Occurrence of N-Acyl Homoserine Lactones in Extracts of Bacterial Strain of Pseudomonas aeruginosa and in Sputum Sample Evaluated by Gas Chromatography–Mass Spectrometry

Susheela Rani, Ashwini Kumar, Ashok Kumar Malik, P. Schmitt-Kopplin

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DOI: 10.4236/ajac.2011.22037                

Abstract

This study presents a fast, accurate and sensitive technique using gas chromatography-mass spectrometry (GC-MS) for the identification and quantification of N-acyl homoserine lactones (AHLs) in the extracts of bacterial strain of Pseudomonas aeruginosa and sputum sample of a cystic fibrosis patient. This method involves direct separation and determination of AHLs by using GC-MS as simultaneous separation and characterization of AHLs were possible without any prior derivatiza-tion. Electron ionization resulted in a common fragmentation pattern with the most common fragment ion at m/z 143 and other minor peaks at 73, 57 and 43. The limit of detection for N-butanoyl, N-hexanoyl, N-octanoyl, N-decanoyl, N-dodecanoyl and N-tetradecanoyl homoserine lactones was 2.14, 3.59, 2.71, 2.10, 2.45 and 2.34 µg/L, respectively. The presence of AHLs in the culture of P. aeruginosa strain and spu-tum of a cystic fibrosis patient was achieved in selected ion monitoring (SIM) mode by using the prominent fragment at m/z 143.

Keywords

Gas Chromatography—Mass Spectrometry, N-Acyl Homoserine Lactone (N-Butanoyl, N-Hexanoyl, N-Octanoyl, N-Decanoyl, N-Dodecanoyl and N-Tetradecanoyl) Homoserine Lactone, Sputum Sample, Bacterial Strain

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	[1] P. Williams, M. Camara, A. Hardman, S. Swift, D. Milton, V. J. Hope, K. Winzer, B. Middleton, D. I. Pritchard and B. W. Bycroft, “Quorum Sensingand the Population-Dependent Control of Virulence,” Philosophical Transactions of the Royal Society B: Biological Sciences, Vol. 355, No. 1397, 2000, pp. 667-680. doi:10.1098/rstb.2000.0607
[2]	S. Swift, P. Williams and G. S. A. B. Stewart, “N-Acylhomo-Serine Lactones and Quorum Sensing in the Proteobacteria,” In: G. M. Dunny and S. C. Winans, Ed., Cell-Cell Signaling in Bacteria, ASM Press, Washington D.C., 1999, pp. 291-313.
[3]	A. M. Hardman, G. S. A. B. Stewart and P. Williams, “Quorum Sensing and the Cell-Cell Communication Dependent Regulation of Gene Expression in Pathogenic and Nonpathogenic Bacteria, Ant. Van Leeuwenhoek,” Journal of Microbiogical Methods, Vol. 74, No. 4, 1998, pp. 199-210.
[4]	N. A. Whitehead, A. M. L. Bernard, H. Slater, N. J. L. Simpson and G. P. C. Salmond, “Quorum-Sensing in Gram-Negative Bacteria,” FEMS Microbilogy Reviews, Vol. 25, No. 4, 2001, pp. 365-404. doi:10.1111/j.1574-6976.2001.tb00583.x
[5]	T. R. De Kievit, B. H. Iglewski, “Bacterial Quorum Sensing in Pathogenic Relationships,” Infection and Immunity, Vol. 68, No. 9, 2000, pp. 4839-4849. doi:10.1128/IAI.68.9.4839-4849.2000
[6]	L. Gram, L. Ravn, M. Rasch, J. B. Bruhn, A. B. Christensen and M. Givskov, “Food Spoilage-Interactions between Food Spoilage Bacteria,” International Journal of Food Microbiology, Vol. 78, No. 1-2, 2002, pp. 79-97. doi:10.1016/S0168-1605(02)00233-7
[7]	C. Fuqua, S. C. Winans and E. P. Greenberg, “Census and Consensus in Bacterial Ecosystems: The LuxR-LuxI Family of Quorum-Sensing Transcriptional Regulators,” Annual Review of Microbiology, Vol. 50, No. 1, 1996, pp. 727-751. doi:10.1146/annurev.micro.50.1.727
[8]	S. Swift, M. K. Winson, P. F. Chan, N. J. Bainton, M. Birdsall, P. J. Reeves, C. E. D. Rees, S. R. Chhabra, P. J. Hill, J. P. Throup, B. W. Bycroft, G. P. C. Salmond, P. Williams and G. S. A. B. Stewart, “A Novel Strategy for the Isolation of Luxl Homologues: Evidence for the Widespread Distribution of a LuxR: Luxl Superfamily in Enteric Bacteria,” Molecular Microbiology, Vol. 10, No. 3, 1993, pp. 511-520. doi:10.1111/j.1365-2958.1993.tb00923.x
[9]	C. Cha, P. Gao, Y. C. Chen, P. D. Shaw and S. K. Farrand, “Production of Acyl-Homoserine Lactone Quorum-Sensing Signals by Gram-Negative Plant-Associated Bacteria,” Molecular Plant-Microbe Interactions, Vol. 11, No. 11, 1998, pp. 1119-1129. doi:10.1094/MPMI.1998.11.11.1119
[10]	L. Gram, A. B. Christensen, L. Ravn, S. Molin and M. Givskov, “Production of Acylatedhomoserine Lactones by Psychrotrophic Enterobacteriaceae Isolated from Foods,” Applied and Environmental Microbiology, Vol. 65, No. 8, 1999, pp. 3458-3463.
[11]	L. Ravn, A. B. Christensen, S. Molin, M. Givskov and L. Gram, “Methods for Detecting Acylatedhomoserine Lactones Produced by Gram-Negative Bacteria and Their Application in Studies of AHL-Production Kinetics,” Journal of Microbiological Methods, Vol. 44, No. 3, 2001, pp. 239-251. doi:10.1016/S0167-7012(01)00217-2
[12]	J. P. Throup, M. Camara, G. S. Briggs, M. K. Winson, S. R. Chhabra, B. W. Bycroft, P. Williams and G. S. Stewart, “Characterisationofthe Yeni/YenR Locus from Yersinia Enterocolitica Mediating the Synthesis of Two N-cylhomoserine Lactone Signal Molecules,” Molecular Microbiology, Vol. 17, No. 2, 1995, pp. 345-356. doi:10.1111/j.1365-2958.1995.mmi_17020345.x
[13]	C. A. Jacobi, A. Bach, L. Eberl, A. Steidle and J. Heesemann, “Detection of N-(3-oxohexanoyl)-l-homoserine lactone with Yersinia Enterocolitica Serotype O8,” Infection and Immunity, Vol. 71, No. 11, 2003, pp. 6624-6626. doi:10.1128/IAI.71.11.6624-6626.2003
[14]	X. Li, A. Fekete, M. Englmann, C. G?tz, M. Rothballer, M. Frommberger, K. Buddrus, J. Fekete, C. Cai, P. Schr?der, A. Hartmann, G. Chen and P. Schmitt-Kopplin, “Development and Application of a Method for the Analysis of N-acylhomoserine Lactones by Solid-Phase Extraction and Ultra High Pressure Liquid Chromatography,” Journal of Chromatography A, Vol. 1134, No. 1-2, 2006, pp. 186-193. doi:10.1016/j.chroma.2006.09.047
[15]	C. V. Delden and B. H. Iglewski, “Cell-to-Cell Signaling and Pseudomonas Aeruginosa Infections,” Emerging Infectious Diseases, Vol. 4, No. 4, 1998, pp. 551-560.
[16]	N. Hoiby and B. Frederiksen “Microbiology of Cystic fibrosis,” In: M. E. Hodson and D. M. Geddes, Eds., Cystic Fibrosis, 2nd Edition, Arnold, London, 2000, pp. 83-107.
[17]	J. B. Lyczak, C. L. Cannon and G. B. Pier, “Lung Infections Associated with Cystic Fibrosis,” Microbiological Reviews, Vol. 15, No. 2, 2002, pp. 194-222. doi:10.1128/CMR.15.2.194-222.2002
[18]	M. Dunn, R. G. Wunderink, “Ventilator-Associated Pneumonia Caused by Pseudomonas Infection,” Clinics in Chest Medicine, Vol. 16, No. 16, 1995, pp. 95-109.
[19]	H. Wu, Z. Song, M. Hentzer, J. B. Anderson, A. Heydorn, K. Mathee, C. Moser, L. Ebrel, S. Molin, N. Hoiby and M. Givskov, “Detection of N-acylhomoserine Lactones in Lung Tissues of Mice Infected with Pseudomonas Aeruginosa,” Microbiology, Vol. 146, No. 10, 2000, pp. 2481- 2493.
[20]	C. E. Chambers, M. B. Visser, U. Schwab and P. A. Sokol, “Identification of N-acylhomoserine Lactones in Mucopurulent Respiratory Secretions from Cystic Fibrosis Patients,” FEMS Microbiology Letters, Vol. 244, No. 2, 2005, pp. 297-304. doi:10.1016/j.femsle.2005.01.055
[21]	B. Middleton, H. C. Rodgers, M. Camara, A. J. Knox, P. Williams and A. Hardman, “Direct Detection of N-Acylhomo-Serine Lactones in Cystic Fibrosis Sputum,” FEMS Microbiology Letters, Vol. 207, No. 1, 2002, pp. 1-7. doi:10.1111/j.1574-6968.2002.tb11019.x
[22]	K. Riedel, M. Hentzer, O. Geisenberger, B. Huber, A. Steidle, H. Wu, N. Hoiby, M. Givskov, S. Molin and L. Eberl, “N-Acyl-L-Homoserinelactone-Mediated Communication between Pseudomonas Aeruginosa and Burkholderia Cepacia in Mixed Biofilms,” Microbiology, Vol. 147, No. 12, 2001, pp. 3249-3262.
[23]	P. D. Shaw, G. Ping, S. L. Daly, J. E. Cronon, K. L. Rinehart and S. K. Farrand, “Detecting and Characterizing NN-Acyl-Homoserine Lactone Signal Molecules by Thin-Layer Chromatography,” Proceedings of the National Academy of Sciences USA, Vol. 94, No. 12, 1997, pp. 6036-6041. doi:10.1073/pnas.94.12.6036
[24]	J. J. Michels, E. J. Allain, S. A. Borchardt, P. Hu and W. F. McCoy, “Degradation Pathway of Homoserine Lactone Bacterial Signal Molecules by Halogen Antimicrobials Identified by Liquid Chromatography with Photodiode Array and Mass Spectrometric Detection,” Journal of Chromatography A, Vol. 898, No. 2, 2000, pp. 153-165. doi:10.1016/S0021-9673(00)00849-9
[25]	T. S. Charlton, R. de Nys, A. Netting, N. Kumar, M. Hentzer, M. Givskov and S. Kjelleberg, “A Novel and Sensitive Method for the Quantification of N-3-Oxoacyl Homoserine Lactones Using Gas Chromatography–Mass Spectrometry: Application to a Model Bacterial Biofilm,” Environmental Microbiology, Vol. 2, No. 5, 2000, pp. 530-541. doi:10.1046/j.1462-2920.2000.00136.x
[26]	D. L. Erickson, R. Endersby, A. Kirkham, K. Stuber, D. D. Vollman, H. R. Robin, I. Mitchell and D. G. Storey, “Pseudomonas Aeruginosa Quorum-Sensing Systems May Control Virulence Factor Expression in the Lungs of Patients with Cystic Fibrosis,” Infection and Immunity, Vol. 70, No. 4, 2002, pp. 1783-1790. doi:10.1128/IAI.70.4.1783-1790.2002
[27]	M. Frommberger, P. Schmitt-Kopplin, F. Menzinger, V. Albrecht, M. Schmid, L. Ebrel, A. Hartman and A. Kettrup, “Analysis of Selected N-Acyl-L-Homoserine Lactones Produced by Soil-Borne Bacteria with Partial Filling Micellar Electrokinetic Chromatography–Electrospray ionization Ion Trap Mass Spectrometry,” Electrophoresis, Vol. 24, No. 17, 2003, pp. 3067-3074. doi:10.1002/elps.200305567
[28]	M. Frommberger, P. Schmitt-Kopplin, G. Ping, H. Frisch, M. Schmid, Y. Zhang, A. Hartman and A. Kettrup, “A Simple and Robust Setup for On-Column Sample Preconcentration-Nano Liquid Chromatography-Electrospray Ionization Mass Spectrometry for the Analysis of N-acylho-Moserine Lactones,” Analytical Bioanalytical Chemistry, Vol. 378, No. 4, 2004, pp. 1014-1020. doi:10.1007/s00216-003-2400-5
[29]	M. Frommberger, N. Hertkorn, M. Englmann, S. Jakoby, A. Hartmann, A. Kettrup and P. Schmitt-Kopplin, “Analysis of N-Acylhomoserine Lactones after Alkaline Hydrolysis and Anion-Exchange Solid-Phase Extraction by Capillary Zone Electrophoresis-Mass Spectrometry,” Electrophoresis, Vol. 26, No. 7-8, 2005, pp. 1523-1532. doi:10.1002/elps.200410365
[30]	T. R. I. Cataldi, G. Bianco, M. Frommberger and Ph. Schimitt-Kopplin, “Direct Analysis of Selected N-Acyl-l- Homoserine Lactones by Gas Chromatography/Mass Spectrometry,” Rapid Communications in Mass Spectrometry, Vol. 18, No. 12, 2004, pp. 1341-1344. doi:10.1002/rcm.1480
[31]	T. R. I. Cataldi, G. Bianco, L. Palazzo, V. Quaranta, “Occurrence of N-Acyl-l-Homoserine Lactones in Extracts of Some Gram Negative Bacteria Evaluated by Gas Chromatography–Mass Spectrometry,” Analytical Biochemistry, Vol. 361, No. 2, 2007, pp. 226-235. doi:10.1016/j.ab.2006.11.037
[32]	J. P. Pearson, L. Passador, B. H. Iglewski and E. P. Greenberg, “A Second N-Acylhomoserine Lactone Signal Produced by Pseudomonas Aeruginosa,” Proceedings of the National Academy of Sciences USA, Vol. 92, No. 5, 1995, pp. 1490-1494. doi:10.1073/pnas.92.5.1490