Antibiotic Resistance Prevalence and Trends in Patients Infected with Helicobacter pylori in the Period 2013–2020: Results of the European Registry on H. pylori Management (Hp-EuReg)

Background: Bacterial antibiotic resistance changes over time depending on multiple factors; therefore, it is essential to monitor the susceptibility trends to reduce the resistance impact on the effectiveness of various treatments. Objective: To conduct a time-trend analysis of Helicobacter pylori resistance to antibiotics in Europe. Methods: The international prospective European Registry on Helicobacter pylori Management (Hp-EuReg) collected data on all infected adult patients diagnosed with culture and antimicrobial susceptibility testing positive results that were registered at AEG-REDCap e-CRF until December 2020. Results: Overall, 41,562 patients were included in the Hp-EuReg. Culture and antimicrobial susceptibility testing were performed on gastric biopsies of 3974 (9.5%) patients, of whom 2852 (7%) were naive cases included for analysis. The number of positive cultures decreased by 35% from the period 2013–2016 to 2017–2020. Concerning naïve patients, no antibiotic resistance was found in 48% of the cases. The most frequent resistances were reported against metronidazole (30%), clarithromycin (25%), and levofloxacin (20%), whereas resistances to tetracycline and amoxicillin were below 1%. Dual and triple resistances were found in 13% and 6% of the cases, respectively. A decrease (p < 0.001) in the metronidazole resistance rate was observed between the 2013–2016 (33%) and 2017–2020 (24%) periods. Conclusion: Culture and antimicrobial susceptibility testing for Helicobacter pylori are scarcely performed (<10%) in Europe. In naïve patients, Helicobacter pylori resistance to clarithromycin remained above 15% throughout the period 2013–2020 and resistance to levofloxacin, as well as dual or triple resistances, were high. A progressive decrease in metronidazole resistance was observed.

E-Test or Agar Dilution for Metronidazole Susceptibility Testing of Helicobacter Pylori: Importance of the Prevalence of Metronidazole Resistance

Background: A number of studies have shown that E-test overestimated the presence of Helicobacter pylori (H. pylori) resistance compared to agar dilution.Objective: The purpose of this study was to explore whether E-test could be an alternative for agar dilution to detect the metronidazole susceptibility of H. pylori.Method: E-test and agar dilution were used to assess susceptibility of H. pylori to metronidazole, clarithromycin and levofloxacin in 281 clinical isolates obtained from China where resistance was high. Cohen kappa analysis, McNemar test, essential and categorical agreement analysis were performed for these two methods. Results: Overall, the result of E-test showed similar prevalence of resistance rate to all antibiotics compared with agar dilution. The essential agreement (EA) of E-test method and agar dilution in the evaluation susceptibility of H. pylori to clarithromycin and levofloxacin were moderate, with 89.0% and 79.7% respectively, but only 45.9% for metronidazole. Results showed categorical agreement (CA) between E-test and agar dilution were 100% for both clarithromycin and levofloxacin. As for metronidazole, the CA was 98.7%, no major error was identified, and rate of very major error was 1.8%.Conclusion: E-test can be an alternative method to detect the metronidazole susceptibility of H. pylori in regions where high-level resistance is common.

The Integrity of Heme Is Essential For Reproducible Detection of Metronidazole-Resistant Clostridioides difficile By Agar Dilution Susceptibility Tests

Metronidazole resistance in clinical Clostridioides difficile is often described as unstable, since resistant strains reportedly appear susceptible following freezer storage or brief passage. This has presented a conundrum for adopting susceptibility testing to accurately evaluate the connection between metronidazole resistance and decreased clinical efficacy of metronidazole in patients with C. difficile infections (CDIs). We discovered that supplementation of microbiological media with the metalloporphyrin heme is crucial for detection of metronidazole-resistant C. difficile using the agar dilution susceptibility testing method. Known metronidazole-resistant strains appeared susceptible to metronidazole in media lacking heme. Similarly, these resistant strains exhibited increased susceptibility to metronidazole when tested on heme-containing agars that were exposed to room light, for more than 1 day, likely due to heme photodecomposition. In parallel experiments, resistance was reproducibly detected when heme-containing agars were either prepared and used on the same day or were protected from light and then used on subsequent days. Notably, heme did not influence the susceptibilities of drug-susceptible strains that were of the same ribotype as the resistant strains. These findings firmly show that the consistent detection of metronidazole-resistant C. difficile is dependent upon heme and its protection from light. Studies are warranted to determine the extent to which this heme-associated metronidazole-resistant phenotype affects the clinical efficacy of metronidazole in CDI and the underlying genetic and biochemical mechanism.

Effect of Temperature on Metronidazole Resistance in Helicobacter pylori

Efficacy of Helicobacter pylori (H. pylori) eradication therapy has declined due to rapid rises in antibiotic resistance. We investigated how increased temperature affected H. pylori (NCTC 11637) growth and its sensitivity to metronidazole in vitro. We performed transcriptomic profiling using RNA-sequencing to identify differentially expressed genes (DEGs) associated with increased temperature. Transcriptional pathways involved in temperature-driven metronidazole resistance changes were analyzed through bioinformatic and literature curation approaches. We showed that H. pylori growth was inhibited at 41°C and inhibition was more apparent with prolonged incubation. Resistance to metronidazole was also reduced—minimum inhibitory concentration for metronidazole decreased from &gt; 256 μg/ml at 37°C to 8 μg/ml at 41°C after culturing for 3 days. RNA-sequencing results, which were highly concordant within treatment conditions, revealed more than one third of genes (583/1,552) to be differentially expressed at increased temperatures with similar proportions up and down-regulated. Quantitative real-time PCR validation for 8 out of 10 DEGs tested gave consistent direction in gene expression changes. We found enrichment for redox and oxygen radical pathways, highlighting a mechanistic pathway driving temperature-related metronidazole resistance. Independent literature review of published genes associated with metronidazole resistance revealed 46 gene candidates, 21 of which showed differential expression and 7 out of 9 DEGs associated with “redox” resistance pathways. Sanger sequencing did not detect any changes in genetic sequences for known resistance genes rdxA, frxA nor fdxB. Our findings suggest that temperature increase can inhibit the growth and reduce H. pylori resistance to metronidazole. Redox pathways are possible potential drivers in metronidazole resistance change induced by temperature. Our study provides insight into potential novel approaches in treating antibiotic resistant H. pylori.

Heme is crucial for medium-dependent metronidazole resistance in clinical isolates of C. difficile

Until recently, metronidazole was the first-line treatment for Clostridioides difficile infection and it is still commonly used. Though resistance has been reported due to the plasmid pCD-METRO, this does not explain all cases. Here, we investigate resistance to metronidazole in a collection of clinical isolates of C. difficile. We find that nearly all isolates demonstrate a heme-dependent increase in the minimal inhibitory concentration for metronidazole, which in some cases leads to isolates being qualified as resistant (MIC > 2 mg/L). Moreover, whole genome sequence analysis reveals a single nucleotide polymorphism in the heme responsive gene hsmA, which defines a metronidazole resistant lineage of PCR ribotype 010 / multilocus sequence type 15 isolates that also includes pCD-METRO containing strains. Together our data demonstrate that heme is crucial for medium-dependent metronidazole resistance in C. difficile.

High prevalence and risk factors of multiple antibiotic resistance in patients who fail first-line Helicobacter pylori therapy in southern China: a municipality-wide, multicentre, prospective cohort study

Background We aimed to study the prevalence of secondary antibiotic resistance of Helicobacter pylori in southern China and its risk factors, particularly geographical and socio-economic factors. Methods This was a municipality-wide, multicentre, prospective cohort study involving five major hospitals. Patients aged ≥18 years who failed first-line bismuth-based quadruple anti-H. pylori therapy between September 2016 and February 2018 were recruited. Participants underwent upper gastrointestinal endoscopy with biopsy from the antrum and body for H. pylori culture and antimicrobial susceptibility testing for six antibiotics (clarithromycin, levofloxacin, metronidazole, amoxicillin, tetracycline and furazolidone). Patients with failure of H. pylori culture were excluded. Participants completed a questionnaire profiling 22 potential risk factors of H. pylori infection and antibiotic resistance, including medical, social, household and birthplace factors. Results A total of 1113 patients failed first-line therapy, with successful H. pylori culture in 791 (71.1%) [male = 433 (54.7%); median age = 43 years]. Secondary resistance rates of dual antibiotics (clarithromycin + metronidazole and levofloxacin + metronidazole) and triple antibiotics (clarithromycin + levofloxacin + metronidazole) were 34.0%, 38.7% and 17.8%, respectively. Risk factors for clarithromycin + metronidazole resistance were history of ≥2 courses of H. pylori therapies [adjusted OR (aOR) = 1.71; 95% CI = 1.17–2.54], ≥3 household members (aOR = 2.00; 95% CI = 1.07–3.90) and family history of gastric cancer (aOR = 1.85; 95% CI = 1.18–2.85). Risk factors for levofloxacin + metronidazole resistance were age ≥40 years (aOR = 1.94; 95% CI = 1.37–2.75), lower gross domestic product per capita (aOR = 0.29; 95% CI = 0.10–0.80) and higher number of doctors/10 000 population (aOR = 1.59; 95% CI = 1.07–2.39). A higher human development index was of borderline significance (aOR = 2.79; 95% CI = 0.97–8.70). Conclusions The rates of secondary resistance of H. pylori to multiple antibiotics were high in southern China. Certain population-level risk factors were associated with levofloxacin + metronidazole resistance.

Mutations of Helicobacter pylori RdxA are mainly related to the phylogenetic origin of the strain and not to metronidazole resistance

Objectives Drug resistance of Helicobacter pylori is a major clinical problem worldwide. The objective of the present study was to investigate the prevalence of antibiotic-resistant H. pylori in the city of Shenzhen in China, as well as to identify the genetic mutations specifically associated with drug resistance rather than unrelated phylogenetic signals. Methods Antibiotic susceptibility testing was performed on 238 clinical strains successfully isolated from H. pylori-positive dyspeptic patients who underwent gastroscopy at the Department of Gastroenterology in Shenzhen People’s Second Hospital. Following WGS of all strains using Illumina technology, mutation and phylogenetic analyses were performed. Results The resistance rates were 84.9%, 35.3%, 25.2% and 2.1% for metronidazole, clarithromycin, ciprofloxacin and rifampicin, respectively. An A2143G conversion in the 23S rRNA gene was the primary mutation observed in clarithromycin-resistant strains, whilst N87K/I and D91G/N/Y in GyrA were detected in ciprofloxacin-resistant strains. In RdxA, our results demonstrated that only R16H/C and M21A are significant contributors to metronidazole resistance; there were 15 other sites, but these are phylogenetically related and thus unrelated to metronidazole resistance. Conclusions There is a high prevalence of metronidazole, clarithromycin and ciprofloxacin resistance and a low prevalence of rifampicin resistance in H. pylori from Shenzhen, China. Omission of phylogenetically related sites will help to improve identification of sites genuinely related to antibiotic resistance in H. pylori and, we believe, other species.