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Visible, colorimetric dissemination between pathogenic strains of Staphylococcus aureus and Pseudomonas aeruginosa using fluorescent dye containing lipid vesicles
Biosensors and Bioelectronics 41 (2013) 538–543
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Visible, colorimetric dissemination between pathogenic strains of Staphylococcus aureus and Pseudomonas aeruginosa using fluorescent dye containing lipid vesicles N.T. Thet n, S.H. Hong, S. Marshall, M. Laabei, A. Toby, A. Jenkins Department of Chemistry, University of Bath, BA2 7AY, Bath, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 June 2012 Received in revised form 15 August 2012 Accepted 13 September 2012 Available online 23 September 2012
This paper describes a biosensing concept for exotoxins secreted by Staphylococcus aureus and Pseudomonas aeruginosa based on the toxin mediated breakdown and subsequent fluorescent dye release from phospholipid vesicles (liposomes). The sensitivity of vesicles to toxins was tuned by altering the lipid and fatty acid composition of the membranes such that vesicles could be engineered to respond to toxins/enzymes from S. aureus only; P. aeruginosa only; and both S. aureus and P. aeruginosa. Nineteen types of vesicle were made with varying compositions of phosphocholine (PC), phosphoethanolamine (PE), cholesterol and the photo-polymerizable ampiphile 10,12-tricosadiynoic acid (TCDA). The selectivity of the vesicles was measured via a simple fluorescence ‘‘switch on’’ assay. Sensitivity of the vesicles to 40 clinically derived strains of S. aureus and P. aeruginosa was also demonstrated. This work suggests that this technology could be utilised in a diagnostic tool to discriminate between the species of S. aureus and P. aeruginosa in wound dressings. & 2012 Elsevier B.V. All rights reserved.
Keywords: Pseudomonas aeruginosa Staphylococcus aureus Carboxyfluorescein Lipid vesicles Selective sensitivity Toxins
1. Introduction Bacterial infection in wounds, especially burn-related wounds in children, is a major clinical concern in hospitals (Church et al., 2006). Young children suffering from burns are particularly vulnerable to, and at risk of infection, largely due to an immature immune system and thin dermal layer. Early detection of infection in children with burns is difficult, as the common symptoms: pyrexia, raised C-reactive protein and white blood cell count are all found in children with systemic inflammatory response syndrome (SIRS) following a major burn. Technology exists for diagnosing infection, but this is generally based on culturing of wound swabs, with results coming from central hospital reference laboratories in 24–48 hours (in the UK). However, young children can become acutely sick in as little as 8 hours, due to complications resulting from infection such as toxic shock syndrome (Young and Thornton, 2007; Jones, 2006; Laabei et al., 2012). Nucleic acid based sensing of bacteria such as PCR (Kubista et al., 2006) has a role, but requires highly skilled operatives, expensive equipment and is prone to contamination. Again, due to the organisation of hospital microbiology services, results from such a test often take 24 hours or more. This requires a reliable and robust sensor that can monitor the microbial state of wounds to detect the infection, and if possible to provide the identity of pathogenic species involved in case of wound infection in situ.
n
Corresponding author. Tel.: þ44 1225 38 6893; fax: þ44 1225 38 6231. E-mail address: [email protected] (N.T. Thet).
0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.09.019
Two main species of pathogenic bacteria commonly found in burn wound infections are Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) (Branski et al., 2009). S. aureus is the gram-positive pathogen persistently colonized on human skin as a part of the normal flora. Most S. aureus strains are known to be pathogenic due to their secreted virulence factors including Pore Forming Toxins (PFTs) such as alpha, gamma and delta toxins (Song et al., 1996; Potrich et al., 2009; Verdon et al., 2009), Panton– Valentine Leukocidin (PVL) exoproteins (Tseng et al., 2009), epidermolytic toxins (Bailey and Redpath, 1992) and Toxic Shock Toxins (TST) (Dinges et al., 2000; Frame et al., 1985). P. aeruginosa is the gram-negative opportunistic pathogen responsible for fatal infections in patients with cystic fibrosis and immuno-suppression (Branski et al., 2009). It is also found in human skin flora and associated with virulence factors for the suspected lysis of healthy eukaryotic cells and tissue matrices upon infection. PFTs of S. aureus and lipid degrading enzymes such as esterase and phospholipases of P. aeruginosa are shown to be able to lyse the cell membrane of healthy mammalian cells in vivo and in vitro (Songer, 1997; Liu, 1974; Dinges et al., 2000; Arpigny and Jaeger, 1999). The different mode of action of toxins associated with S. aureus and P. aeruginosa in terms of their interaction with phospholipid membranes has been investigated previously using a Tethered Bilayer Lipid Membrane (TBLM) on gold surface using spectroscopic and optical analysis (Thet and Jenkins, 2010; Thet et al., 2011). The differential response of bacterial toxins with the membrane gave information on the mechanism of membrane degradation by primarily S. aureus toxins and primarily P. aeruginosa toxins.
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In the vesicle preparation, lipids, cholesterol and TCDA were individually mixed in chloroform to 100 mmol dm-3. They were then mixed together to a desired composition to a total volume of 100 ml and dried under nitrogen before putting inside the vacuum chamber at 10-3 bar for 1 hour. Thoroughly dried lipid mixture was then rehydrated with 5 ml of HEPES buffer with pH 7.3 containing 50 mmol dm 3 carboxyfluorescein. After rehydration, the lipid solution was heated in a hot water bath at 75 1C for 10 min before three freeze-thaw cycles. The lipid-dye solution was extruded three times through a polycarbonate membrane of 100 nm diameter pore size using a LF-50 Lipofast extruder (Avestin, USA). Finally, the extruded vesicles were purified using a DNA grade Sephadex NAP-25 column (GE Healthcare, UK). Lipid vesicle compositions containing TCDA were placed in a quartz vial and exposed to UV-light (254 nm), for a total of 12 s in a commercial flood exposure UV source (Hamamatsu, Japan), after storage at 4 1C for at least 2 hours. (Flow chart describing the process of vesicle preparation is presented in Fig. S9 in Supplementary information section).
Recently the development of a prototype wound dressing using lipid membranes to detect bacterial toxins and thus detect wound infection by pathogenic bacteria was demonstrated by chemical attachment of the lipid vesicles containing fluorescence dyes onto nonwoven fabrics (Zhou et al., 2010, 2011; Jenkins et al., 2011; Jenkins and Young, 2010). In this paper, we report that by changing the lipid and fatty acid composition of phospholipid vesicles it is possible to ’tune’ their response such that they are primarily susceptible to secretion toxins such as phospholipase from P. aeruginosa, or delta toxin secreted by S. aureus or to both toxin types. Hence, it is possible to obtain some initial information on the toxins produced by the bacteria – information that could be used by clinicians to help inform diagnosis and choice of antibiotic/antimicrobial in the treatment pathway.
2. Materials and methods 2.1. Materials and vesicle synthesis
2.2. Type of vesicles
Lipids used in making vesicles were 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) from Avanti Polar Lipids, USA. Cholesterol, 10,12-tricosadiynoic acid (TCDA) and 5(6)-carboxyfluorescein were purchased from Sigma–Aldrich, UK. TCDA is a synthetic polymerizable lipid which has been used to stabilise the vesicles by lateral cross-linking within the lipid bilayer after exposure to UV light. All the lipids, cholesterol and TCDA were used without further purifications. Triton X-100 (Sigma–Aldrich, UK) is a non-ionic surfactant which solubilises, disintegrates and reduces the lipid vesicles into micelles with simultaneous release of encapsulated carboxyfluorescein. A ten-fold dilution of 0.1% solution of triton X-100 was used as a positive control during the plate reading assay experiments. HEPES buffer was prepared according to standard protocol and used as a negative control for the stability of vesicles in the absence of bacteria and Triton X-100.
In order to empirically elucidate the role of the various vesicle components on their sensitivity to different bacteria, five classes of vesicles were studied (A–E, Table 1), each with one component being varied. Type A vesicles were primarily composed of shorter chain DPPC lipids, with varying concentration of cholesterol (0, 10, 20 and 30 mol%); Type B were as Type A but with the addition of a photopolymerizable component TCDA; Type C were composed of longer chain DSPC lipids with varying cholesterol concentration; Type D were also DSPC lipid based, with a fixed mol% of cholesterol and varying TCDA concentration; and finally Type E were composed of DSPC lipids, with a fixed mol% of TCDA and varying cholesterol concentration. Mean increment in fluorescence of respective vesicles before and after inoculation in Hepes buffer, P. aeruginosa PAO1 and S. aureus MSSA476 at 37 1C was also tabulated in Table 1 (see Section 2.4 for experimental detail). The rationale of this work is that cholesterol, chain length and presence of TCDA all profoundly
Table 1 Vesicles with mol% of their phospholipid/fatty acid content, and their fluorescent response in Hepes buffer and pathogenic bacteria (Arbitrary classification A–E depending on principal lipid component and variable i.e. cholesterol or TCDA; vesicle 17 belongs to both type D and E, and vesicle 11 belongs to both type C and D). Vesicles (Types and lipid compositions in mol%)
Mean fluorescent increment after 18 h (a.u.) in medium
Class
Vesicle types
DPPC
DSPC
Cholesterol
TCDA
Hepes (buffer)
PAO1
MSSA476
A
Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves
88 78 68 73 63 53 43 33 23 – – – – – – – – – –
– – – – – – – – 88 78 68 73 68 63 58 53 63 43
10 20 30 0 10 20 30 40 50 10 20 30 20 20 20 20 20 10 30
– – – 25 25 25 25 25 25 – – – 5 10 15 20 25 25 25
35,995 2,035 1,126 23,593 34,885 13,522 1,019 834 85 863 758 563 29 531 767 2,686 1,989 3,186 2,154
1,18,388 1,03,842 41,546 1,38,137 1,03,529 99,245 35,204 14,087 11,430 19,356 52,905 7,816 60,060 51,096 42,839 55,310 60,864 36,058 25,689
76,558 39,610 10,905 1,19,276 84,549 83,047 76,162 81,173 83,671 5,620 9,818 3,321 34,819 41,539 62,152 87,173 91,885 33,910 44,310
B
C
D
E
1 2 3 4b 5b 6ab 7b 8b 9ab 10 11a 12 13b 14b 15b 16b 17b 18b 19b
All DPPC and DSPC vesicles contain 2 mol% of DPPE and DSPE, respectively. Black bold numbers in table describe the distinctive composition of lipid vesicles which give the best selective fluorescence responses between S. aureus and P. aeruginosa. a b
Vesicles used for selective sensitivity tests. Vesicles required UV cross-linking.
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affect membrane properties, such as fluidity and phase transition temperature, which in turn have significant effects on membrane interaction with bacteria and toxins (Potrich et al., 2009). 2.3. Pathogenic bacteria and their culture 21 clinical strains of pathogenic S. aureus were provided from John Radcliffe Hospital, Oxford, UK; 19 out of 21 strains were Methicillin sensitive S. aureus (MSSA) and 2 were Methicillin resistant S. aureus (MRSA). All MSSA and MRSA candidates studied in this work were community acquired virulent strains with suspected virulence factors, which were susceptible for cell lysis (Peacock et al., 2002). 21 clinical strains of P. aeruginosa provided from AmpliPhi BioSciences Corporation, UK and Southmead Hospital, Bristol, UK. Eight strains of P. aeruginosa acquired from Southmead Hospital were extracted from either acute wounds or blood agar of infected patients, the remaining 13 strains were clinical extracts from chronic wounds with known pathogenic effects on human hosts. All the strains studied in this work are listed in Table 2. For the non-pathogenic control, we used Escherichia coli DH5a; a laboratory strain with most of its virulence factors removed (Hanahan, 1985). Luria Broth (LB) and Tryptic Soy Broth (TSB) were prepared in MilliQ water according to the standard protocol and autoclaved after preparation for bacterial culture. E. coli DH5a and P. aeruginosa strains were cultured in LB, and S. aureus strains were cultured in TSB. Cultures were grown overnight in a shaking incubator at 37 1C. Optical Density (OD) was measured at 600 nm. OD of diluted bacteria was related to colony forming units per millilitre (CFU ml 1) by conventional plating and colony counting. Initial concentration of bacteria inoculated with vesicles in experiments was 104 CFU ml 1. 2.4. Fluorescent plate reading assay Fluorescent assays were carried out on a FLUOstar Omega microplate reader (BMG Labtech) using a 96 well plate. Excitation and emission of 485712 and 520 nm were used, respectively, with a gain of 650. Into each well, 50 ml of each vesicle was inoculated with 100 ml of bacteria-broth solution. Each vesicle-bacteria combination was repeated six times. Additionally, positive and negative controls were carried out with 100 ml Triton X-100 and HEPES buffer, respectively. Fluorescent responses of lipid vesicles inoculated with bacteria, HEPES buffer and Triton X-100 were continuously measured at 37 1C for 18 h. For each type of vesicle-bacteria combination, mean and standard deviation of fluorescence from six parallel wells were used to calculate the response in term of fluorescent increase at the end of each measurement for the data presentation.
3. Results and discussion 3.1. Effect of cholesterol and TCDA on stability and sensitivity of vesicles One of the principle differences in membrane components between eukaryotic and prokaryotic cells is the presence or absence of cholesterol. Cholesterol is the essential eukaryotic cell
membrane component, comprising as much as 25% of the membrane in human erythrocytes and as little as a mere trace amount in disk membrane (bovine) (Gennis, 1989). Apart from its physiological role, cholesterol, maintains the integrity and lateral fluidity of cell membranes for normal cell functioning (Ohvo-Rekil¨a et al., 2002; Koyama et al., 1999). Artificial lipid bilayer membranes containing cholesterol exhibit a similar change in membrane fluidity, line tension and formation of rafts; especially if the membrane contains sphingolipids (Veatch and Keller, 2005). Importantly, cholesterol plays an important role in the interaction of cell membranes with bacterial toxins either directly or indirectly (Palmer, 2004), and its presence or absence in cell membranes influences the susceptibility of the cells to bacterial toxins. This hypothesis was verified by designing lipid vesicles with cholesterol of varying mol%; and the effect of cholesterol on sensitivity of vesicles to pathogenic bacteria was examined. Additionally, the photo-polymerizable agent TCDA was incorporated in lipid bilayer to create laterally cross-linked polymer networks within the membrane to enhance the thermodynamic stability of the lipid membrane at elevated temperatures (Zhou et al., 2011). Carbon atoms in the hydrophobic tail of TCDA has reduced from 21 to 17 after lateral cross-linking by UV-light; forming a more favourable chain length for interactions with DPPC and DSPC. Individual and combined effects of cholesterol and TCDA on lipid bilayer stability and sensitivity were also explored. 3.1.1. DPPC vesicles (type A and B) Type A vesicles indicated that increasing the cholesterol concentration decreased the passive leakage in Hepes buffer, however, there was a decreased response with both P. aeruginosa and S. aureus (Table 1) (Fig. S1 in Supplementary information). The maximum measured fluorescence response of Type A vesicles to bacteria, taking into account minimal passive leakage (subtracting Hepes only control numerical values from the bacteria samples and accounting for standard deviations), was for 20 mol % cholesterol with more than twice ( 2.7fold) the response for P. aeruginosa than S. aureus. Type B vesicles, incorporating the photo-polymerizable cross-linker TCDA at a fixed 25 mol%, and varied cholesterol concentration showed increasing stability at high cholesterol concentrations. Sensitivity to P. aeruginosa decreased with increasing concentration of cholesterol but importantly, stable and almost exclusive response was only seen in S. aureus at cholesterol concentrations of 30 mol% and above (Fig. S2 in Supplementary information). 3.1.2. DSPC vesicles (types C and E) Type C vesicles, utilising the longer lipid chain length DSPC showed decreased passive leakage compared to DPPC vesicles. Cholesterol concentration had a large effect on vesicle response to bacterial toxins, with 20 mol% cholesterol having greater than 4 fold ( 4.6 fold) response to P. aeruginosa compared to S. aureus, (Table 1) (Fig. S3 in Supplementary information). Type E vesicles (DSPC with 25 mol% TCDA) showed a slightly greater response to S. aureus over P. aeruginosa, which became more distinguishable at higher cholesterol concentrations (Fig. S4 in Supplementary information).
Table 2 Bacterial strains used for selective sensitivity. Species
Strain numbers
Remarks
S. aureus P. aeruginosa
2, 3, 16, 21a, 25, 38, 49, 52, 56, 67, 69a, 101, 112, 114, 126, 160, 233, 253, 279, 295, 476 259, 260, 739, 854, 855, 856, 887, 889, 927, 935, 936, 937, 45291b, 45311b, 45400b, 45445b, 45468b, 45506b, 45666b, 45701b, PAO1
a
MRSA strainsRemaining are MSSA strains Strains extracted from acute infected wounds. Remaining are clinical extracts from infected chronic wounds
b
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3.1.3. DSPC Vesicles (type D) Type D vesicles, composed of DSPC with 20 mol% cholesterol and varying TCDA compositions (0–25 mol%) showed minimal passive leakage in Hepes buffer. A significant increase in response to S. aureus with increasing concentration of TCDA was observed, while the response to P. aeruginosa remained almost the same regardless of TCDA mol% (Table 1) (Fig. S5 in Supplementary information). These results indicated that vesicles composed primarily of DPPC lipids are more sensitive to bacteria than those composed primarily of DSPC lipids. The optimum cholesterol concentration in both cases was found to be 20 mol%. In the absence of TCDA, all types of vesicles showed a higher response to P. aeruginosa than S. aureus. However, this was not the case if TCDA was included; a relatively higher response to S. aureus with increasing TCDA mol% was measured. The reduced sensitivity of DSPC vesicles could be partly understood in terms of differences in the gel–liquid phase transition temperature (Tm): at the relatively higher Tm of DSPC (55 1C) the lipid bilayer would still be in a gel phase with minimal fluidity at 37 1C. This could not only reduce the passive leakage but also hinder the successful binding and subsequent activation of bacterial toxins onto
in HEPES E. coli DH5α P. aeruginosa PAO1 S. aureus MSSA476
Fluorescence increment / a.u.
70000 60000 50000 40000 30000 20000 10000 0 Vesicle 6
Vesicle 11
Vesicle 9
Fig. 1. Selective fluorescent response of three types of vesicles to pathogenic and non-pathogenic bacteria (vesicle 6 is sensitive to both pathogenic strains; P. aeruginosa PAO1 and S. aureus MSSA476 but not to non-pathogenic E. coli DH5a while vesicles 11 and 9 show selective sensitivity to either P. aeruginosa PAO1 or S. aureus MSSA 476, respectively).
E. coli DH5α
(non-pathogenic) negative control
P. aerunginosa
PAO1 (pathogenic)
541
vesicles. The increased response of S. aureus in the presence of TCDA has not been fully explained as there was no evidence of interaction between polymerizable agents and bacterial toxins (Jelinek and Kolusheva, 2007). It is possible that the formation of TCDA polymer networks could create local domains rich in lipids and cholesterol, which would provide pre-defined targets for local concentration of bacterial toxins (Verdon et al., 2009). The experimental results (Figs. 1 and 2) imply that the sensitivity of vesicles to either P. aeruginosa or S. aureus or both could be achieved by adjusting the percentage composition of DPPC, DSPC, cholesterol and TCDA. Three types of vesicles (Table 1) were chosen for the selective dissemination response test; vesicle 6 for detection of both bacteria, vesicle 11 and 9 for selective detection of P. aeruginosa and S. aureus, respectively.
3.2. Selectivity to pathogenic and non-pathogenic bacteria Determination of vesicle response to the pathogenic bacteria; P. aeruginosa PAO1 and S. aureus MSSA476, compared to a nonpathogenic control, E. coli DH5a was investigated. Inoculation with bacteria, to the vesicles, occurred during its stationary growth phase at 104 CFU ml 1. It took approximately 6 h for the bacteria to enter exponential growth phase, release toxins, and lyse vesicles giving a measured fluorescence response. (Fig. S6 in Supplementary information). Vesicle 6 produced 510 fold greater fluorescent response to both P. aeruginosa and S. aureus relative to the negative controls (Hepes buffer and E. coli DH5a) (Fig. 1). Further selection is possible between the two pathogenic strains (Fig. 1); vesicle 11 only responded to P. aeruginosa PA01, while vesicle 9 only responded to S. aureus MSSA476. This selectivity could be explained by lipid formulation of vesicles, lipid availability and membrane fluidity as well as the toxins involved with pathogenic bacteria. It was experimentally observed that the activities of PFTs of S. aureus increased nonlinearly with an increase in membrane fluidity, especially in membranes with lipids of shorter acyl chains (Potrich et al., 2009). With DPPC, cholesterol and TCDA, vesicle 6 maintained a higher membrane fluidity especially at 37 1C, ensuring promotion of nonspecific binding of toxins with lipids, thus enabling sensitivity to both pathogens. The vesicle composed of DSPC and cholesterol only (vesicle 11), had decreased membrane fluidity at 37 1C, forming gel-like phases, blocking accessibility of S. aureus toxins to the relatively S. aureus MSSA476
(pathogenic)
Triton X-100
Positive control
Vesicle type 6
Vesicle type 11
Vesicle type 9
Fig. 2. Visible fluorescent response of vesicles 6, 11 and 9 after inoculation with bacteria in 96 well plate (Vesicle 6 in two top rows shows fluorescence ‘‘switch on’’ with pathogenic strains; vesicle 11 in two middle rows shows response to P. aeruginosa PAO1 only, while vesicle 9 in two bottom rows indicates response to S. aureus MSSA476 only. Note: all vesicle types do not respond to non-pathogenic E. coli DH5a; inset – photograph of the plate after initial inoculation with bacteria).
N.T. Thet et al. / Biosensors and Bioelectronics 41 (2013) 538–543
Ves 6 (sensitive to both P. aeruginosa and S. aureus) Ves 11 (less sensitive to S. aureus) Ves 9 (more sensitive to S. aureus)
80000 60000 40000 20000 0
Strains of S. aureus Fig. 4. Selectivity of vesicle 9 over vesicle 11 (in terms of increase in fluorescence) in screening with 20 clinical virulent strains of S. aureus.
60000
data indicated that the strains 856 and 887 did not grow during the experiment, and consequently the positive responses were not shown, in contrast to the rest of tested strains. The reason for suppressed growth of strains 856 and 887 is not clear, despite the fact that all the strains of P. aeruginosa grew successfully in an incubator. It is possible that the growth condition of these particular strains in 96 well plate may not be optimum to support growth and hence toxin expression, and subsequently no lysis of vesicles was observed. Selectivity of vesicle 11 over vesicle 9 for P. aeruginosa strains over S. aureus, in terms of the relative increase in fluorescence intensity was observed between 2 and 6 fold (Fig. 3), is clearly distinguishable by eye when observed under UV light. Vesicle 9 showed a greater response to all 20 strains of S. aureus than vesicle 11 (Fig. 4). Two to ten fold increases in fluorescence of vesicle 9 against vesicle 11 was clearly observed, and all tested strains of S. aureus grew well in the experiment according to absorbance data. In terms of fluorescence signal strength, responses of vesicle 9 to S. aureus were slightly less than those of vesicle 11 to P. aeruginosa. This relatively low fluorescence ‘‘switch on’’ could be due to the involvement of particular toxins secreted by the strains of S. aureus which might not necessarily lyse the vesicles completely. If this is the reason, it implies that the toxins produced by P. aeruginosa have relatively stronger lipolytic action than those produced by S. aureus. Nevertheless, visible and selective responses of vesicle 9 against vesicle 11 to all strains of S. aureus were clearly observed. It was evidenced according to the experiments that 90% of all P. aeruginosa strains and 100% of all S. aureus strains tested were selectively distinguishable from each other.
40000
4. Conclusion
3.3. Selective sensitivity between 40 strains of P. aeruginosa and S. aureus In order to evaluate the sensitivity and the selectivity over a range of bacterial strains, the three selected vesicles (vesicles 6, 9 and 11), were tested against 40 additional clinical strains (20 each) of S. aureus and P. aeruginosa. The fluorescence and absorbance of the three replicas of each vesicle-bacterium combination were measured at the beginning and end of each experiment, and the results were plotted in Figs. 3 and 4 for P. aeruginosa and S. aureus, respectively. Vesicle 11 showed positive responses to all the P. aeruginosa strains, with the exception of strains 856 and 887 (Fig. 3). Bacterial optical density (OD600)
100000
Ves 6 (sensitive to both P. aeruginosa and S. aureus) Ves 11 (more sensitive to P. aeruginosa) Ves 9 (less sensitive to P. aeruginosa)
80000
20000 0
259 260 739 854 855 856 887 889 927 936 937 935 45291 45311 45400 45445 45468 45506 45666 45701
Fluorescence increment / a.u.
100000
2 3 16 21 25 38 49 52 56 67 69 101 112 114 126 160 233 253 279 295
stiff membrane. However P. aeruginosa toxins appear unaffected by the low membrane fluidity (Fig. 1), probably due to the associated toxins and their modes of action. P. aeruginosa secrete enzymes that degrade lipids, such as phospholipase C, which targets and hydrolyses the phosphate head group of phospholipids (Songer, 1997; Titball, 1993). Such toxins require only lipid head group availability without depending on membrane fluidity for their activation, which may explain why vesicle 11 responded only to P. aeruginosa secretion toxins and not S. aureus toxins. (Fig. S7 in Supplementary information) Vesicle 9 was composed of 50 mol% cholesterol and 25 mol% TCDA; the remaining 25 mol% was composed of DPPC and DPPE (Table 1). TCDA and cholesterol-rich zones could form raft-like domains, coexisting alongside with the lipid-rich domains, within the membrane (Garcia-Saez and Schwille, 2010; Mateo et al., 1995). The liquid-ordered state of cholesterol-rich domains could potentially enable targeting of S. aureus toxins and their activities in the remaining DPPC lipid-rich liquid-disordered domains at 37 1C. P. aeruginosa toxins primarily require lipids to activate its lipid-degrading enzymes. As a result of the small lipid composition (25 mol%) in vesicle 9, minimal response by P. aeruginosa toxins on lipid membrane is observed. The high presence of cholesterol and TCDA resulted in increased selectivity of vesicle 9 to S. aureus (Fig. S8 in Supplementary information). The fluorescence response of the vesicles to the bacteria and Hepes buffer (Fig. 1) and the visible colorimetric selection among E. coli DH5a, P. aeruginosa PAO1 and S. aureus MSSA476 using three types of vesicles (Fig. 2) indicates a potential discriminatory tool for identifying between two important pathogenic bacteria species.
Fluorescence increment / a.u.
542
Strains of P. aeruginosa Fig. 3. Selectivity of vesicle 11 over vesicle 9 (in terms of increase in fluorescence) in screening with 20 clinical virulent strains of P. aeruginosa.
We have demonstrated the selective detection between non-pathogenic bacteria E. coli DH5a and pathogenic strains of S. aureus MSSA476 and P. aeruginosa PAO1 using lipid vesicles containing carboxyfluorescein dye. There was a clear, visual selection between pathogenic and non-pathogenic strains of bacteria in the form of a fluorescence colour change; switching from relatively pale to bright green colour, after inoculation of lipid vesicles with bacteria and incubation at 37 1C. More importantly, by engineering the desired compositions of lipids, cholesterol and polymerizable agent, the vesicles showed further
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selective discrimination between two pathogenic strains of P. aeruginosa and S. aureus. Additionally, selectivity over a wide range of two common pathogenic species was demonstrated using 40 clinical strains of P. aeruginosa and S. aureus. 90% of P. aeruginosa strains and all of the S. aureus strains were selectively distinguished with visible fluorescence colour change in the order of two to ten fold relative to each other. Vesicle 6 showed virtually no discrimination for S. aureus over P. aeruginosa (or vice versa) and responded to the majority of bacterial strains of both species with a fluorescent intensity change similar to that of vesicles 9 and 11. By integration of responsive lipid vesicles onto a wound dressing, we believed that the current selective and discriminative sensing system has potential as an indicator for bacterial infections, with additional information regarding the likely toxic mode of action of the infection in wounds, without requiring the early removal of the wound dressing during the wound healing process.
Acknowledgement We would like to thank Professor Mark C. Enright (AmpliPhi BioSciences Corporation, Bedfordshire, UK), Southmead Hospital (Bristol, UK) and John Radcliffe Hospital (Oxford, UK) for the bacteria strains. We also acknowledge the European Commission’s 7th Frame-work programme for funding via the EC-FP7 consortium project no. 245500 Bacteriosafe.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2012.09.019.
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Church, D., Elsayed, S., Reid, O., Winston, B., Lindsay, R., 2006. Clinical Microbiology Reviews 19, 403–434. Dinges, M.M., Orwin, P.M., Schlievert, P.M., 2000. Clinical Microbiology Reviews 13, 16–34. Frame, J.D., Eve, M.D., Hackett, M.E.J., Dowsett, E.G., Brain, A.N., Gault, D.T., Wilmshurst, A.D., 1985. Burns 11, 234–241. Garcia-Saez, A.J., Schwille, P., 2010. FEBS Letters 584, 1653–1658. Gennis, R.B., 1989. Biomembranes: Molecular Structure and Function. SpringerVerlag, New York. Hanahan, D., 1985. DNA Cloning (A practical approach), vol. 1. IRL Press, Oxford, UK. Jelinek, R., Kolusheva, S., 2007. Topics in Current Chemistry 277, 155–180. Jenkins, A.T.A., Thet, N.T., Zhou, J., Hong, S.H., Marshall, S., 2011. Burns 37S, S5. Jenkins, A.T.A., Young, A., 2010. Expert Review of Anti-infective Therapy 8, 1063–1065. Jones, V.E., 2006. Wounds UK 2, 66–73. Koyama, T.M., Stevens, C.R., Borda, E.J., Grobe, K.J., Cleary, D.A., 1999. The Chemical Educator 4, 12–15. Kubista, M., Andrade, J.M., Bengtsson, M., Forootan, A., Jona´k, J., Lind, K., Sindelka, ¨ ¨ ¨ ˚ R., Sjoback, R., Sjogreen, B., Strombom, R., Stahlberg, A., Zoric, N., 2006. Molecular Aspects of Medicine 27, 95–125. Laabei, M., Young, A., Jenkins, A.T.A., 2012. The Pediatric Infectious Disease Journal 31, e73–e77. Liu, P.V., 1974. Journal of Infectious Diseases 130, S94–S99. ˇ C.R., Brochon, J-.C., A.U., 1995. Biophysical Journal 68, 978–987. Mateo, C.R., Acuna, ¨ H., Ramstedt, B., Leppimaki, ¨ Ohvo-Rekila, P., Slotte, J.P., 2002. Progress in Lipid Research 41, 66–97. Palmer, M., 2004. FEMS Microbiology Letters 238, 281–289. Peacock, S.J., Moore, C.E., Justic, A., Kantzanou, M., Story, L., Mackie, K., O’Neill, G., Day, N.P.J., 2002. Infection and Immunity 70, 4987–4996. Potrich, C., Bastiani, H., Colin, D.A., Huck, S., Pre´vost, G., Serra, M.D., 2009. Journal of Membrane Biology 227, 13–24. Song, L., Hobaugh, M.R., Shustak, C., Cheley, S., Bayley, H., Gouaux, J.E., 1996. Science 274, 1859–1866. Songer, J.G., 1997. Trends in Microbiology 5, 156–161. Titball, R.W., 1993. Microbiological Reviews 57, 347–366. Thet, N.T., Cameron, P.J., Jenkins, A.T.A., 2011. Biosensors and Bioelectronics 28, 227–231. Thet, N.T., Jenkins, A.T.A., 2010. Electrochemistry Communications 12, 1411–1415. Tseng, C.W., Kyme, P., Low, J., Rocha, M.A., Alsabeh, R., Miller, L.G., Otto, M., Arditi, M., Diep, B.A., Nizet, V., Doherty, T.M., Beenhouwer, D.O., Liu, G.Y., 2009. Public Library of Science ONE 4 (e6387), 1–10. Veatch, S.L., Keller, S.L., 2005. Biochimica et Biophysica Acta 1746, 172–185. Verdon, J., Girardin, N., Lacombe, C., Berjeaud, J-.M., Hechard, Y., 2009. Peptides 30, 817–823. Young, A.E., Thornton, K.L., 2007. Archives of Disease in Childhood. Education and Practice Edition 92, ep97–ep100. Zhou, J., Loftus, A.L., Mulley, G., Jenkins, A.T.A., 2010. Journal of American Chemical Society 132, 6566–6570. Zhou, J., Thet, N.T., Hong, S.H., Mercer-Chalmers, J.D., Laabei, M., Young, A.E.R., Jenkins, A.T.A., 2011. Biosensors and Bioelectronics 30, 67–72.
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Visible, colorimetric dissemination between pathogenic strains of Staphylococcus aureus and Pseudomonas aeruginosa using fluorescent dye containing lipid vesicles N.T. Thet n, S.H. Hong, S. Marshall, M. Laabei, A. Toby, A. Jenkins Department of Chemistry, University of Bath, BA2 7AY, Bath, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history: Received 4 June 2012 Received in revised form 15 August 2012 Accepted 13 September 2012 Available online 23 September 2012
This paper describes a biosensing concept for exotoxins secreted by Staphylococcus aureus and Pseudomonas aeruginosa based on the toxin mediated breakdown and subsequent fluorescent dye release from phospholipid vesicles (liposomes). The sensitivity of vesicles to toxins was tuned by altering the lipid and fatty acid composition of the membranes such that vesicles could be engineered to respond to toxins/enzymes from S. aureus only; P. aeruginosa only; and both S. aureus and P. aeruginosa. Nineteen types of vesicle were made with varying compositions of phosphocholine (PC), phosphoethanolamine (PE), cholesterol and the photo-polymerizable ampiphile 10,12-tricosadiynoic acid (TCDA). The selectivity of the vesicles was measured via a simple fluorescence ‘‘switch on’’ assay. Sensitivity of the vesicles to 40 clinically derived strains of S. aureus and P. aeruginosa was also demonstrated. This work suggests that this technology could be utilised in a diagnostic tool to discriminate between the species of S. aureus and P. aeruginosa in wound dressings. & 2012 Elsevier B.V. All rights reserved.
Keywords: Pseudomonas aeruginosa Staphylococcus aureus Carboxyfluorescein Lipid vesicles Selective sensitivity Toxins
1. Introduction Bacterial infection in wounds, especially burn-related wounds in children, is a major clinical concern in hospitals (Church et al., 2006). Young children suffering from burns are particularly vulnerable to, and at risk of infection, largely due to an immature immune system and thin dermal layer. Early detection of infection in children with burns is difficult, as the common symptoms: pyrexia, raised C-reactive protein and white blood cell count are all found in children with systemic inflammatory response syndrome (SIRS) following a major burn. Technology exists for diagnosing infection, but this is generally based on culturing of wound swabs, with results coming from central hospital reference laboratories in 24–48 hours (in the UK). However, young children can become acutely sick in as little as 8 hours, due to complications resulting from infection such as toxic shock syndrome (Young and Thornton, 2007; Jones, 2006; Laabei et al., 2012). Nucleic acid based sensing of bacteria such as PCR (Kubista et al., 2006) has a role, but requires highly skilled operatives, expensive equipment and is prone to contamination. Again, due to the organisation of hospital microbiology services, results from such a test often take 24 hours or more. This requires a reliable and robust sensor that can monitor the microbial state of wounds to detect the infection, and if possible to provide the identity of pathogenic species involved in case of wound infection in situ.
n
Corresponding author. Tel.: þ44 1225 38 6893; fax: þ44 1225 38 6231. E-mail address: [email protected] (N.T. Thet).
0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.09.019
Two main species of pathogenic bacteria commonly found in burn wound infections are Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa) (Branski et al., 2009). S. aureus is the gram-positive pathogen persistently colonized on human skin as a part of the normal flora. Most S. aureus strains are known to be pathogenic due to their secreted virulence factors including Pore Forming Toxins (PFTs) such as alpha, gamma and delta toxins (Song et al., 1996; Potrich et al., 2009; Verdon et al., 2009), Panton– Valentine Leukocidin (PVL) exoproteins (Tseng et al., 2009), epidermolytic toxins (Bailey and Redpath, 1992) and Toxic Shock Toxins (TST) (Dinges et al., 2000; Frame et al., 1985). P. aeruginosa is the gram-negative opportunistic pathogen responsible for fatal infections in patients with cystic fibrosis and immuno-suppression (Branski et al., 2009). It is also found in human skin flora and associated with virulence factors for the suspected lysis of healthy eukaryotic cells and tissue matrices upon infection. PFTs of S. aureus and lipid degrading enzymes such as esterase and phospholipases of P. aeruginosa are shown to be able to lyse the cell membrane of healthy mammalian cells in vivo and in vitro (Songer, 1997; Liu, 1974; Dinges et al., 2000; Arpigny and Jaeger, 1999). The different mode of action of toxins associated with S. aureus and P. aeruginosa in terms of their interaction with phospholipid membranes has been investigated previously using a Tethered Bilayer Lipid Membrane (TBLM) on gold surface using spectroscopic and optical analysis (Thet and Jenkins, 2010; Thet et al., 2011). The differential response of bacterial toxins with the membrane gave information on the mechanism of membrane degradation by primarily S. aureus toxins and primarily P. aeruginosa toxins.
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In the vesicle preparation, lipids, cholesterol and TCDA were individually mixed in chloroform to 100 mmol dm-3. They were then mixed together to a desired composition to a total volume of 100 ml and dried under nitrogen before putting inside the vacuum chamber at 10-3 bar for 1 hour. Thoroughly dried lipid mixture was then rehydrated with 5 ml of HEPES buffer with pH 7.3 containing 50 mmol dm 3 carboxyfluorescein. After rehydration, the lipid solution was heated in a hot water bath at 75 1C for 10 min before three freeze-thaw cycles. The lipid-dye solution was extruded three times through a polycarbonate membrane of 100 nm diameter pore size using a LF-50 Lipofast extruder (Avestin, USA). Finally, the extruded vesicles were purified using a DNA grade Sephadex NAP-25 column (GE Healthcare, UK). Lipid vesicle compositions containing TCDA were placed in a quartz vial and exposed to UV-light (254 nm), for a total of 12 s in a commercial flood exposure UV source (Hamamatsu, Japan), after storage at 4 1C for at least 2 hours. (Flow chart describing the process of vesicle preparation is presented in Fig. S9 in Supplementary information section).
Recently the development of a prototype wound dressing using lipid membranes to detect bacterial toxins and thus detect wound infection by pathogenic bacteria was demonstrated by chemical attachment of the lipid vesicles containing fluorescence dyes onto nonwoven fabrics (Zhou et al., 2010, 2011; Jenkins et al., 2011; Jenkins and Young, 2010). In this paper, we report that by changing the lipid and fatty acid composition of phospholipid vesicles it is possible to ’tune’ their response such that they are primarily susceptible to secretion toxins such as phospholipase from P. aeruginosa, or delta toxin secreted by S. aureus or to both toxin types. Hence, it is possible to obtain some initial information on the toxins produced by the bacteria – information that could be used by clinicians to help inform diagnosis and choice of antibiotic/antimicrobial in the treatment pathway.
2. Materials and methods 2.1. Materials and vesicle synthesis
2.2. Type of vesicles
Lipids used in making vesicles were 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) from Avanti Polar Lipids, USA. Cholesterol, 10,12-tricosadiynoic acid (TCDA) and 5(6)-carboxyfluorescein were purchased from Sigma–Aldrich, UK. TCDA is a synthetic polymerizable lipid which has been used to stabilise the vesicles by lateral cross-linking within the lipid bilayer after exposure to UV light. All the lipids, cholesterol and TCDA were used without further purifications. Triton X-100 (Sigma–Aldrich, UK) is a non-ionic surfactant which solubilises, disintegrates and reduces the lipid vesicles into micelles with simultaneous release of encapsulated carboxyfluorescein. A ten-fold dilution of 0.1% solution of triton X-100 was used as a positive control during the plate reading assay experiments. HEPES buffer was prepared according to standard protocol and used as a negative control for the stability of vesicles in the absence of bacteria and Triton X-100.
In order to empirically elucidate the role of the various vesicle components on their sensitivity to different bacteria, five classes of vesicles were studied (A–E, Table 1), each with one component being varied. Type A vesicles were primarily composed of shorter chain DPPC lipids, with varying concentration of cholesterol (0, 10, 20 and 30 mol%); Type B were as Type A but with the addition of a photopolymerizable component TCDA; Type C were composed of longer chain DSPC lipids with varying cholesterol concentration; Type D were also DSPC lipid based, with a fixed mol% of cholesterol and varying TCDA concentration; and finally Type E were composed of DSPC lipids, with a fixed mol% of TCDA and varying cholesterol concentration. Mean increment in fluorescence of respective vesicles before and after inoculation in Hepes buffer, P. aeruginosa PAO1 and S. aureus MSSA476 at 37 1C was also tabulated in Table 1 (see Section 2.4 for experimental detail). The rationale of this work is that cholesterol, chain length and presence of TCDA all profoundly
Table 1 Vesicles with mol% of their phospholipid/fatty acid content, and their fluorescent response in Hepes buffer and pathogenic bacteria (Arbitrary classification A–E depending on principal lipid component and variable i.e. cholesterol or TCDA; vesicle 17 belongs to both type D and E, and vesicle 11 belongs to both type C and D). Vesicles (Types and lipid compositions in mol%)
Mean fluorescent increment after 18 h (a.u.) in medium
Class
Vesicle types
DPPC
DSPC
Cholesterol
TCDA
Hepes (buffer)
PAO1
MSSA476
A
Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves Ves
88 78 68 73 63 53 43 33 23 – – – – – – – – – –
– – – – – – – – 88 78 68 73 68 63 58 53 63 43
10 20 30 0 10 20 30 40 50 10 20 30 20 20 20 20 20 10 30
– – – 25 25 25 25 25 25 – – – 5 10 15 20 25 25 25
35,995 2,035 1,126 23,593 34,885 13,522 1,019 834 85 863 758 563 29 531 767 2,686 1,989 3,186 2,154
1,18,388 1,03,842 41,546 1,38,137 1,03,529 99,245 35,204 14,087 11,430 19,356 52,905 7,816 60,060 51,096 42,839 55,310 60,864 36,058 25,689
76,558 39,610 10,905 1,19,276 84,549 83,047 76,162 81,173 83,671 5,620 9,818 3,321 34,819 41,539 62,152 87,173 91,885 33,910 44,310
B
C
D
E
1 2 3 4b 5b 6ab 7b 8b 9ab 10 11a 12 13b 14b 15b 16b 17b 18b 19b
All DPPC and DSPC vesicles contain 2 mol% of DPPE and DSPE, respectively. Black bold numbers in table describe the distinctive composition of lipid vesicles which give the best selective fluorescence responses between S. aureus and P. aeruginosa. a b
Vesicles used for selective sensitivity tests. Vesicles required UV cross-linking.
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affect membrane properties, such as fluidity and phase transition temperature, which in turn have significant effects on membrane interaction with bacteria and toxins (Potrich et al., 2009). 2.3. Pathogenic bacteria and their culture 21 clinical strains of pathogenic S. aureus were provided from John Radcliffe Hospital, Oxford, UK; 19 out of 21 strains were Methicillin sensitive S. aureus (MSSA) and 2 were Methicillin resistant S. aureus (MRSA). All MSSA and MRSA candidates studied in this work were community acquired virulent strains with suspected virulence factors, which were susceptible for cell lysis (Peacock et al., 2002). 21 clinical strains of P. aeruginosa provided from AmpliPhi BioSciences Corporation, UK and Southmead Hospital, Bristol, UK. Eight strains of P. aeruginosa acquired from Southmead Hospital were extracted from either acute wounds or blood agar of infected patients, the remaining 13 strains were clinical extracts from chronic wounds with known pathogenic effects on human hosts. All the strains studied in this work are listed in Table 2. For the non-pathogenic control, we used Escherichia coli DH5a; a laboratory strain with most of its virulence factors removed (Hanahan, 1985). Luria Broth (LB) and Tryptic Soy Broth (TSB) were prepared in MilliQ water according to the standard protocol and autoclaved after preparation for bacterial culture. E. coli DH5a and P. aeruginosa strains were cultured in LB, and S. aureus strains were cultured in TSB. Cultures were grown overnight in a shaking incubator at 37 1C. Optical Density (OD) was measured at 600 nm. OD of diluted bacteria was related to colony forming units per millilitre (CFU ml 1) by conventional plating and colony counting. Initial concentration of bacteria inoculated with vesicles in experiments was 104 CFU ml 1. 2.4. Fluorescent plate reading assay Fluorescent assays were carried out on a FLUOstar Omega microplate reader (BMG Labtech) using a 96 well plate. Excitation and emission of 485712 and 520 nm were used, respectively, with a gain of 650. Into each well, 50 ml of each vesicle was inoculated with 100 ml of bacteria-broth solution. Each vesicle-bacteria combination was repeated six times. Additionally, positive and negative controls were carried out with 100 ml Triton X-100 and HEPES buffer, respectively. Fluorescent responses of lipid vesicles inoculated with bacteria, HEPES buffer and Triton X-100 were continuously measured at 37 1C for 18 h. For each type of vesicle-bacteria combination, mean and standard deviation of fluorescence from six parallel wells were used to calculate the response in term of fluorescent increase at the end of each measurement for the data presentation.
3. Results and discussion 3.1. Effect of cholesterol and TCDA on stability and sensitivity of vesicles One of the principle differences in membrane components between eukaryotic and prokaryotic cells is the presence or absence of cholesterol. Cholesterol is the essential eukaryotic cell
membrane component, comprising as much as 25% of the membrane in human erythrocytes and as little as a mere trace amount in disk membrane (bovine) (Gennis, 1989). Apart from its physiological role, cholesterol, maintains the integrity and lateral fluidity of cell membranes for normal cell functioning (Ohvo-Rekil¨a et al., 2002; Koyama et al., 1999). Artificial lipid bilayer membranes containing cholesterol exhibit a similar change in membrane fluidity, line tension and formation of rafts; especially if the membrane contains sphingolipids (Veatch and Keller, 2005). Importantly, cholesterol plays an important role in the interaction of cell membranes with bacterial toxins either directly or indirectly (Palmer, 2004), and its presence or absence in cell membranes influences the susceptibility of the cells to bacterial toxins. This hypothesis was verified by designing lipid vesicles with cholesterol of varying mol%; and the effect of cholesterol on sensitivity of vesicles to pathogenic bacteria was examined. Additionally, the photo-polymerizable agent TCDA was incorporated in lipid bilayer to create laterally cross-linked polymer networks within the membrane to enhance the thermodynamic stability of the lipid membrane at elevated temperatures (Zhou et al., 2011). Carbon atoms in the hydrophobic tail of TCDA has reduced from 21 to 17 after lateral cross-linking by UV-light; forming a more favourable chain length for interactions with DPPC and DSPC. Individual and combined effects of cholesterol and TCDA on lipid bilayer stability and sensitivity were also explored. 3.1.1. DPPC vesicles (type A and B) Type A vesicles indicated that increasing the cholesterol concentration decreased the passive leakage in Hepes buffer, however, there was a decreased response with both P. aeruginosa and S. aureus (Table 1) (Fig. S1 in Supplementary information). The maximum measured fluorescence response of Type A vesicles to bacteria, taking into account minimal passive leakage (subtracting Hepes only control numerical values from the bacteria samples and accounting for standard deviations), was for 20 mol % cholesterol with more than twice ( 2.7fold) the response for P. aeruginosa than S. aureus. Type B vesicles, incorporating the photo-polymerizable cross-linker TCDA at a fixed 25 mol%, and varied cholesterol concentration showed increasing stability at high cholesterol concentrations. Sensitivity to P. aeruginosa decreased with increasing concentration of cholesterol but importantly, stable and almost exclusive response was only seen in S. aureus at cholesterol concentrations of 30 mol% and above (Fig. S2 in Supplementary information). 3.1.2. DSPC vesicles (types C and E) Type C vesicles, utilising the longer lipid chain length DSPC showed decreased passive leakage compared to DPPC vesicles. Cholesterol concentration had a large effect on vesicle response to bacterial toxins, with 20 mol% cholesterol having greater than 4 fold ( 4.6 fold) response to P. aeruginosa compared to S. aureus, (Table 1) (Fig. S3 in Supplementary information). Type E vesicles (DSPC with 25 mol% TCDA) showed a slightly greater response to S. aureus over P. aeruginosa, which became more distinguishable at higher cholesterol concentrations (Fig. S4 in Supplementary information).
Table 2 Bacterial strains used for selective sensitivity. Species
Strain numbers
Remarks
S. aureus P. aeruginosa
2, 3, 16, 21a, 25, 38, 49, 52, 56, 67, 69a, 101, 112, 114, 126, 160, 233, 253, 279, 295, 476 259, 260, 739, 854, 855, 856, 887, 889, 927, 935, 936, 937, 45291b, 45311b, 45400b, 45445b, 45468b, 45506b, 45666b, 45701b, PAO1
a
MRSA strainsRemaining are MSSA strains Strains extracted from acute infected wounds. Remaining are clinical extracts from infected chronic wounds
b
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3.1.3. DSPC Vesicles (type D) Type D vesicles, composed of DSPC with 20 mol% cholesterol and varying TCDA compositions (0–25 mol%) showed minimal passive leakage in Hepes buffer. A significant increase in response to S. aureus with increasing concentration of TCDA was observed, while the response to P. aeruginosa remained almost the same regardless of TCDA mol% (Table 1) (Fig. S5 in Supplementary information). These results indicated that vesicles composed primarily of DPPC lipids are more sensitive to bacteria than those composed primarily of DSPC lipids. The optimum cholesterol concentration in both cases was found to be 20 mol%. In the absence of TCDA, all types of vesicles showed a higher response to P. aeruginosa than S. aureus. However, this was not the case if TCDA was included; a relatively higher response to S. aureus with increasing TCDA mol% was measured. The reduced sensitivity of DSPC vesicles could be partly understood in terms of differences in the gel–liquid phase transition temperature (Tm): at the relatively higher Tm of DSPC (55 1C) the lipid bilayer would still be in a gel phase with minimal fluidity at 37 1C. This could not only reduce the passive leakage but also hinder the successful binding and subsequent activation of bacterial toxins onto
in HEPES E. coli DH5α P. aeruginosa PAO1 S. aureus MSSA476
Fluorescence increment / a.u.
70000 60000 50000 40000 30000 20000 10000 0 Vesicle 6
Vesicle 11
Vesicle 9
Fig. 1. Selective fluorescent response of three types of vesicles to pathogenic and non-pathogenic bacteria (vesicle 6 is sensitive to both pathogenic strains; P. aeruginosa PAO1 and S. aureus MSSA476 but not to non-pathogenic E. coli DH5a while vesicles 11 and 9 show selective sensitivity to either P. aeruginosa PAO1 or S. aureus MSSA 476, respectively).
E. coli DH5α
(non-pathogenic) negative control
P. aerunginosa
PAO1 (pathogenic)
541
vesicles. The increased response of S. aureus in the presence of TCDA has not been fully explained as there was no evidence of interaction between polymerizable agents and bacterial toxins (Jelinek and Kolusheva, 2007). It is possible that the formation of TCDA polymer networks could create local domains rich in lipids and cholesterol, which would provide pre-defined targets for local concentration of bacterial toxins (Verdon et al., 2009). The experimental results (Figs. 1 and 2) imply that the sensitivity of vesicles to either P. aeruginosa or S. aureus or both could be achieved by adjusting the percentage composition of DPPC, DSPC, cholesterol and TCDA. Three types of vesicles (Table 1) were chosen for the selective dissemination response test; vesicle 6 for detection of both bacteria, vesicle 11 and 9 for selective detection of P. aeruginosa and S. aureus, respectively.
3.2. Selectivity to pathogenic and non-pathogenic bacteria Determination of vesicle response to the pathogenic bacteria; P. aeruginosa PAO1 and S. aureus MSSA476, compared to a nonpathogenic control, E. coli DH5a was investigated. Inoculation with bacteria, to the vesicles, occurred during its stationary growth phase at 104 CFU ml 1. It took approximately 6 h for the bacteria to enter exponential growth phase, release toxins, and lyse vesicles giving a measured fluorescence response. (Fig. S6 in Supplementary information). Vesicle 6 produced 510 fold greater fluorescent response to both P. aeruginosa and S. aureus relative to the negative controls (Hepes buffer and E. coli DH5a) (Fig. 1). Further selection is possible between the two pathogenic strains (Fig. 1); vesicle 11 only responded to P. aeruginosa PA01, while vesicle 9 only responded to S. aureus MSSA476. This selectivity could be explained by lipid formulation of vesicles, lipid availability and membrane fluidity as well as the toxins involved with pathogenic bacteria. It was experimentally observed that the activities of PFTs of S. aureus increased nonlinearly with an increase in membrane fluidity, especially in membranes with lipids of shorter acyl chains (Potrich et al., 2009). With DPPC, cholesterol and TCDA, vesicle 6 maintained a higher membrane fluidity especially at 37 1C, ensuring promotion of nonspecific binding of toxins with lipids, thus enabling sensitivity to both pathogens. The vesicle composed of DSPC and cholesterol only (vesicle 11), had decreased membrane fluidity at 37 1C, forming gel-like phases, blocking accessibility of S. aureus toxins to the relatively S. aureus MSSA476
(pathogenic)
Triton X-100
Positive control
Vesicle type 6
Vesicle type 11
Vesicle type 9
Fig. 2. Visible fluorescent response of vesicles 6, 11 and 9 after inoculation with bacteria in 96 well plate (Vesicle 6 in two top rows shows fluorescence ‘‘switch on’’ with pathogenic strains; vesicle 11 in two middle rows shows response to P. aeruginosa PAO1 only, while vesicle 9 in two bottom rows indicates response to S. aureus MSSA476 only. Note: all vesicle types do not respond to non-pathogenic E. coli DH5a; inset – photograph of the plate after initial inoculation with bacteria).
N.T. Thet et al. / Biosensors and Bioelectronics 41 (2013) 538–543
Ves 6 (sensitive to both P. aeruginosa and S. aureus) Ves 11 (less sensitive to S. aureus) Ves 9 (more sensitive to S. aureus)
80000 60000 40000 20000 0
Strains of S. aureus Fig. 4. Selectivity of vesicle 9 over vesicle 11 (in terms of increase in fluorescence) in screening with 20 clinical virulent strains of S. aureus.
60000
data indicated that the strains 856 and 887 did not grow during the experiment, and consequently the positive responses were not shown, in contrast to the rest of tested strains. The reason for suppressed growth of strains 856 and 887 is not clear, despite the fact that all the strains of P. aeruginosa grew successfully in an incubator. It is possible that the growth condition of these particular strains in 96 well plate may not be optimum to support growth and hence toxin expression, and subsequently no lysis of vesicles was observed. Selectivity of vesicle 11 over vesicle 9 for P. aeruginosa strains over S. aureus, in terms of the relative increase in fluorescence intensity was observed between 2 and 6 fold (Fig. 3), is clearly distinguishable by eye when observed under UV light. Vesicle 9 showed a greater response to all 20 strains of S. aureus than vesicle 11 (Fig. 4). Two to ten fold increases in fluorescence of vesicle 9 against vesicle 11 was clearly observed, and all tested strains of S. aureus grew well in the experiment according to absorbance data. In terms of fluorescence signal strength, responses of vesicle 9 to S. aureus were slightly less than those of vesicle 11 to P. aeruginosa. This relatively low fluorescence ‘‘switch on’’ could be due to the involvement of particular toxins secreted by the strains of S. aureus which might not necessarily lyse the vesicles completely. If this is the reason, it implies that the toxins produced by P. aeruginosa have relatively stronger lipolytic action than those produced by S. aureus. Nevertheless, visible and selective responses of vesicle 9 against vesicle 11 to all strains of S. aureus were clearly observed. It was evidenced according to the experiments that 90% of all P. aeruginosa strains and 100% of all S. aureus strains tested were selectively distinguishable from each other.
40000
4. Conclusion
3.3. Selective sensitivity between 40 strains of P. aeruginosa and S. aureus In order to evaluate the sensitivity and the selectivity over a range of bacterial strains, the three selected vesicles (vesicles 6, 9 and 11), were tested against 40 additional clinical strains (20 each) of S. aureus and P. aeruginosa. The fluorescence and absorbance of the three replicas of each vesicle-bacterium combination were measured at the beginning and end of each experiment, and the results were plotted in Figs. 3 and 4 for P. aeruginosa and S. aureus, respectively. Vesicle 11 showed positive responses to all the P. aeruginosa strains, with the exception of strains 856 and 887 (Fig. 3). Bacterial optical density (OD600)
100000
Ves 6 (sensitive to both P. aeruginosa and S. aureus) Ves 11 (more sensitive to P. aeruginosa) Ves 9 (less sensitive to P. aeruginosa)
80000
20000 0
259 260 739 854 855 856 887 889 927 936 937 935 45291 45311 45400 45445 45468 45506 45666 45701
Fluorescence increment / a.u.
100000
2 3 16 21 25 38 49 52 56 67 69 101 112 114 126 160 233 253 279 295
stiff membrane. However P. aeruginosa toxins appear unaffected by the low membrane fluidity (Fig. 1), probably due to the associated toxins and their modes of action. P. aeruginosa secrete enzymes that degrade lipids, such as phospholipase C, which targets and hydrolyses the phosphate head group of phospholipids (Songer, 1997; Titball, 1993). Such toxins require only lipid head group availability without depending on membrane fluidity for their activation, which may explain why vesicle 11 responded only to P. aeruginosa secretion toxins and not S. aureus toxins. (Fig. S7 in Supplementary information) Vesicle 9 was composed of 50 mol% cholesterol and 25 mol% TCDA; the remaining 25 mol% was composed of DPPC and DPPE (Table 1). TCDA and cholesterol-rich zones could form raft-like domains, coexisting alongside with the lipid-rich domains, within the membrane (Garcia-Saez and Schwille, 2010; Mateo et al., 1995). The liquid-ordered state of cholesterol-rich domains could potentially enable targeting of S. aureus toxins and their activities in the remaining DPPC lipid-rich liquid-disordered domains at 37 1C. P. aeruginosa toxins primarily require lipids to activate its lipid-degrading enzymes. As a result of the small lipid composition (25 mol%) in vesicle 9, minimal response by P. aeruginosa toxins on lipid membrane is observed. The high presence of cholesterol and TCDA resulted in increased selectivity of vesicle 9 to S. aureus (Fig. S8 in Supplementary information). The fluorescence response of the vesicles to the bacteria and Hepes buffer (Fig. 1) and the visible colorimetric selection among E. coli DH5a, P. aeruginosa PAO1 and S. aureus MSSA476 using three types of vesicles (Fig. 2) indicates a potential discriminatory tool for identifying between two important pathogenic bacteria species.
Fluorescence increment / a.u.
542
Strains of P. aeruginosa Fig. 3. Selectivity of vesicle 11 over vesicle 9 (in terms of increase in fluorescence) in screening with 20 clinical virulent strains of P. aeruginosa.
We have demonstrated the selective detection between non-pathogenic bacteria E. coli DH5a and pathogenic strains of S. aureus MSSA476 and P. aeruginosa PAO1 using lipid vesicles containing carboxyfluorescein dye. There was a clear, visual selection between pathogenic and non-pathogenic strains of bacteria in the form of a fluorescence colour change; switching from relatively pale to bright green colour, after inoculation of lipid vesicles with bacteria and incubation at 37 1C. More importantly, by engineering the desired compositions of lipids, cholesterol and polymerizable agent, the vesicles showed further
N.T. Thet et al. / Biosensors and Bioelectronics 41 (2013) 538–543
selective discrimination between two pathogenic strains of P. aeruginosa and S. aureus. Additionally, selectivity over a wide range of two common pathogenic species was demonstrated using 40 clinical strains of P. aeruginosa and S. aureus. 90% of P. aeruginosa strains and all of the S. aureus strains were selectively distinguished with visible fluorescence colour change in the order of two to ten fold relative to each other. Vesicle 6 showed virtually no discrimination for S. aureus over P. aeruginosa (or vice versa) and responded to the majority of bacterial strains of both species with a fluorescent intensity change similar to that of vesicles 9 and 11. By integration of responsive lipid vesicles onto a wound dressing, we believed that the current selective and discriminative sensing system has potential as an indicator for bacterial infections, with additional information regarding the likely toxic mode of action of the infection in wounds, without requiring the early removal of the wound dressing during the wound healing process.
Acknowledgement We would like to thank Professor Mark C. Enright (AmpliPhi BioSciences Corporation, Bedfordshire, UK), Southmead Hospital (Bristol, UK) and John Radcliffe Hospital (Oxford, UK) for the bacteria strains. We also acknowledge the European Commission’s 7th Frame-work programme for funding via the EC-FP7 consortium project no. 245500 Bacteriosafe.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2012.09.019.
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