First Detection in Helicobacter suis of a Mutation Conferring Resistance to Clarithromycin in Helicobacter pylori : Case Report and Review of the Literature

Introduction

Since 1984, Helicobacter pylori is widely recognized as a human pathogen of the stomach. This bacterium infects >50% of population worldwide, with prevalence rates <40% in developed countries, as in Europe. ¹ Other Helicobacter species have been recently described as able to infect the stomach of many mammals and sometimes humans. These non-H. pylori Helicobacter (NHPH) are detected in 0.5–6% of patients who undergo an endoscopy. ² NHPH infections of the human stomach are associated with active chronic gastritis. ³ Furthermore, these have been frequently associated with gastric ulcers or low-grade mucosa-associated lymphoid tissue (MALT) lymphoma. These NHPH have a typical morphology, consisting in large spiral shape, and very fastidious culture conditions that distinguishes them from H. pylori. Based on phylogenetic analysis of their 16S rRNA gene, they are subdivided into two different groups. ³ The first type, Type 1 NHPH, is homogeneous and corresponds to Helicobacter suis usually colonizing the stomach of pigs. The second one, Type 2 NHPH, is a more heterogeneous group of species, including Helicobacter felis, Helicobacter bizzozeronii, and Helicobacter salomonis. ³ Only few research groups have succeeded in cultivating NHPH from the gastric mucosa of two patients. ⁴ The diagnosis of NHPH infections requires a meticulous direct examination of biopsy smears with the detection of bacteria of particular shape associated with a negative culture. ² The amplification and sequencing of Helicobacter genus-specific 16S rDNA fragments from gastric biopsies is now crucial to diagnosis of NHPH infections.

As NHPH remain hardly cultivable, few data are available on their resistance to antibiotics. Moreover, as no guidelines on specific antibiotic treatment of NHPH exist, the usual treatment is empirically based on the reference treatment for H. pylori. ⁴ The standard therapy (amoxicillin and clarithromycin combined with a proton pump inhibitor [PPI]) has resulted in a good relief of symptoms and eradication of the bacterium in children. In contrast, in another reported case, treatment of NHPH infection by clarithromycin, metronidazole, and PPI in a patient with a duodenal ulcer disease had no effect, whereas treatment with metronidazole and tetracycline was successful. ⁵ From the latter, clarithromycin resistance could be suspected in NHPH. In H. pylori, clarithromycin resistance reaches almost 25% of strains in many countries and is the main risk factor for treatment failure. Clarithromycin resistance is due to a decrease in antibiotic binding to the bacterial ribosome due to three independent single nucleotide mutations within the 23S rRNA peptidyl transferase-encoding region that is, A2143G, A2142G, A2142C. ⁶ These resistances can be detected either by phenotypic or genotypic methods to adapt the clinical management to avoid therapeutic failures. The sequence similarity between the different Helicobacter species (7.5% sequence divergence on 23S rRNA gene between H. pylori and H. suis) suggests that the mechanism of action and resistance to clarithromycin in H. pylori should be identical in NHPH. ⁷ Therefore, because the determination of phenotypic resistance to clarithromycin in NHPH remains incompatible with a routine practice, due to the difficulties of culture, detection by molecular techniques of these mutations represents the main test used in routine diagnostic. We describe in this study a clinical case illustrating the benefit to use molecular diagnosis in these infections.

Clinical History and Phylogenic Analysis

A 43-year-old obese woman has beneficiated from a surgical treatment (subtotal gastrectomy, i.e., gastric sleeve). During her preoperative assessment, a gastrointestinal endoscopy was realized with gastric biopsies to detect infection by H. pylori. Fundic and antral gastritis were observed during endoscopy, confirmed by histological analysis (atrophic gastritis with moderate intensity and presence of very long and spiral-shaped bacilli). Bacteriological direct examination after Gram staining of biopsy smear found typically shaped bacilli suggestive of NHPH. Rapid urease test was performed onto the biopsy and was positive. Culture remained negative, even with 2 weeks of incubation on blood medium supplemented with antibiotics under microaerophilic conditions at 37°C. In front of this absence of culture, molecular analysis was performed after DNA extraction of biopsies (amplification of a 1,079 nucleotides-long 16S rDNA fragment). ⁸ During identification of the bacterial strain present in this biopsy, the more proximal cluster was H. suis (99% of identity). Systematic detection of clarithromycin resistance was performed by PCR (according to previously published method) and found a A2143G mutation associated to clarithromycin-resistant H. pylori. ⁹ To confirm the presence of a clarithromycin resistance associated mutation produced amplicons were then Sanger sequenced on ABI 3730 (Thermo Fisher Scientific, MA) (Fig. 1). ⁶ According to this result, patient's management consists in an association of PPI, amoxicillin, and metronidazole for a week (without clarithromycin). Efficacy of this eradication protocol was confirmed by the negativity of the ¹³ C urea breath test 6 weeks later (<1‰, mass spectrometry after ingestion of HELIKIT^®, according to Mayoly Spindler recommendations).

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FIG. 1.

Phylogenic analysis of 23S genes of Helicobacter strains. (A) Phylogenic tree. Maximum likelihood method with 1,000 replicates. (B) Sequence alignment. 1: Mutations associated with clarithromycin resistance; 2: main informative sites. Color images are available online.

The produced sequence was retrospectively analyzed using SeaView Version 4.7 to compare the bacteria strain with the sequences published in the literature. Sequences were deposited onto GenBank (Accession number SAMN12273698).

Discussion

Herein we have described, for the first time in H. suis, the presence of a mutation in the 23s rRNA gene conferring clarithromycin resistance in H. pylori. ⁶ Even in the absence of direct antibiotic susceptibility testing, due to culture conditions, this genomic data suggested that this clinical strain is clarithromycin resistant. Indeed, initially extensively described in H. pylori, this mutation has also been described in a wide range of different bacterial strain, including gram-positive (Streptococcus, Staphylococcus, …), gram-negative (Enterobacteriaceae, Campylobacter, Neisseria, …) bacteria, but also acid-fast bacilli (Mycobacterium) or intracellular bacteria (Mycoplasma, …). This large repartition suggest that this resistance mutation and mechanism could be almost universal. ¹⁰ Indeed, these nucleotides are implicated in the secondary structure of the 30 regions of 23S ribosomal RNA (rRNA) domain V., and described mutations could yield 14/15-membered, 16-membered macrolide, and/or ketolide resistance.^11,12

Although the clinical consequences have not been evaluated yet (due to the low prevalence of this mutation), published data on H. pylori have led us, by analogy, not to treat the patient using clarithromycin. In the present case and even if some studies observed a lack of sensitivity for the ¹³ C urea breath test in infection by H. suis, the patient's results are far below the threshold (<1‰ for a cutoff value of 4‰), indicating a probable success for bacterial eradication. ¹³ As described and because of the difficulty to efficiently culture NHPH, identification and quantification of resistance to clarithromycin by phenotypic methods remain impossible for routine diagnostic purposes. In this context, genotypic detection of mutations (identified in H. pylori) is the unique possible test in NHPH. This detection has a real impact in the therapeutic management of NHPH-infected patients. Indeed, if these patients represent a small percentage of Helicobacter-infected patients, a potential evolution to gastric ulcer or gastric MALT lymphoma must be considered.

According to the national report of the antibiotic use in veterinary medicine, in France, in 2017, even considering a dramatic decrease, macrolides is the fourth antibiotic family use in swine breeding. This wide use could explain the emergence of NHPH clarithromycin resistance and strengthen the concept of “One Health” in antibioresistance for veterinary and human medicines.