<p>Antibiotic resistance is often linked to human use of antibiotics. However, antibiotics and antibiotic biosynthetic pathways have been evolving for millions of years suggesting that antibiotic resistance is an ancient phenomenon. As of now, there has been no systematic survey of environmental microbes proven to exist in the absence of human influence and Lechuguilla cave offers such environment<em>. </em> Resistance diversity in strains isolated from this cave was analyzed by a phenotypic screen against a panel of 26 different antibiotics. Resistant strains were further investigated through determination of minimal inhibitory concentration (MIC) and inactivation studies. Of particular interest was strain LC044 (<em>Brachybacterium paraconglomeratum</em>), observed to inactivate macrolide antibiotics by phosphorylation. Genome sequencing and bioinformatics (BLAST analysis) identified a putative macrolide phosphotransferase (MPH) in strain LC044 and biochemical characterization of the purified recombinant protein confirmed its macrolide inactivating properties. To investigate if characterized MPH was unique to cave isolate, available terrestrial <em>Brachybacterium faecium</em> DSM 4810 genome was mined for presence of MPH-like protein. The top hit to the MPH from LC044 (a protein with 282 amino acids and 72% identity) was heterologously expressed and purified. Complete biochemical analysis of this enzyme revealed (i) MPH-activity, despite its annotation as aminoglycoside phosphotransferase (APH), and (ii) no significant differences in substrate specificities or kinetic parameters between these two enzymes suggesting that these two enzymes were equally effective resistance enzymes. This work highlights the prevalence of antibiotic resistance in a pristine, cave ecosystem and provides further support for the theory that antibiotic resistance is everywhere. Furthermore, the <em>mph</em> resistance determinant found in cave isolate and closely related terrestrial isolate show homology to clinical<em> mph</em> genes, suggesting that environmental <em>mph</em> genes could have served as reservoir of clinical determinants.</p> / Master of Science (MSc)