Tagbo NIEPA, et al.
Electrochemical
Disinfection
https://www.eurekalert.org/pub_releases/2019-10/uop-an102219.php
A
'shocking' new way to treat infections
New Pitt research uses electrochemical approach to treat
infections of metal-based implants
Titanium has many properties that make it a great choice for use
in implants. Its low density, high stiffness, high
biomechnanical strength-to-weight ratio, and corrosion
resistance have led to its use in several types of implants,
from dental to joints. However, a persistent problem plagues
metal-based implants: the surface is also a perfect home for
microbes to accumulate, causing chronic infections and
inflammation in the surrounding tissue. Consequently, five to 10
percent of dental implants fail and must be removed within 10-15
years to prevent infection in the blood and other organs.
New research from the University of Pittsburgh's Swanson School
of Engineering introduces a revolutionary treatment for these
infections. The group, led by Tagbo Niepa, PhD, is utilizing
electrochemical therapy (ECT) to enhance the ability of
antibiotics to eradicate the microbes.
"We live in a crisis with antibiotics: most of them are failing.
Because of the drug- resistance that most microbes develop,
antimicrobials stop working, especially with recurring
infections," says Dr. Niepa, author on the paper and assistant
professor of chemical and petroleum engineering at the Swanson
School, with secondary appointments in civil and environmental
engineering and bioengineering. "With this technique, the
current doesn't discriminate as it damages the microbe cell
membrane. It's more likely that antibiotics will be more
effective if the cells are simultaneously challenged by the
permeabilizing effects of the currents. This would allow even
drug-resistant cells to become susceptible to treatment and be
eradicated."
The novel method passes a weak electrical current through the
metal-based implant, damaging the attached microbe's cell
membrane but not harming the surrounding healthy tissue. This
damage increases permeability, making the microbe more
susceptible to antibiotics. Since most antibiotics specifically
work on cells that are going to replicate, they do not work on
dormant microbes, which is how infections can recur. The ECT
causes electrochemical stress in all the cells to sensitize
them, making them more susceptible to antibiotics.
The researchers hope this technology will change how infections
are treated. Researchers focused their research on Candida
albicans (C. albicans), one of the most common and harmful
fungal infections associated with dental implants. But while
dental implants are one exciting application for this new
technology, Niepa says it has other potential applications, such
as in wound dressings.
https://pubs.acs.org/doi/10.1021/acsami.9b09977
ACS Applied Materials & Interfaces
DOI: 10.1021/acsami.9b09977
Electrochemical
Strategy for Eradicating Fluconazole-Tolerant Candida
albicans using Implantable Titanium
Eloise
Eyo Parry-Nweye, et al.
Abstract
A persistent problem in modern healthcare derives from the
overwhelming presence of antibiotic-resistant microbes on
biomaterials, more specifically, fungal growth on metal-based
implants. This study seeks to investigate the antifungal
properties of low-level electrochemical treatments delivered
using titanium electrodes against Candida albicans. We show that
C. albicans can be readily controlled with electrical
currents/potentials, reducing the number of viable planktonic
cells by 99.7% and biofilm cells by 96.0-99.99%. Additionally,
this study explores the ability of the electrochemical
treatments to potentiate fluconazole, a clinically used
antifungal drug. We have found that electrochemical treatment
substantially enhances fluconazole killing activity. While
fluconazole alone exhibits a low efficiency against the
stationary phase and biofilms C. albicans cells, complete
eradication corresponding to 7-log killing is achieved when the
antifungal drug is provided subsequently to the electrochemical
treatment. Further mechanistic analyses have revealed that the
sequential treatment shows a complex multi-modal action,
including disruption of cell wall integrity and permeability,
impaired metabolic functions, and enhanced susceptibility to
fluconazole, while altering the biofilm structure. Altogether,
we have developed and optimized a new therapeutic strategy to
sensitize and facilitate the eradication of fluconazole-tolerant
microbes from implantable materials. This work is expected to
help advance the use of electrochemical approaches in the
treatment of infections caused by C. albicans in both nosocomial
and clinical cases.
System
And Method For Controlling Bacterial Cells With Weak
Electric Currents
US8663914
[ PDF ]
A system and method for treating bacterial cells with an
electrochemical process, alone or in combination with
antibiotics. Weak electric currents are used to effectively
eliminate bacterial cells. The method may be adapted for novel
therapies of chronic infections and strategies to control
persistent biofouling. The system has broad spectrum
applications in treating chronic and drug resistant infections,
such as those caused by Pseudomonas aeruginosa, Mycobacterium
tuberculosis and methicillin resistant Staphylococcus aureus,
and may also be used for decontamination of medical devices.
BACKGROUND
OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to electrochemical control
of bacterial cells and, more particularly, the effect of weak
electric currents on bacterial cells.
[0004] 2.
Description of the Related Art
[0005] Previous studies of persister cells have led to important
discoveries that are shifting the paradigm of research in
microbiology and antimicrobial therapy. It is now well
recognized that subpopulations of bacterial cells in a culture
can enter a dormant (non-growing) state that are extremely
tolerant to a variety of unrelated stresses such as antibiotics
and heat. Such heterogeneity has been reported to exist in even
well mixed shake flask cultures at exponential phase. This
phenotypic variation can lead to three subpopulations in a given
culture including the normal cells, type I persister cells from
the stationary inoculums and type II persister cells that are
generated during growth. Persister cells are not mutants with
drug resistant genes, but rather phenotypic variants of the
wild-type strain. Persister cells neither die nor grow in the
presence of an antibiotic, and when reinoculated, they grow into
a normal culture with a similar percentage of cells as
persisters, leading to high antibiotic tolerance.
[0006] Although persister cells normally only make up a small
portion of the population, they play a critical role in
antibiotic tolerance. Most antibiotics inhibit bacteria by
targeting growth related cellular activities, e.g., protein,
DNA, and cell wall syntheses. They can eliminate the majority of
bacterial population by killing the normal cells. For persister
cells, however, antibiotics can only repress but not eliminate
this subpopulation because persister cells are non-growing
dormant cells. Thus, the seeming disadvantage of being dormant
in normal environment becomes an advantage for persister cells
when being challenged by antibiotics. When the treatment is
stopped, some persister cells revert back to normal cells and
reestablish the population. Such tolerance leads to reoccurrence
of infections and facilitate the development and spread of
multidrug resistance through true mutations.
[0007] Recent research has demonstrated that persister cell
formation increases significantly in stationary-phase cultures
and the surface-attached highly hydrated structures known as
biofilms. Formed in a dynamic process, mature biofilms typically
have mushroom-like structures with cells embedded in a
polysaccharide matrix secreted by the bound bacterial cells.
Biofilm cells are up to 1000 times more tolerant to antibiotics
and disinfectants compared to their planktonic counterparts.
Thus, deleterious biofilms cause serious problems such as
chronic infections in humans as well as persistent corrosion and
equipment failure in industry. Although not completely
understood at the molecular level, the biofilm-associated
tolerance is due to several factors acting in concert. Bacterial
cells in biofilm produce a polysaccharide matrix, which creates
a physical barrier that retards or blocks the toxic compounds
from reaching the cells. However, protection by the
polysaccharide matrix can only partially explain the tolerance
because at least some antibiotics can readily penetrate the
matrix yet still can not eliminate biofilm cells. Biofilm mode
of growth is also associated with changes in bacterial membrane
structure and reduction in cell growth rate. The changes in
membrane structure could reduce the permeability to toxic
compounds, while the reduction in growth rate can lead to higher
tolerance to growth-dependent killing by antibiotics. Increasing
evidence suggests that the slow growth, especially that
associated with persister cells, is the most challenging
mechanism for treating chronic infections.
[0008] The rapid development and spread of multidrug resistant
infections present an increasing challenge to public health and
disease therapy. As an intrinsic mechanism of drug resistance,
biofilm formation renders bacteria up to 1000 times less
susceptible to antibiotics than their planktonic (free-swimming)
counterparts of the same genotype. Such intrinsic resistance
also facilitates the development of resistance through acquired
mechanisms that are based on genetic mutations or drug
resistance genes. Consistently, excessive antibiotic treatment
of biofilm infections at sublethal concentrations has been shown
to generate antibiotic-tolerant strains. It is estimated that
biofilms are responsible for at least 65% of human bacterial
infections. For example, it is estimated that in the United
States 25% of urinary catheters become infected with a biofilm
within one week of a hospital stay, with a cumulative 5% chance
each subsequent day. Biofilms are also detected on implanted
devices and are a major cause of explanation. Orthopedic
implants showed a 4.3% infection rate, or approximately 112,000
infections per year in the U.S. This rate increases to 7.4% for
cardiovascular implants, and anywhere from 5%-11% for dental
implants.
[0009] In the biofilm state, bacteria undergo significant
changes in gene expression leading to phenotypic changes that
serve to enhance their ability to survive challenging
environments. Although not completely understood, the tolerance
to antibiotic treatments is thought to arise from a combination
of limited antibiotic diffusion through the extracellular
polymeric substances (EPS), decreased growth rate of biofilm
cells, and increased expression of antibiotic resistance genes
in biofilm cells (10). Treatments that are capable of removing
biofilms from a surface are by necessity harsh and often
unsuitable for use due to medical or environmental concerns. It
is evident that alternative methods of treating bacterial
infections, and most notably biofilms, are required.
[0010] Electric currents/voltages are known to affect cells.
However, most of the studies have been focused on high voltages
and current levels such as eletctroporation, electrophoresis,
iontophoresis, and electrofusion except for a few studies about
biofilm control using weak electric currents. In 1994, Costerton
and colleagues reported an interesting synergistic effect
between low level direct currents (DCs) and tobramycin in
killing Pseudomonas aeruginosa biofilm cells grown in a
continuous-flow chamber. This synergistic phenomenon was termed
the “bioelectric effect.” In addition to P. aeruginosa,
bioelectric effects have also been reported for Klebsiella
pneumoniae, Escherichia coli, Staphylococcus aureus, P.
fluorescens, as well as mixed species biofilms. Although the
impact of electric currents on bacterial susceptibility to
antibiotics and biocides is well accepted, there is little
understanding about the mechanism of bioelectric effect.
[0011] An electric current at an electrode surface can trigger
ion flux in the solution as well as electrochemical reactions of
the electrode materials and redox species with electrolyte and
generate many different chemical species, e.g. metal ions,
H<+> and OH<−>. Although pH change has been shown to
cause contraction of the biofilm formed on the cathodic
electrode, change of medium pH to which prevails during
electrolysis did not enhance the activity of antibiotics.
Consistent with this observation, buffering the pH of the medium
during electrolysis fails to eliminate bioelectric effect.
Another finding suggesting the existence of other factors is
that the bioelectric effect has been observed for biofilms
formed in the middle of an electric field, but not in contact
with either the working electrode or counter electrode. Since
the electrochemically-generated ions accumulate around the
electrodes, the biofilms in the middle of an electric field are
not experiencing significant changes in pH or other products of
electrochemical reactions. This is also evidenced by the report
that radio frequency alternating electric current can enhance
antibiotic efficacy. Since no electrochemically generated
molecules or ions will likely accumulate with alternating
currents, other factors may play a critical role.
[0012] The bioelectric effect was also observed when the growth
medium only contained glucose and two phosphate compounds. This
observation eliminates the electrochemical reaction of salts as
an indispensable factor of bioelectric effect. Previous studies
have also ruled out the impact of temperature change during
electrolysis (less than 0.2° C.). Although these studies
provided useful information about bioelectric effect, its
mechanism is still unknown. The exact factors causing
bioelectric effect and their roles in this phenomenon remain
elusive. Compared to biofilms, even less is known about the
effects of weak electric currents on planktonic cells.
[0013] It is important to note that many aspects of cellular
functions are electrochemical in nature. That is, the redox
state of cells is related to membrane status, oxidative status,
energy generation and utilization and other factors. Therefore,
it is possible that redox state of cells may be affected by
electrochemical currents (henceforth ECs). To better understand
the effects of ECs on planktonic and biofilm cells, we conducted
a systematic study of the effects of weak ECs on the planktonic
and biofilm cells of the model Gram-positive bacterium Bacillus
subtilis. Gram-positive bacteria are responsible for 50% of
infections in the United States, and 60% of nosocomial
infections. With the emergence and wide spread of multidrug
resistant bacteria, effective methods to eliminate both
planktonic bacteria and those embedded in surface-attached
biofilms are badly needed.
BRIEF
SUMMARY OF THE INVENTION
[0014] The present invention provides a system and method for
treating persister cells with an electrochemical process, alone
or in combination with antibiotics. The present invention also
includes an electrochemical cell for treating persister cells.
Weak electric currents are used to effectively eliminate
persister cells and the efficacy can be further improved through
synergistic effects with antibiotics. The present invention
demonstrates unprecedented efficacy in controlling persister
cells and the present invention may be adapted for novel
therapies of chronic infections and strategies to control
persistent biofouling. The present invention has a broad
spectrum applications in treating chronic and drug resistant
infections, such as those caused by Pseudomonas aeruginosa,
Mycobacterium tuberculosis and MRSA (Methicillin resistant
Staphylococcus aureus). The present invention may also be used
for decontamination of medical devices.
[0015] According to a first aspect of the present invention is
an electrochemical method for killing persister cells, the
method comprising the step of applying a weak electrical current
to a bacterial culture, either planktonic culture or a biofilm,
wherein the current is between 1 and 500 microamperes per square
centimeter. According to a preferred embodiment, the current is
a direct current of approximately 75 microampheres per square
centimeter.
[0016] According to a second aspect of the present invention is
an electrochemical method for killing persister cells, the
method comprising the step of applying an electrical current to
a bacterial culture, either planktonic culture or a biofilm,
wherein the current is between 1 and 500 microamperes per square
centimeter, and where the medium is an electrically-conductive
saline solution such as 0.85% NaCl.
[0017] According to a third aspect of the present invention is
an electrochemical method for killing persister cells, the
method comprising the step of applying an electrical current to
a bacterial culture, either planktonic culture or a biofilm,
wherein the current is between 1 and 500 microamperes per square
centimeter, and wherein the medium also contains an effective
amount of an antimicrobial compound such as an antibiotic. The
concentration of the antibiotics can be significantly lower than
what it is required to work in the absence of a current.
[0018] According to a fourth aspect of the present invention is
a method for treating an item comprising a biofilm, the method
comprising the steps of: (i) placing the item at least partially
in a medium; and (ii) applying an electrical current of between
1 and 500 microamperes per square centimeter to the medium.
[0019] According to a fifth aspect of the present invention is a
system for killing persister cells, the system comprising: (i) a
treatment cell with a treatment area for receiving an item and
which contains a reference electrode, a working electrode, a
counter electrode; (ii) a medium (liquid or cream) that at least
partially fills the treatment area and is in communication with
the reference electrode, the working electrode, and the counter
electrode. The treatment cell applies an electrical current
between 1 and 500 microamperes per square centimeter to the
medium in order to kill the persister cells.
[0020] According to a sixth aspect of the present invention is a
system for killing persister cells, the system comprising: (i) a
treatment cell with a treatment area for receiving an item and
which contains a reference electrode, a working electrode, a
counter electrode; (ii) a medium that at least partially fills
the treatment area and is in connection with the reference
electrode, the working electrode, and the counter electrode; and
(ii) an effective amount of an antimicrobial compound such as an
antibiotic. The concentration of the antibiotics can be
significantly lower than what it is required to work in the
absence of a current. The treatment cell applies an electrical
current between 1 and 500 microamperes per square centimeter to
the medium in order to kill the persister cells.
[0021] In another embodiment of the present invention, Bacillus
subtilis was used as the model Gram-positive species to
systematically investigate the effects of
electrochemically-based currents on bacteria including the
morphology, viability, and gene expression of planktonic cells,
and viability of biofilm cells. The data suggest that the weak
electrochemical currents can effectively eliminate Bacillus
subtilis both as planktonic cells and in biofilms attached to
surfaces in a dose-dependent manner. DNA microarray results
indicated that the genes associated with oxidative stress
response, nutrient starvation, membrane functions, and
sporulation were induced by electrochemical currents. These
findings suggest that ions and oxidative species generated by
electrochemical reactions might be responsible for the cidal
effects of these currents.
BRIEF
DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0022] The present invention will be more fully understood and
appreciated by reading the following Detailed Description in
conjunction with the accompanying drawings, in which:
[0023] FIG. 1 is a schematic of biofilm formation;
[0024] FIG. 2 is a schematic of an electrochemical cell
according to the present invention;
[0025] FIG. 3A is a graph illustrating the effects of electric
currents and antibiotics on the persister cells of E. coli HM22,
where the graph depicts the results of treatment with 75
μA/cm<2 >DC alone in 0.85% NaCl buffer using 304L
stainless steel as working and counter electrodes, and current
was generated using graphite working and counter electrodes in
0.85% NaCl buffer;
[0026] FIG. 3B is a graph illustrating the effects of electric
currents and antibiotics on the persister cells of E. coli HM22,
where the graph depicts treatment with antibiotic only, 75
μA/cm<2 >DC only, or co-treatment with current and
antibiotic, and the current was generated using graphite working
and counter electrodes in 0.85% NaCl buffer;
[0027] FIG. 4 is a graph showing the effects of current and Tob
on E. coli biofilm cells when treated the biofilm as an anodic
electrode. Bars indicate the numbers of viable persister cells
of E. coli HM22. Biofilms were grown on stainless 304L steel
electrodes and treated with 75 μA/cm<2 >DC and/or 20 μg/mL
Tob for 60 min.
[0028] FIG. 5 is a graph of E. coli HM22 persister cell survival
following treatment with 15 μA/cm<2 >direct current alone,
H2O2 alone, or both;
[0029]FIG. 6 is schematic of a flow cell system for studying
bioelectric effect;
[0030] FIG. 7 is an image of the removal of detached E. coli
biofilm cells by flow;
[0031] FIG. 8 is a schematic of the overall operation of the
present invention;
[0032] FIG. 9A is a graph of the membrane potential of E. coli
HM22 persister cells compared to normal cells;
[0033] FIG. 9B is a graph of the membrane potential of E. coli
HM22 normal cells following treatment of with 15-45 μA/cm<2
>direct current using graphite electrodes in 0.85% NaCl
buffer;
[0034] FIG. 9C is a graph of the membrane potential of E. coli
HM22 persister cells following treatment of with 15-45
μA/cm<2 >direct current using graphite electrodes in 0.85%
NaCl buffer;
[0035] FIG. 10 is a graph of the effects of tobramycin alone,
electric current alone, or both on P. aeruginosa PAO1 cells at
exponential phase;
[0036] FIG. 11 is a graph of the effects of ciprofloxacin on P.
aeruginosa PAO1 cells;
[0037] FIG. 12 is a graph of the effects of tobramycin alone,
electric current alone, or both on P. aeruginosa PAO1 persister
cells;
[0038] FIG. 13 is a graph of the effects of pretreated buffer on
persister cells of P. aeruginosa PAO1 cells where the 0.85% NaCl
buffer was treated with the same level and duration of electric
currentas used in current-treatment experiments, and where the
cells were incubated in the pretreated buffers to evaluate the
effects of released ions in the absence of a current;
[0039] FIG. 14 is a graph showing the comparison of killing
effects on P. aeruginosa PAO1 persister cells using 304
stainless steel electrodes and carbon electrodes;
[0040] FIG. 15 is a graph of the effect of electric currents on
P. aeruginosa PAO1 persister cells in the presence of 0.85% NaCl
buffer pretreated with 75 μA/cm<2 >current using 304
stainless steel electrodes.
[0041] FIG. 16 is a schematic of the electrochemical cell used
in this study. The reference electrode is Ag/AgCl wire inserted
in a thin glass tube to prevent contact with the working or
counter electrode. Biofilms grown on flat steel or carbon
electrodes can be clipped onto the side; the liquid level is
about 1 cm below the top of the cuvette when full (3 mL).
[0042] FIG. 17 is an image of the contact mode AFM images of
cells treated with 500 μA total DC current (83 μA/cm<2>).
Deflection mode images of planktonic B. subtilis 168 incubated
with pre-treated LB medium at 25 μm (A), 5 μm (B) field size; or
treated with 25 μA/cm<2 >applied total current at 25 μm
(C), 5 μm (D) field size. Scan line errors are from movement of
material on the slide by the cantilever.
[0043] FIG. 18 is a series of images showing the effects of DC
and pre-treated medium on planktonic cells of B. subtilis 168.
Planktonic cells were sub-cultured to an OD600 of 0.8, and 3 mL
sub-culture was treated for 15 min at 37° C. with no current,
pre-treated medium, or applied current. CFUs were counted to
determined cell viability after each treatment.
[0044] FIG. 19 is a graph of the effects of DC and pre-treated
medium on biofilms of B. subtilis 168. Biofilms grown for 2 days
on 304L stainless steel electrodes at 37° C. were treated with
pre-treated LB medium or total applied current for 15 min as
indicated. Cell density of the biofilms was calculated from the
CFU data.
[0045] FIG. 20 is a graph of the effects of ampicillin on
biofilms of B. subtilis 168. Biofilms were treated with varying
concentrations of ampicillin and 500 μA total DC current (83
μA/cm<2>) concurrently for 15 min at 37° C.
[0046] FIG. 21 is a graph of the effects of electrode material
and medium composition on the biofilm cells under DC treatment.
Biofilms were grown on graphite electrodes and treated with 500
μA DC current with and without 50 μg/mL ampicillin for 15 min at
37° C. as indicated. Modified M56 buffer without chlorine was
also tested as the electrolyte solution instead of NaCl buffer
or LB medium.
DETAILED
DESCRIPTION OF THE INVENTION
[0047] Referring now to the drawings, wherein like reference
numerals refer to like parts throughout, the present invention
provide a system and method for the elimination of persister
cells by electric currents and synergy with antibiotics.
Example I
[0048] The present invention was tested using an electrochemical
cell seen in FIG. 2 and with the use of E. coli HM22 constructed
by the pioneer work of Moyed and Bertrand because it produces
1000 times more persister cells in exponential-phase cultures
than the wild-type E. coli strains and has been used in most
studies of persister cells. To evaluate the effects of electric
currents, the persister cells were first isolated as described
previously. Briefly, the exponential culture of HM22 at optical
density at 600 nm (OD600) of 0.3 in LB medium was treated with
100 μg/mL ampicillin for 3 h to kill and lyse the normal cells.
The persister cells were then collected by centrifugation at
8000 rpm at 4° C. for 10 min and resuspended in 0.85% NaCl
buffer. The persister cells were then treated in a customized
electrochemical cell, shown in FIG. 2. Electrodes with a
dimension of 1 cm×5.6 cm were cut from a flat 304L stainless
steel sheet (MSC; Melville, N.Y.) or graphite sheet
(McMaster—CARR, Santa Fe Springs, Calif.). The same material was
used for both the counter electrode and working electrode, which
were placed into a 4.5 mL standard-style polystyrene cuvette
(Fisher Scientific; Hampton, N.H.). A 0.48 mm diameter silver
wire (A-M Systems; Sequim, Wash.) was placed in bleach for 30
min to produce an Ag/AgCl reference electrode. The bottom 1″ of
a borosilicate glass Pasteur pipette (Fisher) was cut and the
reference wire was placed inside to prevent contact with the
working or counter electrode. An AFCBP1 potentiostat/galvanostat
(Pine Instrument Company, Grove City, Pa.) was connected via
alligator clamps to the electrodes and used to control the
current. The volume of medium in the fully-constructed
electrochemical cell was 3 mL (see FIG. 2).
[0049] Significant killing of persister cells was observed both
with stainless steel and graphite electrodes. For example,
treatment with 75 μA/cm<2 >(voltage around 1V) for 50 min
in 0.85% NaCl buffer caused complete killing of persister cells
(a 6 log reduction in viability, FIG. 3A) by counting colony
forming units (CFUs) before and after treatment. The killing was
not simply caused by the products of electrochemical reactions
since incubation with the pre-treatment buffer (0.85% NaCl
buffer treated with the same level and duration of current) did
not cause any apparent killing (data not shown). Interestingly,
the electric current was more effective in killing persister
cells than normal cells. As shown in FIG. 3A, the same treatment
of normal cells only caused a 3 log reduction in the number of
viable cells. Effective killing of persister cells was also
observed using graphite electrodes. As shown in FIG. 3B,
treatment with the same current level (75 μA/cm<2>) for 60
min caused a 3 log reduction of viable persister cells, whereas
pre-treated medium only reduced the viable cells by less than 1
log. Furthermore, the efficacy of persister control can be
improved through synergistic effects with antibiotics. The
graphite electrode was used for this experiment since it does
not cause complete killing, allowing the synergistic effects to
be observed. As shown in FIG. 3B, application of 75 μA/cm<2
>current or 20 μg/mL cinoxacin (Cin) alone caused a 3 log or
no apparent reduction in the number of viable persister cells,
respectively. When these two treatments were applied together,
however, nearly complete killing (more than 5 log reduction) was
observed. Such synergistic effects have not been reported for
persister cells. It is also worth noticing that the synergy is
not only limited to Cin since tobramycin (Tob) also exhibited
synergistic effect with electric current (see FIG. 3B).
[0050] To determine if electric currents are also effective in
killing persister cells in biofilms, E. coli HM22 biofilms were
cultured on 304L stainless steel coupons. The biofilm-coated
coupons were then used as anodic or cathodic electrode, and
treated with direct current alone or with tobramycin together.
Immediately after treatment, the cells were removed from the
biofilm-coated coupons by sonication and vertexing. A portion of
the cells was directly plated on LB+DPA plates to quantify the
total number of viable cells by counting CFU, the other part of
the sample was treated with 100 μg/mL ampicillin for another 3 h
and plated on LB+DPA plates to quantify the number of the viable
persister cells. This approach allowed us to study the killing
effects on normal and persister cells separately.
[0051] As shown in these FIG. 4, when treating biofilm
persisters with tobramycin alone (20 μg/mL or 150 μg/mL), there
was no significant reduction in total number of viable cells and
number of viable persister cells compared to the untreated
control sample. These results are consistent with the knowledge
that biofilms have significantly enhanced tolerance to
antibiotics compared to planktonic cells. However, treatments
with 75 μA/cm<2 >alone for 60 min reduced the number of
viable persister cells by 3.5 logs. After treating biofilms with
currents and tobramycin together for 60 min, the number of
viable persister cells was reduced by 5.4 log (nearly complete
killing, FIG. 4). Thus, synergy between electric currents and
antibiotics also exist for killing persister cells in biofilms.
[0052] With the capability to quantify the expression level of
each gene at the genome-wide scale, DNA microarrays have been
extensively used to monitor global gene expression profiles in
response to different stimuli including persister formation and
biofilm formation. However, currently there are no reported data
about the effects of weak electric currents on bacterial gene
expression at the genome-wide scale. To identify the effects of
electric currents on cell physiology of persister cells and
normal cells at the genetic level, the present invention
utilized two experiments that revealed clues about the effects
of weak electric currents on bacterial cells.
[0053] In the first experiment, persister cells and normal cells
of E. coli HM22 harvested using the same method as describe
above were treated with and without 75 μA/cm<2 >DC for 15
min in 0.85% NaCl buffer. In a parallel experiment, the
persister cells were also treated with M56 buffer with the same
level and duration of the current. After harvesting HM22 normal
and persister cells, they were concentrated 40 times and
resuspended in 6 mL 0.85% NaCl buffer and 6 mL M56 buffer
respectively. Both samples were separated into two equal
aliquots: one was left untreated, meanwhile the other one was
treated with 75 μA/cm<2 >DC. After 15 min incubation with
and without current, all of the cells were centrifuged
immediately for 30 s at 13,200 rpm and 4° C. to harvest the
cells. For RNA isolation, each cell pellet was resuspended in 1
mL of TRIzol reagent buffer (Invitrogen Co., Carlsbad, Calif.)
and beaten rigorously at 4,800 beats per min for 30 s in a
closed bead beater tube with 200 μl of silicon beads using a
mini bead beater (Biospec Products Inc., Bartlesville, Okla.).
The following isolation steps were conducted by following Trizol
reagent protocol and the total extracted RNA was subsequently
purified using RNeasy Mini kit (QIAGEN Inc., Valencia, Calif.).
The quality and quantity of the total RNA samples were evaluated
using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara,
Calif.) and the microarray hybridizations were performed using
E. coli Genome 2.0 Arrays (Affymetrix, Inc., Santa Clara,
Calif.). Both were performed using the DNA microarray core
facilities at the SUNY Upstate Medical University (Syracuse,
N.Y.).
[0054] Stringent criteria were applied to select the
induced/repressed genes based on p-values (<0.0025 or
>0.9975) calculated using the Wilcoxon signed rank test and
Tukey By weight. The applied current in 085% NaCl buffer was
found to induce 9 genes and repressed 36 genes in E. coli HM22
persister cells (see Table 1). While 27 of these genes have
unknown functions, the treatment did induce the genes of the trp
operon (trpEL), acyl carrier protein phosphodiesterase (acpD),
L-serine dehydratase (sdaB), oxidative stress response (oxyS),
and repressed the cys operon (cysCDJKNP), production of
tryptophanase (tnaL) and nitrite extrusion (narU) (see Tables
1-5). In comparison, treatment with the same current level in
M56 buffer induced 15 genes (yibP, cysU, csgD, nrdE, narW, hisL,
oxyS, etc) and repressed only 4 genes of persister cells (see
Tables 1-5). Interestingly, the induced genes have functions of
central intermediary metabolism, protease for cell division, PTS
system, sulfate transport, surface structure, DNA synthesis, his
operon, oxidative stress response and unknown functions. Three
of the four repressed genes have unknown functions, while the
forth gene uvrB has functions of DNA damage recognition and
repair. These data suggest that weak electric currents are able
to activate certain cellular activities including those related
to oxidative response, membrane structures and functions.
[0000]
TABLE 1
Number of induced/repressed genes of E. coli HM22 in response
to15-min treatment with 75 μA/cm<2 >
current using graphite electrodes.
Persister cells Persister Normal in
0.85% cells cells
NaCl buffer in M-56 in M-56
Number of induced genes 9 54 379
Number of repressed genes 36 1 25
[0000]
TABLE 2
Genes of E. coli HM22 persister cells induced bytreatment with
75 μA/cm<2 >
DC for 15 min in M56 buffer.
Gene Expression ratio
Name (with DC/no DC) Functions
Environmental information processing
yadM 1.32 Putative fimbrial-like protein
yehB 8.57 Putative outer membrane protein
cysU 6.50 Sulfate transport system permease protein
CysT
yjdL 1.62 Putative peptide transporter
Genetic information processing, transcription factors
C0336 4.29 PTS system, mannitol (Cryptic)-specific
IIA component
oxyS 2.00 Global regulatory RNA OxyS
hisL 2.00 His operon leader peptide
J02459 1.52 Lambda K, tail component
gltF 1.52 Regulator of gltBDF operon, induction of
Ntr enzymes
micF 1.23 Regulatory antisense RNA affecting ompF
expression
trpL 1.23 Trp operon leader peptide
Metabolism, enzyme
narW 24.25 Respiratory nitrate reductase 2 delta
chain
nrdE 7.46 Ribonucleoside-diphosphate reductase 2
alpha chain
acpD 1.52 Acyl carrier protein phosphodiesterase
yhjN 1.52 Cyclic di-GMP binding protein precursor
trpE 1.41 Anthranilate synthase component I
grxA 1.41 Glutaredoxin1 redox coenzyme for
glutathione-
dependent ribonucleotide reductase
yhhW 1.41 Protein YhhW
trxC 1.41 Putative thioredoxin-like protein
pyr I 1.32 Aspartate carbamoyltransferase,
regulatory subunit
cynT 1.32 Carbonic anhydrase
dcp 1.32 Peptidyl-dipeptidase Dcp
maeB 1.87 Putative membrane protein
yibP 2.83 Putative head-tail adaptor
cellular processes, receptors and channels
tsx 1.15 Nucleoside channel; receptor of phage T6
and colicin K
The numbers show the range of fold changes for the induced and
repressed genes in the same operon.
[0000]
TABLE 3
Gene of E. coli HM22 persister cells repressed bytreatment with
75 μA/cm<2 >
DC for 15 min in M56 buffer.
Gene Expression ratio
Name (with DC/no DC) Functions
cspC 0.47 stress protein, member of the CspA-Family
The number shows the range of fold changes for the induced and
repressed genes in the same operon.
[0000]
TABLE 4
Genes of E. coli HM22 persister cells induced by treatmentwith
75 μA/cm<2 >
DC for 15 min in 0.85% NaCl buffer.
Gene Expression ratio
Name (with DC/no DC) Functions
Genetic information processing, transcription factors
oxyS 1.32 Global regulatory RNA OxyS
trpL 1.23 Trp operon leader peptide
Metabolism, enzyme
acpD 1.41 Acyl carrier protein phosphodiesterase
trpE 1.32 Anthranilate synthase component I
sdaB 1.23 L-serine dehydratase (deaminase), L-SD2
yhhW 1.32 Protein YhhW
Unknown function, hypothetical protein
yqjF 1.23 Hypothetical protein YqjF
ybiJ 1.74 Orf, hypothetical protein
yeiH 1.15 Orf, hypothetical protein
The numbers show the range of fold changes for the induced and
repressed genes in the same operon.
[0000]
TABLE 5
Genes of E. coli HM22 persister cells repressed by
treatmentwith 75 μA/cm<2 >
DC for 15 min in 0.85% NaCl buffer.
Gene Expression ratio
Name (with DC/no DC) Functions
Environmental information processing
yeeE 0.50 Putative transport system permease protein
cysP 0.09 Thiosulfate binding protein
narU 0.54 Nitrite extrusion protein 2
Z1375 0.81 Putative tail component encoded by
cryptic
prophage CP-933M
Genetic information processing, transcription factors
tnaL 0.66 Tryptophanase leader peptide
Metabolism, enzyme
wrbA 0.76 Amino terminal fragment of WrbA
cysD 0.71 ATP: sulfurylase, subunit 2
cysN 0.57 ATP-sulfurylase, subunit 1
cysK 0.66 Cysteine synthase A, O-acetylserine
sulfhydrolase A
cysJ 0.76 Sulfite reductase (NADPH), flavoprotein
beta subunit
cysC 0.81 Adenosine 5-phosphosulfate kinase
b1772 0.76 Putative kinase
The numbers show the range of fold changes for the induced and
repressed genes in the same operon.
[0055] The effects on cell membrane functions are corroborated
by a parallel but more complete study regarding the effects of
electric currents on the Gram-positive bacterium Bacillus
subtilis 168. In this experiment, the cells of B. subtilis 168
in late exponential phase was treated for 15 min in LB medium
with 42, 139 or 417 μA/cm<2 >DC using 304L stainless steel
as electrodes in the electrochemical cell shown FIG. 2. Each
condition was tested in duplicate and the data was analyzed
using cluster analysis. To differentiate the effects of currents
from those of the electrochemical reaction products, the control
samples were incubated for 15 min in the LB medium that was
pre-treated with the same level and duration of the current.
Since the control samples were prepared in pre-treated LB medium
containing all the electrochemical reaction products, the gene
expression changes are mainly caused by the currents as well as
the movement and gradient of chemical species, e.g. ions. The
genes that were induced or repressed in all conditions are
listed in Table 6. There were also 839 genes induced under some
but not all conditions, such as transport genes encoding glycine
betaine/carnitine/choline ABC transporters, amino acid
transporters, and putative monovalent cation/H+ antiporters
(gene list not shown). Overall, the microarray results suggest
that electric current and associated ion movement/gradient have
significant influence on cellular activities of bacteria
especially metabolism and membrane functions.
[0000]
TABLE 6
B. subtilis 168 genes consistently induced/repressedby 15
min treatments of 42, 139 and 417 μA/cm<2 >
DC.
Expression
Cluster Genes ratio Function/gene product
Genes cydABCD 2.1-3.5 cytochrome bd oxidase
up-regulated gltACT 1.9-3.2 glutamate/cation
uptake symporter
at all tested hisBCDGHZ 1.7-2.8 histidine
biosynthesis
currents narGHIJK 3.2-7.5 nitrate reductase:
nitrite extrusion
purEKRT 2.3-2.8 purine synthesis and
metabolism
tuaABCD 2.3-5.7 teichuronic acid synthesis
yfkDE 3.0 cation resistance
mtnKUW 1.9-2.5 methylthioribulose recycling
pstSAC, BA, BB 2.8-8.0 PhoPR regulated Pi
transporter
yusU 2.6 unknown function
Genes cotIKS (−4.0)-(−14.0) spore coat
proteins
down-regulated yomBDIP (−1.9)-(8.0) unknown
functionat all testedcurrents
The numbers show the range of fold changes for the induced and
repressed genes in the same operon.
[0056] The DNA microarray data suggests that treatment with
electric currents may lead to generation and accumulation of
reactive oxygen species (“ROS”) (e.g., induction of oxyS, a
global regulatory RNA). Thus, the treatment could render the
persister cells more susceptible to external ROS. To test this
hypothesis, we treated E. coli HM22 persister cells with 100 μA
for 20 min and followed by treatment with and without H2O2 (500
μM) for 1 h. These cells were then split into two parts: one for
microscopic analysis and the other for CFU count. For
microscopic study, cells were treated with 200 μM
dichlorodihydrofluorescein diacetate (H2DCFDA, Sigma-Aldrich,
St. Louis, Mo.) for 30 min in dark at room temperature. After
incubation, cells were spin down and resuspended in PBS buffer
for visualization using a fluorescence microscope (Axio Imager
M1, ZEISS, Jena, Germany). The dye H2DCFDA can penetrate
bacterial cells and get cleaved by cellular esterase to produce
H2DCF. If there is any ROS present, this H2DCF will be converted
to DCF and give fluorescence (Invitrogen, USA). The results
showed that treatment with 15 μA/cm<2 >direct current,
similar to treatment with H2O2 (500 μM) caused accumulation of
ROS in persister cells. For the CFU count, cells were plated on
LB plates supplemented with DPA and incubated overnight at 37°
C. The CFU data further confirmed that the treatment with
electric current rendered the persister cells more sensitive to
H2O2 since treatment with electric current followed by H2O2
killed more persister cells than either the EC or H2O2 alone
(see FIG. 5).
[0057] Construction and use of a flow cell system is possible to
directly visualize the effects of electric currents on biofilm
cells. To directly visualize the effect of electric currents on
biofilm cells and biofilm structure, the FC81 flow cell system
(BioSurface Technologies Corporation, Bozeman Mont.) was
modified to deliver electric current. The flow cell contains two
slides to form a channel with a dimension of 47.5×12.7 mm and
1.6 mm space between the two slides. The cover glass was coated
with 50 Å Ti followed by 70 Å Au. This engineered surface is
transparent and conductive, allowing the direct visualization of
bioelectric effect with microscopy. An Ag/AgCl reference
electrode was also inserted at the exit of the flow cell
(through a Y-junction) without touching the other two
electrodes. The bottom surface could be made with any material
of interest and cut into the dimension of regular glass slides
(2.54 cm by 7.62 cm). The flow cell was assembled with a
gold-coated slide as the counter electrode and the bottom plate
as the working electrode (see FIG. 6). A Y-junction was attached
at the exit of the flow cell, with one line for insertion of
reference electrode and the other for the effluent of biofilm
culture. The tubing that holds the reference electrode was
clamped as a dead-end to prevent any leakage.
[0058] The electrodes were connected to a model AFCBP1
potentiostat/galvanostat (PINE Research Instrumentation) by Cu
wires. This is the first flow cell system containing reference
electrode to allow precise control of the potential and current.
The mature one-day biofilm of E. coli RP437/pRSH103 expressing
red fluorescent protein (RFP) constitutively was treated with 50
μA/cm<2 >DC for 1 h. The flow of LB medium (63) at 10 mL/h
was stopped before the treatment with current and resumed after
the treatment. Significant detachment of biofilm cells by
electric current was observed (see FIG. 7). This flow cell
system is an ideal tool for studying the effects of electric
currents on biofilm-associated persister cells.
[0059] It is well documented that persister cells are
metabolically inactive compared to normal cells. Conceivably, an
approach that can target this difference could have high
efficacy. As shown in FIG. 3A, some of the conditions are more
effective in killing persister cells than normal cells. Thus,
the treatment conditions may be fine tuned to selectively kill
this population that is highly resistant to antibiotics. All
living cells need to maintain a membrane potential for
metabolism and transfer of nutrients. If the membrane potential
is disrupted, the cells could lose the capability to maintain
the ion gradients and cell death will occur. Normal cells may
have higher membrane potential than persister cells due to
higher metabolic activities. In this sense, the persister cells
could be more sensitive to reduction of membrane potential. This
is evidenced by recent mechanistic studies of pyrazinamide for
tuberculosis therapy. Unlike conventional antibiotics that are
more active against growing cells, pyrazinamide is more
effective in killing non-growing bacilli. A recent study has
shown that pyrazinamide kills cells by disrupting the membrane
energetics and transport function at acid pH. An applied
electric current can either positively or negatively influence
the membrane potential, which consequently affects the viability
of persister cells and susceptibility to antibiotics (FIG. 8).
If the membrane potential is reduced by the applied current,
direct killing of persister cells can be expected. If the
membrane potential is positively affected by the current,
however, it may work as a “wake up” call of the persister cells
to enter a metabolically more active stage and therefore render
the cells more susceptible to antibiotics. The membrane
permeability to antibiotics may also be affected by the applied
current. The exact impact on persister cells may rely on the
current level, material of the electrodes and the associated
ions released, medium composition and the antibiotics applied.
[0060] Membrane potential can be measured using either
florescent or radioactive methods. The persister cells of E.
coli HM22 and P. aeruginosa PAO1 cells at different growth
phases may be treated with electric currents; and the cells
before and after treatments may be analyzed to evaluate the
effects of electric currents on membrane potential.
[0061] In light of the above, the membrane potentials of E. coli
HM 22 normal and persister cells were compared. Briefly,
approximately 1×10<6 >persister cells per mL were washed
with PBS buffer (10 mM sodium phosphate, 145 mM sodium chloride,
pH 7.4) followed by addition of carbocyanine dye DiOC2
(Invitrogen, Carlsbad, Calif.) to 30 μM and incubation at room
temperature for 30 min. Fluorescence was determined using a LSR
II flow cytometer (Becton Dickinson, San Jose, Calif.), with
excitation at 495 nm and emission at 575 nm. The red/green
ratiometric parameter was set according to the manufacturer's
instructions for histogram analysis. The ratiometric parameter
was calculated as [(red value)−(green value)+384]. The overlay
histogram of membrane potential analysis was obtained using CXP
software. As shown in FIG. 9A, the membrane potential of
persister cells is lower than that of normal cells. To our
knowledge, this is the first direct comparison of membrane
potential between normal and persister cells of E. coli. In
addition, treatment with 15, 30 and 45 μA/cm<2 >direct
current significantly reduced the membrane potential of
persister cells, but not that of normal cells (FIGS. 9B and 9C).
These data confirm our hypothesis and suggest that membrane
potential is a potential target of new therapies. Further study
on this finding could help understand the mechanism of persister
control by electric current and synergistic effects with
antibiotics.
[0062] For the conditions that exhibit synergistic effects with
antibiotics, the membrane permeability may also be tested using
radioactively labeled antibiotics. In particular, the
intracellular concentration of benzyl-14C-penicillin (potassium)
and <3>H-oxytetracycline (American Radiolabeled Chemicals,
Inc., St. Louis, Mo.) may be measured after incubation with
cells for 30 min in the presence or absence of a current using a
liquid scintillation counter. These data are expected to
corroborate the results regarding the effects of current on
membrane potential and permeability. It will be integrated with
the results in the following study to get insight into the
mechanism of persister control with electric currents.
[0063] As described in the results above, the present invention
is premised on promising evidence that weak electric currents
have significant effects on gene expression of both persister
cells and normal cells of bacteria. As a result, gene expression
in response to electric currents may be further studied to
understand the mechanism at the genetic level by identifying the
differentially expressed genes and pathways.
[0064] First, E. coli HM22 may be used to prepare persister
cells as described above. The harvested persister cells may be
treated with different levels of electric currents (75, 150 and
300 μA/cm<2 >DC) using graphite electrodes in 0.85% NaCl
buffer or M56 buffer. The gene expression of these cells may be
compared with that of persister cells incubated in the buffer
pre-treated with the same level and duration of current. In
addition, normal cells of HM22 may be treated with the same
conditions to identify the persister-specific genes and pathways
affected by electric currents. Similar experiments may also be
performed to treat P. aeruginosa PAO1 cells at exponential and
stationary phases. The treatment time may be 15 min and extended
if more profound changes are needed to identify the pathways.
Each experiment may be conducted in duplicate and the data may
be analyzed using cluster analysis to identify the gene
expression patterns and the pathways involved in response to
current treatments. The representative induced/repressed genes
may be confirmed by RNA dot blotting.
[0065] Compared to other stimuli, e.g., starvation and
temperature change, electric currents (especially constant DCs)
are not the common challenges or evolutionary pressures that
bacteria experience in nature. Thus, the expression patterns may
provide unique information for understanding bacterial
physiology in general, and for developing better control
methods. With the gene expression patterns identified, one may
further study to corroborate the results using mutants of the
differentially expressed genes. For example, the mutants of
induced genes could be more sensitive to electric currents.
Electric currents, especially those with higher current levels
and longer duration than described here, have been found to
improve the efficacy of antibiotics in treating biofilms.
However, the mechanism of such effects remains unknown. Since
persister cells play an important role in biofilm-associated
drug tolerance, it is possible that antibiotics and electric
currents are both capable of killing susceptible biofilm cells,
while electric currents can also kill some persister cells and
the efficacy can be enhanced through synergy with antibiotics.
This is supported by the fact that electric current can be more
effective in killing persister cells than normal cells (FIG.
3A). This may create more friendly treatment conditions with
lower current level and shorter treatment time.
[0066] E. coli HM22 and P. aeruginosa PAO1 may be used to
inoculate biofilm cultures using the flow chamber described in
results of the present invention (FIG. 6). As discussed above,
these two are the best-studied strains of persister formation
and many genetic tools are available.
[0067] The preformed biofilms of E. coli HM22 and P. aeruginosa
PAO1 can be treated with electric currents and antibiotics under
the effective conditions identified. The number of viable cells
can be quantified by counting CFUs after collecting biofilm
cells from the surface by sonication and spreading cells on LB
agar plates. Meanwhile, part of the collected cells may be
treated with 100 μg/mL ampicillin (for E. coli HM22) or 200
μg/mL ofloxacin (for P. aeruginosa PAO1) for 3 hours and then
tested using the same CFU method to quantify the viable
persister cells. The CFU data of biofilms with and without
treatment may be compared to evaluate the effects of electric
currents on the viability of persister cells in biofilms. The
adhesion and metabolic activity of biofilm-associated persister
cells may be analyzed in situ using the flow cell system
described above.
[0068] The effects of electric current on biofilm structure may
be followed in situ using a fluorescence microscopy to obtain
the three dimensional information of biofilms. The structural
parameters of biofilms including surface coverage, thickness,
roughness, and biomass may be calculated using the computer
program COMSTAT (31). The dynamic 3-D imaging data may then be
obtained to help elucidate the effects of electric current on
biofilm formation and structure. To visualize biofilm-associated
persister cells three dimensionally, the promoterless gfp(LVA)
gene may be cloned in pCA24N (for E. coli, available at NIGJ)
and pME290 (for P. aeruginosa, available from ATCC) under the
promoter rrnBP1 of E. coli HM22 and P. aeruginosa PAO1,
respectively, and inserted in the corresponding hosts. Thus, the
intensity of GFP will be proportional to the cell growth rate.
In addition, all biofilm cells may be strained with the
BacLight™ Red fluorescent dye (Invitrogen). Thus, all biofilm
cells may be strained red and the green dye can be used to
differentiate persister cells (weak or no green signal) from
normal cells (stronger green signals). Compared to the highly
stable native GFP, the unstable GFP(LVA), which has a half-life
less than 40 min, may be used to allow the dynamic monitoring of
cell growth. The constructed reporters may then be used to study
the effects of electric currents on the adhesion/detachment and
growth of persister cells in three dimensions and in real time
at different stages of biofilm formation (from initial adhesion
to maturation).
[0069] To understand the mechanism of persister control using
electric currents and to develop better biofilm control methods,
the above studies may systematically investigate the effects of
electric currents on physiology of persister cells, gene
expression and pathways, as well as the effects on
biofilm-associated persister cells. These results may be
integrated to develop a model to explain the mechanism. The
results from these studies may also help develop more effective
control methods, e.g., electrically enhanced antibiotic
therapies and anti-biofouling approaches.
[0070] Conceivably, application of an electric current can cause
complex changes to the chemical composition of the medium. The
effects of currents on bacterial physiology may be carefully
compared with pre-treated medium to eliminate the effects of
electrochemical reactions products. In addition, the
electrochemical reactions may be systematically studied to
identify the roles of each reaction product on persister cells.
[0071] Continued experiments, for example, have already shown
that the effects of electric current and synergy with
antibiotics is not species specific, as similar results were
shown using P. aeruginosa. The experiments were conducted in the
same way as described for E. coli HM22. Briefly, an overnight
culture of P. aeruginosa PAO1 was used to inoculate LB medium to
an OD600 of ̃0.005 (1:1000 dilution of an overnight culture with
LB) and incubated till OD600 reached ̃0.7. Then the cells were
washed twice with 0.85% NaCl buffer and treated in the same way
as described for planktonic E. coli cells. As shown in FIG. 10,
treatment with 1.5 μg/mL Tob did not cause any significant
killing. Treatment with 75 μA/cm<2 >for 60 min reduced the
number of viable P. aeruginosa PAO1 cells by 3 logs. When the
two treatments were combined, however, up to 5 logs of killing
was observed. Thus, synergistic effects clearly also exist
between electric current and tobramycin on P. aeruginosa PAO1,
suggesting this effect is not species specific and can
potentially be applied to treated human bacterial infections.
[0072] To identify the condition for isolating P. aeruginosa
PAO1 persister cells, the overnight culture of P. aeruginosa
PAO1 was treated for 3.5 h with various concentrations of
ciprofloxacin (“Cip”) to determine the appropriate concentration
that can kill normal cells. As shown in FIG. 11, the killing of
P. aeruginosa PAO1 increased with Cip concentration up to 50
μg/mL and no further killing was observed even when Cip was
added as 200 μg/mL. Thus, the 1% cells that survived the
treatment were persister cells and treatment with 200 μg/mL Cip
was used in the following experiments to harvest persister cells
and ensure the complete killing of normal cells.
[0073] Synergistic effects were also observed for treatment with
electric current and Tob, similar to the data of normal cells
described above. The results indicate that 1.5 μg/mL Tob was not
able to kill P. aeruginosa PAO1 persister cells. However,
treatment with 75 μA/cm<2 >(500 μA total) current reduced
the number of viable persisters by ̃2.5 logs and another 2 logs
of killing was obtained when treating with Tob together, as
shown in FIG. 12. It is worthy noticing that the efficiency in
killing by electric current and synergistic effects with Tob
were similar for persisters and normal cells. This is a
significant advantage compared to traditional antibiotics, which
commonly fail to kill bacterial cells that are in stationary
phase or are persisters.
[0074] To understand if the killing by electric currents was due
to the ions generated by electrochemical reactions, P.
aeruginosa PAO1 persister cells were also treated with
pretreated buffer, which was prepared by treating 0.85% NaCl
buffer with SS304 stainless steel electrodes for the same
current level and duration as used for the above experiments.
The pretreated buffers were collected after 20, 40 or 60 min of
treatment. P. aeruginosa PAO1 persister cells were collected as
described above and resuspended in the pretreated buffers in the
presence and absence of Tob. The cells were then incubated at
room temperature without shaking for up to 1 h and samples were
collected every 20 min to count CFU. As shown in FIG. 13, the
ions released from the electrode caused less than one log of
killing of persister cells, significantly less than that with
current treatment (2-3 logs), suggesting the movement of ions or
some short-term ions might be essential for the effectively
killing with electric current. The generation of ROS as
described in E. coli HM22 data could be partially responsible
for the killing. In addition, no synergy was observed between
pretreated buffer and 1.5 μg/mL Tob. This finding suggests that
electric current may enhance the penetration of Tob and/or the
susceptibility of persisters.
[0075] In addition to stainless steel, carbon electrodes were
also found to control E. coli persister cells (discussed above).
Here we also compare the effects of stainless steel and carbon
electrodes on P. aeruginosa persister cells. As shown in FIG.
14, killing by about two logs was achieved using carbon
electrodes. It is slightly less than the 3 logs of killing by
stainless steel electrodes; however, it does confirm that the
killing effects are not limited to stainless steel electrodes.
[0076] Since the current treatment with 304 stainless steel
electrodes was more effective than that with carbon electrodes
in killing persisters, another experiment was conducted to treat
P. aeruginosa PAO1 persister cells using carbon electrodes and
0.85% NaCl buffer pretreated with 304 stainless steel
electrodes. As shown in FIG. 15, additional killing was observed
compared to treatment with 304 stainless steel electrodes (FIG.
15) or carbon electrodes (FIG. 14) alone. These results confirm
that ions or charge movement induced by electric current
treatment may be a key factor in killing persister cells. Thus,
a pre-prepared solution or cream containing such chemical
species might be applied for disease therapy with electric
currents.
[0077] Embodiments of the electrically-enhanced control of
bacterial persister cells, both planktonic persisters and those
in biofilms, are described above. The use of a very small
electric current to control persister cells, as well as the
synergistic effects shown when used in conjunction with
antimicrobial agents, is a new phenomenon. The low level of
electric current/voltage required to control persister cells are
believed to be physiologically safe for humans since similar and
higher current/voltage levels have been used to stimulate tissue
and bone growth.
[0078] Further, the effects of electric current and the synergy
with antimicrobial agents is not species-specific, since similar
results were shown using both E. coli strains and P. aeruginosa
strains. Accordingly, the present invention can be used to kill
a wide variety of microbial species.
[0079] The use of low electric current and/or low electric
current together with an antimicrobial agent is a novel means of
controlling persister cells and can be incorporated into devices
or procedures in order to treat chronic infections both inside
and outside the human body. For example, possible applications
include the treatment of chronic wounds, chronic sinusitis,
implanted-device-associated infections, and middle ear
infection, the decontamination of medical devices, or devices
with bare or coated electrodes, among many others.
Example 2
[0080] Bacterial strains and growth media. B. subtilis 168
(trpC2) was used for planktonic studies. B. subtilis BE1500
(trpC2, metB10, lys-3, ΔaprE66, Δnpr-82, ΔsacB::ermC} was
obtained from EI du Pont de Nemours Inc (Wilmington, Del.) and
used for the biofilm studies. Overnight cultures were grown at
37° C. with aeration via shaking on an orbital shaker (Fisher
Scientific; Hampton, N.H.) at 200 rpm. Biofilms were developed
on stainless steel coupons (5.6 cm by 1.0 cm) in batch culture
at 37° C. in 100 mm petri dishes (Fisher Scientific; Hampton,
N.H.) for 48 hours. Luria-Bertani (LB) medium consisting of 10
g/L NaCl, 10 g/L tryptone, and 5 g/L yeast extract (all from
Fisher Scientific; Hampton, N.H.) was used for both planktonic
and biofilm cultures. LB agar plates were prepared by adding 15
g/L Bacto agar (Fisher Scientific) to LB medium prior to
autoclaving and pouring into 100 mm petri dishes (Fisher
Scientific).
[0081] Poly-γ-glutamic acid (PGA) is a viscous protein produced
predominantly by members of the taxonomic order Bacillales.
However, B. subtilis 168 does not produce PGA, due to mutations
in the degQ promoter region and the gene swrA. This protein is
required in B. subtilis for biofilm formation, and
re-introduction of the wild-type genes into B. subtilis 168
allowed biofilm growth. B. subtilis BE1500 is a strain which
produces PGA and therefore form relatively good biofilms, and is
therefore suitable for the study of B. subtilis biofilms.
[0082] Electrochemical Cell Construction. Electrodes with a
dimension of 1 cm×5.6 cm were cut from a 30.5 cm by 30.5 cm flat
304L stainless steel sheet (<0.08% C, 17.5-20% Cr, 8-11% Ni,
<2% Mn, <1% Si, <0.045% P, <0.03% S; MSC; Melville,
N.Y.). Counter electrodes were bent at the end to form a hook
shape (see FIG. 16). A counter electrode and working electrode
were placed into a 4.5 mL standard-style polystyrene cuvette
(Fisher Scientific; Hampton, N.H.). A 0.015″ diameter silver
wire (A-M Systems; Sequim, Wash.) was placed in bleach for 30
min to generate an Ag/AgCl reference electrode. The bottom 1″ of
a borosilicate glass Pasteur pipette (Fisher Scientific) was cut
and the reference wire was placed inside to prevent accidental
contact with the working or counter electrode. A
potentiostat/galvanostat (Model #AFCBP1, Pine Instrument
Company, Grove City, Pa.) was connected via alligator clamps to
the electrodes and used to control the voltage and current. The
volume of medium in the fully-constructed electrochemical cell
was 3 mL. A schematic of the system is shown in FIG. 16.
[0083] Determination of Minimum Inhibitory Concentration and
Minimum Bactericidal Concentrations. To determine the minimum
inhibitory concentrations (MICs) of ampicillin on planktonic
cells, B. subtilis 168 and B. subtilis BE1500 were cultured in
LB medium overnight as described above. The overnight cultures
were subcultured by a 1:1000 dilution in LB medium containing
various concentrations of ampicillin with seven replicates in a
96-well plate and allowed to grow at 37° C. with shaking at 200
rpm for 24 hours. The OD600 was measured immediately after
inoculations and at 24 hours after inoculation with a microplate
reader (Model EL808, BioTek Instruments, Winooski, Vt.). The MIC
was defined as the lowest concentration of ampicillin that
completely inhibited growth.
[0084] MIC is not a useful measurement of the response of
biofilms to antibiotics because antibiotics added in the growth
medium before inoculation could kill planktonic cells before
they can form a biofilm. Therefore it is important to
characterize the minimum bactericidal concentration (MBC) of
ampicillin on established biofilms. B. subtilis BE1500 was
cultured overnight as described above. Flat stainless steel
electrodes were placed in a 100 mm petri dish with 20 mL LB
medium, which was inoculated with 20 μL of an overnight culture.
Biofilms were allowed to develop for 48 hours at 37° C. without
shaking. The electrodes with biofilms were gently washed three
times in 0.85% NaCl buffer and immersed in LB medium containing
various concentrations of ampicillin for 15 min. Immediately
after treatment, the electrodes with biofilms were placed in a
15 mL polystyrene test tube (Fisher Scientific) containing 4 mL
0.85% NaCl buffer and sonicated for 2 min to remove the biofilm
cells from the surface. The stainless steel electrode was then
removed and the tube was vortexed for 30 s to break up any
remaining cell clusters. CFUs were counted after spreading the
buffer with cells on LB agar plates and incubated overnight at
37° C.
[0085] Treatment of Planktonic Cells with DCs. B. subtilis 168
was cultured overnight as described above, subcultured by a
1:1000 dilution in LB medium and grown to OD600 of 0.8. Cells
from 3 mL of sub-culture were pelleted at 16.1 rcf for 2 min in
a microcentrifuge (Model 5415R Eppendorf, Westbury, N.Y.), and
resuspended in 0.85% NaCl buffer. This process was repeated
three times to wash the cells, which were then resuspended in 3
mL LB or 3 mL pre-treated LB medium (see below). Samples in LB
medium were treated for 15 min with a total current of 150 μA,
500 μA, or 1500 μA in the electrochemical cell described above.
Pre-treated LB medium was prepared by treating LB medium with
150 μA, 500 μA, or 1500 μA total current (corresponding to 0,
25, 83 and 250 μA/cm<2>, respectively) for 15 minutes in
the electrochemical cell described above. Cells were incubated
in the pre-treated LB medium for 15 min without current to
evaluate the cellular response to the ions generated by the
currents, serving as control samples. Immediately after
treatment, cells were aliquoted into microcentrifuge tubes,
pelleted for 1 min at 16.1 rcf and 4° C., and the supernatant
decanted off. Cells used for DNA microarray analysis were frozen
immediately after decanting in a dry ice-ethanol bath and then
stored at −80° C.
[0086] RNA Extraction. RNA extraction was performed using the
Qiagen RNeasy Mini Kit (Qiagen, Valencia, Calif.) by following
the manufacturer's protocol with slight modifications. Briefly,
the homogenization was performed with a model 3110BX mini bead
beater and 0.1 mm diameter Zirconia/Silica beads (both from
Biospec Products, Bartlesville, Okla.) for 1 min. On-column DNA
digestion was performed with 120 μL DNase I; and wash with RPE
buffer was repeated three times rather than once. The isolated
RNA was stored at −80° C. until DNA microarray analysis.
[0087] DNA Microarray Analysis. The total RNA samples were sent
to the DNA Microarray Core Facilities at SUNY Upstate Medical
University for hybridization to Affymetrix DNA microarrays
(Affymetrix; Santa Clara, Calif.). The hybridizations was
performed by following the Prokaryotic Target Preparation
protocol in the GeneChip Expression Analysis Technical Manual
(Affymetrix). cDNA was hybridized on GeneChip B. subtilis Genome
Arrays (Affymetrix; Santa Clara, Calif.) for 16 hours at 45° C.
in an Model 640 Hybridization Oven (Affymetrix). The arrays were
washed and stained using the F5450—0004 protocol on an
Affymetrix Fluidics Station 450, and then scanned with an Model
7G Plus GeneChip Scanner (Affymetrix). For each data set, genes
with a p-value between 0.05 and 0.95 were considered as
statistically insignificant. Cluster analysis was performed with
the TIGR MultiExperiment Viewer (MeV) software (J. Craig Venter
Institute; Rockville, Md.) using a k-means sorting with the
default parameters. A hierarchical tree was also constructed.
[0088] Treatment of Biofilm Cultures with Ampicillin and DC. B.
subtilis BE1500 biofilms were prepared as described for MBC
experiments. Prior to treatment, biofilms were gently washed
three times with 0.85% NaCl buffer. Each stainless steel coupon
with biofilm was placed as the working electrode in the
electrochemical cell cuvette shown in FIG. 16. Prior to placing
the electrode with biofilm in the cuvette, 3 mL LB medium was
added to the cuvette to prevent the biofilm from drying out.
Samples were treated for 15 min with 0, 25, 83 and 250
μA/cm<2>. Immediately after treatment, the biofilms were
placed in a 15 mL polystyrene test tube containing 4 mL 0.85%
NaCl buffer and sonicated for 2 min to remove the biofilm from
electrode. The stainless steel electrode was then removed and
the tube containing the cells and buffer was vortexed for 30 s
to break up any remaining cell clusters. Cell densities were
determined by plating the cultures on LB/agar plates and
counting CFUs. The effect of current-generated ions was tested
in the same way except that the cells were incubated in
pre-treated LB in the absence of a current.
[0089] Atomic Force Microscopy. B. subtilis 168 planktonic cells
were cultured and treated with electric currents as described
above. Immediately after pelleting, the cells were centrifuged
at 16.1 rcf for 2 min at 4° C. and the supernatant was decanted.
Cell pellets were re-suspended in de-ionized (DI) water and
centrifuged at 16.1 rcf for 2 min at 4° C. to wash away ions.
The washing was repeated twice, and the pellet was resuspended
in DI water. To prepare the samples for AFM analysis, 2 μL of
suspended cells was placed on a piece of No. 2 borosilicate
cover glass (VWR, West Chester, Pa.) and placed in a vacuum
dessicator (Fisher Scientific) to dry for 15 min. Samples were
examined using the contact mode of an atomic force microscope
(Veeco Instruments; Malvern, Pa.). Both height and displacement
images were captured at field widths of 50, 25, 10 and 5 μm.
[0090] Effects of DCs on planktonic cells. To determine the
effect of electric currents on planktonic cells, B. subtilis 168
cultures were grown overnight and treated in the electrochemical
cell (FIG. 16) with total currents of 0, 150, 500 or 1500 μA,
corresponding to 0, 25, 83 and 250 μA/cm<2>, respectively.
To make a distinction between the effect of metal cations
generated by electrochemical reactions and electric current on
the planktonic cells, cells were also incubated for 15 min in LB
medium pre-treated with the same current level and duration
(pre-treated LB medium). The number of viable cells was
determined by CFU counts as described in the Materials and
Methods section.
[0091] Planktonic cells exposed to pre-treated medium and
applied current both showed a dose-dependent reduction of cell
viability (FIG. 17). At 25 μA/cm<2 >and 83 μA/cm<2>,
both pre-treated LB medium and LB medium with applied current
resulted in similar reduction of cell viability. For example,
cell viability was reduced approximately 1 log by 25
μA/cm<2>, and 2 logs by 83 μA/cm<2 >versus the
untreated control. At 250 μA/cm<2 >level, however, the
pre-treated medium appeared to kill more cells (4-log reduction)
than current treatment (3-log reduction).
[0092] AFM analysis. To identify if DC treatments caused any
physical damage to the cells, AFM analysis was performed to
determine the effects of electric currents on planktonic cell
morphology. The images suggest the width of the flagella to be
less than 100 nm, the length to be at least 10 μm, and the
wavelength to be approximately 2.5 μm. These numbers are in
agreement with measurement of flagellar dimensions in the
literature, suggesting that AFM is suitable for detecting
changes in cell morphology. AFM images of B. subtilis 168 in
FIG. 18 showed no apparent membrane features, appearing to be
relatively smooth, consistent with an earlier report of AFM
study that the membrane surface of B. subtilis W23 was observed
to be smooth.
[0093] Treatments with DC did not cause apparent changes in cell
morphology (FIG. 18). Interestingly, during AFM and light
microscopy, debris of an unknown type was observed, particularly
in samples treated with 83 and 250 μA/cm<2 >currents (FIG.
18). To determine if this debris originated from the cells or
from electrochemical reactions, LB medium without cells was
treated with the same currents, washed, and analyzed in the same
procedure. AFM images were taken at several resolutions (images
not shown). There was an apparent increase in debris as applied
current increased. This debris was similar to the debris
observed for samples containing cells. The apparent increase in
debris with current suggests that these precipitates may be
electrochemical reaction products and the results of their
interactions with the components of LB medium. This finding
suggests that the killing by DC is not only through direct
physical forces of the currents. The effects of such debris on
bacterial cells, however, remain to be determined.
[0094] DNA microarray analysis. To understand the effect of
electric currents on B. subtilis at the genetic level, RNA from
planktonic B. subtilis 168 treated with applied currents or
pre-treated LB media were analyzed using GeneChip B. subtilis
Genome Arrays (Affymetrix). B. subtilis 168 treated with
pre-treated LB medium was used as a control so as to minimize
the influence of electrochemical products on gene expression.
Cluster analysis was performed to categorize the gene expression
patterns. Five clusters were found, corresponding to
up-regulation at only one current level (25, 83 or 250
μA/cm<2>), up-regulation at all current levels, and
down-regulation at all current levels.
[0095] A selected list of the genes can be seen in Table 7
below, where the genes were selected based on operons with
multiple genes showing altered regulation, as well as those
showing high levels of regulation. SLR is given as a range for
operons that showed similar trends. For the genes in Cluster 4
that were up-regulated at all tested currents, the SLR range is
given for the 1500 μA testing condition. For Cluster 5, negative
numbers are in parenthesis for clarity.
[0000]
TABLE 7
Representative Genes Showing Altered Regulation in Response to
DC Currents.
[0096] At all current levels, the genes tuaABCD from the tua
operon was induced by current treatment. Additionally, at 250
μA/cm<2 >two more genes from the same operon, tuaF and
tuaG also showed increased expression. The tua operon is
responsible for the synthesis of teichuronic acid, an anionic
polymer found in the cell membrane only under phosphate-limited
conditions. The up-regulation of genes related to envelope
synthesis suggests that the cell membranes may have been damaged
or altered in some manner, perhaps related to a loss of
phosphate. Although AFM analysis did not reveal any significant
change in cell morphology, the cells appeared to be more
sensitive to the shear force of the AFM tip after treatment with
250 μA/cm<2 >(images not shown). Further study at protein
level will be helpful for understanding the mechanism.
[0097] The pathway for teichuronic acid synthesis is controlled
by the Pho regulon, responsible for response to
phosphate-limited conditions. The gene ydhF, encoding a
lipoprotein that showed increased expression at all tested
currents, is also controlled by the PhoPR regulation system.
These findings suggest that phosphate limitation may have
occurred due to current treatments.
[0098] Effects of DC treatments on biofilms. To determine the
effect of electric currents on biofilms, B. subtilis biofilms
were developed on 304L stainless steel electrodes and treated
with the same total applied current as described for the
planktonic cells (0, 25, 83, and 250 μA/cm<2>). To
determine the effects of electrochemical reactions on biofilms,
biofilms were also treated with pre-treated LB medium as with
the planktonic cells. Immediately after treatment the biofilm
cells were detached via sonication, washed with 0.85% NaCl
buffer, and plated on LB-agar plates to quantify the viable
cells by counting CFUs. A decrease in cell viability was seen
for biofilm cells treated with current as well as those treated
with pre-treated LB medium (FIG. 19). At each tested current
level, treatment with pre-treated LB medium reduced cell
viability by only 8-10%. Biofilms treated with current showed a
further reduction in viability compared to those exposed to
pre-treated LB medium; e.g., treatment with 25, 83 and 250
μA/cm<2 >decreased cell viability by 97%, 88% and 98.5%,
respectively.
[0099] Consistent with the general knowledge that biofilms are
highly resistant to antibiotics, treatment of B. subtilis BE1500
biofilms with 1000 μg/mL ampicillin for 15 min only killed 59%
of biofilm cells; while the MIC for planktonic B. subtilis
BE1500 was found to be ≦2 μg/mL (data not shown), comparable to
the MIC for B. subtilis 168 of 0.2 μg/mL reported in the
literature. To determine if electric currents can improve
biofilm control with antibiotics, biofilms grown on stainless
steel electrodes were treated simultaneously with 0, 50, 100,
and 1000 μg/mL ampicillin and 83 μA/cm<2 >DC current for
15 min at 37° C. As discussed above, treatment with 83
μA/cm<2 >DC current for 15 min alone decreased cell
viability by 88%. In comparison, treatment with 50, 100 or 1000
μg/mL ampicillin in the presence of 83 μA/cm<2 >DC
decreased cell viability by 93%, 79%, and 86% versus antibiotic
alone, respectively (FIG. 20). Thus, no apparent synergy was
found when treated with 83 μA/cm<2 >DC and ampicillin
together.
[0100] Complex electrochemical reactions occur at the surface of
electrodes when an external voltage is applied. Ionic species
can be generated from the electrode, and these may interact with
the medium, antibiotics, and bacterial cells. The grade of
stainless steel used in this study contains <0.08% C,
17.5-20% Cr, 8-11% Ni, <2% Mn, <1% Si, <0.045% P, and
<0.03% S. Ions and compounds of some of these components
could be toxic. For example Cr(VI), found in chromate and
dichromate ions, is highly toxic to cells. To determine the
effects of metal ions generated during treatment, biofilms were
also grown on graphite electrodes rather than stainless steel
(FIG. 21). Treatment with 500 μA DC current for 15 min decreased
biofilm cell viability by 57% on graphite electrodes versus 88%
on stainless steel. Treatment with 500 μA DC current and 50
μg/mL ampicillin decreased cell viability by 44% on graphite
electrodes versus 93% on stainless steel.
[0101] The electrochemical generation of chlorine-containing
species such as hypochlorite (ClO<−>), chlorite
(ClO2<−>), and chloramines (NH2Cl, NHCl2, NCl3) by DC
current in the medium has been implicated in the killing of
biofilm cells. Increases in viability of biofilm cells grown and
treated on graphite electrodes compared to that on stainless
steel suggest that metallic ions released from the latter have
stronger bactericidal effects on B. subtilis biofilms. To
understand if killing was partially due to hypochlorite
generated by DC current, biofilms grown on graphite electrodes
were also treated with chlorine-free M56 buffer. The viability
of biofilm cells (with untreated control normalized as 100%) in
M56 was 50% when treated with 500 μA DC current alone, and 74%
when treated with 500 μA DC current with 50 μg/mL ampicillin.
Biofilms grown on stainless steel and treated with current with
or without ampicillin in chlorine-free M56 buffer did not show
significant difference in cell viability compared to those grown
on stainless steel and treated in LB medium. This finding
implies that the majority of killing of biofilm cells on
stainless steel surfaces in LB medium was through the activity
of metal ions, and only minimally through chloride ions.
[0102] Treatment with low level DCs can effectively reduce the
viability of B. subtilis cells. When biofilms were grown on
graphite electrodes and subjected to current treatment, however,
only a slight decrease in viability was seen. This finding
suggests that certain metal cations interacted with biofilm
cells and caused the decreased viability. Biofilms subjected to
the metal cations released in pre-treated LB medium showed a
slight decrease in cell viability versus the control. However,
there was less killing of biofilm cells by incubating in the
pre-treated medium than when the current was directly applied,
especially for biofilms treated with 250 μA/cm<2 >(FIG.
19). Thus, movement of ions may be partially responsible for the
killing of biofilm cells.
[0103] In contrast to the biofilm samples, planktonic cells were
much more susceptible to the effects of electric current.
However, planktonic cells exposed to current and to pre-treated
medium showed similar reduction in cell viability. It is
possible that the presence of the biofilm matrix could affect
the chemical reaction of current-generated ions. The majority of
the planktonic cells are not likely to be attaching to the
electrode surface, especially given the vertical positioning of
the electrodes (the turbidity in the cuvette appeared to be
homogeneous). In contrast, biofilms are formed on the surface of
the electrodes, positioned vertically, and held there by EPS.
When the current is applied directly, biofilm cells are in
direct contact with the metal cations, possibly for the entire
period of treatment as the ions were generated from the working
electrode and diffused through the biofilm matrix. In the
pre-treated LB medium, metal cations may have been converted to
more inert metal compounds relatively rapidly through reactions
with water, oxygen, and hydroxide. In addition, biofilms treated
with pre-treated LB medium were not exposed to current directly;
this may lead to a decreased exposure to metal cations, which
were released from the anodic electrode. This can probably
explain why treatments of biofilms with applied currents were
more effective than using the pre-treated media prepared with
the same level and duration of DC, especially at 250
μA/cm<2>. Precipitation of metal complex may also explain
the additional killing by treating planktonic cells with 25 and
83 μA/cm<2 >DC compared to pre-treated media. At
μA/cm<2>, however, applied DC was less effective than
pre-treated media. This is probably due to the changes in
electrochemistry, which may generate metal complex that are more
effective than ions moving in an electric field as existed for
treatments with DC. The exact nature of these reactions,
however, remain to be determined.
[0104] During electrochemical reactions involving stainless
steel as the working electrode, a multitude of ions and other
chemical species can be formed depending on the voltage and
current levels and composition of the medium. In particular, the
chemical species formed of four key elements are of particular
interest with regards to cell viability include iron, chromium,
chlorine, oxygen and hydrogen (pH). Fe<2+> ions can be
generated during electrochemical reactions with stainless steel
or graphite as an electrode. This effect may be intensified by
the presence of biofilms on the stainless steel due to an
increase in the resistance of the system, leading to an
increased voltage when current is held constant. Ferrous ion can
react with hydrogen peroxide via the Fenton reaction, resulting
in the production ferric ion, hydroxide ion, and the hydroxyl
radical. This reaction has been reported to kill bacteria
through further formation of the superoxide radicals. In B.
subtilis, oxidative stress due to H2O2 causes several genes to
be up-regulated based on the response by the per regulon. The
up-regulation of katA by 25 μA/cm<2 >and 83 μA/cm<2
>and of the hemAXCDBL operon by 83 μA/cm<2 >suggests
oxidative stress due to hydrogen peroxide may have been present.
The decreased cell viability in biofilms treated with current
may be in part due to oxidative stress as a result of the
products of the Fenton reaction.
[0105] The second-most abundant metal in stainless steel is
chromium, at amounts of up to 20% in 304L. Chromium ions,
specifically Cr(VI) in chromate and dichromate, are highly toxic
to bacterial cells. The presence and concentration of Cr(VI) in
our system during treatment is unknown. B. subtilis 168 has a
metabolic pathway by which it can reduce Cr(VI) to the much less
toxic Cr(III) that functions when chromate ions are present in
concentrations of up to 0.5 mM. However, genes for chromate
reduction (ywrAB, ycnD) did not show significant changes in
expression under our experimental conditions. Genes related to
oxidative stress, such as the hemX operon, however, were
up-regulated, providing a possible alternative mechanism for
protection against chromium. It has also been reported that the
presence of heavy metals, such as zinc, cadmium, and copper, can
inhibit chromate reduction by B. subtilis. Genes related to
zinc, cadmium, and copper toxicity (cadA, copA) were
up-regulated in the presence of 250 μA/cm<2 >current in
our study. This suggests that ions of some heavy metals may be
present in our system when using stainless steel as electrodes.
Chromium reduction can also occur by chemical processes in
solution, and can be enhanced or inhibited by other chemical
species in the medium. Most significantly, the presence of
Fe<2+> enables the reduction of Cr(VI) to Cr(III), at a
ratio of 3 Fe<2+> to 1 Cr<6+>, possibly forming
Fe/Cr complexes. However, the presence of organic ligands can
modify this reaction; ligands specific for Fe<2+> inhibit
the reaction, while those for Fe<3+> enhance it. In
summary, the interactions of chromium within the system are
complex, and killing via hexavalent chromium can not be ruled
out. However, the significant killing of B. subtilis using
graphite electrodes suggests that the Cr(VI) ions are not
indispensable for the cidal effects of electric currents.
[0106] If metal cations are responsible for a loss of cell
viability, one would expect to see genes up-regulated that are
related to metal tolerance. Indeed, six metal resistance genes
were up-regulated—arsBCR at 83 μA/cm<2>, and cadA and copA
at 250 μA/cm<2>. The arsBCR operon is responsible for the
transport of arsenate, arsenite, and antimonite. These molecules
bear little resemblance to divalent iron or hexavalent chromium
compounds. It is interesting to note that arsenic is in the same
group as phosphorous. It is possible that up-regulation of this
operon may be related to the phosphate starvation. Notably
absent were putative genes responsible for chromium
reduction—ywrAB. It is possible that chromate and dichromate are
not being produced in quantities that would result in a cellular
response, or that they are neutralized by other ions in the
solution.
[0107] In the absence of metal ions in solution as charge
carriers, chloride ions in solution can react with hydroxyl ions
to form hypochlorite, which is well known to be toxic to cells.
Experiments with graphite electrodes in M56 medium that did not
contain chlorine showed that there was no significant decrease
in the viability of the cells after treatment with 83 μA/cm<2
>current compared to the untreated sample. This finding
suggests that chlorine containing compounds, most notably
hypochlorite, are partially responsible for significant
decreases in cell viability in our electrochemical system.
[0108] The bioelectric effect suggests that electric currents
will have a synergistic effect with antibiotics to improve the
overall efficacy of bacterial killing. Surprisingly, when
ampicillin was added to the solution with current, the amount of
killing was not significantly altered versus current alone. In
the case of biofilms grown on graphite electrodes and treated in
chlorine-free M56 buffer with 50 μg/mL ampicillin and 500 μA
current there was even a slight decrease in killing. It is well
documented that iron can interfere with the action of
antibiotics, including ampicillin, through a variety of
mechanisms including chelation of ferric cations by antibiotics.
It is possible that the presence of iron and other metal cations
is inhibiting ampicillin activity through chelation mechanisms.
Such interaction may be dependent on the nature of antibiotics
since some other antibiotics do show synergy with electric
currents in killing biofilm cells. It is also important to note
that in the present invention employed a shorter treatment time
(15 min) than Costerton and others (24 h).
[0109] In summary, the present invention involved a detailed
study of the effects of weak EC on viability, gene expression
and morphology of B. subtilis and revealed that the ions and
oxidative species generated by electrochemical reactions have
significant influence on bacterial gene expression and
viability. Further testing with additional conditions and
different antibiotics will help unveil the mechanism of
bioelectric effects
[0110] Although the present invention has been described in
connection with a preferred embodiment, it should be understood
that modifications, alterations, and additions can be made to
the invention without departing from the scope of the invention
as defined by the claims.
SYSTEM
AND METHOD FOR CONTROLLING BACTERIAL PERSISTER CELLS WITH
WEAK ELECTRIC CURRENTS
US8569027
[ PDF ]
A system and method for treating persister cells with an
electrochemical process, alone or in combination with
antibiotics. Weak electric currents are used to effectively
eliminate persister cells and the efficacy can be further
improved through synergistic effects with antibiotics. The
method may be adapted for novel therapies of chronic infections
and strategies to control persistent biofouling. The system has
a broad spectrum applications in treating chronic and drug
resistant infections, such as those caused by Pseudomonas
aeruginosa, Mycobacterium tuberculosis and methicillin resistant
Staphylococcus aureus, and may also be used for decontamination
of medical devices.
BACKGROUND
OF THE INVENTION
1. Field of
the Invention
The present invention relates to electrochemical control of
bacterial persister cells and, more particularly, the
synergistic effect between weak electric currents and
antibiotics on persister cells.
2.
Description of the Related Art
Previous studies of persister cells have led to important
discoveries that are shifting the paradigm of research in
microbiology and antimicrobial therapy. It is now well
recognized that subpopulations of bacterial cells in a culture
can enter a dormant (non-growing) state that are extremely
tolerant to a variety of unrelated stresses such as antibiotics
and heat. Such heterogeneity has been reported to exist in even
well mixed shake flask cultures at exponential phase. This
phenotypic variation can lead to three subpopulations in a given
culture including the normal cells, type I persister cells from
the stationary inoculums and type II persister cells that are
generated during growth. Persister cells are not mutants with
drug resistant genes, but rather phenotypic variants of the
wild-type strain. Persister cells neither die nor grow in the
presence of an antibiotic, and when reinoculated, they grow into
a normal culture with a similar percentage of cells as
persisters, leading to high antibiotic tolerance.
Although persister cells normally only make up a small portion
of the population, they play a critical role in antibiotic
tolerance. Most antibiotics inhibit bacteria by targeting growth
related cellular activities, e.g., protein, DNA, and cell wall
syntheses. They can eliminate the majority of bacterial
population by killing the normal cells. For persister cells,
however, antibiotics can only repress but not eliminate this
subpopulation because persister cells are non-growing dormant
cells. Thus, the seeming disadvantage of being dormant in normal
environment becomes an advantage for persister cells when being
challenged by antibiotics. When the treatment is stopped, some
persister cells revert back to normal cells and reestablish the
population. Such tolerance leads to reoccurrence of infections
and facilitate the development and spread of multidrug
resistance through true mutations.
Recent research has demonstrated that persister cell formation
increases significantly in stationary-phase cultures and the
surface-attached highly hydrated structures known as biofilms.
Formed in a dynamic process, mature biofilms typically have
mushroom-like structures with cells embedded in a polysaccharide
matrix secreted by the bound bacterial cells. Biofilm cells are
up to 1000 times more tolerant to antibiotics and disinfectants
compared to their planktonic counterparts. Thus, deleterious
biofilms cause serious problems such as chronic infections in
humans as well as persistent corrosion and equipment failure in
industry. Although not completely understood at the molecular
level, the biofilm-associated tolerance is due to several
factors acting in concert. Bacterial cells in biofilm produce a
polysaccharide matrix, which creates a physical barrier that
retards or blocks the toxic compounds from reaching the cells.
However, protection by the polysaccharide matrix can only
partially explain the tolerance because at least some
antibiotics can readily penetrate the matrix yet still can not
eliminate biofilm cells. Biofilm mode of growth is also
associated with changes in bacterial membrane structure and
reduction in cell growth rate. The changes in membrane structure
could reduce the permeability to toxic compounds, while the
reduction in growth rate can lead to higher tolerance to
growth-dependent killing by antibiotics. Increasing evidence
suggests that the slow growth, especially that associated with
persister cells, is the most challenging mechanism for treating
chronic infections.
BRIEF
SUMMARY OF THE INVENTION
The present invention provides a system and method for treating
persister cells with an electrochemical process, alone or in
combination with antibiotics. The present invention also
includes an electrochemical cell for treating persister cells.
Weak electric currents are used to effectively eliminate
persister cells and the efficacy can be further improved through
synergistic effects with antibiotics. The present invention
demonstrates unprecedented efficacy in controlling persister
cells and the present invention may be adapted for novel
therapies of chronic infections and strategies to control
persistent biofouling. The present invention has a broad
spectrum applications in treating chronic and drug resistant
infections, such as those caused by Pseudomonas aeruginosa,
Mycobacterium tuberculosis and MRSA (Methicillin resistant
Staphylococcus aureus). The present invention may also be used
for decontamination of medical devices.
According to a first aspect of the present invention is an
electrochemical method for killing persister cells, the method
comprising the step of applying a weak electrical current to a
bacterial culture, either planktonic culture or a biofilm,
wherein the current is between 1 and 500 microamperes per square
centimeter. According to a preferred embodiment, the current is
a direct current of approximately 75 microampheres per square
centimeter.
According to a second aspect of the present invention is an
electrochemical method for killing persister cells, the method
comprising the step of applying an electrical current to a
bacterial culture, either planktonic culture or a biofilm,
wherein the current is between 1 and 500 microamperes per square
centimeter, and where the medium is an electrically-conductive
saline solution such as 0.85% NaCl.
According to a third aspect of the present invention is an
electrochemical method for killing persister cells, the method
comprising the step of applying an electrical current to a
bacterial culture, either planktonic culture or a biofilm,
wherein the current is between 1 and 500 microamperes per square
centimeter, and wherein the medium also contains an effective
amount of an antimicrobial compound such as an antibiotic. The
concentration of the antibiotics can be significantly lower than
what it is required to work in the absence of a current.
According to a fourth aspect of the present invention is a
method for treating an item comprising a biofilm, the method
comprising the steps of: (i) placing the item at least partially
in a medium; and (ii) applying an electrical current of between
1 and 500 microamperes per square centimeter to the medium.
According to a fifth aspect of the present invention is a system
for killing persister cells, the system comprising: (i) a
treatment cell with a treatment area for receiving an item and
which contains a reference electrode, a working electrode, a
counter electrode; (ii) a medium (liquid or cream) that at least
partially fills the treatment area and is in communication with
the reference electrode, the working electrode, and the counter
electrode. The treatment cell applies an electrical current
between 1 and 500 microamperes per square centimeter to the
medium in order to kill the persister cells.
According to a sixth aspect of the present invention is a system
for killing persister cells, the system comprising: (i) a
treatment cell with a treatment area for receiving an item and
which contains a reference electrode, a working electrode, a
counter electrode; (ii) a medium that at least partially fills
the treatment area and is in connection with the reference
electrode, the working electrode, and the counter electrode; and
(ii) an effective amount of an antimicrobial compound such as an
antibiotic. The concentration of the antibiotics can be
significantly lower than what it is required to work in the
absence of a current. The treatment cell applies an electrical
current between 1 and 500 microamperes per square centimeter to
the medium in order to kill the persister cells.
BRIEF
DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
The present invention will be more fully understood and
appreciated by reading the following Detailed Description in
conjunction with the accompanying drawings, in which:
FIG. 1 is a schematic of biofilm formation;
FIG. 2 is a schematic of an electrochemical cell according to
the present invention;
FIG. 3A is a graph illustrating the effects of electric currents
and antibiotics on the persister cells of E. coli HM22, where
the graph depicts the results of treatment with 75 μA/cm<2
>DC alone in 0.85% NaCl buffer using 304L stainless steel as
working and counter electrodes, and current was generated using
graphite working and counter electrodes in 0.85% NaCl buffer;
FIG. 3B is a graph illustrating the effects of electric currents
and antibiotics on the persister cells of E. coli HM22, where
the graph depicts treatment with antibiotic only, 75 μA/cm<2
>DC only, or co-treatment with current and antibiotic, and
the current was generated using graphite working and counter
electrodes in 0.85% NaCl buffer;
FIG. 4 is a graph showing the effects of current and Tob on E.
coli biofilm cells when treated the biofilm as an anodic
electrode. Bars indicate the numbers of viable persister cells
of E. coli HM22. Biofilms were grown on stainless 304L steel
electrodes and treated with 75 μA/cm<2 >DC and/or 20 μg/mL
Tob for 60 min.
FIG. 5 is a graph of E. coli HM22 persister cell survival
following treatment with 15 μA/cm<2 >direct current alone,
H2O2 alone, or both;
FIG. 6 is schematic of a flow cell system for studying
bioelectric effect;
FIG. 7 is an image of the removal of detached E. coli biofilm
cells by flow;
FIG. 8 is a schematic of the overall operation of the present
invention;
FIG. 9A is a graph of the membrane potential of E. coli HM22
persister cells compared to normal cells;
FIG. 9B is a graph of the membrane potential of E. coli HM22
normal cells following treatment of with 15-45 μA/cm<2
>direct current using graphite electrodes in 0.85% NaCl
buffer;
FIG. 9C is a graph of the membrane potential of E. coli HM22
persister cells following treatment of with 15-45 μA/cm<2
>direct current using graphite electrodes in 0.85% NaCl
buffer;
FIG. 10 is a graph of the effects of tobramycin alone, electric
current alone, or both on P. aeruginosa PAO1 cells at
exponential phase;
FIG. 11 is a graph of the effects of ciprofloxacin on P.
aeruginosa PAO1 cells;
FIG. 12 is a graph of the effects of tobramycin alone, electric
current alone, or both on P. aeruginosa PAO1 persister cells;
FIG. 13 is a graph of the effects of pretreated buffer on
persister cells of P. aeruginosa PAO1 cells where the 0.85% NaCl
buffer was treated with the same level and duration of electric
current as used in current-treatment experiments, and where the
cells were incubated in the pretreated buffers to evaluate the
effects of released ions in the absence of a current;
FIG. 14 is a graph showing the comparison of killing effects on
P. aeruginosa PAO1 persister cells using 304 stainless steel
electrodes and carbon electrodes;
FIG. 15 is a graph of the effect of electric currents on P.
aeruginosa PAO1 persister cells in the presence of 0.85% NaCl
buffer pretreated with 75 μA/cm<2 >current using 304
stainless steel electrodes.
DETAILED
DESCRIPTION OF THE INVENTION
Referring now to the drawings, wherein like reference numerals
refer to like parts throughout, the present invention provide a
system and method for the elimination of persister cells by
electric currents and synergy with antibiotics. The present
invention was tested using an electrochemical cell seen in FIG.
2 and with the use of E. coli HM22 constructed by the pioneer
work of Moyed and Bertrand because it produces 1000 times more
persister cells in exponential-phase cultures than the wild-type
E. coli strains and has been used in most studies of persister
cells. To evaluate the effects of electric currents, the
persister cells were first isolated as described previously.
Briefly, the exponential culture of HM22 at optical density at
600 nm (OD600) of 0.3 in LB medium was treated with 100 μg/mL
ampicillin for 3 h to kill and lyse the normal cells. The
persister cells were then collected by centrifugation at 8000
rpm at 4° C. for 10 min and resuspended in 0.85% NaCl buffer.
The persister cells were then treated in a customized
electrochemical cell, shown in FIG. 2. Electrodes with a
dimension of 1 cm×5.6 cm were cut from a flat 304L stainless
steel sheet (MSC; Melville, N.Y.) or graphite sheet
(McMaster—CARR, Santa Fe Springs, Calif.). The same material was
used for both the counter electrode and working electrode, which
were placed into a 4.5 mL standard-style polystyrene cuvette
(Fisher Scientific; Hampton, N.H.). A 0.48 mm diameter silver
wire (A-M Systems; Sequim, Wash.) was placed in bleach for 30
min to produce an Ag/AgCl reference electrode. The bottom 1″ of
a borosilicate glass Pasteur pipette (Fisher) was cut and the
reference wire was placed inside to prevent contact with the
working or counter electrode. An AFCBP1 potentiostat/galvanostat
(Pine Instrument Company, Grove City, Pa.) was connected via
alligator clamps to the electrodes and used to control the
current. The volume of medium in the fully-constructed
electrochemical cell was 3 mL (see FIG. 2).
Significant killing of persister cells was observed both with
stainless steel and graphite electrodes. For example, treatment
with 75 μA/cm<2 >(voltage around 1V) for 50 min in 0.85%
NaCl buffer caused complete killing of persister cells (a 6 log
reduction in viability, FIG. 3A) by counting colony forming
units (CFUs) before and after treatment. The killing was not
simply caused by the products of electrochemical reactions since
incubation with the pre-treatment buffer (0.85% NaCl buffer
treated with the same level and duration of current) did not
cause any apparent killing (data not shown). Interestingly, the
electric current was more effective in killing persister cells
than normal cells. As shown in FIG. 3A, the same treatment of
normal cells only caused a 3 log reduction in the number of
viable cells. Effective killing of persister cells was also
observed using graphite electrodes. As shown in FIG. 3B,
treatment with the same current level (75 μA/cm<2>) for 60
min caused a 3 log reduction of viable persister cells, whereas
pre-treated medium only reduced the viable cells by less than 1
log. Furthermore, the efficacy of persister control can be
improved through synergistic effects with antibiotics. The
graphite electrode was used for this experiment since it does
not cause complete killing, allowing the synergistic effects to
be observed. As shown in FIG. 3B, application of 75 μA/cm<2
>current or 20 μg/mL cinoxacin (Cin) alone caused a 3 log or
no apparent reduction in the number of viable persister cells,
respectively. When these two treatments were applied together,
however, nearly complete killing (more than 5 log reduction) was
observed. Such synergistic effects have not been reported for
persister cells. It is also worth noticing that the synergy is
not only limited to Cin since tobramycin (Tob) also exhibited
synergistic effect with electric current (see FIG. 3B).
To determine if electric currents are also effective in killing
persister cells in biofilms, E. coli HM22 biofilms were cultured
on 304L stainless steel coupons. The biofilm-coated coupons were
then used as anodic or cathodic electrode, and treated with
direct current alone or with tobramycin together. Immediately
after treatment, the cells were removed from the biofilm-coated
coupons by sonication and vertexing. A portion of the cells was
directly plated on LB+DPA plates to quantify the total number of
viable cells by counting CFU, the other part of the sample was
treated with 100 μg/mL ampicillin for another 3 h and plated on
LB+DPA plates to quantify the number of the viable persister
cells. This approach allowed us to study the killing effects on
normal and persister cells separately.
As shown in these FIG. 4, when treating biofilm persisters with
tobramycin alone (20 μg/mL or 150 μg/mL), there was no
significant reduction in total number of viable cells and number
of viable persister cells compared to the untreated control
sample. These results are consistent with the knowledge that
biofilms have significantly enhanced tolerance to antibiotics
compared to planktonic cells. However, treatments with 75
μA/cm<2 >alone for 60 min reduced the number of viable
persister cells by 3.5 logs. After treating biofilms with
currents and tobramycin together for 60 min, the number of
viable persister cells was reduced by 5.4 log (nearly complete
killing, FIG. 4). Thus, synergy between electric currents and
antibiotics also exist for killing persister cells in biofilms.
With the capability to quantify the expression level of each
gene at the genome-wide scale, DNA microarrays have been
extensively used to monitor global gene expression profiles in
response to different stimuli including persister formation and
biofilm formation. However, currently there are no reported data
about the effects of weak electric currents on bacterial gene
expression at the genome-wide scale. To identify the effects of
electric currents on cell physiology of persister cells and
normal cells at the genetic level, the present invention
utilized two experiments that revealed clues about the effects
of weak electric currents on bacterial cells.
In the first experiment, persister cells and normal cells of E.
coli HM22 harvested using the same method as describe above were
treated with and without 75 μA/cm<2 >DC for 15 min in
0.85% NaCl buffer. In a parallel experiment, the persister cells
were also treated with M56 buffer with the same level and
duration of the current. After harvesting HM22 normal and
persister cells, they were concentrated 40 times and resuspended
in 6 mL 0.85% NaCl buffer and 6 mL M56 buffer respectively. Both
samples were separated into two equal aliquots: one was left
untreated, meanwhile the other one was treated with 75
μA/cm<2 >DC. After 15 min incubation with and without
current, all of the cells were centrifuged immediately for 30 s
at 13,200 rpm and 4° C. to harvest the cells. For RNA isolation,
each cell pellet was resuspended in 1 mL of TRIzol reagent
buffer (Invitrogen Co., Carlsbad, Calif.) and beaten rigorously
at 4,800 beats per min for 30 s in a closed bead beater tube
with 200 μl of silicon beads using a mini bead beater (Biospec
Products Inc., Bartlesville, Okla.). The following isolation
steps were conducted by following Trizol reagent protocol and
the total extracted RNA was subsequently purified using RNeasy
Mini kit (QIAGEN Inc., Valencia, Calif.). The quality and
quantity of the total RNA samples were evaluated using a 2100
Bioanalyzer (Agilent Technologies, Santa Clara, Calif.) and the
microarray hybridizations were performed using E. coli Genome
2.0 Arrays (Affymetrix, Inc., Santa Clara, Calif.). Both were
performed using the DNA microarray core facilities at the SUNY
Upstate Medical University (Syracuse, N.Y.).
Stringent criteria were applied to select the induced/repressed
genes based on p-values (<0.0025 or >0.9975) calculated
using the Wilcoxon signed rank test and Tukey By weight. The
applied current in 085% NaCl buffer was found to induce 9 genes
and repressed 36 genes in E. coli HM22 persister cells (see
Table 1). While 27 of these genes have unknown functions, the
treatment did induce the genes of the trp operon (trpEL), acyl
carrier protein phosphodiesterase (acpD), L-serine dehydratase
(sdaB), oxidative stress response (oxyS), and repressed the cys
operon (cysCDJKNP), production of tryptophanase (tnaL) and
nitrite extrusion (narU) (see Tables 1-5). In comparison,
treatment with the same current level in M56 buffer induced 15
genes (yibP, cysU, csgD, nrdE, narW, hisL, oxyS, etc) and
repressed only 4 genes of persister cells (see Tables 1-5).
Interestingly, the induced genes have functions of central
intermediary metabolism, protease for cell division, PTS system,
sulfate transport, surface structure, DNA synthesis, his operon,
oxidative stress response and unknown functions. Three of the
four repressed genes have unknown functions, while the forth
gene uvrB has functions of DNA damage recognition and repair.
These data suggest that weak electric currents are able to
activate certain cellular activities including those related to
oxidative response, membrane structures and functions.
TABLE 1
Number of induced/repressed genes of E. coli HM22 in responseto
15-min treatment with 75 μA/cm<2 > current using graphite
electrodes.
TABLE 2
TABLE 3
Gene of E. coli HM22 persister cells repressed by treatmentwith
75 μA/cm<2 >DC for 15 min in M56 buffer.
TABLE 4
Genes of E. coli HM22 persister cells induced by treatment
with75 μA/cm<2 >DC for 15 min in 0.85% NaCl buffer.
TABLE 5
Genes of E. coli HM22 persister cells repressed bytreatment with
75 μA/cm<2 >DC for 15 min in 0.85%
The effects on cell membrane functions are corroborated by a
parallel but more complete study regarding the effects of
electric currents on the Gram-positive bacterium Bacillus
subtilis 168. In this experiment, the cells of B. subtilis 168
in late exponential phase was treated for 15 min in LB medium
with 42, 139 or 417 μA/cm<2 >DC using 304L stainless steel
as electrodes in the electrochemical cell shown FIG. 2. Each
condition was tested in duplicate and the data was analyzed
using cluster analysis. To differentiate the effects of currents
from those of the electrochemical reaction products, the control
samples were incubated for 15 min in the LB medium that was
pre-treated with the same level and duration of the current.
Since the control samples were prepared in pre-treated LB medium
containing all the electrochemical reaction products, the gene
expression changes are mainly caused by the currents as well as
the movement and gradient of chemical species, e.g. ions. The
genes that were induced or repressed in all conditions are
listed in Table 6. There were also 839 genes induced under some
but not all conditions, such as transport genes encoding glycine
betaine/carnitine/choline ABC transporters, amino acid
transporters, and putative monovalent cation/H+ antiporters
(gene list not shown). Overall, the microarray results suggest
that electric current and associated ion movement/gradient have
significant influence on cellular activities of bacteria
especially metabolism and membrane functions.
TABLE 6
B. subtilis 168 genes consistently induced/repressed by 15
mintreatments of 42, 139 and 417 μA/cm<2 >
The DNA microarray data suggests that treatment with electric
currents may lead to generation and accumulation of reactive
oxygen species (“ROS”) (e.g., induction of oxyS, a global
regulatory RNA). Thus, the treatment could render the persister
cells more susceptible to external ROS. To test this hypothesis,
we treated E. coli HM22 persister cells with 100 μA for 20 min
and followed by treatment with and without H2O2 (500 μM) for 1
h. These cells were then split into two parts: one for
microscopic analysis and the other for CFU count. For
microscopic study, cells were treated with 200 μM
dichlorodihydrofluorescein diacetate (H2DCFDA, Sigma-Aldrich,
St. Louis, Mo.) for 30 min in dark at room temperature. After
incubation, cells were spin down and resuspended in PBS buffer
for visualization using a fluorescence microscope (Axio Imager
M1, ZEISS, Jena, Germany). The dye H2DCFDA can penetrate
bacterial cells and get cleaved by cellular esterase to produce
H2DCF. If there is any ROS present, this H2DCF will be converted
to DCF and give fluorescence (Invitrogen, USA). The results
showed that treatment with 15 μA/cm<2 >direct current,
similar to treatment with H2O2 (500 μM) caused accumulation of
ROS in persister cells. For the CFU count, cells were plated on
LB plates supplemented with DPA and incubated overnight at 37°
C. The CFU data further confirmed that the treatment with
electric current rendered the persister cells more sensitive to
H2O2 since treatment with electric current followed by H2O2
killed more persister cells than either the EC or H2O2 alone
(see FIG. 5).
Construction and use of a flow cell system is possible to
directly visualize the effects of electric currents on biofilm
cells. To directly visualize the effect of electric currents on
biofilm cells and biofilm structure, the FC81 flow cell system
(BioSurface Technologies Corporation, Bozeman Mont.) was
modified to deliver electric current. The flow cell contains two
slides to form a channel with a dimension of 47.5×12.7 mm and
1.6 mm space between the two slides. The cover glass was coated
with 50 Å Ti followed by 70 Å Au. This engineered surface is
transparent and conductive, allowing the direct visualization of
bioelectric effect with microscopy. An Ag/AgCl reference
electrode was also inserted at the exit of the flow cell
(through a Y-junction) without touching the other two
electrodes. The bottom surface could be made with any material
of interest and cut into the dimension of regular glass slides
(2.54 cm by 7.62 cm). The flow cell was assembled with a
gold-coated slide as the counter electrode and the bottom plate
as the working electrode (see FIG. 6). A Y-junction was attached
at the exit of the flow cell, with one line for insertion of
reference electrode and the other for the effluent of biofilm
culture. The tubing that holds the reference electrode was
clamped as a dead-end to prevent any leakage.
The electrodes were connected to a model AFCBP1
potentiostat/galvanostat (PINE Research Instrumentation) by Cu
wires. This is the first flow cell system containing reference
electrode to allow precise control of the potential and current.
The mature one-day biofilm of E. coli RP437/pRSH103 expressing
red fluorescent protein (RFP) constitutively was treated with 50
μA/cm<2 >DC for 1 h. The flow of LB medium (63) at 10 mL/h
was stopped before the treatment with current and resumed after
the treatment. Significant detachment of biofilm cells by
electric current was observed (see FIG. 7). This flow cell
system is an ideal tool for studying the effects of electric
currents on biofilm-associated persister cells.
It is well documented that persister cells are metabolically
inactive compared to normal cells. Conceivably, an approach that
can target this difference could have high efficacy. As shown in
FIG. 3A, some of the conditions are more effective in killing
persister cells than normal cells. Thus, the treatment
conditions may be fine tuned to selectively kill this population
that is highly resistant to antibiotics. All living cells need
to maintain a membrane potential for metabolism and transfer of
nutrients. If the membrane potential is disrupted, the cells
could lose the capability to maintain the ion gradients and cell
death will occur. Normal cells may have higher membrane
potential than persister cells due to higher metabolic
activities. In this sense, the persister cells could be more
sensitive to reduction of membrane potential. This is evidenced
by recent mechanistic studies of pyrazinamide for tuberculosis
therapy. Unlike conventional antibiotics that are more active
against growing cells, pyrazinamide is more effective in killing
non-growing bacilli. A recent study has shown that pyrazinamide
kills cells by disrupting the membrane energetics and transport
function at acid pH. An applied electric current can either
positively or negatively influence the membrane potential, which
consequently affects the viability of persister cells and
susceptibility to antibiotics (FIG. 8). If the membrane
potential is reduced by the applied current, direct killing of
persister cells can be expected. If the membrane potential is
positively affected by the current, however, it may work as a
“wake up” call of the persister cells to enter a metabolically
more active stage and therefore render the cells more
susceptible to antibiotics. The membrane permeability to
antibiotics may also be affected by the applied current. The
exact impact on persister cells may rely on the current level,
material of the electrodes and the associated ions released,
medium composition and the antibiotics applied.
Membrane potential can be measured using either florescent or
radioactive methods. The persister cells of E. coli HM22 and P.
aeruginosa PAO1 cells at different growth phases may be treated
with electric currents; and the cells before and after
treatments may be analyzed to evaluate the effects of electric
currents on membrane potential.
In light of the above, the membrane potentials of E. coli HM 22
normal and persister cells were compared. Briefly, approximately
1×10<6 >persister cells per mL were washed with PBS buffer
(10 mM sodium phosphate, 145 mM sodium chloride, pH 7.4)
followed by addition of carbocyanine dye DiOC2 (Invitrogen,
Carlsbad, Calif.) to 30 μM and incubation at room temperature
for 30 min. Fluorescence was determined using a LSR II flow
cytometer (Becton Dickinson, San Jose, Calif.), with excitation
at 495 nm and emission at 575 nm. The red/green ratiometric
parameter was set according to the manufacturer's instructions
for histogram analysis. The ratiometric parameter was calculated
as [(red value)−(green value)+384]. The overlay histogram of
membrane potential analysis was obtained using CXP software. As
shown in FIG. 9A, the membrane potential of persister cells is
lower than that of normal cells. To our knowledge, this is the
first direct comparison of membrane potential between normal and
persister cells of E. coli. In addition, treatment with 15, 30
and 45 μA/cm<2 >direct current significantly reduced the
membrane potential of persister cells, but not that of normal
cells (FIGS. 9B and 9C). These data confirm our hypothesis and
suggest that membrane potential is a potential target of new
therapies. Further study on this finding could help understand
the mechanism of persister control by electric current and
synergistic effects with antibiotics.
For the conditions that exhibit synergistic effects with
antibiotics, the membrane permeability may also be tested using
radioactively labeled antibiotics. In particular, the
intracellular concentration of benzyl-14C-penicillin (potassium)
and <3>H-oxytetracycline (American Radiolabeled Chemicals,
Inc., St. Louis, Mo.) may be measured after incubation with
cells for 30 min in the presence or absence of a current using a
liquid scintillation counter. These data are expected to
corroborate the results regarding the effects of current on
membrane potential and permeability. It will be integrated with
the results in the following study to get insight into the
mechanism of persister control with electric currents.
As described in the results above, the present invention is
premised on promising evidence that weak electric currents have
significant effects on gene expression of both persister cells
and normal cells of bacteria. As a result, gene expression in
response to electric currents may be further studied to
understand the mechanism at the genetic level by identifying the
differentially expressed genes and pathways.
First, E. coli HM22 may be used to prepare persister cells as
described above. The harvested persister cells may be treated
with different levels of electric currents (75, 150 and 300
μA/cm<2 >DC) using graphite electrodes in 0.85% NaCl
buffer or M56 buffer. The gene expression of these cells may be
compared with that of persister cells incubated in the buffer
pre-treated with the same level and duration of current. In
addition, normal cells of HM22 may be treated with the same
conditions to identify the persister-specific genes and pathways
affected by electric currents. Similar experiments may also be
performed to treat P. aeruginosa PAO1 cells at exponential and
stationary phases. The treatment time may be 15 min and extended
if more profound changes are needed to identify the pathways.
Each experiment may be conducted in duplicate and the data may
be analyzed using cluster analysis to identify the gene
expression patterns and the pathways involved in response to
current treatments. The representative induced/repressed genes
may be confirmed by RNA dot blotting.
Compared to other stimuli, e.g., starvation and temperature
change, electric currents (especially constant DCs) are not the
common challenges or evolutionary pressures that bacteria
experience in nature. Thus, the expression patterns may provide
unique information for understanding bacterial physiology in
general, and for developing better control methods. With the
gene expression patterns identified, one may further study to
corroborate the results using mutants of the differentially
expressed genes. For example, the mutants of induced genes could
be more sensitive to electric currents. Electric currents,
especially those with higher current levels and longer duration
than described here, have been found to improve the efficacy of
antibiotics in treating biofilms. However, the mechanism of such
effects remains unknown. Since persister cells play an important
role in biofilm-associated drug tolerance, it is possible that
antibiotics and electric currents are both capable of killing
susceptible biofilm cells, while electric currents can also kill
some persister cells and the efficacy can be enhanced through
synergy with antibiotics. This is supported by the fact that
electric current can be more effective in killing persister
cells than normal cells (FIG. 3A). This may create more friendly
treatment conditions with lower current level and shorter
treatment time.
E. coli HM22 and P. aeruginosa PAO1 may be used to inoculate
biofilm cultures using the flow chamber described in results of
the present invention (FIG. 6). As discussed above, these two
are the best-studied strains of persister formation and many
genetic tools are available.
The preformed biofilms of E. coli HM22 and P. aeruginosa PAO1
can be treated with electric currents and antibiotics under the
effective conditions identified. The number of viable cells can
be quantified by counting CFUs after collecting biofilm cells
from the surface by sonication and spreading cells on LB agar
plates. Meanwhile, part of the collected cells may be treated
with 100 μg/mL ampicillin (for E. coli HM22) or 200 μg/mL
ofloxacin (for P. aeruginosa PAO1) for 3 hours and then tested
using the same CFU method to quantify the viable persister
cells. The CFU data of biofilms with and without treatment may
be compared to evaluate the effects of electric currents on the
viability of persister cells in biofilms. The adhesion and
metabolic activity of biofilm-associated persister cells may be
analyzed in situ using the flow cell system described above.
The effects of electric current on biofilm structure may be
followed in situ using a fluorescence microscopy to obtain the
three dimensional information of biofilms. The structural
parameters of biofilms including surface coverage, thickness,
roughness, and biomass may be calculated using the computer
program COMSTAT (31). The dynamic 3-D imaging data may then be
obtained to help elucidate the effects of electric current on
biofilm formation and structure. To visualize biofilm-associated
persister cells three dimensionally, the promoterless gfp(LVA)
gene may be cloned in pCA24N (for E. coli, available at NIGJ)
and pME290 (for P. aeruginosa, available from ATCC) under the
promoter rrnBP1 of E. coli HM22 and P. aeruginosa PAO1,
respectively, and inserted in the corresponding hosts. Thus, the
intensity of GFP will be proportional to the cell growth rate.
In addition, all biofilm cells may be strained with the
BacLight™ Red fluorescent dye (Invitrogen). Thus, all biofilm
cells may be strained red and the green dye can be used to
differentiate persister cells (weak or no green signal) from
normal cells (stronger green signals). Compared to the highly
stable native GFP, the unstable GFP(LVA), which has a half-life
less than 40 min, may be used to allow the dynamic monitoring of
cell growth. The constructed reporters may then be used to study
the effects of electric currents on the adhesion/detachment and
growth of persister cells in three dimensions and in real time
at different stages of biofilm formation (from initial adhesion
to maturation).
To understand the mechanism of persister control using electric
currents and to develop better biofilm control methods, the
above studies may systematically investigate the effects of
electric currents on physiology of persister cells, gene
expression and pathways, as well as the effects on
biofilm-associated persister cells. These results may be
integrated to develop a model to explain the mechanism. The
results from these studies may also help develop more effective
control methods, e.g., electrically enhanced antibiotic
therapies and anti-biofouling approaches.
Conceivably, application of an electric current can cause
complex changes to the chemical composition of the medium. The
effects of currents on bacterial physiology may be carefully
compared with pre-treated medium to eliminate the effects of
electrochemical reactions products. In addition, the
electrochemical reactions may be systematically studied to
identify the roles of each reaction product on persister cells.
Continued experiments, for example, have already shown that the
effects of electric current and synergy with antibiotics is not
species specific, as similar results were shown using P.
aeruginosa. The experiments were conducted in the same way as
described for E. coli HM22. Briefly, an overnight culture of P.
aeruginosa PAO1 was used to inoculate LB medium to an OD600 of
̃0.005 (1:1000 dilution of an overnight culture with LB) and
incubated till OD600 reached ̃0.7. Then the cells were washed
twice with 0.85% NaCl buffer and treated in the same way as
described for planktonic E. coli cells. As shown in FIG. 10,
treatment with 1.5 μg/mL Tob did not cause any significant
killing. Treatment with 75 μA/cm<2 >for 60 min reduced the
number of viable P. aeruginosa PAO1 cells by 3 logs. When the
two treatments were combined, however, up to 5 logs of killing
was observed. Thus, synergistic effects clearly also exist
between electric current and tobramycin on P. aeruginosa PAO1,
suggesting this effect is not species specific and can
potentially be applied to treated human bacterial infections.
To identify the condition for isolating P. aeruginosa PAO1
persister cells, the overnight culture of P. aeruginosa PAO1 was
treated for 3.5 h with various concentrations of ciprofloxacin
(“Cip”) to determine the appropriate concentration that can kill
normal cells. As shown in FIG. 11, the killing of P. aeruginosa
PAO1 increased with Cip concentration up to 50 μg/mL and no
further killing was observed even when Cip was added as 200
μg/mL. Thus, the 1% cells that survived the treatment were
persister cells and treatment with 200 μg/mL Cip was used in the
following experiments to harvest persister cells and ensure the
complete killing of normal cells.
Synergistic effects were also observed for treatment with
electric current and Tob, similar to the data of normal cells
described above. The results indicate that 1.5 μg/mL Tob was not
able to kill P. aeruginosa PAO1 persister cells. However,
treatment with 75 μA/cm<2 >(500 μA total) current reduced
the number of viable persisters by ̃2.5 logs and another 2 logs
of killing was obtained when treating with Tob together, as
shown in FIG. 12. It is worthy noticing that the efficiency in
killing by electric current and synergistic effects with Tob
were similar for persisters and normal cells. This is a
significant advantage compared to traditional antibiotics, which
commonly fail to kill bacterial cells that are in stationary
phase or are persisters.
To understand if the killing by electric currents was due to the
ions generated by electrochemical reactions, P. aeruginosa PAO1
persister cells were also treated with pretreated buffer, which
was prepared by treating 0.85% NaCl buffer with SS304 stainless
steel electrodes for the same current level and duration as used
for the above experiments. The pretreated buffers were collected
after 20, 40 or 60 min of treatment. P. aeruginosa PAO1
persister cells were collected as described above and
resuspended in the pretreated buffers in the presence and
absence of Tob. The cells were then incubated at room
temperature without shaking for up to 1 h and samples were
collected every 20 min to count CFU. As shown in FIG. 13, the
ions released from the electrode caused less than one log of
killing of persister cells, significantly less than that with
current treatment (2-3 logs), suggesting the movement of ions or
some short-term ions might be essential for the effectively
killing with electric current. The generation of ROS as
described in E. coli HM22 data could be partially responsible
for the killing. In addition, no synergy was observed between
pretreated buffer and 1.5 μg/mL Tob. This finding suggests that
electric current may enhance the penetration of Tob and/or the
susceptibility of persisters.
In addition to stainless steel, carbon electrodes were also
found to control E. coli persister cells (discussed above). Here
we also compare the effects of stainless steel and carbon
electrodes on P. aeruginosa persister cells. As shown in FIG.
14, killing by about two logs was achieved using carbon
electrodes. It is slightly less than the 3 logs of killing by
stainless steel electrodes; however, it does confirm that the
killing effects are not limited to stainless steel electrodes.
Since the current treatment with 304 stainless steel electrodes
was more effective than that with carbon electrodes in killing
persisters, another experiment was conducted to treat P.
aeruginosa PAO1 persister cells using carbon electrodes and
0.85% NaCl buffer pretreated with 304 stainless steel
electrodes. As shown in FIG. 15, additional killing was observed
compared to treatment with 304 stainless steel electrodes (FIG.
15) or carbon electrodes (FIG. 14) alone. These results confirm
that ions or charge movement induced by electric current
treatment may be a key factor in killing persister cells. Thus,
a pre-prepared solution or cream containing such chemical
species might be applied for disease therapy with electric
currents.
Embodiments of the electrically-enhanced control of bacterial
persister cells, both planktonic persisters and those in
biofilms, are described above. The use of a very small electric
current to control persister cells, as well as the synergistic
effects shown when used in conjunction with antimicrobial
agents, is a new phenomenon. The low level of electric
current/voltage required to control persister cells are believed
to be physiologically safe for humans since similar and higher
current/voltage levels have been used to stimulate tissue and
bone growth.
Further, the effects of electric current and the synergy with
antimicrobial agents is not species-specific, since similar
results were shown using both E. coli strains and P. aeruginosa
strains. Accordingly, the present invention can be used to kill
a wide variety of microbial species.
The use of low electric current and/or low electric current
together with an antimicrobial agent is a novel means of
controlling persister cells and can be incorporated into devices
or procedures in order to treat chronic infections both inside
and outside the human body. For example, possible applications
include the treatment of chronic wounds, chronic sinusitis,
implanted-device-associated infections, and middle ear
infection, the decontamination of medical devices, or devices
with bare or coated electrodes, among many others.
Although the present invention has been described in connection
with a preferred embodiment, it should be understood that
modifications, alterations, and additions can be made to the
invention without departing from the scope of the invention as
defined by the claims.
DOOR HANDLE
STERILIZATION SYSTEM
WO2013025894