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Many microbial species express natural resistance to antibiotics, either through the absence or altered structure of the target, or by efficient efflux pumps, which pump out the antibiotic before it reaches toxic levels. There are some mechanisms of resistance that emerge quickly, from a single point mutation in a gene that changes the shape of a target to confer resistance. Yet such resistance is often not transferable, preventing the rapid, global dissemination that is a characteristic of clinically-relevant antibiotic resistance. Neither can such mechanisms account for the novel enzymatic activities observed in the resistance genes identified in infectious organisms. Together these observations suggested that antibiotic resistance emerged from somewhere with a longer evolutionary history. Indeed, if antibiotics themselves provide a competitive advantage in the environment that is ancient, this in turn could provide time and the selective pressure necessary for the evolution of resistance.
To test this hypothesis, investigators began exploring microorganisms from the natural environment that could not have been previously exposed to medical antibiotics. If such pristine environments contained antibiotic resistance, this suggested that environmental microorganisms were the reservoir for these resistance genes - the other half of the chemical arms race for survival in the microbial world. Investigators began isolating bacteria from ancient permafrost, isolated tribes in the Amazon jungle, or in the case of our work, deep caves that have been isolated from surface inputs for millions of years. In all cases, these data suggested that antibiotic resistance could not have evolved in the recent past.
Our research has demonstrated that the nutrient limitation of deep, isolated caves results in a very low biomass and remarkably high microbial diversity is, with hundreds (and potentially thousands) of unique species, including all known predatory bacterial genera, suggesting extreme resource competition in the cave environment. To study antibiotic resistance within this environment, we cultured microorganisms from a pristine region of the cave that had been isolated for over 4 million years. Similar to microorganisms found within surface soils, the bacteria we isolated were highly resistant to a broad range of antibiotic classes. Whole-genome sequencing, functional genomics and biochemical assays indicated that one isolate, Paenibacillus sp. LC231, was resistant to almost all classes of clinically-important antibiotics. These data were transformative, significantly expanding the established age of the evolution of antibiotic resistance from thousands to millions of years. While such work suggests that we will always be on the losing side of the battle against antibiotic resistance. But this is not the case. Our work identified mechanisms of resistance that have not been seen in the medical setting and may take decades to emerge. By identifying these resistance determinants now, we have time to find ways of combating this resistance before it emerges in the clinic. Buying ourselves time to stay ahead in the antibiotic resistance battle.
The Red Queen Hypothesis is used to describe social conflict as a driver of evolution in competing species, including the ‘chemical arms race’ that has driven the emergence of antibiotic resistance. If this hypothesis holds true, as species A produces a new antibiotic, species B would develop resistance. In order to remain competitive, species A would therefore either need to evolve a new antibiotic, or develop a means of overcoming the resistance mechanism of species B. Such resistance breakers (also known as resistance adjuvants) are becoming increasingly recognized as a mechanism to potentially overcome antibiotic resistance. And if antibiotic resistance is selected for by resource limitation, then competition should equally drive the evolution of mechanisms to inhibit resistance. Indeed, such compounds have already been identified from the environment; clavulanic acid was isolated from Streptomyces clavuligerus in the 1970s. We are currently screening the highly competitive microbial ecosystems of Lechuguilla Cave for antibiotic adjuvants using a high-throughput screening approach to test this hypothesis.
Microbial activity can have a profound impact on mineral chemistry and dissolution, providing the potential of microbially-driven cave forming processes: biospeleogenesis. One of the more interesting places to study iron Formation caves (IFCs). The Minas Gerais region of Brazil contains iron (III) ore deposits, ranging from the 38% w/w Fe (III) itabirite deposit to the 62% w/w Fe (III) Canga Formation. The itabirite deposits comprise of amorphous magnetite and hematite deposits interbedded with layers of quartz, while the Canga Formation represents an erosion resistant caprock of cemented iron oxides. Numerous IFCs (>3,000) have been described forming at the contact between these deposits, which is difficult to reconcile with the extremely low solubility of most Fe(III) phases in BIF and canga (hematite has a Ksp of approximately 10-44 at the pH of IFC-associated fluids), which would severely limit solubilisation and export of Fe.
Microorganisms are known to play an important role in the formation of certain caves, such as hypogene sulfuric acid systems; however, rather than a mechanism of dissolution driven by acid, we hypothesize that microbially-mediated dissolution of the IFCs occurs via respiratory Fe(III) reduction to soluble Fe(II). We have evaluated this potential Fe(III) reduction using Shewanella oneidensis MR-1, which can reduce Fe(III) to Fe(II) in all IFC-associated Fe(III) phases. The addition of anthraquinone-2,6-disulfonate (AQDS) accelerates this reduction, suggesting that the natural organic matter may enhance bioreduction. If Fe(III)-reducing microorganisms drive Fe(III) phase dissolution, this could provide a mechanism to link hypogene cavities together, creating the longer caves.
Understanding the drivers of species diversity in microscopic communities has historically been a challenge, particularly as most ecological models have been developed for macroscopic systems, while microorganisms themselves refuse to follow established ecological principles. A case in point is Hutchinson’s ‘Paradox of the Plankton’: the observation that under increasingly nutrient-limited conditions, plankton tends to display a more diverse community structure. We have observed similar, highly diverse microbial community structures in caves under extreme nutrient limitation and one of the primary goals of my research is to describe the potential drivers of such diversity, particularly as it relates to carbon turnover.
We hypothesize that microbial diversity in caves is driven by the formation of novel niche space; rather than directly competing for the same nutrients, bacterial species within caves exploit the metabolic breakdown of products of allochthonous carbon to create discrete populations. This strategy provides a metabolic parallel to r/k selection in animal ecology: fast growing and competitive (r-type) principle heterotrophs utilized a highly reduced, plant-derived polymeric carbon pool, while slow-growing oligotrophs serve as (k-type) successional heterotrophs, survive on the breakdown products of this principle metabolism.. In the case of allochthonous soil-derived carbon, this can include the release of monomeric sugars, alcohols, aldehydes, organohalides, cyclic and aromatic organic molecules; all of which have been shown to support the growth of other, successional heterotrophs. These slow-growth of these species prevents the generation of large pools of offspring to be targeted by predators, even as they express alternate cell architectures that make them impervious to attack from predators or antibiotics compounds. If the Red Queen hypothesis of competitive evolution holds true, then organisms successful in fixing allochthonous organic carbon or through autotrophic growth would serve as the ‘primary nutrient’ promoting the evolution of cheating behaviors (such as antimicrobial production and predatory activity) within the microbial ecosystem. Evidence for both approaches has been observed in numerous bacterial populations found in caves.
In the context of the broader value of cave research, it is also important to bring in researchers with no traditional background in the field. To this end, we have ongoing collaborations with a number of researchers, with projects involving:
Caves, by their nature, lack sunlight and are geologically isolated. It would therefore seem that the microorganisms found in these environments would be of limited interest. Yet it is the isolated nature of these environments that make them so fascinating to study. Not only do caves contain a remarkable and varied microbial ecosystem, but their very geologic isolation allows us to examine processes that have evolved in the absence of energy input from the sun that cannot be studied elsewhere. The absence of disturbance (such as daily, seasonal or meteorological cycles) allows us to study ecosystems that have been in equilibrium for thousands of years and reveal aspects of microbial evolution and physiology that would be impossible to study in surface ecosystems.
Caves represent an important environment for studying microbial community adaptation to the absence of sunlight energy. Through their isolation, microbial community structure is driven by available energy and nutrient sources, from the heterotrophic breakdown of scant allochthonous organic carbon delivered by vadose-zone groundwater to autotrophic growth using in situ redox active compounds. While historically cave microbiology was based on cultivation approaches, the inherent bias of such techniques provided an incomplete view of cave diversity. Modern molecular techniques demonstrate that microbial populations in caves are remarkably diverse and demonstrate both community and organismal adaptations to the resource-limitation of the subsurface. While most studies in caves have focused on the role and diversity of bacterial populations, the fungi and archaea also appear to play important roles in community structure and energetics, albeit at polar ends of the nutrient spectrum. Together these data suggests that current cave microbiology research is starting to reveal the potential for a cave microbiome that represents the core of microbial diversity in caves.
