Kelly Hines (Presenter)
University of Washington
Authorship: Kelly Hines (1), Brian Werth (2), and Libin Xu (1)
(1) Department of Medicinal Chemistry, University of Washington School of Pharmacy, Seattle, WA 98195; (2) Department of Pharmacy, University of Washington School of Pharmacy, Seattle, WA 98195
| Short Abstract Antimicrobial resistance is a rapidly growing public health crisis, affecting over 2 million people in the USA each year. The rapid identification of antimicrobial-resistant infections is critical to preventing their spread and ensuring proper treatment. Using untargeted lipidomics by HILIC-ion mobility-mass spectrometry, we have distinguished daptomycin susceptible and resistant strains of S. aureus, E. faecalis, and C. striatum based on their lipid profiles. Significant alterations in cell membrane lipids such as phosphatidylglycerols and diglycosyldiacylglycerols were observed among the three strains of resistant bacteria, presenting a potential approach for rapid identification of lipopeptide-resistant strains from clinical isolates. |
Long Abstract
Introduction
Antimicrobial resistance is a rapidly growing public health crisis, affecting over 2 million people in the USA each year.[1] The lipopeptide antibiotic daptomycin targets the bacterial cell membrane, and has been used as a last-resort antibiotic against vancomycin resistant enterococci (VRE) and methicillin resistant S. aureus (MRSA) infections.[2-4] However, resistance to daptomycin has been reported in both VRE and MRSA.[5, 6] Recent studies have found a strong correlation between the content of phospholipids in the bacterial cell membrane and daptomycin resistance.[7, 8] Ion mobility-mass spectrometry (IM-MS) is a rapid gas-phase technique in which ions are separated on the basis of size-to-charge and mass-to-charge, and is particularly suited for the identification and quantitation of lipids.[9,10] Using untargeted lipidomics by HILIC-IM-MS, we have distinguished daptomycin susceptible and resistant isolates of S. aureus, E. faecalis, and C. striatum based on their global glycerolipid and glycerophospholipid profiles. The study of the lipidomes of lipopeptide-resistant bacteria by HILIC-IM-MS could elucidate the mechanisms of resistance and lead to new targets for therapeutics and rapid diagnostics.
Methods
Daptomycin susceptible/resistant strain pairs of C. striatum (W40308, MIC 0.125 µg/mL; W49297, MIC > 256 µg/mL) [11], clinically derived daptomycin susceptible/resistant strain pairs of E. faecalis (S613 and R712) [12], and daptomycin-susceptible MRSA strain (N315, MIC 0.125 µg/mL) and an in vitro derivative with 8-fold reduction in daptomycin susceptibility (W1-N315, MIC 1 µg/mL) were evaluated in triplicate. Lipids were extracted from bacteria isolates (1-8 mg dry weight) using the Folch method.[13] LC-IM-MS analysis was performed with a Waters Acquity FTN UPLC connected directly to the ESI source of a Waters Synapt G2-Si ion mobility-mass spectrometer. Hydrophilic interaction liquid chromatography (HILIC) was performed to achieve separation of glycerolipid and glycerophospholipid species using a 12-min gradient of acetonitrile and water containing 5 mM ammonium acetate. Experiments were performed in both positive and negative ionization modes. IM separation was achieved with traveling wave height of 40 V and velocity of 500 m/s in Nitrogen. Collision cross section (CCS) calibration was performed using a set of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) standards in positive and negative modes, respectively.[14] The resulting data were submitted to Progenesis QI (Waters) for alignment, peak picking, and multivariate statistical analysis. Features were normalized to dry pellet weight and filtered by ANOVA p ≤ 0.1 and fold-change ≥ 1.5.
Results
The HILIC-IM-MS lipidomics method revealed alterations in several major lipid classes upon the development of daptomycin resistance. In the daptomycin resistant C. striatum strain W49297, the chromatographic peak containing the predominant phosphatidylglycerol (PG) species, PG 16:0-18:1, was shifted by +0.2 min and had a 22 Da increase in mass. Fragmentation experiments confirmed that this modified PG was also composed of palmitic (16:0) and oleic (18:1) acids, and the 22 Da mass shift was retained in all fragments corresponding to the PG headgroup. These data suggested that the modification was on the PG headgroup rather than the fatty acid tails. While a small amount of this modified PG species was observed in the daptomycin susceptible strains, the PG composition in the daptomycin resistant strains was nearly entirely replaced by the modified PG. The daptomycin resistant E. faecalis strain R712 (2.6-fold, t-test p = 0.023) and the MRSA strain W1-N315 (1.7-fold, t-test p = 0.0026) also had decreased PG levels relative to their respective susceptible parent strains. In the MRSA strain W1-N315, diglycosyldiacylglycerols (DGDGs) were reduced by nearly the same fold-change (1.8-fold, t-test p = 3.1x10-5) as PGs and, despite the reduced total levels of PGs, a trend towards elevated levels of lysyl-phosphatidylglycerols (LysylPG) relative to total PG levels was observed in the W1-N315 daptomycin strain.
Conclusions
Alterations in bacterial cell membrane lipid composition have been identified as a means of developing resistance to the antibiotic daptomycin. We have established a novel HILIC-IM-MS approach for characterizing antimicrobial resistant bacteria based on their lipid profiles. This untargeted approach revealed several classes of lipids that were altered in the development of daptomycin resistance in gram positive bacteria, including DGDGs, PGs, and a novel modified PG species. The identification of common lipid pathways in antimicrobial resistance may reveal new mechanistic insights and lead to the development of new therapeutics and rapid diagnostics.
References & Acknowledgements:
Acknowledgements
Financial support to this work was provided by the National Institutes of Health Grant R00 HD073270 (L.X.) and the startup funds to L.X. from the Department of Medicinal Chemistry, School of Pharmacy of the University of Washington. B.J.W. has received research support from Allergan and Merck.
References
1. CDC. Antibiotic Resistance Threats in the United States, 2013. [cited 2016 October 3]; Available from: https://www.cdc.gov/drugresistance/threat-report-2013/pdf/ar-threats-2013-508.pdf#page=13.
2. Silverman, J.A., N.G. Perlmutter, and H.M. Shapiro, Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 2003. 47(8): p. 2538-2544.
3. Sakoulas, G., et al., Efficacy of daptomycin in experimental endocarditis due to methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 2003. 47(5): p. 1714-1718.
4. Fowler, V.G., et al., Daptomycin versus standard therapy for bacteremia and endocarditis caused by Staphylococcus aureus. New England Journal of Medicine, 2006. 355(7): p. 653-665.
5. Munoz-Price, L.S., K. Lolans, and J.P. Quinn, Emergence of resistance to daptomycin during treatment of vancomycin-resistant Enterococcus faecalis infection. Clinical Infectious Diseases, 2005. 41(4): p. 565-566.
6. Hayden, M.K., et al., Development of Daptomycin resistance in vivo in methicillin-resistant Staphylococcus aureus. Journal of Clinical Microbiology, 2005. 43(10): p. 5285-5287.
7. Mishra, N.N., et al., Daptomycin Resistance in Enterococci Is Associated with Distinct Alterations of Cell Membrane Phospholipid Content. Plos One, 2012. 7(8).
8. Mishra, N.N. and A.S. Bayer, Correlation of Cell Membrane Lipid Profiles with Daptomycin Resistance in Methicillin-Resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy, 2013. 57(2): p. 1082-1085.
9. Fenn, L.S., et al., Characterizing ion mobility-mass spectrometry conformation space for the analysis of complex biological samples. Analytical and Bioanalytical Chemistry, 2009. 394(1): p. 235-244.
10. Kliman, M., J.C. May, and J.A. McLean, Lipid analysis and lipidomics by structurally selective ion mobility-mass spectrometry. Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids, 2011. 1811(11): p. 935-945.
11. Werth, B.J., Hahn, W.O., Butler-Wu, S.M., and Rakita, R.M., Emergence of High-Level Daptomycin Resistance in Corynebacterium striatum in Two Patients with Left Ventricular Assist Device Infections. Microbial Drug Resistance, 2016. 22(3): p. 233-237.
12. Arias, C.A., et al., Genetic basis for in vivo daptomycin resistance in enterococci. N. England Journal of Medicine, 2011. 365(10): p. 892-900.
13. Folch, J., M. Lees, and G.H.S. Stanley, A SIMPLE METHOD FOR THE ISOLATION AND PURIFICATION OF TOTAL LIPIDES FROM ANIMAL TISSUES. Journal of Biological Chemistry, 1957. 226(1): p. 497-509.
14. Hines, K.M., et al., Evaluation of Collision Cross Section Calibrants for Structural Analysis of Lipids by Traveling Wave Ion Mobility-Mass Spectrometry. Analytical Chemistry, 2016. 88(14): p. 7329-7336.
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