Resistance and response to anti-pseudomonas agents and biocides

Pseudomonas: Volume 7: New Aspects of Pseudomonas Biology

Volumn , Issue , 2015, Pages 173-187

  Morita, Yuji ^a   Tomida, Junko ^a   Kawamura, Yoshiaki ^a  

^a Department of Microbiology ^*  (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

EID: 84927559977     PISSN: None     EISSN: None     Source Type: Book    
DOI: 10.1007/978-94-017-9555-5_7     Document Type: Chapter

References (96)

1. Aires JR, Kohler T, Nikaido H, Plesiat P (1999) Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides. Antimicrob Agents Chemother 43:2624-2628
2. Alvarez-Ortega C, Wiegand I, Olivares J, Hancock RE, Martinez JL (2010) Genetic determinants involved in the susceptibility of Pseudomonas aeruginosa to beta-lactam antibiotics. Antimicrob Agents Chemother 54:4159-4167
3. Bagge N, Schuster M, Hentzer M, Ciofu O, Givskov M, Greenberg EP et al (2004) Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta- lactamase and alginate production. Antimicrob Agents Chemother 48:1175-1187
4. Beaudoin T, Zhang L, Hinz AJ, Parr CJ, Mah TF (2012) The biofilm-specific antibiotic resistance gene ndvB is important for expression of ethanol oxidation genes in Pseudomonas aeruginosa biofilms. J Bacteriol 194:3128-3136
5. Blazquez J, Gömez-Gömez JM, Oliver A, Juan C, Kapur V, Martín S (2006 Oct) PBP3 inhibition elicits adaptive responses in Pseudomonas aeruginosa. Mol Microbiol 62(1):84-99. Epub 2006 Aug 31. PubMed PMID:1695-6383.
6. Brazas MD, Hancock RE (2005) Ciprofloxacin induction of a susceptibility determinant in Pseudomonas aeruginosa. Antimicrob Agents Chemother 49:3222-3227
7. Brazas MD, Breidenstein EB, Overhage J, Hancock RE (2007) Role of lon, an ATP-dependent protease homolog, in resistance of Pseudomonas aeruginosa to ciprofloxacin. Antimicrob Agents Chemother 51:4276-4283
8. Breidenstein EB, Khaira BK, Wiegand I, Overhage J, Hancock RE (2008) Complex ciprofloxacin resistome revealed by screening a Pseudomonas aeruginosa mutant library for altered susceptibility. Antimicrob Agents Chemother 52:4486-4491
9. Breidenstein EB, Bains M, Hancock RE (2012) Involvement of the lon protease in the SOS response triggered by ciprofloxacin in Pseudomonas aeruginosa PAO1. Antimicrob Agents Chemother 56:2879-2887
10. Buyck JM, Plesiat P, Traore H, Vanderbist F, Tulkens PM, Van Bambeke F (2012) Increased susceptibility of Pseudomonas aeruginosa to macrolides and ketolides in eukaryotic cell culture media and biological fluids due to decreased expression of oprM and increased outer-mem- brane permeability. Clin Infect Dis 55:534-542
11. Cao L, Srikumar R, Poole K (2004) MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720-PA3719. Mol Microbiol 53:1423-1436
12. Chiang WC, Pamp SJ, Nilsson M, Givskov M, Tolker-Nielsen T (2012) The metabolically active subpopulation in Pseudomonas aeruginosa biofilms survives exposure to membrane-targeting antimicrobials via distinct molecular mechanisms. FEMS Immunol Med Microbiol 65:245256
13. Chuanchuen R, Schweizer HP (2012) Global transcriptional responses to triclosan exposure in Pseudomonas aeruginosa. Int J Antimicrob Agents 40:114-122
14. Cirz RT, O’Neill BM, Hammond JA, Head SR, Romesberg FE (2006) Defining the Pseudomonas aeruginosa SOS response and its role in the global response to the antibiotic ciprofloxacin. J Bacteriol 188:7101-7110
15. Cummins J, Reen FJ, Baysse C, Mooij MJ, O’Gara F (2009) Subinhibitory concentrations of the cationic antimicrobial peptide colistin induce the pseudomonas quinolone signal in Pseudomonas aeruginosa. Microbiology 155:2826-2837
16. Daigle DM, Cao L, Fraud S, Wilke MS, Pacey A, Klinoski R et al (2007) Protein modulator of multidrug efflux gene expression in Pseudomonas aeruginosa. J Bacteriol 189:5441-5451
17. Davis BD (1987) Mechanism of bactericidal action of aminoglycosides. Microbiol Rev 51:341350
18. Fernandez L, Gooderham WJ, Bains M, McPhee JB, Wiegand I, Hancock RE (2010) Adaptive resistance to the “last hope” antibiotics polymyxin B and colistin in Pseudomonas aeruginosa is mediated by the novel two-component regulatory system ParR-ParS. Antimicrob Agents Chemother 54:3372-3382
19. Fernandez L, Jenssen H, Bains M, Wiegand I, Gooderham WJ, Hancock RE (2012) The two- component system CprRS senses cationic peptides and triggers adaptive resistance in Pseudomonas aeruginosa independently of ParRS. Antimicrob Agents Chemother 56:6212-6222
20. Fernandez L, Alvarez-Ortega C, Wiegand I, Olivares J, Kocincova D, Lam JS et al (2013) Characterization of the polymyxin B resistome of Pseudomonas aeruginosa. Antimicrob Agents Chemother 57:110-119
21. Fournier D, Richardot C, Muller E, Robert-Nicoud M, Llanes C, Plesiat P et al (2013) Complexity of resistance mechanisms to imipenem in intensive care unit strains of Pseudomonas aeruginosa. J Antimicrob Chemother 68:1772-1780
22. Fraud S, Campigotto AJ, Chen Z, Poole K (2008) MexCD-OprJ multidrug efflux system of Pseudomonas aeruginosa: involvement in chlorhexidine resistance and induction by membrane- damaging agents dependent upon the AlgU stress response sigma factor. Antimicrob Agents Chemother 52:4478-4482
23. Gallagher LA, Shendure J, Manoil C (2011) Genome-scale identification of resistance functions in Pseudomonas aeruginosa using Tn-seq. mBio 2:e00315-e00310
24. Gellatly SL, Needham B, Madera L, Trent MS, Hancock RE (2012) The Pseudomonas aeruginosa PhoP-PhoQ two-component regulatory system is induced upon interaction with epithelial cells and controls cytotoxicity and inflammation. Infect Immun 80:3122-3131
25. Gutu AD, Sgambati N, Strasbourger P, Brannon MK, Jacobs MA, Haugen E et al (2013) Polymyxin Resistance of Pseudomonas aeruginosa phoQ Mutants Is Dependent on Additional Two- Component Regulatory Systems. Antimicrob Agents Chemother 57:2204-2215
26. Hay T, Fraud S, Lau CH, Gilmour C, Poole K (2013) Antibiotic inducibility of the mexXY multidrug efflux operon of Pseudomonas aeruginosa: involvement of the MexZ anti-repressor ArmZ. PLoS One 8:e56858
27. Hinz A, Lee S, Jacoby K, Manoil C (2011 Sep) Membrane proteases and aminoglycoside antibiotic resistance. J Bacteriol 193(18):4790-7. doi:10.1128/JB.05133-11. Epub 2011 Jul 15. PubMed PMID:21764915; PubMed Central PMCID:PMC3165699.
28. Hocquet D, Nordmann P, El Garch F, Cabanne L, Plesiat P (2006) Involvement of the MexXY- OprM efflux system in emergence of cefepime resistance in clinical strains of Pseudomonas aeruginosa. Antimicrob Agents Chemother 50:1347-1351
29. Hocquet D, Muller A, Blanc K, Plesiat P, Talon D, Monnet DL et al (2008) Relationship between antibiotic use and incidence of MexXY-OprM overproducers among clinical isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother 52:1173-1175
30. Hoffman LR, D’Argenio DA, MacCoss MJ, Zhang Z, Jones RA, Miller SI (2005) Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 436:1171-1175
31. Hooper DC (1998) Clinical applications of quinolones. Biochim Biophys Acta 1400:45-61
32. Jaffe A, Francis J, Rosenthal M, Bush A (1998) Long-term azithromycin may improve lung function in children with cystic fibrosis. The Lancet 351:420
33. Jeannot K, Elsen S, Kohler T, Attree I, van Delden C, Plesiat P (2008) Resistance and virulence of Pseudomonas aeruginosa clinical strains overproducing the MexCD-OprJ efflux pump. Anti- microb Agents Chemother 52:2455-2462
34. Kai T, Tateda K, Kimura S, Ishii Y, Ito H, Yoshida H et al (2009) A low concentration of azithromycin inhibits the mRNA expression of N-acyl homoserine lactone synthesis enzymes, upstream of lasI or rhll, in Pseudomonas aeruginosa. Pulm Pharmacol Ther 22:483-486
35. Khuntayaporn P, Montakantikul P, Santanirand P, Kiratisin P, Chomnawang MT (2013) Molecular investigation of carbapenem resistance among multidrug-resistant Pseudomonas aeruginosa isolated clinically in Thailand. Microbiol Immunol 57:170-178
36. Kindrachuk KN, Fernandez L, Bains M, Hancock RE (2011) Involvement of an ATP-dependent protease, PA0779/AsrA, in inducing heat shock in response to tobramycin in Pseudomonas aeruginosa. Antimicrob Agents Chemother 55:1874-1882
37. Kohler T, Michea-Hamzehpour M, Henze U, Gotoh N, Curty LK, Pechere JC (1997) Characterization of MexE-MexF-OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa. Mol Microbiol 23:345-354
38. Kohler T, Dumas JL, Van Delden C (2007) Ribosome protection prevents azithromycin-mediated quorum-sensing modulation and stationary-phase killing of Pseudomonas aeruginosa. Antimicrob Agents Chemother 51:4243-4248
39. Krahn T, Gilmour C, Tilak J, Fraud S, Kerr N, Lau CH et al (2012) Determinants of intrinsic aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 56:5591-5602
40. Kudoh S, Azuma A, Yamamoto M, Izumi T, Ando M (1998) Improvement of survival in patients with diffuse panbronchiolitis treated with low-dose erythromycin. Am J Respir Crit Care Med 157:1829-1832
41. Lau CH, Fraud S, Jones M, Peterson SN, Poole K (2013) Mutational Activation of the AmgRS two-component system in aminoglycoside-resistant Pseudomonas aeruginosa. Antimicrob Agents Chemother 57:2243-2251
42. Lee S, Hinz A, Bauerle E, Angermeyer A, Juhaszova K, Kaneko Y et al (2009) Targeting a bacterial stress response to enhance antibiotic action. Proc Natl Acad Sci U S A 106:14570-14575
43. Li Y, Mima T, Komori Y, Morita Y, Kuroda T, Mizushima T et al (2003) A new member of the tripartite multidrug efflux pumps, MexVW-OprM, in Pseudomonas aeruginosa. J Antimicrob Chemother 52:572-575
44. Llanes C, Kohler T, Patry I, Dehecq B, van Delden C, Plesiat P (2011) Role of the MexEF-OprN efflux system in low-level resistance of Pseudomonas aeruginosa to ciprofloxacin. Antimicrob Agents Chemother 55:5676-5684
45. Macfarlane EL, Kwasnicka A, Hancock RE (2000) Role of Pseudomonas aeruginosa PhoP-PhoQ in resistance to antimicrobial cationic peptides and aminoglycosides. Microbiology 146 (Pt 10):2543-2554
46. Mah TF, Pitts B, Pellock B, Walker GC, Stewart PS, O’Toole GA (2003) A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance. Nature 426:306-310
47. Mao W, Warren MS, Lee A, Mistry A, Lomovskaya O (2001) MexXY-OprM efflux pump is required for antagonism of aminoglycosides by divalent cations in Pseudomonas aeruginosa. Antimicrob Agents Chemother 45:2001-2007
48. Marr AK, Overhage J, Bains M, Hancock RE (2007 Feb) The Lon protease of Pseudomonas aeruginosa is induced by aminoglycosides and is involved in biofilm formation and motility. Microbiology 153(Pt 2):474-82. PubMed PMID:17259618.
49. Marvig RL, Sondergaard MS, Damkiaer S, Hoiby N, Johansen HK, Molin S et al (2012) Mutations in 23S rRNA confer resistance against azithromycin in Pseudomonas aeruginosa. Antimicrob Agents Chemother 56:4519-4521
50. Mc Cay PH, Ocampo-Sosa AA, Fleming GT (2010) Effect of subinhibitory concentrations of ben- zalkonium chloride on the competitiveness of Pseudomonas aeruginosa grown in continuous culture. Microbiology 156:30-38
51. McPhee JB, Lewenza S, Hancock RE (2003) Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa. Mol Microbiol 50:205-217
52. Miller AK, Brannon MK, Stevens L, Johansen HK, Selgrade SE, Miller SI et al (2011) PhoQ mutations promote lipid A modification and polymyxin resistance of Pseudomonas aeruginosa found in colistin-treated cystic fibrosis patients. Antimicrob Agents Chemother 55:5761-5769
53. Mima T, Joshi S, Gomez-Escalada M, Schweizer HP (2007) Identification and characterization of TriABC-OpmH, a triclosan efflux pump of Pseudomonas aeruginosa requiring two membrane fusion proteins. J Bacteriol 189:7600-7609
54. Mima T, Kohira N, Li Y, Sekiya H, Ogawa W, Kuroda T et al (2009) Gene cloning and characteristics of the RND-type multidrug efflux pump MuxABC-OpmB possessing two RND components in Pseudomonas aeruginosa. Microbiology 155:3509-3517
55. Morita Y, Kodama K, Shiota S, Mine T, Kataoka A, Mizushima T et al (1998) NorM, a putative multidrug efflux protein, of Vibrio parahaemolyticus and its homolog in Escherichia coli. An- timicrob Agents Chemother 42:1778-1782
56. Morita Y, Kimura N, Mima T, Mizushima T, Tsuchiya T (2001) Roles of MexXY- and MexAB- multidrug efflux pumps in intrinsic multidrug resistance of Pseudomonas aeruginosa PAO1. J Gen Appl Microbiol 47:27-32
57. Morita Y, Murata T, Mima T, Shiota S, Kuroda T, Mizushima T et al (2003) Induction of mexCD- oprJ operon for a multidrug efflux pump by disinfectants in wild-type Pseudomonas aeruginosa PAO1. J Antimicrob Chemother 51:991-994
58. Morita Y, Cao L, Gould VC, Avison MB, Poole K (2006a) nalD encodes a second repressor of the mexAB-oprMmultidrug efflux operon of Pseudomonas aeruginosa. J Bacteriol 188:8649-8654
59. Morita Y, Sobel ML, Poole K (2006b) Antibiotic inducibility of the MexXY multidrug efflux system of Pseudomonas aeruginosa: involvement of the antibiotic-inducible PA5471 gene product. J Bacteriol 188:1847-1855
60. Morita Y, Gilmour C, Metcalf D, Poole K (2009) Translational control of the antibiotic inducibility of the PA5471 gene required for mexXY multidrug efflux gene expression in Pseudomonas aeruginosa. J Bacteriol 191:4966-4975
61. Morita Y, Narita S, Tomida J, Tokuda H, Kawamura Y (2010) Application of an inducible system to engineer unmarked conditional mutants of essential genes of Pseudomonas aeruginosa. J Microbiol Methods 82:205-213
62. Morita Y, Tomida J, Kawamura Y (2012a) MexXY multidrug efflux system of Pseudomonas aeruginosa. Front Microbiol 3:408
63. Morita Y, Tomida J, Kawamura Y (2012b) Primary mechanisms mediating aminoglycoside resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7. Microbiology 158:1071-1083
64. Morita Y, Tomida J, Kawamura Y (2014 Jan) Responses of Pseudomonas aeruginosa to antimicrobials. Front Microbiol 8(4):422. doi: 10.3389/fmicb.2013.00422. Review. PubMed PMID:24409175; PubMed Central PMCID:PMC3884212.
65. Moskowitz SM, Brannon MK, Dasgupta N, Pier M, Sgambati N, Miller AK et al (2012) PmrB mutations promote polymyxin resistance of Pseudomonas aeruginosa isolated from colistin- treated cystic fibrosis patients. Antimicrob Agents Chemother 56:1019-1030
66. Moya B, Dotsch A, Juan C, Blazquez J, Zamorano L, Haussler S et al (2009) Beta-lactam resistance response triggered by inactivation of a nonessential penicillin-binding protein. PLoS Pathog 5:e1000353
67. Muller C, Plesiat P, Jeannot K (2011) A two-component regulatory system interconnects resistance to polymyxins, aminoglycosides, fluoroquinolones, and beta-lactams in Pseudomonas aeruginosa. Antimicrob Agents Chemother 55:1211-1221
68. Nalca Y, Jansch L, Bredenbruch F, Geffers R, Buer J, Haussler S (2006) Quorum-sensing antagonistic activities of azithromycin in Pseudomonas aeruginosa PAO1: a global approach. Antimicrob Agents Chemother 50:1680-1688
69. Nde CW, Jang HJ, Toghrol F, Bentley WE (2009) Global transcriptomic response of Pseudomonas aeruginosa to chlorhexidine diacetate. Environ Sci Technol 43:8406-8415
70. Pamp SJ, Gjermansen M, Johansen HK, Tolker-Nielsen T (2008) Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB-oprM genes. Mol Microbiol 68:223-240
71. Perez-Martinez I, Haas D (2011) Azithromycin inhibits expression of the GacA-dependent small RNAs RsmY and RsmZ in Pseudomonas aeruginosa. Antimicrob Agents Chemother 55:3399-3405
72. Poole K (2004) Resistance to beta-lactam antibiotics. Cell Mol Life Sci 61:2200-2223
73. Poole K (2005) Aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob Agents Che- mother 49:479-487
74. Poole K (2011) Pseudomonas aeruginosa: resistance to the max. Front Microbiol 2:65
75. Poole K (2013) “Pseudomonas aeruginosa Efflux Pumps” in Microbial Efflux Pumps: Current Research, edited by Yu EW, Zhang Q, and Brown MH (Caister Academic Press), 175-206.
76. Poole K, Gotoh N, Tsujimoto H, Zhao Q, Wada A, Yamasaki T et al (1996a) Overexpression of the mexC-mexD-oprJ efflux operon in nfB-type multidrug-resistant strains of Pseudomonas aeruginosa. Mol Microbiol 21:713-724
77. Poole K, Tetro K, Zhao Q, Neshat S, Heinrichs DE, Bianco N (1996b) Expression of the multidrug resistance operon mexA-mexB-oprM in Pseudomonas aeruginosa: mexR encodes a regulator of operon expression. Antimicrob Agents Chemother 40:2021-2028
78. Riera E, Cabot G, Mulet X, Garcia-Castillo M, del Campo R, Juan C et al (2011) Pseudomonas aeruginosa carbapenem resistance mechanisms in Spain: impact on the activity of imipenem, meropenem and doripenem. J Antimicrob Chemother 66:2022-2027
79. Sadovskaya I, Vinogradov E, Li J, Hachani A, Kowalska K, Filloux A (2009) High-level antibiotic resistance in Pseudomonas aeruginosa biofilm: the ndvB gene is involved in the production of highly glycerol-phosphorylated ß-(1^3)-glucans, which bind aminoglycosides. Glycobiology 20:895-904
80. Schurek KN, Marr AK, Taylor PK, Wiegand I, Semenec L, Khaira BK et al (2008) Novel genetic determinants of low-level aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 52:4213-4219
81. Sekiya H, Mima T, Morita Y, Kuroda T, Mizushima T, Tsuchiya T (2003) Functional cloning and characterization of a multidrug efflux pump, mexHI-opmD, from a Pseudomonas aeruginosa mutant. Antimicrob Agents Chemother 47:2990-2992
82. Shu JC, Chia JH, Siu LK, Kuo AJ, Huang SH, Su LH et al (2012) Interplay between mutational and horizontally acquired resistance mechanisms and its association with carbapenem resistance amongst extensively drug-resistant Pseudomonas aeruginosa (XDR-PA). Int J Antimi- crob Agents 39:217-222
83. Sobel ML, Neshat S, Poole K (2005) Mutations in PA2491 (mexS) promote MexT-dependent mexEF-oprN expression and multidrug resistance in a clinical strain of Pseudomonas aeruginosa. J Bacteriol 187:1246-1253
84. Srikumar R, Paul CJ, Poole K (2000) Influence of mutations in the mexR repressor gene on expression of the MexA-MexB-oprM multidrug efflux system of Pseudomonas aeruginosa. J Bacteriol 182:1410-1414
85. Struble JM, Gill RT (2009) Genome-scale identification method applied to find cryptic aminoglycoside resistance genes in Pseudomonas aeruginosa. PLoS One 4:e6576
86. Sugimura M, Maseda H, Hanaki H, Nakae T (2008) Macrolide antibiotic-mediated downregu- lation of MexAB-OprM efflux pump expression in Pseudomonas aeruginosa. Antimicrob Agents Chemother 52:4141-4144
87. Tanimoto K, Tomita H, Fujimoto S, Okuzumi K, Ike Y (2008) Fluoroquinolone enhances the mutation frequency for meropenem-selected carbapenem resistance in Pseudomonas aeruginosa, but use of the high-potency drug doripenem inhibits mutant formation. Antimicrob Agents Chemother 52:3795-3800
88. Tateda K, Ishii Y, Matsumoto T, Furuya N, Nagashima M, Matsunaga T et al (1996) Direct evidence for antipseudomonal activity of macrolides: exposure-dependent bactericidal activity and inhibition of protein synthesis by erythromycin, clarithromycin, and azithromycin. Anti- microb Agents Chemother 40:2271-2275
89. Tateda K, Comte R, Pechere JC, Kohler T, Yamaguchi K, Van Delden C (2001) Azithromycin inhibits quorum sensing in Pseudomonas aeruginosa. Antimicrob Agents Chemother 45:19301933
90. Trias J, Nikaido H (1990) Protein D2 channel of the Pseudomonas aeruginosa outer membrane has a binding site for basic amino acids and peptides. J Biol Chem 265:15680-15684
91. Wolter DJ, Schmidtke AJ, Hanson ND, Lister PD (2007) Increased expression of ampC in Pseudomonas aeruginosa mutants selected with ciprofloxacin. Antimicrob Agents Chemother 51:2997-3000
92. Yan A, Guan Z, Raetz CR (2007) An undecaprenyl phosphate-aminoarabinose flippase required for polymyxin resistance in Escherichiacoli. J Biol Chem 282:36077-36089
93. Yang L, Chen L, Shen L, Surette M, Duan K (2011) Inactivation of MuxABC-OpmB transporter system in Pseudomonas aeruginosa leads to increased ampicillin and carbenicillin resistance and decreased virulence. J Microbiol 49:107-114
94. Zhang L, Mah TF (2008) Involvement of a novel efflux system in biofilm-specific resistance to antibiotics. J Bacteriol 190:4447-4452
95. Zhang L, Dhillon P, Yan H, Farmer S, Hancock RE (2000) Interactions of bacterial cationic peptide antibiotics with outer and cytoplasmic membranes of Pseudomonas aeruginosa. Antimicrob Agents Chemother 44:3317-3321
96. Zhu L, Lin J, Ma J, Cronan JE, Wang H (2010 Feb) Triclosan resistance of Pseudomonas aeruginosa PAO1 is due to FabV, a triclosan-resistant enoyl-acyl carrier protein reductase. Antimicrob Agents Chemother. 54(2):689-98. doi:10.1128/AAC.01152-09. Epub 2009 Nov 23. PubMed PMID:19933806; PubMed Central PMCID:PMC2812149.