40Although long thought to be a sterile and inhospitable environment, the stomach is inhabited 41 by diverse microbial communities, co-existing in a dynamic balance. Long-term use of orally 42 administered drugs such as Proton Pump Inhibitors (PPIs), or bacterial infection such as 43 Helicobacter pylori, cause significant microbial alterations. Yet, studies revealing how the 44 commensal bacteria re-organize, due to these perturbations of the gastric environment, are in 45 the early phase. They mainly focus on the most prevalent taxa and rely on linear techniques for 46 multivariate analysis.
47Here we disclose the importance of complementing linear dimensionality reduction techniques 48 such as Principal Component Analysis and Multidimensional Scaling with nonlinear 49 approaches derived from the physics of complex systems. Then, we show the importance to 50 complete multivariate pattern analysis with differential network analysis, to reveal mechanisms 51 of re-organizations which emerge from combinatorial microbial variations induced by a 52 medical treatment (PPIs) or an infectious state (H. pylori). 53 Keywords 54 Proton Pump Inhibitors -Dyspepsia -Helicobacter pylori -Gastric microbiota -Linear and 55 nonlinear unsupervised methods -Mininum Curvilinear Embedding -Nonlinearity -PC-corr 56 network -16S rRNA 57 58 3 Introduction 59The gastric environment with its microbiota is the active gate that regulates access to the whole 60 gastrointestinal tract, and therefore it has a remarkable impact on the correct functionality of 61 the entire human organism. Recent studies have revealed that many orally administered drugs 62 can perturb the elegant balance of the gastric flora 1,2 . However, not all of them cause permanent 63 adverse effects and particular attention should be addressed to drugs that are frequently 64 prescribed and administered for long periods. They can cause permanent unbalance of the 65 gastric microbiota that might generate adverse side effects for the patient's health. Since the 66 introduction of proton pump inhibitors (PPIs) into clinical practice more than 25 years ago, PPIs 67 have become the mainstay in the treatment of gastric-acid-related diseases 3 . PPIs are potent 68 agents that block acid secretion by gastric parietal cells by binding covalently to and inhibiting 69 the hydrogen/potassium (H + /K + )-ATPases (or proton pumps), and additionally they can bind 70 non-gastric H + /K + -ATPases, both on human cells and on bacteria and fungi, such as 71 Helicobacter pylori (H. pylori) 4-6 . 72 PPIs are drugs of first choice for peptic ulcers (PU) and their complications (e.g. bleeding), 73 gastroesophageal reflux disease (GERD), nonsteroidal anti-inflammatory drug (NSAID)-74 induced gastrointestinal (GI) lesions, Zollinger-Ellison syndrome and dyspepsia 3,7,8 . In 75 particular, dyspepsia is a common clinical problem characterized by symptoms (e.g. epigastric 76 pain, burning, postprandial fullness, or early satiation) originating from the gastroduodenal 77 region 9 . The potent gastri...