Emergence of a transferable daptomycin resistance mechanism in Gram-positive bacteria

Daptomycin represents a reserve antibiotic effective against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Daptomycin resistance (Dap-R) and clinical treatment failure has been associated with adaptive chromosomal mutations, but so far not with acquisition of specific resistance genes. We previously isolated Staphylococcus/Mammaliicoccus sciuri strain TS92, which displays high-level Dap-R but lacks reported adaptive mutations. To identify the underlying resistance mechanism, we performed transcription profiling upon daptomycin exposure of TS92, with subsequent subcloning and mutational analysis of candidate genes. In TS92, Dap-R is mediated by a novel two-gene operon (named drcAB), controlled by an adjacent two-component regulatory system (drcRS). Heterologous drc expression demonstrated drcAB to be required and sufficient to mediate Dap-R in S. aureus (including clinically relevant MRSA) and Bacillus subtilis via inactivation of the antibiotic. The drc genes and proteins show homologies to membrane-associated antimicrobial peptide (AMP) transporters of Gram-positive bacteria but are distinct from currently known systems. In TS92, drc is flanked by insertion sequences (IS) and integrated near a staphylococcal cassette chromosome (SCC) element, suggesting mobility and acquisition of the locus from another species. Consistent with this hypothesis, IS-flanked drc was identified in various bacterial backgrounds in database searches. Here we report for the first time a horizontally acquired Dap-R mechanism that appears to circulate among Gram-positive bacteria, including staphylococci and enterococci. Increasing clinical daptomycin usage increases the likelihood that drc may enter S. aureus and other pathogens, which calls for vigilant monitoring of drc spread and prudent use of the antibiotic. To identify potential resistance genes, we treated M. sciuri TS92 bacterial cells with daptomycin for 30 minutes and extracted total RNA. Total RNA was also extracted from untreated cells. We performed transcriptional profiling comparing treated samples with control samples including three biological replicates each.