Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. The origin of the genes associated with this resistance is of significant importance to our understanding of the evolution and dissemination of antibiotic resistance in pathogens. A growing body of evidence implicates environmental organisms as reservoirs of these resistance genes; however, the role of anthropogenic use of antibiotics in the emergence of these genes is controversial. We report a screen of a sample of the culturable microbiome of Lechuguilla Cave, New Mexico, in a region of the cave that has been isolated for over 4 million years. We report that, like surface microbes, these bacteria were highly resistant to antibiotics; some strains were resistant to 14 different commercially available antibiotics. Resistance was detected to a wide range of structurally different antibiotics including daptomycin, an antibiotic of last resort in the treatment of drug resistant Gram-positive pathogens. Enzyme-mediated mechanisms of resistance were also discovered for natural and semi-synthetic macrolide antibiotics via glycosylation and through a kinase-mediated phosphorylation mechanism. Sequencing of the genome of one of the resistant bacteria identified a macrolide kinase encoding gene and characterization of its product revealed it to be related to a known family of kinases circulating in modern drug resistant pathogens. The implications of this study are significant to our understanding of the prevalence of resistance, even in microbiomes isolated from human use of antibiotics. This supports a growing understanding that antibiotic resistance is natural, ancient, and hard wired in the microbial pangenome.

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The majority of caves are formed within limestone rock and hence our understanding of cave microbiology comes from carbonate-buffered systems. In this paper, we describe the microbial diversity of Roraima Sur Cave (RSC), an orthoquartzite (SiO 4 ) cave within Roraima Tepui, Venezuela. The cave contains a high level of microbial activity when compared with other cave systems, as determined by an ATP-based luminescence assay and cell counting. Molecular phylogenetic analysis of microbial diversity within the cave demonstrates the dominance of Actinomycetales and Alphaproteobacteria in endolithic bacterial communities close to the entrance, while communities from deeper in the cave are dominated (82-84%) by a unique clade of Ktedonobacterales within the Chloroflexi. While members of this phylum are commonly found in caves, this is the first identification of members of the Class Ktedonobacterales. An assessment of archaeal species demonstrates the dominance of phylotypes from the Thaumarchaeota Group I.1c (100%), which have previously been associated with acidic environments. While the Thaumarchaeota have been seen in numerous cave systems, the dominance of Group I.1c in RSC is unique and a departure from the traditional archaeal community structure. Geochemical analysis of the cave environment suggests that water entering the cave, rather than the nutrient-limited orthoquartzite rock, provides the carbon and energy necessary for microbial community growth and subsistence, while the poor buffering capacity of quartzite or the low pH of the environment may be selecting for this unusual community structure. Together these data suggest that pH, imparted by the geochemistry of the host rock, can play as important a role in niche-differentiation in caves as in other environmental systems.