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Synergistic Effect between Amoxicillin and Zinc Oxide Nanoparticles Reduced by Oak Gall Extract against Helicobacter pylori.

ABSTRACT: Helicobacter pylori (H. pylori) is a global health threat, and the World Health Organization has included H. pylori among 12 bacterial species that require high priority future strategies for the development of new antibiotics due mainly to its high rates of resistance. Metallic nanoparticles are known for their antimicrobial properties. The FDA (Food and Drug Administration) has approved zinc oxide nanoparticles (ZnONPs) as biocompatible antimicrobials. Green synthesis of ZnONPs was performed based on Oak galls extract (OGE) and was characterized by UV, IR, DLS, TEM, and SEM measurements. In addition, LC-MS/MS was used for the identification of OGE constituents. A checkerboard assay was used to evaluate the activity of synthesized Qi-ZnONPs and OGE against H. pylori, and their synergistic effects with amoxicillin were evaluated. LC-MS/MS analyses identified 20 compounds as major gallic acid conjugates. The ZnONPs had average particle sizes of 5.5 nm (DLS) and 7.99 nm (TEM). Both OGE and Qi-ZnONPs exhibited moderate activity against H. pylori. Amoxicillin and Qi-ZnONPs combinations (1:2 and 1:4 amoxicillin:/Qi-ZnONPs) significantly decreased the MIC₉₀ by two-fold and four-fold, respectively, and FIC values for the combinations were more significant than with OGE alone. OGE is rich in phenolics. The synergism between Qi-ZnONPs and amoxicillin can provide an alternative safe agent of low cost to combat H. Pylori infections.

SUBMITTER: Attia HG

PROVIDER: S-EPMC9320066 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundClarithromycin (CLR) is the most commonly recommended antibiotic in Helicobacter pylori eradication regimens, but the prevalence of CLR-resistant H. pylori is increasing. CLR resistance is associated with mutations in the 23S rRNA gene. However, H. pylori eradication can still be achieved with triple therapy, and an additive effect may occur with multiple antibiotics.MethodsTwenty-six CLR-resistant strains were examined. The MIC of clarithromycin was determined by agar-dilution-testing on Columbia agar, as described elsewhere. The conserved region of the H. pylori 23S rRNA gene between nucleotide positions 1445 and 2846 [GenBank: U27270] was amplified. RFLP and sequence analysis were performed with the 1402-bp PCR product. Synergy between clarithromycin and amoxicillin was assessed using the agar dilution checkerboard technique. To confirm the correlation between mutation and synergistic effect with subinhibitory concentrations of AMX, site-directed mutagenesis was performed in four CLR-susceptible H. pylori isolates.ResultsTwenty-six clarithromycin-resistant strains were examined. The conserved region of the H. pylori 23S rRNA gene was amplified, and the purified PCR product was checked for mutations by restriction fragment length polymorphism (RFLP) analysis and sequencing. A synergistic effect was found in only three of the 12 H. pylori strains (25%) with the A2142G mutation and five of the 10 H. pylori strains (50%) with the A2143G mutation (fractional inhibitory concentration: FIC < 0.5, minimal inhibitory concentration: MIC<2 mg/L) was found. Site-directed mutagenesis was performed in four CLR-susceptible H. pylori isolates.Three of these isolates harboring a mutation in position A2143G grew under selection with CLR (MIC >16 mg/L), and all three strains showed the synergistic effect (FIC<0.5). In contrast, three of the same four strains transformed with DNA fragments with a mutation in position A2142G were resistant to CLR (MIC>16 mg/L) and showed no synergism with amoxicillin (FIC>2).ConclusionsHere we demonstrate that in 100% of the in vitro transformed strains, a mutation at position A2143G leads to a synergistic effect between clarithromycin and amoxicillin, whereas a mutation at position at A2142G had no discernible effect.

Project description:The growing resistance to amoxicillin (AMX)-one of the main antibiotics used in Helicobacter pylori eradication therapy-is an increasing health concern. Several mutations of penicillin-binding protein 1A (PBP1A) are suspected of causing AMX resistance; however, only a limited set of these mutations have been experimentally explored. This study aimed to investigate four PBP1A mutations (i.e., T558S, N562H, T593A, and G595S) carried by strain KIN76, a high-level AMX-resistant clinical H. pylori isolate with an AMX minimal inhibition concentration (MIC) of 2 µg/mL. We transformed a recipient strain 26695 with the DNA containing one to four mutation allele combinations of the pbp1 gene from strain KIN76. Transformants were subjected to genomic exploration and antimicrobial susceptibility testing. The resistance was transformable, and the presence of two to four PBP1A mutations (T558S and N562H, or T593A and G595S), rather than separate single mutations, was necessary to synergistically increase the AMX MIC up to 16-fold compared with the wild-type (WT) strain 26695. An AMX binding assay of PBP1A was performed using these strains, and binding was visualized by chasing Bocillin, a fluorescent penicillin analog. This revealed that all four-mutation allele-transformed strains exhibited decreased affinity to AMX on PBP1A than the WT. Protein structure modeling indicated that functional modifications occur as a result of these amino acid substitutions. This study highlights a new synergistic AMX resistance mechanism and establishes new markers of AMX resistance in H. pylori.IMPORTANCEThe development of resistance to antibiotics, including amoxicillin, is hampering the eradication of Helicobacter pylori infection. The identification of mechanisms driving this resistance is crucial for the development of new therapeutic strategies. We have demonstrated in vitro the synergistic role of novel mutations in the pbp1 gene of H. pylori that is suspected to drive amoxicillin resistance. Also deepening our understanding of amoxicillin resistance mechanisms, this study establishes new molecular markers of amoxicillin resistance that may be useful in molecular-based antibiotic susceptibility testing approaches for clinical practice or epidemiologic investigations.

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Synergistic Effect between Amoxicillin and Zinc Oxide Nanoparticles Reduced by Oak Gall Extract against Helicobacter pylori.

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