Diversity of bacteria within the human gut and its contribution to the functional unity of holobionts

Abstract

The composition of bacteria in the human colon has been a subject of interest since the beginning of microbiology. With the development of methods for culturing strict anaerobic bacteria under multiple culture conditions, it was shown the gut contained more than 400 bacterial species and different people harbor different abundant species. The term “gut microbiome” in this review refers to bacteria studied in stool samples. Molecular methods for determining the bacterial composition of human gut has revealed more than 3000 species and less than 130 genera, indicating that the diversity of human colonic bacteria is concentrated at the species and strain levels. This review concludes with a discussion of how diversity can lead to unity of individual holobionts, between holobionts, and between populations. One of the reasons for the unity is that different bacterial species can have similar functional genes.

Background

Diversity and unity are seemingly opposite entities. However, they have become closely knit throughout history, as the expression “Unity in Diversity” can exemplify. This expression means integrating diverse parts by harmony and integrity between these dissimilar entities. This concept can be traced back to the famous Sufi scholar, Ibn Al Arabi (1165–1240), who termed the metaphysical concept of “unity of being”, further on to different religions, and to philosophers as Gottfried Wilhelm Leibniz (1646–1716), who advanced the term “Harmony” that includes both unity and diversity. Unity in diversity is also discussed within the discipline of psychology via the integration of different theories and in politics as in Italian unification and the European Union. Harmony and integrity between diverse parts exist also in the living world that is immensely diverse but has much in common and is deeply interconnected and interdependent. The same is true about humans who are seemingly so different, but have so much in common physically and mentally, and about the human body that is constructed of millions of cells and includes endless chemical and physical activities, however, all sum up into one unit that we and others term a human holobiont.

As has been suggested previously, humans, as all other animals and plants, are not individuals or single units, but are holobionts (sometimes referred to as metaorganisms¹), namely, each includes the host and all of its symbiotic microorganisms, which live in it and on it ^2,3. In this article, we shall demonstrate how the vast diversity of bacteria in the human body contains within it also an inherent unity. We shall do so by focusing on the human gut, which is the largest container of microorganisms in the body. The term “gut microbiome” in this review refers to bacteria studied in stool samples. Microbial DNA extracted from feces has been demonstrated to be a useful proxy of distal colon microbiome⁴.

The first observations of intestinal bacteria were made by Anthony van Leeuwenhoek and published (after translation) in the Philosophical Transactions of the Royal Society in 1674⁵. He described in detail the size, shape and movement of the microorganisms he observed. However, he was not able to determine if what he observed was due to a single microbe that had many forms or a mixture of different organisms. Only two centuries later, when pure culture techniques were developed, was it possible to conclude that Leeuwenhoek had observed mixtures of different bacteria. Thus, Leeuwenhoek was the first to describe diversity of bacteria in the human gut.

Almost two hundred years later, the German pediatrician Theodor Escherich, who discovered E. coli, published what is considered the first comprehensive study of intestinal bacteria in humans⁶. He referred to the bacteria he had observed in the lower sections of the gut as the “bacterium coli commune”. Escherich would have definitely been pleased by the pivotal role that E. coli played in the development of molecular biology in the 20th century. In 1899, Henri Tissier at the Pasteur Institute in Paris extended Escherich’s research by demonstrating that the vast majority of bacteria in the gut were anaerobes. However, the culturing methods used by him and by others of his time yielded still a large underestimation of the diversity of gut bacteria. Almost a hundred years later, in the early nineteen seventies, the improvement of anaerobic cultivation techniques enabled isolation and cultivation of 88% of microscopic counts⁷. Nevertheless, in many cases, the characterization did not go beyond the level of the genus⁸. According to these authors, the number of microbial species in the human gut (Bacteria, Archaea and Eukarya) was estimated to be, in the beginning of the 1980s, ca. 400⁸. The reasons for this low estimate were (i) the large number of isolates made it technically difficult at the time to describe accurately the findings and define the species; (ii) many of the bacteria were not readily cultivated in vitro, and (iii) only the most abundant bacteria could be isolated and identified. Thus, only with the development of molecular technology, starting in the 1990s was it possible to characterize new bacterial species and determine more accurately their numbers and species distribution. Today, the most quoted numbers of bacterial species found at the individual level, range between one thousand and a few thousand^9–11, 90% of which are eubacteria. The numbers observed depend mainly on the identification techniques used and on the number of humans and feces sampled.

The total number of bacteria in the human gut (colon) of the 70 kg “reference man” is approximately 3.8 × 10¹³, similar to the number of human cells in the body¹². However, because all of the human cells contain the same genome, whereas the bacterial population in the human gut is composed of thousands of different bacterial species and strains, there are many more unique bacterial genes than human genes¹³. Thus, gut bacteria provide the human holobionts with enormous genetic potential and functional variation.

The bacterial composition of the human gut is the outcome of a lengthy and complex coevolution^14–16, which has led to a significant role of the gut bacteria in human holobiont physiology and health¹⁷. The human gut provides protection, food and appropriate growth conditions for the microbiome, and the gut bacteria provide, protection against pathogens^17,18, digestion of complex carbohydrates¹⁹, participation in brain–gut communication²⁰, and development of the immune system^21,22, the bone mass²³ and the blood vessels in the intestinal wall²⁴.

After reviewing the published data on gut bacteria diversity determined by both culture and DNA-based methods, we will discuss the underlying intercalation between unity and diversity within the human holobiont. We shall concentrate on the unity and diversity related to the human microbiome: within the human microbiome itself, between the microbiome and its human host and between the microbiomes of many human holobionts. However, this paper will not discuss the unity and diversity within the human hosts, since this subject has been discussed at length along the years²⁵.

Diversity of bacteria in the human gut

The diversity of gut bacteria is a complex entity that can be expressed in different ways. To begin with, the diversity of bacteria can be discussed in terms of taxonomy, from highest grouping to the lowest: domain (Archaea and Bacteria), phylum, class, order, family, genus, species, and subspecies (strains). Initially, an isolated bacterium was placed in a taxonomic group based on its phenotype. Today, the sequence of its 16S rDNA is the most common taxonomic method for classifying bacteria, including in the human gut²⁶. Diversity of gut bacteria can also be discussed with regard to functions and genes²⁷. Another important consideration when discussing diversity is that it can be examined at different levels, namely, (i) in a specific individual at different times, (ii) between individuals, (iii) within specific groups, and (iv) within entire populations.

Generally, reports of bacterial diversity in the human gut are underestimations because limits of detection are not considered. For example, if 10,000 bacteria are examined, and 600 different species are found among those 10,000 bacteria, the conclusion should be that these 600 species are present in the gut with an abundance of at least 10⁹, or 0.01% of the total bacteria in the gut. Each of these 600 species is an example of a very common species. However, relatively rare bacteria would not have been detected in such a sample, and as difficult as it may be to detect them, they should not be ignored; since when the condition changes, they may multiply and play a significant role in the fitness of the holobiont²⁸, and only then would they have been detected.

Diversity based on culturing methods

Unlike soil and seawater bacteria, gut bacteria are generally not in the viable but not culturable (VBNC) state. Quantitative analysis of fecal samples carried out using a culture-independent method (flow cytometry) and an anaerobic culture-dependent method (plating technique) found a culturability of 87%²⁷. This is not surprising because the stressful conditions that induce the VBNC state, deprivation of essential nutrients, oxidative stress, low temperature, high osmolarity and UV radiation²⁹, do not exist in the gut.

An early approach used to study human gut bacteria employed microbial culture techniques. Since Escherich in the 1880’s till the seventies, many reports were published on various morphological or cultural bacterial groups^30–35, but the main break through began in the early 1970’s. In 1974, Moore and Holdeman⁷ published their detailed leading report in which they examined the feces of 20 healthy adults. All fecal specimens were diluted and cultured under anaerobic conditions on special media, immediately after collection. The average cultural recovery was 93% of direct microscopic counts. Isolates were identified by classical phenotypic characteristics. There were 113 different species of organisms detected among the 1147 isolates examined from the 20 people. Each person was found to contain 10–30 different bacterial species. As discussed above, to detect a specific abundant bacterial specie in the human gut from 1000 isolates, at least 10¹⁰ bacteria of that species must be present in the gut. Thus, in the Moore and Holdeman experiment, it is likely that only abundant bacteria were found. Nevertheless, the data demonstrate clearly an important property of gut bacteria: Different people harbor different abundant species; otherwise, the number in each individual would be similar to the number found in the population. The study also showed a predominance of the genera Bacteroides, Bifidobacterium, Fusobacterium, and Eubacterium.

In a subsequent culturing experiment, 5350 bacterial isolates representing 371 species were detected in 88 fecal specimens³⁶. The most frequent species detected were Eubacterium aerofaciens (8.4%) and Bacteroides vulgatus (7.8%). Escherichia coli was the 22nd most common (1.2%). Approximately half of the isolates were singletons (only seen once in all the samples measured), and represented 0.02% of the 5350 isolates examined.

In 2012, a new strategy was introduced for bacterial isolation and cultivation, referred to as culturomics³⁷. Culturomics employs multiple culture conditions combined with the rapid identification of bacteria by mass spectrometry or 16S rDNA sequencing³⁸. Some of the culture conditions employed inhibit the growth of the most abundant bacteria, allowing for the isolation and study of less abundant species. The culturomics approach has led to the identification of hundreds of previously unidentified bacterial species that are present in the human gut. In 2023, Wan et al. reported that the number of bacterial species identified in the human gut reached 3253, of which 63% were contributed by culturomics¹¹.

One major advantage of cultivating bacteria is the ability to describe in detail their physiology, genetics and interactions with the host and with other members of the gut ecosystem by performing functional experiments. While culture-independent molecular methods can address a range of complex questions³⁹, these approaches cannot make the definitive links to bacterial physiology that culturing does. Consequently, there is an increasing interest in improving culturing techniques for studying the gut microbiota, while combining them with molecular methods.

Diversity Based on Culture-Independent Methods

In 2008, in a key paper, Dethlefsen and co-workers performed a comprehensive study of gut bacteria in three individuals, using five time points for each participant during an 8-month interval, by deep 16S rDNA sequencing⁴⁰. The authors reported the existence of more than 5,600 bacterial species in the human gut of the three individuals tested at 5 points during 8 months. This number greatly exceeded earlier estimates^41,42. Many of the species were present as singletons. At the individual level, 2600–3300 species were found, providing minimal estimates of the number of bacterial strains present in one person over an 8-month interval. The number of unique genera in the combined samples was less than 130, indicating that the diversity of human colonic bacteria is concentrated at the species and strain levels. In the Dethlefsen study, as well as many others⁴³, it has been shown that the human gut is dominated by two phyla, Bacteroidetes and Firmicutes, while Proteobacteria, Fusobacteria, Cyanobacteria, Tenericutes, and Verrucomicrobia are present in minor amounts.

A different approach is metagenomics, which determines gene frequency and not species diversity. This method enables also determining the functions exhibited in the microbiome. A meta-analysis of metagenomes from the human gut, covering 2183 samples from six gut microbiome studies found a large genetic diversity in the dataset, identifying 22,254,436 non-redundant genes in the gut¹³. A non-redundant gene is a gene that appears only once in a single organism. Some genes were found in exactly one sequencing sample (singletons), whereas other genes assembled in multiple samples (non-singletons). The gut gene catalog contained 12,621,933 (56.7%) singletons and 9,632,503 non-singletons. On average, 2.9% of the genes were singletons. Furthermore, they found that non-singletons were enriched for primary metabolic processes, such as the Citric Acid Cycle and amino acid biosynthesis, whereas singletons were enriched for a wide range of diverse biosynthesis and degradation pathways. In sum, these detailed data demonstrate the genetic diversity and richness of bacteria in the gut human microbiome.

Changes with time are one of the major difficulties in determining the diversity of human gut bacteria; other difficulties are comparing different experiments using different methods of analysis and the number of samples examined. A review of several studies reported timescales of gut bacteria dynamics in the presence and absence of external perturbations⁴⁴. These changes can be quantitative (increases or decreases in specific bacteria) or qualitative (complete loss or gain of bacterial species). The authors conclude that on the longer run of months and years the microbiome in an individual is relatively stable, while on a shorter time scale of days and weeks a greater variation is observed.

Perturbations that affect the bacterial composition of the human gut include: (i) Age and developmental stage, (ii) diet^45,46, (iii) pregnancy⁴⁷, (iv) physical activity⁴⁸, (v) stress⁴⁹, (vi) disease^50–52, (vii) bacteriophage⁵³, (viii) smoking⁵⁴, (ix) high altitude environments⁵⁵, (x) medication⁵⁶, and (xi) circadian rhythm⁵⁷. Taking into account all these variables leads to the conclusion that it is extremely difficult to determine accurately the complete microbial diversity in an individual or in a population because of the changing conditions that occur even on an hourly basis. It is also now clear that a comprehensive understanding of the bacterial diversity of the human gut requires a combination of both culture and molecular methods.

Unity within diversity

As discussed above bacterial diversity in the human gut exists within an individual⁵⁸, between individuals⁵⁹, and between different populations. However, the microbiome as a whole is part of a single unit that we define as the holobiont with its hologenome, namely it is a part of a functional unit, a genetic unit, and an evolutionary unit^3,60. The unity of the holobiont and its microbiome manifests itself in three forms: unity of the microorganisms within the microbiome, between the human host and its microbiome and unity within the human holobiont population with respect to its microbiome.

First and foremost, for bacteria to be part of the microbiome of the human gut they must possess several major common properties: all must be able to grow anaerobically at 37 °C under conditions present in the colon, all have to be recognized as “self” by the immune system⁶¹, and all should benefit or at least tolerate other microorganisms in the densely populated microbiome. In addition to these general adaptations, there exist also many specific interactions between specific microbes and the human body. The human body, on the other hand, provides the microbes with diverse nutritional substrates, protection, and constant conditions for growth and proliferation. How can we explain that during evolution highly diverse bacterial populations have been selected that perform important conserved functions within the human holobiont? It has been demonstrated that different bacterial species can perform the same function in the human gut⁶². An optimized collection of bacterial genes and pathways, a functional core, rather than a specific population (core taxa) is suggested to be responsible for many biological functions, including processes that are not carried out by human cells and thus represent a potential basis for symbiotic host–microbial relationships⁶³. Thus, unity of function is achieved via diversity. A specific example of functional redundancy in gut bacteria is biogenic amine degradation, where 74 different biogenic amine degrading bacteria were isolated from the human gut⁶⁴. These isolates were widely spread across different taxa present within the human gut microbiota. One of the explanations for functional diversity is horizontal gene transfer, which is extensive in the human gut⁶⁵, with many phylogenetically unrelated taxa carrying similar genes and performing similar functions⁶⁶.

An important example of how the diversity of bacteria interacts with the human gut to provide a major source of energy and regulatory molecules is the breakdown of complex polysaccharides in plant fiber; dietary fiber contains a variety of polysaccharides with diverse sugars linked by different glycosidic bonds that are not broken down by the digestive human enzymes⁶⁷. The gut microbiota possesses hundreds of different carbohydrate degrading enzymes that are able to break down a large portion of the dietary fiber⁶⁸. The breakdown products of the bacterial enzymatic activity on fiber act not only as energy sources for the microorganisms and for the human body, but also as a source for regulatory molecules⁶⁹. The main metabolites produced by the bacterial fermentation activity are shot-chain fatty acids (SCFA), which contribute energy and regulatory processes mainly to the epithelial cells of the colon. SCFA play a role also in intestinal motility, barrier function, regulation of immunity, and in lipid metabolism⁷⁰. In several cases, gut bacteria benefit by metabolic cross feeding between different bacterial species⁷¹.

In summation, although individuals and populations contain different phylogenic compositions of gut bacteria, they perform the same important functions because the same functional genes are distributed in different bacterial species. The concept that function, rather than taxonomic classification is crucial for unity was expressed by Doolittle and Booth⁷² and Wu et al. ⁷³. It is likely that the same principle of “unity via diversity” applies to other parts of the body, such as the oral microbiome⁷⁴, and with other animals.

Author contributions

E.R. is the sole author and approves the manuscript.

Data availability

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

Competing interests

The author declares no competing interests.