CHAPTER ONE

INTRODUCTION

1.0 BACKGROUND OF STUDY

Microbiome are the home tract of wide range of microorganisms that can be commensal,

symbiotic, or toxic to all multicellular organisms, including plants. The microbiota includes

bacteria, archaea, protists, fungi, and viruses, all of which have been shown to be vital for their

host’s immunologic, hormonal, and metabolic balance (Dekaboruah et al., 2020). Microbial

communities live in multiple body sites in humans and animals (including the stomach, oral

cavity, esophagus, skin, and vagina) and interact with and influence their hosts’ immune system

and metabolism. In addition, microbes have developed alongside humans and are now an

essential component of life, performing a variety of essential roles. Due to changes in

environmental parameters such as temperature, pH, oxygen, and nutrition availability, their

composition varies greatly between body locales and specific biogeography. Although much has

been done to explore its diversity, a full understanding of our microbiomes demands an

evolutionary perspective (De Sordi et al., 2019). At the strain level, microbial evolution may

occur e.g., when advantageous mutations in specific genes drive adaptation to new selection

pressures) selection may also enhance the frequency of a specific microbial taxon, causing the

adaptive microbiome’s microbial taxa to be lost (Walaa and Fadia, 2022). The microbiome can

evolve at two levels: first, each individual microbe is subjected to evolutionary processes

(mutation, selection, migration, drift, speciation, etc.), and second, a host species’ microbiome

can evolve by incorporation and elimination of microbial taxa, or by changes in their relative

abundances as a consequence of these evolutionary processes (Glendinning et al., 2020).

Many studies have highlighted that the commensal microorganism plays an important role in the

development of human diseases. Thus, the term “holobiont” is used to describe the complex

network and system formed by human cells and the commensal microorganism (Inda et al.,

2019). The microbiome is used to describe all microorganisms and their genomes, including

bacteria, archaea, viruses, and fungi. Commensal microbiomes, such as the gut microbiome, skin

microbiome, and vagina microbiome, contain beneficial microbes and pathogens, which

contribute to host homeostasis in different locations. Therefore, modulating the microbiome is

regarded to be an effective way to regulate host homeostasis and defeat diseases (Isabella et al.,

2018). Probiotics are defined as live microorganisms that, when administered in adequate

amounts, confer a health benefit on the host, such as several species from Lactobacillus,

Bifidobacterium, and Streptococcus (Zuo et al., 2020). However, some studies have shown the

risks of adverse effects and ineffectiveness of probiotic use, indicating that more research is

needed for a predictable and effective use of probiotics in medical applications (Hill, 2020).

1.1 Significance of Study

As a result of the widely reported relevance of the microbiome and host health, engineering

microbes for medical usage has become an emerging research direction. Their applications

include modulating host metabolism, regulating the host immune system, combating pathogens,

as a novel diagnosis tool or sensor, and as tools for host microbiome relationship discovery. With

the development of more synthetic biology tools, the obstacles along the way for effective

therapy with engineered microbes have been gradually removed. This purpose of this review is

to present an update on the relevance of human microbiome in health and diseases.

 CHAPTER TWO

LITERATURE REVIEW

2.0 Human Microbiota

The human microbiota is defined as a set of organisms inhabiting and interacting with the human

body (Gao et al., 2019). The various interactions may be commensalistic, mutualistic, or

pathogenic. The human microbiome is referred to as the genomic content of organisms

(microbiota) inhabiting a particular site in the human body. Microorganism colonise various

anatomical body sites such as the skin, the mucosa, gastrointestinal tract, respiratory tract,

urogenital tract, and the mammary gland. They form a complex and discrete ecosystem that

adapts to the environmental conditions of each niche (Galloway and Hanson, 2020). From

childbirth, a steady interaction (symbiosis) between the human body and its indigenous

microbiota begins. These interactions play important roles in maintaining general health and

wellbeing. Thorough coevolution, organisms make up the microbiota, they actively adjust to

their specific habitats and reside in their respective niches within the human body (Mousa et al.,

2022). As a result of their biological activities, these organisms are identified as part of the body,

leading to various changes from conception until death. The human microbiome is constantly

evolving in response to host factors. Factors such as age, nutrition, lifestyle, hormonal changes,

inherited genes, and underlying disease are major determinants of the human microbiome at any

given point in time. However, an alteration in the makeup of the human (dysbiosis) microbiota

can lead to life-threatening illnesses (Guo et al., 2020). A balanced microbiota has shown to play

an important role in health sustenance. The largest concentration of the human microbiome is

found in the gut. These organisms are the major players in maintaining and sustaining the health

of humans. Past studies on the human microbiome project have illustrated that changes in the
immune environment may be directly linked to a dysbiotic flora of the gut. Also, life-threatening

health conditions ranging from cancer, cardiovascular disease, bowel inflammatory disease and

difficult-to-treat bacterial infections due to antibiotic resistance have also been linked with

dysbiosis (Sani et al., 2020).

2.1 Human Body Niches: A Glance from the Simple to More Complex

2.1.1 The Vaginal Microbiome

The vagina is the microbial organ with the least diversity in the human body, with a dominance

of Lactobacillus, a species that impedes the colonization of other bacteria that would otherwise

cause infections. The lactic acid produced by the Lactobacilli provides a protective role by

maintaining an acidic pH (<4.5); serving as a chemical barrier (Valenti et al., 2018). In addition,

Lactobacillus spp. produce bacteriocins, hydrogen peroxide (H2O2), and reactive oxygen species

(ROS), impeding the colonization and adherence of pathogens, organisms that would otherwise

cause recurrent vulvovaginal infections (RVVI) associated with discomfort, odor, discharge,

infertility, and, if pregnant, could even lead to miscarriages. The vaginal microbiota can be

characterized in five Community State-Types (CST), representing different microbial groups

(Zhou et al., 2020). CST-I has a predominant abundance of Lactobacillus crispatus, CST-II has

Lactobacillus gasseri, CST-III has Lactobacillus iners, and CST-V has Lactobacillus jensenii.

CST-IV, on the other hand, has a reduction of Lactobacillus spp. and a higher abundance of

anaerobic bacteria such as Prevotella, Atopobium, Sneathia, and Gardnerella, which have been

associated with bacterial vaginosis (Di Paola et al., 2017). CST profiles in women are known to

vary by ethnicity. Caucasians tend to exhibit a CST-I dominated microbiota, while African-

American and Hispanic women tend to present a CST-IV profile (Di Paola et al., 2017). Having

a non-L. crispatus dominant community does not necessarily mean severe dysbiosis; studies have
shown healthy Latinas who have a L. iners dominant community can be asymptomatic (Godoy-

Vitorino et al., 2018; Vargas-Robles et al., 2020). Hormonal changes across the reproductive

cycle in women can disrupt the microbial equilibrium. When there are high hormonal levels, the

abundance of glycogen increases in the vagina, which is used by bacteria to promote an increase

in diversity (Kaur et al., 2020). At the same time, glycogen is used by Lactobacillus spp. to

produce lactic acid, reducing and stabilizing the diversity present. Puberty, menstruation,

pregnancy, and menopause compose the main stages of the female whole reproductive cycle.

The cervicovaginal microbiota becomes even more dominated by Lactobacillus during

pregnancy, resulting in less diversified profiles than in non-pregnant women (Serrano et al.,

2019). However, as estrogen and progesterone levels fall after menopause, there is a drop in

Lactobacillus spp. and an increase in vaginal pH (Auriemma et al., 2021).

2.1.2 The Skin Microbiota

The skin is an essential element of defense against pathogens. Its physiological and anatomical

properties change throughout the body, shaping microbial composition. Compared to the most

diverse body sites, the skin microbiome has fewer taxa due to its textural characteristics such as

oil, moisture, sebaceous glands, and acidic pH (Chen et al., 2020). External factors implicated in

changes in the skin microbiota include the use of antibiotics, cutaneous burns, skincare, and

hygiene products, and lifestyle habits (Sanjar et al., 2020). The most dominant genus in the skin

is Staphylococcus, Propionibacterium, Corynebacterium, and Streptococcus (Bay et al., 2020).

Furthermore, oilier sites have significant dominance of Propionibacterium species (lipophilic),

whereas in humid niches, Staphylococcus and Corynebacterium species thrive (Bay et al., 2020).

Fungi are also a major component of the microbiome. For example, Malassezia is a major

lipophilic yeast distributed throughout the body; however, other fungi are site-specific such as
Aspergillus spp., Cryptococcus spp., and Rhodotorula spp. who colonizes regions of the feet (Jo

et al., 2017). In addition, shifts in fungal composition can occur with age, as children have a

marked profile of Ascomycetes and lower levels of Malassezia when compared to adults. The

age-associated differences in the skin microbiome is so marked that it has been used to predict an

individual’s age with a range of approximately 4 years. Dysbiosis of the skin microbiome has

been related to skin diseases/conditions. For instance, patients with psoriasis have a higher

abundance of Proteobacteria and Staphylococcus aureus and a decrease in Acinetobacter when

compared to healthy individuals (Chang et al., 2018). Similarly, the skin of patients with atopic

dermatitis is characterized by a higher prevalence of Staphylococcus aureus (Paller et al., 2019).

Staphylococcus aureus and Staphylococcus epidermidis distinguished skin lesions of patients

with systemic lupus erythematosus (SLE), whereas healthy individuals had a higher abundance

of Cutibacterium (Huang et al., 2020). On the other hand, patients with alopecia have an increase

in Propionibacterium acnes and a decrease in common members of the skin microbiome such as

Propionibacterium, Corynebacterium, and Staphylococcus (Ho et al., 2019) (Figure 2).

2.1.3 The Eye Microbiota

The eye has three major microbial niches: the eyelid skin, meibum, and conjunctiva, which differ

in diversity and composition. Similar to the skin microbiome, the microbial composition of the

eyelids is dominated by two skin taxa Staphylococcus and Propionibacterium (Suzuki et al.,

2020). The meibum is characterized by a dominance of Propionibacterium and Pseudomonas,

while the conjunctiva is defined exclusively by Propionibacterium (Suzuki et al., 2020). The

dysbiosis in the conjunctiva microbiome has been associated with different health conditions,

such as keratoconjunctivitis, mucosa-associated lymphoid tissue (MALT) lymphoma, and high

glucose levels on the ocular surface due to diabetes (Asao et al., 2019). Notably, the microbiota
of conjunctiva in MALT lymphoma patients is dominated by Delftia (Asao et al., 2019). When

evaluating the role of the ocular microbiota in relation to diabetes, mice studies reveal a reduced

diversity in Type 2 diabetes (Suzuki et al., 2020), while an increase in Bacteroides and a

decrease in Proteobacteria and Acinetobacter are observed in Type 2 diabetes (T2D) (Li et al.,

2019).

2.1.4 The Ear Microbiota

The microbiota of the ear canal is similar to that found on the skin. Therefore, Corynebacterium,

Staphylococcus, and Propionibacterium genera are prevalent taxa (Jervis-Bardy et al., 2019).

There is still a debate over whether microbes from the nasopharynx colonize the middle ear or if

this is a sterile site. Although previous findings suggested no microbial colonization, a recent

Illumina microbiome profiling study demonstrated that the middle ear is actually colonized by

Proteobacteria, Actinobacteria, and Firmicutes (Jervis-Bardy et al., 2019). Otitis media

infections can be characterized as Acute Otitis Media (AOM) or Chronic Otitis Media with

Effusion (COME). Dysbiotic changes are observed in adults and children who suffer from Otitis

Media inflammation (Lappan et al., 2018). The pathogenesis and development of AOM are

dependent on the microbiome of the nasopharynx, with Haemophilus, Alloiococcus,

Staphylococcus, Turicella, Moraxella, and Streptococcus being taxa normally associated with

this condition (Lappan et al., 2018). In COME patients, it’s been documented that a higher

abundance of Alloiococcus, Haemophilus, Moraxella, Turicella, Stenotrophomonas,

Streptococcus, and Staphylococcus (Kolbe et al., 2019). It’s important to mention that

Alloiococcus and Turicella are not found in the healthy middle ear. In addition, COME is

associated with respiratory illnesses such as asthma and bronchiolitis while reflecting a lower
richness and evenness in comparison with those patients that do not present lower respiratory

diseases (Kolbe et al., 2019).

2.1.5 The Microbiota of the Nasopharyngeal Tract

The nasopharynx is a component of the upper respiratory tract, specifically located at the upper

part of the throat behind the nose. The microbiome of the nasal cavity mucosa is colonized

primarily by Corynebacteriaceae and Staphylococcaceae families, while Peptoniphilaceae and

Carnobacteriacea are in lower abundance (Kang and Kang, 2021). This niche also has a high

abundance of Staphylococcus, Corynebacterium, Alloiococcus, Haemophilus, Streptococcus,

Granulicatella, and Moraxella (Kang and Kang, 2021). Diseases such as asthma, influenza A

virus (IAV), bronchiolitis, and rhinosinusitis acute respiratory illness (ARI) are all associated

with changes in the microbiota. Children with IAV infection have increased microbial diversity,

specifically of Streptococcus—associated with the production of type I interferons during IAV

infection, with a concomitant decrease in Corynebacterium, Moraxella and Dolosigranulum

(Wen et al., 2018). Infants with bronchiolitis have an increasing dominance of Haemophilus,

Moraxella, and Streptococcus when compared with healthy individuals. Chronic rhinosinusitis

(CRS) is also a dysbiosis-related disease, with higher alpha diversity in CRS compared to

healthy individuals and increasing levels of Proteobacteria and Escherichia. Other genera,

including Roseateles, Pseudomonas, and Escherichia, were positively correlated with CRS

symptom severity (Copeland et al., 2018).

2.1.6 The Oral Microbiota

The oral microbiome is one of the most diverse body niches, only preceded by the colon. The

oral cavity is highly diverse due to its many structural and physiological niches harboring a

plethora of different microbial communities. These niches include oral mucosa, tongue, saliva,
soft tissue, hard tissue, and the surfaces of the teeth. Each surface has distinct communities;

hence it provides the conditions and nutrients required for these distinctive microbes. For

example, the flora of the tongue differs from that in plaque or the hard tissues of the oral cavity

due to its specific microenvironment (Chen et al., 2019). Bacterial and fungal communities play

an essential role in the development of many oral diseases such as dental cavities, gingivitis,

periodontitis and, subsequently, tooth loss. Bacterial composition consists mainly of Firmicutes,

Bacteroidetes, Proteobacteria, Actinobacteria, Spirochaetes, and Fusobacteria (Chen et al.,

2019). Among the most dominant bacterial taxa in the oral cavity are Streptococcus, Gemella,

Abiotrophia, Granulicatella, Rothia, Neisseria, and Prevotella (Chen et al., 2018). The fungi

flora is often composed of Candida, being the most abundant, Cladosporium, Aureobasidium,

Saccharomyces, Aspergillus, Fusarium, and Cryptococcus (Chang et al., 2020). Few studies

have analyzed archaeal diversity in the oral cavity; however, methanogenic archaea, like

Methanobrevibacter oralis, increase in abundance as periodontitis progresses. The oral cavity is

sterile before birth (Sulyanto et al., 2019). However, soon after birth, the oral microbiome

changes and evolves through adulthood. After 24 h, the newborn oral cavity will most likely be

colonized by gram-positive cocci like Streptococcus and Staphylococcus (Sulyanto et al., 2019).

Streptococcus salivarius is an initial colonizer because it is capable of adhering to epithelial

cells. From birth to 3 months old, infants have a simple microbial community composed of six

main species: Streptococcus mitis, Rothia mucilaginosa, Veillonella parvula, Streptococcus

salivarius, Gemella haemolysans, and Veillonella (Sulyanto et al., 2019). Between 3 and 6

months old, the infant shows a distinctive microbiota due to solid food ingestion, hygiene, built

environment, and contact with other humans and domestic animals; characterized by an increase

of Prevotella, Granulicatella, and Neisseria (Sulyanto et al., 2019). The acquisition of these
species has been assigned to the emergence of teeth, forming microenvironments, niches, and

new surfaces for bacterial colonization and adherence (Kennedy et al., 2019). Late colonizers

include Prevotella, Porphyromonas, Leptotrichia, and Actinomyces, which colonize infants

around 1 year of age (Kennedy et al., 2019). Children with primary dentition have a higher

prevalence of Pseudomonas, Acinetobacter, Moraxella, and Enhydrobacter (Aabed et al., 2019).

As dentition becomes permanent, populations of Veillonella and Prevotella increase, while

Granulicatella decreases (Alderete et al., 2018). The oral microbiome continues to develop from

puberty to adulthood, and lifestyle habits have an impact on microbial diversity. Mucosal

surfaces and saliva are primarily composed of aerobic bacteria. However, fissures and

supragingival surfaces have a higher abundance of facultative anaerobes, in contrast with the

subgingival plaque, which favors strict anaerobes (Auriemma et al., 2021). During puberty,

many hormonal and nutritional changes take place. These changes often lead to an increase of

gram-negative anaerobes and spirochetes, which may be associated with a higher incidence and

severity of gingivitis. Gingivitis and periodontitis are common bacterial infections that are

caused by host immune responses against pathogenic bacteria, leading to inflammation and

dysbiosis. While gingivitis is a mild reversible inflammation, if left untreated, it could develop

into periodontitis, an irreversible disease that causes chronic inflammation of the gums and

subsequent bone loss (Huang et al., 2019). Research conducted on periodontal health and

changes after therapy found that plaque samples have more abundance of Fusobacteria, while

saliva samples have a higher prevalence of Firmicutes and Proteobacteria, even though the saliva

microbiome is likely affected by conditions other than the periodontal disease (Huang et al.,

2019). Additionally, Bacteroidetes and Spirochaetes were higher in healthy individuals, while
Porphyromonas, Tannerella, Prevotella, and Filifactor were more abundant in participants with

periodontitis (Huang et al., 2019).

2.1.7 The Microbiota of the Gastrointestinal Tract: Stomach, Intestines, and Cecum

The gastrointestinal microbiome is the largest and most diverse reservoir of all the human body

niches. From the mouth to the anal cavity, each digestive organ section provides a specific

environment that allows the growth and colonization of organisms. The most common phyla

across the gut tube are the Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria. In the

esophagus, the most prevalent bacterial taxa are Streptococcus, Veillonella, and Prevotella, a

composition that resembles that of the oral microbiome (Sanna et al., 2019). The microbial

communities of the stomach are dominated by Proteobacteria and Firmicutes. Many studies have

also stipulated that Helicobacter pylori is part of the normal flora found in the stomach, which

was lost through modern lifestyles (Schnadower et al., 2018). Other studies found that positive

H. pylori status was associated with an increased relative abundance of non-Helicobacter

bacteria from the Proteobacteria, Spirochaetes, and Acidobacteria phyla, alongside a decreased

abundance of Actinobacteria, Bacteroidetes, and Firmicutes (Schnadower et al., 2018). Despite

the fact that H. pylori is a causative agent of gastritis and is associated with gastric cancer, other

studies demonstrated how H. pylori infections could lower the risk of celiac disease, asthma and

esophageal adenocarcinoma (Nishida et al., 2021). Gastroesophageal reflux disease (GERD),

Barrett’s esophagus, and esophageal carcinoma are all a result of microbial dysbiosis (Pei et al.,

2004). Persistent GERD that progresses to Barrett’s esophagus, predisposing to an esophageal

carcinoma, has been related to an increase of Veillonella, Fusobacterium, and Prevotella, taxa

that are absent in healthy individuals. The small intestine is characterized by an environment

with high concentrations of oxygen and antimicrobials along with a short transit time that allows
the rapid growth of facultative anaerobes (Rinninella et al., 2019). It absorbs 90% of the host’s

energy from the diet and is divided into three parts: duodenum, jejunum, and ileum. The most

abundant phyla in the duodenum are Firmicutes, Proteobacteria, and Actinobacteria, taxa that

contribute to most of the nutrient digestion, including protein, lipids, and simple sugars

(Angelakis et al., 2015). Particularly, the most dominant genera found are Prevotella,

Stenotrophomonas, Streptococcus, Lactococcus, Bacillus, Solibacillus, Pseudomonas,

Arthrobacter, and Lysinibacillus (Gong et al., 2019).

In the jejunum, Firmicutes and Proteobacteria are the most predominant, while Eschericia coli,

Enterococci, and Lactobacillus were also identified as predominant species of the duodenum and

jejunum (Sundin et al., 2017). The ileal microbiota is dominated by Streptococcus, Eschericia

coli, Clostridium; however, with significant inflammation, members of Fusobacteria and

Proteobacteria increase significantly with a reduction of Bacteroidetes and Spirochaetes (Fan et

al., 2020). The colon, which is the most diverse niche, has an anaerobic environment dominated

by Bacteroidetes, especially in the sigmoid colon (James et al., 2020). The most dominant taxa is

Bacteroides, while Enterococcus is more prevalent in the proximal colon, contrary to the distal

colon, which has higher abundance of Coprobacilus and Escherichia/Shigella (James et al.,

2020). Key biomarkers of health across the human colon have been identified, including

Lactobacillus, Bifidobacterium and Fusobacterium prausnitzii. Other non-bacterial components

of the colon microbiome, which are also important residents, include bacteriophages, fungi such

as Ascomycota and Basidiomycota, and Archaea such as Methanobrevibacter smithii (James et

al., 2020).

The cecum and appendix sections of the large intestine have a similar composition to those

previously described, with a slight reduction in Bacteroides (James et al., 2020). The appendix
has been characterized by high diversity, with the dominance of Firmicutes, Proteobacteria,

Actinobacteria, Bacteroidetes, and Fusobacteria. At the family level, Lachnospiraceae,

Enterobacteriaceae, Bacteroidaceae, Fusobacteriaceae, and Bifidobacteriaceae, specifically the

genus Bifidobacterium were the predominant groups (Iglesias-Vázquez et al., 2020). During

appendicitis, other non-intestinal genus such as Fusobacterium Gemella, or Parvimonas have

been detected. Fecal healthy biomarkers such as Bacteroides, Eubacterium rectale,

Fusobacterium prausnitzii, and Akkermansia muciniphila are inversely related with appendicitis.

Despite being considered an organ that has lost its function throughout evolution, the appendix

has great biological redundancy ensuring gut repopulation in dysbiotic situations after pathogen

colonization, diarrheal diseases, or antibiotic treatments (Iglesias-Vázquez et al., 2020).

2.2 Importance of Human Microbiota

2.2.1 The Healthy Gut Microbiota

Intestinal microbial balance is closely relevant to human diseases and health. Compared with

other regions of the body, the human gastrointestinal (GI) tract contains an abundant microbial

community which gathers ~100 trillion microorganisms (Deo and Deshmukh, 2019). Extensive

studies have been performed to reveal the important relationship between gut microbiota and

basic human biological processes. For example, current advances have shown that human

microbiota is closely involved in nutrient extraction, metabolism, and immunity (Hillman et al.,

2017). Microbiota may affect biological processes via several mechanisms. For energy and

nutrient extraction from food, microbiota plays crucial roles due to the versatile metabolic genes

which provide independent unique enzymes and biochemical pathways (Fan and Pedersen,

2021). Moreover, the biosynthesis of bioactive molecules such as vitamins, amino acids and

lipids, are also highly dependent on the gut microbiota. Regarding the immune system, the
human microbiota not only protects the host from external pathogens by producing antimicrobial

substances but also serves as a significant component in the development of intestinal mucosa

and immune system. In healthy conditions, the gut microbiota exhibits stability, resilience, and

symbiotic interaction with the host. There is a lot of research into the definition of a “healthy”

gut microbiota and its link to host physiological functions. Gut microbiota is composed of

bacteria, yeasts, and viruses (Amabebe et al., 2019). A healthy microbiota community often

demonstrates high taxonomic diversity, high microbial gene richness and stable core microbiota.

However, it should be noted that the relative distribution of microorganisms is unique between

individuals and may undergo variations within the same individual. In human, gut microbiota

may vary due to age and environmental factors (for example, medication usage). Additionally,

gut microbiota varies in different anatomical parts of the GI tract. For example, Proteobacteria

such as Enterobacteriaceae are found in the small intestine but not the colon. Instead,

Bacteriodetes such as Bacteroidaceae, Prevotellaceae and Rikenellaceae are often found in the

colon (Hugenholtz and de Vos, 2018). Such variations are majorly due to the different

environments. In the small intestine, the transit time is short and bile concentration is high, while

in the colon, which has slower flow rates and milder pH, as well as larger microbial

communities, especially anaerobic types, are commonly observed (Park et al., 2020). Besides

spatial distribution, gut microbiota also differs by age. Generally, the microbiota diversity

increases in the time between childhood and adulthood and decreases at older age (over 70).

Before the formation of a relatively stable gut microbiota composition, the diversity of children’s

microbiota is dominated by Akkermansia muciniphila, Bacteroides, Veillonella, Clostridium

coccoides spp., and Clostridium botulinum spp (Park et al., 2020). At about age 3, children’s gut

microbiota becomes comparable to that of adults, with three major microbial phyla including
Firmicutes, Bacteroidetes and Actinobacteria becoming dominant. Subsequently at older age,

dietary and immune system change potentially affect the composition of the human gut

microbiota. Specifically, elder people tend to exhibit decreased Bifidobacterium and increased

Clostridium and Proteobacteria (Coyte and Rakoff-Nahoum, 2019). The decrease in the

anaerobic bacteria Bifidobacterium is considered relevant to deteriorated inflammatory status due

to its role in stimulating the immune system. Since the microbiota plays an important role in

human well-being, also proactively involves in multiple biological processes and disease

development, the research on human microbiota is going beyond compositional studies and

investigation on members’ associations. Specifically, more attention has been paid on explaining

the causality of microbiota functions, especially with the boom of new techniques of high-

throughput sequencing, microbiota interactive modeling and simulation. Overall, further

investigations are still necessary to unveil the roles of human microbiota, in order to support the

development of microbiome-based diagnosis and personalized medicine (Furuyama and Sircili,

2021).

2.2.2 Rodent Models for Human Microbiota Research

The human microbiota has attracted more and more research in recent decades. However, the

studies of local microbiota require invasive sampling methods, with practical or ethical reasons

in concern. Animal models, particularly mouse and rat models, have also been used to study the

pathogenic and therapeutic potential of microbiota with varies diseases (Hugenholtz and de Vos,

2018). With a majority of microbiota research is focusing on gut microbiota, the use of germ-free

(GF) mouse model has become popular due to its translatability. It should be noted that, in order

to translate such generated knowledge from rodent to human, the similarities and differences

between their microbiota profile need to be considered. Chen et al. (2020) reported that, in
murine genome, half of the transcription factor binding sites may not have orthologous

sequences in human genome (Chen et al., 2020). Moreover, the genomic studies have shown a

significant difference in the immune system and its regulation in different species. Since gut

microbiota has major impact to host innate and adaptive immune responses, the translation of

findings from rodents to human should be carefully validated before drawing definite

conclusions. While human and murine gut microbiota has 90% overlapping in phyla and genera

levels, the composition and abundance of microbes have key discrepancies (Park et al., 2018).

For instance, the major difference is the Firmicutes/Bacteroidetes ratio, where it is significantly

higher in human than mice. Particularly, human Bacteroidetes is mainly composed of

Prevotellaceae and Bacteroidaceae, while mice Bacteroidetes are primarily composed of S24-7.

Regarding the Firmicutes, Ruminococcaceae is the major phylum observed in human and

Clostridiales is the major one observed in mice. Moreover, human and mouse each carries

specific genera, such as Faecalibacterium, Megasphera, Asteroleplasma, Succinivibrio,

Paraprevotella in human and Mucispirillum in mouse (Park et al., 2018).

2.2.3 Colonization Resistance

Humans are born with and form a large community of symbiotic and pathogenic microbes,

which inhabit our gut, skin, mucosal passages, and form a stable community that is resistant to

external pathogens. Later, such conclusion was further applied to the phenomenon that current

microbiota could provide resistance the colonization of invading pathogenic species, also from

which researchers recognize (Coyte and Rakoff-Nahoum, 2019). As a result, the microbiota is

crucial shield in protecting us from exogenous microorganisms. Despite the fact that microbiota

colonization resistance has not been fully elucidated, with the advent of GF animal models,

researchers have discovered several potential mechanisms such as nutrient competition,

antimicrobial production, and bacteriophage deployment. Another example of colonization

resistance is the interaction of symbolic and pathogenic Escherichia coli, where indigenous

Eschericia coli strains compete with pathogenic Eschericia coli O157: H7 for the amino acid

proline in consuming nutrients.In this section, we focus on the gut microbiota and colonization

resistance (Ducarmon et al., 2019). The vaginal and skin microbiota and their colonization

resistance are also discussed. The GI tract digests proteins as well as sugars from foods.

Metabolizing polysaccharides and specific proteins requires multiple enzymes produced by

various bacteria. For example, Bacteroides species in the large intestine are responsible for sugar

harvest. Pathogenic Enterobacteriaceae also utilizes sugar and amino acids in gut (Furuyama and

Sircili, 2021).

Probably due to the necessity of competing with foreign bacteria, gut bacteria have developed

various ways of suppressing competitors, including the secretion of diverse bacteriocins. A

contact-dependent competition in the gut, namely type 6 secretion system, was originally

identified in the bacteria secretion system involved with eukaryotic cells, which was later found

relevant to intraspecies killing. The system works by contact cells delivering effectors, such as

degraders of nucleotide, cell walls and membranes, into the cytoplasm (Furuyama and Sircili,

2021). Moreover, this system may also contribute to the abundance of Bacteroides species in the

mouse and human gut. Besides the type 6 system, other systems such as type 7 (or ESX system),

also mediate the intra- and interspecies killing (Davies et al., 2019). Currently, the contact-

dependent systems of gut microbiota inhibition and growth are being increasingly discovered.

The intermediate genes, immunity and effectors may serve as amenable factors which are

modifiable via bioengineering methods. Additionally, they are valuable for studying the

interactions, structure, and dynamics of the gut microbiota. However, the exact role of
bactericidal mechanisms remain poorly understood and further studies are still necessary.

Bacteriophage deployment is another mechanism of colonization resistance in the gut; however

relevant research is still in an immature stage (Amitai and Sorek, 2016). It has been revealed that

two cycles, namely the lytic cycle and the lysogenic cycle, are involved in bacteriophage

infection. Phages duplicate by injecting genomic segment into the bacterial cytoplasm, after

which the two cycles start to branch. Phages in lysogenic stage insert their genome into bacteria

genome and render prophages, which guarantees the replication of phage DNA and entrance into

the lytic cycle. In the lytic cycle, phage DNA starts replication, modification and expression,

resulting in new phage assembly, cell lysis and phage spreading. There are several potential

mechanisms to prevent bacteriophage infection including the blockage of surface receptor

recognition, superinfection exclusion system and abortive infection. For infection prevention,

resistant strains exhibit compositions similar to bacterial surface and thereby could serve as

decoys for attacking phages (Goh and Barrangou, 2019). DNA replication could be prevented by

“restriction-modification” system, mainly by methyl-transferase and restriction endonucleases.

This system serves as the primitive inner bacteria defense system in the human body, despite the

disadvantage of this system that it also damages the host DNA (Greathouse et al., 2018). The

defense system of bacteria also inspired the discovery of Clustered regularly interspaced short

palindromic repeats (CRISPR)-Cas9, which has been extensively reviewed (Griffin et al., 2021).

Later, newer defense systems, such as bacteriophage exclusion have been discovered to work by

preventing DNA replication. The third potential mechanism is the abortive infection, where the

infected cells are killed, and surrounding ones are protected. This mechanism is not yet fully

elucidated, and still needs further exploration (Groussin et al., 2021).

2.2.4 Microbiota in the Development of Immune Systems

Microbiota in different organs exhibits distinct characteristics and compositions. As a result,

microbiota interacts with multiple biological processes of the host. In this section, we introduce

the interactions between human microbiota in gut, oral cavities, lungs, skin, vagina, and the

development of immune systems (Zhao and Elson, 2018). The human immune system is

comprised of innate and adaptive immune responses, both of which have been shown to

extensively interact with microbiota. The innate immune response has critical role in maintaining

a homeostatic environment by eliminating pathogenic bacteria and regulating the adaptive

response to microbiota. These effects are mediated by factors such as secretory IgA (sIgA), toll-

like receptor 5 (TLR5), autophagy, and inflammasomes (Cavaleri and Bashar, 2018). For

instance, slgA can bind and form complexes with commensal bacteria, which selectively presents

the bacterial components to tolerogenic dendritic cells. As an anti-inflammatory molecule, slgA

can reduce the inflammatory response that could result from the immense bacteria load in the

organs. On the other hand, dysbiosis of microbiota can alter the sIgA response and lead to

unregulated bacterial growth. The sIgA induction was confirmed as a gradual response to current

bacterial exposure, suggesting a crosstalk between microbiota and immune system. The adaptive

immune response is another important part to maintain a healthy microbiota and immune

balance. Particularly, the education of adaptive immune response is achieved by differentiation

and maturation of B and T cells and establishment of immune tolerance to microbiota (Zhao and

Elson, 2018). Depending on the bacteria species, the CD4 T cell responses vary significantly,

which leads to the differentiation into distinct subsets and the subsequent pro-inflammatory

cytokine release such as interferon-γ and interleukin IL-17A (Zhao and Elson, 2018).
The GI tract hosts a large number of immune cells, which constantly communicate with the gut

microbiota. The maturation of the immune system needs the development of commensal

microorganism. One of the mechanisms of gut microbiota affecting the immune system is by

mediating neutrophil migration, which subsequently impacts T cell differentiation into various

types such as helper T cells (Th1, Th2, and Th17) and regulatory T cells. Disorders in microbiota

development during the maturation of the immune system could lead to deteriorated

immunological tolerance and autoimmune diseases (Lamont et al., 2018). Additionally,

heterogeneous molecules produced by microbiota may induce immune response and stimulate

inflammation or chronic tissue damage. The general interactions of microbiota and host immune

response during healthy and disease states are depicted. Human immune system is closely related

to the microbiota as a complex symbolic relationship during the co-evolution of vertebrates and

microbiota (Barcik et al., 2018). The vertical transmission from the mother’s microbiota to the

child at birth is considered the initial introduction of microbiota to the child. As a result, infants

born by Cesarean section are colonized with bacteria of the epidermal origin, which might link to

higher risk of developing allergies and asthma compared with infants who received initial

microbiota from the maternal vaginal flora. Such difference in immune system and microbiota

would be gradually eliminated during growth. As mentioned before, the infant’s microbiota

stabilizes at ~1-year-old and resembles that of adults. The neonatal immune system also rapidly

develops under the impact of dynamic microbiota (Garcia et al., 2019). In addition to the

microbiota transmission during birth, breastfeeding also plays crucial roles in the establishment

of infant immune system as well as microbiota. Besides the required nutrients and antimicrobial

proteins, breast milk provides slgA, which is specifically shaped by the maternal microbiota. As

a result, infants’ microbiota is seeded not only by the maternal epidermal or virginal origin but
reinforced by the sIgA shaped by maternal microbiota. Moreover, before the solidification of

infant immune system, the sIgA significantly protects the newborn against pathogens (Garcia et

al., 2019).

2.3 The Human Microbiome and Disease

2.3.1. Cancer

The gut microbiota plays a significant role in affecting the wellbeing of its host. Studies on the

interplay of microbial communities and their respective host suggest that these organisms carry

out biochemical activities influencing carcinogenesis, tumour development, and response to

immune therapy (Peng et al., 2020). According to a well-studied model on factors that may

contribute to dysbiosis in the gut, continuous intra-abdominal infections, antimicrobial drugs, or

both may lead to an increased risk of colorectal cancer. Also, end products released by the gut

microbiota affect the intestinal cell coverage, encouraging carcinogenesis or suppressing

tumourigenesis (Pedrolli et al., 2021). Aside from colorectal cancer, the microbiota of the

intestinal tract has shown to play a role in extraintestinal cancer such as hepatocellular carcinoma

through systematic dissemination of these organisms to other parts of the body (Paller et al.,

2021). In addition, H. pylori contributes to the risk of gastric cancer in humans. Recent findings

on the interplay of the human microbiome and cancer point out Fusobacterium and Clostridium

being overrepresented in individuals having gastric cancer (Perino et al., 2020). In the case of

breast cancer, environmental and host factors directly influence the progression of cancer in the

breast. However, bacterial communities could also induce breast cancer. Individuals with breast

cancer have been more likely to have Bacillus, members of the Enterobacteriaceae, and

Staphylococcus in their breast tissue as compared to healthy individuals. Furthermore,

Escherichia coli and Staphylococcus epidermidis isolated from patients having cancer triggered
a double-stranded break in the DNA of HeLa cells. Lactobacillus spp. which contributes to

diverse health benefits was not found in the breast tissue of individuals with breast cancer (Sun

et al., 2018). Prostate cancer has been implicated with a higher population of Bacteroides

massiliensis. An alteration in the human microbiota has contributed to the complex interactions

between cancer and the human microbiota (Swanson et al., 2020).

2.3.2. Inflammatory Bowel Disease (IBD)

The accumulation of disease-causing organisms induces or triggers an abnormal immune

response against body tissues. This, in fact, contributes to autoimmune diseases, bowel

inflammatory disease, and other life-threatening conditions (Hasain et al., 2020). Due to the

coevolution of the human microbiota and the immune system, a balanced and systematic

interaction occurs over time. However, an alteration in the host-microbiota affects this

interaction leading to impairment in immune response which may result in inflammatory

disorder. Sundin et al. (2017) describe the relationship of the gut microbiome with a compromise

in the integrity of the gastrointestinal barrier in inflammatory bowel disease (IBD) (Sundin et al.,

2017). Barrier around epithelial cells forms a tight junction, which separates tissue space and

controls the movement of solutes across the epithelium. The barrier function of the intestine may

be affected by a damaged mucus layer, leading to a defective cell linkage attachment. A

reduction in gut Firmicutes leads to increased levels of proinflammatory cytokines (IL12, IFN-c)

and reduced anti-inflammatory cytokine (IL-10) levels. Certain helminth infections were

associated with anti-inflammatory organisms that prevent inflammatory bowel disease in IBD

susceptible mice (Hayes et al., 2018).

2.3.3. Cardiovascular Diseases

Production of metabolites by the gut microbiota does not only affect the gut but also act

systemically. The production of trimethylamine N-oxide (TMAO) metabolites by certain

gastrointestinal organisms may be implicated in heart disease (He et al., 2019). The gut

microbiota actively metabolizes trimethylamine from diets rich in l-carnitine, choline, and

phosphatidylcholine to trime-thylamine N-oxide (TMAO) by hepatic flavins containing

monooxygenase. TMAO affects lipid transportation in the body and also induces the release of

precursors which promote foam cell formation and hardening of the arteries in animal models

(Dohlman et al., 2021). Intestinal dysbiosis has been discovered to be associated with

cardiovascular diseases. Dohlman et al. (2021) performed a clinical study on two categories of

individuals: individuals with low risk of cardiovascular disease and those with a risk of

cardiovascular disease. In their findings, a disrupted intestinal flora was associated with

individuals having a higher risk of cardiovascular disease (Dohlman et al., 2021).

Overrepresentation of certain organisms has been found to contribute to cardiovascular diseases.

Faecal transplants from hypertensive patients that had overexpressed Prevotella and Klebsiella

increased blood pressure in germ-free mice animal models. Furthermore, the faecal microbiota of

hypertensive mice showed a significant increase of the Firmicutes to Bacteriodetes ratio in their

stool (Hayes et al., 2018).

2.3.4. Systematic Infections Resulting from Bacterial Translocation

Systematic infection occurs by the continuous movement of bacteria in human hosts from the

intestinal mucosa to other extraintestinal sites. The risk of a systemic disease from translocating

organisms is higher in immune-compromised individuals who are hospitalized, undergoing

surgery, or in trauma (Jiang et al., 2018). Damage in the epithelium of the gut and the abuse of
antibiotics disrupts the microbiota, leading to an increase in facultative anaerobes and a defect in

the host immune responses. Some examples of translocating organisms associated with systemic

infections include Esherichia coli, Klebsiella pneumoniae, Enterobacter spp., Proteus mirabilis,

Enterococcus spp., Streptococcus spp., and Candida albicans. In addition, microbial

translocation contributes to systemic infection by the production of uremic toxins. (is discovery

suggests that an alteration in the microbiota of the gut may lead to the synthesis of nitrogenous

compounds which affect the integrity of the epithelial tight junction, allowing the transfer of

these organisms and its toxins to other parts of the body. Uremic toxins produced by dysbiotic

flora activate systemic inflammatory responses which is a trigger to several diseases (Jiang et al.,

2019).

2.3.5 The Human Microbiome and Allergic Diseases

Possible mechanism of the human microbiome in association with allergic diseases has been

identified. Although, little is known on the effects of the lungs microbiota on immune regulation

of the respiratory tract (Mao et al., 2018). However, the respiratory tract is greatly shaped by a

balanced gut microbiome that affects the mucosa of the lungs. A dysbiotic flora directly affects

the microbiome of the lungs through microaspiration, and this increases the occurrence of

respiratory diseases in humans. Liss et al. (2018) illustrated this finding in germ-free mice.

Experimental mice were devoid of an immune regulatory network which induced respiratory and

allergic diseases. Caesarean (CS) delivery of neonates has also been identified as a risk factor for

allergic diseases. The absence of normal maternal flora during CS predisposes children to such

diseases (Liss et al., 2018). Molecular-based studies have revealed that CS-delivered children

have lower counts of healthy flora (Bacteriodetes) in their gut. This reduces the anti-

inflammatory activities of Bacteriodetes and contributes to local tissue inflammation (asthma and
allergic rhinitis) triggered by genetic and environmental factors. A recent epidemiological study

reported a significant association between dysbiotic gut flora and the production of allergic

antigen (IgE) resulting in airway disease in children (Markowski et al., 2018). Additional studies

establish that children with lower microbial diversity of Bifidobacterium, Akkermansia, and

Faecalibacterium were susceptible to multiple allergen respiratory sensitivity (polysensitisation)

and may contribute to asthma at the age of 4 (Inda et al., 2019). A review by Huang et al. (2019)

further supports these findings. Germ-free mice were more susceptible to allergic airway disease.

After microbial colonization, the susceptibility was reversed and a reduced allergen sensitivity

was observed. Clinical studies on the incidence of allergies in Europe showed that farming

environments containing diverse microbial communities had a lower rate of airway allergies

(Massier et al., 2020). The mechanism behind this phenomenon has been linked to the activation

of the innate immune system in the epithelial cell of the respiratory tract. Exposure to farm dust

containing microbial diversities of Acinetobacter lwoffii and Lactococcus lactis has proven to

reduce respiratory inflammation in mice (Massier et al., 2020).

2.4. The Human Microbiome in Health Sustenance

2.4.1. Maintenance of Homeostasis

The human microbiome plays important roles in the maintenance and development of the human

body. These organisms are responsible for launching the immune system, affecting inflammatory

homeostasis and immune regulation in neonates and young children (May et al., 2019). In 2018,

Mao in his study reviewed that children who develop allergies later in life were identified to have

a higher prevalence of Bacteroidaceae and anaerobic bacteria with a lower count of

Bifidobacterium adolescentis, Bifidobacterium bifidum, and Lactobacillus spp. (Mao et al.,

2018). Continuous research studies on microbiome have also identified that these organisms
interact with and degrade external contaminants such as heavy metals, polycyclic aromatic

hydrocarbons, pesticides, ochratoxins, plastic monomers, and organic compounds. Following

renal filtration in the kidney, the toxins removed from the bloodstream are stored within the

bladder which provides substrates and a conducive environment for the urinary tract microbiota

to deactivate toxic substances (Kadosh et al., 2020). The activities of these organisms interplay

in the outcome of an infection. In the female genital tract, the protective mechanism is initiated

by indigenous microbial flora which is responsible for inducing innate immunity, including the

secretions containing cytokines, antimicrobial peptides, and inhibitory substances (Kadosh et al.,

2020).

2.4.2. Development of Host Immune System

Through coevolution of indigenous microbiota and the immune system, immune responses are

developed and enhanced by the immune system’s ability to differentiate between harmful

pathogens and commensal organisms that must be maintained (Shaikh et al., 2021). In the gut,

the composition of the microbiota influences the developmental aspects of the adaptive immune

system; therefore, the mammalian immune system, which is responsible for controlling micro-

organisms, is shaped by the human microbiota. In recent studies on the functions of the human

microbiome, it has been illustrated that the absence of these organisms or early alteration of

commensal organisms may result in exacerbated type II immunity and allergies as a result of an

abnormal immune functionality. In children, for example, alterations to the microbiota through

epigenetic influences such as caesarean births, an increasingly sedentary lifestyle, environmental

pollution, and Western-type diets have been linked with an increase in cases of childhood

allergic rhinitis (Shiao et al., 2021). Probiotics, breastfeeding, lifestyle changes such as allowing

children to play out in the morning sunshine (to promote vitamin D production), and allergen
specific immunotherapy have been proposed as factors promoting the development of the

immune system of and the prevention of atopy in children. The gut microbiota is responsible for

activating the proinflammatory (17cells and regulatory T-cells (Tregs) in the intestine (Si et al.,

2021). In addition, the human microbiota has significant influence on innate immunity. An

example is neutrophil ageing which reduces proinflammatory properties in the body (Paone and

Cani, 2020). These organisms stimulate neutrophil ageing through Toll-like receptor- (TLR-) and

MyD88-mediated signalling pathways. Microbial alteration leads to reduced circulation of aged

neutrophils and in return results in inflammation-related organ damage in models of sickle cell

disease or endotoxin-induced septic shock. Therefore, these organisms actively control disease-

promoting neutrophil which is necessary for inflammatory diseases (Paone and Cani, 2020).

Furthermore, the intestinal microbiota helps in the defence against pathogenic organisms. They

promote colonization resistance and the synthesis of antimicrobial compounds against invading

pathogens. For example, a balanced gut microbiome may be responsible for regulating antibodies

(CD8-T cells and CD4 cells) which respond to the invasion of influenza virus in the respiratory

tract (Scott et al., 2019). Also, the intestinal microbiota helps to improve and maintain the

gastrointestinal functions. The high concentration of organisms in the gut remains a problem to

the intestinal immune system, as the immune system needs to accept commensal microbiota and

dietary antigens while also retaining its ability to eradicate pathogens. The activation of colonic

regulatory T-cells (Tregs) is important in developing immune homeostasis. There are two types

of Tregs: the thymus-derived and peripherally derived Tregs (pTregs). There is complexity in the

differentiation of these two immune responses, but they have an essential role in immune

regulation. However, the pTregs, in particular, require microbiota to be active in the colon

(Tirosh et al., 2019).

2.4.3. Host Nutrition

The colonic microbiota makes significant contributions to the nutritional requirements of their

host (Zhuang et al., 2019). These organisms actively break down complex dietary constituents

that are indigestible (complex carbohydrates) by the intestinal cells making complex food

materials readily available for absorption and assimilation. In the digestive tract, major end

products of carbohydrates and amino acids are the short-chain fatty acids (SCFAs) which include

acetic, propionic, and butyric acids. These three dietary constituents, upon absorption by colonic

mucosa, serve as energy sources and precursors for the synthesis of mucosal lipids and stimulate

cell growth of the epithelial cell resulting in maintenance of gut integrity (Williams et al., 2019).

Colonic microbiota protects the large bowel against cancer by the production of butyrate from

the fermentation of complex dietary constituents. These important microbial activities in the

colon have resulted in the provision of essential nutrients which are not readily accessible but are

required for colonic health sustenance (Zhuang et al., 2019). In Africa, mothers and infants have

been discovered to contain a high level of Bacteriodetes and SCFAs in their stool as compared

with the European infants whose mothers consume Western diets low in SCFAs. Studies have

shown that the consumption of traditional and fermentable carbohydrate may have contributed to

the prevalence of healthy gut microbiome (Williams et al., 2019). Another important function of

the colonic microbiota is the provision of vitamins necessary for host development. Intestinal

bacteria such as Bifidobacterium spp., Bacteroides spp., and enterobacteria are responsible for

the production of vitamins (Wu et al., 2021). Vitamin K, for instance, is an important coenzyme

responsible for the synthesis of several clotting factors which include prothrombin (a deficiency

of this leads to delayed blood clotting and excessive bleeding). Also, folic acid is an important

precursor for DNA and RNA synthesis. Finally, they are involved in the synthesis of red and
white blood cells. Today, probiotics containing lactobacillus or Bifidobacterium are used in

treatments of allergic diseases. Findings from the use of probiotics as treatment options have

revealed that an enhanced immunomodulatory effect is achieved by reducing or inhibiting

antigen-inducing T-cell activation and also suppressing cell signalling protein (tumour necrosis

factor (TNF) involved in systemic inflammation (Wu et al., 2021).

2.5 Microbiome Intervention and Engineered Microbes on Combating Pathogens

2.5.1 Microbiome Transplantation

Currently, microbiome transplantation is already clinically applied for pathogen infections such

as Clostridioides difficile infection (CDI). CDI is the most important risk factor contributing to

antibiotic-associated diarrhea in hospital, and is prevalent in many developed countries.

Recurrent C. difficile infection (rCDI), in the case of atypical and vancomycin resistance is

difficult to treat, and first-line antibiotics therapy may induce the expansion of antibiotic-

resistant organisms (AGOs) such as VRE (Liao et al., 2019). Kwak et al. (2020) reported that

transplantation of RBX2660, an FMT drug, can significantly recover rCDI patients’ microbiome

diversity and eliminate major AGOs. Besides, Jiang et al. (2018) confirmed that the effectiveness

of oral lyophilized fecal microbiota and FMT by enema on CDI are similar. An exploratory study

demonstrated the usage of vaginal microbiome transplantation to treat symptomatic, intractable,

and recurrent bacterial vaginosis patients. Transplantation of a healthy volunteer microbiome

remarkably improved patients’ symptoms and reconstructed a Lactobacillus-dominated vaginal

microbiome, and relieved patients’ bacterial vaginosis long-term (Lev-Sagie et al., 2019). This

implies the diversity of microbiome transplantation forms that can help expand the application of

microbiome transplantation. In addition, clinical studies of FMT for more difficult multidrug-

resistant (MDR) pathogen infections, such as extended spectrum β-lactamase (ESBL)-producing

and carbapenemase-producing Enterobacteriaceae, vancomycin-resistant Enterococci (VRE), or

methicillin-resistant Staphylococcus aureus, are likewise being conducted, and several clinical

cases reported their efficacy and safety (Lev-Sagie et al., 2019).

2.5.2 Probiotics

The use of probiotics could also treat pathogen infections. Probiotics usage can be considered as

a microbiome transplantation of known microbiome composition, so it may be more applicable

to those infections with clear probiotic-pathogen interactions. For example, the vaginal

microbiome is an important commensal microbiome in women, and participates in vaginal

homeostasis. It is known that a Lactobacillus-dominated vaginal microbiome is beneficial to

prevent vaginal infections. In HPV-infected women, reconstruction of the vaginal microbiome

via Lactobacillus rhamnosus implementation aids the clearance of HPV-infection, and a long-

term (6 months) probiotic group showed a higher percentage of clearance of HPV infection

(Palma et al., 2018) compared to the short-term (3 months) probiotic group (Palma et al., 2018).

In ventilator-associated pneumonia (VAP) patients, the pathogenic enteric bacteria aspirated

from the oropharynx is regarded the main etiology. VAP patients treated with probiotic

Lactobacillus plantarum to reconstruct gut microbiota exhibits the same effectiveness as

antibiotic (chlorhexidine) treatment, implying that the probiotic usage may be an alternative way

to control chronic infection (Klarin et al., 2018). Combining probiotics and prebiotics treatment

(Bifidobacterium breve strain Yakult, Lactobacillus casei strain Shirota, and

galactooligosaccharides) has also been reported to be effective in preventing sepsis patients from

enteritis or VAP (Shimizu et al., 2018). These studies suggest that probiotics could perform as a

microbiome regulator to prevent or treat pathogen infection. Although the current range of

applications is still narrow, as the understanding of microbiome-pathogen interaction improves,

more probiotic drugs will be available in the future as primary or adjunctive agents for a wide

range of infections (Shimizu et al., 2018).

2.5.3 Use of Engineered Microbes for Differential Diagnosis or Detection of Pathogen

Infections

With increased understanding of pathogen-host interactions, engineered microbes can be used as

targeted tools in pathogen infections. First, engineered microbes can be used in vivo for

differential diagnosis or detection of pathogen infections. Unlike traditional time-consuming

diagnostic approaches that require culture, engineered microbes can rapidly detect the infecting

pathogen species by carrying sensors that sense pathogen characteristics and thus provide

guidance for subsequent treatment. The work of Mao et al. (2018) provides a proven paradigm

for engineering microbes to aid in the diagnosis of pathogens. Vibrio cholerae produces the

characteristic quorum-sensing molecule cholera autoinducer 1 (CAI-1). Mao integrated a sensor

that senses CAI-1 with a downstream β-lactamase into Lactococcus lactis as an engineered

microbe that senses Vibrio cholerae. When gavaged to mice, engineered Lactococcus lactis

sensed CAI-1 and initiated β-lactamase expression. Mice feces containing β-lactamase

catabolized nitrocefin and produced a color change, thus suggesting Vibrio cholerae infection.

This study illustrates the feasibility of engineered microbes in pathogen diagnosis and suggests

its effectiveness and simplicity (Mao et al., 2018).

 CHAPTER THREE

3.0 CONCLUSION

The commensal microbiome plays a different role in promoting or inhibiting disease processes

such as metabolic diseases, cancer, and pathogenic invasion. Due to the extensive involvement of

the commensal microbiome in the maintenance of homeostasis in the human body, microbiome

intervention has become an emerging direction in disease treatment with high potential for

clinical application. While traditional methods of microbiome intervention, including antibiotic

treatment and fecal transplantation, have already been used in certain diseases, engineered

microbes have the potential to be the next-generation probiotics due to their precision and

versatility in regulating host physiological activities. In addition, engineered microbes can sense

and record certain signals inside the body to reflect host health status.

3.1 RECOMMENDATION

Microbiome research is transforming our understanding of human biology. There are still there

are still many answers that remain unanswered, including detailed microbiome transmission in

body sites, lifestyle impacts to microbiome transmission, how microbiomes evolve and stabilize

after antibiotics, population resilience and even how other members such as Fungi, Archaea or

even the virome respond to changes in the bacterial biota, from perturbations to new probiotic

therapeutics and if and how these dynamic changes continue longitudinally through time.

Although there is still much research to be done to understand the mechanistic links between the

microbiome and disease, this branch of the microbial sciences is opening vast opportunities for

therapeutic treatments and improving human health.

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