*3.7. Dynamic Light Scattering*

The particle size of the OMVs was measured using dynamic light scattering (DLS). OMVs were diluted with PBS to a protein concentration of 0.05 mg/L and the scatter was recorded using a Zetasizer NanoS (Malvern, PA, USA) at 173◦ with a laser of wavelength 632 nm. Data were analysed with Zetasizer Software (V7.11, Malvern, UK) to obtain the average hydrodynamic radius.

#### **4. Conclusions**

In this study, we show that polymyxin B treatment of the susceptible *K. pneumoniae* strains significantly reduced the glycerophospholipid, fatty acid, lysoglycerophosphate and sphingolipid content of their OMVs, compare to the untreated control. On the other hand, in the OMVs of their paired polymyxin-resistant strains these lipids were increased both intrinsically and in response to polymyxin B treatment. In view of these findings, it is reasonable to hypothesize that the outer membrane remodelling associated with polymyxin-resistance in *K. pneumoniae* entails fortifying the membrane with increased glycerophospholipids, fatty acids, lysoglycerophosphates and sphingolipids, which are lipids to which polymyxins cannot avidly bind. It is important to mention that polymyxins primarily target the lipid A in the Gram-negative outer membrane—hence their narrow spectrum of activity against Gram-negative bacteria that do not express LPS. These outer membrane changes may be accompanied by the modification of the lipid A with cationic moieties and/or a reduction in the lipid A content, which, together with the increased content of the aforementioned lipids, serve to make the *K. pneumoniae* outer membrane and OMVs more impervious to polymyxin attack.

**Supplementary Materials:** Supplementary materials can be found at: http://www.mdpi.com/1422-0067/19/8/ 2356/s1.

**Author Contributions:** R.J., M.-L.H., Y.Z., X.H., M.H.H., Y.-W.L., Q.Z., and C.Y.D.-D. All contributed to the experimental data collection, reporting of the results and write-up of the manuscript. J.L. and T.V. developed the experimental design and concepts and helped write the manuscript.

**Acknowledgments:** J.L. and T.V. are supported by research grants from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (R01 AI132681). J.L. and T.V. are also supported by the Australian National Health and Medical Research Council (NHMRC) as Senior Research and Career Development Level 2 Fellows. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health.

**Conflicts of Interest:** The authors declare no conflict of interest.
