Antibiotic resistance analysis and coping strategies of helicobacter pylori

CHEN Run-xiang, ZHANG Xiao-dong, CHEN Shi-ju, BAI Fei-hu

1. Graduate School of Hainan Medical College, Haikou 570100, China

2. The Second Affiliated Hospital of Hainan Medical University, Haikou 570100, China

3. Hainan Digestive Disease Clinical Research Center, Haikou 570100, China

Keywords:

ABSTRACT Helicobacter pylori (H.pylori) is a Gram-negative bacterium that mainly colonizes the stomach and duodenum, and it can cause gastrointestinal diseases such as gastric inflammation, peptic ulcer and gastric cancer, and eradication of H.pylori can effectively stop the occurrence and development of gastrointestinal diseases.Antibiotics are one of the main drugs used to treat H.pylori.Due to the long-term application of antibiotics, the resistance rate of H.pylori to antibiotics increases year by year, which greatly reduces the eradication rate of H.pylori and increases the difficulty of re-treatment and the economic burden of patients.In this paper, we will review three aspects of H.pylori resistance status, resistance mechanism and treatment to provide reference for the progress of H. pylori resistance research and its treatment strategy.

1.Prevalence of Antibiotic Resistance in Helicobacter pylori

The global antibiotic resistance situation of H.pylori is not optimistic.A variety of antibiotics used to treatH.pyloriare showing high resistance rates, with clarithromycin, metronidazole and levofloxacin being the most prominent, posing a huge challenge to the treatment ofH.pylori.Over the past decade, clarithromycin resistance rates have been increasing, with rates above 20% in most countries and regions, and even a high resistance rate of 90% in Melbourne.[5] The antibiotic resistance rate ofH.pyloriis generally high in China, with 20%～50% in relevant studies, which greatly reduces the eradication efficiency of H.pylori.[6-8] In recent years,consensus and guidelines have not recommended the use of standard triple therapy for the eradication of H.pylori in some areas where the rate of clarithromycin resistance is higher than 15%.In the past decades, metronidazole has been widely used in gynecology and dentistry because of its low price and high bactericidal efficiency,so abuse of metronidazole has occurred in many regions, leading to a dramatic increase in the rate of resistance ofH.pylorito metronidazole.Except for a few developed regions, the resistance rate of metronidazole is above 40% in most countries worldwide.[8-12] A study of 17,731 cases ofH.pyloristrains culturedin vitroin the southeastern coastal region of China found that the resistance rate of H.pylori to metronidazole reached 95.4%, which is one of the most serious areas of metronidazole resistance in recent years.[7] Levofloxacin is one of the commonly used drugs to eradicateH.pylori, and the increase of levofloxacin resistance rate in recent years cannot be ignored.According to the literature, the levofloxacin resistance rate has shown an increasing trend year by year in Beijing and Nanjing, China.In Nanjing[8], the resistance rate increased from 14.7% to 26% in the past 5 years, and in Beijing[6], it increased from 24.2% to 87.8% between 2008 and 2014, nearly tripling.Although the local area does not represent the overall level of antibiotic resistance in the China, it reflects to a certain extent the worsening trend of levofloxacin resistance, so in areas with highH.pyloriresistance rate, levofloxacin should be used with caution.

Amoxicillin, furazolidone and tetracycline generally have low resistance rates, generally below 10% in most regions (Table 1), and have become the first-line antimicrobial agents recommended in many guidelines.[37,42]

In addition to regional differences, factors such as age, gender and ulcer site were associated with H.pylori resistance.A study from Poland showed that clarithromycin resistance was significantly higher in the pediatric group than in the adult group(54.5%vs34.6%,P<0.05),while metronidazole resistance was higher in the adult group than in the pediatric group(46.9%vs31.8%, P<0.05).[13] Researchers collected and analyzed 1,117H.pyloristrains from patients in 13 Chinese provinces and cities, found that younger patients had lower rates of resistance to levofloxacin and clarithromycin compared to middle-aged and older patients; levofloxacin resistance differed significantly by gender and ulcer site, with female patients having higher rates of resistance than males(22.7%vs16.8%, P<0.05)and gastric ulcer patients having significantly higher rates of resistance than duodenal ulcer patients(41.7%vs18.3%, P<0.05).14 However,some studies have concluded that there is no correlation between age and gender and antibiotic resistance, so the association of factors such as age and gender with antibiotic resistance needs to be further validated by more studies.15

With the misuse of antibiotics and irregular treatment, multi-drug resistance of H.pylori has become an increasingly serious problem.The multi-drug resistance rate is higher than 20% in many countries and regions and is on the rise, and the combination of metronidazole and clarithromycin being the most common.8,13 This may be related to the fact that both drugs are commonly used to treat other infectious diseases.It is now believed that the decline in H.pylori eradication efficiency is closely related to antibiotic multi-resistance.Therefore, the indications for antibiotics should be strictly controlled in clinical practice, and in addition, the success rate of the first eradication of H.pylori should be enhanced as much as possible to prevent the development of secondary drug resistance, which makes eradication more difficult.Individualized treatment based on antibiotic sensitivity testing can effectively improve the eradication rate and perhaps further alleviate the severe antibiotic resistance situation, and should be promoted in the clinic.

2.Mechanisms of antibiotic resistance in Helicobacter pylori

2.1 Clarithromycin

In 1996, Versalovic et al.first demonstrated that H.pylori was resistant to macrolide antibiotics due to a point mutation in the V region of its 23SrRNA, which resulted in a change in the conformation of the ribosome and a consequent change in the binding site for macrolide antibiotics, and that the affinity between H.pylori and macrolide antibiotics was reduced.Finally, the drug is unable to stop the protein synthesis of the bacteria and develops antibiotic resistance.[16] Further studies confirmed that A2142G and A2143G in 23SrRNA were the most common point mutations and that these mutations play a major role in clarithromycin resistance.[17-19] One of the reasons whether the detection of mutated genes can be applied in clinical practice has not been fully confirmed yet is the existence of imprecision in gene mutations, such as the difference in gene mutations between primary and secondary resistance, which may be used in the future to replace clarithromycin drug sensitivity testing by detecting these mutated genes for guiding individualized clinical treatment.Zhu Zhenhua et al.studied 42 clarithromycinresistant H.pylori strains isolated in Nanchang, China, from 2002 to 2006 and found that primary clarithromycin-resistant strains had two major mutation loci, A2143G and T2182C, while secondary resistance appeared with four mutation loci, A2143T, T2182C,G2172T and G2254T, indicating that antibiotic application may mutate bacterial resistance genes.[20] In addition, different mutated genes have different sensitivity and specificity for determining clarithromycin resistance, which may be one of the reasons hindering the use of genetic testing in clinical practice.A meta-analysis ofH.pylori resistance genes found that the combination of mutant loci A2142C, A2142G and A2143G had high sensitivity and specificity in detecting clarithromycin resistance.[21] Perhaps we can improve the accuracy of determining H.pylori resistance to clarithromycin by combining multiple mutated genes for better clinical application.

2.2 Metronidazole

It was found that mutations in the rdxA and frxA genes of H.pylori caused a weakened enzymatic reduction reaction, reduced metabolite production of metronidazole, and diminished antibacterial effects,ultimately leading to metronidazole resistance in H.pylori.Among them,rdxAgene is responsible for encoding oxygen-insensitive NADPH-nitroreductase andfrxAis responsible for encoding NADPH-flavin oxidoreductase, and mutations in these two genes are highly associated with metronidazole resistance.[22,23] The literature reports repeat mutations at positions 118, 131, 172 and 183 ofrdxAand positions 72, 73, 110, 126 and 193 of frxA, and mutations areprevalent in both genes[22], suggesting that it is difficult for us to identify metronidazole resistance by mutations, thus limiting the application of clinical PCR to detect metronidazole resistance.Another study reported that rdxA mutations are closely associated with eradication failure(P=0.002) and nonsense mutations inrdxAreduce eradication efficiency(P=0.009), perhaps by detecting the above genes to guide clinical dosing[23].rdxA and frxA gene mutations correlation with metronidazole resistance needs further study to provide theoretical basis for clinical application.

Tab 1 H.pylori resistance in different regions

2.3 Levofloxacin

In addition to being involved in DNA replication, recombination and transcription, H.pylori gyrase is essential for maintaining the helical structure of DNA, and it is by inhibiting the function of DNA gyrase in bacteria that fluoroquinolones exert their antibacterial effects.[24] Gyrase is a tetramer composed of two A and two B subunits, encoded by the gyrA and gyrB genes, respectively, and it is currently believed that point mutations in the quinolone resistance determining region (QRDR) of thegyrAgene prevent the binding between the antibiotic and the enzyme, allowing bacteria to develop antibiotic.[25] Although antibiotic sensitivity testing plays a crucial role in the treatment ofH.pylori, traditional methods have had difficulty detecting heterogeneous resistance, leading to increased prevalence of resistant strains and eradication failure.When 694gyrAgene-positive biopsy samples were analyzed in the study, 99(14.3%) showed heterogeneous resistance genotypes, and 68.1%of heterogeneous resistant samples were found to be resistant to levofloxacin based on the drug sensitivity results.[26] This suggests that the detection rate of levofloxacin resistance can be improved by drug sensitivity testing and genetic testing to better guide clinical drug use.In addition,gyrBgene mutation loci are variable, and the association with levofloxacin resistance is not as close as that ofgyrA.[27] We expect more studies to elucidate the relationship betweengyrBgene and levofloxacin in the future.

2.4 Amoxicillin

β-lactam antibiotics exert their antibacterial effects by binding tightly to penicillin binding proteins (PBPs), which inhibit bacterial cell wall synthesis and lead to lysis of bacteria.From a genetic point of view, mutations inpbp1are a common mechanism forH.pylorito develop moderate or low levels of resistance to amoxicillin,while high levels of amoxicillin resistance are associated with the production of β-lactamases byH.pylori.[28] The current rate of amoxicillin resistance is low, but recent reports in the literature have found that with the failure of H.pylori eradication, the MIC90of amoxicillin are increased at a double rate[29], so the indications for amoxicillin should be strictly controlled in clinical work to avoid causing further secondary resistance.Gastric mucosal specimens were transported frozen to an off-site laboratory for in vitro culture and drug sensitivity testing, which could lead to a decrease in amoxicillin resistance and affect the experimental results, thus reducing the success rate ofH.pylorieradication; in addition, it was found that the decrease in amoxicillin resistance was closely related to the disappearance of several genetic mutations, among which PBP1 and HefC played a major role.[30]

2.5 Multiple antibiotic resistance mechanisms

Recent studies have found that biofilm is also an important reason for the failure of H.pylori eradication.Biofilm is an important component ofH.pylori, which can form biofilms both in vitro and on the gastric mucosa, and antibiotic resistance ofH.pyloriincreases dramatically after biofilm formation.[31] Studies have shown that high levels of biofilm-forming strains are more resistant to all antibiotics than low levels of biofilm-forming strains, with the difference being more pronounced in clarithromycin.[32] In addition, overexpression of drug efflux pumps is one of the important mechanisms of bacterial multi-drug resistance.Bacteria discharge harmful substances through the efflux pump to regulate the stability of their internal environment to prevent damage to themselves, the existence of the efflux pump hinders the killing effect of antibiotics on bacteria, the bacterial efflux pump is composed of a series of transport proteins, which is an important structural basis for bacteria to discharge harmful substances.[33] Although there are many transporter proteins involved in constituting the efflux pump, only a few of them are involved in the formation of resistance[34], such as transporter proteins HP0939,HP0497 and HP0471, which are not only involved in the formation of the efflux pump but also in the formation of biofilm and are highly expressed in multi-drug resistant strains, suggesting that they are closely associated with multi-drug resistance inH.pylori.[35]The emergence of multi-drug resistance exacerbates the difficulty of clinical eradication ofH.pyloriand increases the economic burden on patients.Therefore, regulating the use of antibiotics, treatment based on local resistance and drug sensitivity testing are proven methods to prevent biofilm formation and overexpression of efflux pumps, and ultimately to prevent multi-drug resistance.

3.Treatment of Helicobacter pylori

3.1 Dual, triple and quadruple therapy

H.pylori eradication regimens include antibiotics and Proton Pump Inhibitors (PPI), in which antibiotics play a major role in bactericide,while PPI acts to enhance the eradication efficiency of antibiotics by inhibiting gastric acid secretion and providing a suitable intragastric environment for antibiotics to work[36], and commonly used domestic and international treatment regimens include dual, triple and quadruple therapy.

In many parts of the world, triple therapy consisting of a proton pump inhibitor with amoxicillin and clarithromycin (PPI-AC)remains the most commonly used first-line therapy.The main determinant of the eradication success of this regimen is pretreatment clarithromycin resistance, and the prevalence of antibiotic resistance, especially clarithromycin resistance, varies widely around the world (Table 1).Therefore, the Maastricht V [37]recommends that triple therapy should be abandoned as a first-line treatment option in areas with high rates of clarithromycin resistance (more than 15～20%).The optimal timing of treatment is a controversial issue.[38,39] Studies of systematic reviews have found that longer regimens usually have higher eradication rates, and therefore recommend 14 d triple therapy in areas with high rates of clarithromycin resistance.However, in resource-poor countries, longer regimens imply increased treatment costs and a significantly increased risk of adverse effects with prolonged antibiotic use, in addition to reduced patient compliance.In contrast, some countries have reported acceptable eradication rates with 1-week PPI-AC treatment.[38,40] Therefore,eradication regimens should be developed not only depending on the resistance patterns and eradication rates in a region, but also taking into account the local economic level.

With the increasing problem of clarithromycin resistance,quadruple therapy containing bismuth is used as a first-line treatment option in an increasing number of countries and regions.Most foreign quadruple regimens consist of PPI, bismuth, tetracycline, and metronidazole because it delivers reliable and acceptable eradication rates regardless of clarithromycin and metronidazole resistance.[41]The main drawbacks of this therapy are the excessive number of daily doses and the high rate of adverse reactions, which can affect patient compliance and treatment integrity.Seven different 14 d bismuth quadruple regimens are mainly recommended as the firstline regimen forH.pylorieradication in China, and the bismuth quadruple regimen has been clinically proven to be a practical regimen in China and abroad[42,43] ,it is recommended by the Maastricht-5 consensus for clinical application.[37]A prospective randomized trial enrolling 232 patients randomized to 14 days of diptych or quadruple therapy found no significant difference in eradication rates between diptych and quadruple therapy, and that diptych had a lower overall adverse effect rate and was less costly.[44] The high-dose amoxicillin and PPI duo regimen has a high eradication rate and low adverse effect rate, effectively improving patient compliance, and therefore may be used as a firstline treatment option in the future.

3.2 New Drugs

In order to improve the eradication efficiency and reduce the side effects caused by the treatment process, many new drugs have been developed in recent years, such as Vonorazan and probiotics, both of which have achieved good expected results in clinical trials.

As a potassium ion-competitive acid blocker (P-CAB), Vonorazan is unaffected by CYP2C19 gene polymorphism compared to proton pump inhibitors (PPI), and has a long-lasting and stable inhibitory effect on gastric acid secretion.Japanese researchers found that the pH of gastric acid could reach 7.0 within about three hours after dosing, indicating that Vonorazan could achieve ideal gastric acid eradication conditions forH.pyloriin a short period of time.[45] In a randomized, double-blind phase III clinical study, a 7-day triple therapy with vonorazan had a significantly higher eradication rate than a triple therapy with lansoprazole(92.6%vs75.9%).[46] Compared to PPI and amoxicillin, vonorazan-containing triple therapy achieved higher eradication rates at lower doses and shorter dosing times.[47]Considering that most of the study data of Vonorazan are from Asian countries, further studies are needed to determine whether drug resistance in different country regions may affect the eradication efficiency of Vonorazan.

The application of antibiotics enhances the efficiency of H.pylori eradication, but side effects are also common, such as imbalance of intestinal flora, and patients experience constipation and diarrhea.Adding probiotics to the eradication regimen can effectively reduce side effects, enhance compliance, and improve the efficiency ofH.pylorieradication.[48] Therefore, probiotics can be used as a complementary treatment for H.pylori to reduce the occurrence of complications.

3.3 Individualized treatment

The emergence of antibiotic resistance has seriously affected the eradication efficiency of H.pylori, and individualized treatment based on drug sensitivity testing has received clinical attention.A meta-analysis showed that the eradication rate of individualized treatment was superior to that of empirical standard triple therapy and bismuth quadruple therapy.[49] In addition, individualized protocols have lower rates of adverse reactions than empirical therapies.[50] A multicenter double-blind randomized controlled trial recruited 467 H.pylori-positive patients and randomly assigned them to three different treatment regimens, including individualized triple therapy, empirical quadruple therapy and individualized quadruple therapy, with eradication rates of 74.64%, 68.49% and 91.22%, respectively, with the individualized quadruple regimen showing much higher eradication rates than the individualized triple regimen(P<0.001).[51] Although the advantages of individualized treatment protocols are obvious, there are certain limitations, such as invasive examinations, prolonged treatment courses, increased costs and technical limitations, which are important reasons limiting their clinical application.Therefore, a comprehensive consideration of local medical conditions, patients’ economic conditions and medical expectations is needed in the development of treatment plans.

4.Summary

H.pylori is a bacterium that is both infectious and pathogenic,leading to high infection rates and many diseases worldwide.An in-depth study of the mechanism ofH.pyloriresistance provides a theoretical basis for detecting antibiotic resistance.Despite the availability of multiple therapeutic agents, antibiotics remain one of the main agents for eradication ofH.pylori, and triple or quadruple therapy remains the first-line regimen for the treatment of H.pylori.Considering the serious situation of H.pylori resistance, the selection of antibiotics based on drug sensitivity testing is a proven method to improve eradication efficiency.

Authors’ Contribution

Chen Run-xiang: wrote the paper and revised it; Chen Shi-ju and Zhang Xiao-dong were responsible for graph production, collected literature and participated in part of the writing; Bai Fei-hu proposed revisions to the article and reviewed it.

Author Conflict of Interest Public Statement:

No potential conflicts of interest were identified by all authors during the study.