Compositions and methods for disrupting biofilm formation and maintenance

Abstract

A method of treating a biofilm on a surface, comprising: providing a surface having a biofilm; and administering to the surface a treatment that reduces a concentration of pyruvate of the biofilm, comprising pyruvate produced by at least a portion the biofilm, under conditions effective reducing maintenance of the biofilm on the surface. A composition, comprising purified enzyme, within a particle, effective for reducing pyruvate concentration in an aqueous suspension of the composition.

Claims

1. A method of inducing a dispersion of sessile organisms within a biofilm in an aqueous medium, comprising: enzymatically depleting pyruvate in the aqueous medium by supplying at least 5 mU/ml of an enzyme having a pyruvate substrate specificity, to a sufficient amount and for a sufficient time to induce hypoxic stress and a resulting dispersion response of the sessile organisms within the biofilm.

2. The method according to claim 1, wherein the enzymatically altering comprises decarboxylation.

3. The method according to claim 1, wherein the enzymatically altering comprises phosphorylation.

4. The method according to claim 1, further comprising adding at least cis-2-decenoic acid.

5. The method according to claim 1, further comprising adding at least nitric oxide.

6. The method according to claim 1, wherein the depleting comprises absorbing pyruvate to an insoluble matrix.

7. The method according to claim 1, wherein the depleting comprises chemically transforming the pyruvate to another chemical species.

8. The method according to claim 1, wherein the depleting comprises an imine-forming reaction.

9. The method according to claim 1, wherein the depleting comprises operation of pyruvate decarboxylase.

10. The method according to claim 1, wherein the depleting comprises use of a bioreactor comprising pyruvate fermentative organisms.

11. The method according to claim 1, wherein the depleting comprises selectively administering an enzyme preparation selected from the group consisting of pyruvate dehydrogenase; pyruvate oxidase; lactate dehydrogenase; and a transaminase.

12. The method according to claim 1, further comprising administering an antibiotic to the biofilm.

13. A method of treating a biofilm, comprising administering a formulation comprising an enzyme having a pyruvate substrate specificity to the biofilm at a level of at least 5 mU/ml, the enzyme being at least one of encapsulated and immobilized, under conditions effective for reducing pyruvate in an environment surrounding the biofilm, and causing a sufficient hypoxic stress of the cells associated with the biofilm to induce a dispersion response.

14. The method according to claim 13, further comprising administering a bacterial biofilm dispersion inducer to a subject having the biofilm.

15. The method according to claim 13, further comprising administering an antibiotic to the biofilm, wherein an antibacterial activity of the antibiotic is enhanced by the dispersion response.

16. The method according to claim 13, wherein the enzyme having the pyruvate specificity is pyruvate dehydrogenase, is stabilized with respect to a soluble form of pyruvate dehydrogenase by encapsulation.

17. The method according to claim 16, wherein the encapsulation comprises encapsulating the pyruvate dehydrogenase in chitosan nanoparticles.

18. A method of inducing a dispersion of sessile bacteria in a biofilm, comprising: enzymatically depleting pyruvate in an aqueous medium surrounding the biofilm, to induce hypoxic stress in the sessile bacteria and resulting is a dispersion response of the sessile organisms, with an enzyme that is encapsulated, which is stabilized with respect to a corresponding soluble form of the enzyme by the encapsulation to retain at least 50% of its initial activity for 48 hrs after hydration at 37° C. to a concentration of at least 5 mU/ml.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows that depletion of pyruvate from the growth medium via pyruvate dehydrogenase (PDH) impairs biofilm formation.

(2) FIGS. 2A-2H show that pyruvate depletion induces biofilm dispersion within 24 h of exposure to PDH or heat-inactivated (HK) PDH, as indicated by (2A) crystal violet (CV) staining of the attached biofilm biomass, (2B) increased turbidity and (2C) number of viable cells in the supernatant. (2D) CV stained biofilms obtained in the absence (top wells) and presence (bottom wells) of PDH. (2E) Confocal images of biofilms in the absence (2E1)/presence of active (2E3) or heat-inactivated (2E2) PDH. (2F1, 2F2) intact and dispersed biofilm microcolony. (2G, 2H) Dispersion proportion and microcolony diameter in absence, and presence of active or heat-inactivated PDH. Error bars represent standard deviation.

(3) FIGS. 3A and 3B show that pyruvate depletion-induced dispersion is dependent on the diameter of microcolonies. Microcolony diameter of (3A) dispersed and (3B) non-dispersed biofilm microcolonies following exposure to PDH. Untreated biofilms were used as control. On average, 100 microcolonies per biofilm were evaluated.

(4) FIGS. 4A-4D4 show that excess pyruvate or inactivation of genes involved in pyruvate fermentation processes abrogate the dispersion response. (4A) Remaining PAO1 biofilm biomass post PDH treatment in the absence/presence of excess lactate and pyruvate. Inactivation of (4B) ldhA or (4C) mifR encoding lactate dehydrogenase and microcolony formation regulator MifR, respectively, renders mutant biofilms insensitive to pyruvate depletion. Error bars represent standard deviation. (4D) Confocal images of untreated (4D1, 4D3) or PDH-treated biofilms by AmifR (4D2) and AmifR/pJN-mifR (4D4).

(5) FIGS. 5A and 5B show that PDH treatment increases the efficacy of tobramycin in killing PAO1 biofilms. (5A) Number of viable biofilm cells remaining post treatment. (5B) Log reduction. Biofilms were treated with tobramycin (Tob, 150 μg/mL). Error bars represent standard deviation.

(6) FIG. 6 shows that PDH treatment induces dispersion of biofilms by clinical isolate. P aeruginosa clinical isolates were isolated from indicated sites. Error bars represent standard deviation.

(7) FIGS. 7A-7C show CV staining (see 7A inset) of microcolonies, showing (7A) biofilm biomass after treatment with LB media, control, 5, 10 and 20 mU/ml PDH. (7B) brightfield microscopy. (7C) biofim biomass for control, 5 and 10 mU/ml.

(8) FIGS. 8A-8B show CV staining biomass for (8A) PDH and cofactors, and (8B) exogenous pyruvate.

(9) FIGS. 9A and 9B show CV staining, (9A) biomass and (9B) brightfield microscopy, of PAO1 and mutants for control and PDH.

(10) FIGS. 10A and 10B show control and PDH of (10A) confocal fluorescence microscopy and (10B) biofilm biomass for PA14 and mutants.

(11) FIG. 11 shows CV staining biofilm biomass for PAO1, PA14, CF lung, chronic wound, and using P aeruginosa clinical isolates with and without PDH.

(12) FIGS. 12A-12C show the effect of (12A) tobramycin alone, or with PDH, (12B, 12C) tobramycin, tobramycin with PDH or PDH on log reduction of biofilm CFU.

(13) FIG. 13 shows the effect of nitrate on CV staining biofilm mass, for control and PDH treated microcolonies.

(14) FIGS. 14A and 14B show the in vivo relevance of the technology.

(15) FIGS. 15A-15F show the effect of PDH nanoparticles (NP) on biofilms.

(16) FIG. 16 shows that nitric oxide can enhance pyruvate dehydrogenase-induced dispersion in biofilms.

(17) FIGS. 17A and 17B shows reduction in biofilm burden in wounds by reduction in colony forming units (CFU) (17A) and log.sub.10 reduction (17B) by use of pyruvate dehydrogenase (PDH) alone, silver sulfadiazine (SSD) cream alone, and PDH and SSD together.

(18) FIG. 18 shows biochemical pathways of pyruvate.

(19) FIG. 19 shows the pyruvate decarboxylase mechanism.

(20) FIG. 20 shows the ylide resonance form of thiamine pyrophosphate (TPP).

DETAILED DESCRIPTION

(21) A hallmark of biofilms is their extreme tolerance to antimicrobial agents, rendering infections by biofilms to conventional treatment therapies. This has brought on the realization that successful treatment of biofilm infections will require the development of novel treatment strategies. It is thus not surprising that biofilm dispersion, a regulatory response to environmental cues, allowing bacterial cells to convert to the planktonic state, has become a major focus of recent research endeavors to combat biofilms. However, while much attention has been paid to agents inducing biofilm dispersion, little is known about the mechanism underlying dispersion.

(22) Depletion of Pyruvate Coincides With Reduced Biofilm Biomass

(23) P. aeruginosa has been demonstrated to require autogenously produced pyruvate and pyruvate fermentative processes as a means of redox balancing to form microcolonies, with depletion of pyruvate or inactivation of components of the pyruvate fermentation pathway impairing biofilm formation. Considering that transition to the free-living state is initiated within microcolonies as indicated by microcolonies having central voids, pyruvate availability was hypothesized to play a role in dispersion. Enzymatic depletion of pyruvate from the growth medium of established P. aeruginosa biofilms coincided with a significant decrease in biofilm biomass, and central hollowing of microcolonies indicative of dispersion.

(24) No dispersion was noted by strains inactivated in components of the pyruvate fermentation pathway, in the presence of excess pyruvate or heat-inactivated enzyme. Moreover, pyruvate depletion-induced dispersion coincided with enhanced killing of biofilm cells by the aminoglycoside tobramycin.

(25) Pyruvate plays an essential role in the formation of biofilms, as continuous depletion of pyruvate (via pyruvate dehydrogenase plus cofactors) from the growth medium prevented biofilm formation. Given the role of pyruvate in establishing biofilms characterized by a three-dimensional architecture, the requirement for pyruvate by biofilms to remain surface-attached and maintain their three-dimensional architecture was investigated.

(26) FIG. 1 shows that depletion of pyruvate from the growth medium via pyruvate dehydrogenase (PDH) impairs biofilm formation.

(27) FIGS. 2A-2H show that pyruvate depletion induces biofilm dispersion within 24 h of exposure to PDH or heat-inactivated (HK) PDH, as indicated by (2A) crystal violet (CV) staining of the attached biofilm biomass, (2B) increased turbidity and (2C) number of viable cells in the supernatant. (2D) CV stained biofilms obtained in the absence (top wells) and presence (bottom wells) of PDH. (2E) Confocal images of biofilms in the absence (2E1)/presence of active (2E3) or heat-inactivated (2E2) PDH. (2F1, 2F2) intact and dispersed biofilm microcolony. (2G, 2H) Dispersion proportion and microcolony diameter in absence, and presence of active or heat-inactivated PDH. Error bars represent standard deviation.

(28) FIGS. 3A and 3B show that pyruvate depletion-induced dispersion is dependent on the diameter of microcolonies. Microcolony diameter of (3A) dispersed and (3B) non-dispersed biofilm microcolonies following exposure to PDH. Untreated biofilms were used as control. On average, 100 microcolonies per biofilm were evaluated.

(29) FIGS. 4A-4D4 show that excess pyruvate or inactivation of genes involved in pyruvate fermentation processes abrogate the dispersion response. (4A) Remaining PAO1 biofilm biomass post PDH treatment in the absence/presence of excess lactate and pyruvate. Inactivation of (4B) ldhA or (4C) mifR encoding lactate dehydrogenase and microcolony formation regulator MifR, respectively, renders mutant biofilms insensitive to pyruvate depletion. Error bars represent standard deviation. (4D) Confocal images of untreated (4D1, 4D3) or PDH-treated biofilms by AmifR (4D2) and AmifR/pJN-mifR (4D4).

(30) FIGS. 5A and 5B show that PDH treatment increases the efficacy of tobramycin in killing PAO1 biofilms. (5A) Number of viable biofilm cells remaining post treatment. (5B) Log reduction. Biofilms were treated with tobramycin (Tob, 150 μg/mL). Error bars represent standard deviation.

(31) FIG. 6 shows that PDH treatment induces dispersion of biofilms by clinical isolate. P aeruginosa clinical isolates were isolated from indicated sites. Error bars represent standard deviation.

(32) P. aeruginosa biofilms grown for 4 days in 24-well plates were exposed to pyruvate-depleting conditions. This was accomplished by exposing biofilms to increasing concentration of the enzyme pyruvate dehydrogenase (PDH) having a specific activity of 0.57 U/mg. PDH catalyzes the conversion of pyruvate to acetyl-CoA in the presence of CoA and NAD.sup.+. Specifically, biofilms were exposed to 5, 10, and 20 mU (8.7, 17.4, and 32.8 mg enzyme) of PDH in the presence of NAD.sup.+ and CoA. Biofilms grown in LB but left untreated were used as controls. Following overnight incubation, the remaining biofilm was stained using crystal violet. Relative to untreated biofilms, PDH treatment coincided with a significant loss in the CV-stainable biofilm biomass, with exposure to 5 mU resulting in a 2.2-fold reduction in the biofilm biomass while exposure to 10 and 20 mU resulted on average in a 2.9-fold reduction (FIG. 7A).

(33) To determine whether biofilms of different age are susceptible to pyruvate depleting conditions, biofilms grown for 2, 5, and 6 days were exposed to pyruvate depleting conditions, by treating biofilms with 10 mU PDH in the presence of cofactors. Following incubation for 16 hr, microscopic evaluation of the remaining biofilms indicated exposure of biofilms to PDH and thus, pyruvate depleting conditions, to coincide with a significant reduction in the biofilm biomass relative to the biofilms that were left untreated (FIG. 7B).

(34) These findings suggested exposure to PDH contributes to the loss of biofilm biomass regardless of the biofilm age, likely by inducing pyruvate-depleting conditions. This was further supported by the finding that exposure of biofilms to increasing concentrations of heat-inactivated PDH had no effect on the biofilm biomass relative to untreated biofilms (FIG. 7C).

(35) Pyruvate-depletion induced dispersion is inhibited by pyruvate but not by acetyl-CoA or lactate.

(36) The effect of cofactors on the biofilm biomass was determined to ensure that PDH affects the biofilm biomass by inducing pyruvate-depleting conditions. PDH requires NAD.sup.+ and CoA as cofactors to enzymatically convert pyruvate to acetyl-CoA and NADH. Exposure of biofilms to NAD.sup.+ and CoA or NAD+ alone had no effect on the biofilm biomass accumulation (FIG. 8A). In addition, to determine whether exposure to PDH is due to pyruvate depletion or the accumulation of the end products of the PDH catalyzed reaction, acetyl-CoA and NADH, biofilms were exposed to 0.2 mM acetyl-CoA or 2 mM NADH. Analysis of the biofilm biomass relative to untreated biofilms indicated that exposure of biofilms to acetyl-CoA or NADH did not result in increased biofilm biomass accumulation (FIG. 8A). As in P. aeruginosa, pyruvate dehydrogenase contributes to the formation of lactate, the effect of lactate on the biofilm biomass was evaluated. However, relative no untreated biofilms, no difference in the biofilms following exposure to 10 mM lactate was noted (FIG. 8A).

(37) Biofilms were exposed to increasing concentrations of pyruvate in the absence or presence of pyruvate to ensure PDH-induced loss of the biofilm biomass is due to pyruvate depletion. If PDH induces dispersion by depleting pyruvate, the presence of additional pyruvate would overwhelm PDH and thus reduce the efficacy of PDH in inducing loss of biofilm biomass. Biofilms were exposed to increasing concentrations of exogenously added pyruvate (1-100 mM), in the presence of 10 mM PDH and cofactors. Relative to untreated biofilms, treatment with PDH in the presence of 1 and 10 mM pyruvate significantly reduced the crystal violet-stainable biofilm biomass (FIG. 8B). However, while no difference in the fold reduction of the biofilm biomass was noted in the presence of 1 mM pyruvate, the fold reduction in the biofilm biomass decreased to less than 2-fold in the presence of 10 mM pyruvate. In contrast, no difference in the biofilm biomass was noted in the presence of 100 mM pyruvate relative to untreated biofilms (FIG. 8B). These findings strongly suggest PDH to reduce the biofilm biomass in a manner dependent on pyruvate.

(38) Depletion of Pyruvate Coincides With Dispersion Events.

(39) Crystal Violet staining of biofilms in the presence and absence of pyruvate-depleting conditions suggested to significantly reduce the biofilm biomass (FIGS. 7A-B). To determine how exposure to PDH accomplished a reduction in the biofilm biomass, the remaining biofilm architecture was visually analyzed by confocal microscopy. Relative to untreated biofilms, biofilms exposed to PDH were not only characterized by an overall reduced biofilm biomass but by microcolonies having central voids (FIGS. 2E3 and 2F2). Void formation has previously linked with biofilm dispersion, a process in which sessile, surface-attached organisms liberate themselves from the biofilm to return to the planktonic state. Overall, more than 60% of the detectable microcolonies present in PDH treated biofilms showed signs of dispersion apparent by central voids (FIG. 2E3). In contrast, the vast majority of microcolonies by untreated biofilms were intact (FIG. 2E1), with only less than 8% of the microcolonies featuring central void formation (FIG. 2G). Similar results were obtained when biofilms were treated with heat-inactivated PDH (FIG. 2E2).

(40) Previous findings suggested microcolonies of P. aeruginosa form hollow voids at their center when they attain a minimum diameter of 40 microns and thickness of 10 microns, with the microcolony size within which these voids form being dependent on the fluid flow rate. Given that exposure to PDH was found to coincide with a larger percentage of microcolonies showing void formation, the effect of pyruvate-depleting conditions on the minimum diameter of microcolonies that disperse was investigated. Analysis of the microcolony size in untreated biofilms suggested that microcolonies having an average diameter of 90 microns were non-dispersed while larger microcolonies having an average size of 210 microns showed signs of dispersion (FIG. 2H). Exposure of biofilms to heat-inactivated PDH (HK_PDH) had little to no effect on the microcolony size of dispersed and non-dispersed microcolonies (FIG. 2H). Moreover, no significant difference in the size of non-dispersed microcolonies following exposure to PDH was noted. In contrast, however, an overall significant increase in the size of dispersed microcolonies of PDH treated biofilms was observed (FIG. 2H). Based on visual observations, the increase in the size of dispersed microcolonies is likely due to “sagging”, with the remaining microcolony structure bulging downward under weight or pressure or through lack of strength.

(41) Overall, these findings suggest exposure of biofilms to PDH and thus, pyruvate depleting conditions to coincide with dispersion events. Considering that PDH treatment does not affect the overall size of microcolonies that remain intact, these findings furthermore suggest that pyruvate depletion enhances dispersion.

(42) Pyruvate-Depletion Induced Dispersion is Independent of Previously Described Factors Contributing to Dispersion

(43) Considering that PDH exposure of biofilms coincided with dispersion events, factors previously demonstrated to be important in the dispersion response following exposure to nitric oxide or nutrients was required for pyruvate-depletion induced dispersion. Specifically, the role of chemotaxis transducer protein BdlA, and two phosphodiesterases, RbdA and DipA, in the pyruvate-depletion induced dispersion response was investigated. The factors were chosen as they appear to play a central role in the dispersion response by P. aeruginosa biofilms, with inactivation of bdlA, rbdA, and dipA impairing the dispersion by P. aeruginosa biofilms in response to various dispersion cues including nutrients, NO, ammonium chloride, and heavy metals (16-18).

(44) Biofilms by strains ΔbdlA, ΔdipA, and ΔrbdA were grown for 4 days in 24-well plates, and subsequently exposed to 10 mU PDH to induce pyruvate-depleting conditions. Biofilms grown in LB but left untreated were used as controls. Following overnight incubation, the remaining biofilm was stained using crystal violet. Relative to untreated biofilms, PDH treatment coincided with a significant loss in the crystal violet-stainable biofilm biomass, with exposure of ΔdipA, and ΔrbdA biofilms to PDH resulting in a >60% loss of the biofilm biomass (FIG. 9A). Under the conditions analyzed, the reduction of the biofilm biomass was comparable or exceeded to the loss noted for wild-type biofilms (FIG. 9A). In contrast, the crystal violet-stainable biomass by ΔbdlA biofilms was only reduced by 2-fold (FIG. 9A). However, the reduction in the biofilm biomass was significant. The reduction in biomass resulting from PDH treatment on strains ΔbdlA, ΔdipA, and ΔrbdA were confirmed visually using brightfield microscopy (FIG. 9B).

(45) Pyruvate-Depletion Induced Dispersion is Dependent on Lactate Dehydrogenase LdhA and the Microcolony Formation Regulator MifR

(46) These findings suggested that dispersion induced by pyruvate depletion is independent of previously identified factors playing a role in the dispersion of P. aeruginosa biofilms in response to previously described dispersion cues. However, this raised the question of how pyruvate-depleting conditions contribute to biofilm dispersion. The formation of biofilms by P. aeruginosa was previously demonstrated to require pyruvate and pyruvate fermentation, with the biofilm-dependent utilization of pyruvate requiring lactate dehydrogenase LdhA and the microcolony formation regulator MifR. These findings furthermore demonstrated that biofilm formation is associated with stressful, oxygen-limiting but electron-rich conditions, suggesting pyruvate to be required to cope with stressful, oxygen-limiting but electron-rich conditions, referred to as ‘reductive stress’ (too much NADH/electrons, not enough O.sub.2) present in biofilms.

(47) Biofilms by mutant strains ΔldhA and ΔmifR were exposed to PDH, and the biofilm structure of the respective mutant strains exposed to PDH analyzed relative to untreated biofilms, to determine whether the factors contributing to the formation of biofilms by P. aeruginosa also play a role in dispersion. Based on visual comparison, no difference in the biofilm architecture by ΔldhA and ΔmifR in the presence of absence of PDH was noted (FIG. 10A). However, dispersion in response to pyruvate-depleting conditions was restored upon complementation, apparent by biofilms by the complemented strains ΔldhA/pMJT-ldhA and ΔmifR/pJN-mifR demonstrating voids upon exposure to PDH (FIG. 10A).

(48) Visual observations of inactivation of ldhA and mifR showed impaired pyruvate-depletion induced dispersion response, were confirmed using crystal violet staining. Using biofilms by P. aeruginosa wild-type PA14 and corresponding isogenic mutant strains ΔldhA and ΔmifR, no reduction in the biofilm biomass was noted upon exposure of biofilms by the mutant strains ΔldhA and ΔmifR to PDH (FIG. 10B). In contrast, complementation of the ΔldhA and ΔmifR mutant strains coincided with a reduction in the CV-stainable biofilm biomass in a manner similar to the loss of biofilm biomass noted for P. aeruginosa wild-type PA14 (FIG. 10B). These findings strongly suggest exposure to PDH and thus, the pyruvate-depletion induced dispersion response, to require LdhA and MifR. These findings furthermore suggest that dispersion induced by pyruvate depletion may be in response to biofilms no longer being capable of coping with stressful, oxygen-limiting but electron-rich conditions, referred to as ‘reductive stress’ (too much NADH/electrons, not enough O.sub.2) present in biofilms. FIG. 13 shows the effect of 10 mM nitrate under oxic and anoxic conditions, on biofilm crystal violet staining.

(49) Pyruvate-Depletion Induced Dispersion is Not Limited to the P. aeruginosa Laboratory Strain PAO1

(50) The findings suggest that P. aeruginosa biofilms disperse in response to PDH. Given that both P. aeruginosa strain PAO1 and strain PA14 dispersed under the conditions tested (FIGS. 10A & 10B), dispersion in response to PDH was predicted not to be limited to laboratory strains. Instead, three P. aeruginosa clinical strains, isolated from various infection sites, including the urinary tract, burn wounds, and the lungs of cystic fibrosis patients, exhibited dispersion upon exposure to PDH (FIG. 11). It is of interest to note, however, that the efficiency by PDH in reducing the biofilm biomass varied between 40-60%. The variability in the extent of the loss of crystal violet-stainable biomass noted for clinical isolates, however, was within the range of the biofilm biomass loss noted for biofilms by the laboratory strains PAO1 and PA14 (FIG. 11).

(51) To determine whether dispersion upon depletion of pyruvate is limited to biofilms by P. aeruginosa, two facultative anaerobic bacteria, the Gram-negative bacterium Escherichia coli BW25113 and the Gram-positive Staphylococcus aureus, that represent significant burden on the healthcare system, were used. S. aureus is a major cause of nosocomial and community-acquired infections. E. coli is considered the major causative agent for recurrent urinary tract infections, with E. coli biofilm also being responsible for indwelling medical device-related infectivity. Biofilms by E. coli were grown in 24-well plates, and subsequently exposed to 10 mU PDH. Likewise, biofilms by S. aureus were grown in 24-well plates, and exposed to PDH. Following overnight incubation, the remaining biofilm was stained using crystal violet. Relative to untreated biofilms, PDH treatment coincided with a significant loss in the crystal violet-stainable biofilm biomass, with exposure to 10 mU resulting in a reduction in the biomass of E. coli biofilm while exposure of S. aureus biofilms to 10 mU also resulted on average in a reduction. Dispersion in response to PDH was confirmed by microscopic evaluation of the remaining biofilms. Exposure of S. aureus and E. coli biofilms to PDH and thus, pyruvate depleting conditions, coincided with a reduction in the biofilm biomass relative to the biofilms that were left untreated.

(52) Pyruvate-depletion induced dispersion coincides with biofilms being rendered susceptible to tobramycin.

(53) It is well established that planktonic cells are more susceptible to antimicrobial agents than their counterparts growing as a biofilm, and that dispersion coincides with bacteria transitioning to the planktonic mode of growth. Considering that PDH treatment resulted in biofilm dispersion, treatment with dispersion-inducing PDH was investigated to see if it coincides with biofilms being more susceptible to antimicrobial agents. P. aeruginosa biofilms grown for 4 days in 24-well plates were exposed to 100 μg/ml tobramycin in the absence of presence of PDH. Following overnight incubation, the number of viable biofilm cells were determined using viability count. Treatment of biofilms with tobramycin alone resulted in a 2.5-log reduction of the overall biofilm biomass, effectively reducing the number of viable cells from 2.4×10.sup.8 to 1.2×10.sup.5 cells per biofilm (FIG. 12A). Tobramycin in the presence of 10 mU PDH coincided with a reduction in the biofilm biomass to 3.5×10.sup.3 cells/biofilm (FIG. 12A), with the reduction in the viable cells being equivalent to an overall 5.9-log reduction in the biofilm biomass relative to untreated biofilms. These findings clearly indicate that co-treatment with tobramycin in the presence of pyruvate-depleting conditions resulting in biofilm dispersion, renders the antibiotic tobramycin more effective in killing biofilm cells. Overall, co-treatment enhanced the efficacy of tobramycin by 2.4-logs.

(54) Pyruvate-Depletion Reduces the Biofilm Burden in Porcine Burn Wounds and Enhances the Efficacy of Tobramycin in Killing Biofilm Cells.

(55) The data indicate that PDH treatment to induce pyruvate depletion is capable of inducing dispersion of established biofilms and render biofilms more susceptible to the antibiotic tobramycin relative to tobramycin alone. Pyruvate depletion was then investigated in vivo as an anti-biofilm strategy to reduce the bacterial burden of established biofilms, and enhance killing of biofilm cells and thus, reduce or eliminate biofilm-related infections. As P. aeruginosa is considered the 2.sup.nd leading cause of biofilm infections, and is one of the principal pathogens associated with wound infections, a wound model of infection was employed. A porcine rather than a rodent model was employed, as pig skin is more similar to human skin, including similar epidermal/dermal-epidermal thickness ratios, dermal collagen, dermal elastic content similar patterns of hair follicles, blood vessels, and physical and molecular responses to various growth factors. Moreover, while rodent models heal primarily by contraction, pig skin heals in a manner similar to human skin by epithelialization. The well-known clinical burn wound isolate P. aeruginosa ATCC 27312 was employed.

(56) Burn wounds were inoculated with 25 μl of a standardized P. aeruginosa suspension harboring 10.sup.6 CFU/ml and allowed to establish biofilms for 24 h. Infected wounds were subsequently treated daily with 100 and 200 mU PDH in the absence of presence of 100 μg/ml tobramycin. Untreated wounds and wounds only exposed to carrier solution were used as controls. Following 3 and 6 days of treatment, bacterial cells present in wounds were harvested using a flush and scrub technique that separates biofilm bacteria from planktonic bacteria, by flushing the non-adherent (planktonic) bacteria off the wound, followed by scrubbing of the wound to remove adherent biofilm-associated bacteria from the wound bed. Relative to untreated wound biofilms, exposure to 100 mU PDH coincided with up to 2-log reduction in scrub fraction. Similar results for 200 mU PDH (FIG. 12B). Treatment with tobramycin likewise coincided with a 2-log reduction in viable biofilm cells (scrub) relative to untreated biofilms (FIG. 12B). Co-treatment significantly increased the efficacy of tobramycin, apparent by an average reduction in the biofilm CFU/wound of 3.5 and 4-log reduction in the presence of 100 mU and 200 mU PDH, respectively (FIG. 12B). Increased killing of bacteria present in flush in wounds treated with PDH alone and wounds treated with PDH and tobramycin (FIG. 12C) was noted.

(57) The data indicate pyruvate to act as a switch to control biofilm formation, biofilm dispersion, and tolerance, with depletion of pyruvate coinciding with prevention of biofilm formation, disaggregation of existing biofilms, and dispersed biofilms being rendered susceptible to lower doses of antibiotics relative to biofilms.

(58) Pyruvate Depletion is an Effective Anti-Biofilm Therapy, Capable of Controlling and Eradicating Biofilms in Wounds, by Enhancing the Efficacy of Antibiotics and the Immune System.

(59) A treatment strategy based on pyruvate depletion has several added benefits. In P. aeruginosa, pyruvate (i) is an energy source, does not promote growth (not a carbon source), and (ii) has only been linked to biofilm growth and long-term bacterial survival under oxygen limiting conditions. (iii) Long-term survival and biofilm studies have not given rise to pyruvate-insensitive mutants. (iv) While pyruvate insensitivity has been noted upon inactivation of genes contributing to pyruvate fermentation (e.g. acnA, ldhA, mifR), these mutants were incapable of coping with the reductive stress and therefore, were unable to form biofilms. (v) Pyruvate depletion by PDH does not affect bacterial growth in liquid or susceptibility of planktonic cells, All of the above lessen the possible selection of pyruvate-insensitive bacteria.

(60) Experimental Procedure

(61) Biofilms were grown in a 24-well plate system modified from the procedure described by Caiazza and O'Toole (2004) to elucidate the role of pyruvate in biofilm formation and biofilm dispersion.

(62) Overnight cultures grown in LB medium were adjusted to an OD600 of 0.05 in fresh 5-fold diluted LB VBMM and grown at 37° C. and rotated at 220 r.p.m. in 24-well microtiter plates at a 45° angle, ensuring that the bottom of the wells is at the air-liquid interface, with the medium exchanged every 12 h.

(63) For biofilm prevention, the growth medium contained 10 mU porcine pyruvate dehydrogenase, cofactors (1 mM b-NAD, 1 mM sodium Co-A, 20 μM thiamine phosphate (TPP)) at the time of inoculation.

(64) For biofilm dispersion, biofilms were first grown for 4 days after which time the growth medium was supplemented with 10 mU porcine pyruvate dehydrogenase, cofactors (1 mM b-NAD, 1 mM sodium Co-A, 20 μM TPP) and allowed to incubate for an additional 16 h.

(65) For susceptibility assays, biofilms were grown as for dispersion assays, but following 4 days of growth, the growth medium was supplemented the aminoglycoside antibiotic tobramycin (100 μg/ml), 10 mU porcine pyruvate dehydrogenase, and cofactors (1 mM b-NAD, 1 mM sodium Co-A, 20 μM TPP). Untreated biofilms or biofilms only exposed to cofactors alone were used as controls.

(66) Differences in the biofilm architecture were visualized by crystal violet staining or microscopy (brightfield, confocal laser scanning). Differences in drug susceptibility were evaluated by viability count.

(67) In vivo biofilms such as those present in porcine burn wounds were found to require increased pyruvate dehydrogenase activity. A pyruvate dehydrogenase activity of 100-200 mU appeared to have maximal activity in dispersing biofilms as well as having maximal adjunctive activity when used in combination with 100 μg/ml tobramycin in eradicating biofilms present in wounds.

(68) Encapsulation of Pyruvate Dehydrogenase

(69) Pyruvate dehydrogenase can be encapsulated in a poly(lactic-co-glycolic acid) (PLGA) particle formulation while maintaining enzymatic activity. PLGA is FDA-approved, degradable, and can protect encapsulated proteins from proteolysis and immune attack.

(70) Pyruvate dehydrogenase-loaded PLGA nanoparticles were made by water-in-oil-in-water double emulsion and had an average size of 360±10 nm and a zeta-potential of −11±3 mV. Based on Western blots, the encapsulation efficiency was approximately 10%. PLGA-immobilization had several benefits:

(71) Free/Unencapsulated pyruvate dehydrogenase was inactive when stored for 2 days at 37° C., yet PLGA-immobilized pyruvate dehydrogenase was still active after 4 days at 4-37° C. PLGA-immobilized pyruvate dehydrogenase was as effective in inducing biofilm dispersion as free pyruvate dehydrogenase (stored at −20° C. until use). PLGA-immobilized pyruvate dehydrogenase particles adhere to biofilm cells.

(72) PLGA-immobilized PDH maintained 100% of its activity when stored for 4 days at −20° C., 4° C., or 20° C. and approximately 75% of its activity when stored at 37° C. (FIG. 15A).

(73) PLGA-immobilized PDH was as effective in inducing biofilm dispersion as free PDH (stored at −20° C. until use, FIGS. 15B, 15E). PLGA-immobilized PDH adhered to peripheral biofilm cells (FIG. 15D), which produce and secrete pyruvate.

(74) An alternative particle substrate is chitosan, a natural, biodegradable polysaccharide with high biocompatibility and mucoadhesive properties. Chitosan nanoparticles were produced encapsulating pyruvate dehydrogenase, formed through ionic gelation. N-trimethyl chitosan chloride (TMC) particles were prepared by ionic crosslinking with tripolyphosphate. Chitosan particles encapsulating pyruvate dehydrogenase had similar activity (ability to convert pyruvate) as PLGA particles.

(75) Therefore, pyruvate dehydrogenase can be encapsulated in a variety of forms, while retaining activity and gaining stability.

(76) Pyruvate dehydrogenase-containing nanoparticles or microparticles may be provided in conjunction with numerous medical applications, including, for example, deposition on medical devices, in wounds or on wound dressings, or as a therapy for cystic fibrosis patients, such as an inhaled micronized or nanoparticle powder inhalant. A nebulizer may also be used for administration.

(77) FIGS. 14A and 14B show the in vivo relevance of the technology. FIG. 14A shows PDH treatment (5 mU) disperses biofilms by clinical isolates, as indicated by CV staining. FIG. 14B shows PDH treatment (5 mU) renders in vitro biofilms by P. aeruginosa and S. aureus and biofilms by P. aeruginosa burn wound isolate ATCC27312 in porcine wounds significantly more susceptible to antimicrobial agents (Ab) In vitro biofilms were treated with tobramycin (150 μg/ml, P. aeruginosa) or vancomycin (100 μg/ml, S. aureus) for 1 h; in vivo wound biofilms were treated with silver sulfadiazine for 1 d. Log reduction refers to reduction in viable cells post treatment. All experiments were done in triplicate.

(78) FIGS. 15A-15F show the effect of PDH nanoparticles (NP) on biofilms. FIG. 15A shows PDH activity, determined spectrophotometrically via NADH conversion, is affected by storage temperature, storage time, and NP encapsulation (NP-PDH). FIG. 15B shows PLGA-immobilized PDH (NP) is as effective in reducing biofilm biomass as free PDH. Biofilm biomass was quantitatively determined by confocal microscopy and COMSTAT analysis prior/post 24 h PDH treatment. FIG. 15C shows an SEM image of NPs, scale bar=500 nm. (15D) Confocal images of NPs (red) surrounding biofilm cells (green) FIG. 15E shows biofilm microcolonies (bacteria in green) prior/post dispersion (arrows mark voids indicative of dispersion). FIG. 15F shows PDH-induced dispersion is not affected by excess lactate and pyruvate (10 mM), as determined using CV staining of remaining biofilm biomass. PDH, 5 mU. Inset, corresponding CV-stained biofilms post indicated treatment. Experiments done in triplicate.

(79) FIG. 16 shows that nitric oxide (NO) can enhance PDH-induced dispersion of biofilms. Biofilms were grown for 4 days in 24-well polystyrene plates in five-fold diluted LB. Post 4 days, biofilms were either left untreated or exposed to pyruvate dehydrogenase (PDH, 10 mU) or PDH plus 500 uM SNP. SNP was used as a source of nitric oxide (NO). PDH treatment was done in the presence of CoA, B-NAD+, TPP, and MgSO4. Following 16 h of treatment, biofilms were viewed by confocal microscopy to determine the number of microcolonies showing void formation indicative of dispersion. Number of microcolonies as percent of the total colonies counted per treatment group is shown. All experiments were carried out in triplicate. Error bars denote standard deviation.

(80) FIGS. 17A and 17B shows reduction of biofilm burden in wounds. Second degree porcine burn wounds were infected with P. aeruginosa ATCC® 27312™. Wounds were either left untreated or exposed to pyruvate dehydrogenase (PDH, 200 mU, plus cofactors), silver sulfadiazine cream (SSD), or SSD plus PDH for 24 h. Then, bacterial cells were removed from the wounds and the number of viable cells, as shown in (CFU) per wound determined using viability counts. FIG. 17A shows the number of bacterial cells present in wounds. FIG. 17B shows the Log.sub.10 reduction was determined relative to untreated biofilms (based on data shown in FIG. 17A). Experiments are representative data obtained using 3 wounds per treatment group (n=3). Error bars represent standard deviation. Pretreatment with PDH and SSD should have ability to prevent infection.

(81) Encapsulated PDH demonstrates improved thermal stability. The specific depletion of pyruvate can be accomplished enzymatically using pyruvate dehydrogenase (PDH) or lactate dehydrogenase (LDH). PDH requires NAD.sup.+ and CoA as cofactors to enzymatically convert pyruvate to acetyl-CoA while LDH requires NADH to convert pyruvate to lactate. PDH is preferred over LDH for three reasons: (i) NAD.sup.+ is more stable than NADH, (ii) wound exudates have been reported to contain ≥10 mM lactate (but low pyruvate and no acetyl-CoA) with the presence of lactate likely resulting in LDH producing pyruvate in vivo, rather than depleting it (since LDH is reversible), and (iii) PDH is not inhibited by the presence of lactate (FIG. 15F). However, most if not all enzymes are relatively unstable and rapidly lose activity when exposed to temperatures relevant to in vivo applications (body temperature=37° C., FIG. 5A). Free PDH is no exception and is rendered inactive within less than 1 day when stored at 37° C. (FIG. 5A).

(82) PLGA particles can be formulated with varying PLGA molecular weight, synthesis method, and the loading of PDH. The PLGA molecular weight affects the crystallinity, hydrophobicity, and degradation rate of the particles—factors likely to affect PDH stability over time. NPs may be synthesized using either a water-in-oil-in-water (W/O/W, FIG. 15C) double emulsion method or nanoprecipitation method.

(83) W/O/W Synthesis: for a 1×batch, 400 μl of concentrated porcine PDH (Sigma Aldrich, 5.8 mU/μl) in phosphate buffered saline MOPS is rapidly mixed with 4 ml of acetone containing 100 mg PLGA (either 38 kDa or 60 kDa) and sonicated for 40 s. The solution is then added to 8 ml of 0.1% v/v polyvinyl alcohol solution with sonication. The emulsion is diluted, acetone is evaporated, and unencapsulated PDH is removed via centrifugation. Particles are frozen and freeze dried.

(84) Nanoprecipitation Synthesis: In particle synthesis, the avoidance of solvents with potential effects on protein integrity is desirable and may increase the activity of encapsulated PDH. Formulations are created by by nanoprecipitation using glycofurol 67,68, which has low toxicity 69-74. PDH (100 μL) is added to 300 μL of 12% PLGA (38 kDa or 60 kDa) in glycofurol and mixed with 100 μL of ethanol and 1.5 mL of 1% Poloxamer 188. Particles are centrifuged, frozen, and freeze dried.

(85) Chitosan NPs: While PLGA has been used extensively in drug delivery applications and has successfully delivered several proteins without substantial loss of activity and worked well in preliminary experiments, the breakdown of PLGA into its acidic constituents can potentially cause protein instability. As an alternative, chitosan is a natural, biodegradable polysaccharide with high biocompatibility and mucoadhesive properties. Chitosan NPs have been used for encapsulation of proteins and can be simply formed through ionic gelation. N-trimethyl chitosan chloride (TMC) particles may be prepared by ionic crosslinking with tripolyphosphate. Chitosan particles may be formulated with the following variables: chitosan molecular weight (low and medium, Sigma) and PDH loading.

(86) PEGylation of particles: The widely used biocompatible polymer PEG may be incorporated to increase the hydrophilicity of the NP to preserve the biological activity of PDH38 and to render the particles less likely to be immunogenic. In order to avoid affecting the activity of already encapsulated PDH, PEG-5k may be be linked to the already-synthesized particles' surface. Carbodiimide chemistry (EDC/NHS) may then be used to create a bond between an amine group on PEG (mPEG5k-NH2) and PLGA-COOH 32. For chitosan particles, a carboxylic acid group on PEG (mPEG5k-COOH) bonds an amine on the chitosan that is present after reducing the C═N with NaBH.sub.4.

(87) Wound temperatures have been reported by Shorrock et al. to range from 32-41° C. Furthermore, wounds have been shown to be a proteolytic environment and to have an average pH of 7.1-7.5, with measurements taken at the wound center indicating an average pH of 7.6±0.6 and areas on the epitheliated wound borders showing physiological pH values of 5.9±0.4.

(88) While free PDH activity decreases significantly upon storage, PLGA-encapsulated PDH remains active for 4 days (FIG. 15A).

(89) PLGA-encapsulated PDH adheres to biofilm cells (FIG. 15D). This characteristic may be important for effective biofilm dispersion (FIG. 15E, compare 10 mU PDH to PDH-NP-induced dispersion response).

(90) Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.