The Age of Next-Generation Therapeutic-Microbe Discovery: Exploiting Microbe-Microbe and Host-Microbe Interactions For Disease Prevention

MINIREVIEW
The Age of Next-Generation Therapeutic-Microbe Discovery:

Exploiting Microbe-Microbe and Host-Microbe Interactions for
Disease Prevention
Nathan Cruz,a George A. Abernathy,a Armand E. K. Dichosa,a Anand Kumara
B-10: Biosecurity and Public Health Group, Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, USA
a
Nathan Cruz and George A. Abernathy contributed equally to this article. Author order was determined by level of contribution during preparation of manuscript.
ABSTRACT Humans are considered “superorganisms,” harboring a diverse microbial col-

lective that outnumbers human cells 10 to 1. Complex and gravely understudied host-
and microbe-microbe interactions—the product of millions of years of host-microbe
coevolution—govern the superorganism in almost every aspect of life functions and over-
all well-being. Abruptly disrupting these interactions via extrinsic factors has undesirable
consequences for the host. On the other hand, supplementing commensal or beneficial
microbes may mitigate perturbed interactions or enhance the interactive relationships
that ultimately benefit all parties. Hence, immense efforts have focused on dissecting the
innumerable host- and microbe-microbe relationships to characterize if a “positive” or
“negative” interaction is at play and to exploit such behavior for broader implications. For
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example, microbiome research has worked to identify and isolate naturally antipathogenic
microbes that may offer therapeutic potential either in a direct, one-on-one application or
by leveraging its unique metabolic properties. However, the discovery and isolation of
such desired therapeutic microbes from complex microbiota have proven challenging.
Currently, there is no conventional technique to universally and functionally screen for
these microbes. With this said, we first describe in this review the historical (probiotics)
and current (fecal microbiota or defined consortia) perspectives on therapeutic microbes,
present the discoveries of therapeutic microbes through exploiting microbe-microbe and
host-microbe interactions, and detail our team’s efforts in discovering therapeutic
microbes via our novel microbiome screening platform. We conclude this minireview by
briefly discussing challenges and possible solutions with therapeutic microbes’ applications
and paths ahead for discovery.
KEYWORDS probiotics, fecal microbiota transplant, defined microbial consortia, thera-

peutic microbes, microbe-microbe interactions, host-microbe interactions, next-genera-
tion sequencing, unculturables
E very animal (and plant) on Earth is a result of multitudinous host-microbe interac-

tions, wherein at least one-member (microbe and/or host) benefits. Microbes assist
their animal hosts in various physiological processes: metabolizing and digesting food
nutrients, stimulating mucosal immune responses, producing essential vitamins, ener-
Editor Karen M. Ottemann, University of
gizing physioanatomical development, and providing resistance to colonization by California, Santa Cruz
intruding pathogens (1–3). Without microbial interactions, the host becomes predis- This is a work of the U.S. Government and is
posed to various disease conditions. This intellection can be appreciated, as germfree not subject to copyright protection in the
United States. Foreign copyrights may apply.
animals exhibit anatomical and functional differences in the gut, liver, lungs, enteric Address correspondence to Anand Kumar,
nervous system, and suboptimal immunity and endocrine functions (4, 5). However, akumar@lanl.gov.
the mechanistic aspects of microbe-microbe interactions (MMIs) and host-microbe The authors declare no conflict of interest.
interactions (HMIs) have been a major challenge to dissect. Many external factors (e.g., Published 6 April 2022
antimicrobials, diet, and environment) and internal factors (e.g., genotype) influence
May 2022 Volume 90 Issue 5 10.1128/iai.00589-21 1

Minireview Infection and Immunity
MMIs and HMIs (6). Studies have correlated perturbed HMIs with acute (7, 8) and chronic
(9) conditions; furthermore, the duration of HMI abruption and host age covary with the
severity of outcome (10). For example, vaginal delivery is an essential process for seeding
selective species of microbes on the skin surface and internal gut of newborn babies. In
contrast, caesarean births preclude mother-child microbe transfer, and such microbes are
replaced with environmentally acquired microbes; thereupon, newborns are prone to de-
velop chronic diseases in adulthood such as diabetes, obesity, asthma, and neurological
disorders (11, 12). During adulthood, broad-spectrum-antibiotic usage drastically affects
HMIs, leading to acute discomfort (i.e., antibiotic-associated diarrhea [13]). Hence, where
HMI imbalance is inevitable (e.g., caesarean birth and septicemia), limiting the duration of
disruption by supplying essential and beneficial microbes can potentially ameliorate the
deleterious health outcomes associated with perturbed HMIs (14).
The emergence of modern medical practices, germ theory, and the hygiene hypoth-
esis have erroneously imprinted on humanity that all microbes are pathogens. Such
ideologies have contemporized an increase in hygiene practices and overuse of antimi-
crobial products in attempts to eradicate the microbial collective. Indeed, this notion
has prevented millions of morbidities from infectious diseases across the world.
However, a paradigm shift is occurring with newly acquired knowledge of microbial
communities (i.e., the microbiome and microbiota) and their role in health. Emerging
evidence regarding microbes is revealing that not all microorganisms are necessarily
pathogens. In fact, only a limited fraction of known microbes are responsible for the
development of disease (15). Any given microbial species in a complex community will
likely have various kinds of interactions with its neighboring native organisms. Some of
these interactions may provide advantages and/or disadvantages to a given cohabiting
neighbor. Therefore, harnessing such microbial species by dissecting MMIs and HMIs is
an opportune path for discovery of next-generation therapeutics (16).
It is important to mention that the term “therapeutic microbes” can also refer to ge-

netically engineered microbes that sense and respond to their environmental cues by
secreting therapeutic molecules, and such organisms are not covered in this review
(17, 18). Unlike conventional antimicrobials, therapeutic microbes have many favorable
traits, such as consisting of live cells, having self-renewal abilities, being unlikely to
induce resistance, employing broad mechanisms to inhibit pathogens, being pathogen
specific, and inducing minimal side effects (19). In this minireview, we present the his-
torical integration of therapeutic microbes (probiotics), the current perception of their
use in therapy (i.e., microbiome based or rationally selected defined consortia), discov-
ery through exploiting MMI and HMI, and our team’s efforts in banking therapeutic
microbes by use of a novel microbiome-screening platform. We conclude by briefly dis-
cussing challenges and possible solutions with therapeutic microbes’ applications and
present anticipated future directions of discovery.
EARLY HISTORY OF THERAPEUTIC MICROBES

Bipedalism, tool design, domestication of fire, transition to an agriculturist society, and
the development of written language are the decorated hallmarks of human evolution.
Subsequently, the human adaptations have coincided with climate variation, thereby bias-
ing distinct physiological and socioeconomic characteristics that have promoted our health
(20–23). An often-underappreciated feat in human evolution is the resourcefulness and
implementation of microbiology practices (24). The first serendipitous applications origi-
nated during early civilization with the realization of the beneficial health effects of fer-
mented foods. Although humans were unaware of the existence of microscopic organisms,
breakthroughs in fermentation increased food preservation and enhanced recovery from
some diseases (25, 26). Detailed archeological methodologies (analogical, ethnographical,
archaeobotanical, etc.) and chemical analyses (mass spectrometry [MS], nuclear magnetic
resonance [NMR], high-pressure liquid chromatography [HPLC], etc.) of vessel sherds have
dated the earliest uses of fermented dairy, vegetables, bread, and beverages to 8000 BCE
(Fig. 1) (27–29). Furthermore, collections from bronze vessels over the succeeding

FIG 1 Milestones in the discovery of potential therapeutic microbes in human evolution (80, 81, 91, 99, 135–138).
5,000 years reveal optimizations of the fermentation process, resulting in improved

wines, breads, and medicine. As material trade expanded, numerous civilizations
(Mesopotamian, Egyptian, Greek, and Roman) traded knowledge of the dynamic
use of fermentation (30). As such, fermentation expanded across continental

distances, and in the 21st century, these practices have been adapted with human
culture.
With the definitive falsification of spontaneous generation in 1863 by Louis Pasteur’s
germ theory, microbes have been recognized in society as being influential. At the begin-
ning of the 19th century, marketing tools promoting health introduced microbes to the
public. For example, groups advertised and differentiated their milk from competitors by
advertising increased health benefits through their use of beneficial microbes (now identi-
fied as Streptococcus thermophilus and Lactobacillus delbrueckii) (31). In Spain in 1919, Isaac
Carasso made a similar push with marketing tactics to increase yogurt sales. At the same
time, viruses—particularly bacteriophages, which specifically infect and kill bacterial spe-
cies—were used as treatments for Shigella dysenteries in humans (32). Interestingly, mod-
ern history attributes the first eukaryotic therapeutic microbe to the accidental discovery of
the mold Penicillium notatum, which inhibited the growth of pathogenic Staphylococcus
aureus. However, earlier records suggest the Greeks and Indians used molds to treat infec-
tions prior to this. Ironically, the rise of interest in conducive therapeutic microbes (bacteria,
viruses, and fungi) rapidly halted with the emergence of antibiotics. Carrying over to the
next century (1930 to the present), antibiotics became ubiquitous and were viewed as a
panacea. However, the overuse of antibiotics has escalated the prevalence of antibiotic re-
sistance in bacterial pathogens.
As a response to the alarming rise of antibiotic resistance in bacteria, the 1980s
marked a global shift for discovering novel therapeutic microbes and public sponsor-
ship as a route for disease mitigation (33). At the same time, probiotics were touted as
beneficial microbes with a plethora of supported health claims. In 1991, the Office of
Alternative Medicine was established to evaluate and provide scientific data on the use
of probiotics in an effort to prevent customer exploitation by ungoverned marketing
strategies. Intense microbiome research over the last decade has indicated that unex-
plored Archaea species that make up ;1.5% of the human gut microbiota also possess

therapeutic potential in remediating atherosclerosis and kidney disease by metaboliz-
ing trimethylamine N-oxide (TMAO) levels in blood (19, 34). In summary, the interde-
pendency between humans and microbe interactions has given rise to a long record of
accomplishment in the use of therapeutic microbes to maximize health.
CURRENT METHODS OF PROBIOTIC DISCOVERY

A myriad of in vitro assays exist to evaluate a microbe’s inherent antagonist activities to-
ward known microbial pathogens. Such assays focus on determining a microbe’s potential
antagonism via direct interaction or through collected extracts (e.g., exoproteins). In vitro
methods have been routinely used to discover organisms that display antimicrobial charac-
teristics by using semisolid medium platforms (Table 1) (35). Live-cell antimicrobial activity
is assessed by coculturing two species but does not distinguish whether antagonism is a
product of diffusible inhibitors or direct contact (36, 37). Alternatively, layered-agar-based
methods can proliferate probiotics followed by harvested cell-free supernatant to minimize
confounding variables, thereby attributing antagonistic activity to secreted diffusible pro-
teins. In this method, inoculated agar plates containing the pathogen are seeded with a
layer of the microbe’s extract, where both pathogen and extract are titrated (35, 38).
Gelatin- and agar-based methods crudely express antimicrobial activity as either inhibition
zones (in square millimeters) or arbitrary units (AU) per milliliter. Notably, semisolid-sub-
strate techniques demand large amounts of materials and present reproducibility issues,
whereas novel high-throughput microdilution techniques provide elevated reproducibility
and rapid microbe screening. Microdilution protocols employ microwell plates, where each
well contains an inoculum of a predefined concentration of a pathogenic strain. Thereafter,
dilutions of the probiotic extracts are introduced to evaluate dose-dependent inhibition of
microbial growth (35, 39). Furthermore, the microdilution method provides a quantitative
estimate of a suspected therapeutic microbe’s lowest concentration at which it inhibits
complete growth of the pathogen to determine the minimum inhibitory concentration,
which is the universally accepted metric to quantify the lowest concentration of an

TABLE 1 Summarized attributes of therapeutic microbesa
Attribute Probiotics Fecal microbiota transplantb Defined consortiumc Therapeutic microbesc

Minireview
Definition “Live microorganisms which when Screened fecal transplant from healthy Collection of characterized microbes for Adequately characterized, or
administered in adequate amounts donor to recipient (90). Contains therapeutic use (111). May contain a engineered, single and/or collections
confer a health benefit on the host” (41). bacteria, virus, fungi and/or metabolites pure or mixed culture of bacteria and/or of microbes with targeted
Contains mainly bacteria and yeast fungi therapeutic applications
May contain single or a cocktail of
May 2022 Volume 90 Issue 5

bacteria, virus, and/or fungi
species
Discovery and/or rationale Beneficial relationship through the Administration of fecal suspensions Depleted commensal microbe First instance of antagonistic MMI:
fermentation of food that promoted performed by Ge Hong from healthy collections and/or cocktails of probiotic lytic phages against Shigella species
health individual to diarrheic patient (82, 83) species with beneficial effect (100–102) and Penicillium notatum against S.
aureus
Methods Diffusiona (41, 147–155) Screening: identify donors satisfying NGS and other omics approaches Engineering strain via synthetic
Agar disk prerequisite criteria followed by bioinformatics analysis to biology approach (17, 18, 120)
Etest Preparation: samples suspended in shortlist essential microbes (100, 101, Development of microbiome
Agar well saline 103). screening platform [HiSCI] (115)
Agar plug Administration: colonoscopy,
Cross streak nasogastric, oral capsules (139, 143,
TLCa (156–158) 144, 159, 160)
Agar diffusion
Direct bioautography
Agar overlay bioassay
FDA categorization (Table 2) Dietary supplement Biological product Biological product Drug
Applications Allergy (161–164) Selected noninfectious diseases Recurrent C. difficile infections Engineered strain: familial
Carcinogenicity (165) Psychological (144) adenomatous polyposis, urea cycle
Carcinogenicity and mutagenicity (166, Obesity (97, 141) disorder, cirrhosis, phenylketonuria,
167) Drug-induced dysbiosis (142) type 1 diabetes mellitus, oral
Cholesterol reduction (168) Gastrointestinal (143) mucositis (120)
Gastrointestinal (169, 170) Selected infectious diseases Therapeutic microbes: C. scindens
Endotoxemia (171) Rotavirus (88) and C. amalonaticus (103, 119)
Hypertension (135) Bacterial infections (87)
Immunomodulation (136) Fungal infections (58)
Kidney stone (oxaluria) (137) Parasitic prevention (173, 174)
Lactose tolerance (140)
Urinary tract infections (172)
Mechanism/microbes Direct Direct Direct and indirect Direct
involved Cholesterol clearance Establishing eubiosis May employ probiotic or FMT Engineered strain: sense and
Nutrient competition Novel antibacterial species mechanisms respond to external stimuli (i.e.,
Toxin suppression Pathogen-specific lytic bacteriophages pathogen presence) (17, 18)
AMP production Beneficial fungi Therapeutic microbes: may employ
Inhibitory peptide production Unidentified microbial metabolites multiple mechanisms to inhibit or
Competitive exclusion suppress pathogen of interest:
Indirect nutrient competition, toxin
Immunomodulation suppression, AMP secretion,
Gut barrier reinforcement inhibitory peptide production,
Enterocyte inflammatory stimulation competitive exclusion
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Infection and Immunity
(Continued on next page)

TABLE 1 (Continued)
Attribute Probiotics Fecal microbiota transplantb Defined consortiumc Therapeutic microbesc
Minireview
Indirect Indirect
Stimulation of host immunity and Stimulate host immunity and
physiology (see probiotics physiology
mechanisms)
Advantages Derived from human and animals Minimal side effects in contrast to Absence of known pathogens Target specific and minimal side
Consistent history of overall safe use antibiotic treatment (77) Minimal vertical or horizontal antibiotic effect (19)
May 2022 Volume 90 Issue 5

Considered GRASE by FDA Multifaceted clinical applications (77) resistant gene transfer High success rate in pathogen
.70% eradication rate against Known mechanism of action eradication
multidrug-resistant organisms (77) Reduced patient anxiety compared to Well-characterized and defined
FMT mechanism of action towards
Reflects patient-driven health care (175) target and host
No risk of transferable antibiotic
resistance genes (17, 18)
Disadvantages/limitations Limited data of mechanisms of action Relatively high failure rates, non-C. Difficult to discover and isolate essential Technology development in early
Exhaustive list of prerequisite criteria difficile conditions (10–20%) (91) microbes from microbiota stages
(Table 2) Difficulty in finding compatible donor(s) Currently less efficient than FMT in Requires personalized medicine
Various beneficial effect dependent on (92) disease remediation approach
species and strain Limited longitudinal studies for Lack of inclusion of other essential Standard treatment guidelines are
Batch-to-batch variation in large-scale safety, recipient compatibility, and microbes not yet established (121)
manufacturing its benefits (93) Biocontainment, strain stability, and
May harbor and transfer antibiotic Inadequate screening for pathogens quality control assurances have
resistant genes (101) not been proposed or attempted
Vaguely regulated by FDA Transplantation of uncharacterized (121)
microorganisms with unknown
interactions (102)
Enforced discretion by FDA for
temporary use against recurrent
C. difficile and non-C. difficile infection
(107)
Laborious process and elevated patient
anxiety
aNot an exhaustive list of current methodologies. MMI, microbe-microbe interaction; TLC, thin-layer chromatography; GRASE, generally recognized as safe and effective; FDA, Federal Drug Administration. Underlines indicate
appropriate subgroupings that the topic within the matrix's element can be divided into.
bThe terms “fecal microbiota transplant” (FMT) and “fecal transplant” (FT) are used interchangeably in literature but refer to the same concept. Sometimes “bacteriotherapy” is also used in place of “FMT” or “FT,” but this is not
accurate, as “bacteriotherapy” refers to the use of bacterial species for therapy, and FMT/FT contains other microbial organisms, like viruses, archaea, and fungi.
cDefined consortium and therapeutic microbes may appear to be the same but differ in selection, strain generation, and discovery approach; therefore, we put therapeutic microbes in a separate therapeutic class.
10.1128/iai.00589-21
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Infection and Immunity

antimicrobial compound that inhibits bacterial growth. Different versions of these two
forms of assays have been utilized since the discovery of bacteria, but these methods
remain time intensive, have relatively low throughput, possess limited application in evalu-
ating MMI, and presume underlying characteristics of cultured strains (e.g., high
penetrance).
Historical probiotic discoveries, during the inception of germ theory and early agar-based
experiments, are limited to accidental identification. Refinements of methods, specifically
ones that illustrate direct microbe-microbe inhibition, marked an increase in the prospects
for detection of beneficial bacteria. Until recently, testing probiotics has been restricted by
generic methods and biased selection of known bacterial species (e.g., Lactobacillus,
Bifidobacterium, etc.). The combination of interdisciplinary efforts, specifically through com-
putational analysis and culture independent sequencing, fostered the rapid discovery of the
next generation of beneficial microbes by predicting the existence of exploitable microbes
found in the most unusual places: sewage, soil, and the gut (40).
DEFINING PROBIOTICS: FDA

The Food and Agriculture Organization of the United Nations (FAO), the U.S. Food and
Drug Administration (FDA), and the World Health Organization (WHO) define a probiotic as
“a live microorganism(s) which when administered in adequate amounts confer a health
benefit on the host” (41). The FDA monitors marketed use, premarketing requirements,
and endpoint analysis; with a level of constraint based on intended use of the product. To
this end, there is still great confusion as to how the FDA views probiotics. With convoluted
definitions and liberal use of health claim benefits, manufacturers have been bypassing
regulatory categories—many of which promote a level of safety, function, and usability.
Manufacturers often include disclaimers such as “These statements have not been eval-
uated by the FDA” printed in nearly illegible font size to bypass additional testing require-
ments. Adding a disclosure statement on the product permits manufactures to apply con-

jectural assertions in advertisement. For example, businesses may cite relief of nonspecific
symptoms, thereby potentially putting consumers at great risk by delaying professional
medical attention. From Table 2, we can see that as a company transitions from advertising
“management” to “prevention” of a disease condition in their product’s health claim(s),
supervision and board approval from the FDA concomitantly increase. Greater efforts are
required when a probiotic ventures into increasing health claims. The advertisement of a
product’s therapeutic quality requires years of data accumulation, investigative new drug
applications, and review board approval, among other requirements, to be recognized as
generally safe and effective for human use. Thus, manufacturers are finding loopholes in
current FDA policies for profit, thereby prompting a need to refine existing guidelines in
order to protect the consumer.
APPLICATIONS OF PROBIOTICS
Numerous studies on probiotic research have been published; however, only a fraction
have shown the product to produce efficacious outcomes in disease remission that are
supported by mechanistic evidence (Table 1) (42, 43). Applicable probiotics commonly
demonstrate at least one of the following characteristics; (i) antagonism to pathogen prolif-
eration, (ii) competitive inhibition or outcompetition for nutrients; (iii) beneficial immuno-
modulatory effects on the host; and (iv) inhibition of endotoxin production (44–46).
Altogether, these mechanisms present a probiotic’s advantage directly via association with
the infectious agent or indirectly by enhancement of a host’s immunity.
Investigation into direct effects of a probiotic on other microbes convey two primary
mechanisms of action. Lactobacilli, abundantly available in dairy products, are known to
induce their antimicrobial effects by the production of bacteriocins (47, 48). Release of
these low-molecular-weight lipids (LMWL) induces its antimicrobial influence through
depolarization of its target organism, consequently introducing pores into the cytoplasmic
membrane and thus compromising cell viability. Structure-specific toxicity of LMWL results
from the influx of fatty acids coinciding with a pathogen’s inability to rapidly metabolize

TABLE 2 Tentative FDA categorization of foods and drugs based on intended use and their premarketing prerequisites, according to the 2021
Amended Federal Food, Drug, and Cosmetic Acta
Category [U.S. Code] Intended use Premarketing requirements
Medical food [Title 21, chapter 9, Used under the supervision of a physician If GRASE, no clinical studies required, but prior
section 360ee; 2011] and is administered “internally” with the research is needed; otherwise, IRB approval and
intended application for the “management clinical studies required.
of a disease or condition”
Dietary management
Food [Title 21, chapter 9, section 321 Articles used for food, drink, and its Typically, GRASE
(f); 2011] components If not GRASE, requires FDA disclosure
Permits “health claims” to appear on foods
and supplements
Includes natural foodborne microorganisms
Limited claim to “help maintain (micro)
flora”
Dietary supplement [Title 21, chapter A product, excluding tobacco, used to Not required for ingredient marketed before 15
9, section 321(ff); 2011] “supplement the diet,” intended for October 1994
ingestion, and “not present for use as a May be placed on the market with limited claims
conventional food” Manufacturer must disclose to FDA intended
Application of “new dietary ingredient is claims; however, the FDA may request
required” if product not marketed before substantial evidence
15 October 1994 If manufacturer makes claims regarding
ingredients’ use in health remediation, then
premarketing clearance is required
Cannot be marketed as “drug-like” or a disease
preventative
Drug [Title 21, chapter 9, section 321 Claims to “diagnose, mitigate, treat, cure, or IRB approval before preclinical and clinical trials
(g)(1); 2011] prevent a specific disease or class of Must complete NDA and IND trials before
diseases” marketing
Product not GRASE Possession of biologics license
Therapeutic microbes (single strain or Formal communication with FDA prior to clinical
consortium) with the above claims fall testing

within the definition of a “new drug” Submission of test protocols and endpoint
measurements 30 days prior
Transcript of investigational plan
Requires IRB review and approval
Biological product [Title 42, chapter “Application for prevention, treatment and/ Submission and approval of biologics license
6A, section 262; 2010] or prevention of a disease condition” application
Article contains no living cells but can IND studies
include any of the following: “virus,
therapeutic serum, toxin, antitoxin,
vaccine, blood, blood component or
derivative, or allergenic”
aIND, investigative new drug; IRB, institutional review board; GRASE, generally recognized as safe and effective; NDA, new drug application.
LMWL (49). Second, probiotics (e.g., Lactobacillus and Bifidobacterium) exhibit antimicrobial
properties by secreting conjugated bile acids in concert with LMWL (50, 51). Detergent
properties of bile acids result in dissolution of bacterial membranes by membrane frag-
mentation and cytosolic organelle dissolution (52–54). In light of these properties, bacterio-
cin-producing probiotics present a major advantage in disease mitigation and treatment
by interacting directly with the target pathogen.
Alternatively, probiotics may present benefits by conditioning the host immune system.
Immunomodulatory effects of the intestinal microbiome facilitate development of toler-
ance to environmental antigens and govern immunological responses against pathogens,
in effect diminishing autoaggressive or allergic reactions. For example, although not in
direct contact with mucosa-resident bacteria, resident M cells are able to take up and
undergo transcytosis of bacterial fragments from the luminal surface to the subepithelial
surface (55, 56). Nearby dendritic cells collect these fragments and present them to naive
CD41 cells. In an ex vivo study, coculturing dendritic cells with Lactobacillus rhamnosus on
exposure to T cells led to a significant decrease in production of T-cell-specific cytokines
(interleukin 2 [IL-2], IL-4, and IL-10). This suggests that potential immunomodulatory inter-
actions could occur between the resident microbiota and the host (57). In similar studies,

TABLE 3 Prerequisite traits for probiotic selectiona

Measure Criterion
Safety GRASE
No evidence of competition or parasitism with its host
Open-accessible data
Produced by GMP
Commensal or mutualistic to native microflora
Minimal threat of vertical and horizontal transfer of antibiotic resistant genes
Phenotypic and genotypic identification of strain
Resistance to bacteriophage
Functionality Characterized mechanism of action

Resistant to low pH and bile acids produced by host
Usability Manufacturing capabilities

Long-term storage
QA for manufacturing and distribution
Genetic stability over high biomass production
aGRASE, generally recognized as safe and effective; GMP, good manufacturing practices; QA, quality assurance.
the probiotic Escherichia coli Nissle 1917 was able to negate deleterious effects of chemi-
cally induced colitis by strengthening the mucosal gut barrier (58). Mouse models and sup-
porting in vitro experiments reported positive correlations of zonula occludens proteins 1
and 2 with resistance to enteropathogenic E. coli (59, 60). In summary, probiotics’ benefits
have either direct action on a pathogen or indirect host immunomodulation effects; hence,
possession of any of these features flags a microbe as a potential therapeutic.
The aforementioned examples demonstrate why probiotics have taken the limelight
and how they are currently being discussed as the next generation of medicine (61). A

recent clinical trial illustrated the effective use of probiotics to substantially diminish the
overall presence of antimicrobial resistance genes and the bacteria that harbor them (62).
Systematic searches for underlying mechanisms support probiotics’ potential to mitigate
infection, combat cancer, and stabilize/enhance a host’s microbiome. However, the issue of
identifying a particular probiotic strain with the desired specificity remains, as the beneficial
effect may not be conserved across probiotic strains (63).
LIMITATIONS OF PROBIOTICS AND REQUIREMENTS

The reemergence of probiotics research over the last 45 years varies jointly with ele-
vated interest among researchers and consumers. This billion-dollar industry is based
on four predominant market factors: (i) an increased demand for alternative therapeu-
tics to combat the accelerating rise of antibiotic resistance in bacteria, (ii) shifts toward
patient-driven health care, (iii) elevated interest in natural products to minimize side
effects associated with conventional therapies, and (iv) increasing evidence of strain-
specific beneficial effects (64–66). Hence, agencies like the FDA, European Medicines
Agency (EMA), and Codex Alimentarius (a joint FAO/WHO Food Standards Program)
are extending their influence in the realm of probiotics development to provide assur-
ance to users. As a result, these agencies are requiring demonstrations of safety, func-
tionality, and manufacturing usability before marketing (Table 3) (67).
Most probiotics are generally recognized as safe and effective (GRASE) but do not
necessarily have similar beneficial effects. Safety assessments focus on identifying anti-
biotic-resistant populations, in addition to the likelihood of the traits being transferred
to cohabiting species (68–70). With this in mind, vertical gene transfer, measured by
shifts in antibiotic median effective concentration (EC50), is extensively recorded over
succeeding generations (71). Species and strains that rapidly develop antibiotic resist-
ance are disregarded as probiotic candidates. Defining functional mechanisms regard-
ing a probiotic’s endpoint aims to provide empirical data regarding its ability to miti-
gate disease and requires substantial efforts. Before application, probiotic strains’

antagonism toward the pathogen and the mechanistic pathway of action are required
to be validated (69). Finally, new standards for manufacturing practices may disqualify
or delay some identified therapeutic candidates. For example, the probiotic must have
a stable shelf life and be genetically stable over repeated generations of culturing.
Probiotics being investigated are undergoing elevated surveillance and, depending on
the declared application, are subject to various degrees of certification (Table 2), all of
which are geared toward products safety, function, and usability, thereby delaying the
translation to specific clinical application.
CURRENT NOTION OF THERAPEUTIC MICROBES

FMT. In humans, the lack of a heathy gut microbiota due to an imbalance of the normal
gut fauna (also called dysbiosis) is associated with various acute and chronic conditions (72–
75). This idea is not novel, as the Greek physician Hippocrates postulated that “all disease
begins in the gut” nearly 2,500 years ago (76). Recently, physicians and scientists have been
exploring ways to reestablish baseline biodiversity in a dysbiotic gut microbiome through
the use of fecal microbiota transplantation (FMT) to correct the underlying condition (77).
The most common practice is to derive fecal samples from healthy individuals who meet
the stringent donor criteria (78). These samples are then used to inoculate alimentary tracts
of patients exhibiting dysbiosis (79, 80). As mentioned, fecal transplant practices are not
novel, and variations have been used for centuries, with the earliest known implementation
having been dated to 300 CE. Ge Hong in China performed the first iterations of FMT to
treat gut diseases, wherein he orally administered fecal material mixed with water (“yellow
soup”) to patients suffering from diarrhea or food poisoning (81, 82). For unknown reasons,
over the next 1,200 years, no records of the practice are found, and references to FMT resur-
faced in the 16th century during the Ming Dynasty (81, 83). Discussion of fecal microbiota
transplants continued in veterinary medicine into the 17th century (84). Interestingly, the
use of FMT overlapped the era of microbe discovery, thus providing a primitive explanation

for its beneficial outcomes (85). The compounding evidence of historical FMT usage and
modern experimentation in treating recurrent Clostridioides difficile (previously known as
Clostridium difficile) infections, in addition to other gut conditions, has further cemented the
concept of therapeutic microbes.
Investigating microbial communities has elucidated covariation between dysbiosis
and gut infections. Many patients treated with favorable biodiverse microfloras, via
FMTs, have experienced remediation of infectious and noninfectious conditions. Figure
2 presents a graphical summary of recorded FMT applications for review. Under infec-
tious-disease conditions, FMT improved fungal (Candida), viral (rotavirus), and bacterial
(MRSA) infectious outcomes (58, 77, 86–88). Notably, C. difficile infection (CDI)—among
the most commonly diagnosed and well-studied gastrointestinal diseases—is currently
the main target for FMT. A 2019 Centers for Disease Control and Prevention (CDC)
report lists CDI as one of the top threats in the United States, with an estimated annual
224,000 cases and 13,000 deaths (89). Physicians have turned to FMT treatment to
combat recurrent CDI and its associated secondary infections. Preliminary evidence
from FMT-treated patients reported substantial relief from acute CDI symptoms (22).
Further randomized trials of FMT against CDI resulted in 81% remission of symptoms,
in contrast to an average 27% in groups receiving antibiotics (90). These initial suc-
cesses of FMT heavily influenced the FDA to authorize it as a recommended treatment
against recurrent CDI and waived prerequisites for investigational new drug (IND) (91).
Despite the expeditious route of FMT to clinical application, the agency categorizes
FMT as an investigational therapeutic drug based on limited empirical data.
Limitations and risks associated with FMT. Fecal microbiota transplants remain ex-
perimental in composition, efficacy, and safety with respect to non-CDI conditions. For exam-
ple, FMT failure rates are reportedly on average 18.6% in non-CDI bacterial infections, such as
those caused by Enterobacteriaceae, enterococci, and MRSA (77, 92). Another concern involves
the potential failure to screen for pathogens (such as Shiga toxin-producing Escherichia coli)
and/or transferable antibiotic resistance genes (such as those for ampicillin, ciprofloxacin, and
nitrofurantoin resistance) during donor selection. To emphasize the importance of screening,

FIG 2 FMT applications in selected noninfectious conditions and infectious diseases (58, 89, 91, 133, 139–142).
These include gastrointestinal illness (143), obesity (141), autism (144), dysbiosis resulting from
antibiotics (142), and infection with rotavirus (88), Candida (58), and C. difficile (90, 145).
recent clinical trials reported eight patients becoming severely ill, with one death, due to the
presence of multidrug-resistant pathogens (93, 94). Similarly, noninfectious diseases (i.e., neu-

rological dysregulation and obesity), in the absence of diligent screening, have been reported
to result in transfer of pathogens from donor to recipient (95–98). Unfortunately, the symbiosis
between donor and patient, their resident microbiotas, and the target pathogen remains
uncharacterized for FMT. The above-mentioned examples of FMT highlight issues that have
come to light from the limited research and screening practices. While FMT effectively demon-
strates the therapeutic potential of certain gut-derived microbes to benefit human health,
considerable work remains warranted, not only to understand the mechanisms leading to its
therapeutic outcome but also for the refinement of standard practices of FMT. Furthermore,
recipients’ genetics, immunity, microbiota, and lifestyle also shown to influence the clinical effi-
cacy of FMT in infectious and noninfectious conditions. Therefore, a list of strategies must be
taken into consideration to improve FMT treatment: (i) optimization of donor-recipient match-
ing in terms of genetics, immunity, and microbiota compatibility; (ii) preparation of the gut
environment using antibiotics and bowel cleaning, (iii) targeting the recipient’s immune sys-
tem with immunosuppressive therapy, and (iv) controlling the recipient’s lifestyle through die-
tary intervention (99).
Defined microbial consortia. One noteworthy alternative to FMT is the use of a
defined microbial consortium (DMC), which integrates a rationally selected collection of
known microbes at specific abundances for treatment of disease conditions. Thus far, authors
have used a genome-guided approach to design a DMC, which on administration to germfree
mice provided resistance against colonization by pathogenic Salmonella enterica serovar
Typhimurium (100). The isolates acquired by genomic sequencing and antibiotic susceptibility
testing can be combined to generate DMC against specific pathogens. In another study,
researchers prepared a DMC by characterizing a healthy donor fecal sample through culturing
of favorable bacteria and then administered it to individuals with recurrent CDI. Patients given
the characterized DMC exhibited remission of CDI symptoms over a 24-week span (101). In a
similar fashion, researchers provided 55 individuals with recurrent CDI a collection of defined
probiotics (Lactobacillus, Bacteroides, Clostridium, etc.) and reported 80% remission in acute
cases, with no recurrence for 30 days (102). Recent phase II trials investigated the efficacy and

safety of nonpathogenic C. difficile spore administration for the inhibition of pathogenic C. dif-
ficile strains and observed 89% remission in patients receiving DMC treatment (103, 104). In a
novel pursuit for CDI treatment, data from antibiotic therapy and 16S rRNA profiling revealed
that Clostridium scindens to exert an inhibitory influence on C. difficile colonization via the pro-
duction of secondary bile acids (105). To understand the mechanism behind resistance to col-
onization by pathogenic C. difficile strains, authors characterized the unique presence of baiCD
genes, encoding bile acid metabolism in C. scindens (105, 106). Collectively, these studies indi-
cate that specific microbes with specific genes present in DMC have antagonistic activity to-
ward target pathogens. Hence, DMC has a number of advantages over FMT, such as (i) mini-
mum safety concerns, (ii) lack of pathogenic organisms, and (iii) rationally tailored therapeutic
microbes.
Limitations associated with defined consortia. Defined consortia are not without
limitations and concerns. The removal of potential pathogens or antibiotic-resistant
organisms is largely beneficial; other aspects of DMC (preparation and efficacy) are
questionable. The selection of microbes to include in consortia can be resource inten-
sive and warrant metagenomics analyses, safety testing, and postengraftment evalua-
tion to determine the efficacy of treatment (101). Second, rationally generated DMC
may be inferior to FMT due to missed key microbes within the consortia. A recent
randomized trial reported 52% efficacy with DMC compared to 76% with FMT engraft-
ments (107). These data suggest the possibility of excluding therapeutic microbes
when creating DMC, resulting in a subefficacious outcome. Alternatively, advanced cul-
tivation and identification methods like culturomics—a high-throughput bacterial cul-
turing method that comprehensively identifies strains/species from a complex micro-
biota and acts as a complement to metagenomics—will be beneficial in discovering
and isolating novel therapeutic microbes (108–110). Attesting to the utility of this
method, a recent study discovered 174 novel human gut commensals, including rare,
previously unidentified bacteria like Deinococcus-Thermus and Synergistetes (108).

Finally, consortia rarely include beneficial fungi or viruses, which may also be of thera-
peutic value. In an interesting study, sterile-filtered stool, which retained virus-like par-
ticles, effectively cured six recurrent CDI patients (111). In essence, symbiotic interac-
tions between therapeutic microbes, host, commensals, and pathogens are difficult to
characterize due to their complexity. We cannot ignore the limitations of DMC and
must consider them if these therapies are utilized in medicine. Preparing DMC with the
inclusion of other beneficial microbial organisms (i.e., fungi or viruses), validation of
their safety and efficacy, and understanding the mechanisms of action are the path-
ways for bringing DMC into real clinical practice.
EXPLOITING MMIs TO DISCOVER THERAPEUTIC MICROBES

Microbe-microbe interactions are ubiquitous in nature, occupy all ecosystems, can be
extremely complex, and usually involve exchange of chemical signaling, nutrients, gene
transfer, and other relevant mechanisms (112). Microbial interactions are broadly classified
as positive (mutualism, syntrophism, protocooperation, and commensalism) and negative
(amensalism, parasitism, predation, competition) (Table 4) (15). Exploiting inter- and intra-
species MMIs that promote or suppress targeted organisms can provide opportunities to
revolutionize medicine and improve patient quality of life with minimal side effects.
Conveniently, diversity-rich multispecies ecosystems grant an opening to study microbial
communication in an ecosystem and detect therapeutic candidates.
The classical approach to investigate MMIs, derived from Robert Koch’s postulates,
involves obtaining an isolated microbial species that is then one-to-one probed in vitro
to appreciate positive and negative interactions. Even today, such is the gold standard
to investigate MMIs and etiologies of disease. Given that less than 2% of microbes are
culturable using conventional methods (113, 114), only a few candidate organisms can
be evaluated with assays; hence, investigating culture-recalcitrant microbial species
appears impossible. Additionally, microbial communities may differ conspicuously in
species richness. For this reason, we face rapidly increasing dimensions of MMIs that

TABLE 4 Interactions among species grouped broadly into two categories depended if interaction is positive or negativea
Effect on Effect on
Type Interaction Definition/description species 1 species 2
Positive Mutualismb Each species benefits from one another 1 1
Species 1 is irreplaceable to species 2 and vice versa
One species not dependent on other species metabolic product
Proto-cooperationc Both species benefit, but an individual can survive in the absence 1 1
of the other
Syntrophismb Species 1 is dependent on the metabolic product of species 2 1 1
Commensalism (commensal and host) 1 1
Negative Amensalism 1 2
Parasitism (parasite/pathogen and host)b,d An individual species derives nutrients from the host species 1 2
In most cases the parasite does not kill the host
Predationd One organism kills and consumes the other 1 2
Competition Intraspecific competition (species strain 1a = species strain 1b) 2 2
Interspecific competition (species 1 = species 2)
a1, neutral effect on species.
bObligatory interaction (mutualist and host are metabolically dependent).
cNonobligatory interaction (independent).
dAntagonistic interaction.
are quickly becoming laborious to untangle (115). Nonetheless, culture-independent,

next-generation sequencing (NGS), and bioinformatics analyses followed by predictive
modeling have to some extent addressed these challenges. Bioinformatics have simpli-
fied MMIs’ principal components and identified candidates for exerting positive or neg-
ative influences on microbes of interest that theoretically exist (115). However, the
problem of how to isolate lead microbes that have therapeutic potential from their
complex microbial community remains.

As just mentioned, postponement of in-depth research of MMIs is due to the current limi-
tations in cultivation technologies and lack of screening methods, ultimately delaying clinical
application of therapeutic microbes. Of the cultivable 2% of microbes, therapeutic microbes
were haphazardly identified, easily isolated, and fortuitously cultivated using archaic methods.
For example, in 1917, Alfred Nissle sampled fecal content from a dysentery-resistant individual
and correlated resistance with an abundance of an E. coli biovar. Afterward, he successfully iso-
lated and cultivated the particular E. coli strain exhibiting antagonistic activity against tested
enteropathogens. This biovar, named E.coli Nissle 1917, became widely tested as a probiotic
of choice for various enteric conditions (116).
In an effort to overcome the “growth barrier,” our team is investigating MMIs in a rela-
tively high-throughput fashion to identify and isolate therapeutic microbes by screening
different microbiotas. For a phenotype-based microbiome screening approach, we have
developed a high-throughput screening of cell-to-cell interactions (HiSCI) platform by inte-
grating microfluidics, cocultivation methods, flow cytometry, and sequencing techniques.
The HiSCI platform has a number of advantages: (i) it creates millions of microdroplets
(MDs) comprising defined growth medium and effectively representing an independent
petri dish at a picoliter scale, (ii) it preserves microbe-microbe interactions confined in each
MD as observed in a complex community, (iii) it facilitates high-throughput screening of
phenotypes postcultivation via fluorescence-activated cell sorting (FACS), and (iv) it helps
in recovery of microbes exhibiting a desired phenotype(s). We have successfully applied
this platform to identify and isolate a bacterial species that promotes (positive interaction)
the growth and biomass of Chlorella sorokiniana algae (117). In this study, we isolated bac-
teria from freshwater ponds and randomly cocaptured a few bacterial cells (#8 cells) with
a single algal cell suspended in MDs containing growth medium. With the help of FACS,
enrichment of tens of millions of MDs followed for samples containing algal chlorophyll flu-
orescence. Down-selected algal-bacterial MDs were cocultivated in a single chip under
defined dark-light cycles. Subsequently, via FACS, we rapidly identified and recovered the
MDs having increased algal growth with greater fluorescent signals than single captured

algal MD controls. We then isolated and propagated the bacterial MDs and subjected
them to subsequent rounds of algal cocapture and screening, until the same bacterial spe-
cies were repeatedly identified. Ultimately, this set of bacterial pond isolates promoted
faster and improved algal biomass via conventional growth assays. This first successful
demonstration of the HiSCI platform led us to tailor its application for investigating human
microbiota interactions. Recently, similar ultrahigh-throughput methods have been devel-
oped and applied to identify oral microbiota species with antagonistic characteristics to-
ward pathogenic S. aureus (118, 119).
Currently, we are using the modified version of the HiSCI platform to address the
public health antibiotic resistance crisis. Specifically, we have adapted our current
HiSCI protocols to identify therapeutic microbes that naturally inhibit multidrug-resist-
ant pathogens like C. difficile and methicillin-resistant S. aureus (MRSA) (Fig. 3). In the
former project, we are cocapturing microbes harvested from healthy human fecal sam-
ples and suspending them with a single C. difficile cell in millions of MDs. In the latter
project, a similar method is used to harvest human skin microbes to be cocaptured
with a single MRSA cell. Subsequently, cocaptured MDs are cultured under optimal
growth conditions—anaerobic (gut) or aerobic (skin)—that mimic natural community
conditions. Using flow cytometry with differential staining techniques (antibody stain-
ing or fluorescent pathogen strain generation), we sort only MDs where significant
growth inhibition of the pathogen strain occurred, as indicated by a low fluorescent
signal. At present, we are in the process of identifying associated bacterial species by
sequencing efforts (e.g., bacterial 16S rRNA phylotyping), which will, in turn, aid in
identifying and isolating the desired therapeutic bacterial species after iterative rounds
of selection. Finally, we are in the process of developing an analogous platform
wherein we propose to screen for bacterial-bacteriophage interactions in order to rap-
idly and efficiently identify lytic bacteriophages against MRSA.
Limitations of the HiSCI platform. Our developed platforms have many advan-

tages in identifying lead microbes; however, we also observed a small number of nota-
ble challenges. (i) The current process of capturing microbial cells in MDs is highly inef-
ficient, random, and nonuniform. (ii) Due to randomness, at best 10% of the microbial
cell input is captured with the desired cell number inside the MDs, thereby losing most
of the microbial consortium. (iii) Microdroplets with diameters greater than 70 m m are
discarded, as they are too large to sort via flow cytometry, which in turn contributes to
additional loss of the captured cells (117). (iv) Microspheres are made of biocompatible
materials, such as agarose or collagen, that limit the capture of certain microbial spe-
cies that naturally degrade MDs through their enzymatic activities (e.g., agarases). (v)
Because of MD volume, only early MMIs are able to be investigated, screened, and phe-
notyped (117). (vi) HiSCI is biased in favor of rapidly multiplying microbial species, thus
ignoring slow growers. (vii) Bacterial species that resist broth culturing cannot be ana-
lyzed, as the HiSCI platform is dependent on liquid culturing.
EXPLOITING HMIs TO DISCOVER THERAPEUTIC MICROBES

Like MMIs, HMIs are equally multifaceted and crucial for complex eukaryotic life (e.g., ani-
mals, humans, etc.). In fact, higher-order eukaryotic species are believed to contain more
microorganisms than eukaryotic cells; therefore, the term “holobiont” or “superorganism”
refers to a host plus all the associated microbes in a complex eukaryotic species (120, 121).
The concepts of positive and negative interactions applied to MMIs remain consistent for
HMIs, with subtle difference in terminology (Table 4) (122). Unlike the pathways of MMIs,
where therapeutic microbes indirectly benefit the host by suppressing pathogens or bene-
fitting indigenous microflora, investigating HMI leads to the discovery of microbes that
directly benefit the host. This is similar to the case with oncolytic microbes (oncolytic viro-
therapy and bacterial therapy), wherein therapeutic microbes selectively infect and kill can-
cerous cells while stimulating a more robust anticancer immune response (123). In 1867,
the German physician Wilhelm Busch was the first to observe the phenomenon of cancer
remission in patients infected with the agent of erysipelas (now known as Streptococcus

FIG 3 Schematic representation of a microbiome screening platform to identify therapeutic microbes against the
antibiotic-resistant pathogens C. difficile and MRSA (146).

pyogenes) (124). Subsequently, William Coley championed the same practice of using killed
S. pyrogens with another killed bacterial species (now identified as Serratia marcescens)
(123) to successfully treat a number of cancer patients (125). Currently, various genera of
modified bacteria and viruses are being tested in preclinical and clinical studies as oncolytic
microbes (123).
Although Edward Jenner was the first to immunize against the smallpox virus using mate-
rial from the pustules of cowpox-infected patients, modern history remembers Louis Pasteur
and Robert Koch as the pioneers of investigating HMIs. Pasteur’s and Koch’s work led to revo-
lutions in microbial control and the understanding of disease, such as “pasteurization” and
Koch’s postulates (126). Pasteur’s research further contributed to medicine through the devel-
opment of inoculation strategies using live and dead microbial cells to prevent anthrax in
sheep, cattle, and chicken; cholera in fowl; and rabies in humans and dogs (126). The outcome
from generations of refinements of the technique have led to a myriad of models, including
in vitro models such as cell culture, organoids, bioreactors, and organ-on-a-chip (127); ex vivo
models (e.g., harvested living tissue); and in vivo models (e.g., invertebrate and vertebrate
models). Notably, the digital revolution and the search for unculturable microbials have made
some conventional methods obsolete. With the inception of NGS, machine learning, time-se-
ries predictive modeling, and HiSCI, we are able to identify millions of potential therapeutic
microbes and extrapolate outcomes in complex life forms. However, the conventional meth-
ods still have a degree of applicability in the digital age, specifically validating computational
models with observable biological outcomes. For example, authors have isolated and identi-
fied a C. scindens strain and demonstrated with metagenomic and time-series analysis model-
ing in a mouse that this strain inhibited the growth of clinically relevant C. difficile (105).
Furthermore, in a recent study using mouse models, researchers identified and demonstrated
the potential therapeutic effect of Citrobacter amalonaticus on inhibiting the growth of
Citrobacter rodentium, an opportunistic pathogen of humans (128).
Orchestrating computational and laborious in vivo HMI models has made it possible to

identify, isolate, and determine the potential of therapeutic microbes. Furthermore, these stud-
ies suggest that commensal bacterial species in the presence of pathogens can act as thera-
peutic microbes by conferring resistance to colonization. Overall, exploiting either HMIs or
MMIs to investigate microbes antagonistic toward disease-causing pathogens serves as a
novel area for future medicine.
CHALLENGES AND POSSIBLE SOLUTIONS TO THERAPEUTIC MICROBES’ APPLICATIONS

Technologies to discover therapeutic microbes are still being pioneered and, as such, have
elucidated many challenges to overcome. The first concerns live cells and growth rate. Viable
cells with self-renewal properties complicate calculation of pharmacokinetics and pharmaco-
dynamics during clinical testing (129). For example, the growth rate of these microbes is de-
pendent on person-specific factors like food intake, native microbiota composition, and dis-
ease status. To address person-to-person variability, we can screen individuals and group
them based on microbiome composition, diet, and physical activities, etc. To achieve this the
current population-based medicine approach (i.e., one size fits all) needs to be replaced with
personalized medicine. The second challenge involves mixed strains, complex interactions,
and downstream effects. Multistrain therapeutic microbes have another layer of complexity—
multiple off-target interactions (129). Cultivation of mixed strains and strain integrity will chal-
lenge the manufacturing process, wherein genotypes can be highly variable from batch to
batch. Evolving or developing a species with target specificity, gene stability, an inherited self-
destructive kill switch, and a built-in strain characterization can address these challenges and
ensure safety. The third challenge is that of drug delivery and dosing. The standard pharmaco-
logical processes—absorption, distribution, metabolism, and excretion—have limited applica-
tion in the use of therapeutic microbe products. Instead, gastrointestinal distribution, attach-
ment (colonization), multiplication, and shedding (clearance) need consideration. (129). The
fourth challenge concerns manufacturing and quality assurance. Therapeutic microbes need
special consideration regarding temperature, pH, salt concentration, etc., to maintain
the product’s quality. The use of enteric-coated gelatin capsules filled with buffers


FIG 4 Circular flow diagram highlighting the critical steps needed for therapeutic-microbe discovery. (1) Therapeutic-microbe source: human,
animal, or environment. (2) Next-generation culturing methodologies (ichip, large-cohort cultivation, etc.) to collect unculturable microbes.
(3) Isolating, cataloguing, and preservation of cultured novel microbial species to generate a large microbial biobank. (4) Generating and
sharing high-density microbial cell arrays for microbe-pathogen screening. (5) Concomitant analysis of real-time genome sequencing and
high-resolution microscopy to identify potential therapeutic microbes. (6) Preclinical testing of isolated therapeutic microbes. (7) Initiation
of clinical trials to appreciate efficacy of therapeutic microbes and subsequent mass production, per FDA guidelines. A.I., artificial
intelligence.
and low-temperature storage are possibilities for mitigating these concerns. Last, there is
the question of FDA regulation. The FDA is currently working with leading startup com-
panies in this area and developing regulation for such products (e.g., SER-109; Seres
Therapeutics). Given that information, placement in the FDA’s categorization regarding
IND application status is still in debate (129, 130).
PROSPECTS OF THERAPEUTIC-MICROBE DISCOVERY

We are just beginning to appreciate the value of microbial ecosystems in dictating
human health and to be able to gauge the usefulness of microbes for therapeutic and
diagnostic applications. In order to truly understand and exploit microbiotas for

therapeutic applications, we need extensive interdisciplinary efforts (Fig. 4). Advances

in any of the following avenues of research, alone or in combination, will be a step for-
ward in discovering therapeutic microbes: (i) next-generation culturing methods to
grow unculturable organisms (e.g., in situ culturing via an isolation chip [ichip] [131],
targeted phenotypic culturing [132], etc.), (ii) extensive collection of a microbe biobank
for species-specific and tissue-specific microbiotas (e.g., skin microbiome, gut micro-
biome, etc.), (iii) advanced microfluidics (pico- or nanovolume ichips) culturing (117,
133), (iv) highly sensitive and specific rapid screening assays, (v) high-density microbial
cell arrays (134) that can readily be used to screen against any pathogens, (vi) real-time
sequencing and data analysis, and finally, (vii) artificial intelligence (AI)-integrated
advanced and rapid imaging techniques.
CONCLUSION
Due to limited technologies, exploiting a microbial community’s structure and func-
tion for the discovery of favorable therapeutic microbes remains a daunting task.
Recent bursts of microbiome research have ignited this research field, but it needs sig-
nificant interdisciplinary collaborative efforts to address discovery and application chal-
lenges. We expect living therapeutics and diagnostics to be the next medical revolu-
tion, similar to the discovery of antibiotics in the early 19th century.
ACKNOWLEDGMENTS
Anand Kumar’s research work is supported by a Los Alamos National Laboratory
(LANL) internal grant [20210612ECR: A high-throughput (RapidPhage) platform for the
discovery of lytic bacteriophages against multidrug resistance pathogens]. The majority
of Anand Kumar’s previous research was also funded by two internal grants, Using
therapeutic bacteria to treat human diseases (grant 20160340ER) and Developing a
unique technology to discover a novel gut microbial cocktail to treat antibiotic

resistance pathogens (grant 20170671PRD2), to develop and establish a microbiome
screening platform.
Conceptualization and project administration, A.K.; minireview design, A.K., N.C.,
G.A.A.; writing—original draft preparation, N.C., A.K., G.A.; writing—review and editing,
N.C., A.K., G.A.A., A.E.K.D.; supervision, A.K., visualization, G.A.A., N.C. All authors have
read and agreed to the published version of the manuscript.
We declare no conflict of interest.
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Nathan Cruz is a post-master’s student at Los Armand E. K. Dichosa, Ph.D., M.S., is a staff
Alamos National Laboratory (LANL). He holds a scientist in Bioscience Division at LANL. He
B.S. in psychological sciences and a M.S. in biol- received both his M.S. and Ph.D. from the
ogy from Northern Arizona University (NAU). University of New Mexico in biology, investi-
During his undergraduate studies, he assisted gating extremophile archaeal and bacterial
Dr. Christopher Woodruff in recording neuron communities and their potential roles in their
activity using electroencephalography in hu- respective environments. His core research
mans to understand the neuroscience of empa- interests focus on the genomic and viable re-
thy. In his senior year, he joined Dr. Robert covery of novel bacterial species from various
Kellar’s regenerative medicine laboratory to environments and determining their potential
gain experience in mammalian cell culture tech- beneficial or adverse impacts on human
niques. By securing the NAU Research Initiative for Scientific Enhancement health, national security, and the environment. Dr. Dichosa pioneered the
scholarship, he enrolled in a M.S. program and continued research under Dr. integration of gel microdroplets with single-cell genomics to dissect com-
Kellar. With cell culture experience, in his M.S. studies he used an in vitro model plex microbiomes for various research efforts involving biodegradation, pro-
to investigate the nocuous effects of uranyl nitrate on wound healing. While biotic therapies, and novel bacterial characterization. He collaborates on var-
analyzing his research data, Mr. Cruz realized how important statistical tools are ious projects with Dr. Anand Kumar and his team to discover therapeutic
in hypothesis-driven biological research. This led him to simultaneously gradu- microbes. Dr. Dichosa is involved in promoting global cooperatives for mi-
ate with a certification in applied statistics. At LANL, he works with Dr. Anand crobial biosurveillance through genomics and bioinformatics outreach
Kumar in basic and applied aspects of STEM research. Concurrently, he is sup- efforts, as well as leading microbiology-genomics STEM efforts for our
porting the development of a microfluidic-based bacteriophage discovery plat- nation’s high school students.
form. In the next few years, his ambition is to pursue a Ph.D. in Applied
Mathematics to continue his passion in data-driven medical research. Anand Kumar, D.V.M., Ph.D., is a staff scien-
tist in the Bioscience Division at LANL. He

George A. Abernathy is a post-master’s stu- received his D.V.M. in India and M.S. and
dent working with Dr. Anand Kumar at LANL. Ph.D. degrees from the Ohio State University.
He holds a B.S. in genetics and biotechnology His Ph.D. research focused on understanding
from New Mexico State University and an M.S. host responses to probiotics as well as investi-
in integrative genomics from Black Hills State gating the impact of diet and rotavirus infec-
University. While an undergraduate student, tion on a humanized pig model. During his
he received a research scholarship from the M.S. research, he investigated Campylobacter
Howard Hughes Medical Institute to acquire pathogenesis and developed prophylactic
basic microbiological skills. After obtaining his (nanoparticle vaccine) and therapeutic (small-
B.S., he worked with veterinary physicians as a molecule discovery) measures against it. During his postdoctoral tenure at
full-time assistant to support surgeries and LANL, he led several efforts to establish and develop microbiome-screening
routine care for small and exotic animals. During his five years of veterinary platforms. Dr. Kumar’s research is currently geared toward microbiome en-
assistantship, he developed a passion for research with the ultimate goal of gineering by (i) discovering and developing novel microbiome-based ther-
promoting animal health. He enrolled in a graduate program and conducted apeutics to tackle the threat of emerging bacterial pathogens, (ii) engineer-
research under the guidance of Dr. Cynthia Anderson. His research focused ing probiotic strains to create a living diagnostic-therapeutic system, and
on the effect of a natural antifungal agent against fungal species pathogenic (iii) evolving a bioreactor model of the human gut to investigate host-
to humans and animals. With his M.S. in integrative genomics, he joined Dr. microbe interactions.
Kumar’s team at LANL to develop an innovative and rapid phage technology
to discover an effective lytic phage cocktail against antibiotic resistant patho-
gens. Mr. Abernathy plans to pursue an integrated D.V.M. research program
to conduct translational research that benefits human and animal health.

The Age of Next-Generation Therapeutic-Microbe Discovery: Exploiting Microbe-Microbe and Host-Microbe Interactions For Disease Prevention

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The Age of Next-Generation Therapeutic-Microbe Discovery: Exploiting Microbe-Microbe and Host-Microbe Interactions For Disease Prevention

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MINIREVIEW

The Age of Next-Generation Therapeutic-Microbe Discovery:

ABSTRACT Humans are considered “superorganisms,” harboring a diverse microbial col-

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KEYWORDS probiotics, fecal microbiota transplant, deﬁned microbial consortia, thera-

E very animal (and plant) on Earth is a result of multitudinous host-microbe interac-

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EARLY HISTORY OF THERAPEUTIC MICROBES

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5,000 years reveal optimizations of the fermentation process, resulting in improved

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CURRENT METHODS OF PROBIOTIC DISCOVERY

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Attribute Probiotics Fecal microbiota transplantb Deﬁned consortiumc Therapeutic microbesc

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(Continued on next page)

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DEFINING PROBIOTICS: FDA

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TABLE 3 Prerequisite traits for probiotic selectiona

Functionality Characterized mechanism of action

Usability Manufacturing capabilities

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LIMITATIONS OF PROBIOTICS AND REQUIREMENTS

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CURRENT NOTION OF THERAPEUTIC MICROBES

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EXPLOITING MMIs TO DISCOVER THERAPEUTIC MICROBES

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are quickly becoming laborious to untangle (115). Nonetheless, culture-independent,

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EXPLOITING HMIs TO DISCOVER THERAPEUTIC MICROBES

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CHALLENGES AND POSSIBLE SOLUTIONS TO THERAPEUTIC MICROBES’ APPLICATIONS

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PROSPECTS OF THERAPEUTIC-MICROBE DISCOVERY

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therapeutic applications, we need extensive interdisciplinary efforts (Fig. 4). Advances

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