23Treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections is challenging and is 24 associated with high rates of therapeutic failure. The glycopeptide (GP) vancomycin and the lipopeptide (LP) 25 daptomycin are still relied upon to manage invasive MRSA infections; however, resistance to these antibiotics 26 has emerged and there is evidence of cross-resistance between them. It has been observed that the susceptibility 27 of MRSA to beta-lactams increases as susceptibility to GPs and LPs decreases, a phenomenon termed the seesaw 28 effect. Recent efforts to understand the mechanism underlying the seesaw effect have focused on the penicillin 29 binding proteins (PBPs). However, while daptomycin resistance is largely mediated by remodeling of membrane 30 lipid composition, the role of membrane lipids in producing cross-resistance and the seesaw effect has not yet 31 been investigated. Here, we evaluate the lipid profiles, cross susceptibilities, and beta-lactam susceptibilities of a 32 collection of isogenic MRSA strains selected against daptomycin, vancomycin or dalbavancin (a 33 lipoglycopeptide; LGP) to assess the relationship between membrane composition, cross-resistance, and the 34 seesaw effect. We found that modification of membrane composition occurs not only in daptomycin-selected 35 strains, but also vancomycin-and dalbavancin-selected strains. Significantly, we observed that typically the levels 36 of short-chain phosphatidylglycerols (PGs) negatively correlate with MICs of GP/LP/LGP and positively 37 correlate with MIC of certain beta-lactams, the latter being dependent on the primary PBP target of the particular 38 beta-lactam. Furthermore, changes to certain PGs with long-chain fatty acids correlate well with presence of the 39 seesaw effect. These studies demonstrate a major association between membrane remodeling and the seesaw 40 effect. 41 42 43 44 45 46 47 48 Hines et al, In Preparation for Antimicrob. Agents Chemother. 3 49 INTRODUCTION 50Staphylococcus aureus remains a serious public health concern that is responsible for nearly 120,000 51 infections and 20,000 deaths in the USA annually (1). Methicillin resistant S. aureus (MRSA) is particularly 52 challenging to treat and nearly 40% of infected patients will fail therapy with the first-line antibiotic, vancomycin, 53 a glycopeptide (GP) (2-7). Many factors contribute to poor outcomes in these patients including the emergence 54 of reduced susceptibility phenotypes such as vancomycin intermediate S. aureus (VISA) and heterogeneous VISA 55 (hVISA) (8, 9). Several alternatives for vancomycin are available, including the lipoglycopeptides (LGPs) 56 dalbavancin, telavancin and oritavancin, and the lipopeptide (LP) daptomycin. However, the minimum inhibitory 57 concentration (MIC) of vancomycin is positively correlated with MICs for LGPs and LPs, posing the risk of 58 cross-resistance establishing among these therapies without the patient having been exposed to them (10, 11). 59Many studies have observed that the susceptibil...

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