (H53stop) was found in one sample; and an insertion of A at nucleotide 186, leading to a frameshift resulting in N73stop, was found in a different sample. Therefore, two samples harbored truncating mutations in rdxA. One sample had an in-frame deletion of 39 nucleotides which causes the deletion of amino acids 80 to 92 (ASALMVVCSLKPS). Table 1 shows the amino acid variations in rdxA found in ClaR samples.

A total of 29 out of the 93 H.pylori positive samples resulted resistant to clarithomycin (31.2%). This estimation is higher than the 20% of ClaR reported by Garrido and Toledo in Chile [7]. Taken together, ClaR resistance reported for Chile is above the suggested 15% threshold to abandon triple-therapy, according to the recommendation of Maastricht V/Florence consensus [1].

Among 28 ClaR samples, two of them harbored truncating mutations and one an in-frame deletion in rdxA, and therefore are likely resistant to metronidazole. A total of 22 amino acid substitutions was found. Thirteen of them (Q6H, T31E, H53R, L62 V, R90K, H97T, H97Y, G98S, A118T, V123 T, V172I, E175Q, A183V) have been previously observed in metronidazole-resistant (MtzR) as well as in metronidazole-sensitive strains [13‐17]. Therefore, those substitutions probably do not confer resistance to metronidazole. Four substitutions (R16H, A67V, S88P and G162R) were found in 8 samples. R16H and S88P have been observed only in MtzR strains [13‐15]. In fact, Arg 16 is one of the residues of RdxA that interacts with the cofactor flavin mononucleotide (FMN) [18]. A67V corresponds to the replacement of a small alanine by a bulky valine in the protein core, and is associated with resistance [8, 19]. Analysis from the crystallographic data of RdxA reveals that G162 is involved in FMN binding [18]. Hence, missense mutations R16H, A67V, S88P and G162R likely cause MtzR. Nevertheless, phenotypic testing is needed to confirm that those mutations actually cause MtzR.

Five amino acid changes in RdxA (T49 M, E75Q, V151 L, P166A and P166S) have not been described previously in the literature. None of them have been observed as an important amino acid for the RdxA function. We predicted the effect of those substitutions using the crystallographic data of RdxA protein (Protein Data Bank accession number 3QDL) in the I-Mutant Suite server [20], which estimates protein stability changes (expressed as delta delta G values) upon single-point mutations from the protein structure. Delta-delta G values and the prediction were as follows: T49 M = − 0.41 (neutral stability), E75Q = − 0.78 (large decrease of stability), V151 L = − 0.44 (neutral stability), P166A = − 1.28 (large decrease in stability), P166S = − 1.50 (large decrease in stability). Thus, we propose that E75Q (1 sample), P166A (1 sample), and P166S (1 sample) could confer MtzR. However, a phenotypic testing shloud be carry out to confirm it.

We estimated a prevalence of ClaR of 31.2% in Santiago, Chile, which is above the suggested 15% threshold to abandon triple-therapy, according to the recommendation of Maastricht V/Florence consensus. Three out of 28 ClaR samples harboring inactivating mutations in rdxA may be clearly MtzR, and 11 of them had missense mutations in rdxA likely confering MtzR. Further phenotypic testing is needed to obtain a clinically significant conclusion from rdxA sequencing results. Nevertheless, the estimated prevalence of ClaR is significant as an approximation in re-evaluating the triple-therapy for H.pylori eradication currently used in Chile.