Helicobacter pylori Infections in the Bronx, New York: Surveying Antibiotic Susceptibility and Strain Lineage by Whole-Genome Sequencing

Abstract

The emergence of drug resistance in Helicobacter pylori has resulted in a greater need for susceptibility-guided treatment. While the alleles associated with resistance to clarithromycin and levofloxacin have been defined, there are limited data regarding the molecular mechanisms underlying resistance to other antimicrobials. Using H. pylori isolates from 42 clinical specimens, we compared phenotypic and whole-genome sequencing (WGS)-based detection of resistance. Phenotypic resistance correlated with the presence of alleles of 23S rRNA (A2142G/A2143G) for clarithromycin (kappa coefficient, 0.84; 95% confidence interval [CI], 0.67 to 1.0) and gyrA (N87I/N87K/D91Y/D91N/D91G/D99N) for levofloxacin (kappa coefficient, 0.90; 95% CI, 0.77 to 1.0). Phenotypic resistance to amoxicillin in three isolates correlated with mutations in pbp1, pbp2, and/or pbp3 within coding regions near known amoxicillin binding motifs. All isolates were phenotypically susceptible to tetracycline, although four bore a mutation in 16S rRNA (A926G). For metronidazole, nonsense mutations and R16H substitutions in rdxA correlated with phenotypic resistance (kappa coefficient, 0.76; 95% CI, 0.56 to 0.96). Previously identified mutations in the rpoB rifampin resistance-determining region (RRDR) were not present, but 14 novel mutations outside the RRDR were found in rifampin-resistant isolates. WGS also allowed for strain lineage determination, which may be important for future studies in associating precise MICs with specific resistance alleles. In summary, WGS allows for broad analyses of H. pylori isolates, and our findings support the use of WGS for the detection of clarithromycin and levofloxacin resistance. Additional studies are warranted to better define mutations conferring resistance to amoxicillin, tetracycline, and rifampin, but combinatorial analyses for rdxA gene truncations and R16H mutations have utility for determining metronidazole resistance.

Review: Diagnosis of Helicobacter pylori infection

“…The same good correlation was also found in Cambodia 21 . Other studies, using WGS analysis of H pylori strains isolated from gastric biopsies, described new mutations or identification of multiple mutations conferring resistance to rifampicin, metronidazole and amoxicillin 22‐24 …”

Next Generation Sequencing for the Prediction of the Antibiotic Resistance in Helicobacter pylori: A Literature Review

“…Investigating clarithromycin resistance with traditional molecular methods, a high number of mutations have been detected in the macrolide-binding site of 23SrRNA gene, i.e., A2142G, A2142C, A2143G, T1942C, G1939A, C2147G, G2172T, T2182C, A2116G, A2144G/T, A2115G, G2111A, T2717C, T2289C, G2224A, and C2245T (in accordance to Taylor et al numbering) [ 4 , 5 , 6 ]. In general, NGS has confirmed that clarithromycin resistance is mainly based on point mutations in nucleotide positions 2142 (A to G/C) and 2143 (A to G) in the 23SrRNA gene [ 7 , 8 , 9 ]. The commercially available qPCR (quantitative Polymerase Chain Reaction) assays for the detection of A2142C/G and A2143G, would be sufficient to monitor CLA resistance in clinical practice; but only a limited number of nucleotide position can be analysed with this tool, while whole genome sequencing (WGS) delivers a more comprehensive description of resistance determinants present in a clinical isolate and may detect new mutations potentially conferring drug resistance.…”

“…In particular, frameshift mutations (i.e., at codon positions 105, 149 or 192 in frxA and 18, 38 and 112 in rdxA ) and point mutations resulting in amino acid exchanges (i.e., A67V, A68E, K64N, P106S, R90S and R16C/H in the rdxA gene) were observed only in metronidazole resistant strains [ 25 , 26 ]. Other mutations (i.e., at codon positions 18 in frxA and 62, 96 and 162 in rdxA ) were distributed between resistant and susceptible strains [ 5 , 7 , 25 ]. Null mutations in rdxa functional sites (frameshift, premature stop, large deletion, large sequence insertions ending with a stop and point-mutations) leading to a loss of binding sites for FMN (flavin mononucleotide) are predictive of MZ-resistance.…”

Genotypic and Phenotypic Resistance to Clarithromycin in Helicobacter pylori Strains

“…Moreover, visual interpretations of MIC vary widely among individuals, which may lead to intra-and inter-observer variability. Various molecular methods, including next-generation sequencing, have enabled the detection of genetic mutations of H. pylori and suggested association between genotype and phenotype for antimicrobial resistance [ 21 , 22 , 23 , 24 ]. In the present study, whole-genome sequencing of the 23S rRNA gene was performed, and 42 mutations were detected.…”