Antibiotic resistance characteristics and risk factors analysis of Helicobacter pylori strains isolated from patients in Liaoning Province, an area in North China

Introduction

Helicobacter pylori (H. pylori) is a ubiquitous bacterium which is well resistant to the gastric acid. It was estimated that the global prevalence of H. pylori was approximately 50% (Hooi et al., 2017). Previous studies established that the colonization of H. pylori was directly involved in the occurrence of various gastrointestinal diseases (Kishikawa et al., 2020; Yamaoka, 2018). Emerging evidence has suggested that H. pylori eradication could cure gastrointestinal lesions (Lanas & Chan, 2017; Sonnenberg, Turner & Genta, 2020) , delay the development of gastric cancer (Kosunen et al., 2011) , and reducing the recurrence of peptic ulcers and gastric cancers (Chey et al., 2017; Malfertheiner et al., 2017). Thus, it is essential to conduct eradication treatment of this bacteria.

Currently there are three main antibiotic-based eradication strategies of H. pylori infection: bismuth quadruple, triple therapy and sequential therapy (Chey et al., 2017). H. pylori treatment guidelines recommend different regimens in various countries. Cho & Jin (2022) discussed in detail recent guidelines for H. pylori treatment among China, Japan and South Korea, summarized the eradication rates of numerous clinical trials in each country. They revealed current situation of clinical regimen chosen in various countries. The most commonly selected antibiotics in China are furazolidone (AOZ), tetracycline (TC), levofloxacin (LFX), metronidazole (MET), clarithromycin (CLA), and amoxicillin (AMX). Unfortunately, the eradication rate of H. pylori has fallen due to ever increasing antibiotic resistance during the last decades. A systematic review and meta-analysis found the overall eradication rate of H. pylori resistant to antibiotics was significantly more than 16% lower than that sensitive to antibiotics (Zou et al., 2020). Scientists reported that primary resistance to LFX and MET respectively was more than 15% worldwide (Savoldi et al., 2018). Further analyses concordantly showed evident regional differences in antibiotic resistance to H. pylori (Megraud et al., 2021). For example, one study has shown that the antibiotic resistance to CLA was 31.1% in the Japanese population (Okamura et al., 2014). While in Portugal and in the southeast area of Vietnam, the rate of CLA-resistance was reported to be 42% and 72.6%, respectively (Dang et al., 2020; Lopo et al., 2018). Meanwhile, in China, a large country in Asia, the infection rate of H. pylori was almost 55.8%, and the antibiotic resistance differs greatly in various areas (Hooi et al., 2017). The resistance rate of MET was up to 95.5% in Jiaxing City, whereas in Beijing the rate was only 66.8% in the samples obtained during 2009 and 2010 (Ji et al., 2016; Zhang et al., 2015b). Till now, the H. pylori antibiotic resistance rates and patterns in Liaoning, an area of north China, remain unclear.

Risk factors for H. pylori antibiotic resistance have guiding significance in clinical practice, several studies were conducted to explore associations between related factors and their regional antibiotic resistance situation (Boyanova et al., 2012; Ji et al., 2016). By assessing whether infected patients have or how many factors they have, physicians would be instructed to evaluate possible antibiotic resistance. Therefore, analysing correlations between them in separated regions is of great significance to guide clinical drug use for physicians and prevent the occurrence of drug resistance for patients.

In the current study, we investigated the antibiotic resistance to AOZ, TC, LFX, MET, CLA, and AMX in Liaoning Province. Risk factors for antibiotic resistance were further evaluated. Our study intended to provide guidance for the individualized treatment of H. pylori, and thus to improve the eradication rate in Liaoning Province.

Materials & Methods

Results

Among 178 patients determined as H. pylori positive by Urea breath test (UBT), successful bacterial cultures were obtained for 163 (91.57%) patients.

Antibiotic resistance rates in H. pylori isolates

No resistance to AOZ or TC was observed in the population. The overall resistance rates to both MET and CLA remained at about the same high level (79.14% for MET, followed by 71.78% for CLA), with AMX having the lowest rate, but still at 22.09% (Fig. 1). CLA (70.59%) owned the highest antibiotic resistance rate among the 17 isolates without H. pylori treatment history, along with MET (64.71%), LFX (47.06%), and AMX (17.65%). For 146 subjects once accepted H. pylori treatment, resistance rates in descending order were MET (80.82%), CLA (71.92%), LFX (40.41%), and AMX (22.60%), remaining in the same order as the overall resistance rates.

Discussion

H. pylori antibiotic resistance is a major threat to affect the success of current therapies and regional difference has been an ordinary issue in the antibiotic resistance to H. pylori. This issue could be alleviated by antimicrobial susceptibility testing for H. pylori, but with harsh growth environment requirements, operational safety and economic benefits and risks to be further evaluated, so this testing method almost remains unavailable in clinical practice (Jiao, Wang & Ma, 2021; Jones et al., 2017). Under this circumstance, profiling regional antibiotic resistance patterns and specific characteristics is an extremely important measure for guiding the choice of therapeutic regimens. It is urgent to supervise respective antibiotic resistance patterns in different areas to solve the problem of indiscriminate and excessive use of antibiotics.

The resistance rate to antibiotics of H. pylori in China has been increasing with an upward trend during several years. From two studies, primary resistance rates to LFX, MET, CLA and AMX were all much higher than 5 years ago (Chen et al., 2022; Hu, Zhu & Lu, 2017) , as evidence of the difficult eradication today. The data about antimicrobial resistance in Liaoning were the first to be reported and revealed a situation that the overall resistance rates to LFX, MET, CLA, and AMX were higher than those in China (Chen et al., 2022) , especially for CLA (71.78% further higher than 30%), except for TC and AOZ with undetected results. These results were consistent with the conclusion by Liu et al. (2019). Such different prevalence might be correlated well with urbanization level, socioeconomic status, sanitary level and so on (Hooi et al., 2017). We found that both primary and overall antibiotic resistance rates to CLA (70.59% and 71.78%, respectively) were much higher than those in China (34% and 30%, respectively) (Chen et al., 2022) , possibly because the cold weather in north areas could cause colds and flu, and macrolides are widely prescribed for respiratory infections (Megraud et al., 2021). CLA is still widely used in many regions as an antibiotic in the standard triple therapy for H. pylori eradication (Fallone et al., 2016). It was recommended to avoid the usage of CLA if the local resistance to CLA was over 15% (Malfertheiner et al., 2017; Mascellino et al., 2017). Therefore, in the studied region, regimens including CLA was not appropriate for empirical treatment unless the prior antibiotic susceptibility test revealed a sensitive result with CLA. For AMX, all the AMX-resistant patients were multi-resistant. We found the primary AMX-resistant rate in our research (17.65%) is relatively higher than those in China (3.1%) (Hooi et al., 2017) or other places in China, such as Zhang et al. (2015b) in Beijing (4.4%) between 2013 and 2014 and Jiang et al. (2021) in Nanjing (1.83%). Nonetheless, the AMX-resistance rate was still the lowest among the four antimicrobials in this study. Such phenomena revealed less utilization of AMX in this area. AMX-based regimens might have the potential to be adopted as an empirical clinical treatment prescription in the future. MET-resistance rates differ all over the world. In western countries, it gained improvements after regulated uses, such as 16%, 25% and 27% respectively in Austria (Zollner-Schwetz et al., 2016), Portugal (Lopo et al., 2018) and Spain (Macias-Garcia et al., 2017). Meanwhile, in China, the use of inexpensive, easily obtained prescription medicine is easier to develop resistance (70%) (Chen et al., 2022). Within China itself, compared to other areas (Yu et al., 2019; Zhang et al., 2015b), the present study reported a high overall MET-resistance rate (79.14%). Though the typical empirical regimen(proton pump inhibitor, MET, CLA, and AMX) with increased dosage of MET or AMX might be an alternative to partly overcome the resistance to MET (Chen et al., 2019; Liang et al., 2013; Zhang et al., 2015a) , increased incidence of adverse side effects such as vomiting, nausea and rashes require consideration (Graham & Lee, 2015). Therefore, when prior susceptibility testing is not available, we suggest empirical treatment regimens containing MET be preferably abandoned. Resistance rate to LFX was up high to 41.10% in this study, higher than in China (31%), but almost similar to those in Shanghai (40.7%) (Yu et al., 2019) and Shenzhen (39.8%) (Lyu et al., 2020). Recently, LFX has been widely applied in H. pylori eradication treatment for the high CLA- and MET-resistance. A research discovered LFX-resistance were significant different between women and men (40.5% versus 21.5%), probably relating to the extent of use for urinary tract infections (Boyanova et al., 2012). We also found that the resistance to LFX presented a higher rate in female patients (27.61%) than that in male patients (13.50%). Such results suggested that we should reduce the frequency of LFX application among women population. Resistance to AOZ or TC was not detected in this study. Similar phenomenon has been observed in a population living in Sichuan Province, where the primary resistance rate of AOZ or TC was 0.8% (Tang et al., 2020). TC is not a common prescription in China, which means that semisynthetic TC derivatives such as minocycline might replace TC to gain good efficiency. Further randomized controlled trials need to be conducted.

In terms of resistance patterns, among strains with no antibiotic resistance, treatment-naïve strains accounted for a larger proportion. In other resistance patterns, treatment-experienced group was the majority. Dual resistance was an exception, treatment-naïve population (41.18%) were more frequently found than the other(29.45%). Dual resistance occupied the largest ratio (30.67%) of resistance patterns. Isolates exhibiting simultaneous resistance to CLA and MET had the largest prevalence (19.63%), followed by CLA+LFX(6.13%). Dual antibiotic resistance rate to CLA and MET from an Iran study (n = 12, 12%) were similar to ours and demonstrated the combination of those two antibiotics were not recommended as the first-line treatment (Hamidi et al., 2020). Based on our data, the frequency of triple antibiotic resistance was 25.77%, with LFX+MET+CLA predominating (n = 31, 19.02%). This might result in the failure eradication in this region and indicate advance drug susceptibility tests are required for the use of these drugs.

Some factors were investigated to influence antibiotic resistance profiles in the present study. A significant relationship was found between CLA-resistance and endoscopic diagnosis (P = 0.010), we further analysed that MALToma (P = 0.021) was associated with resistance to CLA (Table 4). It has been reported that 60–80% of MALToma patients with H. pylori would be in remission after eliminating the bacteria (Nakamura & Matsumoto, 2013), and long-term CLA use or the immunomodulatory drug lenalidomide also work well (Raderer & Kiesewetter, 2020). An Australia study detected MALToma patients with H. pylori from 1997 to 2014 and concluded CLA-resistance rate was as low as 15%, which supported eradication regimens containing CLA (Bilgilier et al., 2016). However, the present high CLA-resistance rate and its close association with MALToma suggest us antimicrobial susceptibility tests should be required among those patients to select the appropriate antimicrobial eradication therapies or refined immunomodulatory strategies. We found a significant difference between MET resistance and age (P < 0.001). MET is a high prescription rate medicine (Ghotaslou et al., 2018). Choe et al. (2022) concluded that previous MET exposure caused a significant reduction in eradication rate of bismuth quadruple therapy. Another study (Lee et al., 2020) calculated that the odds ratio of past MET use leading to eradication failure was 3.468. Therefore, MET resistance might be related with an increased exposure of drugs. Antibiotics should be chosen more cautiously among patients of all ages. Other factors including gender, BMI, hypertension, diabetes mellitus and so on had no significant relationship with antibiotic resistance. The results of the above data might be due to the tiny sample size, which required to be further explored by expansion of the sample size.

Our study also had limitations. There were limited primary treatment subjects in this study. This is because in clinical practice, antibiotic sensitivity tests are usually applied to patients who have failed eradication several times. Patients without previous treatment history tend to choose empirical treatment regimen, so it is difficult to collect samples from first-treatment subjects, which is also a real problem. But we still offered an updated profile of H. pylori infectious patients in Liaoning. Researchers could carry this study continuously, by combining multiple hospitals and expanding the sample size, especially the number of subjects without experienced treatment history, and to explore a more stable antibiotic resistance profile in Liaoning. It is vital for clinical physicians to modify prescriptions, prevent unnecessary antibiotic consumption and evaluate the effectiveness of empirical treatment in time to improve eradication rate.

Conclusions

In conclusion, among this north China population, the primary resistance rates of LFX, MET, CLA, and AMX were relatively high and different in comparison with the overall or regional resistant data in China. To achieve good success rates, prior antimicrobial resistant tests are suggested before antibiotic prescription in H. pylori eradication to achieve good success rates and avoid severe antibiotic resistance.

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