Resistance Mechanisms of Antimicrobial Drugs Against Helicobacter Pylori and Their Novel Treatment Methods

At the end of 2021, the U.S. Department of Health and Human Services released the 15th edition of its Report on Carcinogens, listing chronic infection with Helicobacter pylori (Hp) as a definite carcinogen. Since then, the topic of Helicobacter pylori has frequently trended on Weibo's hot search. Why is Helicobacter pylori receiving so much attention? Why is it so difficult to treat? This is actually closely related to the increasing drug resistance rate and decreasing eradication rate of Helicobacter pylori treatment.

Helicobacter pylori (Hp) is sensitive to various antibiotics in vitro, but only a few antibiotics can be used to treat it in vivo. During the application of antibiotics, the limited choice of antibiotics and the frequent use of anti-Hp treatments have exacerbated the development of Hp resistance. The most common Hp resistance clinically is triple resistance to clarithromycin, metronidazole, and fluoroquinolones. Research has found that the resistance rates of Hp to amoxicillin, clarithromycin, metronidazole, and levofloxacin are 4.55%, 27.22%, 39.66%, and 22.48%, respectively.

Drug Resistance Mechanisms

1.Target Gene Mutation

Studies have shown that fluoroquinolones are effective DNA gyrase inhibitors, which can cause irreversible bacterial DNA damage. The DNA gyrase gene consists of two subunits, GyrA and GyrB. Mutations in the Hp DNA gyrase gene, especially GyrA gene mutations, can prevent fluoroquinolones from specifically binding to the enzyme, leading to bacterial resistance. Mutations in the RNA polymerase gene cause conformational changes in the antibacterial drug target site on the RNA polymerase, preventing the antibacterial drug from specifically binding to the enzyme and thus losing its antibacterial effect. Mutations in the B subunit encoded by the RNA polymerase Pro gene are the main cause of Hp resistance to rifampicin.

The anti-Hp mechanism of macrolides involves their reversible binding to the peptidyl transferase loop in the V domain of 23S rRNA, thus preventing the extension of the peptide chain. However, point mutations in the V region of the bacterial 23S rRNA coding gene can alter the ribosome conformation, reducing the affinity between the antibacterial drug and the ribosome. This allows the peptide chain within the ribosome to extend, enabling the bacteria to complete protein synthesis while the antibacterial drug is unable to fully exert its antibacterial effect.

Bacterial penicillin-binding proteins (PBPs) are a class of membrane proteins present on the bacterial surface that play a crucial role in the synthesis of peptidoglycan in the cell wall of Hp, as well as in bacterial growth and reproduction. Under normal conditions, amoxicillin covalently binds to the polybrominated biphenyls in PBPs, blocking the synthesis of peptidoglycan. This results in defects in the bacterial cell wall, causing the bacteria to lose their protective barrier, expand, and rupture, thus exerting the antibacterial effect of amoxicillin. Mutations in PBPs encoding genes weaken the affinity between amoxicillin and PBPs, leading to drug resistance.

2.Changes in Cell Barrier or Efflux System

① Changes in Outer Membrane Proteins (OMPs)

Studies have shown that after being stimulated by antibacterial drugs, Hp can upregulate the expression of OMPs genes. High expression of OMPs can enhance the protective effect of the bacterial outer membrane barrier, further reducing the permeability of antibacterial drugs. This is another mechanism of refractory resistance to amoxicillin and clarithromycin. OMPs genes HopB and HopC can lead to refractory resistance to amoxicillin, while HopB, HofC, and OMP31 can cause resistance to clarithromycin.

② Formation of Bacterial Biofilms

Research has shown that bacterial strains with biofilm formation can exhibit an increased resistance to antibacterial drugs by 10 to 1000 times. This high level of resistance may be related to multiple resistance mechanisms mediated by biofilms. Firstly, similar to high expression of OMPs, the formation of biofilms provides stronger barrier protection for bacteria. Furthermore, studies have shown that biofilm formation can promote changes in OMPs, further enhancing the bacteria's defense capabilities against external factors. Secondly, bacteria located in the deep layers of biofilms enter a dormant state due to a lack of nutrients and oxygen. Generally, antibacterial drugs only have good bacteriostatic and bactericidal effects on bacteria in the active phase, while bacteria in the dormant state deep within biofilms are difficult to kill. Lastly, bacterial biofilms can work in conjunction with efflux pumps to pump out antibacterial drugs that have entered the biofilm, such as clarithromycin.

③ Involvement of Efflux Pumps

Bacterial efflux pumps are a class of multidrug transporters located on the cell membrane that can pump antibacterial drugs into the bacterial cell out of the cell, reducing the concentration of antibacterial drugs within the cell and thus exerting a resistant effect. The efflux pump system can participate in the efflux of multiple antibacterial drugs such as amoxicillin, metronidazole, clarithromycin, tetracycline, etc., significantly increasing the probability of bacteria developing dual or multiple resistance.

In addition, a series of reasons such as bacterial spheroplasty and the secretion of enzymes related to resistance can also allow bacteria to evade the activity of antibacterial drugs, leading to bacterial resistance. Spheroplasty is one of the important reasons for Hp resistance, long-term chronic infection of Hp, and recurrence after eradication. When the external environment is unfavorable for the growth and reproduction of Hp, such as lack of nutrients or oxygen, changes in pH, antibacterial drug intervention, etc., the spiral-shaped Hp will undergo spheroplasty and become L-shaped Hp. Spherical Hp exists in two forms: one is already dead or degenerated, while the other is in a non-active state with relatively weakened pathogenicity and insensitivity to antibacterial drugs. During antibacterial drug treatment, spherical Hp in the non-active state initiates a resistance escape mechanism, significantly reducing the sterilizing activity of antibacterial drugs. Spherical Hp can restore its original morphology and activity after stopping antibacterial drugs for 2 to 4 weeks.

New Treatment Methods

1. Bismuth-Containing Quadruple Therapy (BQT)

The standard triple therapy, consisting of a proton pump inhibitor (PPI), amoxicillin, and clarithromycin or metronidazole, is one of the most commonly used treatments. This regimen requires a duration of 10 to 14 days to achieve the best effect for Hp eradication, and it only shows significant results in low-resistance regions. In some areas, the resistance rate of Hp to macrolide antibiotics has reached or even exceeded the recognized threshold, making clarithromycin triple therapy no longer a suitable first-line empirical treatment option. Bismuth-based quadruple therapy, which combines bismuth agents with antibiotics, has shown high success rates in Hp eradication and has been proven to be an effective regimen, especially for antibiotic-resistant strains. Studies have found that due to the increasing resistance rate of Hp to clarithromycin, bismuth-based quadruple therapy lasting more than 10 days is more preferable in terms of efficacy and safety compared to 14-day triple therapy.

2.Vonoprazan Treatment

The novel potassium-competitive acid blocker vonoprazan has been used to eradicate Hp. As a new and effective acid suppressant, vonoprazan exhibits faster, stronger, and longer-lasting inhibition of gastric acid compared to conventional PPIs. Additionally, it does not require pharmacological activation, has a longer half-life, exhibits strong and long-lasting inhibition of gastric acid secretion, and creates a neutral environment in the stomach unfavorable for Hp growth. Studies have shown that vonoprazan-based triple therapy is more effective and better tolerated than PPI-based triple therapy, while also reducing the incidence of adverse events.

3.Sequential Therapy

In first-line treatment, levofloxacin or macrolide-containing alternative therapies can be selected. Studies have compared the efficacy of 10-day and 14-day sequential therapy with 14-day triple therapy, finding that sequential therapy is superior to standard triple therapy in eradicating Hp infection, indicating that sequential therapy can be used as a standard first-line treatment for Hp infection. Meanwhile, 10-day sequential therapy can be a standard treatment option for Hp infection in primary patients. Other studies have also found that comparing different Hp eradication regimens, the use of probiotics combined with triple therapy for 10-14 days is a better Hp eradication scheme.

4.Probiotics

Peptides and non-peptide anti-pathogen substances produced by probiotics such as Lactococcus lactis, Lactobacillus reuteri, and Lactobacillus bulgaricus can inhibit the growth and adhesion of Hp. In addition, probiotics can provide beneficial effects on adverse reactions that may occur during Hp eradication, such as nausea, vomiting, diarrhea, and taste disorders. The possible mechanisms include inhibiting the colonization and adhesion of Hp, reducing inflammation caused by Hp, regulating the immune response to Hp, and decreasing the incidence of adverse reactions. Researchers have found that the use of Lactobacillus as an adjunctive therapy to triple therapy can also improve the efficiency of triple therapy and reduce the incidence of diarrhea related to triple therapy in adults and children.

5.Other Approaches

Anti-Hp compounds may be effective drugs to alleviate Hp's resistance to antibiotics. Recent studies have found that apigenin, chrysin, kaempferol, and hesperidin exhibit high bactericidal activity against Hp. Among them, chrysin has a synergistic effect with clarithromycin or metronidazole, while hesperidin exhibits additive or synergistic effects with clarithromycin or metronidazole, suggesting their potential role as adjunctive therapeutic options, especially in the treatment of multidrug-resistant Hp strains.

The research results indicate that a series of aflatoxin inhibitors based on nitrobenzoxadiazole exhibit low toxicity and are effective against Hp strains resistant to metronidazole, clarithromycin, and rifampicin. These inhibitors are also able to reduce the colonization rate of Hp in the stomach and can eradicate Hp in 60% of infected mice. The results suggest that these aflatoxin inhibitors constitute a new family of specific antibacterial agents that may help address the issue of Hp resistance to antibiotics in the future.

Additionally, research has found that oral preparations based on Saccharomyces cerevisiae can effectively reduce the bacterial load after Hp infection. The results indicate that Saccharomyces cerevisiae-based preparations can serve as promising candidates for future development of bacterial oral therapeutics.

References

[1] Hou Chong, Liu Yipin. Research Progress on the Molecular Mechanisms of Antimicrobial Drug Resistance of Helicobacter pylori [J]. Journal of Gastroenterology, 2021, 26(03): 171-175.

[2] Chen Meihong, Yan Jin, Dang Yini, Zhang Guoxin. Research Progress on Antimicrobial Drug Resistance of Helicobacter pylori [J]. Journal of Gastroenterology, 2019, 24(02): 115-118.

[3] Zhang Xiaotian, Ke Chongwei. Research Progress in the Treatment of Drug-resistant Helicobacter pylori Infection [J]. Shanghai Medical & Pharmaceutical Journal, 2022, (Issue 1).

About the Author: 

Xiaomichong, a researcher in pharmaceutical quality, has been dedicated to pharmaceutical quality research and verification of drug analysis methods for a long time. Currently, she works in a large domestic pharmaceutical research and development company, engaged in drug inspection analysis and verification of analytical methods.