A nanomechanical study of the effects of colistin on the Klebsiella pneumoniae AJ218 capsule
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Atomic force microscopy measurements of capsule thickness revealed that that the wild-type Klebsiella pneumoniae AJ218 capsular polysaccharides were rearranged by exposure to colistin. The increase in capsule thickness measured near minimum inhibitory/bactericidal concentration (MIC/MBC) is consistent with the idea that colistin displaces the divalent cations that cross-bridge adjacent lipopolysaccharide (LPS) molecules through the capsule network. Cryo-electron microscopy demonstrated that the measured capsule thickness at near MIC/MBC of 1.2 μM was inflated by the disrupted outer membrane, through which the capsule is excreted and LPS is bound. Since wild-type and capsule-deficient strains of K. pneumoniae AJ218 have equivalent MICs and MBCs, the presence of the capsule appeared to confer no protection against colistin in AJ218. A spontaneously arising colistin mutant showed a tenfold increase in resistance to colistin; genetic analysis identified a single amino acid substitution (Q95P) in the PmrB sensor kinase in this colistin-resistant K. pneumoniae AJ218. Modification of the lipid A component of the LPS could result in a reduction of the net-negative charge of the outer membrane, which could hinder binding of colistin to the outer membrane and displacement of the divalent cations that bridge adjacent LPS molecules throughout the capsular polysaccharide network. Retention of the cross-linking divalent cations may explain why measurements of capsule thickness did not change significantly in the colistin-resistant strain after colistin exposure. These results contrast with those for other K. pneumoniae strains that suggest that the capsule confers colistin resistance.
KeywordsAntimicrobial peptide Atomic force microscopy Capsular polysaccharide Colistin Klebsiella pneumoniae Polymyxin
Atomic force microscopy
Minimum bactericidal concentration
Minimum inhibitory concentration
The authors gratefully acknowledge the support of the Australian Research Council and the National Health and Medical Research Council (Program Grant 606788) and Dr. Michelle Gee, who together with JL, initially suggested the AFM study of the effect of colistin on K. pneumoniae. AM received an Australian Postgraduate Award and a David Hay Postgraduate Writing-Up Award. JL is an Australian NHMRC Senior Research Fellow and is supported by a research grant from the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (R01 AI111965). 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. All AFM work was conducted at the Nanomaterials Platform, University of Melbourne. Electron microscopy was carried out at the Bio21 Advanced Microscopy Facility, University of Melbourne.
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