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.

Because the ferric uptake regulator (fur) appears to be an essential gene in Pseudomonas aeruginosa, resistance to manganese was used as an enrichment to isolate strains carrying point mutations in the fur gene in order to assess its role in the co-ordinate expression of siderophores and exotoxin A (ETA). This report describes a detailed molecular and phenotypic characterization of four mutants and one revertant, which carry point mutations in the fur gene. Two parental strains were used in this study. Three mutants were isolated from the widely used strain, PAO1. One of these, CS (cold sensitive), has a mutation in the 5' non-coding region of the fur gene while the two other mutants derived from this parent have mutations resulting in the following deduced changes in Fur: mutant A2, H86-->R; mutant A4, H86-->Y. The other mutant (C6) and its revertant (C6Rv) were derived from PAO6261, a mutant of PAO1 with a deletion in the anr gene (anaerobic regulation of arginine deiminase and nitrate reduction) that controls anaerobic respiration in P. aeruginosa. Fur from the C6 mutant has an A10-->G mutation while in the C6Rv spontaneous revertant the mutant Gly residue has been changed to Ser at this position. All mutants were examined for alterations in the iron-regulated expression of siderophores and ETA. The A2 and A4 mutants expressed higher levels of siderophores in iron-deficient media and in iron-replete media. The CS mutant constitutively expressed siderophores at 25 degrees C. At 42 degrees C siderophore biosynthesis was iron repressed as in the parental strain PAO1. The deletion of anr in PAO6261 had no apparent effect on the iron-mediated regulation of siderophore synthesis, but the C6 mutant derived from this strain produces siderophores constitutively. The iron-regulated production of siderophores by C6Rv was similar to the parental strain PAO6261 and PAO1. Because one of the parental strains used in this study is an Anr mutant, regulation of ETA production was assessed under aerobic and microaerobic conditions. Iron-dependent repression of ETA synthesis in both parental strains and A2 and A4 mutants was found to be 50-100-fold under aerobic and microaerobic conditions, as assayed by quantitative Western dot-blot assays. By contrast in the CS and C6 mutants, while iron-dependent repression os ETA synthesis was similar to both parental strains under aerobic conditions, ETA production in these mutants was constitutive in a microaerobic environment. RNase protection analysis of toxA and regAB transcription in PAO1, PAO6261 and the C6 mutant corroborated the results of quantitative dot-blot assays of ETA. The mutant Fur proteins were purified and examined for their ability to bind to the promoter of a gene (pvdS) that positively regulates the expression of siderophores and ETA. Fur from the A2 and A4 mutants and from the C6Rv revertant was able to bind to the target DNA, but with reduced affinity by comparison to wild-type Fur. Fur from the C6 mutant in DNase I footprint experiments failed to protect the promoter...

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Definitive diagnosis of the bat disease white-nose syndrome (WNS) requires histologic analysis to identify the cutaneous erosions caused by the fungal pathogen Pseudogymnoascus [formerly Geomyces] destructans (Pd). Gross visual inspection does not distinguish bats with or without WNS, and no nonlethal, on-site, preliminary screening methods are available for WNS in bats. We demonstrate that long-wave ultraviolet (UV) light (wavelength 366-385 nm) elicits a distinct orange-yellow fluorescence in bat-wing membranes (skin) that corresponds directly with the fungal cupping erosions in histologic sections of skin that are the current gold standard for diagnosis of WNS. Between March 2009 and April 2012, wing membranes from 168 North American bat carcasses submitted to the US Geological Survey National Wildlife Health Center were examined with the use of both UV light and histology. Comparison of these techniques showed that 98.8% of the bats with foci of orange-yellow wing fluorescence (n=80) were WNS-positive based on histologic diagnosis; bat wings that did not fluoresce under UV light (n=88) were all histologically negative for WNS lesions. Punch biopsy samples as small as 3 mm taken from areas of wing with UV fluorescence were effective for identifying lesions diagnostic for WNS by histopathology. In a nonlethal biopsy-based study of 62 bats sampled (4-mm diameter) in hibernacula of the Czech Republic during 2012, 95.5% of fluorescent (n=22) and 100% of nonfluorescent (n=40) wing samples were confirmed by histopathology to be WNS positive and negative, respectively. This evidence supports use of long-wave UV light as a nonlethal and field-applicable method to screen bats for lesions indicative of WNS. Further, UV fluorescence can be used to guide targeted, nonlethal biopsy sampling for follow-up molecular testing, fungal culture analysis, and histologic confirmation of WNS.