Unraveling the Novel Effect of Patchouli Alcohol Against the Antibiotic Resistance of Helicobacter pylori

Abstract

The long-term colonization of Helicobacter pylori can cause various gastrointestinal diseases, and its high genetic variability is prone to antibiotic resistance and leads to failure of clinical treatment. Intracellular survival also contributes to the drug tolerance of H. pylori. Patchouli alcohol (PA) shows a highly efficient activity against H. pylori in vitro and in vivo. And this study aims to explore whether PA can reduce the resistance of H. pylori and determine the underlying mechanism. Checkerboard and time-kill bactericidal curve assay reveal that the combination of PA and clarithromycin (CLR) promoted the inhibition and bactericidal effect against H. pylori. Stimulation of CLR leads to the internalization of H. pylori, but PA can effectively inhibit the invasion induced by CLR. Compared with antibiotics, PA remarkably eradicated the intracellular H. pylori, and this intracellular sterilized ability was further improved in combination with antibiotics (CLR and metronidazole). The expression of H. pylori efflux pump genes (hp0605, hp1327, and hp1489) was dose-dependently downregulated by PA. Digital droplet PCR indicated that the H. pylori mutant of A2143G can be inhibited by PA. Cellular uptake and transport assays showed that PA is rapidly absorbed, which promotes its activity against intracellular bacteria. Therefore, PA can act synergistically with CLR as a candidate treatment against drug-resistant H. pylori.

Keywords: Helicobacter pylori; clarithromycin; intracellular; patchouli alcohol; resistance.

FIGURE 1

Chemical structure of PA.

FIGURE 2

Combined effect of PA and antibiotics against H. pylori. (A) Combined effect tested by the checkerboard method of PA and antibiotics against the H. pylori standard strain NCTC11637 and the drug-resistant strain Hp1870 as determined by FICI (n = 4). (B) Combined effect tested by time–kill curves of PA and CLR against the H. pylori strains NCTC11637 and Hp1870; the synergy is defined as a ≥2-log₁₀ (n = 3). (C) Bacteria imaging by Live/Dead BacLight Bacterial Viability kits to observe the bactericidal capacity of PA and CLR against H. pylori NCTC11637 and Hp1870.

FIGURE 3

Number of intracellular H. pylori stimulated by antibiotics and PA. (A) Influence of low-dose CLR and MTZ on the number of intracellular H. pylori. (B) Antagonism effect of PA on the increased invasion induced by CLR; a gentamycin protection assay was conducted to measure the CFU of intracellular H. pylori. (C) Quantitative results of the fluorescent area of H. pylori (green area) with treatment of PA and CLR. (D) Representative confocal images of intracellular H. pylori after treatment with PA and CLR. The above results are expressed as the mean ± SD (n = 6). ^#P < 0.05, and ^##P < 0.01 (compared with the results for the control group).

FIGURE 4

Killing ability for intracellular H. pylori. (A) Intracellular H. pylori standard strain NCTC11637 and drug-resistant strains Hp1869 and Hp1870 were treated with different doses of PA, CLR, and MTZ, and the number of bacteria was presented as CFU/ml (n = 3). Compared with the control group, a 99% decrease in the intracellular strain viability was defined as an effective extermination. (B) Representative confocal images by the immunofluorescence method to observe the ability of PA (2 MIC), CLR (20 MIC), and MTZ (25 MIC) to kill intracellular H. pylori NCTC11637; the bacteria were stained by anti- H. pylori antibody ab20459 (green).

FIGURE 5

Combined effect of PA and antibiotics against intracellular H. pylori. (A) Killing ability of MTZ alone and in combination with PA (25 μg/ml) against the intracellular H. pylori standard strain NCTC11637 and drug-resistant strains Hp1869 and Hp1870 (n = 3). (B) Killing ability of CLR alone and in combination with PA (25 μg/ml) against intracellular the H. pylori standard strain NCTC11637 and drug-resistant strains Hp1869 and Hp1870 (n = 3). Compared with the control group, a 99% decrease in the intracellular strain viability was defined as an effective extermination.

FIGURE 6

Expression of efflux effect gene. (A) Expression of different efflux effect homolog genes of Hp1870 after being incubated with DMSO, CLR, and PA. (B) Expression of hp0605 after being stimulated with different doses of CLR and PA for different times. The results are expressed as the mean ± SD (n = 6). *P < 0.05 and **P < 0.01, compared with the results for the control group; ^#P < 0.05 and ^##P < 0.01, compared with the results for the model group.

FIGURE 7

Determination of the mutation site and expression of the mutant gene in the drug-resistant strain Hp1870. (A) First-generation sequencing to determine the mutation of Hp1870. (B) Copy number of A2143G after being treated with DMSO, CLR, and PA. The blue droplets were droplets positive for A2143G, and the copy number value is shown in the upper right corner of each figure.

FIGURE 8

Uptake and transport of PA in Caco-2 monolayers. (A, B) Effect of concentrations, time, temperature, and pH on uptake of PA by Caco-2 cells. (C, D) Effect of time, efflux transporter inhibitor, and concentrations on transport of PA by Caco-2 cells. The data were expressed as the mean ± SD (n = 4). *P < 0.05 and **P < 0.01, compared with control group.