Evolutionary consequences of antibiotic use for the resistome, mobilome and microbial pangenome

1The discovery of antibiotics and their use in the treatment of bacterial infections was one of the major scientific achievements of the 20th century. However, over the last 60 years, there has been a spectacular and rapid evolution of antibiotic resistant strains of bacteria. This has culminated in the appearance of pathogens with resistance to a wide range of antibiotics, and a rise of similarly resistant opportunistic organisms. Antibiotic resistance is a critical problem for humans and their domestic animals, and is recognized as such by workers in government, clinical practice, research, and industry.

2The focus of research and thinking about this problem has mainly been from an anthropogenic viewpoint. However, at its heart, antibiotic resistance is an environmental and evolutionary problem. Humans are now the greatest evolutionary force on the planet, and it would help us understand and manage the resistance problem to investigate resistance from a broader perspective, most notably in terms of the interplay between ecology, evolutionary dynamics, and natural selection. A number of authors are now thinking about the ecology and natural history of antibiotics and resistance genes. This approach will inform clinical and veterinary practices and will improve our understanding of evolutionary processes.

3There is evidence that the antibiotic revolution may have second order effects, and is influencing the evolution of the entire microbial biosphere. This paper attempts to place antibiotics, resistance genes, their vectors, and their hosts into an evolutionary perspective. By understanding the evolutionary history of these genes and molecules, we place ourselves in a better position to predict their future.

4The term “antibiotic” reflects our anthropocentric viewpoint. Antibiotics are not a discrete class of molecules, but rather, encompass a broad range of structural and molecular families, united by their ability to inhibit microbial growth at high concentrations. The original use of the word “antibiotic” was a generic term that simply reflected the outcome of a laboratory test. In modern terms, an antibiotic has broadly come to mean any synthetic or naturally occurring low molecular weight molecule that inhibits bacterial growth.

5It is clear that there are millions of low molecular weight compounds in natural environments, and that in high enough concentrations some of these could exhibit an antibiotic effect. However, it is unlikely that such compounds ever reach inhibitory concentrations in nature, and there is a growing realization that their primary role is not necessarily in cross-species warfare, but may largely lie in cell–cell communication. This change in viewpoint began with the discovery of bacterial quorum sensing, a system that allows communication between cells of the same or different species via signaling molecules called auto-inducers. Quorum sensing is found in diverse organisms, and the scale and extent of signaling systems in general has led to the revelation that the microbial world is in a state of constant and complex communication. Consequently, many of the small molecules produced by bacteria may be involved in additional forms of communication between cells. The compounds we know as “antibiotics” are a minor subset of this world of small molecules.

6The growth-inhibiting concentrations of antibiotics used in clinical practice are unlikely to ever be reached via natural synthesis, and consequently, antibiotics probably have significant effects at sub-inhibitory concentrations, without affecting bacterial growth rates. It has now been shown that sub-inhibitory concentrations of various antibiotic classes have effects on gene transcription. Up regulation and down regulation of diverse genes has been demonstrated, with some estimates suggesting that as many as 5% of gene promoters might be affected. Some antibiotic classes modulate expression of particular genes. The types of functions affected are diverse, including genes for protein synthesis, carbohydrate metabolism, transport/binding proteins and genes of as yet unknown function.

7The cascade of gene expression induced by sub-inhibitory concentrations of antibiotics leads to phenotypes of adaptive significance. These include flagella biosynthesis and the ability to colonize biotic and abiotic surfaces. As might be expected, some of these phenotypes have direct relevance for species–species interactions and responses to host cells, such as virulence, biofilm formation, and motility. Collectively, these observations strongly suggest that the antibiotics used in medical treatment are a small subset of the diverse secondary metabolites used in the microbial world for signaling and other cell–cell interactions. Their clinical use is based on a serendipitous ability to inhibit growth at unnaturally high concentrations. There is a growing realization that the discovery, clinical use, and evolution of antibiotics must be considered against the background of their roles in natural environments.

8Thinking about antibiotics as a subset of signaling molecules allows us to construct simple conceptual models that might establish some general principles about antibiotics and antibiotic resistance (Figure 1). A signaling molecule must be made by a metabolic pathway, whose enzymes are encoded by genes (A, B, and C in Figure 1). Any intermediate small molecule in this pathway can be exported as a signaling molecule, most probably using an efflux pump. Small molecules then diffuse into environmental space, where they can bind to receptors on the cell surface of the producing species (intra-species signaling), or to cell surface receptors of different species (inter-species signaling). Alternatively, small molecules can be imported into cells via a membrane transport protein, and then bind to a target within the cell (Figure 1). Binding of signaling molecules can influence transcription or biochemical pathways, and thus affect the phenotypic attributes of the receptor cell.

9This conceptual model (Figure 1) allows us to consider antibiotics and resistance from a different and broader perspective. Antibiotics can be thought of as a subset of small biosynthetic molecules, albeit those which happen to have the property of inhibiting bacterial growth at high concentrations. Given the fact that bacteria have been evolving and interacting for some 3.8 billion years, it should not be surprising that the genes encoding biosynthetic and catabolic pathways for small molecules have an ancient evolutionary history. Pathways for synthesis of erythromycin and streptomycin date to more than 600 million years ago, and it has been estimated that the serine β-lactamases date back 2 billion years. Under this view “antibiotics” predate humans by some billions of years.

10Note that the use of an intermediate in the biosynthetic pathway as a signaling molecule (Figure 1, triangle) means that the producing cell already encodes an enzyme capable of modifying the signal molecule. Under other circumstances, such a gene might confer resistance to high concentrations of the agent. Many biosynthetic gene clusters that make “antibiotics” are also known contain genes that confer “resistance” to those same antibiotics. Given that gene clusters for secreted molecules are frequently subject to lateral gene transfer (LGT;; ), it should not be surprising that there is clear evidence for lateral transfer of antibiotic resistance genes from environmental bacteria into pathogens.

11Small molecules can exit the producing cell via a wide diversity of membrane bound efflux pumps (Figure 1;;;; ), each of which, in turn, may be able to export diverse molecular species. Such pumps have evolved as mechanisms to export natural substances produced internally, or those that are produced by other cells. Efflux pumps may also be essential for colonizing and persisting in eukaryotic hosts. It is thus clear that antibiotic resistance mediated by efflux pumps is a side-effect of a more general molecular export system.

12Genes for efflux pumps are commonly found in bacteria that produce antibiotics, and these genes are often found embedded within biosynthetic gene clusters for antibiotics, thus conferring efflux ability (and “resistance”) on the producing organism. Again, the “resistance” gene is preexistent, and located within a gene cluster characterized by LGT.

13Once exported from the cell, a signaling molecule is free to bind with receptors on the surface of neighboring cells (Figure 1). Point mutations in the genes for these receptors might alter the binding site and thus generate a “resistance” phenotype. Alternatively, the signaling molecule may be taken up by a membrane transport protein and imported into a new cell. A surprising diversity of bacteria make use of such transport proteins to import small molecules such as antibiotics, which they can then use as a sole carbon source. Lateral transfer of the genes for such catabolic processes would confer resistance on the recipient cell. Resistance could also be generated by mutation to the gene encoding the membrane transport protein, preventing entry of the signaling molecule. Once inside a recipient cell, a signaling molecule may bind to a target site, and thus exert an effect. Mutation of the gene encoding the target site may abolish binding and prevent the effect. In the case of an antibiotic, such a change would be called a resistance mutation.

14Although the model I have presented above is simplistic, nevertheless it has good explanatory power for understanding the ecology and evolution of both antibiotics and their corresponding resistance genes. Antibiotics are seen as a subset of a diverse group of small molecules whose primary function is in cell–cell interactions. Genes for synthesizing, transporting, and catabolizing these small molecules can be co-opted as resistance genes when cells are exposed to unnaturally high concentrations of these compounds. The high frequency of lateral exchange of biosynthetic gene clusters for small molecules (together with their associated efflux pumps and catabolic functions) explains the penetration of efflux pumps and degradative enzymes into species unrelated to the original “antibiotic” producer.

15In thinking about the ecology and evolution of antibiotics and resistance genes, we can summarize the sub-components upon which selection can act in a series of Venn diagrams (Figure 2). The largest category is the Global Microbiome, encompassing all the prokaryotic cells in the biosphere. This may contain 4–6 × 1030 cells and hold a significant proportion of the carbon, nitrogen, and phosphorus found in living things. The phenotypes exhibited by the global microbiome are encoded by the microbial Pangenome, that is, the set of genes present in all the genomes of all the prokaryotes in the biosphere. Estimates of the composition of the pangenome have been made, based on the rapid accumulation of bacterial genome and metagenome sequences. Some 250 gene families are common to all bacterial genomes (the extended core genome) and about 8,000 gene families are niche-specific genes essential for survival in particular environments (the character genome). The bulk of pangenomic diversity comprises more than 139,000 gene families that occur as accessory genes, these being found dispersed amongst single strains, serovars or species. The combined coding capacity of the pangenome is expressed as the Panproteome, this being the sum of all the proteins encoded and produced by the microbial realm.

16A proportion of the pangenome is highly mobile, and able to move comparatively freely between prokaryote species in the process known as LGT. Elements that specialize in moving DNA within and between genomes include plasmids, transposons, integrons, insertion sequences, and integrative conjugative elements. These elements and the genes they carry are collectively known as the Mobilome (Figure 2;; ). In principle, and over evolutionary timescales, any part of the pangenome can be mobilized by LGT. The mobilome helps to generate enormous diversity within prokaryote genomes by recombination between various mobile elements to create a series of complex mosaic structures.

17The Resistome is defined as the collection of all genes that could contribute to a phenotype of antibiotic resistance. This is a purely functional definition made from a human and medical point of view, for as we have seen, the resistome encompasses diverse genes whose original functions were not simply for avoiding the effects of antibiotics. Nevertheless, it is a useful concept, because it underscores the point that resistance genes originate from environmental bacteria. A database of antibiotic resistance genes has been developed, but the 20,000 genes already listed are only a small proportion of the resistome, since investigation of soil metagenomes has revealed diverse and divergent resistance genes capable of dealing with multiple classes of antibiotic. Genes encoding antibiotic resistance are ancient components of the pangenome, since they have been recovered from 30,000 year old permafrost, and from a cave microbiome that has been isolated for 4 million years. Note that the resistome overlaps with the mobilome (Figure 2), because many antibiotic resistance genes are found on mobile elements, allowing the resistome to be widely disseminated by LGT. Also note that the resistance genes and mobile elements that are of concern for clinical antibiotic resistance (cross-hatched box, left hand side Figure 2) are just a fraction of the resistome and mobilome.

18The Parvome is the world of small bioactive molecules produced by cells (also sometimes called secondary metabolites). The parvome includes important classes of molecules such as polyketides, aminoglycosides, terpenoids, alkaloids, and non-ribosomal peptides, many of which have antimicrobial activity. The parvome overlaps with the proteome, because some of these small bioactive molecules are peptides. Note however, that only a small proportion of the parvome is utilized by humans for its antimicrobial properties (cross-hatched box, right hand side Figure 2). Commercial production of antibiotics overwhelms their natural synthesis, such that humans are now the major source of antibiotics in the general environment (Figure 2;; ).

19Having examined the relationships between the key components of relevance to antibiotic production and resistance (Figure 2), we can now examine how each might be affected by human activity. There are several potential dimensions to these effects, including changes to the abundance and diversity of each component, and whether the effects are transient or permanent.

20Antibiotics are released into the environment via human waste streams because a significant proportion of prophylactic antibiotics are excreted essentially unchanged. Although it is difficult to obtain precise estimates for commercial production of antibiotics, it probably amounts to millions of metric tons per year. Because of the volume of antibiotics generated by human activity, the majority of the environmental load of antibiotics now originates from this commercial production (Figure 2; ). Certainly the local concentrations of this subset of the parvome have markedly increased due to human activity. Further, the diversity of small bioactive molecules has increased, because many antibiotics are synthetic modifications of natural structures. There is now a zone of influence around all human activities where the abundance and diversity of the parvome is increased. Such zones increase selective pressures on the local microbiome, not just the intended targets of antibiotic therapy, and may also interfere with cell–cell communication.

21Increasing the concentration of antibiotics in the environment has effects on both the diversity and the abundance of genes belonging to the resistome. Selection for cells that carry resistance determinants increases their relative abundance, and thus increases the abundance of genes that confer resistance. Further, the ability of many elements of the resistome to undergo LGT, means that identical resistance genes can now be found in diverse bacterial species, from clinical contexts, from domestic animals, wild animals, and in locations apparently distant from the influence of developed societies, such as the Arctic, Antarctica, and the Amazonian jungle. That humans are responsible for this phenomenon is demonstrated by the positive correlation between the abundance of resistome elements of clinical significance and proximity to human activity.

22Selection pressure is particularly acute in human waste streams, where resistance genes are shed, mixed with high concentrations of antibiotics and other selective agents. Exposure of environmental microorganisms to this mixture encourages fixation of LGT events, spreading elements of the resistome into diverse strains and species, thus further increasing their abundance, and their penetration into new hosts and niches.

23Exposure to sub-inhibitory concentrations of antibiotics induces expression of error-prone DNA polymerases, increasing the basal mutation rate. This has the effect of generating additional genomic diversity, and of co-opting additional genes into the resistome via mutational changes to housekeeping or accessory genes whose original function was not antibiotic resistance. As a second order effect, constant low level antibiotic exposure selects for lineages with inherently higher rates of mutation, generating additional diversity across the entire pangenome via drift and selection.

24Human activity has increased the abundance of resistome elements by selection, as shown by their increase in frequency in soils collected over the last 70 years, and by their lateral transfer to diverse species. We have also increased the diversity and membership of the resistome by fixation of de novo mutations and by the co-option of genes as resistance determinants. Thus the size of the resistome, as a proportion of the pangenome, is probably becoming larger (Figure 2).

25The clinical use and environmental dissemination of antibiotics has had significant effects on the abundance and diversity of elements within the mobilome. Selection for antibiotic resistance has fixed lineages carrying diverse resistance determinants on a similarly diverse array of mobile elements. Such complex mobile elements now occur at high frequency in human-dominated systems, and in more natural ecosystems, where they can move by LGT into environmental organisms.

26The very use of antibiotics may itself increase the frequency of LGT, and thus the penetration of elements of the mobilome into new bacterial hosts. This effect is driven by the bacterial SOS response, which temporarily increases both the basal rate of LGT and of recombination. There is also evidence that human activities actually select for bacteria with a permanently increased propensity for LGT. Consequently, antibiotic pollution creates hotspots for the assembly of complex, mosaic mobile elements from diverse sources, and provides a selective force for their subsequent fixation in diverse lineages.

27The accumulation of diverse mobile elements within single plasmids or at single loci provides opportunities for complex rearrangements and recombination events that in turn, generate more diversity. The emergent properties that arise as mobile elements gain more components means that they can effectively increase their own complexity. This phenomenon is evident in the increasing complexity and phenotypic plasticity of genomic islands and integrative conjugative elements in emerging nosocomial pathogens such as Pseudomonas aeruginosa and Acinetobacter baumannii.

28The selective forces acting upon complex mobile elements are not restricted to antibiotic pressure. Mobile DNAs often carry genes conferring resistance to other selective agents, such as heavy metals, arsenic, and disinfectants. Exposure to any one of these agents selects for lineages containing mobile elements with appropriate resistance genes, and simultaneously fixes all the other genes in physical linkage on that element, including antibiotic resistance determinants. Such co-selection of antibiotic resistance has long been thought to arise as a consequence of pollution with heavy metals. Disinfectant use also results in co-selection of antibiotic resistance, and probably had a role in the origin of the class 1 integrons responsible for the widespread dissemination of antibiotic resistance amongst gram negative organisms.

29High rates of LGT occur between organisms in similar Phyla, organisms with similar GC content, and organisms that occupy the same environmental niches. This activity creates lateral exchange communities, where DNA can be exchanged with relative ease. Shared niches, and in particular, biofilms, promote such exchanges, and both conjugation and natural transformation appear to be important in the process. While evolutionary distance and difference in GC content can restrict LGT, there are mechanisms for bypassing these barriers, and genes can make their way between distantly related organisms via a sequential series of lateral transfers.

30Human use of antibiotics is therefore having multiple effects on the mobile components of the bacterial pangenome. One would predict that the size of the mobilome as a proportion of the pangenome is becoming larger, as more genes are recruited onto mobile elements (Figure 2). The number of resistome elements now residing on mobile DNA is also increasing, given that plasmids collected before the antibiotic era had no resistance determinants, and modern plasmids of similar structure have acquired a wide range of resistance genes dealing with various selective agents in addition to antibiotics. Consequently, there is now a greater overlap between the resistome and mobilome, particularly with respect to genes of concern for human health and welfare (Figure 2).

31Human antibiotic use has pervasive effects on both the pangenome and the local composition of the microbiome, largely because antibiotics have non-target effects at gene, cell, and population levels. Exposure of cells to antibiotics induces an SOS response that has widespread effects on bacterial genomes, including raising the rates of mutation, recombination, and LGT. This increase in basal rates of evolution applies to all the genes and cells in the exposed environment, not just the targets of antibiotic therapy. Consequently, the release of antibiotics into natural environments will affect the diversity of the pangenome and the composition and ecology of the global microbiota.

32Effects in particular environments have been demonstrated experimentally. Short term antibiotic treatments lead to changes in abundance, richness, and diversity of the human gut microbiota, which can remain perturbed for years after treatment. The composition and functional diversity of soil and sediment communities is affected by exposure to antibiotics, and persistence of antibiotic residues in sediments or in the water column leads to alteration of microbiota and in situ selection for antibiotic resistance. While pulses of exposure to antibiotics may only produce transient selection events, alterations to community composition, and the fixation of resistance genes and their mobile vectors may be permanent, with unpredictable consequences for the whole microbiome.

33Antibiotics and their resistance genes originated from natural environments, but human use of antibiotics has perturbed the dynamics of this natural system. The high concentration of antibiotics used prophylactically by humans has two main consequences. Firstly, it leads to antibiotic contamination of human waste streams, and secondly, the selection imposed by antibiotic use has fixed ever more complex genetic elements in commensals and pathogens. These new “xenogenetic” elements are also released via human waste streams. While antibiotics might be treated as simple pollutants, xenogenetic mobile elements are capable of replication, and are thus more akin to invasive species.

34Between 30 and 90% of the antibiotics given for human or veterinary use are excreted essentially unchanged. These compounds can be both persistent and mobile, and are often not removed during sewage treatment. Antibiotics are also released in high concentrations from facilities where antibiotics are produced, and are disseminated during application of manure to agricultural land. The consequences of pollution with antibiotics, particularly for aquatic systems, are being actively examined, and there is a call for development of policies to reduce the release of antibiotics and bacteria via human waste streams.

35Waste streams that release antibiotics into the environment also disseminate antibiotic resistance genes and mobile DNA elements, which should similarly be regarded as pollutants emanating from human activity. The abundance of resistance genes in soils has been increasing since the introduction of antibiotics in the 1940s, in parallel with the increasing concentrations of more conventional chemical pollutants.

36Resistance genes and complex mobile elements are commonly reported from wastewater and sewage treatment plants. Wastewater is regarded as a hotspot for interactions between mobile elements and for their lateral transfer between pathogens, commensals, and environmental bacteria. Resistome and mobilome elements are not necessarily removed by water treatment, allowing resistance genes to be used as markers of human influence on aquatic ecosystems.

37Domestic and agricultural animals are a source of significant quantities of antibiotics and resistance genes. Animal waste and pig slurry are used to manure soils, with the consequent introduction of resistance genes and resistant bacteria. The long term fate of antibiotics and resistance genes is difficult to predict without quantitative measurements over appropriate timescales. However, it is clear that antibiotic resistant bacteria will increase in abundance, lateral transfers to soil organisms will occur, and resistance genes will be sequestered by diverse elements of the mobilome.

38Human use of antibiotics for medicine and agriculture may have consequences beyond their intended applications. Large quantities of antibiotics now emanate from human waste streams, as do the xenogenetic elements fixed in human ecosystems by antibiotic selection. Much more attention needs to be paid to the origins and fates of such pollutants. The antibiotic revolution may be having effects across the entire microbial biosphere, changing the basal rate of bacterial evolution, altering the composition of the resistome and mobilome, and promoting lateral transfer of mobile genetic elements. Antibiotic contamination promotes the fixation and mobilization of resistance genes between environmental and clinical microbiota, and resistance genes are now widely spread through the biosphere.

39Above all, we need to address the antibiotic resistance problem from a broader evolutionary and ecological perspective. The ability of natural selection to shape species and communities is the same for microorganisms as it is for larger species, and the ecological theory of community assembly developed for multicellular organisms can be applied to the microbiome. The risk associated with the environmental spread of resistance genes with known adverse consequences for human welfare has had little attention, nor has the potential for pollution with antibiotics to widely affect the global microbiome. In comparison, the potential escape of resistance gene markers used in the generation of genetically modified plants has been the subject of considerable research. It is time to pay more attention to the bioactive molecules that humans release into the environment.

40The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.