Gut Microbiota Composition Before and After Use of Proton Pump Inhibitors

The prevalence of gastroesophageal reflux disease (GERD) has increased worldwide, most likely due to changes in dietary patterns and increasing obesity [1]. The increasing prevalence of GERD has been associated with decreased prevalence of Helicobacter pylori infection, especially in Japan [2]. Proton pump inhibitors (PPIs) that suppress acid production and result in increased gastric pH are the most frequently used drugs for treatment of GERD [3]. The prophylactic use of aspirin to prevent coronary heart disease and cerebrovascular disease is commonly recommended [4], and PPIs are used to reduce upper gastrointestinal injuries associated with aspirin [5]. With the global growth of older populations, the prevalence of bone fractures [6] and osteoarthritis is increasing [7]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are often used as painkillers to alleviate pain associated with these diseases, and PPIs are also used to prevent NSAID-induced ulcers [8]. Moreover, in clinical trials, the tolerability of PPIs was similar to that of placebo, and PPIs have been concluded to be very safe drugs [9, 10]. Accordingly, the numbers of prescriptions for PPIs have increased significantly, and the duration of treatment has also increased.

Recently, problems associated with PPI use have begun to surface. Long-term PPI use may affect nutrient absorption including calcium malabsorption and the resulting increased risk of bone fracture [11]. PPI use may increase the risk of enteric infections, such as Clostridium difficile and Campylobacter, as well as community-acquired pneumonia [12‐14]. PPI use may also increase the incidence of small intestinal bacterial overgrowth [15]. In addition, PPI use is potentially associated with development of spontaneous bacterial peritonitis in cirrhotic patients with ascites or cryptogenic liver abscess, which might be caused by bacterial translocation [16, 17]. The gut microbiota plays an important role in host resistance against colonization by exogenous enteric microbes and overgrowth of indigenous commensals [18]. Several observation and intervention studies found that PPIs altered the gut microbiota composition [19‐23]. Accordingly, the increased risk of enteric infections in PPI users may be caused by the influence of PPIs on the gut microbiota. Moreover, intestinal bacterial overgrowth promotes bacterial translocation [24]; PPIs are therefore likely to be one of the risk factors for bacterial translocation.

Organic acids have various pathophysiological effects on mucosal blood flow in the gastrointestinal tract [25], intestinal epithelial proliferation [26], intestinal motility [27], and control of the intraluminal pH [28] and are major energy sources for intestinal epithelial cells [29]. Since organic acids are produced by colonic bacteria, any alteration in gut microbiota composition may be associated with a change in organic acid composition [30].

In this current study, we quantitatively investigated the fecal microbiota composition, fecal organic acid concentrations and pH, and the gut bacteria in the blood in the same patients before and after PPI use.

The results of the present study showed that significant differences in the numbers of Lactobacillus, which is a subdominant population in the intestine, were observed between pre- and post-PPI treatment, while significant differences in the numbers of each predominant obligate anaerobe in the feces of PPI users between pre- and post-treatment were not observed. Recently, bacterial rRNA gene-based, metagenomic analyses have been conducted to analyze the composition of the human gut microbiome [22, 23, 42, 43]. However, it is difficult to quantify subdominant important intestinal genera using this approach. Therefore, we used the YIF-SCAN^® system to perform bacterial analysis. The YIF-SCAN^® system can quantify the abundance of the targeted bacterial population, including subdominant populations and dominant populations, with high sensitivity and has the ability to detect viable bacteria [32, 33, 36].

Previous large cohort studies of PPI users and intervention studies of PPI use for 4 weeks reported increases in bacteria from the genus Streptococcus [19, 20, 22, 23]. In the present study, we also observed an increase in the genus Streptococcus. Members of the genus Streptococcus are commensals of the human oral cavity, throat, and nasal cavity. Gastric acidity is known to inactivate ingested microorganisms [44]. Therefore, gastric acid may act as a barrier against bacterial influx down into the lower gastrointestinal tract from upper regions such as the oral cavity. Because PPIs reduce stomach acidity, the barrier function becomes weakened. This may explain the finding of increased Streptococcus counts detected in this study. Our results corroborate the findings of previous studies that employed metagenomic analyses. In addition, for the first time, the current pilot study verified the results of previous studies by using RT-qPCR. No significant differences in blood bacterial detection rates were observed before or after treatment; however, the mean counts of Streptococcus in blood before and after PPI treatment were 5 and 10 cells/mL, respectively. The Streptococcus spp. detected after PPI treatment were S. salivarius and S. oralis, which are commensals of the human oral cavity [45, 46]. This finding shows that bacteria present in the human oral cavity, throat, and nasal cavity increased in the intestine, implying that bacterial translocation may have occurred. Therefore, PPI use may be associated with bacterial translocation. PPI use may also increase the risk of sepsis [47‐49], as well as enteric infections. Four patients had bacteria in the blood before treatment. Although chronic alcohol consumption or diabetes is associated with bacterial translocation [50], neither alcohol nor diabetes was significantly associated with the presence of bacteria in the blood. The reason for bacteremia before treatment is not currently clear. Further, because the method used for counting bacteria in this study is highly sensitive, a very small amount of bacteria in the blood due to unknown cause might be detected. The results of the present study show that PPI use may cause an increase in indigenous lactobacilli because patients who had received drugs and/or dietary items that affect the gut microbiota, such as antibiotics, living bacterial preparations, or yogurt, were excluded from the present study. Among lactobacilli, counts of the L. gasseri subgroup, L. fermentum, the L. reuteri subgroup, and the L. ruminis subgroup were significantly increased after PPI treatment. It is generally considered that these bacteria have probiotic influences on human health [51‐54]. However, there are several case reports demonstrating that lactobacilli caused serious infections such as bacteremia and liver abscesses in susceptible immunocompromised patients [55, 56]. In addition to our results, increased numbers of Lactobacillus were also observed in patients with diseases such as diabetes mellitus type 2 [36] and Parkinson’s disease [57]. The biological effects of probiotics are strain specific [58], and whether these bacteria have probiotic features is determined by a coevolutionary relationship between the bacteria and their hosts [59]. Therefore, it is not clear at present whether increased numbers of such bacteria that are associated with PPI treatment provide harmful or beneficial influences on human health. However, if bacterial translocation was caused by PPI use, then PPI use would have a negative influence on human health.

With regard to total and individual organic acid concentrations and pH values, there were no significant differences between values at pre-treatment and post-8 weeks of PPI treatment. Although formic acid and butyric acid concentrations were significantly increased after 4 weeks of treatment compared with concentrations before treatment, significant differences disappeared after 8 weeks of treatment. PPI treatment therefore did not cause long-lasting changes in fecal organic acid concentrations. The reasons underlying the increases in formic acid and butyric acid concentrations after 4 weeks of treatment are currently not clear.

Regarding the limitations of this study, major drawbacks include the small sample size and short treatment period. Twenty patients and 8 weeks of treatment may be insufficient to evaluate the effects of PPI treatment. Moreover, because we used the proprietary YIF-SCAN^® system, which may have biased results, studies using alternative approaches are needed.

In conclusion, our results by RT-qPCR demonstrate that gut dysbiosis was caused by PPI use, corroborating results obtained by previous metagenomic analyses. Further large-scale studies on longer-term PPI use, substantial effects of PPI use on human health caused by gut dysbiosis, and whether PPI use causes bacterial translocation to blood are needed.