rexresearch.com
Tagbo NIEPA, et
al.
Electrochemical Disinfection
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.
AbstractEloise Eyo Parry-Nweye, et al.
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.
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 ]
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.
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