International Journal of Antimicrobial Agents 43 (2014) 26–31 Contents lists available at ScienceDirect International Journal of Antimicrobial Agents j o ur nal homep age : ht tp://www.elsevier.com/locate/ijantimicag dltA overexpression: A strain-independent keystone of daptomycin resistance in methicillin-resistant Staphylococcus aureus Viviana Cafiso a , Taschia Bertuccio a , Simona Purrello a , Floriana Campanile a , Caterina Mammina b , Assunta Sartor c , Annibale Raglio d , Stefania Stefani a,∗ a Department of Biomedical Sciences–Microbiology, University of Catania, Via Androne 81, 95124 Catania, Italy b Department of Sciences for Healthy Promotion ‘G. D’Alessandro’, University of Palermo, Palermo, Italy c Microbiology Unit, Santa Maria della Misericordia University Hospital, Udine, Italy d USC Microbiologia e Virologia, AO Ospedali Riuniti, Bergamo, Italy a r t i c l e i n f o Article history: Received 26 July 2013 Accepted 4 October 2013 Keywords: MRSA Daptomycin Glycopeptides Real-time qPCR Sequencing a b s t r a c t The mechanisms leading to reduced susceptibility to daptomycin (DAP) are multifactorial and have not been fully elucidated. We analysed, by sequencing and expression studies, the role of the major molec- ular targets (cell-envelope charge genes, dltA, mprF, cls2; cell-wall turnover and autolysis genes, sceD, atl) involved in the emergence of DAP resistance in three series of isogenic clinical methicillin-resistant Staphylococcus aureus (MRSA) in which DAP resistance emerged after a heterogeneous glycopeptide- intermediate S. aureus (hGISA) step under teicoplanin and DAP therapy. All of the isolates had different genotypes and were -haemolysin negative, reflecting a strain proclivity to acquire DAP/glycopeptide non-susceptibility under antibiotic pressure. DAP exposure led to the emergence of DAP resistance after an hGISA step probably in parallel with the timing of the two antimicrobial administrations and, in two of three cases, in conditions of DAP underdosage. Real-time qPCR data revealed that all DAP-resistant (DAP- R) isolates had dltA overexpression, whereas mprF upregulation was found only in DAP-R strains with the S295L and T345I amino acid substitutions. Strains that were heteroresistant to DAP did not possess DAP-R-like characteristics. DAP-R strains presented high cls2 expression and no known cls2 mutations, and moreover exhibited sceD and atl upregulation. In conclusion, these findings highlight that dltA over- expression is the common pathway of resistance among genotypically different series of isolates and may represent the keystone of DAP resistance in MRSA, leading to electrostatic repulsion and, indirectly, to a reduction of autolysin activity. mprF mutations related to increased transcription may play a role in this complex phenomenon. © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. 1. Introduction Daptomycin (DAP) has been increasingly used in the treat- ment of various types of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) [1]. However, since 2005 cases of DAP-resistant (DAP-R) isolates have been described in the litera- ture [2,3]. Although the incidence remains low, non-susceptibility to DAP appears to be the result of single or combined strain- related and/or infection-related events. Strain-related causes of DAP resistance 1 are thought to be due to accumulation of mul- tiple mutations in different targets, affecting different cell wall and membrane pathways [4], whilst infection-related causes can be due to: (i) potential exposure of the micro-organisms ∗ Corresponding author. Tel.: +39 095 250 4714/311 352; fax: +39 095 250 4733. E-mail addresses: stefanis@unict.it, simona.purrello@unict.it (S. Stefani). 1 In this study, although the official terminology is ‘daptomycin nonsusceptibility’, the term ‘daptomycin resistance’ was used for ease of understanding. to subinhibitory concentrations of the drug owing to large variations in serum peak and trough levels at the currently recommended doses [5]; (ii) prior exposure to other antimi- crobial agents, particularly vancomycin (VAN) [6,7]; (iii) a high bacterial inoculum and prolonged antibiotic exposure and/or biofilm-related infections, such as infectious endocarditis or bone infections [8]; and (iv) exposure to host-derived cationic peptides [9]. The emergence of S. aureus with diminished DAP susceptibility during glycopeptide [VAN or teicoplanin (TEC)] therapy represents a challenge for the medical community, as VAN treatment may account for loss of DAP susceptibility [2,7]. The European Committee on Antimicrobial Susceptibility Test- ing (EUCAST) [10] and the Clinical and Laboratory Standards Institute (CLSI) [11] define an S. aureus strain as DAP-R or DAP non-susceptible at a minimum inhibitory concentration (MIC) cut- off value of >1 mg/L. Strains with a heterogeneous phenotype, i.e. strains with subpopulations growing at antibiotic concentrations above the MIC, have also been described [12]. 0924-8579/$ – see front matter © 2013 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. http://dx.doi.org/10.1016/j.ijantimicag.2013.10.001