Exposure of Helicobacter pylori to clarithromycin in vitro resulting in the development of resistance and triggers metabolic reprogramming associated with virulence and pathogenicity.

In H. pylori infection, antibiotic-resistance is one of the most common causes of treatment failure. Bacterial metabolic activities, such as energy production, bacterial growth, cell wall construction, and cell-cell communication, all play important roles in antimicrobial resistance mechanisms. Identification of microbial metabolites may result in the discovery of novel antimicrobial therapeutic targets and treatments. The purpose of this work is to assess H. pylori metabolomic reprogramming in order to reveal the underlying mechanisms associated with the development of clarithromycin resistance. Previously, four H. pylori isolates were induced to become resistant to clarithromycin in vitro by incrementally increasing the concentrations of clarithromycin. Bacterial metabolites were extracted using the Bligh and Dyer technique and analyzed using metabolomic fingerprinting based on Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-Q-ToF-MS). The data was processed and analyzed using the MassHunter Qualitative Analysis and Mass Profiler Professional software. In parental sensitivity (S), breakpoint isolates (B), and induced resistance isolates (R) H. pylori isolates, 982 metabolites were found. Furthermore, based on accurate mass, isotope ratios, abundances, and spacing, 292 metabolites matched the metabolites in the Agilent METLIN precise Mass-Personal Metabolite Database and Library (AM-PCDL). Several metabolites associated with bacterial virulence, pathogenicity, survival, and proliferation (L-leucine, Pyridoxone [Vitamine B6], D-Mannitol, Sphingolipids, Indoleacrylic acid, Dulcitol, and D-Proline) were found to be elevated in generated resistant H. pylori isolates when compared to parental sensitive isolates. The elevated metabolites could be part of antibiotics resistance mechanisms. Understanding the fundamental metabolome changes in the course of progressing from clarithromycin-sensitive to breakpoint to resistant in H. pylori clinical isolates may be a promising strategy for discovering novel alternatives therapeutic targets.

Polymorphism of virulence genes and biofilm associated with in vitro induced resistance to clarithromycin in Helicobacter pylori.

BACKGROUND: Clarithromycin-containing triple therapy is commonly used to treat Helicobacter pylori infections. Clarithromycin resistance is the leading cause of H. pylori treatment failure. Understanding the specific mutations that occur in H. pylori strains that have evolved antibiotic resistance can help create a more effective and individualised antibiotic treatment plan. However, little is understood about the genetic reprogramming linked to clarithromycin exposure and the emergence of antibiotic resistance in H. pylori. Therefore, this study aims to identify compensatory mutations and biofilm formation associated with the development of clarithromycin resistance in H. pylori. Clarithromycin-sensitive H. pylori clinical isolates were induced to develop clarithromycin resistance through in vitro exposure to incrementally increasing concentration of the antibiotic. The genomes of the origin sensitive isolates (S), isogenic breakpoint (B), and resistant isolates (R) were sequenced. Single nucleotide variations (SNVs), and insertions or deletions (InDels) associated with the development of clarithromycin resistance were identified. Growth and biofilm production were also assessed. RESULTS: The S isolates with A2143G mutation in the 23S rRNA gene were successfully induced to be resistant. According to the data, antibiotic exposure may alter the expression of certain genes, including those that code for the Cag4/Cag protein, the vacuolating cytotoxin domain-containing protein, the sel1 repeat family protein, and the rsmh gene, which may increase the risk of developing and enhances virulence in H. pylori. Enhanced biofilm formation was detected among R isolates compared to B and S isolates. Furthermore, high polymorphism was also detected among the genes associated with biofilm production. CONCLUSIONS: Therefore, this study suggests that H. pylori may acquire virulence factors while also developing antibiotic resistance due to clarithromycin exposure.