In this study, we have performed a comprehensive review of the dynamic changes of human gut microbiota and ARGs in patients who underwent various H. pylori eradication therapies using the whole genome sequencing method. Overall, we observed a transient but dramatic shift in ARG richness, ARG class level, and unique ARGs at 6-week after the eradication, especially in patients treated with levofloxacin-based therapy. All of these changes on ARGs were restored at 6-month after eradication therapy.
In addition to the change of ARGs, we also found significant changes in gut microbiota. The alpha diversity significantly decreased at 6-week in all three treatment groups, which all restored at 6-month. The microbiota community structure (beta diversity) was significantly separated at 6-week after different eradication therapies among the three groups. Moreover, microbiota difference was observed at 6-week after the treatment at different taxonomy levels and partially restored at 6-month. For treatment naïve patients, those with higher ARG richness and ErmF gene abundance at baseline were prone to failure of clarithromycin-based triple therapy, and there was also high diversity and abundance of unique ARGs 6-month after the eradication therapy.
Our results showed that several ARGs (MLS, tetracycline, and multidrug resistance genes) were differentially present in the LEVO and the OTHER group compared to the CLA group (treatment naïve) at baseline. The species diversity was lower in the OTHER group and there were several differentially abundant genera compared with the CLA group. These findings may indicate that after previously failed H. pylori eradication therapies, there was emergence of antibiotic resistance, especially for clarithromycin and tetracycline, which was associated with reconstructions of gut microbiota. Moreover, we found that those who failed eradication therapy in the CLA group had higher number of unique ARGs and abundance of ErmF (MLS resistance genes) at baseline. In our patients, we found that the tetracycline (tetQ, tetO, and tetW) and MLS (ErmF and ErmB) resistance genes were highly abundant and prevalent in human fecal samples (Figure S8). Notably, it was also found that the vancomycin resistance genes (VanRG and VanRA) were also highly prevalent and abundant in some populations such as Danish, Spanish, and Chinese individuals.
One of the major issues related to H. pylori eradication therapy was antimicrobial resistance. Prior antibiotic treatment can induce multidrug resistance (MDR) , which is increasing worldwide and could hamper the success rate of conventional H. pylori eradication therapy . The effect of different antibiotics used in various H. pylori eradication therapies on gut ARGs however remains unclear. In this study, we showed a transient increase in ErmF (macrolide resistance) after exposure to clarithromycin and amoxicillin. Besides, the relative abundance of the ErmF gene was overexpressed at baseline in patients who failed multiple previous treatments compared with those treatment naïve patients. A recent Russian study revealed that the ErmB, CFX group (beta-lactam), and tetQ genes were increased after clarithromycin-based quadruple eradication therapy . Consistently, a small study reported that the macrolide resistance gene ErmB gene increased immediately after treatment and remained at a high level four years after clarithromycin-containing triple therapy using the 16S rRNA sequencing method . It would be interesting to explore in future studies whether baseline scarriage of ErmF or ErmB gene can be a predictor of treatment failure.
Escherichia coli are commensals, commonly found in the lower part of the intestine and usually harmless, while virulent isolates are associated with diarrhea and colitis [37, 38]. A Japanese study showed that fluoroquinolone consumption was closely associated with E. coli resistance . Our study showed that the relative abundance of E. coli significantly increased 6 weeks after the levofloxacin-based eradication therapy. In addition, resistant E. coli carried numerous genes that confer resistance to beta-lactam (Escherichia coli ampH beta-lactam), aminoglycoside (acrD), fosfomycin (GlpT), sulfonamide (sul1), phenicol (catS) , multidrug (Escherichia coli EF-Tu mutant, acrA, mdfA, soxR) [41–43]. E. coli could also gain high-level resistance to various antibiotics after levofloxacin and tetracycline exposure by inducing the efflux system during the biofilms formation, especially the emrY/K (tetracycline resistance) and evgS/A (multidrug resistance) pumps . Consistent with the previous study, the relative abundance of those ARGs together with other multidrug resistance genes significantly increased 6-week after levofloxacin-containing therapy compared with baseline.
One recent systematic review reported a remarkable rise in the resistance rate of levofloxacin from 17–27% from 2006 to 2015 in the Asia-Pacific region and this may affect the efficacy of levofloxacin-containing therapies. In our study, levofloxacin-based therapy has the lowest eradication success rate as second-line therapy. Therefore, alternative second-line therapy like bismuth quadruple therapy (PPI, bismuth subsalicylate, metronidazole, and tetracycline) should be considered. Bismuth salt has a synergistic effect with antibiotics and confer no resistance. Recent data also confirmed that 14-day bismuth combining quadruple therapy is a highly effective (over 90% cure rate) and safe second-line option in patients with previous treatment failure. Thus, bismuth quadruple therapy may be preferred in view of the post-treatment ARG profiles.
Previous studies have shown that the consumption of antibiotics leads to immediate[22, 48], short-term[8, 49, 50] and long-term[50–52] alterations in the human gut microbiota. However, most of the studies used 16S rRNA sequencing methods and few of them had employed the more detailed metagenomic sequencing methods. In our study, we found that the relative abundance of Firmicutes increased at 6-week in the CLA and LEVO group but significantly decreased in the OTHER group. Notably, it was reported that the relative abundance of Bacteroidetes and Firmicutes phylum decreased significantly whereas Proteobacteria increased immediately after the eradication therapy. All phylum restored to baseline level at 8-week[8, 22, 50, 53, 54]. The relative abundance of Firmicutes phylum was also found to be decreased 4-week after clarithromycin-based triple therapy in a recent study. In contrast, another study showed an opposite trend of Firmicutes phylum, which increased 6-week after bismuth quadruple therapy. It thus appears that different antibiotics may cause distinct short-term effects on the gut microbiota. The bacteria from Firmicutes phylum can ferment carbohydrates into a variety of short-chain fatty acids (SCFAs), which can increase the intestinal barrier function. The dramatic alteration of Firmicutes phylum after the eradication therapy may seriously affected gut ecosystem and the inflammation recovery process.
Significant changes at the genus level were also identified and most of which were involved in the production of SCFAs. Previous studies have shown that some butyrate-producing bacteria, the Lachnoclostridium, Roseburia, Eubacteria hallii, Erysipelatoclostridium[56, 57] displayed protective effects by generating butyrate (SCFAs) to suppress chronic intestinal inflammation. Besides, Bacteroides spp., which produce acetate and propionate (SCAFs), could also protect against gut inflammation. As expected, these beneficial bacteria were found to be enriched 6-week after different eradication therapies, especially, Lachnoclostridium genus was found to have high-level 6-month after eradication therapy, which was similar to previous findings that Lachnoclostridium enriched 26 weeks after bismuth quadruple therapy. Taken together, those data demonstrate the temporary microbiota perturbation caused by antibiotic exposure and potential long-term protection of bismuth-containing eradication therapy, which was related to the recovery of gut inflammation.
Our study has several strengths. Other than looking at the ARG richness, we demonstrated dynamic changes in antimicrobial classes and unique ARGs under different eradication antibiotics exposure. Besides, we used shotgun whole genome sequencing (WGS) which had better detection of bacterial species, deeper sequencing depth, and identification of potential ARGs compared with the 16S rRNA sequencing method. This study also included both patients who were naïve to and had failed previous eradication therapies to examine difference at baseline and after treatment.
There are some limitations of this study. First, as there are three different treatment groups with samples collected at three different time points, each group had a relatively small sample size which may hinder the application of the results. Second, this study only focused on the changes of gut microbiota and ARG from the metagenomics level, further studies should evaluate the metabolomics and proteomics for more integrated analysis, which can further reveal the consequences of receiving different H. pylori eradication therapies. Third, gut microbiota could change with lifestyle modifications, for instance, after exercise training and dietary intervention. Since no dietary intervention or physical exercise was enforced in this study, the impacts of these lifestyle changes could not be analyzed. However, as most changes restored to baseline at 6-month, suggesting these factors are unlikely to be playing a significant role. Finally, considering the regional variations in antibiotic consumption and resistance, the applicability of our findings may need further validation in a more diverse population, particularly in an area with high background antibiotics resistance.