Nutrition, Microbiota and Noncommunicable Diseases Printed Edition of the Special Issue Published in Nutrients www.mdpi.com/journal/nutrients Julio Plaza-Díaz Edited by Nutrition, Microbiota and Noncommunicable Diseases Nutrition, Microbiota and Noncommunicable Diseases Editor Julio Plaza-D ́ ıaz MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin Editor Julio Plaza-D ́ ıaz Univ Granada Biomed Res Ctr, Inst Nutr & Food Technol Jose Mataix Spain Editorial Office MDPI St. Alban-Anlage 66 4052 Basel, Switzerland This is a reprint of articles from the Special Issue published online in the open access journal Nutrients (ISSN 2072-6643) (available at: https://www.mdpi.com/journal/nutrients/special issues/ Microbiota Noncommunicable Diseases). For citation purposes, cite each article independently as indicated on the article page online and as indicated below: LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. Journal Name Year , Article Number , Page Range. ISBN 978-3-03936-916-4 ( H bk) ISBN 978-3-03936-917-1 (PDF) c © 2020 by the authors. Articles in this book are Open Access and distributed under the Creative Commons Attribution (CC BY) license, which allows users to download, copy and build upon published articles, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. The book as a whole is distributed by MDPI under the terms and conditions of the Creative Commons license CC BY-NC-ND. Contents About the Editor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Julio Plaza-Diaz Nutrition, Microbiota and Noncommunicable Diseases Reprinted from: Nutrients 2020 , 12 , 1971, doi:10.3390/nu12071971 . . . . . . . . . . . . . . . . . . 1 Clara Yieh Lin Chong, David Orr, Lindsay D. Plank, Tommi Vatanen, Justin M. O’Sullivan and Rinki Murphy Randomised Double-Blind Placebo-Controlled Trial of Inulin with Metronidazole in Non-Alcoholic Fatty Liver Disease (NAFLD) Reprinted from: Nutrients 2020 , 12 , 937, doi:10.3390/nu12040937 . . . . . . . . . . . . . . . . . . 5 Yoomin Ahn, Myung Hee Nam and Eungbin Kim Relationship Between the Gastrointestinal Side Effects of an Anti-Hypertensive Medication and Changes in the Serum Lipid Metabolome Reprinted from: Nutrients 2020 , 12 , 205, doi:10.3390/nu12010205 . . . . . . . . . . . . . . . . . . 21 Konomi Tamura, Hiroyuki Sasaki, Kazuto Shiga, Hiroki Miyakawa and Shigenobu Shibata The Timing Effects of Soy Protein Intake on Mice Gut Microbiota Reprinted from: Nutrients 2020 , 12 , 87, doi:10.3390/nu12010087 . . . . . . . . . . . . . . . . . . . 33 Alessandro Dalla Via, Giorgio Gargari, Valentina Taverniti, Greta Rondini, Ilaria Velardi, Veniero Gambaro, Giacomo Luca Visconti, Valerio De Vitis, Claudio Gardana, Enzio Ragg, Andrea Pinto, Patrizia Riso and Simone Guglielmetti Urinary TMAO Levels Are Associated with the Taxonomic Composition of the Gut Microbiota and with the Choline TMA-Lyase Gene ( cutC ) Harbored by Enterobacteriaceae Reprinted from: Nutrients 2020 , 12 , 62, doi:10.3390/nu12010062 . . . . . . . . . . . . . . . . . . . 55 Hiroyuki Sasaki, Hiroki Miyakawa, Aya Watanabe, Yuki Nakayama, Yijin Lyu, Koki Hama and Shigenobu Shibata Mice Microbiota Composition Changes by Inulin Feeding with a Long Fasting Period under a Two-Meals-Per-Day Schedule Reprinted from: Nutrients 2019 , 11 , 2802, doi:10.3390/nu11112802 . . . . . . . . . . . . . . . . . . 69 Manon Dominique, Romain Legrand, Marie Galmiche, Sa ̈ ıda Azhar, Camille Deroissart, Charl` ene Gu ́ erin, Jean-Luc do Rego, Fatima Leon, S ́ everine Nobis, Gr ́ egory Lambert, Nicolas Lucas and Pierre D ́ echelotte Changes in Microbiota and Bacterial Protein Caseinolytic Peptidase B During Food Restriction in Mice: Relevance for the Onset and Perpetuation of Anorexia Nervosa Reprinted from: Nutrients 2019 , 11 , 2514, doi:10.3390/nu11102514 . . . . . . . . . . . . . . . . . . 89 Xuejun Kong, Jun Liu, Murat Cetinbas, Ruslan Sadreyev, Madelyn Koh, Hui Huang, Adetaye Adeseye, Puhan He, Junli Zhu, Hugh Russell, Clara Hobbie, Kevi Liu and Andrew B. Onderdonk New and Preliminary Evidence on Altered Oral and Gut Microbiota in Individuals with Autism Spectrum Disorder (ASD): Implications for ASD Diagnosis and Subtyping Based on Microbial Biomarkers Reprinted from: Nutrients 2019 , 11 , 2128, doi:10.3390/nu11092128 . . . . . . . . . . . . . . . . . . 103 v Ezgi BELLIKCI-KOYU, Banu Pınar SARER-YUREKLI, Yakut AKYON, Fadime AYDIN-KOSE, Cem KARAGOZLU, Ahmet Gokhan OZGEN, Annika BRINKMANN, Andreas NITSCHE, Koray ERGUNAY, Engin YILMAZ and Zehra BUYUKTUNCER Effects of Regular Kefir Consumption on Gut Microbiota in Patients with Metabolic Syndrome: A Parallel-Group, Randomized, Controlled Study Reprinted from: Nutrients 2019 , 11 , 2089, doi:10.3390/nu11092089 . . . . . . . . . . . . . . . . . . 129 Chien-Ning Hsu, Chih-Yao Hou, Chien-Te Lee, Julie Y.H. Chan and You-Lin Tain The Interplay between Maternal and Post-Weaning High-Fat Diet and Gut Microbiota in the Developmental Programming of Hypertension Reprinted from: Nutrients 2019 , 11 , 1982, doi:10.3390/nu11091982 . . . . . . . . . . . . . . . . . . 153 Carmen Tenorio-Jim ́ enez, Mar ́ ıa Jose ́ Mart ́ ınez-Ram ́ ırez, Isabel Del Castillo-Codes, Carmen Arraiza-Irigoyen, Mercedes Tercero-Lozano, Jos ́ e Camacho, Natalia Chueca, Federico Garc ́ ıa, Josune Olza, Julio Plaza-D ́ ıaz, Luis Fontana, M ́ onica Olivares, ́ Angel Gil and Carolina G ́ omez-Llorente Lactobacillus reuteri V3401 Reduces Inflammatory Biomarkers and Modifies the Gastrointestinal Microbiome in Adults with Metabolic Syndrome: The PROSIR Study Reprinted from: Nutrients 2019 , 11 , 1761, doi:10.3390/nu11081761 . . . . . . . . . . . . . . . . . . 169 Eva Lau, Jo ̃ ao S ́ ergio Neves, Manuel Ferreira-Magalh ̃ aes, Davide Carvalho and Paula Freitas Probiotic Ingestion, Obesity, and Metabolic-Related Disorders: Results from NHANES, 1999–2014 Reprinted from: Nutrients 2019 , 11 , 1482, doi:10.3390/nu11071482 . . . . . . . . . . . . . . . . . . 183 Emiko Morita, Hisayo Yokoyama, Daiki Imai, Ryosuke Takeda, Akemi Ota, Eriko Kawai, Takayoshi Hisada, Masanori Emoto, Yuta Suzuki and Kazunobu Okazaki Aerobic Exercise Training with Brisk Walking Increases Intestinal Bacteroides in Healthy Elderly Women Reprinted from: Nutrients 2019 , 11 , 868, doi:10.3390/nu11040868 . . . . . . . . . . . . . . . . . . . 195 David G. Valcarce, Marta F. Riesco, Juan M. Mart ́ ınez-V ́ azquez and Vanesa Robles Diet Supplemented with Antioxidant and Anti-Inflammatory Probiotics Improves Sperm Quality after Only One Spermatogenic Cycle in Zebrafish Model Reprinted from: Nutrients 2019 , 11 , 843, doi:10.3390/nu11040843 . . . . . . . . . . . . . . . . . . . 213 Julio Plaza-Diaz, Bel ́ en Pastor-Villaescusa, Ascensi ́ on Rueda-Robles, Francisco Abadia-Molina and Francisco Javier Ruiz-Ojeda Plausible Biological Interactions of Low- and Non-Calorie Sweeteners with the Intestinal Microbiota: An Update of Recent Studies Reprinted from: Nutrients 2020 , 12 , 1153, doi:10.3390/nu12041153 . . . . . . . . . . . . . . . . . . 229 Estefania Sanchez-Rodriguez, Alejandro Egea-Zorrilla, Julio Plaza-D ́ ıaz, Jer ́ onimo Arag ́ on-Vela, Sergio Mu ̃ noz-Quezada, Luis Tercedor-S ́ anchez and Francisco Abadia-Molina The Gut Microbiota and Its Implication in the Development of Atherosclerosis and Related Cardiovascular Diseases Reprinted from: Nutrients 2020 , 12 , 605, doi:10.3390/nu12030605 . . . . . . . . . . . . . . . . . . 245 Antonio Barrientos-Dur ́ an, Ana Fuentes-L ́ opez, Adolfo de Salazar, Julio Plaza-D ́ ıaz and Federico Garc ́ ıa Reviewing the Composition of Vaginal Microbiota: Inclusion of Nutrition and Probiotic Factors in the Maintenance of Eubiosis Reprinted from: Nutrients 2020 , 12 , 419, doi:10.3390/nu12020419 . . . . . . . . . . . . . . . . . . 269 vi Mar ́ ıa Eugenia Cornide-Petronio, Ana Isabel ́ Alvarez-Mercado, M ́ onica B. Jim ́ enez-Castro and Carmen Peralta Current Knowledge about the Effect of Nutritional Status, Supplemented Nutrition Diet, and Gut Microbiota on Hepatic Ischemia-Reperfusion and Regeneration in Liver Surgery Reprinted from: Nutrients 2020 , 12 , 284, doi:10.3390/nu12020284 . . . . . . . . . . . . . . . . . . . 299 Mar ́ ıa Dolores Mesa, Bego ̃ na Loureiro, Iris Iglesia, Sergi Fernandez Gonzalez, Elisa Llurba Oliv ́ e, Oscar Garc ́ ıa Algar, Mar ́ ıa Jos ́ e Solana, M a Jes ́ us Cabero Perez, Talia Sainz, Leopoldo Martinez, Diana Escuder-Vieco, Anna Parra-Llorca, Mar ́ ıa S ́ anchez-Campillo, Gerardo Rodriguez Martinez, Dolores G ́ omez Roig, Myriam Perez Gruz, Vicente Andreu-Fern ́ andez, Jordi Clotet, Sebastian Sailer, Isabel Iglesias-Platas, Jes ́ us L ́ opez-Herce, Rosa Aras, Carmen Pall ́ as-Alonso, Miguel Saenz de Pipaon, M ́ aximo Vento, Mar ́ ıa Gormaz, Elvira Larqu ́ e Daza, Cristina Calvo and Fernando Caba ̃ nas The Evolving Microbiome from Pregnancy to Early Infancy: A Comprehensive Review Reprinted from: Nutrients 2020 , 12 , 133, doi:10.3390/nu12010133 . . . . . . . . . . . . . . . . . . 323 Krista Salli, Markus J. Lehtinen, Kirsti Tiihonen and Arthur C. Ouwehand Xylitol’s Health Benefits beyond Dental Health: A Comprehensive Review Reprinted from: Nutrients 2019 , 11 , 1813, doi:10.3390/nu11081813 . . . . . . . . . . . . . . . . . . 345 Ronald D. Hills Jr., Benjamin A. Pontefract, Hillary R. Mishcon, Cody A. Black, Steven C. Sutton and Cory R. Theberge Gut Microbiome: Profound Implications for Diet and Disease Reprinted from: Nutrients 2019 , 11 , 1613, doi:10.3390/nu11071613 . . . . . . . . . . . . . . . . . . 365 vii About the Editor Julio Plaza-D ́ ıaz has a degree in pharmacy (2008) and PhD in biochemistry and molecular biology (2014) from the University of Granada, Spain. He has been a researcher at the University of Granada since 2011 and has participated in more than 10 funded research projects. He is the author or coauthor of 50 peer-reviewed publications in the field of identification, characterization, and evaluation of new probiotic/microbiome studies and its impact on health. ix nutrients Editorial Nutrition, Microbiota and Noncommunicable Diseases Julio Plaza-Diaz 1,2,3 1 Department of Biochemistry and Molecular Biology II, School of Pharmacy, University of Granada, 18,071 Granada, Spain; jrplaza@ugr.es; Tel.: + 34-958-241-599 2 Instituto de Investigaci ó n Biosanitaria IBS.GRANADA, Complejo Hospitalario Universitario de Granada, 18,014 Granada, Spain 3 Children’s Hospital of Eastern Ontario Research Institute, Ottawa, ON K1H 8L1, Canada Received: 21 June 2020; Accepted: 29 June 2020; Published: 2 July 2020 The advent of new sequencing technologies has inspired the foundation of novel research to ascertain the connections between the microbial communities that reside in our gut and some physiological and pathological conditions. The microbiota, defined as the full collection of microbes (bacteria, fungi, and viruses, among others) that naturally exist within a particular biological niche, is estimated to contain 500–1000 species [1–4]. This Special Issue of Nutrients , “Nutrition, Microbiota, and Noncommunicable Diseases” contains 13 original publications and seven reviews investigating the contribution of intestinal microbiota on relevant health outcomes in a variety of populations, and animal studies which suggest the growing and extensive interests of research on this topic. Seven studies were published examining the changes in intestinal microbiota in the human population. Two of these studies recruited patients with metabolic syndrome. Tenorio-Jimenez et al. [5] reported the anthropometric variables and biochemical and inflammatory biomarkers as well as the gastrointestinal microbiome composition changes in a randomized, crossover, placebo-controlled, single-center trial in adult patients newly diagnosed with metabolic syndrome treated either with Lactobacillus reuteri V3401 or a placebo during 12 weeks. L. reuteri V3401 administration improved selected inflammatory parameters and modified the gastrointestinal microbiome, especially Verrucomicrobia [ 5 ], and Bellikci-Koyu et al. [ 6 ] investigated the e ff ects of regular kefir consumption on gut microbiota composition, and their relation with the components of metabolic syndrome in a parallel-group, randomized, controlled clinical trial for 12 weeks. Gut microbiota analysis showed that regular kefir consumption resulted in a significant increase only in Actinobacteria abundance [6]. In two more additional studies, one with healthy elderly women and another with patients with non-alcoholic fatty liver disease (NAFLD), Morita et al. [ 7 ] examined the e ff ect of an exercise intervention (12 weeks, trunk muscle training or aerobic exercise training) on the composition of the intestinal microbiota in healthy elderly women. Bacteroides abundance was significantly increased only in the aerobic exercise group, particularly in subjects showing increases in the time spent in brisk walking [ 7 ], and Chong et al. [ 8 ] determined whether inulin supplementation after brief metronidazole therapy is e ff ective in reducing alanine aminotransferase and maintaining weight loss achieved through a very-low-calorie diet among people with NAFLD. Treatment decreased the ratio of Firmicutes / Bacteroidetes [8]. Lau et al. [ 9 ] evaluated the association of probiotic ingestion with obesity, type 2 diabetes, hypertension, and dyslipidemia using data from the National Health and Nutrition Examination Survey, 1999–2014. Probiotic supplementation or yogurt consumption were associated with a lower prevalence of obesity and hypertension [ 9 ]. In another study with humans, Dalla Via et al. [ 10 ] verified whether trimethylamine-N-oxide urinary levels may be associated with the fecal relative abundance of specific bacterial taxa and the bacterial choline trimethylamine-lyase gene cutC in human fecal Nutrients 2020 , 12 , 1971; doi:10.3390 / nu12071971 www.mdpi.com / journal / nutrients 1 Nutrients 2020 , 12 , 1971 samples. Correlation analysis showed that the cut-Kp gene cluster was significantly associated with Enterobacteriaceae [10]. Finally, in one study with the pediatric population, Kong et al. [ 11 ] reported both oral and intestinal microbiota in patients with autism spectrum disorder and controls, with specific microbial patterns [11]. Regarding animal studies, six studies were published examining the changes in intestinal microbiota. Probiotic supplementation, high-fat diet, use of anorexic mice, fiber, and soy intake and antihypertensive e ff ect in metabolomics profiles were analyzed in these studies. Valcarce et al. [12] reported the e ff ect of a short-time probiotic supplementation consisting of a mixture of two probiotic bacteria with proven antioxidant and anti-inflammatory activities on zebrafish sperm quality and male behavior [ 12 ]. Hsu et al. [ 13 ] examined the alterations of gut microbiota, mediation of short-chain fatty acids (SCFAs) and their receptors, and downregulation of nutrient-sensing signals e ff ects in rats that received a high-fat diet. Increased Firmicutes to Bacteroidetes ratio, Akkermansia and Verrucomicrobia , and reduced abundance in the genus Lactobacillus were associated with blood pressure elevation [ 13 ]. Dominique et al. [ 14 ] investigated the role of the microbiome and the ClpB protein in the deregulation and self-maintenance of anorexia pathology in mice. Plasma concentration of ClpB was increased in both limited food access and activity-based anorexia mice and it was correlated with the proportion of Enterobacteriaceae in the animal feces [ 14 ]. Sasaki et al. [ 15 ] investigated the e ff ects of fiber intake timing on metabolism. Data have suggested that inulin is more easily digested by fecal microbiota during the active period than the inactive period. Inulin consumption at breakfast has a greater e ff ect on the microbiota [ 15 ]. Tamura et al. [ 16 ] investigated soy protein intake e ff ects on intestinal microbiota. Soy protein intake whether in the morning or evening led to a greater microbiota diversity and a decrease in cecal pH resulting from SCFA production compared with casein intake [ 16 ]. Finally, Ahn et al. [ 17 ] investigated the metabolomics changes in rats that received amlodipine. Serum levels of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, triglycerides with large numbers of double bonds, cholesterol, sterol derivatives, and cholesterol esters were increased. Amlodipine-induced compositional changes in the gut microbiota are a causal factor in inflammation [17]. Seven reviews investigating the impact of intestinal microbiota on relevant health outcomes in a variety of populations were published. Hills Jr. et al. [ 18 ] described a general vision about the gut microbiome and its important role in human health. Salli et al. [ 19 ] reported the health benefits of xylitol. The other reviews have described the intestinal microbiota changes in specific conditions, early infancy, hepatic ischemia-reperfusion and regeneration in liver surgery, vaginal microbiota, and cardiovascular diseases. Mesa et al. [ 20 ] reported the microbiome changes and how those modulate the inflammatory mechanisms related to physiological and pathological processes that are involved in the perinatal progress. Cornide-Petronio et al. [ 21 ] summarized the role of starvation, supplemented nutrition diet, nutritional status, and alterations in microbiota on hepatic ischemia / reperfusion and regeneration. Barrientos-Duran et al. [ 22 ] examined the most important aspect in the vaginal microbiota, with special emphasis in bacterial vaginosis, and the maintenance of eubiosis, and Sanchez-Rodriguez et al. [23] discussed how external factors such as dietary and physical activity habits influence host microbiota and atherogenesis, the potential mechanisms of the influence of gut microbiota in host blood pressure, and the alterations in the prevalence of those bacterial genera a ff ecting vascular tone and the development of hypertension. Finally, Plaza-Diaz et al. [24] revisited the e ff ects of sweeteners on gut microbiota. The present Special Issue provides a summary of the progress on the topic of intestinal microbiota and its important role in human health in di ff erent populations, which will be of interest from a clinical and public health perspective. Nevertheless, more studies with more samples and comparable methods are necessary to understand the actual function of intestinal microbiota in disease development and health maintenance. 2 Nutrients 2020 , 12 , 1971 Funding: This research received no external funding. Acknowledgments: Julio Plaza-Diaz is part of the University of Granada, Plan Propio de Investigaci ó n 2016, Excellence actions: Units of Excellence; Unit of Excellence on Exercise and Health (UCEES). Conflicts of Interest: The author declares no conflict of interest. References 1. Plaza-Diaz, J.; Ruiz-Ojeda, F.J.; Vilchez-Padial, L.M.; Gil, A. Evidence of the Anti-Inflammatory E ff ects of Probiotics and Synbiotics in Intestinal Chronic Diseases. Nutrients 2017 , 9 , 555. [CrossRef] 2. Plaza-Diaz, J.; Ruiz-Ojeda, F.J.; Gil-Campos, M.; Gil, A. Immune-Mediated Mechanisms of Action of Probiotics and Synbiotics in Treating Pediatric Intestinal Diseases. Nutrients 2018 , 10 , 42. [CrossRef] [PubMed] 3. Plaza-Diaz, J.; Gomez-Fernandez, A.; Chueca, N.; Torre-Aguilar, M.J.; Gil, A.; Perez-Navero, J.L.; Flores-Rojas, K.; Martin-Borreguero, P.; Solis-Urra, P.; Ruiz-Ojeda, F.J.; et al. Autism Spectrum Disorder (ASD) with and without Mental Regression is Associated with Changes in the Fecal Microbiota. Nutrients 2019 , 11 , 337. [CrossRef] 4. Á lvarez-Mercado, A.I.; Navarro-Oliveros, M.; Robles-S á nchez, C.; Plaza-D í az, J.; S á ez-Lara, M.J.; Muñoz-Quezada, S.; Fontana, L.; Abad í a-Molina, F. Microbial population changes and their relationship with human health and disease. Microorganisms 2019 , 7 , 68. [CrossRef] 5. Tenorio-Jimenez, C.; Martinez-Ramirez, M.J.; Del Castillo-Codes, I.; Arraiza-Irigoyen, C.; Tercero-Lozano, M.; Camacho, J.; Chueca, N.; Garcia, F.; Olza, J.; Plaza-Diaz, J.; et al. Lactobacillus reuteri V3401 Reduces Inflammatory Biomarkers and Modifies the Gastrointestinal Microbiome in Adults with Metabolic Syndrome: The PROSIR Study. Nutrients 2019 , 11 , 1761. [CrossRef] [PubMed] 6. Bellikci-Koyu, E.; Sarer-Yurekli, B.P.; Akyon, Y.; Aydin-Kose, F.; Karagozlu, C.; Ozgen, A.G.; Brinkmann, A.; Nitsche, A.; Ergunay, K.; Yilmaz, E.; et al. E ff ects of Regular Kefir Consumption on Gut Microbiota in Patients with Metabolic Syndrome: A Parallel-Group, Randomized, Controlled Study. Nutrients 2019 , 11 , 2089. [CrossRef] [PubMed] 7. Morita, E.; Yokoyama, H.; Imai, D.; Takeda, R.; Ota, A.; Kawai, E.; Hisada, T.; Emoto, M.; Suzuki, Y.; Okazaki, K. Aerobic Exercise Training with Brisk Walking Increases Intestinal Bacteroides in Healthy Elderly Women. Nutrients 2019 , 11 , 868. [CrossRef] [PubMed] 8. Chong, C.Y.L.; Orr, D.; Plank, L.D.; Vatanen, T.; O’Sullivan, J.M.; Murphy, R. Randomised Double-Blind Placebo-Controlled Trial of Inulin with Metronidazole in Non-Alcoholic Fatty Liver Disease (NAFLD). Nutrients 2020 , 12 , 937. [CrossRef] [PubMed] 9. Lau, E.; Neves, J.S.; Ferreira-Magalhaes, M.; Carvalho, D.; Freitas, P. Probiotic Ingestion, Obesity, and Metabolic-Related Disorders: Results from NHANES, 1999–2014. Nutrients 2019 , 11 , 1482. [CrossRef] 10. Dalla Via, A.; Gargari, G.; Taverniti, V.; Rondini, G.; Velardi, I.; Gambaro, V.; Visconti, G.L.; De Vitis, V.; Gardana, C.; Ragg, E.; et al. Urinary TMAO Levels Are Associated with the Taxonomic Composition of the Gut Microbiota and with the Choline TMA-Lyase Gene (cutC) Harbored by Enterobacteriaceae. Nutrients 2019 , 12 , 62. [CrossRef] 11. Kong, X.; Liu, J.; Cetinbas, M.; Sadreyev, R.; Koh, M.; Huang, H.; Adeseye, A.; He, P.; Zhu, J.; Russell, H.; et al. New and Preliminary Evidence on Altered Oral and Gut Microbiota in Individuals with Autism Spectrum Disorder (ASD): Implications for ASD Diagnosis and Subtyping Based on Microbial Biomarkers. Nutrients 2019 , 11 , 2128. [CrossRef] [PubMed] 12. Valcarce, D.G.; Riesco, M.F.; Martinez-Vazquez, J.M.; Robles, V. Diet Supplemented with Antioxidant and Anti-Inflammatory Probiotics Improves Sperm Quality after Only One Spermatogenic Cycle in Zebrafish Model. Nutrients 2019 , 11 , 843. [CrossRef] [PubMed] 13. Hsu, C.N.; Hou, C.Y.; Lee, C.T.; Chan, J.Y.H.; Tain, Y.L. The Interplay between Maternal and Post-Weaning High-Fat Diet and Gut Microbiota in the Developmental Programming of Hypertension. Nutrients 2019 , 11 , 1982. [CrossRef] [PubMed] 14. Dominique, M.; Legrand, R.; Galmiche, M.; Azhar, S.; Deroissart, C.; Guerin, C.; do Rego, J.L.; Leon, F.; Nobis, S.; Lambert, G.; et al. Changes in Microbiota and Bacterial Protein Caseinolytic Peptidase B During Food Restriction in Mice: Relevance for the Onset and Perpetuation of Anorexia Nervosa. Nutrients 2019 , 11 , 2514. [CrossRef] [PubMed] 3 Nutrients 2020 , 12 , 1971 15. Sasaki, H.; Miyakawa, H.; Watanabe, A.; Nakayama, Y.; Lyu, Y.; Hama, K.; Shibata, S. Mice Microbiota Composition Changes by Inulin Feeding with a Long Fasting Period under a Two-Meals-Per-Day Schedule. Nutrients 2019 , 11 , 2802. [CrossRef] 16. Tamura, K.; Sasaki, H.; Shiga, K.; Miyakawa, H.; Shibata, S. The Timing E ff ects of Soy Protein Intake on Mice Gut Microbiota. Nutrients 2019 , 12 , 87. [CrossRef] 17. Ahn, Y.; Nam, M.H.; Kim, E. Relationship Between the Gastrointestinal Side E ff ects of an Anti-Hypertensive Medication and Changes in the Serum Lipid Metabolome. Nutrients 2020 , 12 , 205. [CrossRef] 18. Hills, R.D., Jr.; Pontefract, B.A.; Mishcon, H.R.; Black, C.A.; Sutton, S.C.; Theberge, C.R. Gut Microbiome: Profound Implications for Diet and Disease. Nutrients 2019 , 11 , 1613. [CrossRef] 19. Salli, K.; Lehtinen, M.J.; Tiihonen, K.; Ouwehand, A.C. Xylitol’s Health Benefits beyond Dental Health: A Comprehensive Review. Nutrients 2019 , 11 , 1813. [CrossRef] 20. Mesa, M.D.; Loureiro, B.; Iglesia, I.; Fernandez Gonzalez, S.; Llurba Olive, E.; Garcia Algar, O.; Solana, M.J.; Cabero Perez, M.J.; Sainz, T.; Martinez, L.; et al. The Evolving Microbiome from Pregnancy to Early Infancy: A Comprehensive Review. Nutrients 2020 , 12 , 133. [CrossRef] 21. Cornide-Petronio, M.E.; Alvarez-Mercado, A.I.; Jimenez-Castro, M.B.; Peralta, C. Current Knowledge about the E ff ect of Nutritional Status, Supplemented Nutrition Diet, and Gut Microbiota on Hepatic Ischemia-Reperfusion and Regeneration in Liver Surgery. Nutrients 2020 , 12 , 284. [CrossRef] [PubMed] 22. Barrientos-Duran, A.; Fuentes-Lopez, A.; de Salazar, A.; Plaza-Diaz, J.; Garcia, F. Reviewing the Composition of Vaginal Microbiota: Inclusion of Nutrition and Probiotic Factors in the Maintenance of Eubiosis. Nutrients 2020 , 12 , 419. [CrossRef] [PubMed] 23. Sanchez-Rodriguez, E.; Egea-Zorrilla, A.; Plaza-Diaz, J.; Aragon-Vela, J.; Munoz-Quezada, S.; Tercedor-Sanchez, L.; Abadia-Molina, F. The Gut Microbiota and Its Implication in the Development of Atherosclerosis and Related Cardiovascular Diseases. Nutrients 2020 , 12 , 605. [CrossRef] 24. Plaza-Diaz, J.; Pastor-Villaescusa, B.; Rueda-Robles, A.; Abadia-Molina, F.; Ruiz-Ojeda, F.J. Plausible Biological Interactions of Low- and Non-Calorie Sweeteners with the Intestinal Microbiota: An Update of Recent Studies. Nutrients 2020 , 12 , 1153. [CrossRef] [PubMed] © 2020 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http: // creativecommons.org / licenses / by / 4.0 / ). 4 nutrients Article Randomised Double-Blind Placebo-Controlled Trial of Inulin with Metronidazole in Non-Alcoholic Fatty Liver Disease (NAFLD) Clara Yieh Lin Chong 1 , David Orr 2, *, Lindsay D. Plank 3 , Tommi Vatanen 1 , Justin M. O’Sullivan 1 and Rinki Murphy 4, * 1 Liggins Institute, The University of Auckland, Auckland 1142, New Zealand; clara.chong@auckland.ac.nz (C.Y.L.C.); t.vatanen@auckland.ac.nz (T.V.); justin.osullivan@auckland.ac.nz (J.M.O.) 2 New Zealand Liver Transplant Unit, Auckland City Hospital, Auckland 1023, New Zealand 3 Department of Surgery, Faculty of Medical and Health Sciences, The University of Auckland, Auckland 1142, New Zealand; l.plank@auckland.ac.nz 4 Department of Medicine, Faculty of Medical and Health Sciences, The University of Auckland, Auckland 1142, New Zealand * Correspondence: DOrr@adhb.govt.nz (D.O.); R.Murphy@auckland.ac.nz (R.M.); Tel.: + 64-9-923-6313 Received: 5 March 2020; Accepted: 24 March 2020; Published: 27 March 2020 Abstract: Background : Non-alcoholic fatty liver disease (NAFLD) can be ameliorated by weight loss although di ffi cult to maintain. Emerging evidence indicates that prebiotics and antibiotics improve NAFLD. Aim : To determine whether inulin supplementation after brief metronidazole therapy is e ff ective in reducing alanine aminotransferase (ALT) and maintaining weight loss achieved through a very-low-calorie diet (VLCD) among people with NAFLD. Methods : Sixty-two people with NAFLD commenced 4-week VLCD using Optifast meal replacements (600 kcal / day). Sixty were then randomised into a 12-week double-blind, placebo-controlled, parallel three-arm trial: (1) 400 mg metronidazole twice daily in Week 1 then inulin 4 g twice daily OR (2) placebo twice daily in week one then inulin OR (3) placebo-placebo. Main outcomes were ALT and body weight at 12 weeks. Fecal microbiota changes were also evaluated. Results : Mean body mass index (BMI) and ALT reduced after VLCD by 2.4 kg / m 2 and 11 U / L, respectively. ALT further decreased after metronidazole-inulin compared to after placebo-placebo (mean ALT change − 19.6 vs. − 0.2 U / L, respectively; p = 0.026); however, weight loss maintenance did not di ff er. VLCD treatment decreased the ratio of Firmicutes / Bacteroidetes ( p = 0.002). Conclusion : Brief metronidazole followed by inulin supplementation can reduce ALT beyond that achieved after VLCD in patients with NAFLD. Keywords: prebiotics; alanine aminotransferase; antibiotic; Optifast; gut microbiome; inulin; metronidazole 1. Introduction Non-alcoholic fatty liver disease (NAFLD) is defined by the pathological accumulation of fat in the liver and is now the leading cause of chronic liver disease [ 1 ]. NAFLD encompasses a spectrum of diseases ranging from simple fatty liver (steatosis) through to non-alcoholic steatohepatitis (NASH), which, in turn, leads to fibrosis, irreversible cirrhosis and, finally, hepatocellular carcinoma (HCC) in a small proportion of people [ 2 , 3 ]. The milder simple steatosis is characterised by the ectopic accumulation of fat in the liver, usually associated with energy-surplus-induced obesity. It is believed that multiple parallel factors (diet, insulin resistance, mitochondrial dysfunction and inflammation), acting synergistically in genetically predisposed individuals, are implicated in the development and progression of NAFLD. Nutrients 2020 , 12 , 937; doi:10.3390 / nu12040937 www.mdpi.com / journal / nutrients 5 Nutrients 2020 , 12 , 937 An accumulating number of animal and human studies suggest a compelling role for gut microbiota in NAFLD, which is both transmitted by gut microbiota and reversed by a combination of ciprofloxacin and metronidazole antibiotics in animal models [ 4 ]. NAFLD is associated with dysbiosis of the gut microbiota, which is thought to lead to increased gut permeability, and abnormal choline and bile acid metabolism, leading to inflammation and increased hepatic fat accumulation [ 5 ]. An indication of the involvement of gut microbiota in NAFLD development was first apparent when hepatic steatosis developed in patients undergoing jejunal–ileal bypass surgery, coinciding with intestinal bacterial overgrowth in the blind loop. The hepatic steatosis regressed once patients were treated with the antibiotic metronidazole [ 6 ] which is commonly used for the treatment of small intestinal bacterial overgrowth [ 7 ]. While small intestinal bacterial overgrowth has been shown to be more prevalent in NAFLD [ 8 – 11 ], antibiotic treatment of NAFLD has not been investigated due to concerns about long-term use being associated with side e ff ects, antimicrobial resistance and uncertain e ffi cacy. The cornerstone of NAFLD treatment currently is to o ff er lifestyle advice that targets 7% to 10% weight loss and is proven to be e ff ective [ 12 , 13 ]. Recent evidence shows that very-low-calorie diets (VLCDs) [ 14 ] and bariatric surgery [ 15 ] are very e ff ective in achieving weight loss and remission of associated comorbidities. Both these strategies alter gut microbiota, but to a lesser extent after dietary modification than after surgery [ 16 – 18 ]. However, the maintenance of weight loss remains a challenge and better alternatives to targeting specific mechanistic dysfunction are needed. Prebiotics, which are nondigestible food ingredients that are fermented in the gut and modulate microbiota in a favourable way for the host, have shown promise in the treatment of NAFLD. A systematic review of 26 randomised controlled trials investigating the metabolic benefits of prebiotics concluded that prebiotics improve satiety, postprandial glucose and insulin in both healthy and obese individuals [ 19 ]. A meta-analysis of nine randomised controlled trials in NAFLD showed a reduction in body mass index (BMI) and an overall improvement in aminotransferase (ALT) with the use of prebiotics [ 20 ]. However, the use of a combination of strategies targeting gut microbiota dysbiosis of NAFLD such as VLCD, metronidazole and prebiotics in succession has not previously been investigated. We hypothesised that the beneficial metabolic e ff ects of short-term VLCD among adults with NAFLD could be enhanced by the brief use of metronidazole to target dysbiotic gut microbiota followed by a period of inulin supplementation to maintain this. We conducted a single centre, randomised, placebo-controlled, double-blind clinical three-arm trial of 12 weeks of inulin supplementation with or without an initial week of metronidazole cotreatment among adults with NAFLD who had all received four weeks of VLCDs. 2. Patients and Methods 2.1. Study Design This study focused on adults with an established diagnosis of NAFLD attending Auckland City Hospital hepatology outpatient clinic. Patients either had histological evidence of NAFLD based on a liver biopsy, a phenotypic diagnosis based on the presence of BMI > 27 kg / m 2 and type 2 diabetes or metabolic syndrome (WHO criteria) with an elevated ALT (male > 40 U / L, female > 30 U / L) and age > 18 years and < 75 years. Exclusion criteria were alcohol consumption of more than 20 g per day for at least 3 consecutive months during the previous 5 years as assessed by a questionnaire. Participants were excluded if they had cirrhosis, hepatitis C or another liver disease, if they were awaiting or had previous bariatric surgery, had an allergy to eggs, nuts or metronidazole, a history of drug and alcohol abuse, a calculated eGFR less than 60 mL / min (MDRD formula) or current participation in other therapeutic trials. Ethics approval was from Health and Disability Ethics Committee NTX / 12 / 05 / 040 / AM02; ANZCTR registration number: 12613001002774, prospectively registered on 10 September 2013. 6 Nutrients 2020 , 12 , 937 2.2. Randomisation and Treatment Groups Sixty-two participants with NAFLD who met all eligibility criteria and provided written informed consent were provided with 3 Optifast meal replacements (600 kcal / day) per day for 4 weeks to initiate weight loss after which the 60 participants who attended the second study visit were randomly assigned to one of three parallel groups (1:1:1; Figure A1). The metronidazole and inulin group (Group MI) received metronidazole (dose of 400 mg twice daily for 7 days) along with inulin (at a dose of 4 g twice daily for 12 weeks); the placebo and inulin group (Group PI) received metronidazole-like placebo (twice daily for 7 days) along with inulin (at a dose of 4 g twice daily for 12 weeks); the placebo and inulin placebo group (Group PP) received metronidazole-like placebo (twice daily for 7 days) along with inulin-like placebo (containing maltodextrin at a dose of 4 g twice daily for 12 weeks). The inulin dose was selected on the basis of previous prebiotic studies and was provided by Cargill Belgium. A metronidazole dose of 400 mg twice daily was selected as slightly lower than the standard dose of 400 mg three times daily used for various medical conditions, such as bacterial vaginosis, dental abscess and giardiasis, for increased adherence than three times daily. Metronidazole and matching placebo-containing maltodextrin were encapsulated by the Auckland Hospital Clinical Trials’ Pharmacy department. All participants, their health care providers and assessment sta ff were blinded to treatment allocation. Participants were asked to take the inulin / matching placebo powder twice daily before breakfast and before dinner using a 4 g measuring spoon and two level spoonfuls dissolved into water. All participants were given a standardised set of recommendations about lifestyle changes and diet following the initial expected weight loss period during VLCD at time of study randomisation In total, there were four time points in this study: baseline (study entry), Week 4 (after 4 weeks VLCD pre-randomisation), Week 16 (post-randomisation, at the end of treatment) and Week 28 (post-treatment follow up phase to evaluate whether there were any persistent e ff ects detected beyond the treatment period) as shown in Figure 1. All participants underwent assessment for body weight, height, waist and hip circumference at each of these 4 timepoints. Blood samples for assessment of fasting lipids, glucose, insulin and liver Fibroscan CAP were obtained at baseline, Week 4 and then Week 16. Figure 1. Assessment and sample collection timeline. 2.3. Stool Sample Collection Stool samples were collected at each time point (Figure 1): baseline, Week 4, Week 16 and Week 28. Study participants collected the stool samples at home, using a sterile collection tube, prior to their hospital visits. Stool samples were stored at − 70 ◦ C from the beginning of the study (2013 / 2015) until DNA extraction was performed (2017). DNA was extracted from stool samples using the QIAamp ® Fast DNA Stool Mini Kit according to the manufacturer’s protocol. Extracted DNA quality and quantity were measured using a NanoPhotometer N60 (IMPLEN, Germany; Table S1) and a Qubit (Invitrogen, US). 7 Nutrients 2020 , 12 , 937 2.4. 16S rRNA Gene Amplicon Sequencing Extracted DNA (mean yield = 6733.4 ng; mean 260 / 280 = 1.97; mean concentration = 33.7 ng / μ L) was sent to the School of Biological Sciences (The University of Auckland, New Zealand) for 16S rRNA amplicon sequencing on an Illumina MiSeq sequencing platform. Sequences are available from SRA project number SUB5068044. Then, 16S rRNA gene amplicon sequencing (16S sequencing) libraries were prepared using the Nextera XT kit (Illumina). V3 and V4 regions were targeted for 16S sequencing by using the 16S Amplicon PCR Forward Primer (TCGT CGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG) and Reverse Primer (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC). All amplicons were sequenced on the Illumina MiSeq 600 cycle run to generate an average of 121,346 sequence reads with paired-end (300 bp each) reads per sample. 2.5. 16S rRNA Amplicon Sequence Analyses The 16S sequencing data were processed using QIIME 2 (v. 2018.4) [ 21 ]. Briefly, sequence quality control and denoising were performed using DADA2 [ 22 ]. The quality control step also included the filtering of PhiX reads and chimeric sequences. The sequences obtained after denoising were then classified using Greengenes 13_8 release data to identify amplicon sequencing variants (ASVs) for sequences with > 99% sequence similarity. Samples that were included in downstream analyses had filtered sequence counts ranging from 12,123 to 109,977 (median 53,071). Three samples with less than 10,000 sequencing reads were removed. 2.6. Primary and Secondary Outcomes The primary outcome was the proportion maintaining a ≥ 7% weight loss at the end of the 12-week variable treatment period compared to their baseline (before the fixed 4-week VLCD treatment period). Secondary outcomes measured at Week 16 (the end of the 12-week variable treatment period) included changes in ALT, glycaemia, lipids, Fibroscan ® CAP from what was achieved at Week 4 (after VLCD treatment period) and the changes in gut microbial community from baseline to 28 weeks. 2.7. Statistical Analysis The planned sample size for this pilot study was 60 subjects with an equal assignment to each of the three study groups (20 per group). We estimated that, with this sample size, the study would have 80% power to detect a di ff erence in the proportion achieving a sustained weight loss of ≥ 7% at the end of the 12-week treatment period which we anticipated would be achieved by 50% of those receiving metronidazole and inulin supplemented diet, compared to 5% in the other two placebo-containing groups, with a two-sided type 1 error of 0.05. The primary outcome was assessed using Fisher’s exact test. Pre-planned analyses for secondary outcomes were comparisons of the changes over the 12-week variable treatment period in the MI and PI groups with those in the PP group. Two-sample t -tests were used for these comparisons for normally distributed data and Mann–Whitney U -tests for non-normal data. Within-cohort changes over the VLCD period were analysed using paired t -test or Wilcoxon signed-rank test as appropriate. Data are presented as mean (SD) or