**6. Factors affecting the gastric microbiota**

As described, *H. pylori* is the most significant species that colonizes the stomach and a key factor for the gastric microbial diversity but is far from sufficient to provide a wholesome understanding of the factors that determine its dynamics. Major factors that influence and define the dynamics of the gastric microbiota include gastric acidity, inflammation of the gastric mucosa, dietary habits, and use of medications.

#### **6.1 Gastric acidity**

The human gastric juice has an interprandial pH of between 1 and 2 in the gastric lumen, whereas with food ingestion it can reach up to pH 5. pH also varies in the different anatomical regions of the stomach, with most acidic being the fundus and the least being the antrum. The mucus lining the gastric mucosa establishes also a pH gradient from the lumen to the surface of the epithelium. This mucus consists of two sublayers—an inner mucus layer that is firmly attached to the epithelium and a variable mucus layer directly interacting with the lumen [7, 8]. Thus, across the mucus layer, the pH ranges from about 5.5 to 6.8 or even 7 at the surface of the gastric epithelial cells [5, 6]. It was already discussed that the low pH, caused by the hydrochloric acid, restricts the quantity of microorganisms and reduces the risk of infection by pathogens. Hence, sites with higher pH are significantly more hospitable to colonization and have a higher microbial density. Considerable fluctuations in the microbial density have been described with respect to the pH in the stomach, whereby both the quantity and the proportion of genera also fluctuate [43, 44]. Bacteria and bacterial DNA, which are isolated from gastric juice, differ from bacterial isolates adhering to the mucosa. During abnormal conditions, this balance may be different.

#### **6.2 Dietary habits**

While many studies document the effects of diet on the gut microbiota composition in humans, [45–49] there are only a few, mainly animal model studies,

addressing the influence of diet on the gastric microbiota. An example is an in vivo study that compared the gastric microbiota of mice fed a non-purified diet (natural source-derived food) to mice fed a purified diet (refined food) and found higher levels of total aerobes, total anaerobes, and *Lactobacillus* in the stomach of the mice on a non-purified diet [50]. Nevertheless, it is well established how dietary factors affect the gastric microclimate, and since the microbiota is an inseparable part of this microclimate, it is not farfetched to suspect that diet affects the gastric microbial communities. However, more research is needed.

### **6.3 Use of medications**

The long-term use of proton pump inhibitors (PPIs) and H2 antagonists affects the composition of the gastric microbiota by inducing a non-*H. pylori* bacterial overgrowth [51]. This is not surprising, considering that normally the non-*H. pylori* gastric microbiota is suppressed by the significantly acidic gastric environment. Suppression of gastric acidity will alter the bacterial flora of the upper GI tract, and studies have confirmed that PPIs do alter the bacterial population in the stomach [52]. This is mainly due to oral bacteria that survive instead of being killed in the normally acidic stomach. It has also been suspected that by causing alteration and overgrowth of the microbiota, acid-suppressive treatments may increase the risk of gastric cancer [52]. It has also been shown that a previously antrum-dominant *H. pylori* infection after treatment with acid inhibitors changed to a more corpus predominant infection [51]. The less acidic corpus allows *H. pylori* to penetrate deeper in the crypts and increase the inflammation, which causes faster progression to atrophy [53]. Treatment with acid inhibitors has by culture-dependent methods been shown to affect the survival of bacteria in the stomach. However, no significant differences have been found regarding diversity and composition of the microbiota by using culture methods [10].

Antibiotics are well known to have suppressive effects on the gastrointestinal microflora. *H. pylori* eradication is dependent on combined antibiotic treatment. However, certain antibiotic treatments can have negative effects on the "healthy" gastric microbiota. Animal studies indicate that treatment with penicillins reduces *Lactobacillus* populations and promotes yeast colonization of the gastric epithelium. Furthermore, Mason et al. [54] showed that cefoperazone treatment in humans causes long-term alteration of the gastric microbiota, such as a significant reduction in the number of *Lactobacillus* and overgrowth of *Enterococcus*.
