Field of the Invention

The present invention generally relates to the technical field of treatment or prevention of infections. More particularly, the present invention is in the technical field of low-amperage electrical current by means of electrolysis under conditions of physiologic saline and generation of hypochlorous acid 'in situ' to treat and prevent antimicrobial resistant infections and biofilm infections.

Background of the Invention

Access to effective antibiotics is essential in all health systems. Their use has reduced childhood mortality and increased life expectancy, and they are crucial for invasive surgery and treatments such as cancer chemotherapy and solid organ transplantation. Antimicrobial Resistance (AMR) is a concept rather than a disease like in itself and despite its dramatic rising, is not given the same attention as acute infectious threats like SARS, Pandemic flu or Ebola, nor the same attention as the three major infectious diseases HIV, Tuberculosis and malaria 1. AMR is a serious global menace, affecting global economic, social and public health. The most recent World Economic Forum Global Risks reports have listed AMR as one of the greatest societal risks threats to human health.

Among the most important AMR bacteria in terms of causing infections in hospitalized patients are the so- called 'ESKAPE' pathogens. These are E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter spp. The most prominent threat of AMR is the rapidly rising tide of resistance among AMR, 'ESKAPE' bacteria that cause hospital-based infections in the last years. In addition to the ESKAPE pathogens, AMR E. coli, remains the main cause of mortality in hospitalized patients. In countries with high levels of MDR resistance, including resistance to carbapenems, and in the case of infections due to carbapanem resistant ^.aeruginosa (MDR/XDR ¥ .aeruginosa incidence 25-50%) only a few therapeutic options are available, among these are polymyxins. In these countries and in the case of MDR/XDR P. aeruginosa, presence of resistance to polymyxins or aminoglycosides is an important warning that options for the treatment of infected patients are becoming even more limited. Then, few antibiotics are effective enough for therapy. The antibiotics that still do work, frequently have major side effects, are less efficacious, or are very expensive (tigecycicline). AMR is not only costly in terms of human suffering but also in monetary terms. AMR currently claim at least 50,000 lives each year across Europe and the US alone and about 700,000 lives worlwide; at the estimated cost of more than 1.5 billion EUR or 35$ billion annually.

In addition to increased resistance to existing agents, there is a lack of new antibiotics in development. For many years, the pharmaceutical industry has been successfully churning out new antibacterial drugs. However, it is becoming more difficult to find novel antibiotics, and many large drug companies have withdrawn from antibiotic development programs because the process is extremely costly, and often fruitless. Alarmingly, existing antibiotics are losing their potency due to the spread of resistance at an alarming rate while few new antibiotics are being developed. Therefore, we are facing a paradoxical situation, as a perfect storm, with increased levels of resistant bacteria along with a descending trend in antibiotic development.

The spread of AMR bacteria could dramatically set back modern medicine to the dark age of the pre- antibiotic era; achievements in modern medicine, such as decrease in the safety of childbirth, caesarean sections, treatment of preterm babies, major or even dirty minor surgery, treatment of pneumonia, sexual transmitted diseases, organ transplantation and cancer chemotherapy, which we today take for granted, would not be possible without access to effective treatment for bacterial infections with antibiotics.

Moreover, difficult-to treat chronic infections associated with medical devices such as joint replacements and other types of orthopaedic instrumentation, prosthetic heart valves, pacemakers, implantable defibrillators, urinary catheters and stents, peritoneal dialysis catheters, intravascular catheters, cerebrospinal fluid shunts, breast implants, and vascular grafts and stents are common in today's medical practice. When these devices become infected, they must often be removed to successfully cure the associated infection. Device removal is associated with significant morbidity, cost, and, in some cases, mortality. Devices such as stents, shunts, prostheses, implants, endotracheal tubes, pacemakers, and various types of catheters, to name a few, have all been shown to support colonization and biofilm formation by different bacterial species or Candida spp. These bacterial species or Candida biofilms are 30 to 2,000 times more resistant than planktonic cells to antimicrobial agents.

EP 2190442B1 discloses an apparatus for treatment and prevention of infectious disease and a method for the treatment of bacterial diseases based on the antibacterial characteristic of the electrically released of silver ions. The method includes the implantation of a small electrode including silver in an infected bone, embedding another small electrode including another metallic element subcutaneously, and initiating the release of silver ions by applying a small current between the two electrodes. The initiation current is applied for a very short time and then discontinued. After this a continuous release of the silver ions will take place for a long time while spreading the silver ions to the surrounding tissue as well. This process assures a long-term antibacterial effect, which after accomplishing the infection healing will also provide preventive care. The apparatus comprises all of the components necessary to carry out the method. EP 2403588B1 discloses methods for the treatment of bone, cartilage and other types of hard tissue. The treatments, which are suitable for extended treatment, include the treatment and prevention of pathologies through the controllable use of silver, iron, zinc, or magnesium ions. These pathologies may include a pathology which is at least partially induced or aggravated by an infectious disease, for example a bacterial disease. In this case the electrically released ions are silver ions, which are known to have antibacterial properties. WO 2011078511 refers to a sterilization method and apparatus for medical instruments that proposes preparing a solution containing chlorine and having a temperature of about 60° C. or more, disposing at least one electrode in a container containing the solution and immersing at least one medical instrument in the solution such that the medical instrument is disposed over the electrode; and electrolyzing the solution by applying a current to the electrode to generate sterilizing components of free chlorine comprising hypochlorous acid, hydrogen peroxide (H202), OH radical, and ozone (03) and thus sterilize the medical instrument by the sterilizing components which move up in the opposite direction of gravity from the electrode.

New methods and systems for 'in situ' treating or preventing antimicrobial resistant infections, and also infections difficult to treat, are therefore needed, to preserve antibiotic effectiveness.

Description of the Invention

To that end, embodiments of the present invention provide according to a first aspect a system for 'in situ' treatment or prevention of antimicrobial resistant infections or infections difficult to be treated or at risk to be infected. The proposed system comprises a delivering unit configured to deliver during a treatment period an amount of a saline solution (0.9% NaCl) to a local area of a patient infected by antimicrobial resistant microorganisms or microorganisms growing in biofilms or at risk to be infected, a device configured to be placed to the local area of the patient, an electrical power source and a control unit operatively connected to the device and/or to the electrical power source.

Preferably, the device includes two or more conductive electrodes configured to be placed in contact with the amount of saline solution to produce hypochlorous acid during the treatment period; one or more sensors configured to provide information about temperature and pH in the local area during the treatment period; and a support structure to hold the conductive electrodes and the sensors.

Besides, the electrical power source is configured to generate and apply via the two or more conductive electrodes an electric current to the local area during a given period of time of the treatment period depending on the type and severity of the infection to be treated, thus producing the hypochlorous acid at the local area via the saline solution, and the control unit is configured to control the extent of said given period of time in which the electric current is applied and the value of the electric current.

Preferably, the electric current is kept at a value between 1 mA and 20 mA, that is, being a low-amperage electrical current. For instance, a continuous direct current provided by a DC source. Moreover, the value of the applied electric current is controlled by the control unit based on the temperature and pH information provided by the one or more sensors.

In addition, the given period of time in which the electric current is applied can be comprised between 1 and 30 minutes. Preferably, the temperature during the treatment period is kept to not exceed 40-42 °C and the pH is kept between 6.5 and 8.5.

The control unit can be an independent unit to the electrical power source or can be integrated therein.

Embodiments of the present invention also provides according to a second aspect, a method for treatment and prevention "in situ" of antimicrobial resistant infections or difficult to treat infections by electric current, in particular low-amperage electrical current, to generate electrolysis under conditions of physiologic saline and generation of hypochlorous acid.

For that reason, the method comprises placing a device to a local area of a patient infected by antimicrobial resistant microorganisms or microorganisms growing in biofilms or at risk to be infected, said device preferably including two or more conductive electrodes to be in contact with the local area of the patient during a treatment period; one or more sensors at least providing information about temperature and pH in said local area during the treatment period; and a support structure to hold the conductive electrodes and the sensor(s).

In addition, the method further comprises delivering, by a delivering unit, an amount of a saline solution, such as 0.9% NaCl, to the local area to be in contact with the conductive electrodes; connecting the two or more conductive electrodes to an electrical power source which generates the electric current to be applied to the local area during a given period of time of the treatment period, depending on the type and severity or difficulty of the infection to be treated, producing the hypochlorous acid via the saline solution at said local area. Moreover, a control unit is configured to control the extent of the given period of time in which the electrical current is applied and its value, preferably based on the temperature and pH information.

Preferably, the temperature during the treatment period is kept to not exceed 40-42 °C and the pH is kept between 6.5 and 8.5.

According to an embodiment, if clinically indicated, an antimicrobial treatment is further provided to the patient. The proposed method is capable of creating "in situ" hypochlorous acid, a potent oxidant with antimicrobial capacity in order to kill microorganisms avoiding volatility losses occurring in case the hypochlorous acid being conveyed from a remote point, i.e. not produced at the local area of infection. Thus, the present invention simulates one of the similar effects to that produced by the human innate immune system against microorganisms, as hypochlorous acid (HOC1), as one of the ROS, is released by activated neutrophils by myeloperoxidase-mediated peroxidation of chloride ions, and contributes to the destruction of microorganisms.

The electric current can be applied in a single period of time, i.e. a shot, or in various periods of time, different shots, depending on the severity of the infection to be treated. The infection to be treated can be kept under control by means of common diagnostic techniques of infection (for example imaging or microbiology), or alternatively, by means of acquisition and control of said temperature and pH information.

Preferably, the delivering of the saline solution (0.9% NaCl) is performed under a controlled release over said local area.

According to the proposed method, at least antimicrobial treatment of the infection is not precluded .

The disclosed method/technology can be used, provided as a third aspect of the present invention, as adjuvant therapy of antimicrobial/surgical therapy of difficult to treat infections as abscesses (cerebral, subdural empyema, epidural, pulmonary, pleural, hepatic, splenic, nephritic or perinephric, gynecological, intraperitoneal, muscular, subcutaneous), mediastinitis, acute and chronic osteomyelitis, diabetic foot infections, prosthetic infection (e.g. orthopedic implant-associated Infections, vascular graft infection, tracheal stent infection), lock therapy of long-term catheter-related bloodstream infections, chronic prostatitis, decolonization or disinfection of infected or colonized endotracheal tube or urinary catheters, Cerebrospinal Fluid Shunt and Drain Infections, Cellulitis, and Subcutaneous Tissue Infections. Moreover, may be used as adjunctive treatment of invasive fungal diseases in the immunocompromised host as p.e. invasive aspergillosis or invasive fungal diseases caused by Zygomycetes. Furthermore, the disclosed method can be used as adjunctive therapy of difficult to treat Candida spp. biofilm infections

As per another embodiment, the method can also be used to treat or prevent gram negative rod (GNR) bacterial infections caused by MDR/XDR Pseudomonas aeruginosa, MDR/XDR Klebsiella pneumoniae, MDR/XDR Acinetobacter baumanii, Escherichia coli and others GNR resistant to more than two classes of antimicrobials. Moreover, it would be effective against MDR gram positive cocci bacterial infections caused by methicillin-resistant Staphylococcus aureus. Furthermore, may be used to treat infections due to Candida spp or bacteria or growing in biofilms, especially coagulase-negative staphylococci, Staphylococcus aureus and Pseudomonas aeruginosa. Moreover, the method may be used to treat MDR or extreme resistant Mycobacterium tuberculosis or other atypical mycobacteria difficult to treat.

As per another embodiment, the method is also suitable to be used to treat fungal infections including, among others, Candida spp., Aspergillus spp and Zygomycetes.

Brief Description of the Drawings

The previous and other advantages and features will be more fully understood from the following detailed description of embodiments, with reference to the attached figures, which must be considered in an illustrative and non-limiting manner, in which:

Figs. 1A and IB illustrate two different embodiments of the proposed system. Fig. 2 illustrates, according to an embodiment, the effect of the present invention with different electric current applied in TSB/BFfl or NSS by a) Pa3 b) Kp3 c) Abll d) CA176 e) CP54 and f) Af4751 g) R0868 strains on planktonic growth.

Fig. 3 illustrates the effect of the present invention applied in TSB or NSS at different time-point by Kp3 strain.

Fig. 4 illustrates the effect of the present invention in preventing both microorgani sm adhesion and biofilm formation by P. aeruginosa and S. epidermidis on silicone discs.

Fig. 5 illustrates the effect of the present invention by PAOl strain on silicone discs.

Fig. 6 illustrates the effect of the present invention applied in TSB/BHI or NSS by a) PAOl b) MRSA 15 c) MRSA 16 d) SE 14 e) SE 94 and f) CA176 strains on silicone discs.

Fig. 7 illustrates the effect of the present invention by PAOl, MRSA 15, MRSA 16 and CA176 strains on silicone discs visualized using the LIVE/DEAD® staining viability kit.

Fig. 8 illustrates the effect of the present invention by a) PAOl and b) MRSA 15 strains on titanium discs.

Detailed Description of the Invention Present invention is intended to treat or prevent "in situ' Antimicrobial Resistant Infections or difficult to treat infections or microorganisms growing in biofilms by electric current (in particular electrolysis).

Fig. 1A illustrates a first embodiment of the proposed system. As may be seen in the figure, the system of this first embodiment comprises a device which is configured to be placed to a local area 1 of a patient infected by antimicrobial resistant microorganisms or microorganisms growing in biofilms or at risk to be infected. According to this particular embodiment, the device includes a delivering unit 11 which is configured to deliver an amount of a saline solution to the local area 1. In addition the device also has two conductive electrodes 12, 13, such as platinum electrodes, among others, configured to be in contact with the amount of saline solution to produce hypochlorous acid. The device also includes two sensors 14, 15 configured to provide information about temperature and pH in the local area during the treatment period. All the elements of the device are held on a support structure 16 which is kept on the local area 1 during the treatment.

The system of Fig. 1 further includes an electrical power source 20 configured to generate the electric current via the two conductive electrodes 12, 13 during a given or determined period of time, and a control unit (not shown). In this case, the control unit is integrated in the electrical power source 20, however, in alternative embodiments of the invention, not illustrated, the control unit may be an independent unit connected to the device and/or to the electrical power source. The control unit is configured to control the extent of said period of time in which the electric current is applied and the value of the electric current, preferably, based on the temperature and pH information provided by the sensors 14, 15.

Preferably, said period of time in which the electric current is applied "in situ" is comprised between 1 and 30 minutes, and said electric current is kept between 1 mA and 20 mA (i.e. low-amperage current). Furthermore, the temperature is kept during the treatment to not exceed 40-42 °C, and the pH is kept between 6.5 and 8.5.

The delivering system 11, alternatively to the embodiment of Fig. 1, not illustrated, could be a reservoir or cavity included in the support 16 itself.

Fig. IB illustrates a second embodiment of the proposed system. In this case, contrary to the first embodiment, the device only comprises the two conductive electrodes 12, 13, the two sensors 14, 15 and the support 16, being the delivering unit 21 independent of the device. In this particular embodiment the delivering unit 21 is a syringe. The performance of the electrical power source 20 and of the control unit is in this case the same as in the first embodiment.

In alternative embodiments of the invention, not illustrated, instead of the two sensors 14, 15 the device could include only one sensor configured to provide the temperature and the pH. Besides, the device could only include the two conductive electrodes 12, 13 hold on the support 16, being the sensors 14, 15 (or the single sensor) independents of the device. The support 16 in any of the embodiments of the invention and depending on the local area or the patient to be applied may have different shapes and may be of different materials. Following a detailed explanation of embodiments of the proposed method will be made. i) Parameters

It involved the application of low-amperage electric current to each well containing a culture of antibiotic resistant microorganisms or microorganisms growing in biofilms using the above mentioned electrical power source 20 via the conductive electrodes 12, 13. A constant current level was maintained while voltage fluctuated slightly with changes in the resistive loads across each well. Experiments were performed in a common chamber model. ii) Bacterial strain and growth conditions

For planktonic susceptibility studies, one extensively drug-resistant (XDR) clinical isolate of P. aeruginosa (Pa3; XDR strain harbouring a VIM-2 carbapenemase, only susceptible to colistin and isolate disseminated worldwide (ST235) (Table 1), one XDR clinical isolate of K. pneumonia (Kp3; producing KPC, only susceptible to gentamycin and colistin) (Table 2), one XDR clinical isolate of A. baumannii (Abll; isolate harbouring a NDM-2 and an OXA-51, only susceptible to colistin and tigecycline (ST 102) (Table 3), one C. albicans (CA176), one C. parapsilosis (CP54), one A, fumigatus (Af4751; resistant to voriconazole) and one R. oryzae (R0868) were used.

For biofilm susceptibility studies, one laboratory reference strain of P. aeruginosa (PAOl), and four clinical strains of S. epidermidis (SE94), Methicillin-Resistant S. aureus (MRSA 15 and MRSA 16), and 5 C. albicans (CA176) were studied. All of the strains were well-characterized biofilm-producing.

All strains were stored in skim milk at -80°C in cryovial storage containers. Prior to each experiment, strains were subcultured in Trypticase Soy Agar (TSA) for 24h at 37°C.

Table 1. Susceptibility of P. aeruginosa strain

MIC (mg/L) and clinical category (CLSI breakpi

ID ^a sr Profile β-lactam resistant CST AMK TOB LVX ATM IPM mechanism

Pa3 235 XDR 0.125 32 64 (R) 32 (R) 16 (1) 128

VIM-2

(S) (R) (R)

a ID, strain identification number, ^b CST, colistin; AMK, amikacin; TOB, tobramycin; LVX, levofloxacin; ATM, aztreonam; IPM, imipenem. ⁰ ST, sequence type; S, susceptible; R, resistant; I, intermediate. susceptibility and clinical category (CLSI

Strain Profile β-lactam

FE IM ME

resistant AMP CAZ CIP GEN AK T/S FOS CST

J pr p r M

mechanism

Kp3 XDR KPC R R R R R R S R R R s

Table 2. Susceptibility of Klebsiella pneumoniae strain

aS, susceptible; R, resistant; AMP, ampicillin; CAZ, ceftazidime; FEP, cefepime; IMP, imipenem; MEM, meropenem; CIP, ciprofloxacin; GEN, gentamicin; AK, amikacin; T/S, trimethoprim/sulfamethoxazole; FOS, fosfomycin; CST, colistin.

Table 3. Phenotypic and genotypic features of Acinetobacter baumannii isogenic strain

ST: sequence type; SG: sequence group; CAZ, ceftazidime; FEP, cefepime; IMP, imipenem; MEM, meropenem; CIP, ciprofloxacin; LEV, levofloxacin; GEN, gentamicin; AK, amikacin; T/S, trimethoprim/sulfamethoxazole; TIG: 5 Tigecycline; CO: Colistin. Aminoacidic changes involved in high-level resistance to fluoroquinolones; AGLR, resistance to aminoglycosides by aacA4/aph6A; CHDL, class D carbapenem-hydrolizing β-lactamase. iii) Antimicrobial effect on planktonic growth

Testing different values of electric current and applying different electric currents (shots): The 10 efficacy of the proposed method on planktonic was evaluated in Pa3, Kp3, Abll, Cal 76, Cp54, Af4751 and R0868 strains. Electric current was applied in a 12-well plate containing 4 mL of both conditions; in Tryptic Soy Broth (TSB) for bacteria/Brain Heart Infusion (BFfl) for Candida or in NSS (0.9% NaCl). In this case, the proposed method involved the application of electric current of 2 and 20 mA values during 5 min with 1, 2 or 3 repetitions in periods of 30 min between each electric current. Then waited 30 min and 15 after 24h at 37°C and viable count was then determined.

Testing one value of electric current in different time points: The efficacy of the proposed method on planktonic was evaluated in Kp3 strain. Electric current was applied in 12-well plate containing 4 mL of both conditions; in TSB BHI or in NSS. The proposed method involved the application of 2 mA electric current in different time-points: 20, 40 seconds and 1, 2, 3 and 5 minutes. Then waited 30 min at 37°C and 20 viable count was then determined. iv) Biofilm growth on discs

For the biofilm growth on silicone discs the protocol described by Chandra et al. "In vitro growth and analysis of Candida biofilms" was followed with slight modifications. The microorganisms were grown overnight in TSB at 37°C in case of bacterias or Yeast Nitrogen Base medium with 50 mM dextrose

25 (YNBD) for yeasts. The cultures were centrifuged, washed twice with sterile Phosphate Buffer Solution (PBS) and then re-suspended to a final concentration of 0.5 McFarland for adjusted to the final concentrations (l .OxlO ⁷ cfu/mL for PAOl, MRSA or CA176 strains and 5.0xl0 ⁵ cfu/mL for SE14 and SE94 strains). Then, 4 mL of the culture was placed into each well of the 12-well plates and the silicone/titanium discs were added. The discs were incubated for 90 min at 37°C without shaking and were

30 then transferred to a new plate, containing 4 mL of fresh TSB or Brain Heart Infusion (BHI) (Candidas).

The plates were incubated at 37°C for 24h while being shaken at 60rpm. Afterwards, the biofilms were scraped from the individual wells into a new 12-well plate containing TSB or BHI. The viable count of the dislodged bacterial cells was then determined v) Prevention studies v.i) Antimicrobial effect to prevent microorganism adhesion on silicone discs The efficacy of present invention to prevent microorganism adhesion was evaluated in PAOl and SE94 strains. Electric current was applied (20mA during 15 min) in the 12-well plate containing 4mL of NSS with silicone discs. Then, waited 30 min at 37°C before the addition of the inoculum in each well. After that, next steps were done as was explained in the biofilm growth section (see iv paragraph). Briefly, adhesion step (90 min at 37°C of incubation), growth step (discs were transfer a new 12-well plate with 4 mL fresh TSB and plates were incubated 24h at 37°C while being shaken at 60 rpm) and finally biofilms were scraped from the individual wells into a new 12-well plate containing TSB. The viable count of the dislodged bacterial cells was then determined. v. ii) Antimicrobial effect to prevent biofilm formation on silicone discs The efficacy of present invention to prevent biofilm formation was evaluated in PAOl and SE94 strains. Electric current was applied (20mA during 15 min) after adhesion step and before growth step. The microorganisms were grown overnight in TSB at 37°C. The cultures were centrifuged, washed twice with sterile Phosphate Buffer Solution (PBS) and then re-suspended to a final concentration of 0.5 McFarland for adjusted to the final concentrations (l .OxlO ⁷ cfu/mL for PAOl and MRSA strains and 5.0x10 ⁵ cfu/mL for S. epidermidis strain). Then, 4 mL of the culture was placed into each well of the 12-well plates and the silicone discs were added. The discs were incubated for 90 min at 37°C without shaking and were then transferred to a new plate, containing 4 mL NSS. Electric current was applied at 20 mA during 15 min. Then waited 30 min at 37°C and 0.3 mL of TSB was added to each well to behalf growth step (incubated at 37°C for 24h while being shaken at 60rpm). Afterwards, the biofilms were scraped from the individual wells into a new 12-well plate containing TSB. The viable count of the dislodged bacterial cells was then determined. All experiments were made in triplicate vi) Treatment studies vi. i) Antimicrobial effect to performed biofilm on silicone discs

The efficacy of Present invention to performed biofilm on silicone discs was evaluated in PAOl , MRSA and CA176 strains. Biofilm growth was performed as was explained in the biofilm growth section (see iii paragraph). After biofilm formation, present invention was applied in the following ways:

Testing the same value of electric current with different applications of electric current: it was performed in PAOl strain; 10 mA during 5 min with 1, 2 or 3 electric current applications in periods of 30 minutes between each electric current. Each electric current was applied with fresh NSS in the well. 30 min after last shot the biofilms were scraped from the individual wells into a new 12-well plate containing TSB. The viable count of the dislodged bacterial cells was then determined.

Testing different values of electric current and applying only one electric current: it was performed in PAOl, MRSA 15, MRSA 16, SE 14, SE 94 and CA176 strains; 2 mA during 15 min and 20 mA during 15 min with 1 electric current application. Electric current was applied in two conditions; in TSB/BHI or in NSS. 30 min after, the biofilms were scraped from the individual wells into a new 12-well plate containing TSB/BHI. The viable count of the dislodged bacterial cells was then determined.

Moreover, the effect of present invention was visualized using fluorescence microscope in these different strains: PAOl, MRSA 15, MRSA 16 and CA176. Briefly, discs were placed in a new 12-well plate with 4 mL of medium. Biofilm was scrapped, centrifuged at 10.000G, 5 min at 4°C and the pellet was stained with the mixture of SYTO 9 (3.34 mM solution in DMSO) with propidium iodide (20 mM solution in DMSO), incubated at room temperature in the dark for 15 min. Fluorescent intensities at 535 nm (green emission) and 635 nm (red emission) were measured using a microscope coupled with a camera. All experiments were made in triplicate. vi.ii) Antimicrobial effect to performed biofilm on titanium discs

The efficacy of present invention to performed biofilm on titanium discs was evaluated in PAOl and MRSA 15 strains. Biofilm growth was performed as was explained in the biofilm growth section (see iii paragraph). After biofilm formation, the method was applied in the following way: Testing the same values of electric current with different electric currents applications: it was performed in PAOl and MRSA 15 strains; 10 mA during 5 min with 1, 2 or 3 electric current applications in periods of 30 minutes between each electric current. Each electric current was applied with fresh NSS in the well. 30 min after last shot application of electric current the biofilms were scraped from the individual wells into a new 12-well plate containing TSB. The viable count of the dislodged bacterial cells was then determined. All experiments were made in triplicate. vii) Quantification of HCIO at different intensities of electric current and times of exposition

The aim of the experiment was to evaluate the ability to generate hypochlorous acid with electrolysis of the new system. In this particular case, the two electrodes were used without separating the anodic and cathodic compartments. Then, using the electrodes, has quantified the concentration of electrochemically generated hypochlorous acid in NSS 0.9% NaCl. In the electrolysis experiments 4 ml NSS at 37 ° C was used. Even then, the electrodes introduced and applied the current required during the time required for electrolysis. Quantification of hypochlorous acid has been performed using the spectrophotometric method ASTM 4500-CI. All measurements for each value of current / time applied were performed in triplicate and also, it has been calculated the coefficient of variation.

Results iii) Antimicrobial effect on planktonic growth

Testing different values of electric current and applying different electric currents: Shots were applied at different amperages values (2 and 20 mA) during 5 min in the first electric current in NSS, which significantly (P<0.001) reduced the number of cells with 2 and 20 mA 5 min (logio cfu/mL reduction = 5.3 on Abll 5.8 on CA176, 5.8 on CP54, 5.8 on Af4751 and 5.5 on R0868 compared to the control NSS. Electric current of 2mA 5 min applied in Pa3 and Kp3 need two shots (2 applications of electric current) to achieved significantly lower (P>0.001) compared to Control NSS. Group of 20mA 5 min achieved negative cultures since the first EC (logio cfu/mL reduction = 5.7 on Pa3 and 5.4 on Kp3. Present invention applied in medium wells (TSB or BHI), has had no effect on the reduction of cells in neither strain (in exception of Abll in the 3EC P<0.01) (Fig. 2). Testing one value of electric current in different time points: Shots applied with 2mA at different time-points (20, 40 sec, and 1, 2, 3 and 5 min) showed that with 1 min negative cultures at 3 ^rd EC (P<0.01) were allowed compared to the control NSS. 2 and 3 min are enough to negative cultures at 2 ^nd EC, and with 5 min, in the 1 ^st EC negative cultures were obtained (P<0.01). Present invention applied in TSB wells, has had no effect on the reduction of cells. (Fig. 3) Prevention studies

Antimicrobial effect of present invention to prevent both microorganism adhesion and biofilm formation on silicone discs:

The Present invention applied in different steps of biofilm growth (prior to both microorganism attachment and biofilm growth), significantly (P<0.001) reduced the number of cells (PAOl and SE94; logio cfu/mL reduction = 8.5) of the biofilm of P. aeruginosa PAOl and S. epidermidis SE94 on silicone discs compared to the control discs (Fig. 4).

Treatment studies vi.i) Antimicrobial effect to performed biofilm on silicone discs

Testing the same value of electric current with different electric currents applications (shots): The proposed method applied at 10 mA during 5 min in different electric current applications, significantly (P<0.001) reduced the number of cells (log ₁₀ cfu/mL reduction = 6.8 after 2 or 3 electric currents) of the biofilm of P. aeruginosa PAOl on silicone discs compared to the growth control (Fig. 5). Testing different values of electric current and applying only one electric current (only one shot): The proposed method applied at different amperages values (2 and 20 mA) during 15 min in one electric current in NSS, significantly (PO.001) reduced the number of cells with 20 mA 15 min (logio cfu/mL reduction = 7.1 on the biofilm of PAOl, 6.6 on MRSA 15, 6.6 on MRSA 16, 7 on SE 14, 7 SE 94) on silicone discs compared to the control NSS. Present invention applied in CA 176 achieved significantly lower (P>0.001) negative cultures in both groups (2 and 20 mA) compared to Control NSS. Present invention applied in medium wells (TSB or BHI), has had no effect on the reduction of cells in neither strain (Fig. 6 and Fig. 7). vi.ii) Antimicrobial effect to performed biofilm on titanium discs

Testing the same value of electric current with different electric currents applications: The proposed method applied at 10mA during 5 min in different electric currents applications, significantly (PO.001) reduced the number of cells (PAOl and MRSA 15; logio cfu/mL reduction = 6.6 and 8.1 respectively after 3 electric currents) of the biofilm of PAOl and MRSA 15 on titanium discs compared to the growth control (Fig. 8). HCIO production by means of electrolysis

Planktonic growth:

Table 4. HCIO concentration as a function of applied current and time. Biofilm growth:

Table 5. HCIO concentration as a function of applied current and time.

While the foregoing written description of the invention enables one of ordinary skill to make and use what is considered presently to be the best mode thereof, those of ordinary skill will understand and appreciate the existence of variations, combinations, and equivalents of the specific embodiment, method, and examples herein.

The scope of the present invention is defined in the following set of claims.