Selective Inhibition of Helicobacter pylori Carbonic Anhydrases by Carvacrol and Thymol Could Impair Biofilm Production and the Release of Outer Membrane Vesicles

Abstract

Helicobacter pylori, a Gram-negative neutrophilic pathogen, is the cause of chronic gastritis, peptic ulcers, and gastric cancer in humans. Current therapeutic regimens suffer from an emerging bacterial resistance rate and poor patience compliance. To improve the discovery of compounds targeting bacterial alternative enzymes or essential pathways such as carbonic anhydrases (CAs), we assessed the anti-H. pylori activity of thymol and carvacrol in terms of CA inhibition, isoform selectivity, growth impairment, biofilm production, and release of associated outer membrane vesicles-eDNA. The microbiological results were correlated by the evaluation in vitro of H. pylori CA inhibition, in silico analysis of the structural requirements to display such isoform selectivity, and the assessment of their limited toxicity against three probiotic species with respect to amoxicillin. Carvacrol and thymol could thus be considered as new lead compounds as alternative H. pylori CA inhibitors or to be used in association with current drugs for the management of H. pylori infection and limiting the spread of antibiotic resistance.

Keywords: Helicobacter pylori; amoxicillin; anti-biofilm activity; carbonic anhydrase; carvacrol; molecular modelling; probiotic bacteria; thymol.

Figure 1

Ligand interactions within (A) HpCAα, (B,C) HpCAβ, and (D) MgCA active sites. Carvacrol and thymol are represented in blue and green, respectively. Snapshots from the corresponding MD are shown in each panel together with the trajectories.

Figure 2

Determination of the MBIC of carvacrol, thymol, and amoxicillin versus H. pylori through the alamarBlue assay (A,D,G), the CFU count (B,E,H), and the crystal violet assay (C,F,I). T: treated samples; NT: non-treated samples. ** p < 0.005, *** p < 0.001 vs. the non-treated sample.

Figure 3

Representative images of H. pylori ATCC43629 biofilm after 72 h of incubation. The biofilms have been observed by using a fluorescence microscope. (A,C,E) untreated biofilm and (B) carvacrol-treated sample, (D) thymol-treated sample, and (F) amoxicillin-treated sample. Green fluorescence indicated the presence of live cells, and red fluorescence indicated the presence of dead cells. The presence of dead cells was negligible. Bar: 5 µm.

Figure 4

OMV count performed by flow cytometry. The graphs represent the concentration of total and eDNA-containing pOMVs and bOMVs, obtained from treated (T) and untreated (NT) samples with sub-MIC concentrations of carvacrol (A), thymol (B), and amoxicillin (C,D). * p < 0.05 vs. the non-treated sample.

Figure 5

Phylogenetic analysis of CAs from various microorganisms. The dendrogram was obtained using amino acid sequences of probiotic strains in Table 4, and the α-CAs and β-CAs coming from multiple species of bacteria. Bootstrap values of 100 replicates are reported at branch points. Legend: see Table S2 for sequence accession numbers, CA-class, cryptonyms, and organisms considered.