Occurrence of cross-resistance and ß-lactam seesaw effect in glycopeptide-, lipopeptide- and lipoglycopeptide-resistant MRSA correlates with membrane phosphatidylglycerol levels.

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 ß-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 ß-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 ß-lactams, depending on the PBP targets of the particular ß-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 ß-lactams. CONCLUSIONS: These studies demonstrate a major association between membrane remodelling, cross-resistance and the seesaw effect.

Elucidating the impact of bacterial lipases, human serum albumin, and FASII inhibition on the utilization of exogenous fatty acids by Staphylococcus aureus.

IMPORTANCE: Incorporation of host-derived exogenous fatty acids (eFAs), particularly unsaturated fatty acids (UFAs), by Staphylococcus aureus could affect the bacterial membrane fluidity and susceptibility to antimicrobials. In this work, we found that glycerol ester hydrolase (Geh) is the primary lipase hydrolyzing cholesteryl esters and, to a lesser extent, triglycerides and that human serum albumin (HSA) could serve as a buffer of eFAs, where low levels of HSA facilitate the utilization of eFAs but high levels of HSA inhibit it. The fact that the type II fatty acid synthesis (FASII) inhibitor, AFN-1252, leads to an increase in UFA content even in the absence of eFA suggests that membrane property modulation is part of its mechanism of action. Thus, Geh and/or the FASII system look to be promising targets to enhance S. aureus killing in a host environment by restricting eFA utilization or modulating membrane properties, respectively.

Elucidating the Impact of Bacterial Lipases, Human Serum Albumin, and FASII Inhibition on the Utilization of Exogenous Fatty Acids by Staphylococcus aureus.

Staphylococcus aureus only synthesizes straight-chain or branched-chain saturated fatty acids (SCFAs or BCFAs) via the type II fatty acid synthesis (FASII) pathway, but as a highly adaptive pathogen, S. aureus can also utilize host-derived exogenous fatty acids (eFAs), including SCFAs and unsaturated fatty acids (UFAs). S. aureus secretes three lipases, Geh, sal1, and SAUSA300_0641, which could perform the function of releasing fatty acids from host lipids. Once released, the FAs are phosphorylated by the fatty acid kinase, FakA, and incorporated into the bacterial lipids. In this study, we determined the substrate specificity of S. aureus secreted lipases, the effect of human serum albumin (HSA) on eFA incorporation, and the effect of FASII inhibitor, AFN-1252, on eFA incorporation using comprehensive lipidomics. When grown with major donors of fatty acids, cholesteryl esters (CEs) and triglycerides (TGs), Geh was found to be the primary lipase responsible for hydrolyzing CEs, but other lipases could compensate for the function of Geh in hydrolyzing TGs. Lipidomics showed that eFAs were incorporated into all major S. aureus lipid classes and that fatty acid-containing HSA can serve as a source of eFAs. Furthermore, S. aureus grown with UFAs displayed decreased membrane fluidity and increased production of reactive oxygen species (ROS). Exposure to AFN-1252 enhanced UFAs in the bacterial membrane, even without a source of eFAs, indicating a FASII pathway modification. Thus, the incorporation of eFAs alters the S. aureus lipidome, membrane fluidity, and ROS formation, which could affect host-pathogen interactions and susceptibility to membrane-targeting antimicrobials.

Evolution of cefiderocol resistance in Stenotrophomonas maltophilia using in vitro serial passage techniques.

OBJECTIVES: Cefiderocol is a siderophore cephalosporin active against MDR Gram-negatives including Stenotrophomonas maltophilia. Cefiderocol resistance remains uncommon and incompletely understood. We selected for cefiderocol-resistant S. maltophilia in vitro and characterized the genetic mechanisms and potential for cross-resistance to other antimicrobials. METHODS: We selected cefiderocol resistance in three clinical strains of S. maltophilia by serial passage in escalating concentrations of cefiderocol. Emergent cefiderocol-resistant isolates were subjected to repeat susceptibility testing against a panel of relevant antimicrobials. Isolates with confirmed MIC changes were whole genome sequenced. RESULTS: Each parent strain was initially susceptible to cefiderocol (MICs of 0.03125, 0.03125 and 0.125 mg/L), and one initially tested susceptible to ceftazidime/avibactam (MIC 4 mg/L). We recovered evolved isolates achieving cefiderocol resistance at MICs of 8-32 mg/L from each parental strain. Some cefiderocol resistant isolates reverted following one to four drug-free passages. Ceftazidime/avibactam MICs of passaged isolates repeatedly increased to ≥256 mg/L, and while other MICs were largely unchanged, trimethoprim/sulfamethoxazole MICs declined 4-fold in two strains. WGS revealed one evolved isolate carrying six coding mutations, while four were isogenic mutants of tonB, tolQ, smf-1 and the smeT promoter. Mutation of the smeT promoter downregulated the smeDEF efflux pump and reduced susceptibility to penicillins but increased susceptibility to several other classes including sulphonamides. Other mutations occurred in genes putatively involved in iron metabolism including smlt1148 and cirA. CONCLUSIONS: S. maltophilia strains evolved cefiderocol resistance through different genetic pathways, but often involved iron transport. Future work is required to fully understand the role(s) of other genes in cefiderocol resistance.

Varied Contribution of Phospholipid Shedding From Membrane to Daptomycin Tolerance in Staphylococcus aureus.

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.

Dalbavancin exposure in vitro selects for dalbavancin-non-susceptible and vancomycin-intermediate strains of methicillin-resistant Staphylococcus aureus.

OBJECTIVES: Dalbavancin is a lipoglycopeptide active against methicillin-resistant Staphylococcus aureus (MRSA). Its long half-life (8.5-16 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 µg/mL, ß-elimination t1/2 204 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 ß-lactams with varying penicillin-binding protein (PBP) affinities. RESULTS: Dalbavancin was bactericidal against most strains for days 1-4 before regrowth of less susceptible subpopulations occurred. Isolates with eight-fold increases in dalbavancin MIC were detected as early as day 4 but increased 64-128-fold in all models by day 28. Vancomycin and daptomycin MICs increased 4-16-fold, exceeding the susceptibly breakpoints for both antibiotics; ß-lactam MICs generally decreased by two-to eight-fold, suggesting a dalbavancin-ß-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 ß-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.

Synergy Between Beta-Lactams and Lipo-, Glyco-, and Lipoglycopeptides, Is Independent of the Seesaw Effect in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant S. aureus (MRSA) are resistant to beta-lactams, but synergistic activity between beta-lactams and glycopeptides/lipopeptides is common. Many have attributed this synergy to the beta-lactam-glycopeptide seesaw effect; however, this association has not been rigorously tested. The objective of this study was to determine whether the seesaw effect is necessary for synergy and to measure the impact of beta-lactam exposure on lipid metabolism. We selected for three isogenic strains with reduced susceptibility to vancomycin, daptomycin, and dalbavancin by serial passaging the MRSA strain N315. We used whole genome sequencing to identify genetic variants that emerged and tested for synergy between vancomycin, daptomycin, or dalbavancin in combination with 6 beta-lactams with variable affinity for staphylococcal penicillin binding proteins (PBPs), including nafcillin, meropenem, ceftriaxone, ceftaroline, cephalexin, and cefoxitin, using time-kills. We observed that the seesaw effect with each beta-lactam was variable and the emergence of the seesaw effect for a particular beta-lactam was not necessary for synergy between that beta-lactam and vancomycin, daptomycin, or dalbavancin. Synergy was more commonly observed with vancomycin and daptomycin based combinations than dalbavancin in time-kills. Among the beta-lactams, cefoxitin and nafcillin were the most likely to exhibit synergy using the concentrations tested, while cephalexin was the least likely to exhibit synergy. Synergy was more common among the resistant mutants than the parent strain. Interestingly N315-D1 and N315-DAL0.5 both had mutations in vraTSR and walKR despite their differences in the seesaw effect. Lipidomic analysis of all strains exposed to individual beta-lactams at subinhibitory concentrations suggested that in general, the abundance of cardiolipins (CLs) and most free fatty acids (FFAs) positively correlated with the presence of synergistic effects while abundance of phosphatidylglycerols (PGs) and lysylPGs mostly negatively correlated with synergistic effects. In conclusion, the beta-lactam-glycopeptide seesaw effect and beta-lactam-glycopeptide synergy are distinct phenomena. This suggests that the emergence of the seesaw effect may not have clinical importance in terms of predicting synergy. Further work is warranted to characterize strains that don't exhibit beta-lactam synergy to identify which strains should be targeted with combination therapy and which ones cannot and to further investigate the potential role of CLs in mediating synergy.