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Abstract: Pseudomonas aeruginosa is an opportunistic pathogen that often forms biofilms and causes a significant
number of nosocomial (hospital acquired) infections. Investigations of the processes governing biofilm formation,
persistence, and virulence require a new set of diagnostic tools that can be applied, non-invasively, to prevent
destroying the biofilms structure. This project seeks to explore the prospects of replacing fluorescent stains with
quantum dots (QDs); nanocrystals composed of periodic groups of II-VI, III-V, or IV-VI semiconductor materials.
Unlike traditional fluorochromatic stains, QD luminescence is photostable, with narrow emission spectra that are
size tunable, and broad absorption spectra, which allow the excitation of multiple QDs with a single wavelength.
Employing QDs to either label various bacterial species or to quantify genetic transfer between species requires
background information on the natural uptake of QDs by bacteria. Studies in internalizing high quality
poly(ethylene imine) (PEI) coated QDs using planktonic P. aeruginosa were carried out prior to future studies with
P. aeruginosa biofilms. We studied three distinct internalization methods: (1) passive bacterial incubation with
QDs, (2) CaCl2 treatment transformation, and (3) electroporation. Using epifluorescence microscopy, all three
methods appear to allow for some degree of internalization of QDs within the bacteria, with transformation
efficiencies of 3.7%, 13.9%, and 15.1%, respectively. Fluorescence scanning of supernatants and bacterial solutions
were also performed to test for the presence of the QDs. Further studies will provide supporting evidence that the
QDs are inside the bacteria, not just associated with the surface. Results will provide a link to using QDs to study
the internal mechanisms of the biofilm bacteria without photobleaching.
1. INTRODUCTION
Opportunistic pathogens pose a major health
problem in clinical settings partly due to their
ability to form biofilms. Biofilms consist of a
community of bacteria that are reversibly
adhered to a surface and excrete extracellular
polymeric
substances.
The
community
interaction along with the resilient structure of
biofilms protects these bacteria from host
defense mechanisms. The bacteria within this
community typically carry plasmids that confer
antibiotic resistance [6], allowing for an
increased spread of antibiotic resistance within
the biofilm. Additionally, these plasmids can be
transferred between different species of bacteria.
Therefore, it would be beneficial to study the
transfer of the plasmids within the biofilms to
obtain a better understanding of the increase in
antibiotic resistance seen within these bacterial
communities.
Pseudomonas aeruginosa is a bacterium that
can cause a wide variety of diseases such as
urinary tract infections, chronic lung infections,
eye infections, and soft tissue infections. About
10% of all hospital acquired infections are
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Figure 1. QD internalization by CaCl2 transformation. Arrows point to bacteria that have internalized QDs.
A-D) transmitted light images. E-H) epifluorescent images. A,E) 5 l QD+; B,F) 10 l; C,G) 50 l; D, H) 0
l (negative control). Scale Bar = 10 m.
3. RESULTS
Preliminary studies with both types of QDs
showed that positive QDs (QD+) associated
better with the negatively charged bacteria than
the negatively charged QDs. All further
experiments were conducted with only QD+.
3.1 Cell Viability
Viability of P. aeruginosa before and after
incubation with QDs was assessed with
Live/Dead staining. CaCl2 treatment only killed
20% of the bacterial cells, so there are enough
live bacteria to pick up QDs. Electroporation
killed approximately 70% of the cells, with few
remaining live bacteria. In contrast, simple
bacterial incubation with QDs did not affect cell
viability.
3.2 Epifluorescence Microscopy
In all three internalization methods, QDs were
found to be associated with single bacteria as
was observed with epifluorescence microscopy.
For CaCl2 transformation (Figure 1), there was
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Figure 2. QD internalization by bacterial incubation. Arrows point to bacteria that have internalized
QDs. A-D) transmitted light images. E-H) epifluorescent images. A,E) 5 l QD+; B,F) 10 l; C,G) 50
l; D, H) 0 l (negative control). Scale Bar = 10 m.
Figure 3. QD internalization by electroporation. Arrows point to bacteria that have internalized QDs.
A-D) transmitted light images. E-H) epifluorescent images. A,E) 5 l QD+; B,F) 10 l; C,G) 50 l; D,
H) 0 l (negative control). Scale Bar = 10 m.
t-test
of
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Figure 4: Confocal images of samples that were transformed using CaCl2 with 50 l of QD+. Multiple
slices of specific areas in each sample, from top to bottom (A-H respectively), are shown. Slices
demonstrate different levels within certain bacteria that maintain fluorescence (not only outside).
Depth of slices is 0.15 m. Scale Bar = 10 m.
Figure 5. Confocal images of samples that were passively incubated with 50 l of QD+. Multiple slices of
specific areas in each sample, from top to bottom (A-H respectively), are shown. Slices demonstrate different
levels within certain bacteria that maintain fluorescence (not only outside). Depth of slices is 1.00 m. Scale
Bar = 10 m.
13
Figure 6. Confocal images of samples that were electroporated with 50 l of QD+. Multiple slices of
specific areas in each sample, from top to bottom (A-H respectively), are shown. Slices demonstrate
different levels within certain bacteria that maintain fluorescence (not only outside). Depth of slices is
0.50 m. Scale Bar = 10 m.
14
ACKNOWLEDGEMENTS
A
Bact. Incub.
Control: Bact. Incub.
CaCl2 Trans
Control: CaCl2 Trans
Elect.
Control: Elect.
Fluorescence Intens
70000
60000
50000
40000
30000
20000
10000
0
1
Number of Washes
B
Bact. Incub.
Control: Bact. Incub.
CaCl2 Trans.
Control: CaCl2 Trans.
Elect.
Control: Elect.
70000
Fluorescence Intens
60000
50000
40000
30000
20000
10000
0
1
Number of Washes
C
Bact. Incub.
Control: Bact. Incub.
CaCl2 trans.
Control: CaCl2 trans.
Elect.
Control: Elect.
Fluorescence Intens
50000
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
1
Number of Washes
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