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. 2022 Sep 6:13:962354.
doi: 10.3389/fmicb.2022.962354. eCollection 2022.

Revealing the novel effect of Jinghua Weikang capsule against the antibiotic resistance of Helicobacter pylori

Xiaofen Jia  1 , Qiuyue Huang  1 , Miaomiao Lin  1 , Yingming Chu  1 , Zongming Shi  1 , Xuezhi Zhang  1 , Hui Ye  1
Affiliations

Revealing the novel effect of Jinghua Weikang capsule against the antibiotic resistance of Helicobacter pylori

Xiaofen Jia et al. Front Microbiol. .

Abstract

Background: Helicobacter pylori (H. pylori) infects half of the human population globally. Eradication rates with triple or quadruple therapy have decreased owing to the increasing rate of antibiotic resistance. Jinghua Weikang capsule (JWC) is the first and most popular Chinese patent medicine approved by the state for the treatment of gastritis and peptic ulcers caused by H. pylori infection in China. Previous studies have found that JWC has a certain bactericidal effect on drug-resistant H. pylori and its major component, Chenopodium ambrosioides L. inhibits biofilm formation, but the mechanism remains unclear. This study focused on drug-resistant H. pylori and explored whether JWC could reverse drug resistance and its related mechanisms.

Method: The agar plate dilution method, E-test method, and killing kinetics assay were used to evaluate the bactericidal effect of JWC on antibiotic-resistant H. pylori and its effect on antibiotic resistance. Sanger sequencing was used to detect mutations in drug resistance genes. The crystal violet method, scanning electron microscopy, and confocal laser scanning microscopy were used to evaluate the effects of JWC on biofilms. qPCR was performed to evaluate the effect of JWC on the expression of efflux pump-related genes. qPCR and immunofluorescence were used to evaluate the effects of JWC on H. pylori adhesion.

Results: JWC showed considerable antibacterial activity against drug-resistant H. pylori strains, with minimum inhibitory concentration (MIC) values ranging from 64 to 1,024 μg/ml. The MIC of metronidazole (MTZ) against H. pylori 26,695-16R decreased from 64 to 6 μg/ml after treatment with 1/2 MIC of JWC. The resistance of H. pylori 26,695-16R to MTZ was reversed by JWC, and its effect was better than that of PaβN and CCCP. H. pylori 26,695-16R is a moderate biofilm-forming strain, and JWC (16-64 μg/ml) can inhibit the formation of biofilms in H. pylori 26,695-16R. JWC reduced the expression of HP0605-HP0607 (hefABC), HP0971-HP0969 (hefDEF), HP1327-HP1329 (hefGHI), and HP1489-HP1487. JWC reduced the adhesion of H. pylori to GES-1 cells and the expression of adhesives NapA, SabA, and BabA.

Conclusion: The reversal of MTZ resistance by JWC may be achieved through the adhesin/efflux pump-biofilm pathway.

Keywords: Helicobacter pylori; Jinghua Weikang capsule; adhesion; antibiotic resistance; biofilm; efflux pump; metronidazole.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Inhibiting kinetic curves and killing kinetic curves. (A) Inhibiting kinetics curves of JWC on Helicobacter pylori 26,695–16R. (B) Killing kinetics curves of JWC on H. pylori 26,695–16R. The dotted line represents a 1,000-fold reduction in the number of bacteria compared to the initial inoculation.
Figure 2
Figure 2
G210T point mutation in the MTZ resistance-related gene rdxA of H. pylori 26,695–16R under different drug interventions. A, H. pylori 26,695; B, H. pylori 26,695–16R; C, Treated with PaβN (20 μg/ml); D, Treated with CCCP (1 μg/ml); E, Treated with JWC 16 μg/ml; F, Treated with JWC 32 μg/ml; G, Treated with JWC 64 μg/ml.
Figure 3
Figure 3
Effect of JWC, efflux pump inhibitors and MTZ on the biofilm of H. pylori 26,695–16R. Dc is the OD value of the blank control group, and D is the OD value of the other groups. NC: the blank control group, culture medium without H. pylori 26,695–16R; M, Model group, culture medium with H. pylori 26,695–16R; P, PaβN (20 μg/ml); C, CCCP (1 μg/ml). ***p < 0.001, compared with the results of the control group. #p < 0.05, compared with the results of the model group.
Figure 4
Figure 4
Effect of JWC and efflux pump inhibitors on the biofilm of H. pylori 26,695–16R.
Figure 5
Figure 5
Confocal laser scanning microscope (CLSM) images of H. pylori 26,695–16R strain biofilms. Cells stained with membrane-permeant SYTO 9 (green) and membrane-impermeant propidium iodide (red) were visualized by CLSM. (A) Control group; (B) treated with PaβN (20 μg/ml); (C) treated with CCCP (1 μg/ml); (D) treated with JWC 16 μg/ml; (E) treated with JWC 32 μg/ml; (F) treated with JWC 64 μg/ml.
Figure 6
Figure 6
Expression of different efflux effect genes of H. pylori 26,695–16R after incubation with PaβN, CCCP and different concentrations of JWC. NC, Normal control group; C, CCCP (1 μg/ml); **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with the results of the control group.
Figure 7
Figure 7
Effect of JWC on the adhesion of H. pylori 26,695–16R to GES-1 cells. (A) Immunofluorescence images of H. pylori and GES-1 adhesion after JWC intervention. Control group, only GES-1 cells; Model group, GES-1 and H. pylori. (B) The H. pylori fluorescence area/DAPI fluorescence area calculated by ImageJ software. **p < 0.01, compared with the results of the control group. ##p < 0.01, compared with the results of the model group. Bold values represent a meaningful MIC change of antibiotic and the H. pylori strain used in subsequent experiments.
Figure 8
Figure 8
Effect of JWC on adhesions of H. pylori 26,695–16R. NC, Normal control group, H. pylori 26,695–16R; P, PaβN (20 μg/ml); C, CCCP (1 μg/ml). *p < 0.05, **p < 0.01, ****p < 0.0001, compared with the results of the control group.
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