Abstract
Daptomycin is an extensively used anti-staphylococcal agent due to the rise in methicillin-resistant Staphylococcus aureus, but the mechanism(s) of resistance is poorly understood. Comparative genome sequencing, transcriptomics, ultrastructure, and cell envelope studies were carried out on two relatively higher level (4 and 8 µg/ml−1) laboratory-derived daptomycin-resistant strains (strains CB1541 and CB1540 respectively) compared to their parent strain (CB1118; MW2). Several mutations were found in the strains. Both strains had the same mutations in the two-component system genes walK and agrA. In strain CB1540 mutations were also detected in the ribose phosphate pyrophosphokinase (prs) and polyribonucleotide nucleotidyltransferase genes (pnpA), a hypothetical protein gene, and in an intergenic region. In strain CB1541 there were mutations in clpP, an ATP-dependent protease, and two different hypothetical protein genes. The strain CB1540 transcriptome was characterized by upregulation of cap (capsule) operon genes, genes involved in the accumulation of the compatible solute glycine betaine, ure genes of the urease operon, and mscL encoding a mechanosensitive chanel. Downregulated genes included smpB, femAB and femH involved in the formation of the pentaglycine interpeptide bridge, genes involved in protein synthesis and fermentation, and spa encoding protein A. Genes altered in their expression common to both transcriptomes included some involved in glycine betaine accumulation, mscL, ure genes, femH, spa and smpB. However, the CB1541 transcriptome was further characterized by upregulation of various heat shock chaperone and protease genes, consistent with a mutation in clpP, and lytM and sceD. Both strains showed slow growth, and strongly decreased autolytic activity that appeared to be mainly due to decreased autolysin production. In contrast to previous common findings, we did not find any mutations in phospholipid biosynthesis genes, and it appears there are multiple pathways to and factors in daptomycin resistance.
Highlights
Increases in the number of methicillin-resistant Staphylococcus aureus (MRSA) strains, increases in vancomycin minimum inhibitory concentrations (MICs) in such strains resulting in vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA), have provided impetus to the development of novel antistaphylococcal antimicrobial agents [1]
In this paper we report on genomic, transcriptomic, ultrastructure, and cell wall autolysis studies of two strains, CB1540 and
Initial Characterization of The Strains The daptomycin MIC for strains CB1540 and CB1541 were 8 mg ml21, and 4 mg ml21 respectively compared to 1 mg ml21 for parent strain CB1118 (Table 1)
Summary
Increases in the number of methicillin-resistant Staphylococcus aureus (MRSA) strains, increases in vancomycin minimum inhibitory concentrations (MICs) in such strains resulting in vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA), have provided impetus to the development of novel antistaphylococcal antimicrobial agents [1]. Transcriptional profiling studies of the response to daptomycin have shown induction of the cell wall stress stimulon including its critical two-component regulator vraSR in S. aureus and liaSR in Bacillus subtilis, and genes responsive to membrane depolarization [10,11].
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