Curr Microbiol DOI 10.1007/s00284-014-0599-3
Cell Envelope Phospholipid Composition of Burkholderia multivorans Sallie A. Ruskoski • James W. Bullard Franklin R. Champlin
•
Received: 10 May 2013 / Accepted: 14 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Burkholderia multivorans causes opportunistic pulmonary infections in cystic fibrosis and immunocompromised patients. The purpose of the present study was to determine the nature of the phospholipids and their fatty acid constituents comprising the cell envelope membranes of strains isolated from three disparate sources. A conventional method for obtaining the readily extractable lipids fraction from bacteria was employed to obtain membrane lipids for thin-layer chromatographic and gas chromatography-mass spectrophotometric analyses. Major fatty acid components of the B. multivorans readily extractable lipid fractions included C16:0 (palmitic acid), C16:1 (palmitoleic acid), and C18:1 (oleic acid), while C14:0 (myristic acid), DC17:0 (methylene hexadecanoic acid), C18:0 (stearic acid), and DC19:0 (methylene octadecanoic acid) were present in lesser amounts. Fatty acid composition differed quantitatively among strains with regard to C16:0, C16:1, DC17:0, C18:1, and DC19:0 with the unsaturated:saturated fatty acid ratios being significantly less in a cystic fibrosis type strain than either environmental or
S. A. Ruskoski F. R. Champlin Departments of Biochemistry and Microbiology, Oklahoma State University Center for Health Sciences, 1111 West 17th Street, Tulsa, OK 74107, USA Present Address: S. A. Ruskoski (&) Health Professions/Medical Laboratory Sciences, Northeastern State University, 3100 E. New Orleans, Broken Arrow, OK 74014, USA e-mail: [email protected] J. W. Bullard Department of Forensic Sciences, Oklahoma State University Center for Health Sciences, 1111 West 17th Street, Tulsa, OK 74107, USA
chronic granulomatous disease strains. Phospholipids identified in all B. multivorans strains included lyso-phosphatidylethanolamine, phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol in similar ratios. These data support the conclusion that the cell envelope phospholipid profiles of disparate B. multivorans strains are similar, while their respective fatty acyl substituent profiles differ quantitatively under identical cultivation conditions.
Introduction Burkholderia multivorans is an obligately aerobic gramnegative bacillus classified as one of seventeen phylogenetically related species forming the Burkholderia cepacia complex (Bcc) [15]. Bcc species are environmentally important organisms in that certain species produce antimicrobial agents which protect economically important fruit and crop plants from various mycoses, while others are exploited as bioremediators by virtue of their disparate catabolic properties [12, 23]. B. multivorans is the causative agent of opportunistic infections in cystic fibrosis (CF), chronic granulomatous disease (CGD), and otherwise immunocompromised patients. The bacterium can colonize or infect pulmonary epithelial cells with either no effect on or suppression of pulmonary function, yet it is capable of more serious involvement leading to necrotizing pneumonia and patient fatality known as cepacia syndrome in some patients [1, 24]. B. multivorans accounts for 39 % of all Bcc organisms isolated from CF patients in the United States [18], and many strains have been shown to be genetically identical to environmental isolates [13]. B. multivorans promotes an inflammatory response by stimulating tumor necrosis factor alpha, interleukin-6, and interleukin-8 production by
123
S. A. Ruskoski et al.: Cell Envelope Phospholipid Composition of B. multivorans Table 1 Burkholderia multivorans strain sources Strain
Origin
Generation time (min)a
ATCC 17616
Soil [20]
45.2
ATCC BAA-247 (type strain)
CF patient [22]
47.4
CGD2
CGD patient [25]
46.7
obtained by inoculating 200 ml of LBB with stationaryphase starter cells to an initial optical density at 620 nm (OD620) of 0.025 (Spectronic 20 optical spectrophotometer, Thermo Electron Corp., Madison, WI) and incubating at 37 °C with rotary aeration at 180 rpm (Excella E24 benchtop incubator shaker; New Brunswick Scientific Co., Inc., Edison, NJ, USA) until late exponential phase.
a
Calculated from turbidimetric growth curves during exponential phase in LBB
human monocytes [26]. Moreover, the bacterium is intrinsically resistant to multiple antimicrobial agents [24]. A paucity of information exists in the literature regarding the cell envelope lipid composition of B. multivorans. Krejcˇi and Kroppenstedt [11] utilized the Sherlock microbial identification system (MIDI, Inc., Newark, DE) to determine the major cellular fatty acid components of 17 B. multivorans clinical isolates to be C16:0, C16:1, and C18:1 in a manner consistent with that for the type species B. cepacia [11, 21]. Other research has shown the major phospholipids of B. cepacia to include two forms of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and diphosphatidylglycerol (DPG) [21]. The purpose of the present study was to determine the phospholipid composition of the B. multivorans cell envelope and its substituent fatty acyl content by analyzing readily extractable lipid (REL) fractions of three disparate strains. Environmental, CF, and CGD test strains were selected to represent three important sources of this organism (Table 1). It is anticipated that these data will aid ongoing investigations of B. multivorans pathogenicity as we endeavor to better understand molecular mechanisms underlying its ability to adhere to host tissues and initiate biofilm assembly.
Materials and Methods Bacterial Strains and Cultural Growth Conditions Environmental (ATCC 17616) and clinical (CGD2) B. multivorans strains were obtained from Dr. Adrian Zelazny (NIH-NIAID, Bethesda, MD), while CF clinical type strain (ATCC BAA-247) is maintained as a reference organism in this laboratory. Pseudomonas aeruginosa PA01 (this laboratory) was employed for comparative purposes because of its known cell envelope lipid composition [9]. All cultures were maintained under cryoprotective conditions; working cultures were cultivated on Luria–Bertani Agar (LBA; Difco Laboratories, Detroit, MI, USA); and starter cultures were prepared by inoculating Luria–Bertani Broth (LBB; Difco Laboratories) with cells from working cultures as described previously [6]. Test cultures were
123
Readily Extractable Lipid Fraction Preparation Readily extractable lipid (REL) fractions were obtained from lyophilized cells using a simplified version of the method described by Folch et al. [7] to extract polar lipids from animal cells. This protocol differs from the Bligh and Dyer [2] method often used by bacteriologists in which water is absent during the initial extraction phase. We have found that water causes emulsions to form which interfere with the extraction process, thereby likely explaining the observation that up to 5 % of total polar lipids are not recovered with the second method. Late exponential-phase cells were harvested by centrifugation at 12,0009g and 4 °C for 12 min (Sorvall Legend XTR Refrigerated Superspeed Centrifuge, Thermo-Fisher Scientific, Inc., Pittsburgh, PA), then washed twice in one volume of cold deionized water. Final cell pellets were combined in a minimal volume of cold deionized water, frozen to -80 °C, and lyophilized to dryness. Approximately 30–40 mg of freeze-dried cells were suspended in 5.0 ml of methanol and mixed vigorously under nitrogen using a wrist-action shaker (Burrell Corporation, Pittsburgh, PA) for 10 min at ambient temperature. An additional 10 ml of chloroform was added to the extraction mixture which was agitated under nitrogen for an additional 12–15 h at ambient temperature. Insoluble material was removed by filtration under negative pressure through a chloroform– methanol (2:1) rinsed paper (Whatman no. 1) filter which was rinsed twice with 5.0 ml of chloroform–methanol (2:1). Remaining polar material was removed from the combined extract by adding 0.2 vol of deionized water, mixing using vortex agitation (Fisher Scientific Touch Mixer; Thermo-Fisher Scientific, Inc., Pittsburgh, PA), and facilitating phase separation with centrifugation at 2,5009g and 4 °C for 10 min (Sorvall Legend XTR Centrifuge) in a screw-capped (Teflon-lined) tube (25 9 150 mm). The total REL extract volume (chloroform layer) was reduced to a small volume under flowing nitrogen and stored at -20 °C under nitrogen until needed. REL Fatty Acid Analysis Phospholipid fatty acyl esters in REL fractions were converted to volatile fatty acid methyl esters (FAMEs) using the method of Morrison and Smith [16] as modified in this laboratory [8]
S. A. Ruskoski et al.: Cell Envelope Phospholipid Composition of B. multivorans
and analyzed utilizing gas chromatography-mass spectrometry (GC–MS) [3]. Morrison and Smith [16] reported their onestep boron fluoride-methanol methanolysis procedure rapidly proceeded to an essentially 100 % yield when authentic phospholipid standards were tested. REL fraction samples were reduced to dryness under flowing nitrogen gas, solubilized in 2.5 ml of benzene and 5.0 ml of boron trifluoridemethanol (14 %; Sigma-Aldrich Co., St. Louis, MO), and heated in a boiling water bath for 5.0 min in Parafilm-sealed, screw-capped (Teflon-lined) tubes (25 9 150 mm). After allowing to cool to ambient temperature, 5.0 ml of saturated aqueous NaCl and 5.0 ml of hexane were added, mixed vigorously with vortex agitation (Fisher Scientific Touch Mixer) for 1.0 min at ambient temperature, and allowed to sit until phase separation was complete. The benzene–hexane layer (top) containing FAMEs was transferred to a clean, screwcapped (Teflon-lined) tube (25 9 150 mm). Each extraction sample was washed twice by mixing vigorously with 10 ml of deionized water using vortex agitation and allowed to sit at ambient temperature until phase separation was complete. The benzene–hexane layer (top) containing FAMEs was then transferred to a screw-capped (Teflon-lined) tube (25 9 150 mm), reduced to a volume of approximately 3.0 ml under a gentle stream of nitrogen, and filtered through a pre-rinsed (chloroform–hexane, 1:4) paper (Whatman no. 1) filter containing Na2SO4 to remove residual moisture. The filter was rinsed with an additional 3.0 ml of chloroform-hexane (1:4) and the combined filtrates were evaporated to dryness under a gentle stream of nitrogen gas. The dehydrated FAMES were then dissolved in 0.5 ml of dichloromethane and stored under nitrogen gas at -20 °C until needed. GC–MS analysis was carried out using a Hewlett Packard GC/MS series II plus (GC 5890 MS 5972; Hewlett-Packard Co., Palo Alto, CA). Instrument parameters included an injection volume of 2.0 ll with a flow rate of 20 ml per min, GC injector temperature of 250 °C with a split ratio of 1:20, and an initial oven temperature of 150 °C for 4.0 min which was increased 6 °C per minute until a temperature of 230 °C was reached. Mass spectrophotometric analysis involved electron impact ionization and was programmed for full-scan mode. Authentic fatty acid methyl ester standards of fatty acids C11:0 through C20:0 (47080-U, Supelco, Inc., Bellefonte, PA) were employed to obtain comparative retention times. The lower detection limits of the Hewlett-Packard GC/MS series II were empirically determined to be 100 ng/ll for each authentic fatty acid methyl ester standard employed. REL Phospholipid Analysis REL phospholipid composition was determined using analytical thin-layer chromatography on 250 lm thick
Silica Gel G Uniplates (Analtech, Inc., Newark, DE) as described by Hart and Champlin [9]. Plates were washed in chloroform–methanol water (65:25:4) and heat activated at 100 °C for 30 min immediately prior to use. A 4.0–5.0 ll deposition of each REL fraction sample was applied, and chromatograms were first developed to a height of 10 or 11 cm in chloroform–methanol–acetic acid–water (85:15: 10:3.5), allowed to air dry, and then developed to a height of 14 or 15 cm in hexane-diethyl ether (4:1) and allowed to air dry. Resolved lipids were visualized by exposure to iodine vapor (Mallinckrodt, Inc., St. Louis, MO), which reacts with organic compounds in general, or specific spray reagents using the methods of Skidmore and Entenman [19]. A mixture of lyso-phosphatidylethanolamine (L-PE), PG, PE, DPG, oleic acid, and palmitic acid authentic standards (Sigma-Aldrich Co., St. Louis, MO) was employed for confirmation of identifications on the basis of co-migration. The iodine vapor method used to detect the presence of lipids on analytical thin-layer chromatographic plates was sensitive to less than 3.0 mg [19].
Results and Discussion B. multivorans strains from three disparate sources were selected for this study to provide representation of these important environments and to examine possible intraspecies disparity with regard to cell envelope lipid composition. Their similar generation times under identical laboratory cultural conditions underscore their species unity (Table 1). Major fatty acids esterified to the phospholipids of the B. multivorans REL fractions included C16:0 (palmitic acid), C16:1 (palmitoleic acid), and C18:1 (oleic acid), while C14:0 (myristic acid), DC17:0 (methylene hexadecanoic acid), C18:0 (stearic acid), and DC19:0 (methylene octadecanoic acid) were present in lesser amounts (Table 2). These results are consistent with the total cellular fatty acid content previously reported for B. multivorans [11] and B. cepacia [21]. However, fatty acid composition differed quantitatively among B. multivorans strains with regard to C16:0, C16:1, DC17:0, C18:1, and DC19:0, with the unsaturated:saturated fatty acid ratios being significantly less in the CF type strain than either the environmental or CGD clinical strains. The fatty acids esterified to the phospholipids of P. aeruginosa cell envelope lacked DC17:0 and possessed more C18:1 and less C16:1 than did those of the B. multivorans strains. REL phospholipids identified in all B. multivorans strains included L-PE, PG, PE, and DPG (Table 3) in similar ratios (Fig. 1). These findings are consistent with those previously reported for the closely related organism B. cepacia strain NCTC 10661 which lacks L-PE [21].
123
S. A. Ruskoski et al.: Cell Envelope Phospholipid Composition of B. multivorans Table 2 Fatty acid content of REL fractions from B. multivorans and P. aeruginosa strains % of total fatty acids ± SDa
Fatty acids
B. multivorans ATCC 17616
P. aeruginosa ATCC BAA-247
CGD2
PA01
Saturated 0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
43.1 ± 3.5 3.8 ± 1.7
53.1 ± 0.3 3.1 ± 0.6
b
41.1 ± 1.2 3.4 ± 0.9
41.1 ± 0.1 2.1 ± 1.0
C16:1
20.7 ± 1.3b
25.8 ± 1.0b
23.6 ± 2.2
16.0 ± 0.2
C18:1
27.6 ± 3.2
10.7 ± 0.2c
26.4 ± 3.0
40.4 ± 0.6
DC17:0
3.0 ± 0.4
5.6 ± 0.2c
3.4 ± 0.4
0.0 ± 0.0
DC19:0 Unsaturated:Saturated ratio
1.8 ± 0.2 1.0 ± 0.1
0.0 ± 0.0c 0.6 ± 0.0c
2.1 ± 0.2 1.1 ± 0.0
0.5 ± 0.4 1.3 ± 0.0
C14:0 C16:0 C18:0
1.6 ± 1.4
Unsaturated
Cyclopropane
a
Each value represents the mean obtained from REL fractions of three-to-four independent cultures ± standard deviation (SD). The total values do not necessarily equal 100 % due to rounding b
Values statistically different (P \ 0.01; calculated by an ANOVA one-way analysis of variance)
c
Values statistically different (P \ 0.0001; calculated by an ANOVA one-way analysis of variance)
Table 3 Phospholipid composition of REL fractions from B. multivorans and P. aeruginosa strainsa Phospholipids
Rbf B. multivorans ATCC 17616
P. aeruginosa PA01 ATCC BAA-247
Authentic lipid standards
CGD2
L-PE
0.2 ± 0.1
0.1 ± 0.0
0.2 ± 0.1
0.2 ± 0.0
0.2 ± 0.1
PG
0.3 ± 0.0
0.2 ± 0.0
0.3 ± 0.0
0.3 ± 0.0
0.3 ± 0.1
PE
0.4 ± 0.0
0.4 ± 0.0
0.4 ± 0.0
0.4 ± 0.0
0.5 ± 0.0
DPG
0.5 ± 0.1
0.7 ± 0.0
0.5 ± 0.1
0.6 ± 0.1
0.6 ± 0.1
FFAs
0.9 ± 0.0
0.9 ± 0.0
0.9 ± 0.0
0.9 ± 0.0
0.9 ± 0.0
a
Component identities were established on the basis of thin-layer chromatography co-migration with lipids known to be present in P. aeruginosa, and authentic lipid standards as well as reactivity with spray reagents [8, 19]. Resolved lipids were visualized using iodine vapor (Mallinckrodt, Inc., St. Louis, MO), molybdenum blue (Applied Science Division, Milton Roy Co., State College, PA), ninhydrin (SigmaAldrich Co.), ammoniacal silver nitrate (J.T. Baker Chemical Co., Phillipsburg, NJ), Dragendorff reagent (Sigma-Aldrich Co.), and Bial’s orcinol (Applied Science Division, Milton Roy Company) spray reagents
b Rf retention factor. Each mean was obtained from REL fractions extracted from three-to-four independent cultures ± SD as determined using conventional analytical thin-layer chromatography
Certain strains of B. multivorans contain putative genes coding for hopanoid biosynthesis [14]. None were detected under the conditions employed in this study, despite the fact they have been obtained from other Bcc species by extraction with chloroform–methanol (2:1) [5]. Phospholipid composition of B. multivorans was qualitatively indistinguishable from that of related organism P. aeruginosa PA01 [9] except that the latter possessed an unidentified phosphate-containing lipid (X). The numbers of cell envelope phospholipids and
123
phospholipid-esterified fatty acids reported here for B. multivorans and reference organism P. aeruginosa are in line with those previously reported for the latter bacterium [3, 9]. They are also consistent with the enteric bacteria Escherichia coli and Salmonella typhimurium which are known to possess cell envelope membranes composed of mixtures of C16:0, C1616:1, and C18:1 esterified to PE, PG, and DPG [10]. It can be concluded that L-PE, PG, PE, and DPG are the major phospholipids comprising the cytoplasmic and outer
S. A. Ruskoski et al.: Cell Envelope Phospholipid Composition of B. multivorans
A
B C D
S
FFAs
DPG
PE
PG X L-PE Origin Fig. 1 One-dimensional analytical thin-layer chromatogram of REL fractions from late exponential-phase batch cultures of B. multivorans and P. aeruginosa with mixed authentic lipid standards (S). Lanes ATCC 17616 (A), ATCC BAA-247 (B), CGD2 (C), and P. aeruginosa PA01 (D). The following components were identified on the basis of comigration with authentic lipid standards and specific spray reagents (Table 3): lyso-phosphatidylethanolamine (L-PE), unknown lipid (X), phosphatidylglycerol (PG), phophatidylethanolamine (PE), diphosphatidylglycerol (DPG), and mixed free fatty acids (FFAs)
membranes of the B. multivorans cell envelope. Their fatty acid substituents include C16:0, C16:1, and C18:1 with consistently detectable amounts of C18:0, DC17:0, and DC19:0. The latter two fatty acyl substituents likely represent the cyclopropylization products of C16:1 and C18:1 methylation as cultures transition into the stationary phase of growth as seen in P. aeruginosa [4]. While the qualitative major phospholipid and fatty acid profiles of all three cell envelopes appear extremely similar, the quantitative analysis of their fatty acyl substituents can be seen to differ somewhat. The major difference is seen most notably in the unsaturated to saturated ratios which are significantly less in the CF strain than in either the environmental or CGD isolates. These phospholipid ester data are reflective of the B. multivorans whole cell fatty acid profiles previously reported by Krejcˇi and Kroppenstedt [11], who observed a lesser unsaturated:saturated ratios in a CF strain (BAA-247 in this study) than in other B. multivorans strains. The conclusions reached here are supported by the efficiency of
the extraction method employed, as well as the lower detection limits of the analytical procedures used to find that the phospholipid and fatty acid content of the B. multivorans cell envelope is consistent with that of both closely related and disparate gram-negative organisms. Skidmore and Entenman [19] employed the Folch et al. [7] chloroform–methanol extraction method used in the present study to obtain phosphatidic acid, phosphatidylserine, PE, phosphatidylinositol, phosphatidylcholine, sphingomyelin, and lyso-phosphatidylcholine from rat liver tissue. Moreover, they visualized lipid moieties on thinlayer chromatograms using iodine vapor. O’Fallon et al. [17] also used the Folch et al. [7] extraction method, and the Morrison and Smith [16] boron fluoride-methanol method used here to convert fatty acids to FAMEs. They were able to detect as many as 20 and 23 different fatty acids from fish oil and bovine muscle tissue, respectively. These results validate the methods chosen for the present study when considered in the context of the much simpler lipid composition of the bacterial cell envelope [3, 4, 8–11]. In summation, these data support the conclusion that the major phospholipid composition of three B. multivorans strains of disparate origins is very similar and reflects that of the phylogenetically related organism P. aeruginosa. Moreover, despite possessing essentially identical fatty acyl substituents, some quantitative differences exist among the B. multivorans strains with regard to the amounts esterified to phospholipids. Acknowledgments We gratefully acknowledge Dr. A.M. Zelazny of the NIH National Institute of Allergy and Infectious Diseases for kindly providing B. multivorans environmental and clinical strains. Funding to F.R.C. was provided by the Oklahoma State University Center for Health Sciences.
References 1. Bertot GM, Restelli MA, Galanternik L, Urey RCA, Valvano MA, Grinstein S (2007) Nasal immunization with Burkholderia multivorans outer membrane proteins and the mucosal adjuvant adamantylamide dipeptide confers efficient protection against experimental lung infections with B. multivorans and B. cenocepacia. Infect Immun 75:2740–2752 2. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917 3. Bullard JW, Champlin FR, Burkus J, Millar SY, Conrad RS (2011) Triclosan-induced modification of unsaturated fatty acid metabolism and growth in Pseudomonas aeruginosa PA01. Curr Microbiol 62:697–702 4. Champlin FR, Gilleland JR, Conrad RS (1983) Conversion of phospholipids to free fatty acids in response to acquisition of polymyxin resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemoth 24:5–9 5. Cvejic JH, Putra SR, El-Beltagy A, Hattori R, Hattori T, Rohmer M (2000) Bacterial triterpenoids of the hopane series as biomarkers for the chemotaxonomy of Burkholderia, Pseudomonas, and Ralstonia spp. FEMS Microbiol Lett 183:295–299
123
S. A. Ruskoski et al.: Cell Envelope Phospholipid Composition of B. multivorans 6. Darnell KR, Hart ME, Champlin FR (1987) Variability of cell surface hydrophobicity among Pasteurella multocida somatic serotype and Actinobacillus lignieresii strains. J Clin Microbiol 25:67–71 7. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497–509 8. Fuller CA, Brignac PJ, Champlin FR (1993) Phospholipid fatty acid ester composition of Pasteurella multocida and Actinobacillus lignieresii. Curr Microbiol 27:237–240 9. Hart ME, Champlin FR (1988) Susceptibility to hydrophobic molecules and phospholipid composition in Pasteurella multocida and Actinobacillus lignieresii. Antimicrob Agents Chemother 32:1354–1359 10. Kadner RJ (1996) Cytoplasmic membrane. In: Neidhardt FC (ed) Escherichia coli and Salmonella cellular and molecular biology, vol 1, 2nd edn. ASM Press, Washington, pp 58–87 11. Krejcˇ´ı E, Kroppenstedt RM (2006) Differentiation of species combined into the Burkholderia cepacia complex and related taxa on the basis of their fatty acid patterns. J Clin Microbiol 44:1159–1164 12. Mahenthiralingam E, Urban TA, Goldberg JB (2005) The multifarious, multireplicon Burkholderia cepacia complex. Nat Rev Microbiol 3:144–156 13. Mahenthiralingam E, Baldwin A, Dowson CG (2008) Burkholderia cepacia complex bacteria: opportunistic pathogens with important natural biology. J Appl Microbiol 104:1539–1551 14. Malott RJ, Steen-Kinnaird BR, Lee TD, Speert DP (2012) Identification of hopanoid biosynthesis genes involved in polymyxin resistance in Burkholderia multivorans. Antimicrob Agents Chemother 56:464–471 15. McKeon SA, Nguyen DT, Viteri DF, Zlosnik JEA, Sokol PA (2010) Functional quorum sensing systems are maintained during chronic Burkholderia cepacia complex infections in patients with cystic fibrosis. J Infect Dis 203:383–392 16. Morrison WR, Smith LM (1964) Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoridemethanol. J Lipid Res 5:600–608
123
17. O’Fallon JV, Busboom JR, Nelson ML, Gaskins CT (2007) A direct method for fatty acid methyl esters synthesis: application to wet meat tissues, oils, and feedstuffs. J Anim Sci 85:1511–1521 18. Reik R, Spilker T, LiPuma JL (2005) Distribution of Burkholderia cepacia complex species among isolates recovered from persons with or without cystic fibrosis. J Clin Microbiol 43:2926–2928 19. Skidmore WD, Entenman C (1962) Two-dimensional thin-layer chromatography of rat liver phophatides. J Lipid Res 3:471–475 20. Stanier RY, Palleroni NJ, Doudoroff M (1966) The aerobic pseudomonads: a taxonomic study. J Gen Microbiol 43:159–271 21. Taylor CJ, Anderson AJ, Wilkinson SG (1998) Phenotypic variation of lipid composition in Burkholderia cepacia: a response to increased growth temperature is a greater content of 2-hydroxy acids in phosphatidylethanolamine and ornithine amide lipid. Microbiol 144:1737–1745 22. Vandamme P, Holmes B, Vancanneyt M, Coenye T, Hoste B, Coopman R, Revets H, Lauwers S, Gillis M, Kersters K, Govan JRW (1997) Occurrence of multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of Burkholderia multivorans sp. nov. Int J Syst Bacteriol 47:1188–1200 23. Vial L, Chapalain A, Groleau M-C, De´ziel E (2011) The various lifestyles of the Burkholderia cepacia complex species: a tribute to adaptation. Environ Microbiol 13:1–12 24. Zahariadis G, Levy MH, Burns JL (2003) Cepacia-like syndrome caused by Burkholderia multivorans. Can J Infect Dis 14:123–125 25. Zelazny AM, Ding L, Elloumi HZ, Brinster L, Czapiga M, Ulrich RL, Sampaio EP, Holland SM (2008) Burkholderia multivorans preferential interaction with chronic granulomatous disease leukocytes: cytokine induction and virulence in vivo, abstr D-027. Abstr 108th Gen Meet Am Soc Microbiol American Society for Micrbiology, Boston 26. Zelazny AM, Ding L, Elloumi HZ, Brinster LR, Benedetti F, Czapiga M, Ulrich RL, Ballentine SJ, Goldberg JB, Sampaio EP, Holland SM (2009) Virulence and cellular interactions of Burkholderia multivorans in chronic granulomatous disease. Infect Immun 77:4337–4344
Cell Envelope Phospholipid Composition of Burkholderia multivorans Sallie A. Ruskoski • James W. Bullard Franklin R. Champlin
•
Received: 10 May 2013 / Accepted: 14 March 2014 Ó Springer Science+Business Media New York 2014
Abstract Burkholderia multivorans causes opportunistic pulmonary infections in cystic fibrosis and immunocompromised patients. The purpose of the present study was to determine the nature of the phospholipids and their fatty acid constituents comprising the cell envelope membranes of strains isolated from three disparate sources. A conventional method for obtaining the readily extractable lipids fraction from bacteria was employed to obtain membrane lipids for thin-layer chromatographic and gas chromatography-mass spectrophotometric analyses. Major fatty acid components of the B. multivorans readily extractable lipid fractions included C16:0 (palmitic acid), C16:1 (palmitoleic acid), and C18:1 (oleic acid), while C14:0 (myristic acid), DC17:0 (methylene hexadecanoic acid), C18:0 (stearic acid), and DC19:0 (methylene octadecanoic acid) were present in lesser amounts. Fatty acid composition differed quantitatively among strains with regard to C16:0, C16:1, DC17:0, C18:1, and DC19:0 with the unsaturated:saturated fatty acid ratios being significantly less in a cystic fibrosis type strain than either environmental or
S. A. Ruskoski F. R. Champlin Departments of Biochemistry and Microbiology, Oklahoma State University Center for Health Sciences, 1111 West 17th Street, Tulsa, OK 74107, USA Present Address: S. A. Ruskoski (&) Health Professions/Medical Laboratory Sciences, Northeastern State University, 3100 E. New Orleans, Broken Arrow, OK 74014, USA e-mail: [email protected] J. W. Bullard Department of Forensic Sciences, Oklahoma State University Center for Health Sciences, 1111 West 17th Street, Tulsa, OK 74107, USA
chronic granulomatous disease strains. Phospholipids identified in all B. multivorans strains included lyso-phosphatidylethanolamine, phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol in similar ratios. These data support the conclusion that the cell envelope phospholipid profiles of disparate B. multivorans strains are similar, while their respective fatty acyl substituent profiles differ quantitatively under identical cultivation conditions.
Introduction Burkholderia multivorans is an obligately aerobic gramnegative bacillus classified as one of seventeen phylogenetically related species forming the Burkholderia cepacia complex (Bcc) [15]. Bcc species are environmentally important organisms in that certain species produce antimicrobial agents which protect economically important fruit and crop plants from various mycoses, while others are exploited as bioremediators by virtue of their disparate catabolic properties [12, 23]. B. multivorans is the causative agent of opportunistic infections in cystic fibrosis (CF), chronic granulomatous disease (CGD), and otherwise immunocompromised patients. The bacterium can colonize or infect pulmonary epithelial cells with either no effect on or suppression of pulmonary function, yet it is capable of more serious involvement leading to necrotizing pneumonia and patient fatality known as cepacia syndrome in some patients [1, 24]. B. multivorans accounts for 39 % of all Bcc organisms isolated from CF patients in the United States [18], and many strains have been shown to be genetically identical to environmental isolates [13]. B. multivorans promotes an inflammatory response by stimulating tumor necrosis factor alpha, interleukin-6, and interleukin-8 production by
123
S. A. Ruskoski et al.: Cell Envelope Phospholipid Composition of B. multivorans Table 1 Burkholderia multivorans strain sources Strain
Origin
Generation time (min)a
ATCC 17616
Soil [20]
45.2
ATCC BAA-247 (type strain)
CF patient [22]
47.4
CGD2
CGD patient [25]
46.7
obtained by inoculating 200 ml of LBB with stationaryphase starter cells to an initial optical density at 620 nm (OD620) of 0.025 (Spectronic 20 optical spectrophotometer, Thermo Electron Corp., Madison, WI) and incubating at 37 °C with rotary aeration at 180 rpm (Excella E24 benchtop incubator shaker; New Brunswick Scientific Co., Inc., Edison, NJ, USA) until late exponential phase.
a
Calculated from turbidimetric growth curves during exponential phase in LBB
human monocytes [26]. Moreover, the bacterium is intrinsically resistant to multiple antimicrobial agents [24]. A paucity of information exists in the literature regarding the cell envelope lipid composition of B. multivorans. Krejcˇi and Kroppenstedt [11] utilized the Sherlock microbial identification system (MIDI, Inc., Newark, DE) to determine the major cellular fatty acid components of 17 B. multivorans clinical isolates to be C16:0, C16:1, and C18:1 in a manner consistent with that for the type species B. cepacia [11, 21]. Other research has shown the major phospholipids of B. cepacia to include two forms of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and diphosphatidylglycerol (DPG) [21]. The purpose of the present study was to determine the phospholipid composition of the B. multivorans cell envelope and its substituent fatty acyl content by analyzing readily extractable lipid (REL) fractions of three disparate strains. Environmental, CF, and CGD test strains were selected to represent three important sources of this organism (Table 1). It is anticipated that these data will aid ongoing investigations of B. multivorans pathogenicity as we endeavor to better understand molecular mechanisms underlying its ability to adhere to host tissues and initiate biofilm assembly.
Materials and Methods Bacterial Strains and Cultural Growth Conditions Environmental (ATCC 17616) and clinical (CGD2) B. multivorans strains were obtained from Dr. Adrian Zelazny (NIH-NIAID, Bethesda, MD), while CF clinical type strain (ATCC BAA-247) is maintained as a reference organism in this laboratory. Pseudomonas aeruginosa PA01 (this laboratory) was employed for comparative purposes because of its known cell envelope lipid composition [9]. All cultures were maintained under cryoprotective conditions; working cultures were cultivated on Luria–Bertani Agar (LBA; Difco Laboratories, Detroit, MI, USA); and starter cultures were prepared by inoculating Luria–Bertani Broth (LBB; Difco Laboratories) with cells from working cultures as described previously [6]. Test cultures were
123
Readily Extractable Lipid Fraction Preparation Readily extractable lipid (REL) fractions were obtained from lyophilized cells using a simplified version of the method described by Folch et al. [7] to extract polar lipids from animal cells. This protocol differs from the Bligh and Dyer [2] method often used by bacteriologists in which water is absent during the initial extraction phase. We have found that water causes emulsions to form which interfere with the extraction process, thereby likely explaining the observation that up to 5 % of total polar lipids are not recovered with the second method. Late exponential-phase cells were harvested by centrifugation at 12,0009g and 4 °C for 12 min (Sorvall Legend XTR Refrigerated Superspeed Centrifuge, Thermo-Fisher Scientific, Inc., Pittsburgh, PA), then washed twice in one volume of cold deionized water. Final cell pellets were combined in a minimal volume of cold deionized water, frozen to -80 °C, and lyophilized to dryness. Approximately 30–40 mg of freeze-dried cells were suspended in 5.0 ml of methanol and mixed vigorously under nitrogen using a wrist-action shaker (Burrell Corporation, Pittsburgh, PA) for 10 min at ambient temperature. An additional 10 ml of chloroform was added to the extraction mixture which was agitated under nitrogen for an additional 12–15 h at ambient temperature. Insoluble material was removed by filtration under negative pressure through a chloroform– methanol (2:1) rinsed paper (Whatman no. 1) filter which was rinsed twice with 5.0 ml of chloroform–methanol (2:1). Remaining polar material was removed from the combined extract by adding 0.2 vol of deionized water, mixing using vortex agitation (Fisher Scientific Touch Mixer; Thermo-Fisher Scientific, Inc., Pittsburgh, PA), and facilitating phase separation with centrifugation at 2,5009g and 4 °C for 10 min (Sorvall Legend XTR Centrifuge) in a screw-capped (Teflon-lined) tube (25 9 150 mm). The total REL extract volume (chloroform layer) was reduced to a small volume under flowing nitrogen and stored at -20 °C under nitrogen until needed. REL Fatty Acid Analysis Phospholipid fatty acyl esters in REL fractions were converted to volatile fatty acid methyl esters (FAMEs) using the method of Morrison and Smith [16] as modified in this laboratory [8]
S. A. Ruskoski et al.: Cell Envelope Phospholipid Composition of B. multivorans
and analyzed utilizing gas chromatography-mass spectrometry (GC–MS) [3]. Morrison and Smith [16] reported their onestep boron fluoride-methanol methanolysis procedure rapidly proceeded to an essentially 100 % yield when authentic phospholipid standards were tested. REL fraction samples were reduced to dryness under flowing nitrogen gas, solubilized in 2.5 ml of benzene and 5.0 ml of boron trifluoridemethanol (14 %; Sigma-Aldrich Co., St. Louis, MO), and heated in a boiling water bath for 5.0 min in Parafilm-sealed, screw-capped (Teflon-lined) tubes (25 9 150 mm). After allowing to cool to ambient temperature, 5.0 ml of saturated aqueous NaCl and 5.0 ml of hexane were added, mixed vigorously with vortex agitation (Fisher Scientific Touch Mixer) for 1.0 min at ambient temperature, and allowed to sit until phase separation was complete. The benzene–hexane layer (top) containing FAMEs was transferred to a clean, screwcapped (Teflon-lined) tube (25 9 150 mm). Each extraction sample was washed twice by mixing vigorously with 10 ml of deionized water using vortex agitation and allowed to sit at ambient temperature until phase separation was complete. The benzene–hexane layer (top) containing FAMEs was then transferred to a screw-capped (Teflon-lined) tube (25 9 150 mm), reduced to a volume of approximately 3.0 ml under a gentle stream of nitrogen, and filtered through a pre-rinsed (chloroform–hexane, 1:4) paper (Whatman no. 1) filter containing Na2SO4 to remove residual moisture. The filter was rinsed with an additional 3.0 ml of chloroform-hexane (1:4) and the combined filtrates were evaporated to dryness under a gentle stream of nitrogen gas. The dehydrated FAMES were then dissolved in 0.5 ml of dichloromethane and stored under nitrogen gas at -20 °C until needed. GC–MS analysis was carried out using a Hewlett Packard GC/MS series II plus (GC 5890 MS 5972; Hewlett-Packard Co., Palo Alto, CA). Instrument parameters included an injection volume of 2.0 ll with a flow rate of 20 ml per min, GC injector temperature of 250 °C with a split ratio of 1:20, and an initial oven temperature of 150 °C for 4.0 min which was increased 6 °C per minute until a temperature of 230 °C was reached. Mass spectrophotometric analysis involved electron impact ionization and was programmed for full-scan mode. Authentic fatty acid methyl ester standards of fatty acids C11:0 through C20:0 (47080-U, Supelco, Inc., Bellefonte, PA) were employed to obtain comparative retention times. The lower detection limits of the Hewlett-Packard GC/MS series II were empirically determined to be 100 ng/ll for each authentic fatty acid methyl ester standard employed. REL Phospholipid Analysis REL phospholipid composition was determined using analytical thin-layer chromatography on 250 lm thick
Silica Gel G Uniplates (Analtech, Inc., Newark, DE) as described by Hart and Champlin [9]. Plates were washed in chloroform–methanol water (65:25:4) and heat activated at 100 °C for 30 min immediately prior to use. A 4.0–5.0 ll deposition of each REL fraction sample was applied, and chromatograms were first developed to a height of 10 or 11 cm in chloroform–methanol–acetic acid–water (85:15: 10:3.5), allowed to air dry, and then developed to a height of 14 or 15 cm in hexane-diethyl ether (4:1) and allowed to air dry. Resolved lipids were visualized by exposure to iodine vapor (Mallinckrodt, Inc., St. Louis, MO), which reacts with organic compounds in general, or specific spray reagents using the methods of Skidmore and Entenman [19]. A mixture of lyso-phosphatidylethanolamine (L-PE), PG, PE, DPG, oleic acid, and palmitic acid authentic standards (Sigma-Aldrich Co., St. Louis, MO) was employed for confirmation of identifications on the basis of co-migration. The iodine vapor method used to detect the presence of lipids on analytical thin-layer chromatographic plates was sensitive to less than 3.0 mg [19].
Results and Discussion B. multivorans strains from three disparate sources were selected for this study to provide representation of these important environments and to examine possible intraspecies disparity with regard to cell envelope lipid composition. Their similar generation times under identical laboratory cultural conditions underscore their species unity (Table 1). Major fatty acids esterified to the phospholipids of the B. multivorans REL fractions included C16:0 (palmitic acid), C16:1 (palmitoleic acid), and C18:1 (oleic acid), while C14:0 (myristic acid), DC17:0 (methylene hexadecanoic acid), C18:0 (stearic acid), and DC19:0 (methylene octadecanoic acid) were present in lesser amounts (Table 2). These results are consistent with the total cellular fatty acid content previously reported for B. multivorans [11] and B. cepacia [21]. However, fatty acid composition differed quantitatively among B. multivorans strains with regard to C16:0, C16:1, DC17:0, C18:1, and DC19:0, with the unsaturated:saturated fatty acid ratios being significantly less in the CF type strain than either the environmental or CGD clinical strains. The fatty acids esterified to the phospholipids of P. aeruginosa cell envelope lacked DC17:0 and possessed more C18:1 and less C16:1 than did those of the B. multivorans strains. REL phospholipids identified in all B. multivorans strains included L-PE, PG, PE, and DPG (Table 3) in similar ratios (Fig. 1). These findings are consistent with those previously reported for the closely related organism B. cepacia strain NCTC 10661 which lacks L-PE [21].
123
S. A. Ruskoski et al.: Cell Envelope Phospholipid Composition of B. multivorans Table 2 Fatty acid content of REL fractions from B. multivorans and P. aeruginosa strains % of total fatty acids ± SDa
Fatty acids
B. multivorans ATCC 17616
P. aeruginosa ATCC BAA-247
CGD2
PA01
Saturated 0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
43.1 ± 3.5 3.8 ± 1.7
53.1 ± 0.3 3.1 ± 0.6
b
41.1 ± 1.2 3.4 ± 0.9
41.1 ± 0.1 2.1 ± 1.0
C16:1
20.7 ± 1.3b
25.8 ± 1.0b
23.6 ± 2.2
16.0 ± 0.2
C18:1
27.6 ± 3.2
10.7 ± 0.2c
26.4 ± 3.0
40.4 ± 0.6
DC17:0
3.0 ± 0.4
5.6 ± 0.2c
3.4 ± 0.4
0.0 ± 0.0
DC19:0 Unsaturated:Saturated ratio
1.8 ± 0.2 1.0 ± 0.1
0.0 ± 0.0c 0.6 ± 0.0c
2.1 ± 0.2 1.1 ± 0.0
0.5 ± 0.4 1.3 ± 0.0
C14:0 C16:0 C18:0
1.6 ± 1.4
Unsaturated
Cyclopropane
a
Each value represents the mean obtained from REL fractions of three-to-four independent cultures ± standard deviation (SD). The total values do not necessarily equal 100 % due to rounding b
Values statistically different (P \ 0.01; calculated by an ANOVA one-way analysis of variance)
c
Values statistically different (P \ 0.0001; calculated by an ANOVA one-way analysis of variance)
Table 3 Phospholipid composition of REL fractions from B. multivorans and P. aeruginosa strainsa Phospholipids
Rbf B. multivorans ATCC 17616
P. aeruginosa PA01 ATCC BAA-247
Authentic lipid standards
CGD2
L-PE
0.2 ± 0.1
0.1 ± 0.0
0.2 ± 0.1
0.2 ± 0.0
0.2 ± 0.1
PG
0.3 ± 0.0
0.2 ± 0.0
0.3 ± 0.0
0.3 ± 0.0
0.3 ± 0.1
PE
0.4 ± 0.0
0.4 ± 0.0
0.4 ± 0.0
0.4 ± 0.0
0.5 ± 0.0
DPG
0.5 ± 0.1
0.7 ± 0.0
0.5 ± 0.1
0.6 ± 0.1
0.6 ± 0.1
FFAs
0.9 ± 0.0
0.9 ± 0.0
0.9 ± 0.0
0.9 ± 0.0
0.9 ± 0.0
a
Component identities were established on the basis of thin-layer chromatography co-migration with lipids known to be present in P. aeruginosa, and authentic lipid standards as well as reactivity with spray reagents [8, 19]. Resolved lipids were visualized using iodine vapor (Mallinckrodt, Inc., St. Louis, MO), molybdenum blue (Applied Science Division, Milton Roy Co., State College, PA), ninhydrin (SigmaAldrich Co.), ammoniacal silver nitrate (J.T. Baker Chemical Co., Phillipsburg, NJ), Dragendorff reagent (Sigma-Aldrich Co.), and Bial’s orcinol (Applied Science Division, Milton Roy Company) spray reagents
b Rf retention factor. Each mean was obtained from REL fractions extracted from three-to-four independent cultures ± SD as determined using conventional analytical thin-layer chromatography
Certain strains of B. multivorans contain putative genes coding for hopanoid biosynthesis [14]. None were detected under the conditions employed in this study, despite the fact they have been obtained from other Bcc species by extraction with chloroform–methanol (2:1) [5]. Phospholipid composition of B. multivorans was qualitatively indistinguishable from that of related organism P. aeruginosa PA01 [9] except that the latter possessed an unidentified phosphate-containing lipid (X). The numbers of cell envelope phospholipids and
123
phospholipid-esterified fatty acids reported here for B. multivorans and reference organism P. aeruginosa are in line with those previously reported for the latter bacterium [3, 9]. They are also consistent with the enteric bacteria Escherichia coli and Salmonella typhimurium which are known to possess cell envelope membranes composed of mixtures of C16:0, C1616:1, and C18:1 esterified to PE, PG, and DPG [10]. It can be concluded that L-PE, PG, PE, and DPG are the major phospholipids comprising the cytoplasmic and outer
S. A. Ruskoski et al.: Cell Envelope Phospholipid Composition of B. multivorans
A
B C D
S
FFAs
DPG
PE
PG X L-PE Origin Fig. 1 One-dimensional analytical thin-layer chromatogram of REL fractions from late exponential-phase batch cultures of B. multivorans and P. aeruginosa with mixed authentic lipid standards (S). Lanes ATCC 17616 (A), ATCC BAA-247 (B), CGD2 (C), and P. aeruginosa PA01 (D). The following components were identified on the basis of comigration with authentic lipid standards and specific spray reagents (Table 3): lyso-phosphatidylethanolamine (L-PE), unknown lipid (X), phosphatidylglycerol (PG), phophatidylethanolamine (PE), diphosphatidylglycerol (DPG), and mixed free fatty acids (FFAs)
membranes of the B. multivorans cell envelope. Their fatty acid substituents include C16:0, C16:1, and C18:1 with consistently detectable amounts of C18:0, DC17:0, and DC19:0. The latter two fatty acyl substituents likely represent the cyclopropylization products of C16:1 and C18:1 methylation as cultures transition into the stationary phase of growth as seen in P. aeruginosa [4]. While the qualitative major phospholipid and fatty acid profiles of all three cell envelopes appear extremely similar, the quantitative analysis of their fatty acyl substituents can be seen to differ somewhat. The major difference is seen most notably in the unsaturated to saturated ratios which are significantly less in the CF strain than in either the environmental or CGD isolates. These phospholipid ester data are reflective of the B. multivorans whole cell fatty acid profiles previously reported by Krejcˇi and Kroppenstedt [11], who observed a lesser unsaturated:saturated ratios in a CF strain (BAA-247 in this study) than in other B. multivorans strains. The conclusions reached here are supported by the efficiency of
the extraction method employed, as well as the lower detection limits of the analytical procedures used to find that the phospholipid and fatty acid content of the B. multivorans cell envelope is consistent with that of both closely related and disparate gram-negative organisms. Skidmore and Entenman [19] employed the Folch et al. [7] chloroform–methanol extraction method used in the present study to obtain phosphatidic acid, phosphatidylserine, PE, phosphatidylinositol, phosphatidylcholine, sphingomyelin, and lyso-phosphatidylcholine from rat liver tissue. Moreover, they visualized lipid moieties on thinlayer chromatograms using iodine vapor. O’Fallon et al. [17] also used the Folch et al. [7] extraction method, and the Morrison and Smith [16] boron fluoride-methanol method used here to convert fatty acids to FAMEs. They were able to detect as many as 20 and 23 different fatty acids from fish oil and bovine muscle tissue, respectively. These results validate the methods chosen for the present study when considered in the context of the much simpler lipid composition of the bacterial cell envelope [3, 4, 8–11]. In summation, these data support the conclusion that the major phospholipid composition of three B. multivorans strains of disparate origins is very similar and reflects that of the phylogenetically related organism P. aeruginosa. Moreover, despite possessing essentially identical fatty acyl substituents, some quantitative differences exist among the B. multivorans strains with regard to the amounts esterified to phospholipids. Acknowledgments We gratefully acknowledge Dr. A.M. Zelazny of the NIH National Institute of Allergy and Infectious Diseases for kindly providing B. multivorans environmental and clinical strains. Funding to F.R.C. was provided by the Oklahoma State University Center for Health Sciences.
References 1. Bertot GM, Restelli MA, Galanternik L, Urey RCA, Valvano MA, Grinstein S (2007) Nasal immunization with Burkholderia multivorans outer membrane proteins and the mucosal adjuvant adamantylamide dipeptide confers efficient protection against experimental lung infections with B. multivorans and B. cenocepacia. Infect Immun 75:2740–2752 2. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917 3. Bullard JW, Champlin FR, Burkus J, Millar SY, Conrad RS (2011) Triclosan-induced modification of unsaturated fatty acid metabolism and growth in Pseudomonas aeruginosa PA01. Curr Microbiol 62:697–702 4. Champlin FR, Gilleland JR, Conrad RS (1983) Conversion of phospholipids to free fatty acids in response to acquisition of polymyxin resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemoth 24:5–9 5. Cvejic JH, Putra SR, El-Beltagy A, Hattori R, Hattori T, Rohmer M (2000) Bacterial triterpenoids of the hopane series as biomarkers for the chemotaxonomy of Burkholderia, Pseudomonas, and Ralstonia spp. FEMS Microbiol Lett 183:295–299
123
S. A. Ruskoski et al.: Cell Envelope Phospholipid Composition of B. multivorans 6. Darnell KR, Hart ME, Champlin FR (1987) Variability of cell surface hydrophobicity among Pasteurella multocida somatic serotype and Actinobacillus lignieresii strains. J Clin Microbiol 25:67–71 7. Folch J, Lees M, Sloane Stanley GH (1957) A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226:497–509 8. Fuller CA, Brignac PJ, Champlin FR (1993) Phospholipid fatty acid ester composition of Pasteurella multocida and Actinobacillus lignieresii. Curr Microbiol 27:237–240 9. Hart ME, Champlin FR (1988) Susceptibility to hydrophobic molecules and phospholipid composition in Pasteurella multocida and Actinobacillus lignieresii. Antimicrob Agents Chemother 32:1354–1359 10. Kadner RJ (1996) Cytoplasmic membrane. In: Neidhardt FC (ed) Escherichia coli and Salmonella cellular and molecular biology, vol 1, 2nd edn. ASM Press, Washington, pp 58–87 11. Krejcˇ´ı E, Kroppenstedt RM (2006) Differentiation of species combined into the Burkholderia cepacia complex and related taxa on the basis of their fatty acid patterns. J Clin Microbiol 44:1159–1164 12. Mahenthiralingam E, Urban TA, Goldberg JB (2005) The multifarious, multireplicon Burkholderia cepacia complex. Nat Rev Microbiol 3:144–156 13. Mahenthiralingam E, Baldwin A, Dowson CG (2008) Burkholderia cepacia complex bacteria: opportunistic pathogens with important natural biology. J Appl Microbiol 104:1539–1551 14. Malott RJ, Steen-Kinnaird BR, Lee TD, Speert DP (2012) Identification of hopanoid biosynthesis genes involved in polymyxin resistance in Burkholderia multivorans. Antimicrob Agents Chemother 56:464–471 15. McKeon SA, Nguyen DT, Viteri DF, Zlosnik JEA, Sokol PA (2010) Functional quorum sensing systems are maintained during chronic Burkholderia cepacia complex infections in patients with cystic fibrosis. J Infect Dis 203:383–392 16. Morrison WR, Smith LM (1964) Preparation of fatty acid methyl esters and dimethylacetals from lipids with boron fluoridemethanol. J Lipid Res 5:600–608
123
17. O’Fallon JV, Busboom JR, Nelson ML, Gaskins CT (2007) A direct method for fatty acid methyl esters synthesis: application to wet meat tissues, oils, and feedstuffs. J Anim Sci 85:1511–1521 18. Reik R, Spilker T, LiPuma JL (2005) Distribution of Burkholderia cepacia complex species among isolates recovered from persons with or without cystic fibrosis. J Clin Microbiol 43:2926–2928 19. Skidmore WD, Entenman C (1962) Two-dimensional thin-layer chromatography of rat liver phophatides. J Lipid Res 3:471–475 20. Stanier RY, Palleroni NJ, Doudoroff M (1966) The aerobic pseudomonads: a taxonomic study. J Gen Microbiol 43:159–271 21. Taylor CJ, Anderson AJ, Wilkinson SG (1998) Phenotypic variation of lipid composition in Burkholderia cepacia: a response to increased growth temperature is a greater content of 2-hydroxy acids in phosphatidylethanolamine and ornithine amide lipid. Microbiol 144:1737–1745 22. Vandamme P, Holmes B, Vancanneyt M, Coenye T, Hoste B, Coopman R, Revets H, Lauwers S, Gillis M, Kersters K, Govan JRW (1997) Occurrence of multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of Burkholderia multivorans sp. nov. Int J Syst Bacteriol 47:1188–1200 23. Vial L, Chapalain A, Groleau M-C, De´ziel E (2011) The various lifestyles of the Burkholderia cepacia complex species: a tribute to adaptation. Environ Microbiol 13:1–12 24. Zahariadis G, Levy MH, Burns JL (2003) Cepacia-like syndrome caused by Burkholderia multivorans. Can J Infect Dis 14:123–125 25. Zelazny AM, Ding L, Elloumi HZ, Brinster L, Czapiga M, Ulrich RL, Sampaio EP, Holland SM (2008) Burkholderia multivorans preferential interaction with chronic granulomatous disease leukocytes: cytokine induction and virulence in vivo, abstr D-027. Abstr 108th Gen Meet Am Soc Microbiol American Society for Micrbiology, Boston 26. Zelazny AM, Ding L, Elloumi HZ, Brinster LR, Benedetti F, Czapiga M, Ulrich RL, Ballentine SJ, Goldberg JB, Sampaio EP, Holland SM (2009) Virulence and cellular interactions of Burkholderia multivorans in chronic granulomatous disease. Infect Immun 77:4337–4344
