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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2006, p. 7339–7344
0099-2240/06/$08.00⫹0 doi:10.1128/AEM.01324-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 11
Rapid Chromatic Detection of Bacteria by Use of a New Biomimetic
Polymer Sensor䌤†
Liron Silbert,1 Izek Ben Shlush,1 Elena Israel,2 Angel Porgador,2 Sofiya Kolusheva,1 and Raz Jelinek1*
Department of Chemistry and Ilse Katz Center for Nanotechnology1 and Department of Immunology,2
Ben Gurion University of the Negev, Beer Sheva 84105, Israel
Received 9 June 2006/Accepted 13 September 2006
We present a new platform for visual and spectroscopic detection of bacteria. The detection scheme is based
on the interaction of membrane-active compounds secreted by bacteria with agar-embedded nanoparticles
comprising phospholipids and the chromatic polymer polydiacetylene (PDA). We demonstrate that PDA
undergoes dramatic visible blue-to-red transformations together with an intense fluorescence emission that are
induced by molecules released by multiplying bacteria. The chromatic transitions are easily identified by the
naked eye and can also be recorded by conventional high-throughput screening instruments. Furthermore, the
color and fluorescence changes generally occur in shorter times than the visual appearance of bacterial
colonies on the agar. The chromatic technology is generic and simple, does not require identification a priori
of specific bacterial recognition elements, and can be applied for detection of both gram-negative and grampositive bacteria. We demonstrate applications of the new platform for reporting on bacterial contaminations
in foods and for screening for bacterial antibiotic resistance.
(1). Secretion of pore-forming exotoxins by bacteria is abundant, and endotoxins, such as lipopolysaccharides, which are
often released by gram-negative bacteria, strongly interact with
membrane components of the host cell (21). Here, we describe
a new bacterial detection platform generating dramatic visible
color changes accompanied by intense fluorescence emission
that are induced by membrane-active molecules secreted by
proliferating bacteria.
The emerging global risks of bioterrorism, the recurring
incidents of bacterial food contaminations, and the need to
monitor sterile environments in health care applications and
other industries have pushed the development of methods for
detection of pathogens to the forefront of technological and
scientific research. Numerous technologies for reporting on
bacterial presence have been developed (3, 5, 9, 25, 26). There
are, however, limitations to existing bacterial detection techniques as rapid and generic approaches. Specifically, many
bioanalytical techniques employed for pathogen detection
(such as culture-based methods) provide results after relatively
long time spans (several hours to days) (10). Other currently
employed technologies often involve complex detection mechanisms that require specialized instrumentation, application by
trained personnel, and the need for active operation (addition
of reagents, initiation of chemical reactions, etc.), which overall do not lend their use in settings other than laboratory
environments (3, 6). Furthermore, a prerequisite for many
detection methods is the detailed understanding of the biochemical and structural properties of the bacterial species
sought, limiting applications in the case of unknown pathogens
or variants (9, 31).
Various membrane-active compounds are released by bacteria to their environments (2, 30), a process that often has an
essential functional role as a means for overcoming host defense mechanisms, allowing colony proliferation, and facilitating bacterial communication (17). Membrane-active peptides
and toxins, in particular, are produced by bacteria, for example, pneumolysins secreted by streptococci (27) and ␣-toxin,
which is the major cytolysin emitted by Staphylococcus aureus
MATERIALS AND METHODS
Materials and bacterial strains. Dimyristoylphosphatidylcholine (DMPC),
Tris (Tris base buffer), kanamycin, and streptomycin were purchased from
Sigma. The diacetylenic monomer 10,12-tricosadiynoic acid was purchased from
GFS Chemicals (Powell, OH), dissolved in chloroform, and passed through a
0.45-␮m nylon filter before use. Luria-Bertani (LB) agar was purchased from
Pronadisa (Spain).
The bacteria used in the studies were Salmonella enterica serovar Typhimurium 1a (strain CS093 [22]), Bacillus cereus, Escherichia coli K-12 strains
C600, C600 pMRInv, and MC4100 (provided by Ehud Gazit, Tel Aviv University), and E. coli BL (provided by Dudy Bar-Zvi, Ben Gurion University).
Bacterial growth. Salmonella enterica serovar Typhimurium 1a, E. coli BL, and
E. coli K-12 C600 were grown to saturation at 37°C and streaked. E. coli K-12
MC4100 was grown in LB medium supplemented with streptomycin (10 ␮g/ml).
E. coli K-12 C600 pMRInv was grown in LB medium supplemented with kanamycin (30 ␮g/ml). Bacterial concentrations were determined from absorption at
600 nm on a Jasco V-550 spectrophotometer.
Vesicle preparation. Vesicles containing DMPC and 10,12-tricosadiynoic acid
(2:3 molar ratio) were prepared at a concentration of 1 mM. The lipids were
dried together in vacuo. Following evaporation, distilled water was added and
the suspension was probe sonicated at 70°C. The resultant vesicle solution
was cooled at 4°C overnight and then polymerized by irradiation at 254 nm
for 0.5 min.
Visible spectroscopy. Samples were prepared by adding 30 ␮l bacterial samples
(at the different concentrations tested in the experiments) to 30 ␮l of 1 mM
vesicle solutions, followed by the addition of 30 ␮l Tris base at 50 mM, pH 8, and
dilution in water to 1 ml prior to spectral acquisition. All measurements were
carried out at room temperature on a Jasco V-550 UV/VIS spectrophotometer.
Preparation of chromatic lipid-PDA agar matrix. Unpolymerized DMPCpolydiacetylene (PDA) vesicles at a concentration of 5 mM were added right
after the sonication stage to hot LB agar. The mixture was then cooled to room
temperature and poured into a six-well plate (Cellstar, Greiner Bio-one).
After solidification of the agar, the plate was kept at 4°C for 2 days and
* Corresponding author. Mailing address: Department of Chemistry, Ben Gurion University, Beer Sheva 84105, Israel. Phone: 972-86461747. Fax: 972-8-6472943. E-mail: [email protected].
† Supplemental material for this article may be found at http://aem
.asm.org/.
䌤
Published ahead of print on 22 September 2006.
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FIG. 1. Chromatic sensor concept. (Left) Agar scaffold containing
vesicular nanoparticles composed of phospholipids (black) and PDA
(blue). (Right) Bacterial proliferation (green oval) on the agar surface
causes blue-to-red transformation of embedded vesicles due to bacterially secreted compounds that diffuse through the agar.
polymerized by irradiation (254 nm, 40 s) in a UV cross-linker (UV-8000;
Stratagene, California).
Multiwell fluorescence spectroscopy. On the surface of the chromatic DMPCPDA agar (in each well), we spotted 10-␮l diluted bacterial samples (107 particles), placed the plate in a multiwell fluorescence plate reader (Fluoroscan
Ascent; Thermo, Finland), and kept it at 26°C or 29°C. All measurements were
carried out using 485-nm excitation and 555-nm emission, using LP filters with
normal slits. Acquisition of data was automatically performed every 30 min.
Color scanning. Scanned images were recorded on an Epson Perfection 4990
photo scanner.
All experiments were repeated at least three times.
RESULTS
The chromatic PDA sensor. The new bacterial detection
approach utilizes biomimetic chromatic membranes that undergo colorimetric and fluorescence transformations through
interactions with bacterially secreted substances. A schematic
description of the sensing scheme is shown in Fig. 1: vesicular
nanoparticles comprising phospholipids (forming a biomimetic
membrane bilayer) and polydiacetylene (the colorimetric/fluorescence reporter) (11, 12, 19) are embedded in agar scaffolding containing bacterial growth medium (Fig. 1). The agar
matrix facilitates bacterial multiplication and amplification of
the colorimetric/fluorescent signals. Essentially, molecules released by bacteria that proliferate on the agar surface diffuse
through the semiporous substrate and induce chromatic
changes in response to the bacterial presence.
PDA constitutes the signal-generating module in the new
sensing platform. Cross-linked PDA assemblies exhibit unique
chromatic properties; polymerized PDA appears intense blue
due to the conjugated ene-yne framework (24). Furthermore,
APPL. ENVIRON. MICROBIOL.
PDA aggregates and films have previously been shown to undergo dramatic blue-to-red changes due to conformational
transitions in the conjugated polymer backbone that are induced by external structural perturbations, such as binding of
positively charged ions and molecules or amphiphilic molecules (4, 19). The colorimetric transformations of PDA go
hand in hand with induction of fluorescence; no fluorescence is
emitted by the initially polymerized blue-phase PDA, while the
red-phase PDA strongly fluoresces at 560 nm and at 640 nm
(29). The chromatic transitions of PDA also occur in biological
contexts; recent studies have demonstrated that blue-to-red
and fluorescence transformations could be induced in vesicle
assemblies comprising phospholipid bilayers interspersed
within the PDA matrices by membrane-active compounds (12,
13, 18). Other reports have demonstrated that PDA “nanopatches” attached to surfaces of living cells could report on
local membrane events through the chromatic changes of the
polymer (20). In all such systems, the mechanism for the chromatic transformations corresponds to surface perturbations
and fluidity changes within the lipid domains, which induce the
structural/chromatic transformations of the adjacent PDA matrix through the molecular interface between the two components (13).
The new bacterial sensor exploits the chromatic response
induced upon interaction of lipid-PDA assemblies with membrane-active substances as the underlying mechanism for bacterial detection. Previous studies have employed chemically
derivatized PDA for bacterial sensing; however, those systems
relied mostly on the covalently displaying specific recognition
units on the PDA surface, producing signals only through actual binding of bacterial proteins to the PDA matrix. Such
assemblies require chemical modifications of the PDA units,
and their detection levels are determined by the number of
bacterial particles attaching to the PDA-displayed receptors
(15, 23). A previous study employing PDA for bacterial sensing
exhibited a detection limit of 108 bacterial particles (16). The
concept we describe here is simpler and more generic and
features higher sensitivity. Essentially, we show that bacterially
released substances diffuse through the agar matrix and interact with the incorporated phospholipid-PDA nanoparticles,
consequently inducing the colorimetric/fluorescence transitions that result from bacterial presence.
Color detection of bacteria. Fig. 2 depicts a representative
experiment showing the color transitions induced by bacteria
in DMPC-PDA agar. Specifically, Fig. 2B and C show scanned
FIG. 2. Color transitions induced by bacterial colonies. Scanned images of DMPC-PDA agar plates prior to bacterial growth (A), 18 h after
three colonies of Bacillus cereus were transferred (B), and 18 h after three colonies of E. coli BL were transferred (C). The plates were incubated
at 26°C.
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FIG. 3. Reactions of bacteria with lipid-PDA vesicles. (A) Colors observed after mixing of DMPC-PDA vesicles with the resuspended pellet
(left) and supernatant (right) after Salmonella enterica serovar Typhimurium 1a was grown overnight at 37°C (number of bacteria, 109).
(B) Relationship between the bacterial growth curve and the blue-to-red transformation of PDA. The bacterial growth curve (black) and
blue-to-red transition (blue) of a solution containing Salmonella enterica serovar Typhimurium 1a in growth medium mixed with DMPC-PDA
vesicles (bacteria grown at 26°C) are shown. The extent of the blue-to-red transformation is reflected in the ratio of the intensity of the peak at
500 nm (the red peak in the visible spectrum) to that at 640 nm (the blue peak). Experiments were repeated at least three times. The variability
of color response was between 10% and 15%.
images of DMPC-PDA agar plates onto which we transferred
colonies of the gram-positive Bacillus cereus and the gramnegative Escherichia coli BL strains, respectively, and incubated them at 26°C. The pictures in Fig. 2B and C clearly show
that red hallows form around the bacterial colonies following
incubation (note that the apparent “doublets” in Fig. 2B are
due to the reflection of the scanner light). The blue-to-red
transformation of the matrix was directly related to bacterial
proliferation; each colony was surrounded by an area in which
the blue agar matrix changed color to red, while the remaining
blue agar matrix stayed blue (Fig. 2B and C). The dispersion of
red regions under and around the bacterial colonies indicates
that the color transitions were due to diffusion of substances
released by the bacteria into the surrounding matrix.
To corroborate the relationship between the color transitions of the PDA assemblies and bacterially secreted substances, we investigated the reaction between blue DMPCPDA vesicles in aqueous solutions and either the pellet or the
supernatant extracted after growth of bacteria to saturation
(Fig. 3A). In the experiment whose results are depicted in Fig.
3A, we grew Salmonella enterica serovar Typhimurium 1a to
saturation (20 h at 37°C, number of bacteria was approximately
109), separated the supernatant solution from the pellet, mixed
each extraction with the lipid-PDA vesicles (the pellet was
resuspended and thoroughly washed in water prior to addition
to the vesicles), and incubated the mixtures for 5 min prior to
color recording. The pictures shown in Fig. 3A clearly demonstrate that the bacterial particles themselves did not induce a
noticeable change (matrix remained blue), while the addition
of the supernatant gave rise to an intense purple-red color.
We further examined the time dependence of the colorimetric transformation of the lipid-PDA vesicles during the bacterial growth curve (Fig. 3B). The data depicted in Fig. 3B show
that several hours after the initiation of bacterial growth, the
lipid-PDA vesicle solution appeared increasingly redder (i.e.,
ratio between 500 nm and 640 nm is higher; see Materials and
Methods) as the number of bacteria in the suspension increased. Figure 3B also indicates that the relative intensity of
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FIG. 4. Fluorescence induced by bacterial colonies. Three-dimensional fluorescence intensity distributions, recorded using a multiwell
fluorescence reader, in conventional bacterial growth agar plates used
as a control (A and B) and DMPC-PDA agar plates (C and D) are
shown. The z axes depict the fluorescence intensity (arbitrary units),
while the x and y planes correspond to the well surface area. (A and C)
Initial measurements (time zero). (B and D) Measurements 6 h after
Salmonella enterica serovar Typhimurium 1a colonies were streaked on
the agar surface. Growth was carried out at 26°C.
the 500-nm signal (i.e., extent of “redness” of the solution)
increased significantly only after the bacteria reached the plateau region in the growth curve (i.e., reached saturation). The
time lag between the increase in bacterial concentration and
the occurrence of the blue-to-red colorimetric transition confirms that bacterially released substances, but not the bacterial
particles themselves, are the primary factor responsible for the
blue-to-red transitions. Importantly, the contribution of the
small pH change in the bacterium-vesicle solutions to the color
change was found to be insignificant, indicating that the color
transformations are indeed related to bacterially produced
compounds rather than a pH effect.
Bacterial detection through fluorescence. In addition to
color transformations, the fluorescence properties of PDA can
be exploited as well for bacterial detection using the new assay.
Figure 4 shows three-dimensional fluorescence “topographic
maps” recorded in a conventional multiwell enzyme-linked
immunosorbent assay reader (excitation, 485 nm; emission,
555 nm). In the experiment whose results are depicted in Fig.
4, we compared the fluorescence responses of conventional
agar (used as a control) (Fig. 4A and B) and DMPC-PDA agar
(Fig. 4C and D) following bacterial proliferation. Specifically,
we transferred microscopic colonies of Salmonella enterica
serovar Typhimurium, extracted from a fresh agar plate, onto
the agar surfaces and recorded the fluorescence intensities at
different time points using a multiwell fluorescence reader,
with the temperature maintained at 26°C.
Figure 4D demonstrates that areas of pronounced fluorescence appeared only in the well containing the lipid-PDA agar
matrix. The fluorescence signals, corresponding to the trans-
APPL. ENVIRON. MICROBIOL.
FIG. 5. Chromatic screening of bacterial antibiotic resistance. A
portion of a multiwell plate containing the DMPC-PDA agar matrix,
further incorporating the antibiotic compounds kanamycin (top rows)
and streptomycin (bottom rows), is shown. The bacterial strains (1 ⫻
107 bacterial particles) streaked in the wells were (top left) E. coli K-12
C600 pMRInv (kanamycin resistant), (bottom left) E. coli K-12
MC4100 (streptomycin resistant), and (bottom and top right) E. coli
K-12 C600 (not resistant to either antibiotic). (A and B) Area distributions of fluorescence intensities recorded using a multiwell fluorescence reader (color key for relative intensity units shown on left).
(A) Measurements at time zero. (B) Measurements 8 hours after
streaking. (C) Scanned color image of the plate after an 18-h incubation. Growth was carried out at 26°C.
formed red-phase PDA, appeared exactly where the bacterial
colonies were placed on the agar surface. Significantly, the
fluorescence signals could be detected in a few hours (initial
signals appeared in less than 6 h) and several hours before
actual bacterial colonies were visible to the eye on the agar
surface. Kinetic profiles of the fluorescence emissions induced
by bacterial suspensions at different concentrations are shown
in the supplemental material.
Practical applications. Fig. 5 and 6 depict the results of
experiments in which the new assay was used for evaluation
of bacterial resistance to antibiotics (Fig. 5) and for detection of
bacteria in foods (Fig. 6). In the experiment whose results are
shown in Fig. 5, we streaked E. coli strains exhibiting different
antibiotic resistances onto plates containing the lipid-PDA
agar matrix that further incorporated antibiotic compounds
and incubated the plates at 29°C. Figure 5B features fluorescence distribution maps of the plates recorded 8 hours after
streaking that clearly reveal which of the bacteria were resistant to the antibiotic compound present in the agar matrix. For
example, fluorescence emission appeared in a plate containing
kanamycin-DMPC-PDA agar onto which the kanamycin-resistant E. coli C600 pMRInv strain (7) was streaked (Fig. 5B, top
left), while no fluorescence change occurred when the E. coli
K-12 C600 strain, a bacterium that cannot grow on kanamycincontaining substrates, was streaked (8). Similarly, fluorescence
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FIG. 6. Inspection of bacterial contamination in food. DMPC-PDA agar plates incubated overnight at 26°C are shown. (A) Control. (B) Plate
contacted with exposed liver. (C) Plate contacted with exposed liver that was boiled for 3 min.
induction was recorded only in a streptomycin-DMPC-PDA
agar plate onto which the streptomycin-resistant E. coli K-12
MC4100 strain was streaked (Fig. 5B, bottom left). Importantly, the fluorescence transitions could be detected earlier
than the bacterial colonies, highlighting the usefulness of the
new assembly as a bacterial screening tool. The striking color
transitions induced by proliferation of the antibiotic-resistant
strains (Fig. 5C), which appear exactly in the regions where
fluorescence was recorded, further emphasize the practicality
of the new assay for visual screening of bacterial antibiotic
resistance.
Figure 6 demonstrates the potential of the new colorimetric
assay as a tool for visualizing bacterial contamination in foods.
In the experiment whose results are depicted in Fig. 6, we
placed a meat sample (chicken liver purchased at a store) for
24 h under ambient conditions at room temperature, thus
facilitating bacterial proliferation on the exposed meat sample.
Following this treatment, we contacted the meat sample for 2
to 3 seconds with the surface of the blue DMPC-PDA agar
matrix and incubated the plate overnight at 29°C. As depicted
in Fig. 6B, after incubation the blue matrix was completely
transformed to red. To confirm that the blue-to-red change
was indeed induced by bacteria that were present on the surface of the meat sample and consequently grew on the chromatic agar plate, we carried out a control experiment in which
part of the same liver specimen was boiled for a few minutes,
then placed in contact with the chromatic agar surface, and
incubated overnight (Fig. 6C). As apparent in the scanned
image in Fig. 6C, the boiled liver sample did not give rise to any
color change within the lipid-PDA agar matrix after incubation, most likely due to sterilization of the meat sample
through boiling.
DISCUSSION
This work presents a new generic bacterial detection scheme
based on colorimetric and fluorescence transformations induced within lipid-polymer assemblies by bacterially released
substances. The technology does not require a priori knowledge of the composition or exact identities of the bacterially
secreted compounds. Indeed, because the chromatic response
of the agar-embedded vesicles is due to the general affinity of
the lipid-PDA assemblies to membrane-active, lipophilic, or
positively charged compounds released by bacteria, the new
platform should be able to report, in principle, on the presence
of any bacterial species. An additional important facet of the
new bacterial sensor is the amplification capabilities inherent
in the agar scaffolding, which facilitates bacterial growth and
thereby lowers the detection threshold. The semipermeable
agar additionally functions as an effective filter, reducing background signals by preventing nonbacterially secreted substances, such as ions and components of the growth media,
from interacting with the embedded vesicles.
The sensitivity of the new assay is essentially determined by
the number of bacteria and the time of detection; even a single
bacterium can be detected following its proliferation on the
agar and reaction with PDA. This feature distinguishes this
sensor from existing bacterial sensing approaches and PDAbased assays that rely mostly on specific recognition elements
displayed by the bacteria.
The reported experiments demonstrate that bacterially secreted molecules accumulate and diffuse within the porous
agar scaffolding, subsequently inducing the color and fluorescence transformations of lipid-PDA. The chromatic sensing
capabilities of lipid-PDA assemblies have previously been
demonstrated in various applications; in particular, the vesicles
were previously shown to constitute a useful platform for detection of membrane-active and amphiphilic molecules (28). A
recent study demonstrated the induction of colorimetric transitions in lipid-PDA vesicles by a pore-forming bacterial toxin
(14). In the context of this work, the lipid-PDA nanoparticles
constitute the reporter element within the sensing assembly,
emitting signals in response to bacterial presence.
Utilization of either visual color changes observed by the
naked eye or fluorescence emission as a viable detection
method is an important advantage of the new bacterial sensing
system. While the pronounced color changes should facilitate
bacterial detection by nonexperts in diverse settings, the fluorescence properties of the chromatic matrix could additionally
open the way for employing the platform for high-throughput
screening applications requiring high sensitivity, large sample
quantities, or automation. Indeed, the fluorescence images in
Fig. 3 and 4 underlie the enhanced sensitivity of the platform
to bacterial proliferation and the possibility of detecting bacteria in shorter times than with conventional microbiology
methods.
Microbe selection was designed to demonstrate the general
applicability of the assay, and the choice of bacterium was also
related to the type of experiment presented. Specifically, the
experiments aimed to show the fundamental features of the
technique (Fig. 2 to 4) were carried out with widely used
conventional bacterial species: E. coli (gram negative), B.
cereus (gram positive), and S. enterica serovar Typhimurium
(representing common pathogenic bacteria). In the experi-
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ments depicting specific applications of the technique for antibiotic screening (Fig. 5), we have used E. coli genotypes
containing the desired antibiotic resistance.
The chromatic platform could be employed for diverse practical applications. Utilizing the assay for screening for antibiotic resistance of bacteria is feasible through incorporation of
antibiotic compounds within the chromatic matrix. Figure 5,
for example, demonstrates that evaluation of bacterial sensitivity to antibiotic substances can be carried out in such matrixes by using a conventional fluorescence multiwell reader.
The experiment whose results are depicted in Fig. 5 also points
to applications of the chromatic platform for rapid screening of
putative antibacterial properties of compound libraries. The
color/fluorescence sensitivity of the chromatic assembly and
the amenability to conventional high-throughput screening formats are attractive features of the new system for diagnostic
and industrial utilization. The facile blue-to-red changes following bacterial proliferation could be useful in other applications, such as monitoring food freshness. Figure 6, for example,
demonstrates that visible color transformations provide a direct alert on the presence of bacteria in a contaminated food
specimen.
The new bacterial detection assay is not capable, at this
stage, of differentiating among bacteria. However, the generality of the detection concept and the nonspecificity of the
chromatic platform can, in fact, be advantages in applications
in which reporting on the presence of any type of bacteria is
required, for example, monitoring sterile environments, evaluating food freshness, screening for bacterial resistance to existing and new antibiotic compounds, and others. Research
currently being carried out in our laboratory is exploring avenues for facilitating specificity of detection among bacterial
species by varying the lipid composition within the chromatic
platform.
ACKNOWLEDGMENT
The Human Frontiers Science Program is acknowledged for generous financial support.
REFERENCES
1. Arvand, M., S. Bhakdi, B. Dahlback, and K. T. Preissner. 1990. Staphylococcus aureus alpha-toxin attack on human platelets promotes assembly of
the prothrombinase complex. J. Biol. Chem. 265:14377–14381.
2. Bendtsen, J. D., L. Kiemer, A. Fausboll, and S. Brunak. 2005. Non-classical
protein secretion in bacteria. BMC Microbiol. 5:58.
3. Canhoto, O. F., and N. Magan. 2003. Potential for detection of microorganisms and heavy metals in potable water using electronic nose technology.
Biosens. Bioelectron. 18:751–754.
4. Charych, D., Q. Cheng, A. Reichert, G. Kuziemko, M. Stroh, J. O. Nagy, W.
Spevak, and R. C. Stevens. 1996. A ‘litmus test’ for molecular recognition
using artificial membranes. Chem. Biol. 3:113–120.
5. Gfeller, K. Y., N. Nugaeva, and M. Hegner. 2005. Rapid biosensor for
detection of antibiotic-selective growth of Escherichia coli. Appl. Environ.
Microbiol. 71:2626–2631.
6. Gfeller, K. Y., N. Nugaeva, and M. Hegner. 2005. Micromechanical oscillators as rapid biosensor for the detection of active growth of Escherichia coli.
Biosens. Bioelectron. 21:528–533.
7. Gophna, U., M. Barlev, R. Seijffers, T. A. Oelschlager, J. Hacker, and E. Z.
Ron. 2001. Curli fibers mediate internalization of Escherichia coli by eukaryotic cells. Infect. Immun. 69:2659–2665.
APPL. ENVIRON. MICROBIOL.
8. Gophna, U., T. A. Oelschlaeger, J. Hacker, and E. Z. Ron. 2002. Role of
fibronectin in curli-mediated internalization. FEMS Microbiol. Lett. 212:
55–58.
9. Hobson, N. S., I. Tothill, and A. P. Turner. 1996. Microbial detection.
Biosens. Bioelectron. 11:455–477.
10. Ivnitski, D., I. Abdel-Hamid, P. Atanasov, and E. Wilkins. 1999. Biosensors
for detection of pathogenic bacteria. Biosens. Bioelectron. 14:599–624.
11. Jelinek, R., and S. Kolusheva. 2001. Polymerized lipid vesicles as colorimetric biosensors for biotechnological applications. Biotechnol. Adv. 19:109–
118.
12. Kolusheva, S., L. Boyer, and R. Jelinek. 2000. A colorimetric assay for rapid
screening of antimicrobial peptides. Nat. Biotechnol. 18:225–227.
13. Kolusheva, S., T. Shahal, and R. Jelinek. 2000. Peptide-membrane interactions studied by a new phospholipid/polydiacetylene colorimetric vesicle
assay. Biochemistry 39:15851–15859.
14. Ma, G., and Q. Cheng. 2005. Vesicular polydiacetylene sensor for colorimetric signaling of bacterial pore-forming toxin. Langmuir 21:6123–6126.
15. Ma, Z., J. Li, M. Liu, J. Cao, Z. Zou, J. Tu, and L. Jiang. 1998. Colorimetric
detection of Escherichia coli by polydiacetylene vesicles functionalized with
glycolipid. J. Am. Chem. Soc. 120:12678–12679.
16. Ma, Z., J. Li, and L. Jiang. 2000. Influence of the spacer length of glycolipid
receptors in polydiacetylene vesicles on the colorimetric detection of Escherichia coli. Langmuir 16:7801–7804.
17. Miller, M. B., and B. L. Bassler. 2001. Quorum sensing in bacteria. Annu.
Rev. Microbiol. 55:165–199.
18. Okada, S., R. Jelinek, and D. Charych. 1998. Induced color change of
conjugated polymeric vesicles by interfacial catalysis of phospholipase A2.
Angew. Chem. Int. Ed. Engl. 38:655–659.
19. Okada, S., S. Peng, W. Spevak, and D. Charych. 1998. Color and chromism
of polydiacetylene vesicles. Acc. Chem. Res. 31:229–239.
20. Orynbayeva, Z., S. Kolusheva, E. Livneh, A. Lichtenshtein, I. Nathan, and R.
Jelinek. 2005. Visualization of membrane processes in living cells by surfaceattached chromatic polymer patches. Angew. Chem. Int. Ed. Engl. 44:1092–
1096.
21. Prescott, L. M., J. P. Harley, and D. A. Klein. 2002. Microbiology, 5th ed., p.
799–802. McGraw Hill, New York, N.Y.
22. Qimron, U., N. Madar, H. W. Mittrucker, A. Zilka, I. Yosef, N. Bloushtain,
S. H. Kaufmann, I. Rosenshine, R. N. Apte, and A. Porgador. 2004. Identification of Salmonella typhimurium genes responsible for interference with
peptide presentation on MHC class I molecules: Deltayej Salmonella mutants induce superior CD8⫹ T-cell responses. Cell. Microbiol. 6:1057–1070.
23. Rangin, M., and A. Basu. 2004. Lipopolysaccharide identification with functionalized polydiacetylene liposome sensors. J. Am. Chem. Soc. 126:5038–
5039.
24. Ringsdorf, H., B. Schlarb, and J. Venzmer. 1988. Molecular architecture and
function of polymeric oriented systems: models for the study of organization,
surface recognition, and dynamics of biomembranes. Angew. Chem. Int. Ed.
Engl. 27:113–158.
25. Schmidt, M., C. Weis, J. Heck, T. Montag, S. B. Nicol, M. K. Hourfar, V.
Schaefer, W. Sireis, W. K. Roth, and E. Seifried. 2005. Optimized Scansystem
platelet kit for bacterial detection with enhanced sensitivity: detection within
24 h after spiking. Vox Sang. 89:135–139.
26. Simpson, J. M., and D. V. Lim. 2005. Rapid PCR confirmation of E. coli
O157:H7 after evanescent wave fiber optic biosensor detection. Biosens.
Bioelectron. 21:881–887.
27. Srivastava, A., P. Henneke, A. Visintin, S. C. Morse, V. Martin, C. Watkins,
J. C. Paton, M. R. Wessels, D. T. Golenbock, and R. Malley. 2005. The
apoptotic response to pneumolysin is Toll-like receptor 4 dependent and
protects against pneumococcal disease. Infect. Immun. 73:6479–6487.
28. Su, Y. L., J. R. Li, and L. Jiang. 2004. Effect of amphiphilic molecules upon
chromatic transitions of polydiacetylene vesicles in aqueous solutions. Colloids Surf. B 39:113–118.
29. Takayoshi, K., Y. Masahiko, O. Shuji, M. Hiro, and N. Hachiro. 1997.
Femtosecond spectroscopy of a polydiacetylene with extended conjugation
to acetylenic side groups. Chem. Phys. Lett. 267:472–480.
30. Thanassi, D. G., and S. J. Hultgren. 2000. Multiple pathways allow protein
secretion across the bacterial outer membrane. Curr. Opin. Cell Biol. 12:
420–430.
31. Vollenhofer-Schrumpf, S., R. Buresch, G. Unger, N. Stahl, G. Frnzl, and M.
Schinkingfr. 2005. Detection of Salmonella spp., Escherichia coli O157,
Listeria monocytogenes and Campylobacter spp. in chicken samples by multiplex polymerase chain reaction and hybridization using the GeneGen major
food pathogens detection kit. J. Rapid Methods Autom. Microbiol. 13:148–
176.