Background Glycopeptides (GPs), lipopeptides (LPs) and lipoglycopeptides (LGPs) are related antimicrobials important for the management of invasive MRSA infections. Cross-resistance among these antibiotics in MRSA is well documented, as is the observation that susceptibility of MRSA to β-lactams increases as susceptibility to GPs and LPs decreases (i.e. the seesaw effect). Efforts to understand the relationship between GP/LP/LGP cross-resistance and the seesaw effect have focused on the PBPs, but the role of lipid metabolism has not been investigated. Objectives Since the cell membrane is structurally and metabolically integrated with the cell wall and anchors associated proteins, including PBPs, we examined the relationship between membrane lipid composition and the phenomena of cross-resistance among GPs/LPs/LGPs and the β-lactam seesaw effect. Methods We selected for daptomycin, vancomycin and dalbavancin resistance using the USA300 strain JE2 and evaluated the resulting mutants by WGS, MS-based lipidomics and antimicrobial susceptibility testing to assess the relationship between membrane composition, cross-resistance, and the seesaw effect. Results We observed cross-resistance to GPs/LPs/LGPs among the selected strains and the seesaw effect against various β-lactams, depending on the PBP targets of the particular β-lactam. We found that modification of membrane composition occurs not only in daptomycin-selected strains, but also vancomycin- and dalbavancin-selected strains. Significantly, we observed that the abundance of most phosphatidylglycerols positively correlates with MICs of GPs/LPs/LGPs and negatively correlates with the MICs of β-lactams. Conclusions These studies demonstrate a major association between membrane remodelling, cross-resistance and the seesaw effect.

Objectives: Dalbavancin is a lipoglycopeptide active against methicillin-resistant Staphylococcus aureus (MRSA). Its long half-life (8.5e16 days) allows for once-weekly or single-dose treatments but could prolong the mutant selection window, promoting resistance and cross-resistance to related antimicrobials such as vancomycin. The objective of this study was to evaluate the capacity of post-distributional pharmacokinetic exposures of dalbavancin to select for resistance and cross-resistance in MRSA. Methods: We simulated average, post-distributional exposures of single-dose (1500 mg) dalbavancin (fCmax 9.9 mg/mL, b-elimination t 1/2 204 h) in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model for 28 days (672 h) against five MRSA strains and one methicillin-susceptible strain (MSSA). Samples were collected at least daily, and surviving colonies were enumerated and screened for resistance on drug-free and dalbavancin-supplemented medium respectively. Isolates from resistance screening plates were subjected to whole-genome sequencing (WGS) and susceptibly testing against dalbavancin, vancomycin, daptomycin, and six b-lactams with varying penicillin-binding protein (PBP) affinities. Results: Dalbavancin was bactericidal against most strains for days 1e4 before regrowth of less susceptible subpopulations occurred. Isolates with eight-fold increases in dalbavancin MIC were detected as early as day 4 but increased 64e128-fold in all models by day 28. Vancomycin and daptomycin MICs increased 4e16-fold, exceeding the susceptibly breakpoints for both antibiotics; b-lactam MICs generally decreased by two-to eight-fold, suggesting a dalbavancineb-lactam seesaw effect, but increased by eight-fold or more in certain isolates. Resistant isolates carried mutations in a variety of genes, most commonly walKR, apt, stp1, and atl. Conclusions: In our in vitro system, post-distributional dalbavancin exposures selected for stable mutants with reduced susceptibility to dalbavancin, vancomycin, and daptomycin, and generally increased susceptibility to b-lactams in all strains of MRSA tested. The clinical significance of these findings remains unclear, but created an opportunity to genotype a unique collection of dalbavancin-resistant strains for the first time. Mutations involved genes previously associated with vancomycin intermediate susceptibility and daptomycin non-susceptibility, most commonly walKR-associated genes.

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...

Objectives Tedizolid is an oxazolidinone antimicrobial with activity against Gram-positive bacteria, including MRSA. Tedizolid resistance is uncommon and tedizolid’s capacity to select for cross-resistance to other antimicrobials is incompletely understood. The objective of this study was to further explore the phenotypic and genetic basis of tedizolid resistance in MRSA. Methods We selected for tedizolid resistance in an MRSA laboratory strain, N315, by serial passage until an isolate with an MIC ≥1 log2 dilution above the breakpoint for resistance (≥2 mg/L) was recovered. This isolate was subjected to WGS and susceptibility to a panel of related and unrelated antimicrobials was tested in order to determine cross-resistance. Homology modelling was performed to evaluate the potential impact of the mutation on target protein function. Results After 10 days of serial passage we recovered a phenotypically stable mutant with a tedizolid MIC of 4 mg/L. WGS revealed only one single nucleotide variant (A1345G) in rpoB, corresponding to amino acid substitution D449N. MICs of linezolid, chloramphenicol, retapamulin and quinupristin/dalfopristin increased by ≥2 log2 dilutions, suggesting the emergence of the so-called ‘PhLOPSa’ resistance phenotype. Susceptibility to other drugs, including rifampicin, was largely unchanged. Homology models revealed that the mutated residue of RNA polymerase would be unlikely to directly affect oxazolidinone action. Conclusions To the best of our knowledge, this is the first time that an rpoB mutation has been implicated in resistance to PhLOPSa antimicrobials. The mechanism of resistance remains unclear, but is likely indirect, involving σ-factor binding or other alterations in transcriptional regulation.

It has been suggested that daptomycin can be inactivated by lipids released by Staphylococcus aureus and that this effect is antagonized by phenol soluble modulins (PSMs), which bind to the shed lipids. PSM production is regulated by the Agr system, and others have shown that loss of the Agr function enhances S. aureus survival in the presence of daptomycin. Here we assessed the impact of Agr function on daptomycin activity and lipid metabolism under various conditions. Daptomycin activity was evaluated against three sets of isogenic strain series with wild-type or dysfunctional Agr using static daptomycin time-kills over 24 h and against one strain pair using in vitro pharmacokinetic/pharmacodynamic (PK/PD) models simulating clinical daptomycin exposure for 48 h. We performed comprehensive lipidomics on bacterial membranes and the spent media to correlate lipid shedding with survival. In static time-kill experiments, two agr-deficient strains (SH1000- and USA300 LAC ΔagrA) showed improved survival for 8 h compared with their corresponding wild-type strains as seen in previous studies, but this difference did not persist for 24 h. However, four other agr-deficient strains (SH1001 and JE2 agr KOs) did not demonstrate improved survival compared to isogenic wild-type strains at any time in the time-kills. Lipidomics analysis of SH1000, SH1001, and SH1000- strains showed daptomycin exposure increased lipid shedding compared to growth controls in all strains with phosphatidylglycerols (PGs), lysylPGs and cardiolipins predominating. In the cell pellets, PGs and lysylPGs decreased but cardiolipins were unchanged with daptomycin exposure. The shed lipid profiles in SH1001 and SH1000- were similar, suggesting that the inability to resist daptomycin by SH1001 was not because of differences in lipid shedding. In the PK/PD model, the agr mutant SH1000- strain did not show improved survival relative to SH1000 either. In conclusion, inactivation of daptomycin by shed lipids may be dependent on genetic background, the specific agr mutations, or the techniques used to generate these KOs rather than the overall function of the Agr system, and its contribution to daptomycin tolerance seems to be varied, transient, and growth-condition dependent.