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Vijay Kothari · Prasun Kumar ·
Subhasree Ray Editors

Probiotics,
Prebiotics,
Synbiotics, and
Postbiotics
Human Microbiome and Human Health
Probiotics, Prebiotics, Synbiotics, and Postbiotics
Vijay Kothari • Prasun Kumar • Subhasree Ray
Editors

Probiotics, Prebiotics,
Synbiotics, and Postbiotics
Human Microbiome and Human Health
Editors
Vijay Kothari Prasun Kumar
Institute of Science Chemical Engineering
Nirma University Yeungnam University, Korea
Ahmedabad, India Gyeongsan, Korea (Republic of)

Subhasree Ray
Department of Life Sciences
School of Basic Sciences
& Research, Sharda University
Greater Noida, Uttar Pradesh, India

ISBN 978-981-99-1462-3 ISBN 978-981-99-1463-0 (eBook)

https://doi.org/10.1007/978-981-99-1463-0

© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore
Pte Ltd. 2023
This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether
the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of
illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and
transmission or information storage and retrieval, electronic adaptation, computer software, or by
similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this
book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or
the editors give a warranty, expressed or implied, with respect to the material contained herein or for any
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claims in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd.
The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721,
Singapore
We dedicate this book to all those Teachers
who taught us at any level right from
kindergarten to doctorate.
Preface

With a lot of data being generated on the Human Microbiome, quite many correla-
tions are being uncovered between the microbiome composition and human health/
diseases. Almost every aspect of human health and behavior, e.g., obesity, function-
ing of the immune system and nervous system, susceptibility to various infections,
and mood fluctuations, seems to be correlated with the microbiome composition.
Although the microbiomes of the skin, oral cavity, nasal cavity, and vagina have
their own importance, much research has focused on the microbiome of the intestine
since the gut harbors the greatest number of resident microorganisms within the
human body. As we are developing more and more understanding of the human
microbiome and its role in the regulation of the human system, more interest is being
generated in finding ways of manipulating this microbiome toward the “healthier”
direction. This manipulation of the human microbiome seems to be possible through
the use of the following:
• Probiotics: Live microbial strains which, upon ingestion, are expected to confer
some health benefit.
• Prebiotics: Selectively fermented ingredients causing specific changes in the
composition and/or activity of the gut microbiota, thereby offering beneficial
health effects to the host.
• Synbiotics: A mix of probiotics and prebiotics that can confer some beneficial
effect on the host by improving the survival and activity of beneficial microor-
ganisms in the intestine.
• Postbiotics: Metabolic products of the fermentation by probiotics in the intestine.
There is a hope that if we can gain sufficient knowledge of the human microbiome
composition and of the ways of manipulating it, this can enable us to manage many
of the complex metabolic disorders by careful designing of the diet, i.e., simple
dietary adjustments can be a major part of disease/health management strategy.
In the above-mentioned context, this contributory volume features manuscripts
from researchers working on a different aspect of the Human Microbiome and its
manipulation for better health. In this collection, we have tried to showcase the

vii
viii Preface

current status of research in the field and also to point toward future directions, not
only from an academic but also from an industrial and regulatory perspective.
The editors thank all the contributing authors and acknowledge support from the
publisher.
Wish you all a happy reading!
May we all enjoy a healthy microbiome throughout life!

Ahmedabad, Gujarat, India Vijay Kothari

Gyeongsan, Republic of Korea Prasun Kumar
Greater Noida, Uttar Pradesh, India Subhasree Ray
January 2023
Contents

Part I Current State of Knowledge Regarding the Human

Microbiome Structure, Function, and Diversity
Impact of Dietary Habits, Ethnicity, and Geographical Provenance
in Shaping Human Gut Microbiome Diversity . . . . . . . . . . . . . . . . . . . . 3
Payal G. Patel, Ajay C. Patel, Prasenjit Chakraborty, and Haren B. Gosai
Methods Used for Studying Human Microbiome . . . . . . . . . . . . . . . . . . 29
Chinmayi Joshi and Vijay Kothari
Factors Affecting the Composition of the Human Microbiome . . . . . . . . 49
Madangchanok Imchen, Simi Asma Salim, Ranjith Kumavath,
and Siddhardha Busi

Part II Correlation of the Human Microbiome to Specific

Health/Disease Conditions
Mapping the Microbial Metabolites in Metabolic Disorder with Special
Reference to Type-2 Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Sunny Kumar, Zeel Bhatia, and Sriram Seshadri
Human Microbiome in Malnutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Mehul Chauhan, Priya Mori, and Vijay Kumar
Association of Probiotics and Prebiotics with Human Microbiome and
the Functioning of Immune System . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Pia Dey, Samir Kumar Mukherjee, and Debaprasad Parai
Human Microbiome and the Susceptibility to Infections . . . . . . . . . . . . . 117
V. T. Anju, Siddhardha Busi, Mahima S. Mohan, and Madhu Dyavaiah
Human Microbiome and the Neurological Disorders . . . . . . . . . . . . . . . 139
Rajesh Pamanji and Joseph Selvin

ix
x Contents

Exploring the Unexplored Arena: Butyrate as a Dual Communicator

in Gut–Brain Axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
Zeel Bhatia, Sunny Kumar, and Sriram Seshadri
Human Microbiome and Lifestyle Disorders . . . . . . . . . . . . . . . . . . . . . 165
Ankit Gupta and Abhilasha Jha
Correlation of Human Microbiome and Immune Functioning with
COVID-19 Infections: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Purnima Khatri, Asha Rani, Ramendra Pati Pandey, and Saif Hameed
Exploring the Pathoprofiles of SARS-COV-2 Infected Human
Gut–Lungs Microbiome Crosstalks . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Sisir Nandi, Sarfaraz Ahmed, Aaruni Saxena, and Anil Kumar Saxena
Role of Human Microbiome in Cardiovascular Disease: Therapeutic
Potential and Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
Sathiya Maran, Wendy Wai Yeng Yeo, Kok Song Lai,
and Swee Hua Erin Lim
The Human Microbiome and Respiratory Diseases . . . . . . . . . . . . . . . . 255
Oksana Zolnikova and Vladimir Ivashkin

Part III Manipulation of the Human Microbiome for Better Health

Probiotics: An Emerging Strategy for Oral Health Care . . . . . . . . . . . . 275
Subramani Parasuraman, Venkata Kanthi Vaishnavi Vedam,
and Gokul Shankar Sabesan
Dietary Modulation of the Nervous and Immune System: Role
of Probiotics/Prebiotics/Synbiotics/Postbiotics . . . . . . . . . . . . . . . . . . . . . 307
Priya Mori, Mehul Chauhan, Ishita Modasiya, and Vijay Kumar
Probiotics for Skin Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Chinmayi Joshi, Ritul Suthar, Aryushi Patel, Feni Patel,
and Drashti Makwana
Human Microbiome and Autism-Spectrum Disorders . . . . . . . . . . . . . . 347
Rishi Gupta and Shailendra Raghuvanshi
Psychobiotics as an Emerging Category of Probiotic Products . . . . . . . . 361
Sahdev Choudhary, Kumari Shanu, and Sarita Devi
Probiotics for Vaginal Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393
Emi Grace Mary Gowshika Rajendran
Interactions Between Microbial Therapeutics and the Endogenous
Microbiome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 421
Haley Anne Hallowell, Anne Lulu Gao, Kristen E. Kelly, and Jotham Suez
Contents xi

Part IV Applied and Translational Aspects

Bacillus Endospore Probiotics Are a Promising Intervention for
Mitigation of Metabolic Endotoxemia . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
Kiran Krishnan, Sujit Nair, and Dilip Mehta
Characterization and Authentication of Probiotic Preparations . . . . . . . 479
Vijay Kothari, Anselm de Souza, and Dilip Mehta
A Survey of Commercially Available Probiotics . . . . . . . . . . . . . . . . . . . 489
Swati Misra and Shailendra Raghuwanshi
Regulatory Aspects Relevant to Probiotic Products . . . . . . . . . . . . . . . . 513
Parul Chugh, Swati Misra, Mahesh S. Dhar, and Shailendra Raghuwanshi
Probiotic Identity from Spore: Focus on Bacillus Probiotics . . . . . . . . . . 535
Bhanuramanand K., Veena P. V. S., Haritha Rani B., Dilip Mehta,
Anselm Desouza, and Madhusudhana Rao Nalam
About the Editors

Vijay Kothari Ph.D. is a microbiologist. His current

research is in the areas of AMR (antimicrobial resis-
tance), traditional medicine, prebiotic and immunomod-
ulatory properties of natural products, microbial
response to sonic stimulation, etc. His group is actively
involved in investigating antimicrobial/anti-virulence
potential of natural products as well as synthetic com-
pounds. In recent past, his lab has extensively investi-
gated the anti-pathogenic/prophylactic activity of
various traditional medicine formulations, e.g.,
Panchvalkal, Panchgavya, and Triphala, against differ-
ent antibiotic-resistant bacterial strains. His lab was
awarded SRISTI-DBT-BIRAC Appreciation Award
(2017) for validation of anti-infective potential of a
polyherbal formulation inspired from folk medicine—
Herboheal. He has also been awarded two AIMS (Arti-
ficial Intelligence Molecular Screen) award (2020–
2022) projects by Atomwise Inc., USA, for identifying
novel anti-infective leads. Dr. Kothari has also contrib-
uted substantially to the field as an active editor and
reviewer, and has been conferred the Sentinel of Science
award (2016) by Publons recognizing his contribution
as a peer reviewer. He has 88 research/review/book
chapter publications to his credit. His publications
have enjoyed a wide readership as evident from more
than 253,000 reads from ResearchGate alone. Vijay
derives great satisfaction from the hitherto output of
the M.Sc. dissertations guided by him. During the
period 2007–2019, he guided 84 M.Sc. students for
their dissertation projects, of which 71 (i.e., 84.52%)

xiii
xiv About the Editors

could publish research/review papers in different peer-

reviewed, indexed journals (or a citable preprint), based
on their master’s dissertation.

Prasun Kumar Ph.D. holds a Ph.D. in Biotechnology

from CSIR-Institute of Genomics and Integrative Biol-
ogy, Delhi, India. He is presently working as a Scientific
Officer at DBT-IOC Center for Advanced Bioenergy
Research, Faridabad. Earlier, he was working as an
Assistant Professor at the Department of Chemical Engi-
neering, Yeungnam University, Republic of Korea. He
has over 7 years of experience in applied microbiolog-
ical research including about 2 years of experience in
industrial R&D. His main areas of research are biopoly-
mers, microbial biodiversity, bioenergy, microbial
biofilms, quorum sensing, quorum quenching, and
genomics. His present research is oriented toward valo-
rizing lignocellulosic biowastes into value-added prod-
ucts such as biopolymer, 2G ethanol, bioenergy, and
antibiofilm compounds. To his credit, there are over
34 articles in SCI journals, 5 books, and 11 chapters
with international publishers. He has been serving the
scientific society by reviewing articles for several SCI
journals and delivering guest lectures. Publons awarded
him the peer review award in the year 2018. He also
serves as the editorial board member of a few interna-
tional journals.

Subhasree Ray Ph.D. is currently working as an

Assistant professor at Sharda University, Greater
Noida, Uttar Pradesh, India. She earned her Ph.D.
degree from CSIR-IGIB, Delhi, in 2018. She received
the prestigious CSIR-SRF fellowship. Her main
research was focused on producing biopolymers from
waste biomass. After Ph.D., she joined as a postdoctoral
researcher at Ewha University and the University of
Seoul, South Korea. Here, her main focus was the
anaerobic digestion of food wastes for methane produc-
tion. She also studied methanogenesis at a 4000 L pilot-
scale plant. After the successful completion of 1 year,
she joined another project at Yeungnam University,
South Korea. During that period, she worked on several
fungal toxins and their inhibition from fermented food.
About the Editors xv

She also worked on biofilm inhibition of pathogenic

organisms by natural bioactive compounds. To her
credit, she has 13 research papers published in peer-
reviewed journals and 8 book chapters. In addition,
she is a life member of various scientific societies and
also a member of various committees at Sharda Univer-
sity for Graduate and Undergraduate programs.
 Part I
Current State of Knowledge Regarding
the Human Microbiome Structure,
Function, and Diversity
Impact of Dietary Habits, Ethnicity,
and Geographical Provenance in Shaping
Human Gut Microbiome Diversity

Payal G. Patel, Ajay C. Patel, Prasenjit Chakraborty, and Haren B. Gosai

Abstract Human gut microbiome is comprised of billions of microorganisms that

reside within gastrointestinal tract and form a symbiotic bond with humans. This
unique relationship between microbes and human is possible through gut–brain axis
which enables bidirectional communication between central and enteric nervous
system. The diversity of the gut microbiome, i.e., the composition of the microbes
living in the human gut is influenced by various external and internal factors. The
dietary habits of an individual not only play a major role in determining which kinds
of microbes exist in the gut but also effect the interaction among the different species
of microbes. Dietary habits in turn are shaped by the culture, lifestyle, ethnicity, and
geographical location of a population. People belonging to same ethnicity have
similar dietary habitats; however, their lifestyle and geographical variation may
lead to different microbiome composition. Different ethnic groups within same
geographical location have been observed to have diverse microbiome; meanwhile,
migration has proven to westernize the gut microbiome. The evident data suggests
that ethnicity, dietary habits, and geographical provenance are closely interlinked,
and their interrelationship is a key player in determining gut microbiome diversity.
In this chapter, the authors attempt to elucidate how and to what extent these three
factors impact the microbiome composition and diversity.

Keywords Gut microbiome · Dietary habits · Ethnicity · Geographical provenance

1 Introduction

Microorganisms play a crucial role in the daily life and functioning of human beings.
From microbial products that we use in our day-to-day life to the microorganisms
that reside within our body, we depend on them for a variety of reasons. Microor-
ganisms are ubiquitous and constitute a large part of nature’s living matter, and

P. G. Patel · A. C. Patel · P. Chakraborty · H. B. Gosai (✉)

Department of Biosciences, School of Sciences, Indrashil University, Mehsana, Gujarat, India
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 3
V. Kothari et al. (eds.), Probiotics, Prebiotics, Synbiotics, and Postbiotics,
https://doi.org/10.1007/978-981-99-1463-0_1
4 P. G. Patel et al.

humans are not an exception. Microorganisms have resided synergistically within

our body, undergoing co-evolution with humans, and have continued to influence
human health for centuries (Chauhan 2019; Rackaityte and Lynch 2020). Human
gastrointestinal tract (GI) consists of millions of diverse bacteria, archaea, and
eukaryotes that form a symbiotic relationship with the host (Thursby and Juge
2017). Microorganisms that inhabit the GI form the human ‘gut microbiome’, a
collection of genomes from all the microorganisms living together in the GI (Zhu
et al. 2010). While ‘microbiome’ signifies the entire habitat, ‘microbiota’ refers
solely to the microbes that reside within that specific habitat or GI in this case
(Valdes et al. 2018). Human gut microbiome has also been described by some
researchers as the second genome or metagenome of the human body (Zhu et al.
2010; Grice and Segre 2012). It has been estimated that more than 3.3 million
microbial genes are present within gut microbiome which is approximately
150 times the number of genes in the human genome (Ehrlich 2016). Gut microbiota
also makes up approximately 1.5–2 kg of the total body weight in humans (Mazidi
et al. 2016). Gut microbiome (GM) in addition to prominent bacterial species also
comprises archaea (mostly Methanobrevibacter smithii), yeast, fungi, and viruses
(mostly phages). Moreover, the mycobiome, archaea, and virus provide an extra
dimension to the mutually beneficial interactions between host and microbiota
(Lozupone et al. 2012; Cani 2018). Every individual has a ‘core microbiome’ that
remains similar universally, but the overall gut microbiome is as distinguishable as a
person’s fingerprint (Lozupone et al. 2012; Franzosa et al. 2015). Core GM consists
of more than 1000 species of microorganisms, but three phyla Bacteroidetes,
Firmicutes, and Actinobacteria are most prominent. While Proteobacteria and
Verrucomicrobia make up only small percentage of the core microbiome (Eckburg
et al. 2005; Tap et al. 2009; Kho and Lal 2018). Aside from GM, microorganisms are
also concentrated at other different environments within human body including oral
cavity, oesophagus, vagina, and skin. However, diversity and composition of these
microbiomes differ from that of GM (Gilbert et al. 2018).
Until last decade, the idea that GM could have significant influence on human
health was considered fringe science. However, due to advancements and break-
throughs in the field of metagenomics, it is now possible to elucidate the relationship
between microbiota and host organism (D’Argenio and Salvatore 2015). GM
encodes several genes that enable production of hydrolytic enzymes. These enzymes
breakdown the otherwise indigestible components present in the meal (Flint et al.
2012). Apart from digestion, GM has been proved to be responsible for vitamin
synthesis (Magnúsdóttir et al. 2015), development of immune system (Kau et al.
2011), providing defence against pathogens (Sekirov et al. 2010), behaviour devel-
opment (Cryan and O’Mahony 2011), and promotion of intestinal angiogenesis
(Franks 2013).
GM diversity, i.e., the composition of the microbes living in the human gut is
affected by a wide range of external and internal factors. External factors include
lifestyle choices, dietary habits, geographical location, antibiotic usage, etc., whereas
internal factors refer to host genetics, owing to particular ethnicity, population or
individual genome (van Best et al. 2015; Kho and Lal 2018). ‘We Are What We Eat’
Impact of Dietary Habits, Ethnicity, and Geographical Provenance. . . 5

is a phrase that is often used, but in case of GM this phrase is indeed true. Evidences
have revealed that our diet choices, nutritional uptake, and even frequency of meals
have a correlation with the GM (De Filippis and Ercolini 2018; Wang et al. 2019).
Our daily meals not only provide nutrition to us, but they also supply essential
metabolites to GM. In humans, gut microbiome is established by 2–3 years age, and
the food consumed during these initial ‘first 1000 days’ affect the diversity of GM
(Laursen et al. 2017). However, in adults, type of long-term dietary patterns, e.g.,
westernized or traditional, is also related with particular gut microbial profiles (Wang
et al. 2019). The dietary patterns of an individual are often shaped by their ethnicity
as different culture and civilization have their own food customs and distinctive
cuisine (Low et al. 2021). Ethnic groups are formed over a long period of time as a
result of isolation, adaptation, and migration. As a result, different ethnic groups
have specific inherent genotype, which further influences their GM (Dehingia et al.
2019). Under similar external conditions (socioeconomic status, environment, age),
ethnicity was reported to be the most influential factor in regulating alpha diversity
of GM (Liu et al. 2020).
Apart from that, one cannot dismiss the role of geographical provenance since
they not only mould the lifestyle or diet choices but, in some instances, impact gut
microbiome at physiological level (Senghor et al. 2018; Mazel 2019). Studies have
suggested that individuals from high latitude have higher Firmicutes and lower
Bacteroidetes proportion. These two phyla are associated with increase in body
weight by regulating fat storage and energy extraction from diet (Suzuki and
Worobey 2014). Effects of dietary habits, ethnicity, and geography on GM have
been studied at individual level but limited numbers of reports have focused on the
interrelationship between these three factors. As illustrated in Fig. 1, all three factors
are interlinked and play a key role in shaping GM diversity and composition. In this

Fig. 1 Interaction between dietary habits, ethnicity, and geographical provenance and its correla-
tion with gut microbiome
6 P. G. Patel et al.

chapter, the authors attempt to elucidate this intertwined relationship between

geography, diet, and ethnicity. This chapter also shed light on their individual and
combined impact on diversity and composition of human GM.

2 Significance of GM

It is now a well-established fact that the composition of the GM is responsible for

the overall health and well-being of a particular individual. With each passing year,
the relationship between GM and human health is being increasingly recognized.
The vast majority of these microbes are classified into five different phyla, namely
Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, and Fusobacteria
(Andersson et al. 2008). Though in an infant, the GM lacks any obvious principle
of organization, by the age of 3 years it starts resembling the adult microflora. Even
in adult, temporal and spatial dissimilarities exist in microbial distribution across the
digestive system. Due to their extensive importance, next-generation sequencing
technologies are being employed to study these microorganisms. Gut microbiota
directly interacts with its host to control the host metabolism, along with mainte-
nance of structural integrity of the gut mucosal barrier, and immunomodulation.
Several factors such as diet, antibiotics, age, drug metabolism, and diseases are at
play in determining the make-up of normal gut microbiota. Among them, misuse of
antibiotics is a major cause of concern affecting the normal healthy GM. Generation
of multidrug resistance species and horizontal transfer of these resistance genes
could affect the composition of normal GM. The microbiome present in the gut
has been associated with a large number of diseases, such as inflammatory bowel
diseases, irritable bowel syndrome, allergic disease, neurodevelopmental illnesses,
obesity, and diabetes (Bisgaard et al. 2011; Ferreira et al. 2014; Karlsson et al. 2013;
Kennedy et al. 2014). It has intrigued scientists for decades and is still a field of very
active research. Various microbiome and metagenomics projects carried out by the
USA and European consortiums have established the benefits of having a healthy gut
flora to the genetic level (Gevers et al. 2012; Qin et al. 2010). If we consider the
perspective of an immunologist, microorganisms are foreign pathogens that need to
be eliminated by the immune system of the host. So, according to that logic,
microflora inhabiting our guts should have been eradicated. However, the majority
of the gut bacteria are non-pathogenic and have established a symbiotic relationship
with the enterocytes. This is only possible because these microorganisms and the
immune system have co-evolved to form a symbiotic relationship. Apart from aiding
in various metabolic activities, these healthy microorganisms help to maintain
intestinal barrier function and to prevent the colonization of pathogenic
microorganisms.
There are over 35,000 bacterial species residing in the human gut with approx-
imately ten million non-redundant genes (Frank et al. 2007). It is not just the number
and type of bacterial species, but the total gene count of those species that has huge
implications for health and disease. The high gene count (HGC) of some microbial
Impact of Dietary Habits, Ethnicity, and Geographical Provenance. . . 7

species like Akkermansia sp., Anaerotruncus colihominis, Butyrivibrio crossotus,

and Faecalibacterium sp. is effective in controlling obesity (Le Chatelier et al.
2013). Not only the overall count but also the ratio of certain microbial species is
important. A high ratio of Akkermansia sp.:Ruminococcus gnavus is beneficial for
digestive health. Some of the salient features to look out for in the HGC microbiome
favouring digestive health are
1. The enhanced percentage of butyrate-producing organisms.
2. Increased tendency to produce hydrogen.
3. Establishment of methanogenic/acetogenic ecosystem.
4. A reduction in the capability to produce hydrogen sulfide.
In contrast, individuals with a low gene count (LGC) of the above-mentioned
bacterial species harbour a higher percentage of pro-inflammatory bacteria like
Ruminococcus gnavus and Staphylococcus sp., causing inflammatory bowel disease.
In addition to this, LGC individuals possess bacterial metabolites modules for
degradation of aromatic amino acids and β-glucuronide that are deleterious health
effects. Conversely, HGC individuals have robust gut microbiome functionality with
a lower frequency of metabolic disorders. Now, let us look at the important func-
tional aspects of the gut microbiota in more details.

2.1 Antimicrobial Protection

GM plays a major role in maintaining homeostasis within the gut mucosal immune
system by being tolerant to advantageous commensals yet preventing harmful
pathogens. Unlike the large intestine, antimicrobial proteins (AMP) are of great
importance in the small intestine as the mucus layer in the small intestine is
discontinuous and inadequate. Synthesis of AMP such as C-type lectins,
cathelicidins, and defensins is induced by the gut microbiota, via its structural
components and metabolites (Hooper 2009). Crosstalk between the pattern recog-
nition receptor (PRR) and microbe associated molecular patterns (MAMP) results in
the activation of signalling pathways essential for promoting the production of
AMPs, mucin glycoproteins, mucosal barrier function, and immunoglobulin A
(IgA). Due to its location in the base of the small intestinal crypts, the concentration
of AMPs is maximum in Paneth cells. A healthy and composite microbiota may
seem as a prerequisite for AMP production but Bacteroides thetaiotaomicron and
Lactobacillus innocua are the key individual species that promote production of
AMPs. Bacteroides thetaiotaomicron can also promote the cleavage of prodefensin
to active defensin. Apart from benefitting in the process of digestion, lactic acid
produced by Lactobacillus sp. can enhance the antimicrobial activity of the host
lysozyme. It has been proven beyond doubt that AMP expression is a two-way
interactive mechanism. Bacterial metabolites such as short-chain fatty acids (SCFA)
and lithocholic acid stimulate the expression of short cationic peptides that are part
8 P. G. Patel et al.

of the innate immune system by mechanisms of epigenetic alterations and mitogen-

activated protein kinase/extracellular signal-regulated kinases pathway.

2.2 Nutrients Metabolism

GM is involved in the metabolism of almost all the essential nutrients required by

our body, be it carbohydrates, proteins, lipids, vitamins, and/or polyphenols. Car-
bohydrates are the main source of energy for the microorganisms residing in the gut.
Bacteroides sp., Bifidobacterium sp., Enterobacteria sp., Faecalibacterium sp., and
Roseburia sp. can ferment indigestible carbohydrates to SCFA and provide ample
energy to the host (Samuel et al. 2008). SCFA such as butyrate can avert built-up of
D-lactate, a toxic metabolic by-product. Some of these bacterial species like
Bacteroides thetaiotaomicron encodes more hydrolases than the human genome.
Oxalate is a common by-product obtained from the fermentation of carbohydrates.
Accumulation of oxalate can lead to the formation of kidney stones. Oxalobacter
formigenes along with Bifidobacterium and Lactobacillus species can counter oxa-
late, preventing the formation of kidney stones. Amino acids can enter the gut
microbiota from the intestinal lumen via transporters present on the cell wall of
bacteria. Several enzymes, such as histamine decarboxylase and glutamate
decarboxylases present within the bacteria convert those amino acids into small
signalling molecules and/or bacteriocins. Apart from carbohydrates and protein, gut
microbiota also has a positive influence on lipid metabolism (Hooper et al. 2001).
Bacteroides thetaiotaomicron can upregulate the expression of a colipase required
for lipid digestion. Moreover, some microbes can counteract the inhibition of
lipoprotein lipase activity in adipocytes. Among other major nutrient metabolism
functions of gut microbiota are: (a) synthesis of vitamins, conjugated linoleic acid,
secondary bile acids; (b) increasing the concentrations of pyruvic acid, citric acid,
fumaric acid, and malic acid in serum; (c) breakdown of polyphenols consumed in
the diet (Valdes et al. 2018).

2.3 Immunomodulation

GM can modulate innate as well as adaptive immune systems. Dendritic cells in the
lamina propria, group 3 innate lymphoid cells, gut-associated lymphoid tissues
(GALT), IgA producing plasma B cells, resident macrophages, and T cells are
regulated by microbes inhabiting the human gut. For example, commensals and
pathogens can stimulate the tissue-resident dendritic cells to secrete several factors
required for the production and class switching of antibodies (Jandhyala et al. 2015).
In the past decade, research has provided a comprehensive picture of the crosstalk
between the gut microbiome and regulatory T cells (Zheng et al. 2020). These
regulatory T cells in turn amplify cytotoxic CD8+ T cells, high-affinity antibody
Impact of Dietary Habits, Ethnicity, and Geographical Provenance. . . 9

responses, and memory B cells. An axis of Follicular helper T cells and microbiota
may play significant roles in autoimmune diseases. Crosstalk between the immune
system and gut microbes is a bidirectional communication process. Disturbance in
the microbiome by environmental factors and genetic susceptibility can lead to
immune dysregulation, which in turn can cause more microbiome disturbance.
This can lead to serious diseases like inflammatory bowel disorders. Still, more
mechanistic studies are required to explore the roles of the commensal microbiome
in impacting immunity in health and disease.

2.4 Metabolism of Various Drugs and Xenobiotics

Growing scientific evidences have elucidated the role of the GM in xenobiotics and
drugs metabolism (Valdes et al. 2018; Jandhyala et al. 2015; Zheng et al. 2020). GM
could play a vital role in treatment of several diseases in future. Cardiac glycosides
like digoxin are known to upregulate a cytochrome containing operon in organisms
belonging to Actinobacteria phyla resulting in the inactivation of digoxin. Further-
more, p-Cresol, a metabolite derived from gut microbes, can decrease liver’s metab-
olism against acetaminophen by competitive inhibition of hepatic sulfotransferases.
Microbes residing in our gut can also metabolize anticancer drugs; an interesting
example is the deconjugation of the anticancer drug irinotecan catalysed by the
enzyme ß-glucuronidase.

2.5 Faecal Microbiota Transplantation

Faecal microbiota transplantation (FMT) therapy is emerging as one of the most

intriguing applications of GM studies. Here, faecal microbiota present in stool from a
healthy individual is transplanted into a patient suffering from a gut microbiota-
related disease. The aim is to reconstitute or restore the gut microbiota balance in the
patient to overcome GM dysbiosis (Cammarota et al. 2014). GM of patients who
underwent antibiotic treatment before stem cell transplantation has been successfully
reconstituted through the means of FMT therapy (Taur et al. 2018). Obesity and gut
microbiome dysbiosis have been known to be interlinked but now with the help of
FMT therapy, this connection is being thoroughly investigated. By transplanting
faecal microbiota from a healthy person into an obese person or vice-a-versa, the
immediate effect of gut microbiota can be studied (Kang and Cai 2017). Since
gaining traction within last decade, FMT has been accepted for treatment of Clos-
tridium difficile infection by FDA. Moreover, several stool banks including
OpenBiome have been established for providing faecal microbiota (Smith
et al. 2014).
10 P. G. Patel et al.

3 Dietary Habits and Their Influence on the Gut

Microbiome

In addition to heredity and anthropometry, environmental factors like diet and drugs
also play a major role in determining the composition of GM in an individual
(Goodrich et al. 2014; Rothschild et al. 2018). Among all these factors, the influence
of diet is particularly important, because nourishing the GM with probiotics or
dietary fibre can provide far-reaching health benefits. Almost all the food ingredients
that we consume affect the microbes living in and around the stomach. Rampant use
of pesticides for agriculture is harming the microbiota diversity. The medical
community is considering microbiota as a key component of nutrition as it can be
used to personalize diet plans. To maintain beneficial microbes in the gut, greater
importance has to be given to our dietary habits.
Many diabetic patients and even calorie conscious individuals switch to artificial
and high-intensity sweeteners as alternatives to white sugar. Although food regula-
tory agencies consider them safe, studies on animal models have revealed that they
can cause GM dysbiosis (Nettleton et al. 2016). Sugar substitutes induce metabolic
disturbances and impart glucose intolerance that leads to change in GM population.
Drastic alteration in GM population may cause gut dysbiosis and other health
problems as evident by previously reported studies (Li et al. 2022; Ruiz-Ojeda
et al. 2019). A widely used sugar substitute when fed to rats changed the proportion
of total aerobic bacteria in their guts (Abou-Donia et al. 2008). It also caused a spike
in the amount of cytochrome p-450 and p-glycoprotein in the intestine. Similar
results have been obtained in experiments with mice also, where it was found to
cause liver inflammation (Bian et al. 2017). Even the addition of emulsifiers to the
diet can potentially decrease the diversity of microbial flora. Especially affected are
species belonging to the phylum Verrucomicrobia and order Bacteroidales
(Chassaing et al. 2015). Food additives also cause an increase in the population of
pro-inflammatory gram-negative bacteria belonging to the phylum
Pseudomonadota. This can lead to serious health disorders such as colitis and
metabolic syndrome. Numerous people, especially millennials, are switching to
restrictive diet plans trying to lose weight or for some other reasons. Some of
these diet plans like the low FODMAP (fermentable oligosaccharides, disaccharides,
monosaccharides, and polyols) diet increase the beneficial Actinobacteria richness
and diversity and are used to treat irritable bowel syndrome (Chumpitazi 2020). If
we take the example of people on strict vegan diets and compare it to normal
omnivores, little difference was observed in composition and diversity composition
of microflora, but significant differences in metabolomic profile (Wu et al. 2016).
The gut microbiota helps to ferment many non-digestible substrates like dietary
fibres and endogenous intestinal mucus, in turn providing the main energy source for
human colonocytes (De Vadder et al. 2014). Moreover, the metabolites generated
from this fermentation process can have beneficial effects through neural circuits.
The incorporation of gluten-free bread helps people with coeliac disease and/or
gluten sensitivity (Bevilacqua et al. 2016). The addition of cheese to the diet in
Impact of Dietary Habits, Ethnicity, and Geographical Provenance. . . 11

human observational and interventional studies has been shown to increase the
beneficial Bifidobacteria and decrease Bacteroides and Clostridia associated with
intestinal infections (Montel et al. 2014; Zheng et al. 2015). It also enhances the
production of short-chain fatty acids (SCFA) and reduces the amount of
trimethylamine N-oxide.
Consumption of live microorganisms can influence the abundance of different
types of bacteria in the gut. Mixtures of such live beneficial microbes (bacteria,
yeast, etc.) are referred to as probiotics. It is now a well-established fact that
probiotics have several health benefits. They are often taken as food supplements
all around the globe. The most commonly used microbes as probiotics belong to the
genus Lactobacillus, usually present in the yoghurt. Probiotics also help in the
process of wound healing, improving immune system and decrease in obesity
(Hori et al. 2020; Fijan et al. 2019). Table 1 describes the probiotic products
commercially available worldwide for direct consumption. Prebiotics, however, is

Table 1 Probiotic products available in global market

Probiotic
brand/market Manufacturing Type of
name company probiotic Country Bacterial strain
Yakult Yakult Yoghurt- Japan Lactobacillus casei Shirota
based drink (available
worldwide)
Coco biotic Body ecology Ferment USA Lactobacillus acidophilus,
coconut L. delbrueckii
water drink
GoodBelly NextFoods Fermented USA Lactobacillus plantarum
probiotics fruit juice 299V
Actimel Danone Yoghurt- Worldwide Lactobacillus casei
based drink DN-114001
Tropicana PepsiCo Fermented USA Bifidobacterium lactis
essential fruit juice
probiotics
Toyo K95 foods Fermented India Acetic acid bacteria and yeast
Kombucha black tea consortium
Vitafytea Eko-bio Freeze-dried Netherland Lactobacillus rhamnosus,
Bififlor formula L. acidophilus, and
Bifidobacterium bifidum
Organic gut Farmhouse Fermented USA Lactobacillus (from sauer-
shot CULTURE vegetable kraut and vinegar brine)
drink
PERKii spar- PERKii Soda with Australia Bifidobacterium
kling probi- added bacte-
otic drink rial strain
Lactovit Velbiom Freeze-dried India Bacillus coagulans,
formula Lactobacillus
Vitality Müller dairy Yoghurt- UK Lactobacillus acidophillus,
based drink Bifidobacterium sp.
12 P. G. Patel et al.

defined as substrates for beneficial host microorganisms. Prebiotics include all the
fermentable carbohydrates and dietary fibre used by the GM. So, it is quite obvious
that prebiotics can modulate the population of host microbiota. Observational studies
and experimental models have provided evidence for the therapeutic benefits of
prebiotics in fighting allergies. If we combine prebiotics with probiotics in a specific
formulation, we will get a product termed a synbiotics. Synbiotics provide the
advantages of both probiotics and prebiotics. Apart from adding beneficial organ-
isms to food or feed, it will also stimulate the proliferation of native bacterial strains
already present in the gastrointestinal tract. One of the most popular synbiotics in use
employs a combination of Bifidobacterium or Lactobacillus genus bacteria with
fructooligosaccharides (Markowiak and Ślizewska 2017). The field of probiotics,
prebiotics, and synbiotics has witnessed exponential research growth in the past few
decades. Apart from these, different ethnic groups across the globe have their own
indigenous fermented foods and beverages whose consumption provides live micro-
organisms (Table 2). These food items have several health benefits and some of them
such as Yakult, kimchi, kombucha, etc. have gained popularity in recent years.
Consumption of tempeh, a traditional Indonesian food item made from fermented
soybean has been reported to improve cognitive functions in older people. In a recent
study by Handajani et al. (2020) 90 subjects aged above 60 years with mild cognitive
disorder consumed tempeh for 6 months. At the end of experiment, cognitive
improvement was demonstrated by the subjects. All these studies reinforce a greater
impact of diet in influencing the long-term metabolome derived from the bacterial
community than just the short-term bacterial community. An important point to
consider is that the effect of dietary habits on the microbiome composition may not
be a one-way process, as the microbes in the gut can also influence one’s food
choices and appetite.

4 Role of Ethnicity in Shaping Gut Microbiome

While ethnicity has been studied as one of the factors that shapes human gut
microbiome, it has been often viewed under biological and genetical lenses. Several
times ethnicity is used interchangeably with race when used in context of scientific
studies. However, ethnicity is more than shared genetic factors within a population
(Fortenberry 2013). Ethnic groups refer to the population or group that has similar
culture orientation, history, biology, religious beliefs, and lifestyle patterns.
Thus, study of microbiome profiles required combination of extensive and
intensive differences among various ethnicities (Irvine et al. 2002; Findley et al.
2016). A large percentage of microbiome studies have been focused on GM of
people living in Western countries. Therefore, inclusion of diverse ethnic groups in
microbiome studies could help in elucidating role of GM in health disparity among
various ethnicities (Findley et al. 2016). Studies on infant GM have revealed that
ethnicity is a vital element in early GM development. One longitudinal study by Xu
et al. has included infants from three different ethnic groups, Chinese, Malay, and
Impact of Dietary Habits, Ethnicity, and Geographical Provenance. . . 13

Table 2 Regional fermented foods and beverages with probiotic potential

Fermented Type of
food/ food/ Place of
beverage beverage origin Microflora Health benefit References
Kefir Fermented Caucasus Lactobacillus Antimicrobial Dertli and
milk kefiranofaciens, activity, improves Çon
beverage Enterobacter, lactose digestion, (2017)
Acinetobacter, and helps with
Enterococcus sp. constipation
Tempeh Fermented Indonesia Rhizopus Enhance immune Yulandi
dehulled oligosporus, Lacto- system, improve et al.
and boiled bacillus, Enterococ- cognitive functions (2020)
soybean cus, Streptococcus,
and Weissella
Sauerkraut Fermented Central Leuconostoc, Lacto- Anti-inflamma- Zabat et al.
cabbage and East bacillus, tory, anti-oxidant, (2018)
Europe Lactococcus, and counteracts effects
Enterobacteriaceae of carcinogens
Kimchi Fermented Korea Weissella, Anti-mutagenic Hong et al.
vegetables Pediococcus, and activity, anti- (2016)
Leuconostoc oxidant, anticancer
Natto Fermented Japan Bacillus subtilis Anti-fibrinolytic, Nagai
soybean natto reduces hyperten- (2015)
sion, improves
digestion
Miso Fermented Japan Aspergillus oryzae, Decreased gastro- Allwood
soybean Enterococcus, intestinal reflux, et al.
paste Staphylococcus, lower risk of mor- (2021)
Tetragenococcus, tality, anti-
and Leuconostoc diabetic, anti-
inflammatory
Dahi Fermented South Lactococcus, Helps with intesti- Joishy
milk Asia Leuconostoc, and nal disorders, et al.
yoghurt Lactobacillus improved diges- (2019)
tion, anti-bacterial
effect in gut
Kombucha Fermented China Gluconacetobacter, Anti-diabetic, anti- Marsh
black/ Lactobacillus, carcinogenic, et al.
green tea Lactococcus, and improves gastric (2014)
Zygosaccharomyces ulcers, immune
response
Tuaither Fermented India Lactobacillus, Anti-oxidant, Deka et al.
bamboo Corynebacterium cardioprotective, (2021)
shoots sp., anti-ageing, and
Sphingobacterium weight loss
sp., and Pseudomo-
nas sp.
Boza Fermented Turkey, Lactobacillus, Can- Antimicrobial Botes et al.
cereal- Balkans dida, and Pischia activity, enhance (2007)
based sp. digestion
beverage
14 P. G. Patel et al.

Indian, within same geographical location. Ethnicity was an influential factor at

3 months, before introduction of solid foods and remained so up till 1 year in
Chinese and Indian infants. Microbiota of Chinese infant had high percentage of
Bacteroides and Akkermansia; however, Indian infants had predominantly
Bifidobacterium and Lactobacillus composition (Xu et al. 2020). Another similar
study has compared gut microbiome of South Asian and Caucasian infants born in
Canada. Results suggest that from birth to 12 months of age, both ethnicity and
breast-feeding habits are influential factors in initial establishment and composition
of gut microbiome. South Asian infants had higher abundance of lactic acid bacteria,
while Caucasian infants had higher Clostridiales genera. These differences in gut
microbiome profile have been attributed to variation in maternal and infant diet
among the two ethnic groups (Stearns et al. 2017).
GM composition of several ethnic groups residing in USA were analysed by
Brooks et al. in order to understand the correlation between GM variation and
chronic illnesses that generally plagues ethnical minorities. After studying GM of
1267 individuals from four different ethnicities, 12 taxa were found to reproducibly
vary among the four groups. Christensenellaceae was the most reproducibly herita-
ble taxon along with majority of microbial taxa which indicates a possibility of
genetic inheritance within specific ethnic groups (Brooks et al. 2018). In another
study, geographical variation was ruled out by selecting 25,000 individuals living in
Amsterdam, Netherlands. Ethnicity emerged as the major contributor to gut
microbiome composition and individuals from same ethnic group had similar gut
microbiome. Three main poles demarcated by operational taxonomic unit (OTU)
were identified among different ethnic groups, Clostridiales (Dutch), Prevotella
(Turks, Ghanaians, Moroccans), and Bacteroides (South Asian Surinamese, African
Surinamese) (Deschasaux et al. 2018). Dwiyanto studied the GM profiles in a multi-
ethnic middle-income Malaysian district, Segamat. Members of four different eth-
nicities Malay, Chinese, Indian and, Jakun were included in the study, and ethnicity
displayed major influence over gut microbiome diversity. The differences in lifestyle
patterns and dietary choices were considered as the cause of the dissimilarity within
gut microbiome (Dwiyanto et al. 2021). GM composition of young children has also
been examined in some studies to understand early microbiome development. One
such study by Liu et al. focused on ethnic groups living in Qinghai-Tibet plateau.
141 school children (age 8–12 years) belonging to Tibetan, Han, and Hui population
were tested for their gut microbiome similarities and dissimilarities. Through 16S
sequencing it was proved that Firmicutes (47.61%) and Bacteroides (38.05%) were
the predominant phyla among all three ethnicities. However, the Tibetan population
had highest alpha diversity, with comparatively high percentage of Oscillibacter and
Barnesiella. Since the environment and dietary habits were similar between the
Tibetan, Han, and Hui population, ethnicity was responsible for alpha diversity
(Liu et al. 2020). Khine et al. determined the significance of ethnicity on gut
microbiome when geographical variation is included. Pre-adolescent children from
two ethnic groups (Chinese and Malay) geographically separated across three
different cities (Guangzhou, Penang, and Kelantan) took part in this study. Here,
Impact of Dietary Habits, Ethnicity, and Geographical Provenance. . . 15

differences in GM among the two ethnicities within the same city were due to
variation in diet and food habits (Khine et al. 2019).
Ethnic groups that are isolated from other populations with distinct cultural, food,
and lifestyle patterns have been reported to have a unique GM. These ethnic groups
are often geographically isolated or have limited interaction with other populations.
Nomadic Fulani group living in Nigeria have a pastoral lifestyle that is a combina-
tion of Paleolithic and Neolithic style. On comparing the GM profile of Fulani group
with their urban contemporaries, the Fulani had lower microbial diversity. Notably,
their GM comprised several microbial phyla that are associated with hunter-gatherer
or foraging communities such as Prevotellaceae, Bacteroides, and Spirochaetes.
Fulani had higher percentage of pathogenic microbes within their GM as a result of
their nomadic diet and lifestyle choices (Afolayan et al. 2019). Inuit population
residing in Canada has traditional dietary habits that have been formed over centuries
and responsible for a distinct and dynamic gut microbiome. These dietary choices
have formed as a result of geography and seasonal availability. GM of Inuit
population differs from those of urbanized European population with Western diet
patterns (Dubois et al. 2017). Inuit group had higher abundance of Akkermansia
phyla which is associated with diet as well as geography, observed within Arctic
region (Girard et al. 2017). Assimilation and lifestyle change often lead to variation
within GM of isolated ethnic groups. This pattern was observed when Nicobarese-
tribal community living in remote areas relocated to rural and urban areas. As dietary
and lifestyle patterns were altered the GM composition changed as well. Group from
remote location had higher GM diversity compared to their rural and urban groups.
While the remote group had predominantly Prevotella genus owing to the
carbohydrate-rich diet, the urban group is comprised mainly of Bifidobacterium
(Anwesh et al. 2016). However, in a contradictory study, GM composition of
previously nomadic Irish Traveller community was similar to that of
non-industrialized rural gut microbiome. Since the diet of the community is similar
to their urban contemporaries, non-dietary factors such as living conditions and
horizontal microbiome transfer (Keohane et al. 2020) were assumed to be responsi-
ble for these variations.
Occurrence of gastrointestinal diseases or other similar disorders has been linked
to GM composition. Moreover, certain diseases are prevalent in certain ethnic
groups and through means of GM profiling, this correlation can be elucidated.
Furthermore, this could help in development of personalized treatment for some of
the diseases. Prideaux et al. studied GM of individuals with gastrointestinal diseases
and their results suggest that ethnicity plays a role in altering the GM composition.
Chinese patients of ulcerative colitis had reduced microbiome diversity and inflam-
matory bowel disease (IBD) was more likely to occur in case of Chinese individuals.
Chinese IBD patients tended to have Western style compared to more traditional
Chinese diet of healthier individuals (Prideaux et al. 2013). Mar et al. revealed
though their study on ulcerative colitis patients that distinct microbiome cohort is
present in different ethnic groups that may be responsible for disease severity (Mar
et al. 2016). Association between type-2 diabetes and GM composition has also been
studied in various ethnic groups. Trans-ethnic study between type-2 diabetes patients
16 P. G. Patel et al.

from Sweden and India showed distinct GM composition in the two groups
(Alvarez-Silva et al. 2021). Though, when ethnic groups from same geographical
region were tested for alteration in GM no significant changes were observed. But
when metformin treated patients were analysed, alpha diversity was lower in South
Asian Surinamese subjects compared to African Surinamese and had unique gut
microbiome biomarkers (Balvers et al. 2021).
Chronic kidney disease (CKD), a renal impairment disease, is one of the ailments
that has been associated with dysbiosis of the GM. Changes in GM composition are
responsible for production of uremic toxins (indoles, creatinines, hippuric acid, etc.)
that accumulates over time and induce CKD-related complications (Nallu et al.
2017). Although several attempts have been made to diagnose CKD using GM as
biomarkers, a consensus has yet to be reached. Multiple studies have revealed that
occurrence of CKD may differ based on ethnicity, with South Asians and Africans
more likely to contract CKD than Caucasians (Dreyer et al. 2009; Mathur et al. 2018;
Liyanage et al. 2022). As observed in other metabolic or cardiovascular diseases,
GM composition of CKD patients may differ based on their ethnicity, which may
further hinder identification of specific biomarkers. GM analysis of Han Chinese
population with CKD suggested significant reduction in gut bacteria, with
Bacteroides being the most dominant phyla. Additionally, creatinine and cystatin
C were determined as uremic compounds that altered gut microbiota (Jiang et al.
2017). Role of probiotics in CKD progression has also been evaluated in a meta-
analysis of published reports. Ten trials carried out in eight different countries
revealed that while administration of probiotics reduced urea levels, it did not have
significant impact on uremic acid, C-reactive proteins, and creatinine (Tao et al.
2019).

5 Does Geographical Variation Impact GM?

Every geographical region has different agricultural products, food products, and
various region-specific cultural goods that impact the overall GM development and
diversity (Senghor et al. 2018). The composition of GM is dependent on the
demographics, diets, and even the geographical location (Dwiyanto et al. 2021). In
recent years, studies on GM have become global as shown in Fig. 2 and the
international GM composition database is continuously expanding. The widely
accepted Burgmann’s rule has proved to be applicable on humans as well. As per
the law, in geographical area with high latitude, population has large body mass,
whereas in lower latitude area low body mass is observed. Firmicutes and
Bacteroides phyla are responsible for fat extraction and energy storage and thereby
influence the body. In their study, Suzuki and Worobey found link between these
two microbial phyla composition and geography (Suzuki and Worobey 2014), with
increased proportion of these phyla at high latitude. Physiological factors such as
high altitude can also alter the gut microflora. In high altitude regions, low thermal
energy and reduced oxygen concentration lead to adaptation in blood circulation.
Impact of Dietary Habits, Ethnicity, and Geographical Provenance. . . 17

Stearns et al., 2018

Liu et al., 2016
Lan et al., 2017
Alvarez-Silva et al.,
2021
Xu et al., 2020
Lin et al., 2013
Fontana et al., 2019
Yatsuneko et al.,
2012
Afolayan et al., 2019
Deschasaux et al.,
2018
Kabwe et al., 2020
Shin et al., 2019

Fig. 2 Internationalization of GM studies (edited from Ecklu-Mensah et al. 2022)

Mazel performed experiments to analysis how high altitude affect mouse GM. Their
study demonstrated high prevalence of Prevotella phyla at high altitude environ-
ment. Prevotella are organisms that generate short-chain fatty acids (SCFA), through
saccharolysis of carbohydrates from host diet. High concentration of SCFA
increases nutrition uptake in high altitude region that could improve blood circula-
tion (Mazel 2019). Another similar study exhibited that Bacteroides to Prevotella
ratio increases after exposure to high altitude induced stress. Since the subjects were
initially tested at lower altitude and the diet remained constant throughout the
duration of the study, alterations in GM are due to physiological stress (Karl et al.
2018). Aside from altitude, exposure to cold has also been documented to cause
alteration in GM. Studies on mice models or native model found in colder regions
have indicated that GM plays a key factor in mediating homeostasis during
prolonged cold exposure. Transplantation of acclimatized cold microbiota from a
mice to regular, healthy mice resulted in acquisition of cold-tolerance and other
features (Chevalier et al. 2015). Prolonged cold exposure also reduces potentially
pathogenic as well as beneficial microflora in mice models (Wang et al. 2022).
There are various such studies show that geographical variations also impact the
GM. Lin et al. have conducted GM study on subjects from two geographically
separated countries: USA and Bangladesh. On comparing young children from
Bangladesh and USA, children from Bangladesh had higher GM diversity. The
study indicates that these dissimilarities in GM are due to combination of factors
including environment, dietary choices, lifestyle, genetic factor, and socioeconomic
status (Lin et al. 2013). To understand the diversity within GM across different
continents, Mobeen et al. have studied GM of individuals from 15 different coun-
tries. Firmicutes and Bacteroidetes turned out to be the major phyla in the GM
globally. At the inter-continental level Bacteroides, Bifidobacterium and Prevotella
were reported, while at intra-continental level higher variations were observed. In
case of America—two phyla Bacteroides and Ruminococcaceae, for Europe—four
18 P. G. Patel et al.

phyla Prevotella, Faecalibacterium, Clostrdiales and Bacteroides, and finally for

Asia—three phyla Bacteroides, Bifidobacterium, Prevotella were identified. These
variations within the GM diversity are attributed to environment and climate and
geographical variations (Mobeen et al. 2018). A short pilot study carried out by
Afolayan et al. demonstrated that geographical relocation for a brief time duration
can also alter gut microbiome. This study examined the gut microbiome of subject
after 2 months stay in Italy followed by another 2 months stay in Nigeria. The diet
choices were constant throughout the duration of 4 months. Within 2 months of stay
in Italy, gut microbiome diversity and Firmicutes percentage reduced, which was
quickly restored to the original Prevotella and Treponema composition on return to
Nigeria (Afolayan et al. 2021). Therefore, geography variation has a significant
influence on the gut microbiome even if the separation lasts for short period.
In modern world immigration has become the new normal and people frequently
relocate for various purposes like study, business, and medical treatment. Migration
to another country leads to cultural assimilation that includes new foodstuffs, change
in weather condition, and altered lifestyle. All of these adaptations induce significant
changes in the overall composition of the GM. In USA many immigrants have been
reported to become obese, have increased heart-conditions, chronic diseases, and
gastrointestinal diseases. Rapid lifestyle changes for better cultural assimilation have
been regarded as the major cause of these problems (Sonnenburg and Sonnenburg
2018). Thus, it is important to analyse the microbial diversity of gut as well as
immediate and long-term effect of migration on GM. Most of the studies involving
migration have been focused on USA as they have the largest number of immigrants.
As of 2015, about 21% of their total population is comprised of immigrants (United
Nations 2017). Vangay et al. performed several experiments to determine the effect
of migration on GM of South-East Asian women. They analysed gut microbiomes of
514 healthy females of two different ethnicity originally from Thailand (Hmong
Thai and Karen Thai). European American females were used as control to compare
the changes within GM. Initially the immigrants and control population had different
microflora in gut before migration. However, 6 months after migration, reanalysis of
the GM showed drastic changes in the gut microflora. Originally, they contained
high number of Prevotella phyla which have plant polysaccharide-degrading capac-
ity but after moving to USA Prevotella were displaced by Bacteroides. Second-
generation immigrants lacked Prevotella in their gut and were completely replaced
by Bacteroides (Vangay et al. 2018). Recently, Copeland et al. carried out similar
research on effect of migration on GM of South Asian immigrants in Canada. Here,
first generation immigrant and children born to immigrant parents in Canada
underwent gut microbiome profiling. The analysis demonstrated that South Asians
who immigrated in early life had similar gut microflora as that of second-generation
children born in Canada. However, the subjects that had migrated recently had
different GM composition from that of second-generation South Asians and Cana-
dians. South Asians GM is predominated by Prevotella copri, whereas second-
generation children have gut microflora dominated by Bacteroides spp. or Clostridia
spp. and Dialister invisus. But with the time Bacteroidia spp. replaced the original
Prevotella copri as the effect of immigration becomes more prominent (Copeland
Impact of Dietary Habits, Ethnicity, and Geographical Provenance. . . 19

et al. 2021). Both of these studies prove that geographical relocation due to migra-
tion can completely reshape the fundamental GM composition.
While several studies have revealed geographical provenance to be a major factor
in determining gut microbial diversity, there have been certain contradictory studies
that suggest otherwise. When GM within similar geographical area but with different
ethnicities and socioeconomic status was examined, variation in the gut microbial
profiles was reported. Amaruddin et al. and Chong et al. studied GM of individuals
from Makassar, Indonesia and Perak, Malaysia, respectively. In both cases dissim-
ilarities in GM composition were attributed to either ethnicity or socioeconomic
differences among the subjects (Amaruddin et al. 2020; Chong et al. 2015). Simi-
larly, Quin and Gibson suggest that in case of infants within same geographical
region, human behaviour, i.e., maternal diet, mode of delivery, and infant feeding
habits are responsible for differences in GM (Quin and Gibson 2020).
From the studies mentioned, it is evident that rather than a single determinant, a
combination of factors shapes the overall composition of the human GM. Therefore,
for GM studies instead of concentrating on a single factor, it is more advantageous to
focus on the interconnected relationship between various factors and their combined
impact on gut microbiome. Table 3 here summarizes the most prominent GM phyla
in different populations along with their ethnicity, dietary habits, and geographical
location.

6 Conclusion

The composition and diversity of GM is a complex matter that is interlinked with a

variety of factors. One cannot predict the composition of a particular GM based on a
single factor, rather one must look at how these factors are associated with each
other.
In case of dietary habits, ethnicity, and geographical provenance, change in one
factor leads to change in other factors. Migration, i.e., geographical variation led to
alterations in dietary patterns that ultimately transformed GM. Similarly, individuals
belonging to different ethnic groups had dissimilarities in their GM profile despite
being from same geographical location. Type of diet followed by individuals of
same ethnicity within same location also plays a key role in the overall GM
composition. Two people having drastically different dietary habits will have vari-
ation in their GM profile. Therefore, when studying GM these factors should not be
segregated into distinct categories but instead viewed as an interwoven cycle. Any
interaction within this cycle alters overall balance and how it influences the
GM. Previously, majority of GM-based studies originated from Western countries.
However, within last few years the GM research has become truly global. Nowadays
many studies being published that includes diverse ethnic groups and population
from unexplored regions. Technological advances made within recent years have
helped with the continuous expansion of global GM database. In future it may be
possible to diagnose a specific disease based on dysbiosis within GM. Future studies
 20

Table 3 GM composition of various populations across the globe

Geographical
location Ethnicity Dietary habits GM composition References
Seoul, South Korean 1. Traditional Korean 1. Coprococcus, Blautia, and Weissella Shin et al. (2019)
Korea 2. Western-American 2. Bifidobacterium, Faecalibacterium, Lactobacillus, and
Lachnospira
Papua New Pacific Non-processed plant-based Prevotella (Coriobacteriaceae, Slackia, and Propionibacterium) Martínez et al.
Guinea Islander traditional diet Firmicutes (Streptococcus, Staphylococcus, Eubacterium) (2015)
Tanzania Hazda Seasonal hunting/gathering Prevotellaceae, Succinivibrionaceae, Paraprevotellaceae, and Fragiadakis et al.
tribe based diet Spirochaetaceae (2019)
India Indian 1. Plant-based diet 1. Prevotella Dhakan et al. (2019)
1. North-Central 2. Omnivorous diet 2. Bacteroides, Ruminococcus, and Faecalibacterium
2. South India
Oklahoma, USA Native- Processed carbohydrate and Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria Sankaranarayanan
American protein-based diet et al. (2015)
1. Chey-
enne
2. Arapaho
1. Florence, Italy 1. Italian 1. Typical Western diet 1. Firmicutes and Proteobacteria de Filippo et al.
2. Rural Burkina 2. Mossi 2. Traditional plant-based rural 2. Prevotella, Xylanibacter (Bacteroidetes), and Treponema (2017)
Faso tribe diet (Spirochaetes)
Thailand Thai 1. Mixed diet (tradi- 1. Bifidobacterium spp., Enterobacteriaceae, and Methanogens La-ongkham et al.
1. Central (Bang- tional + processed foods) 2. Lactobacilli, Clostridium coccoides, Eubacterium rectale, (2015)
kok) 2. Traditional Thai diet Clostridium leptum, Prevotella, and Bacteroides fragilis
2. Northeast
(Khon Khaen)
P. G. Patel et al.
Impact of Dietary Habits, Ethnicity, and Geographical Provenance. . . 21

may also focus on developing biomarker for various metabolic or digestive disor-
ders, personalized probiotic treatment, and optimizing diet for a well-balanced GM.

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Methods Used for Studying Human
Microbiome

Chinmayi Joshi and Vijay Kothari

Abstract Human microbiome is a complex and dynamic component of the human

system. Identifying the members of this vast community and elucidating their
functions remains a daunting challenge. Collecting samples from internal body
sites under aseptic conditions is the first challenge, and then equally difficult is to
identify the microorganisms present in these samples, particularly when most of
them are not amenable to conventional laboratory culture. Obligate anaerobes
(e.g. many residents of the large intestine) have always been difficult to grow
routinely in an average microbiology lab. This chapter describes various sampling
methods employed for study of human microbiome and the culture-dependent as
well as culture-independent methods for elucidating the taxonomic and functional
identity of the microbial symbionts of the human system.

Keywords Human microbiome · Symbiont · Sample collection · Metagenomics ·

Metabolome

1 Introduction

‘Human microbiome’ a well-balanced and extremely environment-specific ecosys-

tem constitutes extraordinarily diverse microbial cells, which exist in correlative
associations and reside on or within tissues and biofluids along with the analogous
sites of the human body. Microorganisms such as bacteria, archaea, fungi, protists,
and viruses can reside in the human body, colonise humans, and form a composite
and distinct ecosystem that acclimatises to the specific environment of each niche. A
symbiotic relationship between the human body and its naturally occurring

C. Joshi
Smt. S.S. Patel Nootan Science and Commerce College, Sankalchand Patel University,
Visnagar, Gujarat, India
V. Kothari (✉)
Institute of Science, Nirma University, Ahmedabad, Gujarat, India
e-mail: [email protected]

© The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2023 29
V. Kothari et al. (eds.), Probiotics, Prebiotics, Synbiotics, and Postbiotics,
https://doi.org/10.1007/978-981-99-1463-0_2
30 C. Joshi and V. Kothari

microbiota begins at childbirth, which plays a crucial role in maintaining overall

health. As a result of their biological activities, these organisms are identified as part
of the body and result in a variety of alterations from conception to death. In reaction
to host factors, the human microbiome is continually changing. Host factors such as
age, lifestyle, nutrition, hormonal fluctuations, inherited genes, underlying disease,
etc. are key determining factors of the human microbiome. A change in the human
microbiota’s composition, a dysbiosis, might cause health problems. Interruptions in
human microbiota are being linked to many diseases including inflammatory bowel
disease (IBD), diabetes, cancer, cardiovascular diseases, allergic diseases, and
antibiotic-resistant infections. Moreover, the human microbiome can also serve as
an early detection biomarker and target for curative interface for the treatment of life-
threatening diseases (Castillo et al. 2019; Berg et al. 2020; Ogunrinola et al. 2020).
Historically, members of a microbial community were identified by culture-
dependent methods, which depend on the growth of an organism in the laboratory.
Using this approach, researchers did not get the information about the uncultivable
members of the microbiota. To overcome this issue, DNA-based methods have been
developed in the 1980s. The field of genomics, metagenomics, metatranscriptomics,
and modern high-throughput sequencing technologies has contributed to a great
extent in identification and characterisation of microorganisms present in
microbiota. Additionally, sequencing becomes rapid, easy, and cost-effective, as a
result successive characterisation of the human microbiota to explore changes that
occur in the human microbiome over time would become possible. Though many
questions have been answered using these high-throughput techniques, many ques-
tions about human microbiome are yet to be addressed (https://www.ncbi.nlm.nih.
gov/books/NBK481559/; Iyer 2016).
The different approaches for assessing human microbiome, such as culture-
dependent or culture-independent methodologies (Fig. 1), have their own advan-
tages and limitations. Studies that include human microbiome are costly and difficult
to handle the experiments at different stages, i.e. sample collection, data generation,
and data analysis. In addition to sophisticated high-throughput methods, gnotobiotic
models and inter-disciplinary approaches are also available for assessing human
microbiome. In vitro microbial systems that comprise host cells in the microbial
culture are also obtainable and present well-regulated environments for mechanistic
and molecular profiling. However, continuous culture of many anaerobic organisms
poses difficulties, and in vitro systems are physiologically irrelevant (https://www.
ncbi.nlm.nih.gov/books/NBK481559/).
This chapter reviews the current approaches and methods used for studying the
human microbiome. After describing various sampling methods for accessing
microbiome of different human body sites, the chapter further covers the culture-
dependent in vitro–ex vivo systems, followed by an overview of the high-throughput
methods and data analysis approaches. The chapter concludes with a discussion of
strengths, weaknesses, and gaps in the technologies.
Methods Used for Studying Human Microbiome 31

Human
microbiome

Culture-
Culture-dependent
independent
methods
methods

Whole genome sequencing

Conventional approaches Molecular approaches and High-throughput
sequencing methods

Genetic fingerprinting techniques, Metagenomics

Isolation
DNA microarrays, Metaproteomics
Purification
Microautography, Flowcytometry Metatranscriptomics
Biochemical characterization
Real-time PCR Proteogenomics

Fig. 1 Methods for assessing human microbiome

2 Methods Used for Studying Human Microbiome

2.1 Sample Collection

The human microbiome is the collection of all microbiota that live on different body
parts including the skin, gastrointestinal tract, uterus, mammary glands, oral mucosa,
seminal fluid, lung, ovarian follicles, conjunctiva, saliva, and biliary tract (Table 1).
To study the human microbiome, the first step usually includes the collection of
stabilised microbial biomass specimens from specific sites. The following section
describes the collection methods for each major body site.

Sample Collection to Study the Skin Microbiome

Skin, a body shield, protects the body from the external harms and prevents the
evaporation of body fluids. Skin comprises the commensal bacteria such as
S. epidermidis, C. acnes, and Corynebacterium spp. that play an important role for
skin barrier, immunological reactions, and prevent colonisation of pathogenic bac-
teria. Kong et al. (2017) reviewed multiple methodologies for skin microbiome
sampling, i.e. swabs, surface scrapes, biopsies, cup scrubs, and tape strips. The
selection of the sampling method depends on the criteria such as biomass yield,
sampling depth, and human DNA contribution. Amongst the sampling methods for
32 C. Joshi and V. Kothari

Table 1 Bacteria present on different body parts and common sample collection methods used for
each site
Body part Common microbial residents Sampling method References
Skin Acinetobacter johnsonii, Swabbing and tape stripping Ogai et al.
Corynebacterium spp., methods (2018)
Cutibacterium acnes, Pseudo-
monas aeruginosa, Staphylo-
coccus aureus, S. epidermidis,
S. warneri, Streptococcus
mitis, S. pyogenes
Gastrointestinal Bacteroides, Bifidobacterium, Faeces, biopsy, luminal brush, Tang et al.
tract Clostridium, Eubacterium, laser capture microdissection, (2020)
Fusobacterium, Lactobacillus, catheter aspiration, surgery
Peptococcus,
Peptostreptococcus,
Ruminococcus, Staphylococ-
cus, Streptococcus
Uterus Escherichia spp., Cytobrush, Swab Mitra et al.
Fusobacterium nucleatum, (2017)
Prevotella tannerae,
Bacteroides spp., Streptomy
ces avermitilis, Mycoplasma
spp., Neisseria lactamica,
Neisseria polysaccharea,
Ureaplasma parvum
Mammary Staphylococcus, Streptococ- Milk sampling Taponen
glands cus, Corynebacterium, et al.
Cutibacterium, Lactobacillus, (2019)
Lactococcus, and
Bifidobacterium
Oral mucosa Actinomycetes, Bacillus, Mouth-rinsed water, filter Jo et al.
Firmicutes, Proteobacteria paper sampling (2019)
Seminal fluid Lactobacillus spp. and Semen sample Baud et al.
Prevotella spp. (2019)
Conjunctiva Acinetobacter, Swabbing, corneal epithelial Katzka
Aquabacterium, biopsy et al.
Brevundimonas, Corynebacte- (2021)
rium, Methylobacterium,
Cutibacterium,
Bradyrhizobium, Pseudomo-
nas, Sphingomonas, Staphylo-
cocci, Streptococcus,
Streptophyta
Lung Acinetobacter, Lung tissue Carney
Fusobacterium, Megasphaera, et al.
Prevotella, Pseudomonas, (2020)
Sphingomonas, Staphylococ-
cus, Streptococcus, Veillonella
Ovarian Actinomyces, Lactobacilli Endocervical mucus, cervical Vitale
follicles spp., Cutibacterium swabs and serum, endometrial et al.
biopsy, follicular fluid (2021)
(continued)
Methods Used for Studying Human Microbiome 33

Table 1 (continued)
Body part Common microbial residents Sampling method References
samples and vaginal swab,
cotton swab
Saliva Campylobacter, Corynebacte- Mouthwash, swabbing, and Kaan et al.
rium, Cellulosimicrobium, spitted saliva (2022)
Haemophilus,
Porphyromonas,
Solobacterium, Streptococcus
Biliary tract Proteobacteria and Firmicutes Endoscopic retrograde Binda
cholangiopancreatography et al.
(ERCP), percutaneous biliary (2022)
drainage, and surgical
sampling

skin microbiome, premoistened swabbing is the most established sampling method,

while dry swabbing has not been extensively employed because of the low biomass
collection. Other methods such as tape stripping and scrapes provide a high amount
of biomass, but tape stripping is inappropriate for all body sites due to its dimensions
and sample attainment time, whereas scrapes could be helpful only for low abun-
dance microbes, i.e. fungi. Cup scrubbing and skin punch biopsies are also the
methods which can be useful for microbiome studies. However, these methods are
invasive and cannot be used at multiple sites in patients (Chng et al. 2016; Kong
et al. 2017). Any method can be used for the study of skin microbiomes depending
on the requirement, but it is important to maintain consistency for sample collection
throughout the study to reduce confounders and to maximise the ability to identify
differences in microbiomes. Additionally, location, sampling frequency, and use of
controls are also important aspects of sample collection.

Sample Collection to Study the Gut Microbiome

Gut microbiota comprises 1952 uncultured species (Almeida et al. 2019). In humans,
the composition of gut microbiota varies between the various regions of gastroin-
testinal (GI) tract. Gut microbiota play a major role in prevention of infections and
promotion of the mature immune system, nutritional assimilation and metabolism,
and stimulating anti-cancer functions. Disruption in gut microbiota is associated
with various diseases including Clostridium difficile infection, IBD, and irritable
bowel syndrome (IBS). We can say that gut microbiota is closely associated with
human health, and it is necessary to analyse the association between gut microbiota
and disease occurrence, development, and prognosis (Li et al. 2019; Tang et al.
2020).
In the previous years, gut microbiome analysis depended on the isolation of
anaerobic bacteria that are adequate in the intestine, which affected the precision
of the analysis due to the problems in cultivation of anaerobic organisms
34 C. Joshi and V. Kothari

(Lloyd-Price et al. 2019). In current times, the progress of next-generation sequenc-

ing (NGS) can precisely evaluate microbial components without culture, has fasci-
nated the research on the gut microbiome. Currently, mucosal biopsy, intestinal
aspiration, sample collection from faeces, etc. are the common methods which are
being used for sample collection to study the gut microbiome (Tang et al. 2020).
Most common method is the sampling from stool. It comprises higher microbial
load and minimal human genetic contamination (HMP Consortium 2012a, b) and
contains material that can be assayed by using various molecular techniques. This
technique can be useful, but preservation of samples, i.e. immediately freezing is
required to avoid the change in microbial characteristics due to the environmental
changes. Fixatives allow suitable collection and shipping of samples, but they may
prevent culture and might not be suitable with accomplishing some molecular assays
at afterwards.
Other device-based methods such as endoscopy, biopsy, and luminal brushes can
also be used to investigate the gut microbiota. Some defects are linked with these
methods including invasive procedures, use of laxatives for bowel preparation, and
contamination of sampling tools during the time of penetration in the endoscopic
channel. Amongst the device-based methods, mucosal biopsy covers a little surface
area that may consequence in sampling variation and inaccessibility of rare taxa and
may comprise a large amount of contaminated host DNA, which make difficult to
analyse the molecular data. Another device-based method is the use of luminal
brushing, which is commonly used for the collection of the infectious samples
from the lower respiratory tract. This method is appropriate for the analysis of
infection associated with lower respiratory tract because the brush specimens are
not easily contaminated by microorganisms present in the upper respiratory tract.
This method can reduce the bleeding and infection. However, bowel preparation
influence and invasion are the similar defects of this method as biopsy. In addition to
above sample collection methods, the samples can also be collected using laser
capture microdissection (LCM), ingestible sampling devices, capsule device, intes-
tine microbiome aspiration (IMBA). Samples can also be collected using surgery
and aspirated intestinal fluid. In spite of the availability of numerous sampling
methods, more accurate sampling methods are warranted with reduced invasiveness,
avoiding cross-contamination, sampling at fixed points while minimising distur-
bance to normal physiology of intestine (Song et al. 2016; Tang et al. 2020;
https://www.ncbi.nlm.nih.gov/books/NBK481559/).

Sample Collection Methods for Assessment of Microbiome at Various

Body Sites

To study the respiratory microbiome, samples have been collected from the nasal
passages, sinus cavities, oral cavity and pharyngeal region, and the tracheobronchial
tree. Swabs, aspirates, sputum, lavage, and brushings are the common methods that
are used in respiratory microbiome studies. The respiratory tract has a lower
microbial load, and it is crucial to exploit protocols comprising controlled elements
to lessen sample contamination by non-target tissue (Lauder et al. 2016). In addition
Methods Used for Studying Human Microbiome 35

to respiratory, skin, and gut microbiome, study of vaginal microbiome is also

important. Cytobrush and swab are the common methods, which are used for
sampling. Mitra et al. (2017) demonstrated the use of swab and cytobrush to study
the compare the vaginal microbiota results at all taxonomic levels and identified
unique taxa in cytobrush samples. LefSe analysis identified Proteobacteria,
Betaproteobacteria, Burkholderiales, Burkholderiaceae, and Comamonadaceae to
be overrepresented in the cytobrush-collected samples.
Further, the oral microbiome also has a high biomass and it is also associated with
illness. It is associated with the lung microbiome. The oral microbiome served as a
promising diagnostic tool for SARS-CoV-2 infection detection and a predictor of
disease severity. Several sample collection methods, i.e. raw saliva, oral wash, oral
swabs, and scrapings of dental plaque are being used to study oral microbiome. Yano
et al. (2020) compared a commercially available OMNIgene ORAL kit to three
alternative collection methods including Saccomanno’s fixative, Scope mouthwash,
and non-ethanol mouthwash. The results showed clear differences in oral microbial
communities between the used oral collection methods. In conclusion, they showed
the impact of the sample collection method on oral microbiome analysis. The authors
suggested that one consistent oral collection method should be followed for all oral
microbiome comparisons.
These findings describe that sampling protocols for human microbiome analysis
are sensitive to technical methods. Existing microbial communities, their natural
features, or environmental conditions can change measured microbial communities.
Sample collection and processing can affect the microbiome assays. All these factors
should be kept in mind at the time of study design and data analysis (Sinha et al.
2017). Few considerations are necessary for sample collection. First, a sampling
device or sampling strategy is determined by factors like clinical facility, staff for
sampling, and scientific question behind sampling. After sampling, storage condi-
tions are vital for preserving the samples until further processing. Storage tempera-
ture requirement and time duration are important factors. Sample quantity is an
important parameter for microbiome analysis. For example, stool samples need little
quantity, but consistency of the stool sample is a major contributing factor which
affects the downstream microbiome analysis. Bristol scale is required to check the
consistency of the stool sample. If the sample requires shipping, then shipping
guidelines should be followed and dry ice should be used to preserve the sample
during shipping (https://blog.microbiomeinsights.com/considerations-when-choos
ing-a-collection-device-for-your-fecal-samples).

2.2 Methods for Taxonomic and Functional Profiling

of the Microbiome
Metabolic Profiling to Get Insights of Human Microbiome

The vast variety of bacteria that make up the gut microbiota produce a wide range of
substances that are essential for both the selection of microbes and the development
36 C. Joshi and V. Kothari

of a metabolic signalling network. Environmental triggers have an impact on

microbial activity, resulting in the production of several chemicals that have an
impact on the host metabolome and human health. As a result, metabolite profiles
associated to the gut microbiota can provide in-depth knowledge about how dietary
and lifestyle choices affect both chronic and acute disorders. The host metabolome
and its metabolic processes are impacted by the small molecule metabolites. As a
result, metabolome research is crucial for understanding how the host and gut
microbiota interact. The potential samples for examining host–gut microbiome
interactions include faeces, urine, plasma/serum, saliva, exhaled breaths, cerebro-
spinal fluid (CSF), and tissues from target organs. For metabolome studies, few
aspects are important, i.e. the sample types used for experiment, analytical methods
to be used for analysis, tools to be used for data processing, etc.
For metabolome analysis, careful sample collection and appropriate storage
conditions are required. Low temperature should be maintained to preserve the
samples. With regard to the extraction of hydrophilic metabolites, it is important
to adopt appropriate techniques to enhance the phase transfer of these compounds
from a complex biological sample to a clean extract before analysis. The procedures
utilised for the global metabolic profile of faeces were evaluated by Deda et al.
(2015). Important sample preparation techniques were discussed, including homog-
enisation, filtration, centrifugation, and solvent extraction. The state of the art in
metabolomics research was outlined by Chen et al. (2016), who also provided a more
thorough metabolome coverage through advancements in targeted and untargeted
methodologies, as well as analytical quality control and calibration methods. Gas
chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spec-
trometry (LC-MS), and capillary electrophoresis-mass spectrometry (CE-MS), three
potent technologies that can be used to explore host-gut microbiota interactions.
Gas chromatography combined with mass spectrometry, such as electron impact-
quadrupole (GC-EIMS) or time-of-flight mass spectrometry (GC-TOF MS), has
been used to identify metabolites with thermal stability and volatility. GC-MS can
also detect non-volatile metabolites. The results of giving microorganisms to animals
with IBS were published by Yu et al. in 2018. GC-TOF MS analysis was performed
from the faeces samples, which were prepared using organic solvent extraction
method. According to their research, giving C. butyricum supplements to IBS
mice may have positive effects through altering the metabolism of the host. For
their investigation of the biological consequences of faecal microbiota transplanta-
tion (FMT) in young patients with ulcerative colitis, Nusbaum et al. (2018) used
metagenomics and metabolomics methods. They discovered that the expression of
metabolites such short chain fatty acids, xanthine, oleic acid, putrescine, and
5-aminovaleric acid was changed following FMT according to the results of their
GC-TOF MS investigation. For the study of gut microbial-host associated
co-metabolites in faecal samples, Yin et al. (2017) developed an improved approach
based on the gas chromatography/time-of-flight mass spectrometry (GC-TOFMS)
platform. For the extraction of certain metabolic pathways of interest, a ratio of
chloroform, methanol, and water (W) was utilised. Intestinal microbial metabolite
receptors like the pregnane X receptor (PXR) and the aryl hydrocarbon receptor
Methods Used for Studying Human Microbiome 37

(AHR) activity, as well as enzymes like ornithine decarboxylase, have been identi-
fied as intestinal microbial metabolite receptors and enzymes that may be affected by
host-microbe relationships (ODC). Sixty-two reference standards are used to vali-
date the GC-TOFMS methodology. These illustrations demonstrated the potential of
GC-MS as a tool for host-gut microbiota investigations. Liquid chromatography-
mass spectrometry (LC-MS), in addition to GC-MS, is one of the analytical plat-
forms that is most frequently employed for metabolomics research to examine the
hydrophobic and hydrophilic metabolites. The use of LC-MS for host-gut
microbiota interactions has been shown in numerous investigations. In their 2017
study, Dodd et al. combined genetics and targeted metabolic profiling. Their findings
showed that the bacteria in human guts produce aromatic amino acid metabolites,
which build up in the bloodstream of the host and influence intestinal permeability
and systemic immunity. To explore tryptophan metabolites in urine, which are
connected to gut microbiome metabolism, Pavlova et al. (2017) developed a
UHPLC-MS/MS approach. Rusconi et al. (2018) investigated the effects of
sphingomyelin metabolism on necrotising enterocolitis in new-borns using broad-
range and tailored metabolomics techniques with a focus on ceramides and
sphingomyelins.
Another crucial analytical technique is capillary electrophoresis (CE), which
offers a distinctive mechanism for separation from conventional chromatographic
techniques. Ferrer et al. (2017) reported used of CE for phenotyping of the gut
microbiota. In order to evaluate the effects of xylitol on the gut microbiota and lipid
metabolism, Urbanso et al. (2017) used CE-TOF MS to analyse luminal metabolites
(110 targets). The data were combined with bacterial compositions. Mishima et al.
(2017) examined the effects of the gut microbiota on uremic solute accumulation and
Reno protective effects using CE-TOF MS to analyse mouse metabolites from
plasma, urine, and faeces. For metabolite studies employing biological samples,
mass spectrometry methods such as matrix-assisted laser desorption ionisation mass
spectrometry (MALDI-MS), desorption electrospray ionisation mass spectrometry
(DESI-MS), and Mass Spec Pen are also employed (Cameron and Takáts 2018).
These cutting-edge MS-based analytical systems could be used for gut microbiome
research.

Experimental Methods to Examine Host–Microbiome Interactions In

Vitro and Ex Vivo

In addition to metabolomics, host–microbiome interactions are being studied using

in vitro and ex vivo experimental methods, which improves the management of
experimental settings and the ability to explore interactions that are too complex to
study in vivo. The primary distinction between in vitro and ex vivo is the origin of
the samples used in the investigation. Ex vivo investigations use samples that are
directly obtained from a host organism, whereas in vitro research use cell lines or
laboratory microbe cultures. Co-cultures of microbes with or without host primary
epithelial cells, tissues, or cell lines; microfluidic co-cultures of microorganisms with
38 C. Joshi and V. Kothari

or without engineered tissue, and organoid culture are now being introduced as
in vitro and ex vivo approaches to study host–microbiota interactions. These
co-culture techniques can be used to investigate the bidirectional signalling between
microorganisms, target host tissues or cell types, as well as a body-site microbiome.
Additionally, native or immortalised small intestine or colonic cells are planted on
the apical face of trans well membranes to form polarised epithelial monolayers.
Permeability, transmembrane resistance, active transport, absorption, and excretion
can all be assessed to detect changes in the epithelium layer’s quality. A few
restrictions of the in vitro and ex vivo experimental systems include the absence of
secondary epithelial structures like villi and crypts, the lack of additional epithelial-
cell subtypes, the absence of mucus layers between host and microbial cells, and the
challenge of incorporating realistic multi-organism microbial community
components.
Gut-on-a-chip technology is employed to get over the constraints of the in vitro
and ex vivo experimental methods. This technique investigates the use of
microfluidic platforms to cultivate intestinal epithelial cells and replicate the flow
of fluids through the gut, which encourages the production of intestinal tissue
structures with specialised cell types. Fluids that circulate continuously can support
microbial colonisation that lasts over time. Several technology’s constraints include
the need for specific chip fabrication, specialised tools and technical know-how, and
challenges with adding various microbial components. Chemical exposures, luminal
perfusion, and microbial colonisation can all be carefully controlled using ex vivo
culture systems. Additionally, it is possible to separate intestinal tissues from model
organisms and keep them in ex vivo culture for brief periods of time. Ex vivo
methods produce physiological readouts that closely resemble in vivo
circumstances.
Similar technologies, such as primary airway epithelial cells and cell lines, are
well-developed tools to research the host–microbiome interactions in the respiratory
tract. It is crucial to research the host–microbiome interactions in the respiratory tract
since it is well known that respiratory tract infections (RTIs) are a significant source
of morbidity and mortality in children all over the world. The epidemiological
evidence relating these connections to mechanistic insights was examined by de
Steenhuijsen Piters et al. in 2020.
In order to create a three-dimensional model of the human lung, Dye et al. (2015)
stimulated human stem cells to produce cell types that later grew into complex
tissues in a petri dish. Dye et al. used several signalling pathways that control how
organs grow throughout the development of animal embryos to create these lung
organoids. Lung-on-a-chip and small-airway-on-a-chip technologies, which are like
the gut-on-a-chip platform, were featured by Benam et al. (2016). Animal and
human ex vivo lung-perfusion models are being employed in translational research
on lung illnesses. Additionally, there are synthetic models for studying the skin
microbiota. The model system was created to explore the human stratum corneum by
Van der Krieken et al. (2016). The effects of chemical exposure on skin colonisation
can be evaluated using a commercial three-dimensional in vitro skin model that is
filled with human skin bacteria (Bojar 2015). In order to research how skin
Methods Used for Studying Human Microbiome 39

integration surrounding percutaneous devices protects against bacterial infection,

Bolle et al. (2020) created an in vitro reconstructed human skin comparable model.
An in vitro mixed infection model containing commensal and pathogenic Staph-
ylococci was established by Kohda et al. in 2021 for the investigation of interspecific
interactions and their effects on skin physiology. However, the microbial variety in
the context of biochemical conditions on skin is not covered by the microbiological
adaptability or modelling accuracy of these synthetic systems. The experimental
throughput, physiologic relevance, and experimental control of the in vitro and
ex vivo systems used to study host–microbiota interactions are all different. Tradi-
tional co-culture with primary epithelial cells or cell lines offers for a moderate
amount of precisely controlled and manipulable experimental output.

Use of Model Organisms to Study the Microbiome

Microbiome and its interaction with the human host can also be studied using
nonhuman model systems. These systems can provide good opportunities to get
insights into molecular pathways, physiologic processes, host-specific gut
microbiome traits, and biochemical factors such as metabolite concentrations
(Davenport et al. 2017). Since animal models provide rigorous control of experi-
mental variables and reproducibility, animal models are widely employed to study
the human microbiome. Since humans and other animals share a phylogenetic link
with animal models, many genomic, molecular, cellular, and physiologic features
have been preserved across animal lineages, allowing for the extrapolation of many
findings from animal studies to humans (Turner 2018).
Animal models can be utilised to explore microbiomes using a variety of intel-
ligent experimental strategies. The microbiome makeup of animals can be assessed
using a variety of factors, including host age, host genotype, host body site, nutrition,
and chemical exposure, among others. Animals of the wild type that have been
invaded by intricate microbial communities are used in these laboratory
investigations. Animals having an indigenous microbiome can be treated with
broad-spectrum antibiotics to reduce microbial abundance and change community
composition in order to test whether microbiome makeup influences host character-
istics. That is a relatively quick and inexpensive technique to disrupt the
microbiome, but it has the drawback of not differentiating between the impacts
brought on by the direct use of antibiotics, the surviving antibiotic-resistant
microbes, or the loss of antibiotic-sensitive microorganisms (Morgun et al. 2015).
Gnotobiotic animal models, or germ-free animal models, are being utilised to
investigate how the makeup of a microbiota affects a host. To evaluate the effects
on the host, these animal models can be colonised with the relevant microbial strains.
Strong experimental control can be achieved using gnotobiotic animal models,
although these models are relatively expensive, labour-intensive, and have special
nutritional needs for gnotobiotic animals as well as developmental, immunologic,
and physiologic abnormalities (https://www.ncbi.nlm.nih.gov/books/NBK481559/).
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