Location and geology lcds1 and lceae deep and within

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location and geology: LCDS1 and LCEAE (deep and within the Capitan Formation) and LCEA1 (mid-depth in the Yates Formation). None of the sites received any direct source of water with any moisture likely to come via percolation from the surface or from condensation events within the cave atmosphere. LCDS1 was the deepest sample site (at 2 400 m) within a region known as Deep Secrets and LCEAE was in the proximity of the LCDS1 sample site at the same depth. LCEA1 was chosen as an area that has been closed to human access for the past 20 years, with the only human activity in the area being carried out by scientists. The LCEA1 sample is at the bottom of the Yates Formation, which is high in iron and manganese and is the rock unit in which much of the oil and gas is extracted from the Delaware Basin. As a result, this rock unit is usually associated with organic material from ancient sediments [14]. A total of 20 different culture media were inoculated at each sample site, resulting in the isolation of over 500 unique isolates (Text S1) [20]. Of these, 93 that were able to grow in 50% TSB were chosen at random to screen for antibiotic resistance in this fraction of the cave microbiome (Table S1, Figure S1). These bacteria were phylogenetically classified by sequencing of the 16 S rRNA gene sequence (Table S2) and spanned several Gram- positive and Gram-negatives genera known to be associated with cave environments [21,22,23,24,25,26,27]. Antibiotic Resistance Screen of the Lechuguilla Microbiome The Lechuguilla bacterial collection (93 strains, 33% Gram- positive and 63% Gram-negative) was screened against 26 different antimicrobial agents representing a broad spectrum of natural products, their semi-synthetic derivatives, and completely synthetic molecules (Figure 2). In the primary screen, the level of resistance was assessed by monitoring the growth at 20 m g/ml drug concentration (a high level of antibiotic to select for robust resistance) and resistance in the primary screen was defined as . 50% growth in the presence of antibiotic, consistent with other surveys of resistance [11,28]. Resistant and sensitive strains obtained from the primary screen were further quantitatively analyzed by determination of the minimal inhibitory concentra- tion (MIC) of the antibiotics (Figure 3). Resistance spanned most of the major drug families in both Gram-positive and Gram-negative strains. In the collection of 33 Gram-positive strains, on average, 70% of the strains were resistant to 3–4 different antibiotic classes. Three strains were resistant to 14 antibiotics, all of which were Streptomyces spp. These values are consistent with those found in surface Streptomyces [11]; however, unlike surface strains, we saw no Gram-positive resistance to the synthetic drugs ciprofloxacin and linezolid, the semi synthetic compounds rifampicin and minocycline, and the natural product vancomycin (Figures 2–3).
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