Bugs as Drugs: Therapeutic Microbes for the Prevention and Treatment of Disease

The gastrointestinal tract (GIT) is a diverse and complex ecosystem shaped by continual interactions between host cells, nutrients, and the gut microbiota. The gut microbiome is estimated to contain approximately 1013 bacterial cells and is dominated by the major phyla Firmicutes, Bacteriodetes, Actinobacteria, Proteobacteria, and Verrucomicrobia ( 1 , 2 ). Early colonizers of the GIT include bifidobacteria from the phylum Actinobacteria. These commensal microbes colonize immediately after birth and are speculated to prime the GIT and influence the gut-brain axis ( 3 – 5 ). The infant microbiota is considered to be relatively unstable. Despite dramatic changes in the microbiome structure during early life, the gut microbiota increases in diversity and stability over the first 3 years of life ( 6 ). Following this initial establishment, the microbiomes of children are generally enriched in Bifidobacterium spp., Faecalibacterium spp., and Lachnospiraceae compared to adults ( 7 – 9 ). During adulthood, the gut microbiome is considered to be stable and is dominated by the phyla Firmicutes and Bacteriodetes. While bacterial populations vary between individuals, the fecal microbiota of adults is highly stable through time ( 6 ). This stability is maintained until older age (>65), when the microbiome stability and function begin to decline ( 10 , 11 ).

The genus Lactobacillus includes 177 species (http://www.bacterio.net/lactobacillus.html): they are non-spore-forming, mostly nonmotile, and rod-shaped (although coccobacilli are observed). They generally have a fermentative metabolism (although genome sequence analysis has provided evidence of potential for respiration [ 1 ]) with lactic acid as the main fermentation product.

The genus Bifidobacterium is included within the phylum Actinobacteria, class Actinobacteria (high G+C Gram-positive bacteria), order Bifidobacteriales, and family Bifidobacteriaceae. Currently, this genus contains more than 50 species, including several subspecies; this number rises every year. From a metabolic point of view, the more typical trait of this genus is the catabolism of monosaccharides. Bifidobacteria use a particular route for monosaccharide degradation, the so-called fructose 6-phosphate pathway, or bifid shunt. The fructose 6-phosphate phosphoketolase (Xfp) is the main enzyme of this path. Xfp possesses a dual-substrate specificity on fructose 6-phosphate or xylulose 5-phosphate. The end metabolites of the pathway are acetate, lactate, and ethanol ( 1 ). Xfp activity on fructose 6-phosphate is the most common phenotypic test for bifidobacteria, and for many years it has been the main taxonomic test to identify this genus, since this activity is present in members of the family Bifidobacteriaceae, but it is not present in other Gram-positive intestinal bacteria. However, currently, DNA-sequencing-based analyses are the standard techniques for identification and typing of bifidobacteria.

Colorectal cancer (CRC), the most common form of gastrointestinal (GI) tract cancer, is globally the third leading cause of cancer and is associated with significant mortality ( 1 ). Approximately 90% of CRC cases are sporadic, caused by somatic mutations leading to the progression of invasive carcinomas ( 2 ). There are numerous risk factors associated with the development of CRC, and the disease is more common in industrialized countries than it is in the developing world ( 1 ). Poor diet (in particular, a diet that is low in fiber and high in fat) appears to be a major influencing factor for disease development and progression, and recently it has been recognized that gut microbes may act as the interface between dietary factors and tumor development (reviewed in reference 3 ). This chapter will review the pathways that lead to CRC, what is currently known about microbial involvement in these processes, and how these may be manipulated therapeutically.

Malnutrition, encompassing both excessive and insufficient nutrient intake, is a major public health concern worldwide. On the one hand, overweight and obesity affect more than one-third and one-tenth of the world’s population, respectively. Excessive body weight and fat mass gain are classically linked with several metabolic disorders and cardiometabolic risk factors, including insulin resistance, type 2 diabetes, hypertension, low-grade inflammation, and liver diseases ( Fig. 1 ). Over the past 20 years, researchers have gathered evidence showing the involvement of chronic inflammation in the onset of the metabolic syndrome. Among the plethora of factors involved in the etiology of metabolic disorders, our lab and others have shown that the interplay between a too-rich diet and another environmental factor, namely the gut microbiota, plays a major role (for reviews, see references 1 to 5 ).

In order to design and apply a therapeutic, there must be a vaginal disease or disorder in need of treatment or a condition influenced by the vaginal microbiota. There must also be a way for a new therapeutic agent to function through the microbiome. Those conditions include bacterial vaginosis (BV), aerobic vaginitis (AV), urinary tract infection, urethritis, cervicitis, vulvovaginal candidiasis, vulvodynia, endometriosis, and chorioamnitis. In addition, sexually transmitted infections are included, as arguably some may be prevented by vaginal microbes. Cancer is included, not only because of the association with viral infection, but also because microbial dysbiosis has been associated with cancers at other sites.

In 2007, the Human Microbiome Project (HMP) was added to the National Institutes of Health (NIH) Roadmap for Medical Research, and since then, over $200 million has been invested in the exploration of the human microbiome. Several sites on the human body, including the nares, oral cavity, skin, gastrointestinal tract, and urogenital tract, have been studied by the HMP ( 1 ). The gastrointestinal tract remains the most thoroughly investigated organ-microbiome interaction studied thus far, and its role in shaping the host immune response is rapidly becoming defined within the context of inflammatory response ( 2 – 4 ). Observations have noted associations of specific microbes and the gut microbiome in obesity ( 5 – 8 ), coronary artery disease ( 9 – 12 ), Clostridium difficile colitis ( 13 , 14 ), type 2 diabetes ( 15 , 16 ), and inflammatory bowel disease ( 4 , 17 , 18 ). While the nares and oral cavity were included as locations to be studied for the HMP, the lower airway respiratory system was not included as a location of interest. The microbial community of the oropharynx had been well described even before the advances of next-generation sequencing and multiplexed data ( 19 , 20 ). Microbiome approaches to the upper and lower respiratory systems created a deluge of associations between host and microbes in health and disease ( 21 – 23 ). Over the past few years, our understanding of the airway microbiome has shifted, upending the old adage of the lungs being a sterile field ( 24 ) to a new paradigm of a continuous organ system with a rich and vibrant mucosal surface that embodies complex interactions between the microbiome and its host that can propagate or resist disease ( 21 , 24 ). Moreover, little is known regarding how the respiratory microbiome interacts with the host immune response. While multiple research projects have focused on the effects of the gut microbiota on the immune response of the gastrointestinal mucosa ( 25 – 27 ), few papers have focused on the effects of the lower airway microbiota on the respiratory mucosa that it inhabits ( 23 , 28 – 30 ).

Excessive alcohol consumption and being obese/overweight are the leading causes of chronic liver disease in Western countries. Nonalcoholic fatty liver disease (NAFLD) encompasses all liver lesions that can be observed in overweight/obese patients, ranging from pure steatosis to steatohepatitis (nonalcoholic steatohepatitis [NASH]), fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC). Alcoholic liver disease (ALD) defines liver lesions observed in patients with alcohol abuse and includes steatosis, hepatitis, fibrosis, cirrhosis, and HCC. It usually occurs when alcohol consumption is higher than 70 g/day in men and 60 g/day in women. However, only some chronic alcohol consumers develop liver injury, suggesting that factors other than excessive alcohol consumption play a role in ALD. Moreover, some patients with other liver diseases, such as chronic hepatitis C or NAFLD, also consume alcohol. Alcohol may lead to liver injury at a lower level of consumption in these patients with another underlying liver disease.

The adult human skeleton comprises 206 bones, excluding the sesamoid bones ( 1 ). The bones are subdivided into four general types: long bones, short bones, flat bones, and irregular bones. Long bones such as the femur are composed of a hollow diaphysis which flares at the end to form the metaphysis, the region below the growth plate, and the epiphysis, the region above the growth plate. The diaphysis, also known as the shaft, is mainly composed of dense, solid bone known as cortical bone, whereas the metaphysis and epiphysis contain a honeycomb-like network of interconnected trabecular plates surrounding bone marrow known as cancellous or trabecular bone ( 1 ).

The oral microbiome is formed by hundreds of microbial species, including bacteria, fungi, archaea, and viruses, which coexist in specific and organized arrangements in the different habitats of the oral cavity ( 1 – 8 ). Oral subhabitats include the mucosa, covered by keratinized and nonkeratinized stratified squamous epithelium, the papillary surface of the tongue dorsum, and the hard structures of teeth, which comprise those above (supragingival) and those below (subgingival) the gingival margin. The distinct environmental characteristics found in each of these habitats promote the development of unique microbial communities that, although living in close proximity, can be clearly discriminated from each other ( 9 – 12 ). Moreover, the microbial composition of these communities is critical to oral health, with the main oral diseases characterized by deleterious alterations in microbiome community structure at specific subhabitats ( 13 , 14 ).

Each year in the United States, billions of dollars are spent combating almost half a million Clostridium difficile infections (CDIs) and trying to reduce the ∼29,000 patient deaths in which C. difficile has an attributed role ( 1 ). In Europe, disease prevalence varies by country and level of surveillance, though yearly costs are estimated at €3 billion ( 2 ). One factor contributing to the significant health care burden of C. difficile is the relatively high frequency of recurrent CDIs ( 3 ). Recurrent CDI, i.e., a second episode of symptomatic CDI occurring within 8 weeks of successful initial CDI treatment, occurs in ∼25% of patients, with 35 to 65% of these patients experiencing multiple episodes of recurrent disease ( 4 , 5 ). Using microbial communities to treat recurrent CDI, either as whole fecal transplants or as defined consortia of bacterial isolates, has shown great success (in the case of fecal transplants) or potential promise (in the case of defined consortia of isolates). This review will briefly summarize the epidemiology and physiology of C. difficile infection, describe our current understanding of how fecal microbiota transplants treat recurrent CDI, and outline potential ways that knowledge can be used to rationally design and test alternative microbe-based therapeutics.

The human intestinal tract contains trillions of bacteria, collectively called the gut microbiota. The majority of bacteria belong to the Gram-negative phyla Bacteroidetes and Proteobacteria and the Gram-positive phyla Firmicutes and Actinobacteria ( 1 ). In humans, the diversity of the gut microbiota and the abundance of species increase rapidly after birth and, after 2 to 4 years, remain relatively stable throughout adult life ( 2 ). Nevertheless, shifts in gut microbiota composition may occur, especially after use of antibiotics. Even a short course of antibiotics can result in perturbations that last for several years ( 3 , 4 ).

The genus Enterococcus comprises over 50 species that can be found in diverse environments, from the soil to the gastrointestinal (GI) tract of animals and humans to the hospital environment ( 1 , 2 ; http://www.bacterio.net/enterococcus.html). The first member of this Gram-positive genus was isolated in 1899 from a lethal case of endocarditis ( 3 , 4 ). It was not until 1984 that enterococcal species were seen as genetically distinct from Streptococcus and assigned their own genus ( 3 – 5 ). Enterococci are Gram-positive facultative anaerobes that exist in chains or pairs and do not form spores. They grow optimally at 35°C, hydrolyze esculin in the presence of 40% bile salts, and are catalase negative ( 6 , 7 ). Enterococcal species can be distinguished by phenotypic tests that rely on strains’ ability to form acid in mannitol and sorbose broth and to hydrolyze arginine ( 8 , 9 ).

Genetically engineered bacteria have the potential to diagnose and treat a wide range of diseases linked to the gastrointestinal tract, or gut. Such engineered microbes will be less expensive and invasive than current diagnostics and more effective and safe than current therapeutics. Recent advances in synthetic biology have dramatically improved the reliability with which bacteria can be engineered with the sensors, genetic circuits, and output (actuator) genes necessary for diagnostic and therapeutic functions. However, to deploy such bacteria in vivo, researchers must identify appropriate gut-adapted strains and consider performance metrics such as sensor detection thresholds, circuit computation speed, growth rate effects, and the evolutionary stability of engineered genetic systems. Other recent reviews have focused on engineering bacteria to target cancer ( 1 , 2 ) or genetically modifying the endogenous gut microbiota in situ ( 3 , 4 ). Here, we develop a standard approach for engineering “smart probiotics,” which both diagnose and treat disease, as well as “diagnostic gut bacteria” and “drug factory probiotics,” which perform only the former and latter function, respectively. We focus on the use of cutting-edge synthetic biology tools, gut-specific design considerations, and current and future engineering challenges.

Advances in recombinant technology (e.g., genetic engineering) and in the understanding of the human immune system have led to prodigious advances in the development of novel delivery systems for mucosal administration ( 1 , 2 ). The administration of therapeutic molecules through mucosal routes offers several important advantages over conventional strategies (i.e., systemic injection) such as reduction of secondary effects, easy administration, and the possibility to modulate both systemic and mucosal immune responses ( 3 ). Moreover, it is important for molecules of health interest that exert their effects at mucosal surfaces, the gastrointestinal tract (GIT), for example, to be delivered directly to the appropriate site. Nonetheless, a major disadvantage of the mucosal route of administration is that the actual amount of protein to be administered needs to be large due to the very small quantities of protein that survive degradation at mucosal surfaces such as the GIT ( 1 , 3 ).

With renewed interest in the human microbiome and its role in human health, unique opportunities for using microbes as therapeutics have recently emerged. These opportunities range from the traditional probiotics to the engineering of intestinal mutualistic bacteria to deliver therapeutic proteins ( 1 – 3 ). However, a more in-depth understanding of how intestinal microbes impact human health and how they function in the complex, dynamic environment of the gastrointestinal tract (GIT) requires advancements in genetic tools for nonmodel strains.

For thousands of years, lactic acid bacteria (LAB) have been interwoven with our food supply. The earliest evidence of milk use dates back to 7,000 years BCE ( 1 ), while cheeses were produced as early as 6,000 years BCE ( 2 ). Advances in our understanding of the microbial world in the past couple of centuries have enabled microbiology-based food manufacturing on an industrial scale. Not only are LAB used to ferment dairy products, but they are also applied to pickle vegetables, to cure meats, and to produce alcoholic beverages such as wine and sake ( 3 , 4 ). This long history of safe consumption led to the consideration that many LAB strains are Generally Recognized As Safe (GRAS). As early as 1906, LAB were linked to the promotion of human health. The Russian Nobel laureate Élie Metchnikoff hypothesized that ingestion of yogurt prolonged life in Eastern European populations by reducing “putrefying” [sic] bacteria. His linkage of the perceived longevity of the Eastern European populations with consumption of fermented dairy products ( 5 ) made him the grandfather of modern probiotics. Probiotics are defined as “live microorganisms, which when administrated in adequate amounts, confer a health benefit to the host” ( 6 ). Metchnikoff’s probiotic theory of life prolongation was never directly tested, and researchers reported in 1924 that LAB present in yogurt, specifically Lactobacillus bulgaricus, most likely do not reduce “putrifying” bacteria in the intestine because L. bulgaricus did not survive gastrointestinal (GI) transit ( 7 ). Other groups challenged this finding. Elli et al. demonstrated that both Streptococcus thermophilus and L. bulgaricus were present in human feces after yogurt ingestion ( 8 ).

Our expanding knowledge of the nature and importance of the human microbiota has provided additional impetus to investigate the use of beneficial bacteria to promote improved health. Recent technological developments in both next-generation DNA sequencing and novel bioinformatic analyses have advanced our understanding of the nature of our microbiome and the degree to which its makeup correlates with human health and specific disease states. It is clear that the application of these technologies has the potential to inform the mechanisms of beneficial effects and to identify potentially valuable candidates for intervention. It should be noted, however, that there is a long history of the use of bacteria, initially in the form of naturally fermented foods and later by investigation of the potential roles of individual bacterial strains in disease prevention. For example, the strain Escherichia coli Nissle 1917 was reportedly isolated about 100 years ago from a World War I soldier who, unlike his cohorts, appeared unaffected by bacterial dysentery, and it has remained a subject of study ever since ( 1 ).

Phage therapy is the use of bacterial viruses to reduce or eliminate bacterial infections. As such, phage therapy has been employed clinically for approximately 100 years. It is possible, nevertheless, that more English-language reviews and commentaries on bacteriophage use as antibacterial agents are published yearly (e.g., for 2015 [ 1 – 19 ] and for approximately the first third of 2016 [ 20 – 30 ]), than there are people within the borders of the United States who are subject to officially sanctioned phage treatment. This contrasts with well over 100 million courses of antibiotic that are prescribed per year ( 31 ).

Vertebrates have evolved with dense microbial populations in their gastrointestinal (GI) tract (referred to as the GI microbiome) that contribute to performance and health of the host ( 1 ). Although symbiotic in nature, animal experiments have established that the GI microbiota plays a causative role in the development of chronic noncommunicable diseases (CNCDs) such as obesity, diabetes, cardiovascular disease, colon cancer, autism, autoimmune diseases, allergies, and other atopic diseases including asthma ( Fig. 1 ) ( 2 ). CNCDs are often associated with microbial dysbiosis, which is typically characterized by a reduced diversity, a bloom of facultative taxa (such as enterobacteria), and a lower output of beneficial metabolites ( 3 ). These associations provide a clear rationale for the development of strategies that modulate GI microbiome structure and function for the prevention of CNCDs ( 4 ).