U.S. patent application number 16/437624 was filed with the patent office on 2019-10-24 for diagnostic system and process for rapid bacterial infection diagnosis.
The applicant listed for this patent is Northeastern University. Invention is credited to Edgar D. GOLUCH, Hunter J. SISMAET, Thaddaeus A, WEBSTER.
Application Number | 20190323056 16/437624 |
Document ID | / |
Family ID | 55534007 |
Filed Date | 2019-10-24 |
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United States Patent
Application |
20190323056 |
Kind Code |
A1 |
GOLUCH; Edgar D. ; et
al. |
October 24, 2019 |
Diagnostic System and Process for Rapid Bacterial Infection
Diagnosis
Abstract
Methods and devices for monitoring the viability of a biofilm
comprising Pseudomonas aeruginosa bacteria by detecting pyocyanin
are provided.
Inventors: |
GOLUCH; Edgar D.;
(Somerville, MA) ; SISMAET; Hunter J.; (Boston,
MA) ; WEBSTER; Thaddaeus A,; (Exeter, NH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Northeastern University |
Boston |
MA |
US |
|
|
Family ID: |
55534007 |
Appl. No.: |
16/437624 |
Filed: |
June 11, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15511735 |
Mar 16, 2017 |
10316348 |
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PCT/US2015/050412 |
Sep 16, 2015 |
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16437624 |
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62051099 |
Sep 16, 2014 |
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62215379 |
Sep 8, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/0636 20130101;
B01L 3/502715 20130101; G01N 2333/21 20130101; C12Q 1/18 20130101;
A61K 38/12 20130101; G01N 27/327 20130101; G01N 33/48735 20130101;
C12M 1/34 20130101; A61K 31/496 20130101; G01N 33/48707 20130101;
C12Q 1/04 20130101; C12Q 1/12 20130101; G01N 27/48 20130101; A61P
31/04 20180101; C12Q 1/22 20130101; C12Q 1/02 20130101; B01L
2300/0645 20130101; B01L 3/5027 20130101; B01L 2300/02
20130101 |
International
Class: |
C12Q 1/12 20060101
C12Q001/12; C12Q 1/22 20060101 C12Q001/22; G01N 27/48 20060101
G01N027/48; C12Q 1/02 20060101 C12Q001/02; G01N 27/327 20060101
G01N027/327; G01N 33/487 20060101 G01N033/487; B01L 3/00 20060101
B01L003/00; C12M 1/34 20060101 C12M001/34; A61K 31/496 20060101
A61K031/496; C12Q 1/18 20060101 C12Q001/18; C12Q 1/04 20060101
C12Q001/04; A61K 38/12 20060101 A61K038/12 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] The invention was developed with government support under
Grant No. 1125535 awarded by the National Science Foundation. The
government has certain rights in the invention.
Claims
1. A method of monitoring viability of a biofilm comprising
Pseudomonas aeruginosa bacteria, the method comprising: (a)
introducing a fluid sample into a microfluidic device including a
working electrode and a reference electrode; (b) performing an
electrochemical measurement to detect pyocyanin in the fluid
sample; and (c) determining a concentration of pyocyanin in the
fluid sample by using a previously determined correlation between
pyocyanin concentration and current flow through the working
electrode, the correlation comprising a linear relationship of
increasing current flow with increasing pyocyanin concentration;
wherein the pyocyanin concentration is the fluid sample provides a
measure of the viability of the biofilm.
2. The method of claim 1, wherein the microfluidic device is
disposed in a contact lens case, a urine bag, a urine collection
cup, a medication pump, a water pipe, a bioreactor, or a water
pump.
3. The method of claim 1, further comprising estimating a number of
viable cells of Pseudomonas aeruginosa in the biofilm based on the
concentration of pyocyanin determined in step (c).
4. The method of claim 1, wherein the electrochemical measurement
is selected from the group consisting of squarewave voltammetry,
linear sweep voltammetry, staircase voltammetry, cyclic
voltammetry, normal pulse voltammetry, differential pulse
voltammetry, and chronoamperometry.
5. The method of claim 4, wherein the electrochemical measurement
is square wave voltammetry and the current flow is measured in
response to one or more square wave potentials.
6. The method of claim 1, wherein: the microfluidic device
comprises a second working electrode; the working electrode is one
of an oxidizing electrode and a reducing electrode, and the second
working electrode is the other of the oxidizing electrode and the
reducing electrode; and the concentration of pyocyanin is measured
as current flow through the oxidizing electrode and the reducing
electrode.
7. The method of claim 1, wherein 10 .mu.L or less of the fluid
sample volume is introduced.
8. The method of claim 1, further comprising in step (a),
continuously introducing the fluid sample into the microfluidic
device.
9. The method of claim 8, further comprising repeating steps (a),
(b), and (c).
10. The method of claim 1, wherein a capillary or wicking material
is disposed at or near an inlet of the microfluidic device to draw
the fluid sample into the device.
11. The method of claim 1, wherein the microfluidic device is worn
by a patient or implanted in a patient.
12. The method of claim 1, wherein the microfluidic device is
embedded in a wound dressing or within or adjacent to an absorbent
pad for a wound dressing.
13. The method of claim 1, wherein the fluid sample is from a human
with cystic fibrosis, ventilator-associated pneumonia, a chronic
wound, a burn wound, a surgical implant, or a surgical site.
14. The method of claim 11, wherein the microfluidic device
comprises a second working electrode; the working electrode is one
of an oxidizing electrode and a reducing electrode, and the second
working electrode is the other of the oxidizing electrode and the
reducing electrode; and the concentration of pyocyanin is measured
as current flow through the oxidizing electrode and the reducing
electrode.
15. The method of claim 14, further comprising applying a potential
suitable for oxidizing the pyocyanin at the oxidizing electrode and
a potential suitable for reducing the pyocyanin at the reducing
electrode.
16. The method of claim 14, wherein the oxidizing electrode and the
reducing electrode are separated by a distance of about 200 to 100
nm.
17. The method of claim 1, wherein the microfluidic device is in
communication with a potentiostat operable to control voltage at
the working electrode and the reference electrode.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 .sctn. 119(e) of
U.S. Provisional Application No. 62/051,099 filed on 16 Sep. 2014,
and of U.S. Provisional Application No. 62/215,379 filed on 8 Sep.
2015. This application also is a divisional application of U.S.
application Ser. No. 15/511,735, filed on 16 Mar. 2017, which is
the U.S. national phase of PCT Application PCT/US15/50412, filed on
16 Sep. 2015. The disclosures of each of the aforementioned
applications are hereby incorporated by reference.
BACKGROUND
[0003] For more than a century, the primary clinical identification
method for bacterial infections has been plate cultures where
bacteria are isolated and purified overnight using nutrient-based
agar medium. Although rapid, automated instrumentation has been
widely regarded as the next step towards advancing bacterial
identification, these instruments still require a pure bacterial
colony obtained from a plate culture and thus a lead time of at
least 18-24 hours before any identification can be made.
(Cherkaoui, A., et al., Comparison of two matrix-assisted laser
desorption ionization-time of flight mass spectrometry methods with
conventional phenotypic identification for routine identification
of bacteria to the species level. J Clin Microbiol, 2010. 48(4): p.
1169-75. Holland, R. D., et al., Rapid identification of intact
whole bacteria based on spectral patterns using matrix-assisted
laser desorption/ionization with time-of-flight mass spectrometry.
Rapid Communications in Mass Spectrometry, 1996. 10(10): p.
1227-1232.) Likewise, molecular diagnostics such as polymerase
chain reaction require pure bacterial colonies and hours of
processing. (Meng, J., et al., Polymerase chain reaction for
detecting Escherichia coli 0157: H7. Int J Food Microbiol, 1996.
32(1-2): p. 103-13.) As a result, rapid screens for
clinically-relevant bacterial species have become an attractive
alternative option for hospitals looking for more rapid
point-of-care diagnostics. A rapid screen for Pseudomonas
aeruginosa and other clinically-relevant bacteria would allow
doctors to promptly switch from broad-spectrum antibiotics to
specific directed therapies, lowering hospital expenditures,
minimizing drug resistance, and improving patient care outcomes.
(Trenholme, G. M., et al., Clinical impact of rapid identification
and susceptibility testing of bacterial blood culture isolates. J
Clin Microbiol, 1989. 27(6): p. 1342-5.)
[0004] Additionally, the ability to monitor the effect antibiotics
have on bacteria is important for infection control. The
conventional approach to determining antibiotic efficacy requires
the creation of culture plates with the antibiotic cocktail of
choice at a series of concentrations. (J. M. Andrews, J. Antimicrob
Chemother, 2001, 48, 5-16.) After culturing (for 24 hours or
longer, depending on the strain), the plates are visually inspected
for growth. At a certain concentration, known as the minimum
inhibitory concentration (MIC), no bacterial growth is observed.
This concentration is then used to design an antibiotic schedule
for the patient. This effective approach suffers from the use of
large amounts of reagents required to produce the culture plates.
Furthermore, these screens only measure the effectiveness of the
antibiotic against planktonic cell growth; not removal of biofilms,
which are commonly associated with infections and significantly
more difficult to treat. (P. K. Singh, A. L. Schaefer, M. R.
Parsek, T. O. Moninger, M. J. Welsh and E. P. Greenberg, Nature,
2000, 407, 762-764. C. F. Schierle, M. De la Garza, T. A. Mustoe
and R. D. Galiano, Wound Repair Regen, 2009, 17, 354-359.)
[0005] An alternative is coupling microfluidics, to grow the
bacteria, with antibiotic screens. Kim et al. (2012) utilized a
microfluidic system to simultaneously expose biofilms of
Escherichia coli to eight different concentrations of antibiotics
on a single chip. (J. Kim, M. Hegde, S. H. Kim, T. K. Wood and A.
Jayaraman, Lab chip, 2012, 12, 1157-1163.) The smaller volumes,
inherent in microfluidic devices, along with the ability to produce
multiple concentration gradients provided a faster, cheaper
alternative to current antibiotic susceptibility tests. By flowing
antibiotics over the grown biofilms, researchers more closely
simulated in vivo conditions. Many current microfluidic studies
determine biofilm viability based on the presence of fluorescent
proteins during exposure to antibiotics. (J. Kim, H. D. Park and S.
Chung, Microfluidic approaches to bacterial biofilm formation,
Molecules, 2012, 17, 9818-9834. K. P. Kim, Y. G. Kim, C. H. Choi,
H. E. Kim, S. H. Lee, W. S. Chang and C. S. Lee, Lab chip, 2010,
10, 3296-3299.) While these methods are certainly robust and
promising, the fluorescent signal requires expensive optical
equipment and genetically modified bacteria or selective labels.
(L. Richter, C. Stepper, A. Mak, A. Reinthaler, R. Heer, M. Kast,
H. Bruckl and P. Ertl, Lab Chip, 2007, 7, 1723-1731. H.-Y. N.
Holman, R. Miles, Z. Hao, E. Wozei, L. M. Anderson and H. Yang,
Anal Chem, 2009, 81, 8564-8570. Y. Yawata, K. Toda, E. Setoyama, J.
Fukuda, H. Suzuki, H. Uchiyama and N. Nomura, J Biosci Bioeng,
2010, 110, 130-133. Y. Yawata, K. Toda, E. Setoyama, J. Fukuda, H.
Suzuki, H. Uchiyama and N. Nomura, J Biosci Bioeng, 2010, 110,
377-380.) A cheaper and easier method of determining the relative
amount of live cells in a biofilm under exposure to antibiotics can
be achieved by monitoring the electrochemical response of the
system. Robust bacterial biofilms produce a plethora of molecules
that promote communication, defend the colony, and cause infection.
(M. B. Miller and B. L. Bassler, Annu Rev Microbiol, 2001, 55,
165-199. M. D. P. Willcox, H. Zhu, T. C. R. Conibear, E. B. H.
Hume, M. Givskov, S. Kjelleberg and S. A. Rice, Microbiology, 2008,
154, 2184-2194. G. W. Lau, D. J. Hassett, H. Ran and F. Kong,
Trends Mol Med, 2004, 10, 599-606.) Of interest are molecules that
provide information about the condition of the biofilm, which can
be detected by electrochemical methods.
SUMMARY OF THE INVENTION
[0006] The invention relates to electrochemical methods and devices
that offer a simple and inexpensive alternative for immediate
identification of bacterial infection due to the presence of
Pseudomonas aeruginosa. In some embodiments, an inexpensive,
disposable electrochemical sensor can be used to rapidly screen for
the presence of P. aeruginosa in clinical wound effluent samples.
This technology can be incorporated in a rapid, point-of-care
diagnostic for P. aeruginosa, allowing for better antimicrobial
stewardship and improved patient care outcomes.
[0007] Other aspects of the methods and devices include the
following:
1. A method of monitoring viability of a biofilm comprising
Pseudomonas aeruginosa bacteria in a patient, the method
comprising:
[0008] (a) introducing a fluid sample from the patient into a
microfluidic device including a working electrode and a reference
electrode;
[0009] (b) performing an electrochemical measurement to detect
pyocyanin in the fluid sample; and
[0010] (c) determining a concentration of pyocyanin in the fluid
sample by using a previously determined correlation between
pyocyanin concentration and current flow through the working
electrode;
[0011] wherein the pyocyanin concentration is the fluid sample
provides a measure of the viability of the biofilm.
2. The method of item 1, further comprising estimating a number of
viable cells of Pseudomonas aeruginosa in the biofilm based on the
concentration of pyocyanin determined in step (c). 3. The method of
any of items 1-2, wherein the concentration of pyocyanin is
determined in step (c) based on a linear relationship of the
current flow through the working electrode. 4. The method of item
3, wherein the concentration in .mu.M of pyocyanin is equal to the
current flow in .mu.A through the working electrode divided by
0.18. 5. The method of any of items 1-4, wherein if the current
flow through the working electrode is less than 1 .mu.A, the
pyocyanin concentration is considered to be zero. 6. The method of
any of items 1-5, wherein if the current flow through the working
electrode is less than 1 .mu.A, the biofilm is considered nonviable
or absent. 7. The method of any of items 1-6, wherein the
electrochemical measurement is selected from the group consisting
of squarewave voltammetry, linear sweep voltammetry, staircase
voltammetry, cyclic voltammetry, normal pulse voltammetry,
differential pulse voltammetry, and chronoamperometry. 8. The
method of item 7, wherein the electrochemical measurement is square
wave voltammetry and the current flow is measured in response to
one or more square wave potentials. 9. The method of any of items
1-8, wherein:
[0012] the microfluidic device comprises a second working
electrode;
[0013] the working electrode is one of an oxidizing electrode and a
reducing electrode, and the second working electrode is the other
of the oxidizing electrode and the reducing electrode; and
[0014] the concentration of pyocyanin is measured as current flow
through the oxidizing electrode and the reducing electrode.
10. The method of item 9, further comprising applying a potential
suitable for oxidizing the pyocyanin at the oxidizing electrode and
a potential suitable for reducing the pyocyanin at the reducing
electrode. 11. The method of items 9-10, wherein the oxidizing
electrode and the reducing electrode are separated by a distance of
about 200 to 100 nm. 12. The method of any of items 1-11, wherein
10 .mu.L or less of the fluid sample volume is introduced. 13. The
method of any of items 1-12, further comprising in step (a),
continuously introducing the fluid sample into the microfluidic
device. 14. The method of any of items 1-12, further comprising
repeating steps (a), (b), and (c). 15. The method of item 14,
wherein steps (a), (b), and (c) are repeated at least every 6
hours. 16. The method of any of items 1-15, wherein a capillary or
wicking material is disposed at or near an inlet of the
microfluidic device to draw the fluid sample into the device. 17.
The method of any of items 1-16, wherein the microfluidic device is
in communication with a potentiostat operable to control voltage at
the working electrode and the reference electrode. 18. The method
of item 17, wherein the microfluidic device is connectable to the
potentiostat by a cable. 19. The method of any of items 1-18,
wherein the microfluidic device is disposable. 20. The method of
any of items 1-19, wherein the microfluidic device is worn by the
patient or implanted in the patient. 21. The method of any of items
1-20, wherein the microfluidic device is embedded in a wound
dressing or within or adjacent to an absorbent pad for a wound
dressing. 22. The method of any of items 1-19, wherein the
microfluidic device is present in a wound dressing, a bandage, a
surgical implant, a catheter, a ventilator mask, a face mask, a
surgical mask, or an intubation tube. 23. The method of any of
items 1-19, wherein the microfluidic device is present in a contact
lens case, a urine collection cup, or a urine bag. 24. The method
of any of items 1-23, wherein the microfluidic device is in
wireless communication with a remote monitoring station. 25. The
method of any of items 1-24, wherein the fluid sample is from a
human with cystic fibrosis, ventilator-associated pneumonia, a
chronic wound, a burn wound, a surgical implant, or a surgical
site. 26. The method of any of items 1-25, wherein the fluid sample
is a bodily fluid selected from the group consisting of wound
exudate, bronchial lavage, sputum, urine, saliva, spinal fluid,
tears, and blood. 27. A method of monitoring effectiveness of an
antibiotic treatment of a Pseudomonas aeruginosa infection in a
patient, the method comprising:
[0015] (a) introducing a fluid sample from the patient into a
microfluidic device including a working electrode and a reference
electrode;
[0016] (b) performing an electrochemical measurement to detect
pyocyanin in the fluid sample; and
[0017] (c) determining a concentration of pyocyanin in the fluid
sample by using a previously determined correlation between known
concentrations of the pyocyanin and a current flow through the
working electrode;
[0018] wherein the pyocyanin concentration in the fluid sample
provides a measure of the effectiveness of the antibiotic
treatment.
28. The method of item 27, further comprising administering an
increased dose of the antibiotic if the concentration of the
pyocyanin is above a threshold level. 29. The method of item 28,
wherein the threshold level of pyocyanin is a concentration of at
least 5 .mu.M. 30. The method of item 27, further comprising
administering an increased dose of the antibiotic if the
concentration of the pyocyanin does not drop below a threshold
level after a predetermined time interval. 31. The method of item
30, wherein the predetermined time interval is at least 12 hours.
32. The method of items 30-31, wherein the threshold level of
pyocyanin is a concentration of at least 5 .mu.M. 33. The method of
item 27, further comprising administering a decreased dose of the
antibiotic or stopping the antibiotic if the concentration of the
pyocyanin drops below a threshold level. 34. The method of item 33,
wherein the threshold level of pyocyanin is a concentration of at
least 5 .mu.M. 35. The method of any of items 27-34, wherein the
antibiotic is colistin sulfate or ciprofloxacin. 36. The method of
any of items 27-35 further comprising administering an additional
antibiotic or other pharmaceutical agent. 37. The method of any of
items 27-36, wherein in step (b), continuously introducing a fluid
sample into the microfluidic device. 38. The method of any of items
27-36, further comprising repeating step (b) and step (c) interval.
39. The method of item 38, wherein steps (b) and (c) are repeated
at least every 6 hours. 40. The method of any of items 27-39,
wherein the electrochemical measurement is selected from the group
consisting of squarewave voltammetry, linear sweep voltammetry,
staircase voltammetry, cyclic voltammetry, normal pulse
voltammetry, differential pulse voltammetry, and chronoamperometry.
41. The method of item 40, wherein the current flow is measured in
response to one or more square wave potentials. 42. The method of
any of items 27-41, wherein:
[0019] the microfluidic device comprises a second working
electrode;
[0020] the working electrode is one of an oxidizing electrode and a
reducing electrode, and the second working electrode is the other
of the oxidizing electrode and the reducing electrode; and
[0021] the concentration of pyocyanin is measured as current flow
through the oxidizing electrode and the reducing electrode.
43. The method of item 42, further comprising applying a potential
suitable for oxidizing the pyocyanin at the oxidizing electrode and
a potential suitable for reducing the pyocyanin at the reducing
electrode. 44. The method of any of items 42-43, wherein the
oxidizing electrode and the reducing electrode are separated by a
distance of about 200 to 100 nm. 45. The method of any of items
27-44, wherein a capillary or wicking material is disposed at or
near an inlet to the microfluidic device to draw the fluid sample
into the microfluidic device. 46. The method of any of items 27-45,
wherein the device is in communication with a potentiostat operable
to control voltage at the working electrode and the reference
electrode. 47. The method of item 46, wherein the microfluidic
device is connectable to the potentiostat by a cable. 48. The
method of any of items 27-47, wherein the microfluidic device is
disposable. 49. The method of any of items 27-48, wherein the
microfluidic device is worn by the patient or implanted in the
patient. 50. The method of any of items 27-49, wherein the
microfluidic device is embedded in a wound dressing or within or
adjacent to an absorbent pad for a wound dressing. 51. The method
of any of items 27-49, wherein the microfluidic device is present
in a wound dressing, a surgical implant, a catheter, a ventilator
mask, or an intubation tube. 52. The method of any of items 27-48,
wherein the microfluidic device is present in a contact lens case,
a urine collection cup, or a urine bag. 53. The method of any of
items 27-52, wherein the microfluidic device is in wireless
communication with a remote monitoring station. 54. The method of
any of items 27-53, wherein the fluid sample is from a human with
cystic fibrosis, ventilator-associated pneumonia, a chronic wound,
a burn wound, a surgical implant, or a surgical site. 55. The
method of any of items 27-54, wherein the fluid sample is a bodily
fluid selected from the group consisting of wound exudate,
bronchial lavage, sputum, urine, saliva, spinal fluid, tears, and
blood. 56. A method of screening effectiveness of an antibiotic
against a biofilm comprising Pseudomonas aeruginosa, the method
comprising:
[0022] (a) introducing a sample comprising Pseudomonas aeruginosa
into a growth chamber in a microfluidic device including a working
electrode and a reference electrode;
[0023] (b) allowing the Pseudomonas aeruginosa to grow and form a
biofilm in the growth chamber;
[0024] (c) introducing an antibiotic at a selected concentration
into the growth chamber of the device;
[0025] (d) performing an electrochemical measurement to detect
pyocyanin in the fluid sample;
[0026] (e) determining a concentration of pyocyanin in the sample
by using a previously determined correlation between known
concentrations of the pyocyanin and a current flow through the
working electrode, wherein the concentration of pyocyanin below a
threshold indicates effectiveness of the antibiotic.
57. The method of item 56, wherein the threshold level comprises a
concentration of at least 5 .mu.M. 58. The method of items 56-57,
further comprising providing an indication of a presence of
Pseudomonas aeruginosa when the concentration of pyocyanin is above
the threshold level, the threshold level comprising a concentration
of at least 5 .mu.M. 59. The method of any of items 56-58, further
comprising estimating a number of cells of Pseudomonas aeruginosa
based on the concentration of pyocyanin. 60. The method of any of
items 56-59, wherein step (a) comprises introducing the sample
comprising Pseudomonas aeruginosa into a plurality of growth
chambers in the microfluidic device; and step (c) comprises
simultaneously introducing the antibiotic at selected different
concentrations into each growth chamber to screen the effectiveness
of multiple concentrations of the antibiotic. 61. The method of any
of items 56-60, further comprising repeating steps (a) through (c)
with a different concentration of the antibiotic. 62. The method of
any of items 56-60, further comprising in step (b), continuously
introducing the antibiotic into the growth chamber. 63. The method
of any of items 56-62, wherein the electrochemical measurement is
selected from the group consisting of squarewave voltammetry,
linear sweep voltammetry, staircase voltammetry, cyclic
voltammetry, normal pulse voltammetry, differential pulse
voltammetry, and chronoamperometry. 64. The method of item 63,
wherein the current flow is measured in response to one or more
square wave potentials. 65. The method of any of items 56-62,
wherein:
[0027] the microfluidic device comprises a second working
electrode;
[0028] the working electrode comprises one of an oxidizing
electrode and a reducing electrode, and the second working
electrode comprises the other of the oxidizing electrode and the
reducing electrode; and
[0029] the concentration of pyocyanin is measured as current flow
through the oxidizing electrode and the reducing electrode.
66. The method of item 65, further comprising applying a potential
suitable for oxidizing the pyocyanin at the oxidizing electrode and
a potential suitable for reducing the pyocyanin at the reducing
electrode. 67. The method of items 65-66, wherein the oxidizing
electrode and the reducing electrode are separated by a distance of
about 200 to 100 nm. 68. The method of any of items 56-67, wherein
a capillary or wicking material is disposed at or near an inlet of
the microfluidic device to draw the fluid sample into the device.
69. The method of any of items 56-68, wherein the microfluidic
device is in communication with a potentiostat operable to control
voltage at the working electrode and the reference electrode. 70.
The method of item 69, wherein the microfluidic device is
connectable to the potentiostat by a cable. 71. The method of any
of items 56-70, wherein the microfluidic device is disposable. 72.
A method of monitoring viability of a biofilm comprising
Pseudomonas aeruginosa bacteria, the method comprising:
[0030] (a) introducing a fluid sample into a microfluidic device
including a working electrode and a reference electrode;
[0031] (b) performing an electrochemical measurement to detect
pyocyanin in the fluid sample; and
[0032] (c) determining a concentration of pyocyanin in the fluid
sample by using a previously determined correlation between
pyocyanin concentration and current flow through the working
electrode;
[0033] wherein the pyocyanin concentration is the fluid sample
provides a measure of the viability of the biofilm.
73. The method of item 72, wherein the microfluidic device is
disposed in a contact lens case, a urine bag, a urine collection
cup, a medication pump, a water pipe, a bioreactor, or a water
pump. 74. The method of items 72-73, wherein the electrochemical
measurement is selected from the group consisting of squarewave
voltammetry, linear sweep voltammetry, staircase voltammetry,
cyclic voltammetry, normal pulse voltammetry, differential pulse
voltammetry, and chronoamperometry. 75. The method of item 74,
wherein the electrochemical measurement is square wave voltammetry
and the current flow is measured in response to one or more square
wave potentials. 76. The method of any of items 72-75, wherein:
[0034] the microfluidic device comprises a second working
electrode;
[0035] the working electrode is one of an oxidizing electrode and a
reducing electrode, and the second working electrode is the other
of the oxidizing electrode and the reducing electrode; and
[0036] the concentration of pyocyanin is measured as current flow
through the oxidizing electrode and the reducing electrode.
77. The method of item 76, further comprising applying a potential
suitable for oxidizing the pyocyanin at the oxidizing electrode and
a potential suitable for reducing the pyocyanin at the reducing
electrode. 78. The method of items 76-77, wherein the oxidizing
electrode and the reducing electrode are separated by a distance of
about 200 to 100 nm. 79. The method of any of items 72-78, wherein
a capillary or wicking material is disposed at or near an inlet of
the microfluidic device to draw the fluid sample into the device.
80. The method of any of items 72-79, wherein the microfluidic
device is in communication with a potentiostat operable to control
voltage at the working electrode and the reference electrode. 81.
The method of item 80, wherein the microfluidic device is
connectable to the potentiostat by a cable. 82. The method of items
76-81, wherein the microfluidic device is disposable. 83. A device
for monitoring viability of a biofilm comprising Pseudomonas
aeruginosa, the device comprising:
[0037] a sensor comprising a microfluidic or nanofluidic electrode
assembly comprising a microfluidic or nanofluidic channel disposed
in a substrate, a working electrode disposed in the microfluidic or
nanofluidic channel, and a reference electrode disposed in the
microfluidic or nanofluidic channel;
[0038] a control system comprising a processor and memory,
machine-readable instructions stored in the memory that, upon
execution by the processor, control voltage at the working
electrode and the reference electrode, and determine a
concentration of pyocyanin in a fluid sample in the microfluidic or
nanofluidic channel by using a previously determined correlation
between known concentrations of pyocyanin and current flow through
the working electrode.
84. The device of item 83, wherein the device is operable to
provide an indication of the concentration of pyocyanin over a
range from about 5 .mu.M to about 1 mM. 85. The device of item 84,
wherein the device is operable to provide an indication of the
concentration of pyocyanin over a range from about 5 .mu.M to a
solubility limit of the pyocyanin 86. The device of any of items
83-85, wherein the controller is operable to provide an indication
of a presence of Pseudomonas aeruginosa when the concentration of
pyocyanin is above a threshold level of about 5 .mu.M. 87. The
device of any of items 83-86, wherein the controller is operable to
determine a number of cells of Pseudomonas aeruginosa in the
biofilm based on the determined concentration of pyocyanin. 88. The
device of any of items 83-87, wherein the concentration of
pyocyanin is determined from a linear relationship, stored in the
memory, of the current flow through the working electrode. 89. The
device of item 88, wherein the concentration of pyocyanin in .mu.M
is equal to the current flow in .mu.A through the working electrode
divided by 0.18. 90. The device of any of items 83-89, wherein the
control system further comprises a potentiostat in communication
with the processor and the sensor, the potentiostat operable to
control the voltage at the working electrode and the reference
electrode. 91. The device of item 90, wherein the potentiostat is
in communication with the processor via a hardwired connection, via
a removable cable, or via a wireless connection. 92. The device of
item 91, wherein the potentiostat is battery powered. 93. The
device of any of items 83-92, wherein the sensor is disposable and
is connectable to the potentiostat by a cable. 94. The device of
any of items 83-93, wherein the controller is operable to perform
an electrochemical measurement selected from the group consisting
of squarewave voltammetry, linear sweep voltammetry, staircase
voltammetry, cyclic voltammetry, normal pulse voltammetry,
differential pulse voltammetry, and chronoamperometry. 95. The
device of item 94, wherein the current flow is measured in response
to one or more square wave potentials. 96. The device of any of
items 83-95, further comprising a display in communication with the
controller, the display operable to display one or more of the
determined concentration of pyocyanin, an indication of a presence
of Pseudomonas aeruginosa, and an indication of a number of cells
of Pseudomonas aeruginosa. 97. The device of any of items 83-96,
wherein the sensor is embedded in a wound dressing or within or
adjacent to an absorbent pad for a wound dressing. 98. The device
of any of items 83-96, wherein the sensor is present in a wound
dressing, a surgical implant, a catheter, a ventilator mast, an
intubation tube, or a contact lens case. 99. The device of any of
items 83-99, wherein the controller further includes a
telecommunications network connection.
DESCRIPTION OF THE DRAWINGS
[0039] The invention will be more fully understood from the
following detailed description taken in conjunction with the
accompanying drawings in which:
[0040] FIG. 1 illustrates a disposable, screen-printed electrode
sensor with mesh modification for small-volume analysis.
[0041] FIG. 2 is a graph of square wave voltammograms of wound
fluid exudate in which a pyocyanin peak indicates the presence of
Pseudomonas aeruginosa in the sample.
[0042] FIG. 3 illustrates a series of square wave voltammograms of
wound fluid exudate.
[0043] FIG. 4 illustrates baseline-subtracted square wave
voltammograms of wound fluid exudate of FIG. 3.
[0044] FIG. 5 illustrates square wave voltammograms of wound fluid
exudate for two different volumes tested before (left) and after
(right) baseline subtraction.
[0045] FIG. 6 illustrates square wave voltammograms of wound fluid
exudate of negative control (left), positive controls (middle), and
positive control at different volumes (right).
[0046] FIG. 7 illustrates baseline-subtracted square wave
voltammograms of wound fluid exudate of FIG. 6 of negative control
(left), positive controls (middle), and positive control at
different volumes (right).
[0047] FIG. 8 is a bright-field image of a membrane mesh placed on
top of an electrode sensor for small-volume analysis.
[0048] FIGS. 9A, 9B, and 9C illustrate experimental apparatus used
in monitoring the susceptibility of Pseudomonas aeruginosa
biofilms. A) Finished device connected to a potentiostat. Inlets
and outlets contain filters (pore size 0.2 .mu.m) to prevent PA14
from leaving the channels. B) Schematic of the sensor covered with
a microfluidic chamber (not to scale). Bacteria are trapped in the
chamber while fluid moves in and out. C) Scanning electron
micrograph (SEM) of PA14 grown on top of the carbon working
electrode after overnight growth under stagnant conditions.
Reference, Working, and Counter Electrodes (RE, WE, and CE,
respectively).
[0049] FIG. 10 illustrates square wave voltammetry (SWV) scans of
PA14 and E. coli (solid and dashed lines respectively) cultured in
trypticase soy broth (TSB) after loading 24 .mu.L of overnight
culture after A) 0 h, B) 12 h, C) 22 h, D) 35 h, E) 40 h, and F) 45
h. Flow of fresh TSB at 100 nL/min was initiated at 22 h. SWV scans
performed from -0.5 to 0.2 V at a frequency of 15 Hz and an
amplitude voltage of 50 mV.
[0050] FIG. 11A illustrates response of PA14 biofilms at selected
time points during the 48 hour experiments. Left axis: average peak
current (blank subtracted) measured over time in PA14 cultures
exposed to colistin sulfate at 0 (right slash), 4 (left slash, low
MIC), 16 (crosses, High MIC), and 100 mg/L (no slash lines). Right
axis: approximate pyocyanin concentration based on calibration
curve. * indicates time points where only two replicates were used.
** indicates P<0.05 from ANOVA analysis.
[0051] FIG. 11B illustrates live cell counts from PA14 after
exposure to 0, 4, 100 mg/L colistin sulfate. Error bars are one
standard deviation of mean for 3 samples. ** indicates P<0.05
from ANOVA analysis between the number of cells.
[0052] FIG. 12 illustrates PA14 exposed to 100 mg/L and 4 mg/L
colistin sulfate for 20 hours within PDMS chambers then spotted
onto TSB plates. Photographs of a plate after incubation at
37.degree. C. for A) 4.3 h, B) 6.5 h, and C) 74.3 h (removed from
the incubator after 24 h to avoid drying and grown at room
temperature (.apprxeq.23.degree. C.)). Arrows highlight the
locations of the first observed PA14 colonies. A vertical line
divides the areas on the plate where PA14 exposed to 100 mg/L
(left) and 4 mg/L (right) colistin sulfate were spotted.
[0053] FIG. 13 illustrates maximum current from SWVs of pyocyanin
in 30 g/L TSB from 0 to 50 .mu.M. SWVs were performed from -0.5 to
0 V at a frequency of 15 Hz and an amplitude voltage of 50 mV.
Linear fit: 1/.mu.A=0.18[PYO]/.mu.M; where I=current and
[PYO]=concentration of pyocyanin in TSB.
[0054] FIG. 14 illustrates SEM images of P. aeruginosa grown on the
working electrode of a three electrode cell. From left to right,
SEM images were taken at a magnification of 9,000.times.,
15,000.times., and 30,000.times. at 3 kV. Note the presence of a
large number of cells in all three images interlaced embedded in
extracellular matrix.
[0055] FIG. 15 illustrates SEM images of A) P. aeruginosa cells in
the access hole of a PDMS device leading to the chamber area
containing the sensor, and B) cells attached to the surface of the
PDMS that forms the top of the sensing chamber.
[0056] FIG. 16 illustrates response of PA14 biofilms at selected
time points during the 48 hour experiments. (BL=Bacteria loaded
into the chamber). Left axis: average peak current (blank
subtracted) measured over time in PA14 cultures exposed to colistin
sulfate at 0 (right slash), 4 (left slash, low MIC), 16 (crosses,
High MIC), and 100 mg/L (no slash lines). Right axis: Approximate
pyocyanin concentration based on calibration curve. * indicates
time points where only two replicates were used. ** indicates
P<0.05 from ANOVA analysis of 16 and 100 mg/L antibiotic
concentrations against the control.
[0057] FIG. 17 illustrates SWVs of PA14 (solid lines) and E. coli
(dashed lines) cultured in trypticase soy broth (TSB) after loading
24 .mu.L of overnight culture after A) 0 h, B) 12 h, C) 22 h, D) 35
h, E) 40 h, and F) 45 h. Flow of 4 mg/L colistin sulfate in fresh
TSB at 100 nL/min was initiated at 22 h. SWVs performed from -0.5
to 0.2 V at a frequency of 15 Hz and an amplitude voltage of 50
mV.
[0058] FIG. 18 illustrates SWVs of PA14 (solid lines) and E. coli
(dashed lines) cultured in trypticase soy broth (TSB) after loading
24 .mu.L of overnight culture after A) 0 h, B) 12 h, C) 22 h, D) 35
h, E) 40 h, and F) 45 h. Flow of 16 mg/L colistin sulfate in fresh
TSB at 100 nL/min was initiated at 22 h. SWVs performed from -0.5
to 0.2 V at a frequency of 15 Hz and an amplitude voltage of 50
mV.
[0059] FIG. 19 illustrates SWVs of PA14 (solid lines) and E. coli
(dashed lines) cultured in trypticase soy broth (TSB) after loading
24 .mu.L of overnight culture after A) 0 h, B) 12 h, C) 22 h, D) 35
h, E) 40 h, and F) 45 h. Flow of 100 mg/L colistin sulfate in fresh
TSB at 100 nL/min was initiated at 22 h. SWVs performed from -0.5
to 0.2 V at a frequency of 15 Hz and an amplitude voltage of 50
mV.
[0060] FIG. 20 illustrates SWVs of P. aeruginosa grown in TSB
without flow for A) 12 h. The cells were then exposed to flowing
100 mg/L ampicillin (light lines) or colistin sulfate (dark lines)
in TSB starting at B) 22 h. SWVs C) 35 h and D) 40 h after the
start of the experiment. Baseline signal was subtracted from
resulting scans. SWV performed from -0.5 to 0.2 volts at a
frequency of 15 Hz and an amplitude voltage of 50 mV.
[0061] FIG. 21 illustrates images of PA14 colonies after 20 hours
of growth at 37.degree. C. on cetrimide agar plates mixed with
different concentrations of colistin sulfate.
[0062] FIG. 22 illustrates PA14 streaked onto TSB after exposure to
100 mg/L and 4 mg/L colistin for 20 hours within PDMS flow
chambers. Photographs show the plate after A) 4.33 h, B) 6.5 h, and
C) 74.3 h of incubation. The plate was incubated at 37.degree. C.
for the first 24 hours, then grown at room temperature for the
remaining time to prevent the agar from drying out.
[0063] FIG. 23 is a schematic illustration of one embodiment of a
sensor for monitoring viability of a biofilm comprising Pseudomonas
aeruginosa and performing other methods as described herein.
[0064] FIG. 24 is a schematic illustration of an embodiment of a
device for monitoring viability of a biofilm comprising Pseudomonas
aeruginosa.
DETAILED DESCRIPTION OF THE INVENTION
[0065] This application incorporates by reference the entire
disclosure of U.S. Provisional Application No. 62/051,099 filed on
Sep. 16, 2014, entitled "Diagnostic System and Process for Rapid
Bacterial Infection Diagnosis", and the entire disclosure of U.S.
Provisional Application No. 62/215,379 filed on Sep. 8, 2015,
entitled "Diagnostic System and Process for Rapid Bacterial
Infection Diagnosis".
[0066] The present invention relates to using an inexpensive,
disposable electrochemical sensor for the rapid identification of
Pseudomonas aeruginosa in clinical patient samples. The presence of
P. aeruginosa can be electrochemically determined by its production
of pyocyanin (PYO), a unique, redox-active chemical marker. In some
embodiments, a simple, electrochemical detection strategy is
provided that requires no sample preparation, takes less than a
minute to operate, and requires only 7.5 microliters of sample to
complete the analysis.
[0067] Electrochemically monitoring the viability of P. aeruginosa
cells in a microfluidic system has previously been demonstrated.
(L. Pires, K. Sachsenheimer, T. Kleintschek, A. Waldbaur, T.
Schwartz and B. E. Rapp, Biosens Bioelectron, 2013, 47, 157-163.)
Pires et al. (2013) combined impedance and amperometric
measurements to simultaneously monitor the growth and respiration
of P. aeruginosa cells. This approach emphasizes the potential to
non-destructively observe P. aeruginosa, but it lacks the ability
to measure the produced PYO itself, a potential marker of cell
viability and virulence. (X. Mulet, G. Cabot, A. A. Ocampo-Sosa, M.
A. Dominguez, L. Zamorano, C. Juan, F. Tubau, C. Rodriguez, B.
Moya, C. Pella, L. Martinez-Martinez and A. Oliver, Antimicrob
Agents Chemother, 2013, 57, 5527-5535.) One way of measuring
excreted PYO electrochemically is accomplished by square wave
voltammetry (SWV) over the range of voltages where PYO is reduced
(half wave potential is approximately -250 mV vs. Ag/AgCl
reference) via the following reaction:
[PYO].sub.ox+2H.sup.++2e.sup.-[PYO].sub.red
(T. A. Webster and E. D. Goluch, Lab chip, 2012, 12, 5195-5201.)
The ability to measure a virulence factor as it relates to the
amount of live cells in a biofilm during exposure to antibiotics,
could help in determining effective treatment procedures.
[0068] As used herein, a microfluidic device can include a
nanofluidic device. Also as used herein, the term "nanoscale"
refers to an object or a feature whose size is in the range from
about 1 nm to about 999 nm, or to less than 1 .mu.m. The term
"microscale" refers to an object of feature whose size is in the
range from about 1 .mu.m to about 999 .mu.m, or to less than 1 mm.
A nanofluidic device as used herein is a device having at least one
dimension, such as a channel diameter, of nanoscale size. A
microfluidic device as used herein is a device having at least one
dimension, such as a channel diameter or channel length, in the
microscale range.
[0069] In some embodiments, methods are provided of monitoring
viability of a biofilm comprising Pseudomonas aeruginosa bacteria
based on the detection of pyocyanin. In some embodiments, a method
of monitoring viability of a biofilm comprising Pseudomonas
aeruginosa bacteria in a patient includes the steps of (a)
introducing a fluid sample from the patient into a microfluidic
device including a working electrode and a reference electrode; (b)
performing an electrochemical measurement to detect pyocyanin in
the fluid sample; and (c) determining a concentration of pyocyanin
in the fluid sample by using a previously determined correlation
between pyocyanin concentration and current flow through the
working electrode. The pyocyanin concentration is the fluid sample
provides a measure of the viability of the biofilm.
[0070] More particularly, a fluid sample from a patient in
introduced into a microfluidic device including a working electrode
and a reference electrode. Suitable microfluidic devices are
described in WO/2014/015333 and WO/2015/031798, the disclosures of
which are incorporated by reference herein. An electrochemical
measurement is performed to detect pyocyanin in the fluid sample,
and a concentration of pyocyanin in the fluid sample is determined
by using a previously determined correlation between pyocyanin
concentration and current flow through the working electrode. The
pyocyanin concentration is the fluid sample provides a measure of
the viability of the biofilm. The concentration of pyocyanin can be
determined based on a linear relationship of the current flow
through the working electrode. See, for example, FIG. 13, described
further below. In some embodiments, the concentration in .mu.M of
pyocyanin is equal to the current flow in .mu.A through the working
electrode divided by 0.18. In other embodiments, the current flow
in .mu.A through the working electrode divided by 0.10, 0.14, 0.16,
0.20, 0.22, or 0.26. In some embodiments, if the current flow
through the working electrode is less than 1 .mu.A, the pyocyanin
concentration is considered to be zero.
[0071] In some embodiments, if the current flow through the working
electrode is less than 1 .mu.A, the biofilm is considered nonviable
or absent. In some embodiments, a number of viable cells of
Pseudomonas aeruginosa in the biofilm can be estimated based on the
determined concentration of pyocyanin. (T. A. Webster, H. J.
Sismaet, A. F. Sattler, E. D. Goluch, Improved monitoring of P.
aeruginosa on agar plates, Anal. Methods, 2015, 7, 7150-7155.)
[0072] The electrochemical measurement can be made in any suitable
manner. For example, the electrochemical measurement can be made by
squarewave voltammetry, linear sweep voltammetry, staircase
voltammetry, cyclic voltammetry, normal pulse voltammetry,
differential pulse voltammetry, and chronoamperometry. In some
embodiments, the electrochemical measurement is square wave
voltammetry and the current flow is measured in response to one or
more square wave potentials.
[0073] In some embodiments, the microfluidic device can include an
oxidizing electrode and a reducing electrode (the working
electrodes). The concentration of pyocyanin is measured as current
flow through the oxidizing electrode and the reducing electrode. A
potential suitable for oxidizing the pyocyanin is applied at the
oxidizing electrode and a potential suitable for reducing the
pyocyanin is applied at the reducing electrode.
[0074] The working electrodes can make up a wall or part of a wall
of a channel, such as a microfluidic channel or a nanofluidic
channel, into which the fluid sample is introduced and within which
the redox reaction takes place. In some embodiments, the oxidizing
electrode and the reducing electrode are separated by a distance of
about 200 to 100 nm. In other embodiments, the distance can be from
about 20 nm to about 100 nm, or from about 20 nm to about 40 nm, or
from about 40 nm to about 60 nm, or from about 60 nm to about 80
nm, or from about 80 nm to about 100 nm, or from about 100 nm to
about 150 nm.
[0075] The surface area of the working electrodes can be selected
to accommodate a desired size of the device. A larger surface area
generally improves the signal and sensitivity of the device. For
example, in different embodiments, the surface area of each working
electrode can be about 100, 200, 300, 400, 500, 800, 1000, 200,
3000, 5000, 10000, 50000, 100000, 200000, or 500000 nm.sup.2, or 1,
2, 5, 10 .mu.m.sup.2, or greater.
[0076] In other embodiments, the fluid sample can be introduced
into a well, chamber, or another form of receptacle in which the
reaction can take place. The volume of the channel, well, chamber
or other receptacle can be less than about 50 nanoliters (nL), less
than about 10 nL, less than about 1 nL, less than about 100
picoliters (pL), less than about 50 pL, less than about 10 pL, less
than about 5 pL, or less than about 1 pL.
[0077] In some embodiments, a sample volume that is introduced into
the microfluidic device can be less than 100 .mu.L, less than 50
.mu.L, less than 20 .mu.L, less than 10 .mu.L, less than 5 .mu.L,
less than 2 .mu.L, or less than 1 .mu.L.
[0078] In some embodiments, the fluid sample can be introduced
continuously into the microfluidic device. In other embodiments,
fluid samples are introduced repeated into the microfluidic device.
For example, the steps of introducing a fluid sample into the
device, performing an electrochemical measurement to detect
pyocyanin in the fluid sample, and determining a concentration of
pyocyanin in the fluid sample can be performed repeatedly at time
intervals. In some embodiments, the steps can be repeated at least
every 6 hours, every 12 hours, every 18 hours, every 24 hours, or
every 48 hours.
[0079] In some embodiments, a capillary or wicking material can be
disposed at or near an inlet of the microfluidic device to draw the
fluid sample into the device. Is some embodiments, a matrix
material can be disposed at or near an inlet of a microfluidic
device, for example, to isolate the electrodes from the bacteria
while permitting passage of pyocyanin to access the electrodes.
[0080] One exemplary embodiment of a microfluidic device is
illustrated in FIG. 23. The device includes a first working
electrode 510 and a second working electrode 520. Pyocyanin 400 in
a channel between the electrodes undergoes a reduction-oxidation
cycle at the first 510 and second 520 electrodes, indicated
schematically with arrows.
[0081] In some embodiments, the microfluidic device can be in
communication with a potentiostat operable to control voltage at
the working electrode and the reference electrode. See FIG. 24. The
microfluidic device can be connectable to the potentiostat by a
cable. The microfluidic device can be in wireless communication
with a remote monitoring station. The microfluidic device can be
disposable, such as a disposable sensor that can be disconnected
from the potentiostat to allow a fresh sensor to be used.
[0082] In some embodiments, the microfluidic device can be worn by
the patient or implanted in the patient. For example, in some
embodiments, the microfluidic device can be embedded in a wound
dressing or within or adjacent to an absorbent pad for a wound
dressing. With such embodiments, a wound can be continuously or
repeatedly monitored for the presence of Pseudomonas aeruginosa,
and upon detection of pyocyanin, an appropriate selective
antibiotic can be administered to the patient.
[0083] In other embodiments, the microfluidic device can be present
in a wound dressing, a bandage, a surgical implant, a catheter, a
ventilator mask, a face mask, a surgical mask, or an intubation
tube. In still other embodiments, the microfluidic device can be
present in a contact lens case, a urine collection cup, or a urine
bag.
[0084] The fluid sample can be from a human with cystic fibrosis,
ventilator-associated pneumonia, a chronic wound, a burn wound, a
surgical implant, or a surgical site. The fluid sample can be a
bodily fluid from a wound exudate, bronchial lavage, sputum, urine,
saliva, spinal fluid, tears, and blood.
[0085] In other aspects, methods of monitoring viability of a
biofilm comprising Pseudomonas aeruginosa bacteria can be used in
other applications. For example, a bioreactor for treating medical
or other waste products can become contaminated with bacteria. The
bacteria can be introduced through a variety of mechanisms,
including through the waste products, through a water feed pipe, or
through the addition of other additives used to break down waste.
In some embodiments, a microfluidic device are described and used
herein can be disposed in a contact lens case, a urine bag, a urine
collection cup, a medication pump, a water pipe, a bioreactor, or a
water pump.
[0086] In another aspect of the invention, methods of monitoring
effectiveness of an antibiotic treatment of a Pseudomonas
aeruginosa infection in a patient are provided. In some
embodiments, the method includes steps of (a) introducing a fluid
sample from the patient into a microfluidic device including a
working electrode and a reference electrode; (b) performing an
electrochemical measurement to detect pyocyanin in the fluid
sample; and (c) determining a concentration of pyocyanin in the
fluid sample by using a previously determined correlation between
known concentrations of the pyocyanin and a current flow through
the working electrode. The pyocyanin concentration in the fluid
sample provides a measure of the effectiveness of the antibiotic
treatment.
[0087] An increased dose of the antibiotic can be administered if
the concentration of the pyocyanin is above a threshold level. In
some embodiments, the threshold level of pyocyanin can be a
concentration of at least 1 .mu.M, at least 5 .mu.M, or at least 10
.mu.M. In some embodiments, the methods can include administering
an increased dose of the antibiotic if the concentration of the
pyocyanin does not drop below a threshold level after a
predetermined time interval. The predetermined time interval can be
at least 6 hours, at least 12 hours, at least 18 hours, at least 24
hours, at least 48 hours, or greater. In some embodiments, the
methods can include administering a decreased dose of the
antibiotic or stopping the antibiotic if the concentration of the
pyocyanin drops below a threshold level. In some embodiments, the
threshold level of pyocyanin can be a concentration of at least 1
.mu.M, at least 5 .mu.M, or at least 10 .mu.M.
[0088] In some embodiments, the antibiotic can be colistin sulfate
or ciprofloxacin. In other embodiments, the methods can include
administering an additional antibiotic or other pharmaceutical
agent.
[0089] In a further aspect of the invention, methods of screening
effectiveness of an antibiotic against a biofilm comprising
Pseudomonas aeruginosa are provided. In some embodiments, the
method includes steps of (a) introducing a sample comprising
Pseudomonas aeruginosa into a growth chamber in a microfluidic
device including a working electrode and a reference electrode; (b)
allowing the Pseudomonas aeruginosa to grow and form a biofilm in
the growth chamber; (c) introducing an antibiotic at a selected
concentration into the growth chamber of the device; (d) performing
an electrochemical measurement to detect pyocyanin in the fluid
sample; and (e) determining a concentration of pyocyanin in the
sample by using a previously determined correlation between known
concentrations of the pyocyanin and a current flow through the
working electrode. The concentration of pyocyanin below a threshold
indicates effectiveness of the antibiotic.
[0090] In some embodiments, a threshold level of pyocyanin can be a
concentration below 1 .mu.M, 5 .mu.M, or 10 .mu.M, 20 .mu.M, 30
.mu.M, 40 .mu.M, 50 .mu.M, 100 .mu.M, or 200 .mu.M.
[0091] In some embodiments, the method includes providing an
indication of a presence of Pseudomonas aeruginosa when the
concentration of pyocyanin can be above 1 .mu.M, 5 .mu.M, or 10
.mu.M, or greater. In some embodiments, the method includes
estimating a number of cells of Pseudomonas aeruginosa based on the
concentration of pyocyanin.
[0092] In some embodiments, the method includes introducing a
sample comprising Pseudomonas aeruginosa into a plurality of growth
chambers in the microfluidic device; and simultaneously introducing
the antibiotic at selected different concentrations into each
growth chamber to screen the effectiveness of multiple
concentrations of the antibiotic.
[0093] In some embodiments, the method steps can be repeated with a
different concentration of the antibiotic. In some embodiments, the
methods can include continuously introducing the antibiotic into a
growth chamber.
[0094] A device for carrying out the methods described herein can
be provided.
[0095] In some embodiments, a device for monitoring viability of a
biofilm comprising Pseudomonas aeruginosa can include a sensor
having a microfluidic (or nanofluidic) electrode assembly
comprising a microfluidic (or nanofluidic) channel disposed in a
substrate. A working electrode and a reference electrode can be
disposed in the microfluidic channel. A control system is provided,
including a processor and memory. Machine-readable instructions can
be stored in the memory that, upon execution by the processor,
control the voltage at the working electrode and the reference
electrode, and determine a concentration of pyocyanin in a fluid
sample in the microfluidic channel by using a previously determined
correlation between known concentrations of pyocyanin and current
flow through the working electrode.
[0096] In some embodiments, the device is operable to provide an
indication of the concentration of pyocyanin over a range from
about 1 .mu.M to about 1 mM, or from about 5 .mu.M to about 1 mM.
In some embodiments, the concentration range can have a lower limit
of 1 .mu.M, 5 .mu.M or 10 .mu.M. In some embodiments, the
concentration range can have an upper limit of 50 .mu.M, or about 1
mM. In some embodiments, the concentration range can have an upper
limit of a solubility limit of pyocyanin.
[0097] In some embodiments, the control system is operable to
provide an indication of a presence of Pseudomonas aeruginosa when
the concentration of pyocyanin is above a threshold level. In some
embodiments, the threshold level is about 1 .mu.M, or about 5
.mu.M, or about 10 .mu.M. In some embodiments, the control system
is operable to determine a number of cells of Pseudomonas
aeruginosa in the biofilm based on the determined concentration of
pyocyanin.
[0098] In some embodiments, the concentration of pyocyanin can be
determined from a linear relationship, stored in the memory, of the
current flow through the working electrode.
[0099] In some embodiments, the concentration in .mu.M of pyocyanin
is equal to the current flow in .mu.A through the working electrode
divided by 0.18. In other embodiments, the current flow in .mu.A
through the working electrode divided by 0.10, 0.14, 0.16, 0.20,
0.22, or 0.26. In some embodiments, if the current flow through the
working electrode is less than 1 .mu.A, the pyocyanin concentration
is considered to be zero.
[0100] In some embodiments, the control system includes a
potentiostat in communication with the processor and the sensor.
The potentiostat is operable to control the voltage at the working
electrode and the reference electrode. The potentiostat can be in
communication with the processor via a hardwired connection, via a
removable cable, or via a wireless connection. The potentiostat can
be in communication with the sensor via a cable, which can be
disconnectable. The potentiostat can be is battery powered.
[0101] The control system is operable to perform an electrochemical
measurement using squarewave voltammetry, linear sweep voltammetry,
staircase voltammetry, cyclic voltammetry, normal pulse
voltammetry, differential pulse voltammetry, and chronoamperometry.
In some embodiments, the current flow is measured in response to
one or more square wave potentials.
[0102] The device can also include a display in communication with
the control system, which can be operable to display one or more of
the determined concentration of pyocyanin, an indication of a
presence of Pseudomonas aeruginosa, and an indication of a number
of cells of Pseudomonas aeruginosa.
[0103] In some embodiments, the sensor can be disposable and can be
connectable to the potentiostat by a cable. The sensor,
particularly, a disposable sensor, can be embedded in a wound
dressing or within or adjacent to an absorbent pad for a wound
dressing. In some embodiments, the sensor can be present in a wound
dressing, a surgical implant, a catheter, a ventilator mast, or an
intubation tube. In other embodiments, the sensor can be embedded
in a contact lens case, a urine bag or a urine collection cup.
[0104] The control system can include a telecommunications network
connection, for example, to enable communication to a remote
monitoring station.
[0105] It will be appreciated that the control system can be part
of a computer system that executes programming for controlling the
methods and devices as described herein. The computing system can
be implemented as or can include a computing device that includes a
combination of hardware, software, and firmware that allows the
computing device to run an applications layer or otherwise perform
various processing tasks. Computing devices can include without
limitation personal computers, work stations, servers, laptop
computers, tablet computers, mobile devices, hand-held devices,
wireless devices, smartphones, wearable devices, embedded devices,
microprocessor-based devices, microcontroller-based devices,
programmable consumer electronics, mini-computers, main frame
computers, and the like.
[0106] The computing device can include a basic input/output system
(BIOS) and an operating system as software to manage hardware
components, coordinate the interface between hardware and software,
and manage basic operations such as start up. The computing device
can include one or more processors and memory that cooperate with
the operating system to provide basic functionality for the
computing device. The operating system provides support
functionality for the applications layer and other processing
tasks. The computing device can include a system bus or other bus
(such as memory bus, local bus, peripheral bus, and the like) for
providing communication between the various hardware, software, and
firmware components and with any external devices. Any type of
architecture or infrastructure that allows the components to
communicate and interact with each other can be used.
[0107] Processing tasks can be carried out by one or more
processors. Various types of processing technology can be used,
including a single processor or multiple processors, a central
processing unit (CPU), multicore processors, parallel processors,
or distributed processors. Additional specialized processing
resources such as graphics (e.g., a graphics processing unit or
GPU), video, multimedia, or mathematical processing capabilities
can be provided to perform certain processing tasks. Processing
tasks can be implemented with computer-executable instructions,
such as application programs or other program modules, executed by
the computing device. Application programs and program modules can
include routines, subroutines, programs, drivers, objects,
components, data structures, and the like that perform particular
tasks or operate on data.
[0108] The computing device includes memory or storage, which can
be accessed by the system bus or in any other manner. Memory can
store control logic, instructions, and/or data. Memory can include
transitory memory, such as cache memory, random access memory
(RAM), static random access memory (SRAM), main memory, dynamic
random access memory (DRAM), and memristor memory cells. Memory can
include storage for firmware or microcode, such as programmable
read only memory (PROM) and erasable programmable read only memory
(EPROM). Memory can include non-transitory or nonvolatile or
persistent memory such as read only memory (ROM), hard disk drives,
optical storage devices, compact disc drives, flash drives, floppy
disk drives, magnetic tape drives, memory chips, and memristor
memory cells. Non-transitory memory can be provided on a removable
storage device. A computer-readable medium can include any physical
medium that is capable of encoding instructions and/or storing data
that can be subsequently used by a processor to implement
embodiments of the method and system described herein. Physical
media can include floppy discs, optical discs, CDs, mini-CDs, DVDs,
HD-DVDs, Blu-ray discs, hard drives, tape drives, flash memory, or
memory chips. Any other type of tangible, non-transitory storage
that can provide instructions and/or data to a processor can be
used in these embodiments.
[0109] The computing device can include one or more input/output
interfaces for connecting input and output devices to various other
components of the computing device. Input and output devices can
include, without limitation, keyboards, mice, joysticks,
microphones, displays, monitors, scanners, speakers, and printers.
Interfaces can include universal serial bus (USB) ports, serial
ports, parallel ports, game ports, and the like.
[0110] The computing device can access a network over a network
connection that provides the computing device with
telecommunications capabilities. Network connection enables the
computing device to communicate and interact with any combination
of remote devices, remote networks, and remote entities via a
communications link. The communications link can be any type of
communication link, including without limitation a wired or
wireless link. For example, the network connection can allow the
computing device to communicate with remote devices over a network,
which can be a wired and/or a wireless network, and which can
include any combination of intranet, local area networks (LANs),
enterprise-wide networks, medium area networks, wide area networks
(WANs), the Internet, or the like. Control logic and/or data can be
transmitted to and from the computing device via the network
connection. The network connection can include a modem, a network
interface (such as an Ethernet card), a communication port, a
PCMCIA slot and card, or the like to enable transmission of and
receipt of data via the communications link.
[0111] The computing device can include a browser and a display
that allow a user to browse and view pages or other content served
by a web server over the communications link. A web server, server,
and database can be located at the same or at different locations
and can be part of the same computing device, different computing
devices, or distributed across a network. A data center can be
located at a remote location and accessed by the computing device
over a network.
[0112] The computer system can include architecture distributed
over one or more networks, such as, for example, a cloud computing
architecture. Cloud computing includes without limitation
distributed network architectures for providing, for example,
software as a service (SaaS), infrastructure as a service (IaaS),
platform as a service (PaaS), network as a service (NaaS), data as
a service (DaaS), database as a service (DBaaS), backend as a
service (BaaS), test environment as a service (TEaaS), API as a
service (APIaaS), and integration platform as a service
(IPaaS).
[0113] The methods and devices described herein can be used in a
variety of hospitable and other medical settings, such as post
operation facilities, emergency rooms, ICUs, burn wards, central
laboratories, and outpatient facilities, such as for diabetes
patients.
Example 1
[0114] In one study, the use was evaluated of an inexpensive
disposable electrochemical sensor to screen wound fluid exudate
sampled obtained from patients with chronic wounds for the presence
of Pseudomonas aeruginosa.
[0115] Materials and Methods
[0116] This research was conducted through the Wound Etiology and
Healing (WE-HEAL) Study, a biospecimen and data repository designed
for studying chronic wounds approved by the George Washington
University Institutional Review Board (041408). Subjects are
eligible for this study if they have an open wound at the time of
evaluation and are older than 18 years of age. All subjects gave
written informed consent for collection of specimens and data.
[0117] For this experiment, 14 paired wound fluid and biofilm
samples from 12 patients were selected for analysis. This was a
convenience sample selected based on availability of wound fluid
and wound microbiome samples from the same collection date.
[0118] According to standard operating procedures for the WE-HEAL
Study, wound effluent specimens were collected using the Levine
technique. (Levine, N. S., et al., The quantitative swab culture
and smear: A quick, simple method for determining the number of
viable aerobic bacteria on open wounds. J Trauma, 1976. 16(2): p.
89-94.) This technique has been well validated to ensure
standardization throughout all specimens collected in the WE-HEAL
Study. After collection, the swabs were immediately placed in 0.65
.mu.m pore size centrifugal filters (Ultrafree-MC DV, Merck
Millipore, MA, USA). Samples were centrifuged at 12000 rpm for 4
minutes to extract the wound exudate and remove cellular and
fibrinous debris. Samples were stored at -80.degree. C. until
analysis.
[0119] According to standard operating procedures for the WE-HEAL
Study, wound biofilm specimens were collected by swabbing the wound
with a cotton swab also using the Levine technique. (Levine, N. S.,
et al., The quantitative swab culture and smear: A quick, simple
method for determining the number of viable aerobic bacteria on
open wounds. J Trauma, 1976. 16(2): p. 89-94. Angel, D. E., et al.,
The clinical efficacy of two semi-quantitative wound-swabbing
techniques in identifying the causative organism(s) in infected
cutaneous wounds. Int Wound J, 2011. 8(2): p. 176-85.) Samples were
then stored at -80.degree. C. until analysis.
[0120] Bacterial DNA for 16S sequencing was isolated from biofilm
samples using enzymatic lysis followed by phenol-chloroform isoamyl
alcohol extraction and ethanol precipitation. (Chomczynski, P. and
N. Sacchi, Single-step method of RNA isolation by acid guanidinium
thiocyanate-phenol-chloroform extraction. Anal Biochem, 1987.
162(1): p. 156-9.) Utilizing established 454 FLX sequencing methods
(454 Life Sciences, Roche Inc., Branford, Conn., USA), universal
PCR primers with unique barcode identifiers were used to amplify
the hypervariable regions of the bacterial 16S rRNA gene. Taxonomic
classification was performed using mothur software (University of
Michigan, USA) based on the 16S rRNA gene reference sequences from
the Ribosomal Database Project. (Cole, J. R., et al., The Ribosomal
Database Project: improved alignments and new tools for rRNA
analysis. Nucleic Acids Res, 2009. 37(Database issue): p. D141-5.)
Biofilm specimens were considered to be positive for Pseudomonas
spp. if any Pseudomonas reads were detected in the specimen
regardless of relative abundance.
[0121] Disposable, screen-printed electrode sensors (TE100, Zensor,
Taichung City, Taiwan) were used to detect the presence of
pyocyanin in clinical samples (FIG. 1). The sensors utilize a
3-electrode setup, containing carbon-based working (3 mm diameter
disk) and counter electrodes along with a Ag/AgCl reference
electrode. FIG. 8 illustrates a bright-field image of a membrane
mesh placed on top of the electrode sensor for small volume
analysis. All electrochemical measurements were recorded using a
portable potentiostat (.mu.Stat 200, Dropsens, Parque Tecnologico
de Asturias, Spain). The sensing surface was covered with polymeric
membrane (DRP-MEMB, Dropsens, Parque Tecnologico de Asturias,
Spain) to reduce the amount of sample volume required for
analysis.
[0122] For each test, 7.5 .mu.L of wound exudate was pipetted into
the detector well. Square-wave voltammetric scans were performed at
potentials ranging from -0.7 to 0.0 V at an amplitude voltage of
0.05 V, step voltage of 0.004 V, and a frequency of 15 Hz. (See
FIGS. 2-7).
[0123] Each clinical sample was run in duplicate with a new sensor
being used each time. The investigators were blinded to the
microbiome 16SrRNA results at the time of the sensor detection
experiment. The data was analyzed by two independent investigators
using OriginPro 9.1 (OriginLab Corporation). Baselines were created
for each data set using spline interpolation with 32 base points.
The resulting baseline-subtracted data set was used to identify
peaks in the current and to determine the maximum currents of those
peaks (FIG. 4). From these maximum current values, using a cutoff
of 0.030 .mu.A, a binary determination was made for whether the
probe was detecting pyocyanin (positive) or not (negative) (Table
2).
TABLE-US-00001 TABLE 1 Demographic and clinical characteristics of
patients (n = 12) from whom wound fluid samples were tested. Wound
size (mean .+-. SD) of all wounds with specimens collected (n =
14). All Pseudomonas spp. Pseudomonas spp. patients positive on
16SrRNA negative on 16SrRNA n = 12 n = 6 n = 6 p-value Age (years,
mean .+-. SD) 50.76 (.+-.17.14) 49.85 (.+-.11.57) 51.67 (.+-.22.55)
0.8642 Male sex (n, %) 8 (66%) 4 (66%) 4 (66%) 1.00 Race African
American (n, %) 8 (66%) 5 (83%) 3 (50%) 0.3998 Caucasian (n, %) 3
(25%) 1 (16.7%) 2 (33.3%) Asian (n, %) 1 (8.3%) 1 (16.7%) Smoking
Past 5 2 3 1.00 Never 7 4 3 Current 0 0 0 Diabetes 4 2 2 1.00 Renal
disease 2 1 1 1.00 Wound surface area 85.41 (.+-.177.3) 14.13
(.+-.12.77) 146.5 (.+-.230.9) 0.19 (cm.sup.2, mean .+-. SD)
TABLE-US-00002 TABLE 2 Experimental determinations
(Positive/Negative) for whether clinical samples contained
Pseudomonas aeruginosa based on peak currents obtained from
electrochemical square-wave voltammograms. 16SrRNA sequencing
Determination 16SrRNA results (% Peak current using 0.030 .mu.A
sequencing relative Sample (.mu.A) threshold cutoff results
abundance) 10000111243072303-01A 0.0000 Negative Negative 0
10000111243072303-01B 0.0000 10000281244182304-01A 0.1792 Positive
Negative 0 10000281244182304-01B 0.0000 10000281245152307-01A
0.0000 Positive Positive 0.0479 10000281245152307-01B 0.0743
10000281245152307-01C 0.0094 10000361152152303-01A 0.0360 Negative
N/A N/A 10000361152152303-01B 0.0000 10000371347232399-01A 0.1241
Positive Positive 0.0558 10000371347232399-01B 0.0517
10001641245022307-01A 0.2263 Positive Positive 0.0005
10001641245022307-01B 0.2309 10001681243072302-01A 1.3491 Positive
Positive 0.0027 10001681243072302-01B 0.9303 10002511245142301-01A
0.0195 Negative Positive 0.0005 10002511245142301-01B 0.0163
10003031250192307-01A 0.0000 Negative Positive 0.9779
10003031250192307-01B 0.0000 10003131248152301-01A 0.0090 Negative
Negative 0 10003131248152301-01B 0.0280 10003551344182399-01A
0.0000 Negative Negative 0 10003551344182399-01B 0.0201
10003841344182301-01A 0.0757 Positive Negative 0
10003841344192301-01A 0.0469 Positive Negative 0
10003841344192301-01B 0.0539 10003961346272308-01A 0.0000 Negative
Negative 0 10003961346272308-01B 0.0000 10004351351142399-01A
0.6262 Positive Positive 0.2478 20000721546162301-06 0.6232
Positive Positive N/A wf volume test 6 .mu.L 4.3056
20000721546162301-06 wf volume test 7.5 .mu.L
[0124] Statistical Analysis
[0125] Data was analyzed using GraphPad Prism 5.03 (for Windows,
GraphPad Software, San Diego Calif., USA). Fisher's exact test and
Chi-squared tests were used for categorical variables and Student's
t-test was used for continuous variables. Results are represented
as mean.+-.SD. A p value less than 0.05 indicate statistical
significance; all significance tests were performed and interpreted
in a two-sided manner.
[0126] Results obtained from the microbiome profile generated by
16S Ribosomal RNA sequencing were reviewed and samples with any
positive Pseudomonas reads were considered to test positive for
Pseudomonas. These results were compared to the results from the
pyocyanin detector, and the sensitivity and specificity of the
sensor was calculated.
[0127] Results
[0128] Paired wound effluent and biofilm samples were analyzed from
14 unique samples obtained from 12 patients (2 patients with serial
samples collected at different time points were available). The
mean age of patients was 50.18 years. Of the 14 samples subjected
to microbiome profiling by 16SrRNA sequencing, 7 had detectable
Pseudomonas spp. (sequencing positive). All 14 wounds were
recalcitrant at the time of specimen collection.
[0129] There were no significant differences in age, sex, race, or
comorbidities in the patients whose samples were positive for P.
aeruginosa using 16SrRNA sequencing compared to those that were
negative (Tables 1-2). Wounds that were positive for P. aeruginosa
using 16SrRNA sequencing tended to be larger but this did not reach
statistical significance.
[0130] A positive test on the pyocyanin detector was considered to
be an oxidation peak around -0.25 V vs. a Ag/AgCl reference
electrode with a cutoff of 0.030 .mu.A. (Sismaet, H. J., T. A.
Webster, and E. D. Goluch, Up-regulating pyocyanin production by
amino acid addition for early electrochemical identification of
Pseudomonas aeruginosa. Analyst, 2014. 139(17): p. 4241-6. Bellin,
D. L., et al., Integrated circuit-based electrochemical sensor for
spatially resolved detection of redox-active metabolites in
biofilms. Nat Commun, 2014. 5: p. 3256.) Data was analyzed as the
mean of duplicates. Of the 14 samples, 8 tested positive using the
pyocyanin detector cutoff of 0.030 .mu.A.
[0131] Sensitivity and specificity of the pyocyanin probe for
detecting the samples that contained Pseudomonas spp. based on
microbiome sequencing and results are reported in Table 3. The
probe tested positive in 5 out of 7 samples that were positive for
Pseudomonas on 16SrRNA sequencing and was negative on 4 out of 7
samples with negative 16SrRNA results, giving a sensitivity of 71%
(95% CI 0.29-0.96) and specificity of 57% (95% CI 0.18-0.90).
TABLE-US-00003 TABLE 3 Sensitivity and specificity of pyocyanin
probe compared to 16SrRNA sequencing for Pseudomonas spp. Data
analyzed using Fisher's exact test. 16SrRNA 16SrRNA positive
negative Pyocyanin sensor 5 3 positive Pyocyanin sensor 2 4
negative Sensitivity Specificity Positive predictive Negative
predictive (95% CI) (95% CI) value (95% CI) value (95% CI) 0.71
0.57 0.62 0.66 (0.29-0.96) (0.18-0.90) (0.25-0.91) (0.22-0.95)
[0132] The pyocyanin probe was simple to use and had high
inter-observer agreement regarding interpretation of a positive
result. When compared with a diagnostic gold-standard of 16SrRNA
sequencing, the pyocyanin probe had a sensitivity of 71% and
specificity of 57% indicating that it may be useful as a
point-of-care test in screening for presence of Pseudomonas in
human wound fluid.
[0133] During probe development, one of the concerns raised about
the utility of this probe for testing human samples was that there
may be multiple other organisms generating other molecules which
might interfere with probe performance. Human wound samples often
have polymicrobial flora and this was indeed the case for the
specimens reported here. The results reported showed no other redox
peaks in the reference window for the pyocyanin probe. This
indicates that despite presence of multiple other organisms in
human specimens, there do not appear to be other redox-active
molecules that would impede the probe performance in a clinical
setting. However, small potential shifts were observed in where the
pyocyanin peak occurred. This may be attributed to differences in
the salt and pH concentration of the sample media, and the limited
stability of the Ag/AgCl quasi-reference electrode of the
disposable sensor.
[0134] It was found that most of the samples containing P.
aeruginosa tested pyocyanin positive using the present
electrochemical approach. By lowering the cutoff maximum current
determination, the sensor's sensitivity could have been improved to
85.7% while decreasing specificity to 42.9%. The detection limit of
electrochemical sensors can be improved by switching to micro and
nano-fabricated electrodes. (Zevenbergen, M. A., et al., Stochastic
sensing of single molecules in a nanofluidic electrochemical
device. Nano Lett, 2011. 11(7): p. 2881-6.)
[0135] The testing revealed some false negative results using the
pyocyanin probe. While pyocyanin is a very specific molecule,
produced only by Pseudomonas, there are some environments even in
in vitro culture of Pseudomonas in which pyocyanin production is
low. It is possible that the wound microenvironment may have
impacted pyocyanin production in some of the clinical cases studied
here.
[0136] Additionally, the sample size is small because this was a
pilot study designed to be hypothesis generating. Also, while the
16SrRNA testing is a good gold standard test for determination of
bacterial presence in the wound and relative abundance of specific
bacteria relative to the entire microbiome profile of the specimen,
16SrRNA testing alone does not give information regarding the
quantitative abundance of a particular bacterium in one specimen
relative to other specimens. It is possible that some of the false
positives and false negatives in this study are attributable to
very low Pseudomonas abundance in the sample and further testing is
needed to understand the clinical relevance of false positive and
false negative tests as this device is further refined and
developed with a view to clinical indications. In addition, 16SrRNA
does not discriminate between Pseudomonas species whereas the
pyocyanin probe will only detect the presence of Pseudomonas
aeruginosa. Taking this into account potentially explains one of
our false negatives, where the sequencing may be indicating the
presence of Pseudomonas species other than aeruginosa.
[0137] The results from this study provide useful and unexpected
data regarding detection of P. aeruginosa in clinical samples and
validate this electrochemical approach as a rapid point-of-care
diagnostic.
Example 2
[0138] A study was conducted to look at the killing of cells inside
of a P. aeruginosa biofilm (grown in a microfluidic environment)
via detected PYO, using SWV at a disposable three electrode cell,
when exposed to different concentrations of the antibiotic colistin
sulfate. The condition of cells in Pseudomonas aeruginosa biofilms
was monitored via the electrochemical detection of the
electro-active virulence factor pyocyanin in a fabricated
microfluidic growth chamber coupled with a disposable three
electrode cell. Cells were exposed to 4, 16, and 100 mg/L colistin
sulfate after overnight growth. At the end of testing, the measured
maximum peak current (and therefore pyocyanin concentration) was
reduced by approximately 68% and 82% in P. aeruginosa exposed to 16
and 100 mg/L colistin sulfate, respectively. Samples were removed
from the microfluidic chamber, analyzed for viability using
staining, and streaked onto culture plates to confirm that the P.
aeruginosa cells were affected by the antibiotics. The correlation
between electrical signal drop and the viability of P. aeruginosa
cells after antibiotic exposure highlights the usefulness of this
approach for future low cost antibiotic screening applications.
[0139] Materials and Methods
[0140] P. aeruginosa strain PA14 and m-cherry Escherichia coli
strain K12 were used for all antibiotic tests performed. Trypticase
soy broth (BD 211768) was used as the nutrient source for all
bacteria grown in these tests. Colistin sulfate (Adipogen
AG-CN2-0065-G001) was dissolved in trypticase soy broth (TSB) at 1
g/L and used as a stock solution. When not in use, the stock
solution was stored at 4.degree. C. Polydimethylsiloxane (Ellsworth
Adhesives 184 Sil. Elast. Kit 0.5 kg) was used to prepare all
microfluidic devices. Disposable three electrode cells (Zensor
TE100) were used for all measurements in this study. The
electrochemical cells consist of carbon working and counter
electrodes with a Ag/AgCl paste reference electrode. Tubing and
luer lock fittings for microfluidic connections were purchased from
Amazon Supply (B001GMWZM) and Value Plastic (MTLL230). To prevent
bacteria from leaving the microfluidic growth chambers, Minisart
RC4 0.2 micron regenerated cellulose luer lock syringe filters
(17821K) were attached to the inlets and outlets of the devices via
19 gauge luer lock syringes (NE192PL-25). A syringe pump (Harvard
Apparatus Fusion 200 211097) was used to control the flow rate of
growth media and antibiotic through the microfluidic chamber.
Electrochemical measurements were made using a multipotentiostat
(CHI 1040C A2728).
[0141] Polydimethylsiloxane (PDMS) wells were fabricated from 9
mm.times.9 mm tape molds made on glass slides (3M Scotch Tape, tape
thickness.apprxeq.50 .mu.m) using a standard method. (A. B. Shrirao
and R. Perez-Castillejos, Simple fabrication of microfluidic
devices by replicating scotch-tape masters, Chips Tips, 17 May
2010.) The PDMS wells had a final volume of approximately 4 .mu.L,
and were designed to cover the entire electrochemical cell (FIG.
9A). Inlets and outlets in the wells were drilled and the resulting
microfluidic devices were fabricated by irreversibly bonding PDMS
to the disposable electrochemical cells using air plasma (Anatech
SP-100, 5-7 s at 100 W). The microfluidic channels were filled with
trypticase soy broth (TSB) at a flow rate of 10 .mu.L/min. To
facilitate complete filling of the chamber (removal of air
bubbles), an empty syringe was attached to the outlet. By pulling
and releasing vacuum on the outlet, TSB was pulled through the
chamber displacing any air bubbles.
[0142] For antibiotic testing, cultures of PA14 and E. coli were
grown over night in 3 mL of TSB (concentration approximately
10.sup.11 cells/mL). After overnight growth, samples were
centrifuged for 3 min at 10,000 rpm. The supernatant was discarded
and the cultures were reconstituted in 3 mL of fresh TSB. After
removing the inlet syringe filter, approximately 24 .mu.L, of
reconstituted cell culture was loaded into the growth chamber at a
flow rate of 10 .mu.L/min. At this flow rate the velocity in the
chamber was such that PA14 could not resist flow (P. aeruginosa
speed.apprxeq.30-50 .mu.m/s), while the outlet filter prevented
cells from exiting. (T. S. Murray and B. I. Kazmierczak, J
Bacteriol, 2006, 188, 6995-7004.) After loading cells, the filter
was replaced, sealing the bacteria into the growth chamber.
Biofilms were then allowed to grow at room temperature overnight
under stagnant conditions. Stagnant conditions were chosen to
ensure that the cells had ample time to adhere to the surface and
form a biofilm over the sensor. After overnight growth, flow at 0.1
.mu.L/min was initiated with either TSB or colistin sulfate in TSB
and the electrochemical response was monitored. (K. P. Kim, Y. G.
Kim, C. H. Choi, H. E. Kim, S. H. Lee, W. S. Chang and C. S. Lee,
Lab Chip, 2010, 10, 3296-3299.)
[0143] Samples were scanned from -0.5 to 0.2 V versus the internal
Ag/AgCl reference electrode on the disposable electrochemical cell
(Zensor). Square wave voltammetry (SWV) was used at an amplitude
voltage of 50 mV and a frequency of 15 Hz. SWV was chosen due to
its increased sensitivity and its ability to monitor the
electrochemical peak of PYO compared to other voltammetric and
amperometric techniques. (L. Pires, K. Sachsenheimer, T.
Kleintschek, A. Waldbaur, T. Schwartz and B. E. Rapp, Biosens
Bioelectron, 2013, 47, 157-163. A. J. Bard and L. R. Faulkner,
Electrochemical methods fundamentals and applications, John Wiley
& Sons Inc., 2011.)
[0144] A calibration curve for PA14 grown in trypticase soy broth
(TSB) was obtained, as illustrated in FIG. 13. More particularly,
square wave voltammograms for concentrations of pyocyanin from 0 to
50 .mu.M were obtained using three different disposable Zensor
electrodes. The dilution series was repeated twice and each
concentration was scanned three times per disposable electrode. The
average maximum current for both runs through the dilution series
was averaged and plotted versus current. FIG. 13 illustrates a
maximum current from SWVs of pyocyanin in 30 g/L TSB from 0 to 50
.mu.M. SWVs performed from -0.5 to 0 V at a frequency of 15 Hz and
an amplitude voltage of 50 mV. A linear fit is
I/.mu.A=0.18[PYO]/.mu.M; where I=current and [PYO]=concentration of
pyocyanin in TSB.
[0145] PYO concentration was approximated from the calibration
curve of PYO in TSB (FIG. 13). After loading the PDMS chambers with
TSB, the sample was scanned 10 times and the average taken to get
the mean response of the TSB. All subsequent measurements were then
compared to this response. Three measurements were taken during the
loading of the cells, with additional measurements taken every 30
min during the remainder of the tests. For each concentration of
antibiotic tested, three different microfluidic setups were
used.
[0146] Electrochemical measurements were processed by subtracting
the baseline signal. One way analysis of variation (ANOVA) was used
to determine the statistical significance of resulting
measurements.
[0147] Samples were prepared for SEM imaging by fixing in a 2.5%
glutaraldehyde (EMSDIASUM 16120) in a 0.1 M sodium cacodylate
buffer (EMSDIASUM 11654). After fixing, samples were washed in
cacodylate buffer, and then dehydrated in increasing concentrations
of ethanol (Fisher BP2818-4 30-100%). After dehydration, ethanol
was removed via critical point drying (Samdri-PVT-3D) using liquid
CO.sub.2. The final step in SEM preparation was plasma sputtering
(Cressington Sputter Coater 208HR) 5 nm of palladium metal onto the
samples making them conductive. Once prepared for imaging, samples
were loaded into a Field Emission SEM (Hitachi S-4800) and probed
at an acceleration voltage and emission current of 3 kV and 10 mA,
respectively.
[0148] Cell viability after exposure to colistin sulfate was
assessed using a LIVE/DEAD staining kit (EMD Chemicals Millipore
50-231-0606). Stains were prepared per manufacturer's operating
procedure. After staining, 10 .mu.L of sample was injected into an
INCYTO C-chip disposable hemocytometer (DHC-N01). Cells were imaged
using a fluorescence microscope, and the number of PA14 cells that
were alive after exposure was determined using IMAGE J (ImageJ, U.
S. National Institutes of Health, Bethesda, Md., USA,
http://imagej.nih.gov/ij/).
[0149] Results
[0150] SWV were collected every 30 min from overnight cultures of
PA14 in TSB, starting from the point at which they were loaded into
the PDMS chambers, to determine whether electro-active molecules
were being produced. P. aeruginosa continuously produces PYO as it
grows, in both planktonic and biofilm phenotypes, which can be
monitored electrochemically during the experiments. (D. Sharp, P.
Gladstone, R. B. Smith, S. Forsythe and J. Davis,
Bioelectrochemistry, 2010, 77, 114-119.) The utility of this
approach is highlighted in FIG. 10 where the electrochemical
response of PA14 grown in TSB is monitored over time.
[0151] (P. aeruginosa samples were prepared for SEM analysis to
determine where in the growth chamber the bacteria were collecting.
Samples were prepared by peeling the PDMS from the electrode and
fixing the bacteria using a 2.5% glutaraldehyde in 0.1 M
cacodyalate buffer (pH 7.2) at 4.degree. C. for 2 hours. Samples
were dehydrated in an increasing dilution series of ethanol and
then critical point dried using liquid CO2. Resulting SEMs are
reported in FIGS. 14 and 15 and show the clear presence of biofilms
growing in the chamber.)
[0152] The lack of observable peaks during loading indicated that
no detectable PYO was present initially in the fresh TSB cell
suspension (FIG. 10, graph A). As the biofilm formed under stagnant
conditions, the oxidation peak height increased over time (FIG. 10,
graphs A-B). SEM images of the PDMS growth chamber and the working
electrode substrate showed bacteria carpeting both surfaces (see
FIGS. 14 and 15) after overnight growth under stagnant conditions.
Initiating the flow of fresh TSB into the channels after overnight
growth allowed the biofilm to thrive. Indeed, the electrical signal
increased after TSB flow was initiated (FIG. 2 graphs D-F),
indicating the increased production rate of PYO. The presence of a
second peak at later time points was observed. The first peak is
due to PYO, while the appearance of a second peak is ascribed to
the electrochemical reaction of a second phenazine derivative that
has been reported in the literature as being
5-methylphenazine-1-carboxylic acid or one of its derivatives. (V.
B. Wang, S. L. Chua, B. Cao, T. Seviour, V. J. Nesatyy, E. Marsili,
S. Kjelleberg, M. Givskov, T. Tolker-Nielsen, H. Song, J. S. Loo
and L. PloS One, 2013, 8, e63129. D. L. Bellin, H. Sakhtah, J. k.
Rosenstein, P. M. Levine, J. Thimot, K. Emmett, L. E. Dietrich and
K. L. Shepard, Nat Commun, 2014, 5, #3256.) The change in the
oxidation potential, after the initiation of flow, where the peak
current was measured can be attributed to the internal Ag/AgCl
pellet used as the reference for these studies. Drift due to fluid
flow is an unavoidable consequence of having the reference in
direct contact with the test fluid. (M. W. Shinwari, D.
Zhitomirsky, I. A. Deen, P. R. Selvaganapathy, M. J. Deen and D.
Landheer, Sensors, 2010, 10, 1679-1715. 40. L. Rassaei, K. Mathwig,
E. D. Goluch and S. G. Lemay, J Phy Chem C, 2012, 116,
10913-10916.) The measured peak potential stabilized over time with
constant fluid flow and the peak current at this new potential was
used for calculations. The movement of the peak over time can be
observed in FIGS. 16-17. Measurement of the PA14 cultures with a
traditional Ag/AgCl reference electrode (BASi MW-2030) showed that
the PYO peak current appeared at the expected potential.
[0153] SWVs were taken every 30 minutes during exposure of cells to
colistin sulfate. Pseudomonas aeruginosa and Escherichia coli were
exposed separately to colistin sulfate at 4, 16, and 100 mg/L at a
flow rate of 100 nL/min. Of interest is the complete lack of
discernible peaks from SWVs for E. coli cells exposed to colistin
sulfate compared to P. aeruginosa (Fig. S5-S7). Scans are from one
replicate but are representative all acquired scans.
[0154] While the overall electrical signal increased over time, a
decrease was observed consistently at the initiation of fluid flow.
There are two possibilities for the observed result. First, it can
be an indicator of how firmly the biofilm has adhered to the
surface of the microfluidic channel. The role of shear stress on
cell adhesion has been studied previously; and, the results show
that cells can be removed from surfaces at high shear stresses.
(J.-C. Ochoa, C. Coufort, R. Escudie, A. Line and E. Paul, Chem
Engin Sci, 2007, 62, 3672-3684. Y.-P. Tsai, Biofouling, 2005, 21,
267-277.) As growth media flows through the channel it may remove
bacteria if the biofilm is not firmly attached. (M. M. Salek, S. M.
Jones and R. J. Martinuzzi, Biofouling, 2009, 25, 711-725.) The
removal of bacteria in turn would lead to reduced production of PYO
in the vicinity of the sensor (lowering the electrical signal).
This is unlikely as the applied flow rates in this study are
similar to those used by other groups and should be slow enough to
avoid significant removal of the bacterial biofilm. (J. Kim, H. D.
Park and S. Chung, Microfluidic approaches to bacterial biofilm
formation, Molecules, 2012, 17, 9818-9834. K. P. Kim, Y. G. Kim, C.
H. Choi, H. E. Kim, S. H. Lee, W. S. Chang and C. S. Lee, Lab Chip,
2010, 10, 3296-3299.)
[0155] Second, it is possible that the decrease in signal is due to
PYO in solution being removed during flow, and it is only when a
sufficiently large concentration of PYO is produced, to overcome
convective transport, that the signal rebounds. Koley et al. (2011)
demonstrated the presence of a PYO gradient (eletrocline) in
biofilms of P. aeruginosa using scanning electro-chemical
microscopy. (M. M. R. D. Koley, A. J. Bard, M. Whiteley, Proc Nat
Acad Sci USA, 2011, 108, 19996-20001.) The authors showed that this
electrocline extended hundreds of microns above the biofilm's
surface. The change in the PYO electrocline due to fluid flow is
likely responsible for the initial drop in signal when bulk fluid
flow starts. Regardless, it is clear that even after the initiation
of flow within the microfluidic chamber, the peak current remains
indicating the cells are indeed growing within the chamber (FIGS.
11A, 11B, and 17-19). E. coli in TSB was used as a control since it
is not expected to produce molecules that are redox-active in this
voltage window. (D. Sharp, P. Gladstone, R. B. Smith, S. Forsythe
and J. Davis, Bioelectrochemistry, 2010, 77, 114-119. T. A.
Webster, H. J. Sismaet, J. L. Conte, I. P. J. Chan and E. D.
Goluch, Biosens Bioelectron, 2014, 60, 265-270.) The lack of any
discernible peak confirms that there are no electrochemical
molecules produced by E. coli and that there is no contamination of
the chambers by P. aeruginosa from the environment over the course
of the experiment (see FIGS. 17-19). The absence of oxidation peaks
from E. coli cells highlights the limitations of the proposed
approach to electrochemically monitor the antibiotic susceptibility
of other bacterial species. Alternatively, the ability to
electrochemically measure the viability of PA14 by the production
of PYO can be a useful selective marker of P. aeruginosa in patient
samples. (T. A. Webster, H. J. Sismaet, J. L. Conte, I. P. J. Chan
and E. D. Goluch, Biosens Bioelectron, 2014, 60, 265-270.)
Furthermore the transparent nature of the PDMS used to fabricate
growth chambers facilitates the use of fluorescent bacterial
species and markers as reported in the literature. (J. Kim, H. D.
Park and S. Chung, Microfluidic approaches to bacterial biofilm
formation, Molecules, 2012, 17, 9818-9834. K. P. Kim, Y. G. Kim, C.
H. Choi, H. E. Kim, S. H. Lee, W. S. Chang and C. S. Lee, Lab Chip,
2010, 10, 3296-3299.)
[0156] After overnight growth of P. aeruginosa, 0.100 .mu.L/min
flow of colistin sulfate at 4, 16, and 100 mg/L in TSB was
initiated. These concentrations were chosen to cover the range of
colistin sulfate MIC values that are reported in literature. (J. M.
Andrews, J. Antimicrob Chemother, 2001, 48, 5-16.) SWV measurements
were taken to determine what effect the reported MIC concentrations
of colistin sulfate (4 and 16 mg/L) have on PYO production. This,
in turn, can be an indicator of P. aeruginosa biofilm
susceptibility to colistin sulfate. Three devices per concentration
of colistin sulfate were used and the average peak current reported
(FIGS. 11A and 16). Error bars represent the standard deviation of
the mean for three separate measurements at that time point, unless
otherwise indicated. As a control, E. coli biofilms were exposed to
the same concentrations of colistin sulfate. One replicate per
concentration was performed for these tests. No oxidation peaks
were observed for E. coli exposed to colistin sulfate signifying a
lack of electrochemically active molecules (Fig. S2-S4). (D. Sharp,
P. Gladstone, R. B. Smith, S. Forsythe and J. Davis,
Bioelectrochemistry, 2010, 77, 114-119. E. Kim, T. Gordonov, W. E.
Bentley and G. F. Payne, Anal Chem, 2013, 85, 2102-2108.)
[0157] ANOVA was used to identify significant differences between
the average peak currents of the three antibiotic concentrations
and the control experiment without antibiotic (FIG. 11A). The
analysis showed that the average peak current was significantly
lower (P<0.05) for PA14 exposed to 16 and 100 mg/L colistin
sulfate concentrations when compared against the control. The
average percent decrease in the maximum peak current at the end of
testing for PA14 exposed to 16 and 100 mg/L colistin sulfate was
68% and 82%, respectively, compared to the current produced by the
cells in the control experiment. The average percent decrease in
the measured current, compared to the control cells, was calculated
by % Decrease=100*(I.sub.t-I.sub.c)/I.sub.c where I.sub.t equals
the average peak current at time t and I.sub.c is the average peak
current of the control P. aeruginosa cells. The decreased current
response is directly related to a decrease in the measured PYO,
indicating a correlation between the colistin sulfate concentration
and PYO production. In contrast, the average response for cells
treated with 4 mg/L colistin sulfate showed no significant
difference when compared to biofilms exposed to only TSB,
indicating that the lower MIC value was not significantly affecting
the production of PYO. Importantly, FIGS. 11A and 11B show that
continuous electrochemical monitoring allows the researcher to view
the efficacy of an anti-pseudomonas antibiotic via a reduction in
PYO production. FIG. 20 supports these results by demonstrating
that PA14 exposed to ampicillin, an antibiotic that is not
effective against this species, has no effect on PYO production. By
reducing the amount of PYO produced by the bacteria, the host's
body may be able to more effectively fight off the infection. (G.
M. Denning, L. A. Wollenweber, M. A. Railsback, C. D. Cox, L. L.
Stoll and B. E. Britigan, Infect Immun, 1998, 66, 5777.) (Regarding
FIG. 20, PA14 cells were exposed to 100 mg/L of ampicillin under
the same conditions as those subjected to 100 mg/L colistin sulfate
exposure. A comparison of SWVs with the two antibiotics is shown in
FIG. 20. Of note is that cells exposed to ampicillin continued to
show an electrochemical response compared to those exposed to
colistin sulfate. This was expected since ampicillin is known to be
ineffective at killing P. aeruginosa.)
[0158] The inherent resistance of PA14 to the lowest MIC value used
in this study could explain why the pyocyanin response did not
significantly differ from blank measurements. Liquid samples of
PA14 cultured on 4 mg/L colistin sulfate agar plates were able to
grow indicating that this concentration had no effect on planktonic
cell attachment and growth (FIG. 21). As such, it makes sense that
biofilms of PA14 exposed to this concentration would not be
affected and should produce similar levels of pyocyanin.
[0159] Regarding FIG. 21, liquid cultures of PA14, grown over night
in 3 mL of TSB, were plated directly onto cetrimide agar plates
containing 0, 4, and 100 mg/L of colistin sulfate. These plates
were incubated overnight at 37.degree. C. The plates were then
inspected for bacterial growth (FIG. 21). Plates containing 0 and 4
mg/L colistin sulfate showed growth, whereas plates containing 100
mg/L colistin sulfate showed no colony formation. Colony formation
at the lower MIC value of 4 mg/L colistin sulfate agrees with the
results reported in FIGS. 11A, 11B, and 12.
[0160] The number of living cells measured after exposure to three
different concentrations of colistin sulfate were compared (FIG.
11B). After the biofilm was exposed to antibiotic in the device,
the PDMS chambers were peeled off and 100 .mu.L of fresh TSB was
spotted on the biofilm and pipetted vigorously to remove material
from the surface of the electrode. Removed samples were used to
measure live cell counts, performed with Millipore 3P Live/Dead
Stain, using a haemocytometer. Each measurement was performed in
triplicate and the error bars show one standard deviation of the
mean. A concentration of approximately 4.times.10.sup.5 live
cells/mL was measured in the biofilms not exposed to colistin
sulfate. Biofilms typically have lower concentrations of live cells
than agitated liquid cultures. A statistically significant
reduction in the number of live PA14 cells was measured for samples
exposed to 100 mg/L colistin sulfate compared to cells exposed to 0
and 4 mg/L colistin sulfate. The .about.2.times. reduction in the
number of living cells supports the hypothesis that a reduction in
the PYO signal is correlated with a reduction in the number of
living cells. Recently, Connell et al. (2014) supported these
findings as well by showing a correlation between the number of
cells trapped in a chamber and the concentration of PYO that is
present around the cells. (J. L. Connell, J. Kim, J. B. Shear, A.
J. Bard, and M. Whiteley, Proc Nat Acad Sci USA, 2014, 111,
18255-18260.)
[0161] FIG. 12 shows the culture results at three different time
points for cells exposed to 4 and 100 mg/L colistin sulfate in
microfluidic devices. Growth was observed in samples exposed to 4
mg/L colistin sulfate after only 4.3 h of incubation implying that
this concentration had little effect on the cells' viability (FIG.
22). No growth was observed for cells exposed to 100 mg/L colistin
sulfate after 6.5 h of incubation. Growth was observed for samples
collected from chambers exposed to 100 mg/L colistin sulfate after
74 h, indicating that the complete elimination of viable bacteria
from inside the chamber was not achieved. The qualitative results
of the live cell stain are consistent with the culture plate
experiments. The live cell concentration in biofilms exposed to 100
mg/L colistin sulfate (FIG. 11B), however, is higher than expected
when compared to the reduced rate of colony formation on agar
plates (FIG. 12). Taken together, these results suggest that the
cells exposed to this antibiotic may have reduced their metabolic
activity to make them less susceptible to the antibiotic.
[0162] Regarding FIG. 22, after exposure to colistin sulpahte at 4
and 100 mg/L flowing at 100 nL/min, the PDMS chambers were peeled
from the disposable electrodes. A sterile loop was placed onto the
electrodes, then streaked onto fresh TSB agar plates. Samples were
incubated at 37.degree. C., and monitored for growth. Cells exposed
to 4 mg/L colistin sulfate began producing visible colonies after
only 4 h of incubation. Over 24 h of incubation was required for
colonies to form from cells exposed to 100 mg/L colistin sulfate.
This indicates that cells were affected by the presence of colistin
sulfate in solution at higher concentrations.
[0163] These results support the findings in the literature,
drawing attention to the lower efficacy of reported MICs against
microbial biofilms. (K. P. Kim, Y. G. Kim, C. H. Choi, H. E. Kim,
S. H. Lee, W. S. Chang and C. S. Lee, Lab Chip, 2010, 10,
3296-3299.) It is clear from the results in FIGS. 11A, 11B, and 12
that a reduction in PYO production, under exposure to colistin
sulfate, is correlated with a reduction in the viability of PA14.
This reduction in pyocyanin and inhibited growth rate may allow a
person's immune response to successfully fight off the bacterial
infection. (L. Allen, D. H. Dockrell, T. Pottery, D. G. Lee, P.
Cornelis, P. G. Hellewell and M. K. B. Whyte, J Immunol, 2005, 174,
3643-3649. L. R. Usher, R. A. Lawson, I. Geary, C. J. Taylor, C. D.
Bingle, G. W. Taylor and M. K. B. Whyte, J Immunol, 2002, 168,
1861-1868. R. Wilson, T. Pitt, G. Taylor, D. Watson, J. Macadermot,
D. Sykes, D. Roberts, and P. Cole, J Clin Invest, 1987, 79,
221-229.)
[0164] This study demonstrates, for the first time, the possibility
of using electrochemical sensors to monitor metabolites produced by
a biofilm that is exposed to antibiotics. The time to detection
using this electrochemical approach (.about.45 h) is comparable to
standard culture plate techniques. While simple identification of
bacterial species can be accomplished within 24 hours, sensitivity
tests required an additional 24-72 hours of incubation on several
plates. Biochemical and molecular methods are available
commercially that provide sensitivity information within minutes
after the initial 24 hour colony formation period, but they require
expensive reagents/instrumentation and additional sample
processing. The analysis time of the proposed method may be lowered
by employing miniature microfabricated electrochemical sensors
that, in turn, allow for smaller microfluidic chambers to be
employed compared to those utilized in this current study. Smaller
chambers would potentially decrease the time to detection due to
the confinement imposed on the cells.
[0165] In healthcare situations, such as wound infections, biofilms
form rapidly and require immediate treatment. This approach can
also be utilized to study biofilms that are more mature or exposed
to any number of other experimental variables. Ultimately, an
electrochemical sensor for susceptibility determination may be
valuable for low-resource settings or for monitoring the status of
infections in vivo while they are being treated with
antibiotics.
[0166] It will be appreciated that the various features of the
embodiments described herein can be combined in a variety of ways.
For example, a feature described in conjunction with one embodiment
may be included in another embodiment even if not explicitly
described in conjunction with that embodiment.
[0167] The present invention has been described in conjunction with
certain preferred embodiments. It is to be understood that the
invention is not limited to the exact details of construction,
operation, exact materials or embodiments shown and described, and
that various modifications, substitutions of equivalents,
alterations to the compositions, and other changes to the
embodiments disclosed herein will be apparent to one of skill in
the art.
* * * * *
References