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

ABSTRACT

Humans are considered “superorganisms,” harboring a diverse microbial collective 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 overall 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 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.

INTRODUCTION

Every animal (and plant) on Earth is a result of multitudinous host-microbe interactions, 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, energizing physioanatomical development, and providing resistance to colonization by intruding pathogens (1–3). Without microbial interactions, the host becomes predisposed to various disease conditions. This intellection can be appreciated, as germfree animals exhibit anatomical and functional differences in the gut, liver, lungs, enteric nervous system, and suboptimal immunity and endocrine functions (4, 5). However, the mechanistic aspects of microbe-microbe interactions (MMIs) and host-microbe interactions (HMIs) have been a major challenge to dissect. Many external factors (e.g., antimicrobials, diet, and environment) and internal factors (e.g., genotype) influence 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 develop 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 hypothesis have erroneously imprinted on humanity that all microbes are pathogens. Such ideologies have contemporized an increase in hygiene practices and overuse of antimicrobial 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 genetically 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 historical integration of therapeutic microbes (probiotics), the current perception of their use in therapy (i.e., microbiome based or rationally selected defined consortia), discovery 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 discussing 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 biasing 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 originated during early civilization with the realization of the beneficial health effects of fermented 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 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.

FIG 1.

Milestones in the discovery of potential therapeutic microbes in human evolution (80, 81, 91, 99, 135–138).

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 beginning 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 identified 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 species—were used as treatments for Shigella dysenteries in humans (32). Interestingly, modern 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 infections 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 resistance 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 sponsorship 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 unexplored Archaea species that make up ∼1.5% of the human gut microbiota also possess therapeutic potential in remediating atherosclerosis and kidney disease by metabolizing trimethylamine N-oxide (TMAO) levels in blood (19, 34). In summary, the interdependency 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 toward 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 characteristics 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 proteins. 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-substrate 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 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 evaluating MMI, and presume underlying characteristics of cultured strains (e.g., high penetrance).

TABLE 1.

Summarized attributes of therapeutic microbes^a

Attribute	Probiotics	Fecal microbiota transplantb	Defined consortiumc	Therapeutic microbesc
Definition	“Live microorganisms which when administered in adequate amounts confer a health benefit on the host” (41). Contains mainly bacteria and yeast	Screened fecal transplant from healthy donor to recipient (90). Contains bacteria, virus, fungi and/or metabolites	Collection of characterized microbes for therapeutic use (111). May contain a pure or mixed culture of bacteria and/or fungi	Adequately characterized, or engineered, single and/or collections of microbes with targeted therapeutic applications May contain single or a cocktail of bacteria, virus, and/or fungi species
Discovery and/or rationale	Beneficial relationship through the fermentation of food that promoted health	Administration of fecal suspensions performed by Ge Hong from healthy individual to diarrheic patient (82, 83)	Depleted commensal microbe collections and/or cocktails of probiotic species with beneficial effect (100–102)	First instance of antagonistic MMI: lytic phages against Shigella species and Penicillium notatum against S. aureus
Methods	Diffusiona (41, 147–155) Agar disk Etest Agar well Agar plug Cross streak TLCa (156–158) Agar diffusion Direct bioautography Agar overlay bioassay	Screening: identify donors satisfying prerequisite criteria Preparation: samples suspended in saline Administration: colonoscopy, nasogastric, oral capsules (139, 143, 144, 159, 160)	NGS and other omics approaches followed by bioinformatics analysis to shortlist essential microbes (100, 101, 103).	Engineering strain via synthetic biology approach (17, 18, 120) Development of microbiome screening platform [HiSCI] (115)
FDA categorization (Table 2)	Dietary supplement	Biological product	Biological product	Drug
Applications	Allergy (161–164) Carcinogenicity (165) Carcinogenicity and mutagenicity (166, 167) Cholesterol reduction (168) Gastrointestinal (169, 170) Endotoxemia (171) Hypertension (135) Immunomodulation (136) Kidney stone (oxaluria) (137) Lactose tolerance (140) Urinary tract infections (172)	Selected noninfectious diseases Psychological (144) Obesity (97, 141) Drug-induced dysbiosis (142) Gastrointestinal (143) Selected infectious diseases Rotavirus (88) Bacterial infections (87) Fungal infections (58) Parasitic prevention (173, 174)	Recurrent C. difficile infections	Engineered strain: familial adenomatous polyposis, urea cycle disorder, cirrhosis, phenylketonuria, type 1 diabetes mellitus, oral mucositis (120) Therapeutic microbes: C. scindens and C. amalonaticus (103, 119)
Mechanism/microbes involved	Direct Cholesterol clearance Nutrient competition Toxin suppression AMP production Inhibitory peptide production Competitive exclusion Indirect Immunomodulation Gut barrier reinforcement Enterocyte inflammatory stimulation	Direct Establishing eubiosis Novel antibacterial species Pathogen-specific lytic bacteriophages Beneficial fungi Unidentified microbial metabolites Indirect Stimulation of host immunity and physiology (see probiotics mechanisms)	Direct and indirect  May employ probiotic or FMT  mechanisms	Direct Engineered strain: sense and respond to external stimuli (i.e., pathogen presence) (17, 18) Therapeutic microbes: may employ multiple mechanisms to inhibit or suppress pathogen of interest: nutrient competition, toxin suppression, AMP secretion, inhibitory peptide production, competitive exclusion Indirect Stimulate host immunity and physiology
Advantages	Derived from human and animals Consistent history of overall safe use Considered GRASE by FDA	Minimal side effects in contrast to antibiotic treatment (77) Multifaceted clinical applications (77) >70% eradication rate against multidrug-resistant organisms (77)	Absence of known pathogens Minimal vertical or horizontal antibiotic resistant gene transfer Known mechanism of action Reduced patient anxiety compared to FMT Reflects patient-driven health care (175)	Target specific and minimal side effect (19) High success rate in pathogen eradication Well-characterized and defined mechanism of action towards target and host No risk of transferable antibiotic resistance genes (17, 18)
Disadvantages/limitations	Limited data of mechanisms of action Exhaustive list of prerequisite criteria (Table 2) Various beneficial effect dependent on species and strain Batch-to-batch variation in large-scale manufacturing May harbor and transfer antibiotic resistant genes Vaguely regulated by FDA	Relatively high failure rates, non-C. difficile conditions (10–20%) (91) Difficulty in finding compatible donor(s) (92) Limited longitudinal studies for safety, recipient compatibility, and its benefits (93) Inadequate screening for pathogens (101) Transplantation of uncharacterized 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	Difficult to discover and isolate essential microbes from microbiota Currently less efficient than FMT in disease remediation Lack of inclusion of other essential microbes	Technology development in early stages Requires personalized medicine approach Standard treatment guidelines are not yet established (121) Biocontainment, strain stability, and quality control assurances have not been proposed or attempted (121)

^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.

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 computational 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 evaluated by the FDA” printed in nearly illegible font size to bypass additional testing requirements. Adding a disclosure statement on the product permits manufactures to apply conjectural 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.

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 Act^a

Category [U.S. Code]	Intended use	Premarketing requirements
Medical food [Title 21, chapter 9, section 360ee; 2011]	Used under the supervision of a physician and is administered “internally” with the intended application for the “management of a disease or condition” Dietary management	If GRASE, no clinical studies required, but prior research is needed; otherwise, IRB approval and clinical studies required.
Food [Title 21, chapter 9, section 321(f); 2011]	Articles used for food, drink, and its components Permits “health claims” to appear on foods and supplements Includes natural foodborne microorganisms Limited claim to “help maintain (micro)flora”	Typically, GRASE If not GRASE, requires FDA disclosure
Dietary supplement [Title 21, chapter 9, section 321(ff); 2011]	A product, excluding tobacco, used to “supplement the diet,” intended for ingestion, and “not present for use as a conventional food” Application of “new dietary ingredient is required” if product not marketed before 15 October 1994	Not required for ingredient marketed before 15 October 1994 May be placed on the market with limited claims Manufacturer must disclose to FDA intended claims; however, the FDA may request substantial evidence 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(g)(1); 2011]	Claims to “diagnose, mitigate, treat, cure, or prevent a specific disease or class of diseases” Product not GRASE Therapeutic microbes (single strain or consortium) with the above claims fall within the definition of a “new drug”	IRB approval before preclinical and clinical trials Must complete NDA and IND trials before marketing Possession of biologics license Formal communication with FDA prior to clinical testing Submission of test protocols and endpoint measurements 30 days prior Transcript of investigational plan Requires IRB review and approval
Biological product [Title 42, chapter 6A, section 262; 2010]	“Application for prevention, treatment and/or prevention of a disease condition” Article contains no living cells but can include any of the following: “virus, therapeutic serum, toxin, antitoxin, vaccine, blood, blood component or derivative, or allergenic”	Submission and approval of biologics license application IND studies

^aIND, investigative new drug; IRB, institutional review board; GRASE, generally recognized as safe and effective; NDA, new drug application.

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 proliferation, (ii) competitive inhibition or outcompetition for nutrients; (iii) beneficial immunomodulatory 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 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 fragmentation and cytosolic organelle dissolution (52–54). In light of these properties, bacteriocin-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 tolerance 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 CD4⁺ 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 interactions could occur between the resident microbiota and the host (57). In similar studies, the probiotic Escherichia coli Nissle 1917 was able to negate deleterious effects of chemically induced colitis by strengthening the mucosal gut barrier (58). Mouse models and supporting 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 elevated interest among researchers and consumers. This billion-dollar industry is based on four predominant market factors: (i) an increased demand for alternative therapeutics 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 assurance to users. As a result, these agencies are requiring demonstrations of safety, functionality, and manufacturing usability before marketing (Table 3) (67).

TABLE 3.

Prerequisite traits for probiotic selection^a

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.

Most probiotics are generally recognized as safe and effective (GRASE) but do not necessarily have similar beneficial effects. Safety assessments focus on identifying antibiotic-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 resistance are disregarded as probiotic candidates. Defining functional mechanisms regarding a probiotic’s endpoint aims to provide empirical data regarding its ability to mitigate 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 resurfaced 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 infectious-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 successes 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.

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).

Limitations and risks associated with FMT.

Fecal microbiota transplants remain experimental in composition, efficacy, and safety with respect to non-CDI conditions. For example, 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, 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., neurological 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 demonstrates 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 efficacy 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 matching 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 system with immunosuppressive therapy, and (iv) controlling the recipient’s lifestyle through dietary 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. difficile 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 production of secondary bile acids (105). To understand the mechanism behind resistance to colonization 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 indicate that specific microbes with specific genes present in DMC have antagonistic activity toward target pathogens. Hence, DMC has a number of advantages over FMT, such as (i) minimum 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 intensive and warrant metagenomics analyses, safety testing, and postengraftment evaluation 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 engraftments (107). These data suggest the possibility of excluding therapeutic microbes when creating DMC, resulting in a subefficacious outcome. Alternatively, advanced cultivation and identification methods like culturomics—a high-throughput bacterial culturing method that comprehensively identifies strains/species from a complex microbiota 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 therapeutic value. In an interesting study, sterile-filtered stool, which retained virus-like particles, effectively cured six recurrent CDI patients (111). In essence, symbiotic interactions 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 pathways 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 intraspecies 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.

TABLE 4.

Interactions among species grouped broadly into two categories depended if interaction is positive or negative^a

Type	Interaction	Definition/description	Effect on species 1	Effect on species 2
Positive	Mutualismb	Each species benefits from one another 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 of the other	+	+
	Syntrophismb	Species 1 is dependent on the metabolic product of species 2	+	+
	Commensalism (commensal and host)		+	∅
Negative	Amensalism		∅	−
	Parasitism (parasite/pathogen and host)b,d	An individual species derives nutrients from the host species In most cases the parasite does not kill the host	+	−
	Predationd	One organism kills and consumes the other	+	−
	Competition	Intraspecific competition (species strain 1a ≠ species strain 1b) Interspecific competition (species 1 ≠ species 2)	−	−

^a∅, neutral effect on species.

^bObligatory interaction (mutualist and host are metabolically dependent).

^cNonobligatory interaction (independent).

^dAntagonistic interaction.

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 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 simplified MMIs’ principal components and identified candidates for exerting positive or negative 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 limitations 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 isolated 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 relatively 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 integrating 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 bacteria 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 fluorescence. 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 species 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 developed and applied to identify oral microbiota species with antagonistic characteristics toward 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-resistant 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 samples 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 staining 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 rapidly and efficiently identify lytic bacteriophages against MRSA.

FIG 3.

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

Limitations of the HiSCI platform.

Our developed platforms have many advantages in identifying lead microbes; however, we also observed a small number of notable challenges. (i) The current process of capturing microbial cells in MDs is highly inefficient, 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 μm 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 species 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 phenotyped (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 analyzed, 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., animals, 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 benefitting 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 virotherapy and bacterial therapy), wherein therapeutic microbes selectively infect and kill cancerous 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 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 material 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 revolutions 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 development 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-series predictive modeling, and HiSCI, we are able to identify millions of potential therapeutic microbes and extrapolate outcomes in complex life forms. However, the conventional methods still have a degree of applicability in the digital age, specifically validating computational models with observable biological outcomes. For example, authors have isolated and identified a C. scindens strain and demonstrated with metagenomic and time-series analysis modeling 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 studies suggest that commensal bacterial species in the presence of pathogens can act as therapeutic 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 pharmacodynamics during clinical testing (129). For example, the growth rate of these microbes is dependent on person-specific factors like food intake, native microbiota composition, and disease 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 challenge 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 pharmacological processes—absorption, distribution, metabolism, and excretion—have limited application in the use of therapeutic microbe products. Instead, gastrointestinal distribution, attachment (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 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 companies 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 forward 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 microbiome, 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.

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.

CONCLUSION

Due to limited technologies, exploiting a microbial community’s structure and function for the discovery of favorable therapeutic microbes remains a daunting task. Recent bursts of microbiome research have ignited this research field, but it needs significant interdisciplinary collaborative efforts to address discovery and application challenges. We expect living therapeutics and diagnostics to be the next medical revolution, similar to the discovery of antibiotics in the early 19th century.

Biographies

Nathan Cruz is a post-master’s student at Los Alamos National Laboratory (LANL). He holds a B.S. in psychological sciences and a M.S. in biology from Northern Arizona University (NAU). During his undergraduate studies, he assisted Dr. Christopher Woodruff in recording neuron activity using electroencephalography in humans to understand the neuroscience of empathy. In his senior year, he joined Dr. Robert Kellar’s regenerative medicine laboratory to gain experience in mammalian cell culture techniques. By securing the NAU Research Initiative for Scientific Enhancement scholarship, he enrolled in a M.S. program and continued research under Dr. Kellar. With cell culture experience, in his M.S. studies he used an in vitro model to investigate the nocuous effects of uranyl nitrate on wound healing. While analyzing his research data, Mr. Cruz realized how important statistical tools are in hypothesis-driven biological research. This led him to simultaneously graduate with a certification in applied statistics. At LANL, he works with Dr. Anand Kumar in basic and applied aspects of STEM research. Concurrently, he is supporting the development of a microfluidic-based bacteriophage discovery platform. 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.

George A. Abernathy is a post-master’s student working with Dr. Anand Kumar at LANL. He holds a B.S. in genetics and biotechnology from New Mexico State University and an M.S. in integrative genomics from Black Hills State University. While an undergraduate student, he received a research scholarship from the Howard Hughes Medical Institute to acquire basic microbiological skills. After obtaining his B.S., he worked with veterinary physicians as a full-time assistant to support surgeries and routine care for small and exotic animals. During his five years of veterinary assistantship, he developed a passion for research with the ultimate goal of promoting animal health. He enrolled in a graduate program and conducted research under the guidance of Dr. Cynthia Anderson. His research focused on the effect of a natural antifungal agent against fungal species pathogenic to humans and animals. With his M.S. in integrative genomics, he joined Dr. Kumar’s team at LANL to develop an innovative and rapid phage technology to discover an effective lytic phage cocktail against antibiotic resistant pathogens. Mr. Abernathy plans to pursue an integrated D.V.M. research program to conduct translational research that benefits human and animal health.

Armand E. K. Dichosa, Ph.D., M.S., is a staff scientist in Bioscience Division at LANL. He received both his M.S. and Ph.D. from the University of New Mexico in biology, investigating extremophile archaeal and bacterial communities and their potential roles in their respective environments. His core research interests focus on the genomic and viable recovery of novel bacterial species from various environments and determining their potential beneficial or adverse impacts on human health, national security, and the environment. Dr. Dichosa pioneered the integration of gel microdroplets with single-cell genomics to dissect complex microbiomes for various research efforts involving biodegradation, probiotic therapies, and novel bacterial characterization. He collaborates on various projects with Dr. Anand Kumar and his team to discover therapeutic microbes. Dr. Dichosa is involved in promoting global cooperatives for microbial biosurveillance through genomics and bioinformatics outreach efforts, as well as leading microbiology-genomics STEM efforts for our nation’s high school students.

Anand Kumar, D.V.M., Ph.D., is a staff scientist in the Bioscience Division at LANL. He received his D.V.M. in India and M.S. and Ph.D. degrees from the Ohio State University. His Ph.D. research focused on understanding host responses to probiotics as well as investigating the impact of diet and rotavirus infection on a humanized pig model. During his M.S. research, he investigated Campylobacter pathogenesis and developed prophylactic (nanoparticle vaccine) and therapeutic (small-molecule discovery) measures against it. During his postdoctoral tenure at LANL, he led several efforts to establish and develop microbiome-screening platforms. Dr. Kumar’s research is currently geared toward microbiome engineering by (i) discovering and developing novel microbiome-based therapeutics to tackle the threat of emerging bacterial pathogens, (ii) engineering probiotic strains to create a living diagnostic-therapeutic system, and (iii) evolving a bioreactor model of the human gut to investigate host-microbe interactions.