Advertisement

European Biophysics Journal

, Volume 46, Issue 4, pp 351–361 | Cite as

A nanomechanical study of the effects of colistin on the Klebsiella pneumoniae AJ218 capsule

  • Anna Mularski
  • Jonathan Wilksch
  • Eric Hanssen
  • Jian Li
  • Takehiro Tomita
  • Sacha James Pidot
  • Tim Stinear
  • Frances Separovic
  • Dick Strugnell
Original Article

Abstract

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.

Keywords

Antimicrobial peptide Atomic force microscopy Capsular polysaccharide Colistin Klebsiella pneumoniae Polymyxin 

Abbreviations

AFM

Atomic force microscopy

AMP

Antimicrobial peptide

CFU

Colony-forming unit

ESBL

Extended-spectrum beta-lactamases

LB

Luria–Bertani

LPS

Lipopolysaccharide

MBC

Minimum bactericidal concentration

MIC

Minimum inhibitory concentration

PEI

Polyethylene imine

Notes

Acknowledgments

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.

Supplementary material

249_2016_1178_MOESM1_ESM.docx (55.3 mb)
Supplementary material 1 (DOCX 56632 kb)

References

  1. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Pevzner PA (2012) SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19(5):455–477CrossRefPubMedPubMedCentralGoogle Scholar
  2. Blumenkrantz N, Asboe-Hansen G (1973) New method for quantitative determination of uronic acids. Anal Biochem 54(2):484–489CrossRefPubMedGoogle Scholar
  3. Campos MA, Vargas MA, Regueiro V, Llompart CM, Albertí S, Bengoechea JA (2004) Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides. Infect Immun 72(12):7107–7114CrossRefPubMedPubMedCentralGoogle Scholar
  4. Cannatelli A, Di Pilato V, Giani T, Arena F, Ambretti S, Gaibani P, Rossolini GM (2014) In vivo evolution to colistin resistance by PmrB sensor kinase mutation in KPC-producing Klebsiella pneumoniae is associated with low-dosage colistin treatment. Antimicrob Agents Chemother 58(8):4399–4403CrossRefPubMedPubMedCentralGoogle Scholar
  5. Centers for Disease Control and Prevention (2013) Antibiotic resistance threats in the United States. Centers for Disease Control and Prevention, AtlantaGoogle Scholar
  6. Choi MJ, Ko KS (2014) Mutant prevention concentrations of colistin for Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae clinical isolates. J Antimicrob Chemother 69(1):275–277CrossRefPubMedGoogle Scholar
  7. Clinical and Laboratory Standards Institute (2003) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Clinical and Laboratory Standards Institute, Pennsylvania, pp 14–17Google Scholar
  8. Considine RF, Drummond CJ, Dixon DR (2001) Force of interaction between a biocolloid and an inorganic oxide: complexity of surface deformation, roughness, and brushlike behavior. Langmuir 17(20):6325–6335CrossRefGoogle Scholar
  9. de Lorenzo V, Herrero M, Jakubzik U, Timmis KN (1990) Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative eubacteria. J Bacteriol 172(11):6568–6572CrossRefPubMedPubMedCentralGoogle Scholar
  10. Dupres V, Menozzi FD, Locht C, Clare BH, Abbott NL, Cuenot S, Dufrêne YF (2005) Nanoscale mapping and functional analysis of individual adhesins on living bacteria. Nat Methods 2(7):515–520CrossRefPubMedGoogle Scholar
  11. Dzul SP, Thornton MM, Hohne DN, Stewart EJ, Shah AA, Bortz DM, Younger JG (2011) Contribution of the Klebsiella pneumoniae capsule to bacterial aggregate and biofilm microstructures. Appl Environ Microbiol 77(5):1777–1782CrossRefPubMedPubMedCentralGoogle Scholar
  12. Engel A, Müller DJ (2000) Observing single biomolecules at work with the atomic force microscope. Nat Struct Biol 7(9):715–718CrossRefPubMedGoogle Scholar
  13. Falagas ME, Kasiakou SK, Saravolatz LD (2005) Colistin: the revival of polymyxins for the management of multidrug-resistant Gram-negative bacterial infections. Clin Infect Dis 40(9):1333–1341CrossRefPubMedGoogle Scholar
  14. Formosa C, Herold M, Vidaillac C, Duval RE, Dague E (2015) Unravelling of a mechanism of resistance to colistin in Klebsiella pneumoniae using atomic force microscopy. J Antimicrob Chemother 70(8):2261–2270CrossRefPubMedGoogle Scholar
  15. Gaboriaud F, Bailet S, Dague E, Jorand F (2005) Surface structure and nanomechanical properties of Shewanella putrefaciens bacteria at two pH values (4 and 10) determined by atomic force microscopy. J Bacteriol 187(11):3864–3868CrossRefPubMedPubMedCentralGoogle Scholar
  16. Gaboriaud F, Gee ML, Strugnell R, Duval JFL (2008a) Coupled electrostatic, hydrodynamic, and mechanical properties of bacterial interfaces in aqueous media. Langmuir 24(19):10988–10995CrossRefPubMedGoogle Scholar
  17. Gaboriaud F, Parcha BS, Gee ML, Holden JA, Strugnell RA (2008b) Spatially resolved force spectroscopy of bacterial surfaces using force-volume imaging. Coll Surf B 62(2):206–213CrossRefGoogle Scholar
  18. Hertz H (1896) On the contact of elastic solids Miscellaneous Papers by H. Hertz. Macmillan, LondonGoogle Scholar
  19. Highsmith AK, Jarvis WR (1985) Klebsiella pneumoniae: selected virulence factors that contribute to pathogenicity. Infect Control 6(2):75–77CrossRefPubMedGoogle Scholar
  20. Hutter JL, Bechhoefer J (1993) Calibration of atomic force microscope tips. Rev Sci Instrum 64(7):1868–1873CrossRefGoogle Scholar
  21. Jayol A, Poirel L, Brink A, Villegas MV, Yilmaz M, Nordmann P (2014) Resistance to colistin associated with a single amino acid change in protein PmrB among Klebsiella pneumoniae isolates of worldwide origin. Antimicrob Agents Chemother 58(8):4762–4766CrossRefPubMedPubMedCentralGoogle Scholar
  22. Jenney AW, Clements A, Farn JL, Wijburg OL, McGlinchey A, Spelman DW, Strugnell RA (2006) Seroepidemiology of Klebsiella pneumoniae in an Australian tertiary hospital and its implications for vaccine development. J Clin Microbiol 44(1):102–107CrossRefPubMedPubMedCentralGoogle Scholar
  23. Kao FS, Pan YR, Hsu RQ, Chen HM (2012) Efficacy verification and microscopic observations of an anticancer peptide, CB1a, on single lung cancer cell. Biochimica et Biophysica Acta (BBA) Biomembr 1818(12):2927–2935CrossRefGoogle Scholar
  24. Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, Chaudhary U, Doumith M, Giske CG, Irfan S, Krishnan P, Kumar AV, Maharjan S, Mushtaq S, Noorie T, Paterson DL, Pearson A, Perry C, Pike R, Rao B, Ray U, Sarma JB, Sharma M, Sheridan E, Thirunarayan MA, Turton J, Upadhyay S, Warner M, Welfare W, Livermore DM, Woodford N (2010) Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis 10(9):597–602CrossRefPubMedPubMedCentralGoogle Scholar
  25. Kwon YM, Ricke SC (2000) Efficient amplification of multiple transposon-flanking sequences. J Microbiol Methods 41(3):195–199CrossRefPubMedGoogle Scholar
  26. Li J, Nation RL, Turnidge JD, Milne RW, Coulthard K, Rayner CR, Paterson DL (2006) Colistin: the re-emerging antibiotic for multidrug-resistant Gram-negative bacterial infections. Lancet Infect Dis 6(9):589–601CrossRefPubMedGoogle Scholar
  27. Liu B, Yu HH, Ng TW, Paterson DL, Velkov T, Li J, Fu J (2014a) Nanoscale focused ion beam tomography of single bacterial cells for assessment of antibiotic effects. Microsc Microanal 20(02):537–547CrossRefPubMedGoogle Scholar
  28. Liu BL, Liu YL, Di XD, Zhang XZ, Wang RW, Bai YB, Wang JW (2014b) Colistin and anti-Gram-positive bacterial agents against Acinetobacter baumannii. Rev Soc Bras Med Trop 47(4):451–456CrossRefPubMedGoogle Scholar
  29. Llobet E, Tomas JM, Bengoechea JA (2008) Capsule polysaccharide is a bacterial decoy for antimicrobial peptides. Microbiology 154(Pt 12):3877–3886CrossRefPubMedGoogle Scholar
  30. Lounatmaa K, Nanninga N (1976) Effect of polymyxin on the outer membrane of Salmonella typhimurium: freeze-fracture studies. J Bacteriol 128(2):665–667PubMedPubMedCentralGoogle Scholar
  31. Lounatmaa K, Mäkelä PH, Sarvas M (1976) Effect of polymyxin on the ultrastructure of the outer membrane of wild-type and polymyxin-resistant strain of Salmonella. J Bacteriol 127(3):1400–1407PubMedPubMedCentralGoogle Scholar
  32. Lower SK, Hochella MF, Beveridge TJ (2001) Bacterial recognition of mineral surfaces: nanoscale interactions between Shewanella and α-FeOOH. Science 292(5520):1360–1363CrossRefPubMedGoogle Scholar
  33. Mortensen NP, Fowlkes JD, Sullivan CJ, Allison DP, Larsen NB, Molin S, Doktycz MJ (2009) Effects of colistin on surface ultrastructure and nanomechanics of Pseudomonas aeruginosa cells. Langmuir 25(6):3728–3733CrossRefPubMedGoogle Scholar
  34. Mularski A, Wilksch JJ, Wang H, Hossain MA, Wade JD, Separovic F, Gee ML (2015) Atomic force microscopy reveals the mechanobiology of lytic peptide action on bacteria. Langmuir 31(22):6164–6171CrossRefPubMedGoogle Scholar
  35. Mularski A, Wilksch JJ, Hanssen E, Strugnell RA, Separovic F (2016) Atomic force microscopy of bacteria reveals the mechanobiology of pore forming peptide action. Biochimica et Biophysica Acta (BBA) Biomembr 1858(6):1091–1098CrossRefGoogle Scholar
  36. Poirel L, Jayol A, Bontron S, Villegas MV, Ozdamar M, Türkoglu S, Nordmann P (2015) The mgrB gene as a key target for acquired resistance to colistin in Klebsiella pneumoniae. J Antimicrob Chemother 70(1):75–80CrossRefPubMedGoogle Scholar
  37. Radmacher M (1997) Measuring the elastic properties of biological samples with the AFM. Eng Med Biol Mag IEEE 16(2):47–57CrossRefGoogle Scholar
  38. Radmacher M, Fritz M, Hansma PK (1995) Imaging soft samples with the atomic force microscope: gelatin in water and propanol. Biophys J 69(1):264–270CrossRefPubMedPubMedCentralGoogle Scholar
  39. Schembri MA, Blom J, Krogfelt KA, Klemm P (2005) Capsule and fimbria interaction in Klebsiella pneumoniae. Infect Immun 73(8):4626–4633CrossRefPubMedPubMedCentralGoogle Scholar
  40. Shaw JE, Epand RF, Hsu JCY, Mo GCH, Epand RM, Yip CM (2008) Cationic peptide-induced remodelling of model membranes: direct visualization by in situ atomic force microscopy. J Struct Biol 162(1):121–138CrossRefPubMedGoogle Scholar
  41. Soon RL, Nation RL, Harper M, Adler B, Boyce JD, Tan CH, Larson I (2011) Effect of colistin exposure and growth phase on the surface properties of live Acinetobacter baumannii cells examined by atomic force microscopy. Int J Antimicrob Agents 38(6):493–501CrossRefPubMedPubMedCentralGoogle Scholar
  42. Strauss J, Kadilak A, Cronin C, Mello CM, Camesano TA (2010) Binding, inactivation, and adhesion forces between antimicrobial peptide cecropin P1 and pathogenic E. coli. Colloids Surf B 75(1):156–164CrossRefGoogle Scholar
  43. Struve C, Krogfelt KA (2003) Role of capsule in Klebsiella pneumoniae virulence: lack of correlation between in vitro and in vivo studies. FEMS Microbiol Lett 218(1):149–154CrossRefPubMedGoogle Scholar
  44. Suo Z, Avci R, Deliorman M, Yang X, Pascual DW (2009) Bacteria survive multiple puncturings of their cell walls. Langmuir 25(8):4588–4594CrossRefPubMedGoogle Scholar
  45. Taubes G (2008) The bacteria fight back. Science 321(5887):356–361CrossRefPubMedGoogle Scholar
  46. Velegol SB, Logan BE (2002) Contributions of bacterial surface polymers, electrostatics, and cell elasticity to the shape of AFM force curves. Langmuir 18(13):5256–5262CrossRefGoogle Scholar
  47. Velkov T, Roberts KD, Nation RL, Thompson PE, Li J (2013) Pharmacology of polymyxins: new insights into an ‘old’ class of antibiotics. Future Microbiol 8(6):711CrossRefPubMedGoogle Scholar
  48. Wang H, Wilksch JJ, Lithgow T, Strugnell RA, Gee ML (2013) Nanomechanics measurements of live bacteria reveal a mechanism for bacterial cell protection: the polysaccharide capsule in Klebsiella is a responsive polymer hydrogel that adapts to osmotic stress. Soft Matter 9(31):7560–7567CrossRefGoogle Scholar
  49. Wang H, Wilksch JJ, Strugnell RA, Gee ML (2015) Role of capsular polysaccharides in biofilm formation: an AFM nanomechanics study. ACS Appl Mater Interfaces 7(23):13007–13013CrossRefPubMedGoogle Scholar
  50. Zhang L, Dhillon P, Yan H, Farmer S, Hancock REW (2000) Interactions of bacterial cationic peptide antibiotics with outer and cytoplasmic membranes of Pseudomonas aeruginosa. Antimicrob Agents Chemother 44(12):3317–3321CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2016

Authors and Affiliations

  1. 1.School of ChemistryUniversity of MelbourneMelbourneAustralia
  2. 2.Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and ImmunityUniversity of MelbourneMelbourneAustralia
  3. 3.Advanced Microscopy Unit, Bio21 Molecular Science and Biotechnology InstituteUniversity of MelbourneMelbourneAustralia
  4. 4.Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical SciencesMonash UniversityParkvilleAustralia
  5. 5.Microbiological Diagnostic Unit Public Health Laboratory, The Peter Doherty Institute for Infection and ImmunityUniversity of MelbourneMelbourneAustralia

Personalised recommendations