Antibiotic resistance is manifested through a number of different mechanisms including target alteration, control of drug influx and efflux, and through highly efficient enzyme-mediated inactivation. Resistance can emerge relatively quickly in the case of some mutations in target genes and there is evidence that antibiotics themselves can promote such mutations 
; however, resistance to most antibiotics occurs through the aegis of extremely efficient enzymes, efflux proteins and other transport systems that often are highly specialized towards specific antibiotic molecules. Such elements are the result of evolution through natural selection; this therefore implies that antibiotic resistance has a long evolutionary past. A growing body of evidence suggests that non-pathogenic environmental organisms are a reservoir of resistance genes that have the potential to be transferred to pathogens 
. The problem of antibiotic resistance in clinical settings therefore likely has its origins in the environment.
One of the challenges in studying the evolution and prevalence of resistance is the massive use of antibiotics in the clinic and in agriculture over the past seven decades that makes identifying environments that have not been impacted by anthropogenic antibiotics difficult. Studying resistance in pristine environments that have not been exposed to human antibiotic use provides a critical measure of the genetic diversity of resistance that is essential to our understanding of resistance gene prevalence and evolution. Lechuguilla Cave provides an outstanding ecosystem that has been isolated for over 4 million years. The cave's geologic features, including the impermeable siltstone caprock which prevents rapid influx of surface water, great depth, and long isolation from the surface, rules out the possibility of exposure to anthropogenic use of antibiotics as well as antibiotic contamination through water bodies. As a result Lechuguilla Cave is an ideal ecosystem for investigating microbes that have not been exposed to anthropogenic antibiotics.
We surveyed the antibiotic susceptibility of 93 bacterial strains isolated from Lechuguilla Cave. This was a genetically diverse collection of oligotrophic organisms (Figure S1
), highly adapted to survive in a nutrient limited environment 
. Like surface organisms 
, the majority of these strains were multidrug resistant indicating that antibiotic resistance is a common and widespread phenotype in pristine, unimpacted environments; however, there are differences in the pattern of resistance. For example, we measured little resistance to the synthetic antibiotics ciprofloxacin and linezolid, while resistance to natural product antibiotics was more prevalent. Unlike surface bacteria, we also detected very little resistance to tetracycline, glycopeptide (vancomycin), rifamycin (rifampicin) and lipopeptide (daptomycin) natural product antibiotics. There are several possible reasons for these differences. First, this survey includes multiple bacterial genera across five phyla, while our original sampling focused on actinomycetes 
. As prodigious producers of natural products including antibiotics, it is logical that actinomycetes would also be enriched in resistance elements. Second, the isolate sample size is smaller in this study in comparison to our previous study and we have likely not examined the full resistome of both the culturable and non-culturable microbiome. Further studies of both surface and cave microbiomes including more extensive cultivation and metagenomic analysis are therfore necessary to interpret the results with more confidence.
Aminoglycoside antibiotic resistance was more common in Lechuguilla Cave isolates as compared to surface actinomycetes. This may reflect the biosynthetic capacity of antibiotic producing bacteria in Lechuguilla Cave and the production of these antibiotics by species within the cave. A survey of the actinomycetes in our collection using oligonucleotide primers designed to amplify aminoglycoside biosynthetic genes failed to identify potential aminoglycoside producers; however linking resistance to antibiotic production will require an extensive and systematic survey of the cave microbiome and resistome that is beyond the objectives of this work.
The mechanisms of antibiotic modification and inactivation are evidence of highly specific evolutionary adaptations to evade the cytotoxic action of these antibiotics. The high level of ß-lactam antibiotic resistance by hydrolysis parallels that of surface bacteria and the result of genetically diverse ß-lactamases that are widespread in microbial genomes. Similarly, chloramphenicol acetylation was also detected, an activity that is well established in surface bacterial isolates 
. Nonetheless, the hydrolytic inactivation of daptomycin in isolates of P. lautus
was unexpected. We recently showed that high G+C content actinomycetes use a hydrolytic ring-opening reaction as a common strategy of daptomycin inactivation 
, and this work further exposes the susceptibility of daptomycin's structure to hydrolytic cleavage. We also provide the first evidence that daptomycin inactivation can occur within the low G+C content bacteria Firmicutes
, for which daptomycin's use is approved; a mechanism that clinical microbiologists should be on alert for emergence in pathogens. The inducible activity in P. lautus
is very likely catalyzed by an EDTA-sensitive ring-opening esterase/protease [Figure S3
]. Our efforts to purify the associated protein were unsuccessful due to instability of the activity (possibly by autocatalytic digestion); however, a similar inducible activity was recapitulated in a surface strain of P. lautus
. This is intriguing as it suggests either involvement of a specific receptor for daptomycin or a non-specific response to the physiological impact of daptomycin bioactivity.
The observation of two distinct macrolide inactivation mechanisms in the Lechuguilla bacterial isolates was also intriguing. In the resistant Streptomyces
strains we determined that antibiotic modification by glycosylation was the primary mechanism of inactivation, a mechanism that is known in surface actinomycetes 
. On the other hand, we established that the mechanism of macrolide inactivation in B. paraconglomeratum
is through phosphorylation at position 2′ catalyzed by a member of the MPH class of antibiotic kinases. Previously identified mph
genes are encoded on plasmids found in clinically resistant isolates of the pathogens Escherichia coli
, Staphylococcus aureus
, Pasteurella multocida
and Pseudomonas aeruginosa
. This is the first report of mph
genes from environmental bacteria as a potential source of the genes currently circulating in pathogens. The presence of a transposase-like gene upstream of mph
from a surface strain of B. faecium
points to a potential history of horizontal gene transfer (). It is possible that mph
genes have been circulating among bacterial populations before the cave was sealed off millions of years ago, resulting in an mph
gene that is a shared trait between both terrestrial and cave bacteria.
There are two likely explanations for retention of the mph genes with same biochemical properties despite the long isolation of the Brachybacterium strains: (i) these genes could serve a physiologic or metabolic function unrelated to antibiotic resistance (although the genetic context () does not suggest an obvious role); or (ii) these genes are resistance elements for conferring antibiotic resistance. We could not detect any macrolide biosynthetic gene clusters in bacteria collected in the same region as B. paraconglomeratum (as evidenced by a absence of the signature macrolide D-desosamine biosynthesis gene, eryCVI, not shown); however the actinomycete small sample size does not rule out the possibility of the presence of hitherto undetected macrolide producers within the cave.
This work demonstrates that antibiotic resistance is widespread in the environment even in the absence of anthropogenic antibiotic use. Lechuguilla Cave represents a remarkable ecosystem that has been isolated for millions of years, well before the clinical and agricultural use of antibiotics. The presence of multidrug resistant organisms even in this pristine environment reinforces the notion that the antibiotic resistome is an ancient and pervasive component of the microbial pangenome. Given the nutrient-limited nature of the cave environment, it is likely that competition for resources plays a dominant role in species persistence. This competition may occur through numerous adaptations, from changes in cell physiology and growth, to the production of antimicrobials to outcompete nutritional rivals. Equally important could be the acquisition and development of defense mechanisms to enable flexibility and growth in the presence of noxious bioactive compounds and limit the effectiveness of such competitors in the ecosystem.
Given the relatively small sample size of this study, the observation of two previously unidentified mechanisms of antibiotic resistance (e.g. daptomycin hydrolysis) suggests that significant genetic diversity may be present in the environment and capable of being marshaled in the presence of antimicrobial agents. The available genetic diversity extant in microbial genomes dwarfs our ability to introduce antibiotics in to clinical use. Furthermore, the microbial chemical ecology of bioactive compounds such as antibiotics is not well understood and as such the resistome includes mechanisms that very likely have not been evolved simply to evade the effects of molecules that we have termed antibiotics 
. This fact further underlines the importance of the judicious use of antibiotics to avoid selection of existing resistance elements and their subsequent mobilization through microbial communities thereby limiting the effectiveness of these drugs to treat infectious diseases. The remarkable genetic diversity of the antibiotic resistome, uncovered in this and other studies has additional practical application as an ‘early warning system’ for new drugs introduced into the clinic. Resistance mechanisms in the environmental resistome can emerge in the clinics and the clinical community should be aware of them; for example our discovery of hydrolytic mechanisms of daptomycin resistance. Finally, the diversity in the resistome also suggests that there are a myriad of bioactive molecules with antibiotic properties waiting to be discovered. Some of these may have the potential to be productive leads as antibiotics or as improved scaffolds that can evade existing clinical resistance.