Human Microbiome as Drug Delivery System

Taraj Vyasa, Abhishek S Dhobleb, Meghna Pandeya, and Gautam Singhvia, a Department of Pharmacy, Birla Institute of
Technology and Science (BITS), Pilani Campus, Pilani, Rajasthan, India; and b Department of Chemical Engineering, Birla Institute of
Technology and Science (BITS), Pilani Campus, Pilani, Rajasthan, India
© 2022 Elsevier Inc. All rights reserved.

Introduction 1
The Human Microbiome Project (HMP) 2
Genesis of HMP and Parallel Efforts 2
Phases of HMP 3
Phase I (2007–2014) 3
Phase II (iHMP) (2014–2016) 3
Impact of HMP 3
Relevance of HMP in Microbiome Therapy 3
Advantages and Disadvantages of Using Human Microbiome as Drug Delivery System 3
Motivation Behind Therapeutics 3
How is Microbiome Therapeutics Done? 3
Opportunities in Microbiome Therapeutics 4
Challenges in Microbiome Therapeutics 5
Approaches in Microbiome Therapy 5
Additive Therapy 5
Subtractive Therapy 6
Modulatory Therapy 6
Future Outlook in Microbiome Therapeutics 6
Formulation Strategies 6
Probiotics 7
Microbial Encapsulation 7
Microencapsulation 8
Synthetic Microbes 9
Engineered Consortia 9
Targeted Delivery Through Bacteriophage 10
Bacteriocin Based Delivery 10
Bacterial Surfactants 11
Microbe Derived Extracellular Vesicle 11
US-FDA and “Live Biotherapeutic Products” 12
Future Perspectives 12
References 12

Introduction

Microorganisms are diverse communities that form an ecosystem on the interior as well as the surface of humans to build micro-
biomes. Joshua Lederberg coined the word microbiome for these diverse community of microorganisms. It is reported that more
than 10,000 microorganism species occupy the human microbiota and more than 8 million genes that are linked among the various
microbiome present in the human body. Microorganisms generally inhabit the gastrointestinal tract, saliva, oral mucosa, skin, and
conjunctiva, and among all these regions majority of microbes reside in the colon of the human body. Each of these microbiomes
are distinct from each other as they live in different locations in the body, and the relationship between microorganisms and the
human body is equally advantageous for both of them (Chellappan et al., 2019).
The microbiome plays a principal role in human health by supporting process such as:
1. To sustain the intestinal integrity and supporting the barrier purpose.
2. Provide essential nutrients, vitamins, and useful fatty acids like butyrate by breaking down the food.
3. It maintains the immune functions of the body.
4. Protect from various infections by secretion of antagonizing pathogens and bacteriocins.
5. Regulate the metabolism of the body as well as maintain mental health.

Reference Module in Food Sciences https://doi.org/10.1016/B978-0-12-819265-8.00006-1 1

2 Human Microbiome as Drug Delivery System

The advancement in the field of the genome sequencing technologies and metagenomic analysis has pulled the interest of scientists
toward studying the community of microorganisms in the body. Remarkable variation has been noticed in terms of species that
make up a person’s microbiome (Jimenez et al., 2019).
Research in the microbiome field is shifting our perception toward human biology, and it has now become clear that we must
consider that microbes that play a critical role in health and disease as shown in Fig. 1. Recent research points toward an interesting
perspective that microbes in complex ecosystems ultimately reach a stable point, even after the putative dysbiosis has occurred. This
forms the basis of the overarching ambitious idea of using the microbiome as a drug vehicle or drug itself. Data-driven quantitative
approaches backed by inter and cross-disciplinary approaches like machine learning (ML), artificial intelligence (AI), and the
internet of things (IoT) will significantly facilitate the creation of microbiota-based therapeutics (Petrosino, 2018; Zmora et al.,
2019).

The Human Microbiome Project (HMP)

There are more microbes than human cells. Understanding of microbial genes and their putative role in human survival were also
building up. However, a comprehensive study on the human microbiome, an easily accessible database, and a software toolchain to
access the information were lacking for a long duration of time. Scientists were of the opinion that a comprehensive investigation
and profiling of the human microbiome would significantly enhance our understanding of their role and function, revealing valu-
able insights (Proctor, 2011).

Genesis of HMP and Parallel Efforts

Around 2007, the Human Genome Project (HGP), which was once perceived as the world’s largest international collaborative scien-
tific biological research, got logically, conceptually and experimentally extended into the Human Microbiome Project (HMP) (Turn-
baugh et al., 2007).
In parallel to the HMP, there were concentrated attempts on creating a collaborative pool of datasets to establish the interrela-
tionship between microbiome structure and their function in the human body. One such organized effort is the International
Human Microbiome Consortium (IMHC) (Zhao, 2010). After the success of the Canadian Microbiome Initiative, currently
advanced research is being carried out under Canadian Microbiome Initiative 2 (CMI 2) better to understand the mechanistic links
between dysbiosis and human disease to understand the underlying causes of disease and discover new preventive and therapeutic
interventions (CIHR, 2009).

Sustain the
intesnal
integrity

Provide
Regulate the essenal
metabolism nutrients,
of the body vitamins and
Role of fay acids
human
microbiome

Maintains Protect from

the immune various
funcons infecons

Figure 1 Role of the microbiome in the human body.

 Human Microbiome as Drug Delivery System 3

Various tasks in HMP were carried out in collaboration with various international institutes of repute. These tasks ranged from
datasets to technology development, on one hand, to ethical and legal studies on the other. These tasks lasted for about a decade in
two different but well-focused phases.

Phases of HMP
Phase I (2007–2014)
The HMP-1 was a $157 million, a five-year effort aimed at identifying and characterizing the human microbiome (Nelson et al.,
2010).

Phase II (iHMP) (2014–2016)

There were three projects carried out under iHMP: Project 1 was on pregnancy and early birth; Project 2 explored the beginning of
Inflammatory Bowel Disease (IBD), while Project 3 focused on the onset of type 2 diabetes. All three projects were carried out at
multiple collaborating institutes. Mostly contemporary techniques for microbiome characterization involving the analysis of
marker gene (16S ribosomal RNA being most common) were used. As the costs started decreasing, other “omics” techniques
were also utilized (Franzosa et al., 2015).

Impact of HMP
The impact and achievements of HMP couldn’t just be measured in the number of research papers or patents it produced. As the
phrase goes “Data is new oil,” the most crucial impact of HMP was on the development of first of its kind comprehensive database
which was made available to the scientific community along with advanced analytical toolchain. This type of system allowed effi-
cient use of a large amount of data. This further led to the development of newer protocols for sequencing assembly. Creative algo-
rithms were developed for enhanced utilization of such datasets.
Furthermore, outcomes of some of the projects funded under HMP, e.g., time-laps moving pictures of the human microbiome,
served as a tremendous educational demonstration as well as public outreach material fueling public interest into the project.
Learning from HMP crossed the discipline boundaries with outcomes of the project on recognition of differentiating features for
healthy and problematic gut microbiome leading to parallel research on soil bacterial community and so on (Bergmann et al.,
2011).

Relevance of HMP in Microbiome Therapy

While it was a known fact that structure and function of the human microbiome changed over time, depending on the state of the
patient (healthy vs. unhealthy) as well as whether the patient is on any medication, HMP established that the microbiome ulti-
mately reaches a stable point, even after the putative dysbiosis has occurred (Huttenhower et al., 2012). HMP also helped in estab-
lishing the role of the microbiome in various diseases igniting further research on microbiome therapy. Another area that emerged
out after HMP is on the impact of sterility on pharmaceutical products and establishing the relationship between microbiomes on
entering the human body on the drugs.

Advantages and Disadvantages of Using Human Microbiome as Drug Delivery System

Microbiome plays a vital role in perpetuating health and fitness in the human body. With the successful implementation in two
phases, HMP indeed provided an exciting dataset to work on, opening doors to the drug delivery system using human microbiomes.
Analysis of HMP data for the purpose of exploring its possibility as an efficient drug delivery system is an intriguing field to explore.

Motivation Behind Therapeutics

Disturbances in the structure and function of a microbiome is being construed as a crucial step in the causation of infectious disease.
Broadly speaking, there are two related goals: (1) to elucidate the mechanisms of ecological interactions within a microbial
ecosystem by which resilience toward external perturbation is accrued, and (2) to identify actionable “biomarkers” of diagnostic
and prognostic value in distinguishing between normal and pathological states of the ecosystem. Currently, both approaches
exploit genomic and metagenomic approaches due to the relatively low cost and ample availability of next-generation sequencing
(NGS) technologies (Sarda and Hannenhalli, 2014).

How is Microbiome Therapeutics Done?

Approaches in microbiota-based therapeutics have been summarized in Fig. 2 (Mimee et al., 2016). (A) The first strategy is based on
the “Engineered Microbes”. There is a possibility of on-site disease marker diagnostics as well as the production of the drug on the
instigation of the engineered microbiome into native flora. (B) The second approach works more on the feed-forward strategy,
4 Human Microbiome as Drug Delivery System

Figure 2 Approaches in Microbiota-Based Therapeutics. (A) Engineered microbes have been one strategy for microbiota-based therapies. Gene
circuits are constructed using libraries of genetic parts to enable microbial production of therapeutic proteins. Introduction of these microorganisms
into the endogenous microbiota allows for in situ detection of disease biomarkers and/or drug production. (B) Designer microbial consortia are
informed by community profiling studies of clinical samples from healthy and diseased patients. Clinical isolates from these patients can then be
assembled into a defined mixture of microorganisms that can reprogram the microbial ecology within an individual. (C) Bacteriophages, bacteriocins
and small molecule antibiotics can be used to selectively eliminate deleterious microbes from the microbiota. Consequently, the loss of specific taxa
elicits a global shift in the microbial community as new constituents occupy the niches of the eliminated microbes. The addition of engineered
bacteria together with selective elimination of targeted strains may provide enhanced therapeutic efficacy. Adopted from: Mimee, M., Citorik, R.J., Lu,
T.K., 2016. Microbiome therapeutics d advances and challenges. Adv. Drug Deliv. Rev. 105, 44–54. https://doi.org/10.1016/j.addr.2016.04.032 .

where profiles of healthy and diseased microbial communities were established based on clinical studies. This information gets used
in designing a microbial consortium. This latter gets used to reorient the structure and function of the microbiome within each host.
(C) The last strategy works on the disruptive technique. Bacteriophages, a particular class of antibiotics with small molecules as well
as bacteriocins, can be exploited to preferentially eradicate the targeted sub-group of problematic microbes from the microbiome.
Niche theory kicks in with as different constituents occupying the territory of the eradicated microbes. In summary, a combination
of one of two approaches together, for instance, clubbing preferential eradication of chosen microbe with the incorporation of engi-
neered microbe (Singhvi et al., 2018a,b). The various approaches have been described in detail further in the chapter.

Opportunities in Microbiome Therapeutics

Like the advances in fingerprints and DNA based tests in the field of forensic sciences, recent research has shown that there is
a “microbial fingerprint” unique to every individual, that could be explored further. It has been observed that there is continuing
stability in the microbiome integrated with an interpersonal variation. This permits the identification of more than 80% of individ-
uals. These “microbial fingerprints” have the potential to form the basis of further analysis, such as identification and classification
of healthy vs. unhealthy individuals. This might lead to intervention strategies at the microbiome level, as outlined above.
Newer phenotypic fingerprinting tools could be explored to produce partitions based on informational entropy alone as well as
in combination with relevance to the specific therapeutic strategy. The human microbiome could show significant changes within
a very short span of time. A vital consideration to judge the efficacy of such therapies would be to come up with a quantitative
comparison of normal vs. problematic variation. Microbiome therapy has been building on the latest advancement in the field
of biosensors. Detection of disease-specific biomarkers may lead to the digitalization and objective approaches reducing human
intervention and subjectivity in microbiome therapeutics (Meskó et al., 2017).
 Human Microbiome as Drug Delivery System 5

Challenges in Microbiome Therapeutics

While it is well known that the microbiome structure and function could be altered by immigrant microbes, antimicrobials, disease,
and diet, we are still lacking the quantitative measurement to reach a confidence level required to carry out microbiome-based ther-
apies on a routine basis. In order to study complex microbial communities and their responsiveness/resilience to external pertur-
bation, we need to develop a label-free, sensitive, high throughput technique.
Furthermore, current efforts in developing microbiome therapeutics over-exploit genomic and metagenomic approaches due to
the relative cost-effectiveness and ample availability of next-generation sequencing (NGS) technologies (Kumar et al., 2016).
The fact that majority of microbes are still uncultivable; choosing the correct microbial mixture exogenously is challenging. Acci-
dently injecting the wrong microbe within the microbial chassis might lead to disastrous effects. Lack of fast, in line, automated,
efficient microbiome characterization technique makes it difficult to predict application-specific suitability of microbe(s). On the
other hand, a microbe which is meant for the therapeutic purpose may also be inviable in its new environment. The dynamics
of therapeutic microbes with the commensal/endogenous microbiota may be unknown and hard to predict. Lastly, scientists are
still exploring evolutionary and phenotypic robustness of the microbiome.

Approaches in Microbiome Therapy

With years long experience from HMP and capitalizing on cutting-edge research in establishing the relationship between micro-
biome and health, a new era of research on “microbiome therapy” is emerging. Just like any other therapy, microbiome therapy
also includes intervention in some form or the other. Various strategies have been emerging in this domain. On one end, approach
could be adding or enriching the beneficial strain of microbe while the other extreme could be selectively eliminating the problem-
atic ones. Some indirect approaches could be modulating the native microbiome by creating a favorable condition(s) for one or
more of the strains or species to outperform others. These strategies could be predominantly be classified as shown in Fig. 3
(Singhvi et al., 2018a,b).

Additive Therapy
This approach comprises of augmenting host microbiome with either individual microbe or microbial consortia. This consortia
could be sourced from the natural environment or could be engineered. The rationale behind the addition of exogenous microbiota
is to supplement or boost the function of the host-microbiome when it experiences a deficiency in maintaining the desired output to
carry out essential body functions. Engineered microbes could be developed to release the therapeutic agent.

Figure 3 Different approaches to microbiome therapy. Adopted from: Singhvi, G., Girdhar, V., Patil, S., Gupta, G., Hansbro, P.M., Dua, K., 2018b.
Microbiome as therapeutics in vesicular delivery. Biomed. Pharmacother. 104, 738–741. https://doi.org/10.1016/j.biopha.2018.05.099.
6 Human Microbiome as Drug Delivery System

Until recently, microbial consortia intended for therapeutic use have been from naturally occurring strains. However, now genet-
ically engineered microbes are gaining attention. Such genetically engineered microbes mainly delivered as Monotherapies could be
one such example. Additive microbiota therapy has been demonstrated in the prevention of vaginal simian/human immunodefi-
ciency virus (SHIV) transmission in macaques by a live recombinant Lactobacillus (Lagenaur et al., 2011) (Shen et al., 2015).

Subtractive Therapy
The primary rationale behind subtractive approaches in microbiome therapeutics is the removal of observed pathogenic or puta-
tively problematic microbe(s). This selective removal might be at the level of species, strain, or even higher. In recent years, there
has been increased attention toward such approaches because of the rise of antibiotic resistance. The majority of the currently avail-
able antibiotics may be construed as broad spectrum when it comes to gut microbiota. The reason behind this is that a single anti-
biotic may eradicate the beneficial bacteria along with the problematic one for which it was initially intended. This might lead to
dysbiosis and the essential functions of the gut microbiome during the period of such antibiotic administration might be compro-
mised. This disruptive effect may continue even after the antibiotic therapy is over, leading to the critical question: Is the net effect of
such antibiotics is positive or negative? While there are efforts in developing new antibiotics, drugs, and therapies, but due to highly
constrained regulatory and policy pipeline, it would be a while before these come to the real benefit of patients (Gökalp and Branke,
2020).
One such approach is based on bacteriophages. The use of natural or engineered viral particles is under consideration for selec-
tively killing problematic bacterial species in a complex mixture of the microbial communities (Reardon, 2014). Such bacterio-
phages or “phages” as popularly being addressed are not new. They were discovered approximately 100 years ago. These phages
are nothing but viruses that infect bacteria. The intention behind infecting bacteria is to exploit bacterial cellular resources to
produces multiple copies, sometimes killing the bacterial host in the process. In the context of microbiome therapeutics, such phage
therapies have been demonstrated on the intestinal pathogens (Sulakvelidze et al., 2001). To further develop subtractive micro-
biome therapy based on phages, an insight into the ecology and dynamics in the overall complex microbiomes need to be studied.
Efforts in terms of studies on the coexistence of phage populations in the murine gut might go a long way (Weiss et al., 2009; Maura
et al., 2012).
Another subtractive microbiome therapy approach is based on “bacteriocins.” These are produced by bacteria’s ribosomes and
considered as antimicrobial (Sheth et al., 2016; Chee and Brown, 2020).

Modulatory Therapy
Unlike additive and subtractive approaches, modulatory therapies primarily comprise of administration of non-living agents in the
host microbiome. Popular examples of such non-living agents are probiotics and prebiotics. There has been extensive research in the
past few decades on the modulatory effect of the first generation microbiome therapeutics (like probiotics, prebiotics, and symbi-
otics) on native system. When administered in the right amount and frequency, such non-living modulators could have multiple
benefits in terms of the addition of beneficial substrates for the selective growth of beneficial microbes or even eradicating the prob-
lematic ones by stimulating the body’s immune response (De LeBlanc and LeBlanc, 2014; Varankovich et al., 2015).
Newer antimicrobials are on the rise with selective eradication capabilities. Modulatory approaches might particularly be impor-
tant in metabolic disorders where direct intervention in terms of the addition of live microbe or removal of a species might lead to
severe complications.

Future Outlook in Microbiome Therapeutics

While each of the microbiome therapeutics approaches discussed above have their own benefits, a combination of one or more
approaches might be a way forward. For instance, additive approaches could be clubbed with subtractive or modulatory ones
that could achieve better impact. Phage therapy could be used in the very specific/targeted approaches. Routine therapeutics could
be tailored toward dietary supplementation with prebiotics. The main challenge lying ahead is the lack of rigorous quantitative
approaches and reliable mathematical models in identifying the specific problem and eradicating the same with microbiome ther-
apeutics. Considering complexity of gut microbiome, integrating these biological findings with upcoming state of the art data
analytics toolchain backed by machine learning (ML), artificial intelligence (AI) and internet of things (IoT) may be the way
forward.

Formulation Strategies

For utilization of the human microbiome as a drug delivery system, the microbiota needs to be formulated appropriately so that
maximum benefits can be harvested. In developing a microbiome as a drug delivery system, the three main approaches which are
utilized have already been discussed above, namely, Additive therapy, Subtractive therapy, and Modulatory therapy. All formulation
strategies are based on these three basic approaches. Here, some widely used strategies for formulating microbiome have been
described.
 Human Microbiome as Drug Delivery System 7

Probiotics
Probiotics are live, non-pathogenic microorganisms and are used for having a variety of favorable effects when consumed in suitable
amounts. Probiotics can be used for improving the overall health of a human in a multi-faceted manner, i.e., by enhancing immu-
nity by producing various amino acids, vitamins, etc. or by preventing the growth of other detrimental agents (Hsieh and Versalovic,
2008; Yadav et al., 2018).
Probiotics have found applications in many diseases already, including those related to gut, lung, cardiovascular system, etc.
In Cardiovascular diseases, a decrease in plasma glucose levels and cholesterol levels was observed with the use of conventional
probiotics such as Lactobacilli and Bifidobacteria (Tahri et al., 1996; Yadav et al., 2007). Lactobacilli were found to be useful for mildly
hypercholesterolaemic patients (Costabile et al., 2017). In type 2 diabetes patients, the regular administration of probiotics led to
enhanced antioxidant levels and an improved metabolic profile (Paper, 2013).
Probiotics have also shown promising results in curing Helicobacter Pylori infection. In a clinical trial, it was observed that the
consumption of Lactobacillus casei DN-114 001 as an adjunct to the standard treatment of antibiotics caused faster clearance of Hel-
icobacter Pylori from the body (Study et al., 2005).
A microbial species, A. muciniphila, is presently being explored as a new probiotic candidate as an adjunct for obesity and dia-
betes and other metabolic disorders (Cani and Vos, 2017). The preclinical data obtained from diabetic and obese mice showed
improved metabolism and is suggested to be safe for use in humans (Plovier et al., 2016).
Lactobacillus rhamnosus, GG, L. reuteri, Bifidobacterium, and the yeast “Saccharomyces boulardii” when used as probiotics were found
to be useful in diarrhea and gastroenteritis (Hsieh and Versalovic, 2008). Similar results were obtained for children of various demo-
graphic populations, suffering from diarrhea and acute gastroenteritis, when they were administered with probiotics (Shornikova
et al., 1997; Villarruel et al., 2007; Weizman et al., 2014).
For the development of a new probiotic, two approaches can be utilized:
➢ Either the genes which cause the benefits can be identified, isolated, and then the protein can be expressed through it, or
➢ The entire probiotic strain of organism can be identified and used (Chellappan et al., 2019).
Once the strains of desired microbes are identified, isolated, and produced on a large scale, the next step is administering it. The
formulation prepared, and the administration of the probiotics should be such that they maximize the targeting of the microbes
and also increase the life of the strains in the body.
Prudhviraj and colleagues prepared a fermentable polysaccharide-based formulation of probiotics with sulfasalazine. This was
done with the purpose of site-specific delivery of sulfasalazine to the colon. When these spheroids of probiotics and sulfasalazine
were administered to rats, the desired results were observed (Prudhviraj et al., 2015). The addition of probiotics helps in the replen-
ishment of the gut microbiota, which assists in the degradation of polysaccharide coating. This ultimately leads to the release of
sulfasalazine in the colon, which is responsible for improved results. This is an apt example of adding probiotics to regular therapy
for better results.
In one study, to improve the targeting and increase the survival of Bifidobacterium breve (a probiotic) in the GIT, the bacterial
strain was encapsulated within a multilayered capsule of alginate and chitosan. In vitro results showed an increase in the life of
free cells from <3 log CFU per mL to 8.84 log CFU per mL in a 3 layered matrix. When the number of layers was increased the
formulation of the probiotic was also capable of delivering the viable cells to the intestine (Cook et al., 2013). This shows that
several formulation strategies can be combined to obtain enhanced results.
A term “psychobiotics” has been coined for a specific-class of probiotics which, when administered in adequate doses, shows
therapeutic effect in patients of psychiatric illness (Dinan et al., 2013). In a recent study, Lactococcus rhamnosus, which is a commensal
strain, was administered to mice. It was observed that there was a significant reduction in anxiety and differential expression of the
GABA receptors in the brain (Bravo et al., 2011).

Microbial Encapsulation
Encapsulation is a way to give the microbes protection from the environment of the body so that it can reach the desired site of
action. Also, it aids in overcoming the stability issues in the delivery of microbiome and ultimately leads to an increased shelf-
life. Moreover, it gives protection to the cells from the host immune system. Many companies have attempted to encapsulate
the human microbiota, especially for delivering it to the gut.
In a study conducted on rats, an attempt was made to decrease the bacterial load of external E. coli in the GIT using the admin-
istration of bacteriophages. It was found that out of all the formulations which were administered, the capsules were the most effec-
tive. Encapsulating the bacteria led to a decrease in the E. coli load from 3.1  106 CFU to as low as 2.6  102 CFU within 6 days
(Abdulamir et al., 2014). Thus, it can be safely concluded that encapsulation can be very effective for the delivery of live microbiota
to the gut.
Attempts of encapsulating the bacteria in various bio-polymeric systems have been made, such as in alginate systems, proteins
and polysaccharide mixtures, chitosan-coated alginate systems, cellulose derivatives, in whey protein gel particles and modified algi-
nate systems (Huq et al., 2013). The Assembly Biosciences (Carmel, IN) has introduced the Gemicel capsule, which has two layers,
both of which dissolve at different pH levels in the GI tract. Thus, by using a single capsule, two doses of the drug can be delivered in
the two separate locations of the GI tract.
8 Human Microbiome as Drug Delivery System

Clinical trials for its use in Hepatitis and Ulcerative colitis are already being undertaken. Aquashell (Finch Therapeutics, Somer-
ville, MA) is a polysaccharide coating that is digested by microbes present in the colon. It is also a pH-sensitive formulation that
delivers various gut microbes to the colon and is currently undergoing clinical trials for Ulcerative colitis and Clostridium difficile
infections. Intract Pharma (London, UK) has come up with Phloral and Duocoat, which have been developed for duodenal and
colonal release of human microbes, respectively, and promise site-specific release (Cohen et al., 2019).
Extrusion encapsulation is also one of the methods of encapsulation. In this method, the bacterial cells are encapsulated within
the hydrogel particles. The bacteria are dispersed in the aqueous biopolymer solution, which can be gelled. This solution is later
extruded through a nozzle to form small biopolymer droplets with viable bacterial cells enclosed within it. These droplets are
then stabilized using further coating or gelation methods. The stabilized particles can then be washed, dried, and collected (Yeung
et al., 2016).
Currently, Seres is undertaking trials for encapsulated suspension of Firmicute spores, given the name SER-109 and SER-287 for
the Clostridium difficile infection (Khanna et al., 2016; Jimenez et al., 2019). For the type-1 Diabetes mellitus, ActoBiota is conducting
clinical trials for encapsulated Lactococcus lactis. Evelo is conducting clinical trials on a commensal strain for Psoriasis, Eczema, and
Melanoma. A trial is also being undertaken for capsules of Bifidobacterium breeve in the treatment of Asthma by 4D Pharma. 4D
Pharma is also conducting clinical trials for various encapsulated bacteria such as Bacteroides thetaiotaomicron, Blautia hydrogenotroph-
ica for the treatment of Clostridium difficile infection, and Inflammatory Bowel Syndrome (Jimenez et al., 2019). NuBiyota is also
conducting clinical trials for more than 30 commensal strains of bacteria in a capsule for the treatment of a range of diseases
such as Clostridium difficile infection, obesity, solid tumor, etc. (Jimenez et al., 2019)

Microencapsulation
Microencapsulation is a method to enclose the bacterial cells in the microcapsules using various methods for the purpose similar to
those cited for encapsulation. However, the microcapsules have the added advantage of increased bioavailability (Yeung et al.,
2016). Not only the foreign cells get protection from the host immune systems, but the diffusion of nutrients and viable cells is
not hindered as shown in Fig. 4 (Ausländer et al., 2012).
In an experiment conducted by Dr. Yeung and his colleagues, a strain of Lactococcus lactis was encapsulated in calcium alginate
beads. It was found that the viability of the cells increased as compared to non-encapsulated cells. Less than a two log reduction was
observed in the cell count of encapsulated bacteria, even when stored at room temperature for seven days (Yeung et al., 2016).

Figure 4 Properties of cell capsules: Transplanted or implanted cells can be microencapsulated into a semi-permeable membrane, which provides
permeability of oxygen, nutrients, and growth factors (Chang, 1964). At the same time it prevents high molecular weight complexes like
immunoglobulins and immune cells from reaching the cells. Thus, the surrounding membrane acts as a semi-permeable shield for the encapsulated
cells, protecting the host from these cells and vice versa. HMW: high molecular weight; LMW: low molecular weight. Adopted from: Ausländer, S.,
Wieland, M., Fussenegger, M., 2012. Smart medication through combination of synthetic biology and cell microencapsulation. Metab. Eng. 14 (3),
252–260. https://doi.org/10.1016/j.ymben.2011.06.003.
 Human Microbiome as Drug Delivery System 9

Many research studies have been conducted where bioengineered cells are incorporated in the microcapsules to harness the
benefits of smart cells along with microcapsules (Ausländer et al., 2012). This method is another example of how more than
two strategies can be combined to augment the effect of microbiome-based delivery system.

Synthetic Microbes
The synthetic biology can be used to engineer the commensal microbiome for various therapeutic and diagnostic applications.
Microbial transplantation of re-wired commensal bacteria can be a possible way to eradicate the dysbiosis caused by the existing
commensal bacteria, in diseases such as IBS. Re-engineering the gut genome is an option, but currently, there is a lack of genetic
tools. The benefits of using engineered commensal bacteria over the conventional oral and systemic routes are many. The
commensal bacteria can survive in particular niches in the gut; thus, the drug delivery can be very site-specific. Due to this, the
dose required for the administration will also drastically decrease (Donia, 2015).
Moreover, if the bacteria is controlled by gene expression, administering only one bacteria produces the desired molecule effec-
tively and in sufficient quantities, instead of administering several types of microbes. Further, it will also be more cost-effective as the
expense of formulating the drug molecule will be eliminated, especially when the drug has to be administered through routes other
than the oral route. Mimee and colleagues performed the rewiring of commensal bacterium Bacteroides thetaiotaomicron by using
various tools. These engineered bacteria could effectively, efficiently and accurately detect a signal, integrate this signal using an
intracellular circuit, and respond appropriately and timely to this signal (Mimee et al., 2015).
For demonstrating the signal detection, the LacO/lacI pair (taken from the E. coli bacteria) were induced in the Bacteriodes to
obtain two IPTG-mediated promoters with different induction profiles. Later, it was proved that all these four systems were inde-
pendent of each other, and a change of 8 to 104 times in gene expression was obtained with different carbohydrates (rhamnose,
chondroitin sulfate, arabinogalactan, and IPTG) (Mimee et al., 2015).
For demonstrating signal integration and propagation, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)
interference was used. Here, Cas9 protein, which was catalytically inactive and guided by RNA, was used to block transcription
by binding to the DNA. Thus, CRISPR interference specifically suppressed the gene expression in response to IPTG-induction.
For fine control of the stimuli over gene expression, a library of constitutive promoters and ribosome binding sites that spans 104
fold expression levels were constructed and characterized by the authors. The integration of circuits for signal detection, genetic
memory, and CRISPR interference was functioning as anticipated (Mimee et al., 2015).
Lagenaur and colleagues attempted to engineer the commensal bacteria Lactobacillus jensenii, to decrease the transmission of HIV
in the female macaques through the vaginal route. The bacteria were engineered such that they could produce the HIV-1 entry inhib-
itor Cyanovirin-N. A 63% reduction in transmission of a chimeric simian/HIV (SHIV(SF162P3)) was reported. Moreover, a decrease
in peak viral loads was observed in colonized macaques by six-folds. This method was found to be inexpensive and durable, thus
providing an additional basis to prove the effectiveness of engineered commensal bacteria (Lagenaur et al., 2011).
However, there are currently many limitations to the rewiring of gene expression in the bacteria.
The studies conducted so far mostly use carbohydrates as the inducer or suppressor. The challenge is to find a way to use other
molecules, such as proteins and fats, as an inducer or suppressor. Also, most of the studies have only been conducted on gut micro-
biota; thus, a vast area of human microbiome remains unexplored (Donia, 2015).
Also, a grave concern with engineering the commensal bacteria is the possibility of horizontal gene transfer and hence, bio-
containment. To overcome this, a “SWITCH OFF” module can be induced in the bacteria, as has been done by Mimee and
colleagues (Mimee et al., 2015) so that the gene expression will stop once the disease has been cured, or the deficiency has been
overcome.

Engineered Consortia
Engineering, the entire human microbiome, is being considered a viable option in the imminent future. This approach has the
prospective to treat diseases in the long run by altering the host metabolism, without any significant side effects. It is an approach
that is currently in use for the treatment of recurrent C. difficile infections. Moreover, the fecal microbial transplant is already in use
for the treatment of IBD. A study conducted by Ianiro et al. on patients suffering from IBD and C. difficile infection showed that
a reduction of symptoms was achieved in 71% of patients (Ianiro et al., 2014).
In a study conducted on hyperammonemia induced mice, it was attempted to engineer the microbial consortia to modulate the
metabolic activity urea. Hyperammonemia is a condition where a systemic accumulation of ammonia occurs due to the conversion
of urea to ammonia and carbon dioxide in the gut. The authors first eradicated the natural microbial consortia of the mice by the
use of antibiotics and PEG, as purgative. Following this, they inoculated the mice with altered Schaedler flora (ASF). ASF is a consor-
tium of 8 bacteria with minimal urease gene content. The consortia so established was stable for months and also lead to a decrease
in morbidity and mortality in mice with hepatic injury (Shen et al., 2015).
Currently, the company Rebiotix is undertaking a clinical trial for various Fecal microbiota, formulated as an enema for C. difficile
infections, Urinary tract infection, and Hepatic encephalopathy and another donor-derived microbiota, in the form of capsules
(Jimenez et al., 2019).
However, there are many safety concerns associated with such engineering of entire microbial consortia. One of them being the
contamination of the microbes which are to be inoculated. Also, the microbes collected from the host contain not only the desired
10 Human Microbiome as Drug Delivery System

microbes but also several fungi, pathogenic bacteria as well as parasites. Thus, due to a lack of study trials and adverse events asso-
ciated with engineered consortia, it is yet to be established as a viable treatment option.

Targeted Delivery Through Bacteriophage

Bacteriophages are naturally occurring viral parasites that infect bacteria, which can be used as such or after engineering to kill
specific bacteria in the gut. The targeting is highly specific, which is an advantage. Delivery through bacteriophages is an example
of a subtraction approach, where certain species of microbes are eliminated from the gut. Moreover, it can be a very effective method
to kill pathogenic bacteria, especially when there is an ever-growing threat of antibiotic resistance (Kingwell, 2015).
The engineered-phages are advantageous because of their ability to target specific niches, unlike their naturally occurring coun-
terparts. Moreover, these can also be modified in a way that they remain localized to the gut by increasing their GI residence time.
Barr et al. demonstrated this in an experiment in which the phage to bacteria ratio in the gut mucus was increased by 4.4 folds (from
9:1 to 39:1) as compared to surrounding areas. The authors proposed that increasing the ratio would lead to a decrease in the
account of infections. The reason for the increase in the association of phage to mucus was that some phages have Ig-like protein
domains on their capsids. When infective strains of E. coli were made to infect the host, it was observed that the account of the bacte-
rial pathogen adhering to the mucus surface decreased when the surface of mucosa was pre-treated by the phages (Barr et al., 2013).
In another study, the Saccharomyces cerevisiae was engineered with the genomes of bacteriophages for targeting of Yersinia and
Klebsiella bacteria. It was observed that after treating the bacterial population with the engineered bacteria for an hour, more
than 99.9% Klebsiella sp. 390 and Y. ptb IP2666 was eradicated without any significant effect on the remaining bacterial species.
Similar studies were conducted using phage cocktails, and the same results were achieved (Ando et al., 2015). Studies on animals
have shown that phage treatment might be a treatment for acute respiratory infections, such as Pseudomonas aeruginosa (Debarbieux
et al., 2010) and in the treatment of systemic infections such as those caused by Staphylococcus aureus.
An attempt was made to prepare engineered enzymatic bacteriophage, which could disperse pathogenic biofilms, which play
a significant role in the development of many infections. The bacteriophages were engineered such that they would disrupt the
bacteria present in the biofilms as well as biofilm matrix. The engineered bacteriophage showed a 99.97% removal of bacterial bio-
film (Lu and Collins, 2007).
Bacteriophage therapy can also be used as an adjuvant for the antibiotics, as antibiotic resistance is on the rise. Some studies
showed that the bacteriophages can be used to target the non-SOS networks, thus leading to overexpression of specific genes aiding
the antibiotics (Lu and Collins, 2009). It was also shown that bacteriophages could be engineered to reduce the numbers of
antibiotic-resistant bacteria.
In another research conducted, various solid and semisolid formulations of bacteriophages were prepared to test their lytic
capacity against cutaneous bacteria (Brown et al., 2017). A range of creams and ointments developed in the study were able to
exhibit the lytic activity against the Propionibacterium acnes, due to the presence of the bacteriophage. Phages were also formulated
as pessaries and troches, which showed good lytic activity against the Rhodococcus equi.
Recently, many studies were conducted where the phage therapy has been used orally, or through the parenteral route, through
the pulmonary route for many chronic and acute infections, many of which have been discussed by Malik et al. (Malik et al., 2017).
Moreover, bacteriophages can be engineered using various genetic tools now available. The enzymatic assembly of DNA (Gibson
et al., 2009) and the type I-E CRISPR-Cas system can be used to engineer the bacteriophages efficiently (Kiro et al., 2014).

Bacteriocin Based Delivery

Bacteriocins are the peptides or proteinaceous toxins produced by bacteria produced ribosomally, which inhibit the growth or kill
other bacterial strains. Bacteriocins-based delivery is a subtractive therapy approach. Low toxicity and high potency are the chief
advantages of bacteriocins (Cotter et al., 2013). In a study conducted recently, mice models were used to demonstrate that the
E. coli was engineered to produce bacteriocins, which inhibited the biofilms in the case of the P. aeruginosa gut infection (Hwang
et al., 2017). The P. aeruginosa PAO1 releases a Bacteriocin Pyocin S5, which was engineered by Saeidi et al. to successfully kill
the Pseudomonas bacteria (Saeidi et al., 2011).
Currently, two bacteriocins, Nisin (obtained from Lactococcus lactis) and Carnocyclin A (obtained from Carnobacterium maltar-
omaticum) are available commercially. The bacteriocins from Carnobacterium maltaromaticum are effective against gram-negative
as well as gram-positive bacteria (Martin-Visscher et al., 2011). Nisin and other bacteriocins derived from Lactococcus lactis are effec-
tive against specific strains, including Methicillin-resistant Staphylococcus aureus (MRSA). These bacteriocins can be an alternative
treatment from skin infections caused by S. aureus (Van Staden et al., 2016).
In an experiment conducted, it was demonstrated that bacteriocin Abp118 produced in Lactobacillus salivarius UCC118 was effec-
tive against the Listeria monocytogenes infection in mice models. The mice were fed with the Lactobacillus salivarius UCC118 for 6 days,
after which they were infected with luminescent Listeria monocytogenes. It was observed that the amount of light emitted from the
livers and spleens of placebo was significantly higher (5.910.82 Log10 relative light units per organ) than those fed with the pro-
biotic strains (3.961.18 Log10 relative light units per organ) (Corr et al., 2007).
Bacteriocin lacticin 3147 was effective in killing C. difficile without disrupting other microbiota in the human distal colon model
(Rea et al., 2011). When the concentration of Bacteriocin lacticin 3147 was increased to 270 mmL, the pathogen could not be detected
after 12 h. It was also found that Thuricin CD, which is a narrow-spectrum bacteriocin produced by Bacillus thuringiensis that exhibits
 Human Microbiome as Drug Delivery System 11

potent activity against C. difficile. Its activity is more than that presented by Vancomycin. The added benefit was that no activity
against gut microbiota or any administered probiotic was observed (Rea et al., 2011).
Thus, given the effectiveness and benefits, active research is being undertaken to discover more bacteriocins and formulate them
effectively.

Bacterial Surfactants
Apart from the applications stated above, there are several ways of delivery to and using the human microbiome. One of them is
through bacterial surfactants. These are also known as bio-surfactants or microbial surfactants. Surfactant are one of the major
constituents for dispersion and nanocarriers systems including liposomes, solid lipid nanoparticles, lyotropic liquid crystals, poly-
meric nanoparticles, and many more (Girdhar et al., 2018; Singhvi et al., 2018a; Joseph and Singhvi, 2019; Waghule et al., 2019).
Microbial surfactants are amphiphilic molecules produced by microbes. They are a promising novel tool for delivery to the human
microbiome because of their high biodegradability, high selectivity, activity at extreme temperatures, and low toxicity (Banat et al.,
2010). But apart from these, some bio-surfactants exhibit distinctive characteristics such as antimicrobial, anti-viral, anti-cancer
activities. Some studies show the utility of bio-surfactants in nano-sized vector formulation, such as liposomes, microemulsions,
nanoparticles, etc.
Rhamnolipid (Pseudomonas aeruginosa), Surfactin (Bacillus subtilis), Iturin (Bacillus subtilis), Fengycin/Plipastatin (Bacillus subtilis),
Lichenysin (Bacillus licheniformis) are some examples of the microbial surfactants.
Microbial-surfactants are used in liposome-based vectors for gene transfection. Lipofection is a prospective method for the
delivery of selected genes to the cells using cationic liposomes (Rodrigues, 2015). In a study, biosurfactant Mannosylerythritol lipid
A (MEL-A) produced by microorganisms was used liposomes preparation. The studies showed that the transfection efficiency was
significantly higher by MEL-A then by the other surfactants used (Igarashi et al., 2006).
Microbial surfactants are also being used in the development of nanoparticles. Gold and silver nanoparticles have been synthe-
sized by Reddy et al. using Bacillus subtilis. The authors reported that spherical and stable gold particles were synthesized intra-
cellularly as well as extra-cellularly, while the silver nanoparticles were synthesized only extra-cellularly. The release of surfactin
was credited as the primary reason for the formation of stable nanoparticles. Surfactin is a cyclic lipopeptide synthesized by Bacillus
subtilis, which acts as a bio-surfactant and hence increases the stability of the nanoparticles (Satyanarayana Reddy et al., 2010).
Apart from these systems, it was observed that the bio-surfactants significantly increased the stability of microemulsions. A
system of rhamnolipid, which is a bio-surfactant, along with n-heptane and water, was prepared to produce a nanoparticle-
based emulsion of Nickel oxide. A stable emulsion was obtained, with uniform distribution of nanoparticles. The nanoparticles
so generated with rhamnolipid as biosurfactants were also found to be spherical and stable. However, it was reported that the
size of nanoparticles was sensitive to the pH and temperature (Palanisamy and Raichur, 2009).
Thus, it can be concluded that due to the numerous advantages over their chemical counterparts, they have a vast scope in the
development of various delivery systems. Their ability to form organized aggregates such as bilayers, lamellar aggregates, reverse
cylinder aggregates, etc. offer additional benefits. Also, they are biodegradable and thus are the green alternatives to their chemical
counterparts. However, their nature and stability are two major concerns for their usage. Moreover, these are not cost-effective, and
hence their uses are still limited.

Microbe Derived Extracellular Vesicle

Extracellular vesicle (EV) is used to describe all nanoparticles derived from bacteria formed by lipids and are bi-layered. These trans-
port lipids, proteins, and nucleic acid from cells to the extracellular region (Yang et al., 2018). The EV’s have already been used for
immune modulation, for the transport of active biomolecules, for receptor-mediated host cell entry, etc. Earlier it was thought that
only the gram-negative bacteria produce EV’s, known as Outer Membrane Vesicles (OMV). Studies showed that even gram-positive
bacteria produce EV’s, which are called Membrane Vesicles (MV) (Lee et al., 2009). A few other classes of the EV’s are Eukaryotic cell-
derived exosomes, Microbial apoptotic bodies. EV’s majorly consists of lipids, proteins, nucleic acids, metabolites, etc. each of which
has a different function.
There are many advantages of using exosomes as drug delivery systems. Due to their small size and origin in animals themselves,
they can bypass phagocytosis. Stability in the blood allows the exosomes to travel long-distances in the body. Also, they fuse with
the cell membrane and are not engulfed by lysosomes (Bunggulawa et al., 2018). The loading of drug or endogenous molecules is
done by several methods such as simple mixing electroporation, freeze-thaw, sonication, incubation, extrusion, etc.
Recently, there has been quite some research in the relation of EV and microbiome and their utility. An experiment was conduct-
ed by Tian et al. where the exosomes were loaded with Doxorubicin, an anti-cancer agent, widely used for the treatment of breast
cancer. The drug was loaded in the EV’s using the electroporation method. The EV’s were administered to tumor tissue in BALB/c
nude mice. To increase the targeting capability of the exosomes, they were fused with iRGD (CRGDKGPDC) targeting peptide for av
integrin. It was reported that the targeting of such engineered enzymes (95.4%) was indeed better than blank exosomes (35%). In-
vitro assessment of the iGRD exosomes was done along with other control treatments. A 15 fold increase in tumor growth was
observed in control mice from day 3 to day 21, whereas in the iGRD-exosomes, it saw a rise of just 4 fold (Tian et al., 2014).
In another research study conducted by Sun et al. the effect of anti-inflammatory activity of curcumin was checked after loading it
in exosomes. The drug was loaded using simple mixing with exosomes. As compared to curcumin, exosomal curcumin was more
12 Human Microbiome as Drug Delivery System

stable, i.e., >80% drug was stable even after 150 minutes in phosphate buffer solution. The bioavailability of exosomal-curcumin
was assessed by administering it in C57BL/6j mice intraperitoneally. It was observed by the authors that the exosomal-curcumin
circulated in the mice even after 12 h, whereas when curcumin was administered alone, it eliminated before 12 h. The LPS-
induced shock model of C57BL/6j mice was used to assess the in vivo bioavailability. A distinct survival advantage was observed
in mice administered with exosomal-curcumin (Sun et al., 2010).
Exosomes have also been engineered with liposomes, PEG, etc. for delivering various bio-molecules such as RNAs, miRNAs, and
proteins to cells (Bunggulawa et al., 2018). According to the current scenario, it can be safely stated that as the pathways of biogen-
esis and functions of EV’s become more explicit, their potential will be fully harnessed in more fields such as pharmacognostic,
biomedical sciences, vaccine delivery, etc (Yang et al., 2018).

US-FDA and “Live Biotherapeutic Products”

In March 2012, USFDA published the guidelines for the first time for the Phase I clinical trials for Live Biotherapeutic Products
(LBP’s) under the title “Early Clinical Trials with Live Biotherapeutic Products: Chemistry, Manufacturing, and Control Informa-
tion” (Food and Drug Administration, 2016). There was a revision of these guidelines in 2016. These guidelines provide recommen-
dations for IND submissions for clinical trials with LBP’s. These guidelines apply to all the clinical trials conducted using LBP’s
whether in a clinical setting or commercially or under Title 21 of the Code of Federal Regulations Part 312 (21 CFR Part 312).
The guidelines are for all the foods marketed legally in the United States (US) and proposed for clinical uses regulated under section
351 of the Public Health Service (PHS) Act (42 USC. 262). However, these guidelines are not applicable to the LBP’s to be admin-
istered as gene therapy vectors, to oncolytic bacteria or oncolytic viruses.

Future Perspectives

Microbiome-based delivery systems are being developed for the treatment of multiple diseases, including IBD, Crohn’s disease,
C. difficile infections, Urinary Tract Infections (UTI), Ulcerative colitis, Obesity, etc. Significant work is being done in the field of
Fecal Microbe Transplant (FMT) for various diseases (Dhakan et al., 2019). However, the microbiome-based delivery systems
have not been utilized to their full potential.
The microbiome-based delivery might be the way to precision diagnosis and personalized treatment. Some studies show that the
microbiome if used for personalized medicine, might also be useful in the treatment and early diagnosis of fatal diseases such as
cancer. Personalized phage cocktails, personalized probiotic cocktails, etc. are under study (Rizkallah et al., 2012).
Utilizing commensal bacteria for biotransformation of the drug within the gut is also an area that is being explored. The genet-
ically engineered microbiome might also be exploited in the future for determining the pharmacokinetics of the drug. The
commensal bacteria might be modified to improve the therapeutic response of the patients to existing therapies.
The development of the microbiome as a drug delivery system faces numerous challenges. The primary limitations in this field
are the manufacturing and stability of the delivery system. Moreover, biocontainment and safety issues are another concern. The
development of genetic circuits can be advantageous in this scenario. Furthermore, the data generated from the Human Microbiome
Project (HMP) should be further studied to gain useful information. Also, most of the research in this field focuses majorly on the
diseases of the gut. But commensal microbes are present in the entire body. Furthermore, exploring the bacteria present in other
organ systems has a vast scope.

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