Defining the Microbiome Components (Bacteria,Viruses,Fungi) and Microbiome Geodiversity

Essential Components of the Microbiome

One of the most important historic paradigm shifts in modern medicine is the germ theory based on the nineteenth century work of Robert Koch. This novel discovery that pathologic microbes disseminated and caused infectious diseases sets the stage for antisepsis, antimicrobial agents, as well as source control. As the field of microbiology developed, we discovered that pathogenic microbes were only a small fraction of the species living around, on, and inside the human body. These observations were similarly extended to veterinary medicine in a synergistic fashion. The vast majority of identified microbes were either commensal and often—as increasingly delineated—beneficial to the organism. Modern DNA sequencing technologies helped identify, describe, and characterize microbial communities and their variations within and between individuals and groups. This gave birth to one of the next great paradigm shifts in medicine: the recognition of the microbiome and its role in health and disease.

Inquiry into the composition and function of the human microbiome has expanded greatly. Such studies have documented the massive number of symbiotic microbes that colonize almost every surface of our body including the skin, airways, urinary tract, dentition, and the epithelial lining of the gastrointestinal tract. ¹ These organisms are so abundant that the number of individual genes in the human genome is greatly surpassed by that of the colonizing microbes. This collection of bacteria, archaea, fungi, viruses, and eukaryotes are referred to as microbiota, which together with the individual tissues they inhabit is defined as our individual microbiome. ² This vast community of organisms work with each other, host tissues, and the immune system to influence most biological processes including: nutrition, ³ tissue and immunologic development, pathogen resistance, as well as surgical wound healing. ⁴

The explosion of research into the human microbiome has been made possible with recent technological and bioinformatics advances in high throughput DNA sequencing technologies. These technologies have allowed robust characterization of not just the bacterial components of the microbiome but the viral and fungal components as well. These diverse communities are characterized by the diversity of species present in a given sample and also by the differences in diversity between one sample and another. These quantities are mathematically quantitated as alpha and beta diversity statistics. Alpha diversity summarizes the structure of a given microbial community with respect to the number of taxonomic groups it contains and the distribution of abundances of these groups (within sample diversity). Beta diversity metrics describe how different microbiome samples vary from one another (between sample diversity). ⁵ Taken together, alpha and beta diversity statistics give a measure of the total biodiversity and richness of a given microbiome sample.

Specific Microbiome Components

Bacteria are the most well-studied component of the human microbiome based on next-generation whole-genome sequencing techniques. Most of the bacterial species present in the human body come from four phyla: Bacteroidetes, Firmicutes, Actinobacteria, and the Proteobacteria. ⁶ However, a majority of an individual's microbial diversity, their microbiome's unique signature, derives from thousands of less numerous species. ⁶ There is also diversity within each body system based on the various niches of colonization, thereby demonstrating the crucial role of the host tissues in the composition of the overall microbiome. Cutaneous surfaces, for example, vary in their microbial flora depending on the site of colonization. Sebaceous areas are dominated by Actinobacteria whereas dry skin is predominately colonized by Proteobacteria. ⁶ The composition of flat skin surface differs from in-folded areas such as the armpit or gluteal cleft, which are dominated by Staphylococcus or Corynebacterium species. ⁶ The healthy skin's commensal bacteria benefit the host by producing inhibitory compounds or outcompeting pathogenic strains for limited resources. ⁶ On the other hand, disruptions in normal skin architecture, including inflammatory states such as Hidradenitis suppurativa, are associated with polymicrobial cultures of Staphylococcus aureus and anerobic species that may be maladaptive. ⁷

Although bacteria are the best-characterized components of the microbiome, viruses also play a large role in its composition and function. Viruses are, by definition, parasitic entities that require host cells for replication. They are composed of single- or double-stranded DNA, or RNA, and a protein capsid. Viruses infect nearly every type of cell in existence including eukaryotes, archaea, fungi, and bacteria. Bacteriophages, or viruses that infect bacteria, are the main viral component of the human viral microbiome and exist on all surfaces of the human body. ⁸ Other represented viruses include enteroviruses, rotaviruses, and noroviruses.

The role of viruses in the gut microbiome is not well understood. Nonetheless, some studies suggest that viruses may help to maintain a healthy balance of gut microorganisms and support the immune system. Bacteriophages, in particular, appear to have a profound impact on the composition and functional properties of the bacterial microbiota and in this respect are a unique element that helps shape innate, humoral, and cell mediated immunity. ⁹

Bacteriophages serve as important reservoirs of genetic diversity by acting as vehicles for the horizontal transfer of genetic material between bacteria. In this way, bacteriophages may alter bacterial metabolism and virulence, including antibiotic resistance. They can also impact the overall composition of commensal bacterial populations by enabling aspects of bacterial metabolism that provide a selective survival advantage. Duerkop et al. ⁹ did elegant work studying the chromosomally encoded prophage elements of Enterococcus faecalis V583. When V583 colonizes the intestines of mice, it produces a chromosomally encoded bacteriophage that is harmful to other closely related Enterococcus species thereby conferring a competitive advantage to V583 and thus altering the composition of the microbiome. ¹⁰

Previous methods for studying the microbiome included 16S ribosomal RNA sequencing that were targeted exclusively to bacteria. However, with new whole genome sequencing approaches, fungal elements are increasingly recognized as a vital part of the human microbiome. A diverse group of fungi have been identified that are particularly associated with the digestive tract. These fungi include those from the Candida, Rodotorula, Issatchenkia, Malassezia, and Sarccharomyces genera. ¹¹ Fungi demonstrate roles in both regulating the composition of microbiome organisms and influencing the host immune system in a beneficial fashion during health. On the skin, the predominate fungal genus is Malassezia, which has adapted to their environment by using skin lipids as nutrients. Their presence is beneficial as Malassezia secrete antimicrobial products that deter skin bacterial pathogen growth. ¹²

Fungal elements of the microbiome also appear to modulate the host immune response. ¹³ Candida albicans, by far the most abundant fungus in the gut, plays a crucial role in activating human T helper 17 (T _h 17) cells. The T _h 17 cells orchestrate protective immunity at barrier sites and are principally confined to the intestine and skin. However, dysregulated T _h 17 responses are associated with inflammatory disorders such as inflammatory bowel disease. ¹⁴ Shao et al. ¹⁰ demonstrate that, in mice, intestinal colonization with Candida albicans drives expansion of fungal specific T _h 17 cells systemically and increases circulating neutrophils interleukin (IL)-17 responsiveness. These two linked processes help protects against pathogenic invasive Candida albicans infection by priming the immune system to early recognition and eradication of invasive species. ¹⁵ These observations and others suggest that therapeutic targets tied to specific clinical conditions might be possible. For example, blocking IL-17 is intuitively attractive to manage inflammatory bowel disease, but so doing is unfortunately devoid of clinical benefit in recent randomized trials. ¹⁶ Similar to other attempts to impact a single point in a complicated cascade of interrelationships, failure is not unanticipated. It is likely that alternate control points, or a montage-based approach will be required for clinically relevant impact.

The Geodiversity of the Microbiome

The human microbiome exhibits tremendous geodiversity or change in its composition according to anatomic location and environmental factors (Fig. 1). This is particularly exemplified in the gastrointestinal tract where there is the largest area of interaction between human cells and microbes. Nonetheless, different sites demonstrate different microbiomes and may be derailed from homeostasis to a maladaptive state known as a dysbiome. The dysbiome is generally characterized by a decrease in microbial population diversity and may maladaptively impact a variety of organ functions spanning nutrient processing to neoplasia surveillance.

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FIG. 1.

Human microbiota compositions by anatomic location. Predominant bacterial genera are italicized and presented in decreasing order of predominance.

Oropharynx

Hundreds of bacterial species reside in the mouth, in addition to numerous fungal species, such as Candida or Aspergillus. These organisms colonize either the stable dental surface or proliferative mucosa and play an important role in host digestion and immunity. ⁶ A whole-genome sequencing study of healthy patients' oral microbiomes found that the genera Streptococcus, Neisseria, Prevotella, and Haemophilus were among the most abundant bacterial groups present across all sample sites and methods. ¹⁷ The study also noted that the viral population was dominated by bacteriophages preying on the dominant bacterial strains. ¹⁷ The healthy oral microbiome also appears to be a reservoir for antibiotic resistance genes, including those for macrolides and clindamycin. ¹⁷ These observations will have implications for dental procedures to reduce surgical site infection, oral care to address infections related to oral endotracheal intubation, as well as dental implant manufacture to help retard implant infection.

Upper and Lower Gastrointestinal Tracts

The stomach and small intestine contain fewer species and lower total number of organisms compared to the large intestine by a factor of 10⁸. However, the upper and lower tract share several bacteria taxa during health with Firmicutes, Proteobacteria, Bacteroides, Actinobacteria, and Fusobacteria being the most common in the upper gastrointestinal tract. ¹⁸ The human stomach is a highly acidic environment once thought to be inhospitable to micro-organisms. Yet, although it has several orders of magnitude fewer organisms than the small intestine, it is still a rich and diverse environment where maintenance of a healthy microbiome is emerging to be of significant importance to health. Of particular interest in the stomach, is the potential to be colonized with Helicobacter pylori. Helicobacter pylori is usually thought of as a pathologic organism because of its association with the development of peptic ulcer disease, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. However, recent studies have shown that Helicobacter pylori is a pathobiont, or natural member, of the microbiome that only has pathogenic potential under certain conditions. Although only a small minority of patients colonized with Helicobacter pylori develop disease, those that do develop disease typically exhibit dysbiosis. ¹⁸ Additionally, standard treatment for Helicobacter pylori infection involves antibiotic and proton pump inhibitor therapy, both of which further promote dysbiosis. As we come to understand the role of the gastric pathobiome and its interplay with Helicobacter pylori-induced pathologies, regulation or restoration of a healthy gastric microbiome is emerging as a potential preventative measure or even as therapeutic. ¹⁹

In the large and small intestine, bacterial and fungal species play an important role in host metabolism by degrading undigested complex carbohydrates, synthesizing vitamins, regulating epithelial cell development, and enhancing host immune tolerance. ⁶ For example, Clostridium and Bifidobacterium species are responsible for degrading undigested complex carbohydrates which reach the large intestine into short-chain fatty acids (SCFAs), which are used as fuel, increase nutrient absorption, and regulate the growth and development of the intestinal epithelial cells. ⁶ Other bacterial species synthesize vitamins or increase the absorption of minerals, including calcium and magnesium. ⁶ Maintenance of a healthy microbiome likely protects against pathologic micro-organisms. For example, Lactobacillus acidophilus and other commensal species inhibit pathogenic invasion by occupying epithelial binding sites, consuming limited nutrients, and producing inhibitory bacteriocins. ⁶ On the other hand, disease states such as Crohn's disease and ulcerative colitis appear to be associated with exaggerated immune responses, specifically immunoglobulin G secretion, against commensal bacteria as well as distortions in mucosal associated bacterial communities. ⁶

Pulmonary Tract

The respiratory tract is similar to the gastrointestinal tract in that it contains a gradient of microbial colonizers. The conducting system (nose to bronchi) is heavily colonized with bacteria whereas the respiratory system (respiratory bronchioles and alveoli) contains a lower abundance of microbes under normal circumstances. ⁶ For example, in healthy lungs, the microbial mass is 10³ to 10⁵ bacteria per gram of tissue compared with 10¹¹ to 10¹⁵ bacteria per gram of tissue in the colon. Nevertheless, the lung contains a diverse and important microbial community. Some exposure to microbial organisms appears required for healthy lung development, as germ-free mice were found to have smaller lungs that contained fewer alveoli and lacked lymphoid tissue development prior to Lactobacillus exposure. ¹⁵ Later in life, respiratory commensal species function to out compete pathologic organisms and prime the immune system to detect and respond to pathogen invasion. ¹⁵

The composition of the pulmonary microbiome is controlled by three factors: immigration into the airways via inhalation or microaspirations, the rate of elimination of microbes from the airway through mucociliary clearance and cough, and regional growth conditions in the lung. Although immigration and elimination remain relatively constant, regional growth characteristics can change dramatically during disease. ²⁰ Of particular interest with respect to pulmonary diseases is the concept of a lung–gut axis. This is the finding that dysbiosis of the gut is associated with both pulmonary dysbiosis and pulmonary diseases. For example, factors associated with the pathogenesis of asthma including vaginal birth, exposure to pets, breast-feeding, and antibiotic use in late pregnancy are also strongly associated with gut dysbiosis. In a Canadian study, the presence of Lachnospira, Veillonella, Faecalibacterium, and Rothia in the gut of three-month-old infants correlated with an increased risk of asthma development. ²¹ Although the precise aspects of the lung–gut axis relationshs are not well understood, other pulmonary diseases such as chronic obstructive pulmonary disease, malignancy and bronchiectasis are associated with dysbiosis at both anatomic sites. ²⁰

Bladder

Previously considered sterile, a healthy bladder hosts numerous bacterial and viral species. This key change in perspective flows from next-generation sequencing technology that has identified bladder and lung commensals alike. The bladder microbiomes of males and females are mostly the same, except for more abundant Corynebacterium and Streptococcus species in men and more abundant Lactobacillus species in women. ²² Urinary bacteria may improve host health by producing neurotransmitters capable of interacting with the peripheral nervous system, degrading renally excreted toxins, and prime epithelial and immunologic defenses against pathogenic species. On the other hand, overgrowth of commensal bacteria leading to chronic urinary tract infections as well as the sequestering of toxic compounds that may contribute to the genesis of bladder malignancy. ²³

Vagina

Finally, the vaginal canal contains a large colony of mutualistic microbes. Lactobacillus species represent the major bacterial inhabitant of the vaginal canal and certain species are associated with greater or lesser resistance to dysbiosis and infection. The various species of Lactobacilli maintain their microbiologic niche by producing lactic acid and bacteriotoxins. ²⁴ The vaginal microbiome maintains a tightly controlled balance during health. In contrast, increased vaginal flora diversity is associated with a heightened risk of Gardnerella infection, which in turn crafts a proinflammatory environment. ¹² Augmented local inflammation raises the risk of sexually transmitted infections and well as human papilloma virus persistence. ¹² Chronic vaginal inflammation has some overlap with carcinogenesis by inhibiting the regression of cervical intra-epithelial neoplasia. ²⁴ Chronic inflammation also supports the persistence of pathogenic species such as Prevotella bivia, a bacteria that is related to neoplastic lesion recurrence after local excision. ²⁵ Clearly, similar to other microbiomes, the vaginal microbiome may be influenced to provide host benefit or harm.

Age-Related Changes

The microbiome is neither a static nor uniform community even in healthy humans. For example, the gut microbiome undergoes major changes during the first years of life, beginning as a primarily aerobic community and transitioning to an anaerobe-dominated one by the first year of life. ² Later in life, the gut microbiome shifts again and is characterized by reduced diversity and a rise in pathogenic species. ¹ This later change suggests that ageing induces dysbiosis and raises questions regarding a natural progression toward dysbiosis that may be re-aligned with balance. The microbiome's diversity appears to be unrelated to genetic diversity, as the microbiomes of monozygotic twins are scarcely more similar than those of dizygotic twins. ⁶ On the other hand, cultural and geographic factors may play a more substantial role in gut microbiome diversity. Individuals consuming a European diet high in sugar, starch, and animal proteins were found to have different proportions of certain phyla compared with those consuming an African diet higher in plant fibers. ¹

Other lifestyle variables that impact the microbiome include pet and livestock exposure, which decrease the rates of atopic conditions in children, and enhance microbiome similarity between individuals cohabitating with dogs. Exercise, sleep, and stress all exert effects on the microbiome by varying proportions of bacterial phyla, metabolites, and cytokines. Certainly, exercise and sleep support a healthy microbiome although stress does not, adding a different dimension to the pursuit of wellness. Finally, the factor most associated with changes in the microbiome is antibiotic exposure—whether empiric, therapeutic, or prophylactic—all of which reduce microbial diversity substantially. Reduced microbiome heterogeneity is a common characteristic of the microbiomes of surgical patients after antibiotic administration. ²⁶

Conclusions

In summary, the human microbiome is a community of bacteria, viruses, and fungi interacting with a particular anatomic and environmental niche. Collectively, these microbial populations are emerging as significant influencers of human health. Furthermore, derangements of the normal, balanced, and diverse microbiome are implicated in a variety of clinical conditions including inflammatory disorders, malignancies, and metabolic disorders. Understanding these dynamic interactions and how we can best preserve a healthy microbiome or rehabilitate a deranged one underpins the next paradigm shift in modern healthcare. Such changes are relevant for surgeons as some conditions that currently require surgical management may be entirely managed using microbiome management techniques, or may have the surgical approach altered to address specific aspects of the microbiome such as durable implant design that resists microbial colonization and infection.