One of the challenges in measuring contemporary

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One of the challenges in measuring contemporary environ- mental resistance is rigorously ensuring the absence of anthropo- genic sources of antibiotics as a selective pressure for the acquisition of ‘modern’ resistance genes. A survey of a diverse PLoS ONE | 1 April 2012 | Volume 7 | Issue 4 | e34953
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environmental bacterial population that has never been exposed to modern antibiotics and resistance genes would unequivocally establish the depth of the contemporary environmental resistome: Lechuguilla Cave offers a unique environment for such a study. Lechuguilla Cave, located within Carlsbad Caverns National Park (USA), was formed in the Capitan Formation of the Delaware Basin by hypogenic (ascending water) sulfuric acid speleogenesis in the last 7 million years [14,15,16], resulting in the formation of a very large ( . 200 km) and deep ( . 500 m), maze- like cave system [17]. While the cave is still connected to the artesian aquifer that formed it (Figure 1), the majority of the cave became isolated as the aquifer dropped [18]. Due to its isolated hypogene nature, extensive cave development occurred primarily at 2 300 to 2 400 m, while the impermeable Yates Formation that overlies the cave limits vadose water from entering the system [14,17,19]. The deep recesses of Lechuguilla Cave, isolated from surface input for the past 4–7 million years, therefore provide a unique environment to study the presence and prevalence of antibiotic resistance elements. We report here a sampling of the culturable Lechuguilla Cave resistome – the antibiotic resistance genes found in a culture collection of isolates from this cave environment. In addition to finding a wide array of resistance in this culture collection, we also describe a novel macrolide kinase variant and an inducible daptomycin hydrolase, demonstrating not only that resistance is prevalent in the absence of anthropogenic antibiotics, but hitherto undiscovered mechanisms of resistance that have potentially important clinical implications are prevalent in the environment. Figure 1. Plan and profile maps of Lechuguilla Cave, Carlsbad Caverns National Park, New Mexico. The sites where microbial strains were collected (LCECE, LCDS1 and LCEA1) are shown relative to the entrance and depth. tN represents true North on the plan, while the profile has an exaggerated vertical profile of 1.5 6 . doi:10.1371/journal.pone.0034953.g001 Antibiotic Resistance in Cave Bacteria PLoS ONE | 2 April 2012 | Volume 7 | Issue 4 | e34953
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Results Sampling of the Lechuguilla Cave Culturable Microbiome Since its discovery in 1986, Lechuguilla Cave has been closed to human access without a permit. The sample areas were chosen off the designated trail through the cave, in areas that have experienced very little human impact (National Park records suggest that a maximum of 4–6 people have been in the vicinity of each sample site) (Figure 1). Specific sample locations were chosen due to the unlikelihood that these areas were actually exposed to human visitation: absence of footprints and scuffmarks that usually indicate human activity. Three sample sites were chosen, based on
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