ES1274434U - BIOFILM FORMATION PREVENTION SYSTEM FOR RESPIRATORY MEDICAL DEVICES. (Machine-translation by Google Translate, not legally binding) - Google Patents

Abstract

Biofilm formation prevention system for respiratory medical devices, comprising the system a thermal radiator (2) adapted to engage in fluid communication with a respiratory medical device, to raise the temperature of the air flow entering the respiratory medical device, wherein the thermal radiator (2) is an elongated metallic body having first and second ends (2a, 2b), and a plurality of channels (4', 4) extending longitudinally along the interior of the body and establish a fluid communication between the first and second ends (2a, 2b) for the passage of ventilation air from one end to the other end, the system further comprising at least one heating wire (21) wound around the thermal radiator (2) to heat the air flowing through the channels (4). (Machine-translation by Google Translate, not legally binding)

Description

DESCRIPTION

BIOFILM FORMATION PREVENTION SYSTEM FOR DEVICES

RESPIRATORY DOCTORS

Field and object of the invention

The present invention relates generally to biofilm prevention techniques.

More specifically, an object of the invention is to provide a device that prevents biofilm formation in respiratory medical devices, such as endotracheal tubes or respiratory face masks.

A further object of the invention is to provide a biofilm prevention system that is simple to use and has no side effects.

Background of the invention

Access to effective antibiotics is essential in all healthcare systems. Their use has reduced infant 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 in itself, and despite its dramatic increase, it is not receiving the same attention as acute infectious threats such as severe acute respiratory syndrome (SARS), the pandemic flu or Ebola, or the three main infectious diseases: acquired immunodeficiency syndrome (AIDS), tuberculosis and malaria. AMR is a serious global threat that affects the global economy and public and collective health. The most recent Global Risks reports from the World Economic Forum have cited AMR as one of the greatest risks to society that threatens human health.

AMR is not only expensive in terms of human suffering, but also in economic terms. AMR currently claims at least 50,000 lives each year in Europe and the United States alone and approximately 700,000 lives worldwide, at an estimated cost of more than € 1.5 billion or $ 35 billion annually. .

Among the most important AMR bacteria in causing infections in hospitalized patients are the so-called "ESKAPE" pathogens. These microorganisms are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. The most prominent threat from AMR is the rapid increase in resistance among “ESKAPE” bacteria that have caused hospital-acquired infections in recent years. In addition to the ESKAPE pathogens, Escherichia coli with AMR remains the leading cause of death from severe sepsis in hospitalized patients with limitations. In countries with high levels of multidrug resistance (MDR), including carbapenemase resistance, only a few therapeutic options are available against infections caused by carbapenem-resistant P. aeruginosa (incidence of MDR / extremely drug-resistant P. aeruginosa (XDR) 25-50%), among which are polymyxins. In these countries and in the case of P. aeruginosa with MDR / XDR, the presence of resistance to polymyxins or aminoglycosides is an important warning because the options for the treatment of infected patients are even more limited. So few antibiotics are effective enough for therapy. Antibiotics that still work, which often have side effects, are less effective, or are very expensive, such as tigecycline.

Furthermore, some ESKAPE pathogens can grow as a community of microorganisms surrounded by a self-produced extracellular polymeric matrix attached to a surface. This community is known as biofilm and has the ability to develop resistance mechanisms against antimicrobial agents and cells of the immune system. According to a statement made by the National Institutes of Health of the United States, microorganisms that produce biofilm are crucial from a medical point of view because they cause 80% of infections in our body. Among nosocomial infections, which represent a major health care problem due to their associated high mortality, infections related to medical devices account for almost a quarter of nosocomial infections. Ventilator-associated pneumonia (VAP) is considered the most serious form of nosocomial pneumonia and contributes to an increased risk to the patient. The prevalence of VAP varies between 9-65% and has a mortality rate of 15-76%. Endotracheal tubes (ETTs) have been reported to act as a reservoir for microbial pathogens, being a perfect environment for the colonization and adhesion of microorganisms at the distal end of the endotracheal tube, forming the biofilm. This biofilm can develop rapidly after intubation, forming detectable antibiotic-tolerant structures within 24 h, or they can even break off and re-enter the lung, causing critical infections. Although some preventive measures for biofilm formation have been reported in TTE, they have not been implemented as they do not demonstrate significant evidence of VAP reduction. Therefore, it is necessary to develop new preventive or treatment strategies in order to reduce the high risk of TTE-related infections in critically ill patients.

In addition to increased resistance to existing agents, there are no new antibiotics in development. For many years, the pharmaceutical industry has been successfully mass producing new antibacterial drugs. However, it is becoming increasingly difficult to find novel antibiotics and many large pharmaceutical companies have abandoned antibiotic development programs because the procedure is extremely expensive and often with useless drugs. Furthermore, existing antibiotics are losing their potency due to the expansion of resistance at an alarming rate, while very few antibiotics are being developed. Therefore, we are faced with a paradoxical situation, like a perfect storm, with increased levels of resistant bacteria coupled with a downward trend in the development of antibiotics.

The spread of bacteria with AMR could dramatically roll back modern medicine to the dark years of the pre-antibiotic era; achievements in modern medicine, such as decreased safety of deliveries, cesarean sections, treatment of premature babies and major or even minor dirty surgery, treatment of pneumonia, sexually transmitted diseases, transplantation of organs and cancer chemotherapy, which are now taken for granted without access to effective treatment for bacterial infections.

The urgent need to fund relevant research to develop new antibiotics and alternatives to treat AMR and biofilm-producing strains is now recognized. Therefore, it is essential to increase financial incentives to develop urgently needed antibiotic or antimicrobial strategies, as well as different policies at different levels, both in the human and animal health sector to preserve the efficacy of antibiotics.

Heat has been used as an antibacterial strategy for hundreds of years, such as the sterilization of liquid foods, the pasteurization of milk, or even the sterilization of medical equipment surfaces by autoclaving. Some authors have reported the heat inactivation of bacteria, however, most of them have focused on planktonic bacteria. In addition, there is some evidence for heat antibiofilm activity. A possible explanation for this result could be that heat shock can destroy the bacterial membrane structure by altering hydrogen bonds and respiratory enzymes.

Given this, there is a critical situation, since the extraction of TTEs from patients and reintubation contributes to a risk factor for suffering from pneumonia.

Therefore, there is a demand to inhibit biofilm formation within ETTs in a simple way, without side effects and without depending on antibiotics.

Description of the invention

The present invention is defined in the attached independent claim and successfully solves the demands of the prior art by providing a technique that prevents biofilm formation by homogeneously heating the air flowing through a respiratory device and at a suitable temperature.

The system incorporates a heating device that can be coupled upstream of a respiratory medical device, and that can also be coupled with air ventilation equipment, in order to homogeneously raise the temperature of an air flow entering a device respiratory system, to effectively prevent biofilm formation within the respiratory device.

Preferably, the temperature of the air flowing through a breathing device is kept within the range of 37 ° C to 50 ° C, and preferably around 42 ° C.

In a preferred embodiment of the invention, the system is specially adapted to be coupled upstream of an endotracheal tube, and to forced ventilation medical equipment.

Therefore, one aspect of the invention relates to a biofilm formation prevention system comprising a thermal radiator adapted to be coupled in fluid communication with a respiratory medical device, to raise the temperature of the air flow entering the respiratory medical device. The thermal radiator is an elongated metallic body that has first and second ends, which incorporates a plurality of channels that extend longitudinally along the interior of the body and that establish a fluid communication between the first and second ends for the passage of air from ventilation through it.

In a preferred embodiment, the system is adapted to be used in conjunction with endotracheal tubes, such that one end of the thermal radiator is adapted to mate with an endotracheal tube of conventional dimensions and a second end is adapted to mate with a source of ventilating air. .

Preferably, one end of the radiator is configured as a male connecting tube and another end is configured as a female connecting tube.

The system further comprises at least one electrical resistance implemented as a heating wire wound around the thermal radiator to heat the same and to heat the air flowing through the channels. The heating wire is wound on the external surface of the thermal radiator, so that the heating wire extends around the channels. Preferably, the entire length of the channels is covered by the heating wire.

Preferably, the heating wire includes first and second filaments, wherein the first and second filaments are made from different metals or metal alloys. In a preferred implementation, the first filament is a nickel-chromium alloy and the second filament is a nickel-aluminum alloy. These two resistor-forming filaments are connected in parallel and significantly reduce manufacturing cost compared to using a single filament of rated electrical power.

Preferably, the system includes a thermal insulation cover, such that the heat sink and wire are enclosed within the insulation cover.

In a preferred embodiment, the radiator has a central channel arranged axially, and a set of channels arranged circumferentially around the central channel and also extending longitudinally within the radiator, such that the channels arranged circumferentially are parallel to the central channel. This arrangement of the Channels inside the thermal radiator allow the homogenization and uniformity of the air temperature at the radiator outlet. The electrical resistance has enough power to compensate for any loss of temperature when the air enters the thermal radiator. Preferably, the radiator has a cylindrical configuration.

The system further comprises at least one temperature sensor, thermally coupled with the thermal radiator to measure its temperature. Furthermore, the system comprises a temperature controller operatively communicated with the temperature sensor and with the heating wire, and adapted to maintain the temperature of the radiator within the temperature range 80-90 ° C.

Furthermore, the temperature controller is adapted to maintain a temperature within an endotracheal tube connected to the radiator within the range of 37 ° C to 50 ° C, and preferably about 42 ° C, in order to prevent biofilm formation. inside the tube.

The temperature controller includes a timing device, adapted to activate the heating wire for predefined periods of time at predefined time intervals. Preferably, the time period is within the range of 10 to 20 minutes, and preferably about 15 minutes, and the time range is within the range of 20 to 40, and preferably 30 minutes.

Brief description of the drawings

The preferred embodiments of the invention are described below with reference to the accompanying drawings, in which:

Figure 1.- shows a perspective view of the thermal radiator and the thermal insulation cover.

Figure 2.- shows in figure A a longitudinal section view of the thermal radiator, in figure 2B a longitudinal section view of the sleeve and in figure C a cross-sectional view of the thermal radiator.

Figure 3.- shows a longitudinal section view of the thermal radiator and the insulation cover coupled to each other.

Figure 4.- shows in a perspective view the heating system in use, connected to ventilation equipment and an endotracheal tube.

Figure 5.- shows an electrical diagram of the temperature controller.

Figure 6.- shows in graphs A and B the efficacy of the heating system of the invention against a P. aeruginosa strain (Pa1016) and a K. pneumoniae strain (Kp16) that grow in silicone discs, determined by culture quantitative.

Figure 7.- shows in graphs A and B the effectiveness of the heating system of the invention against a Pa1016 strain that grows on silicone discs by applying 1, 2 or 3 shocks of 45 ° C for 15 min.

Figure 8.- shows in graphs A to H the efficiency of the heating system of the invention against different strains that grow in PVC discs by applying 1, 2 or 3 discharges of 42 ° C for 15 min.

Figure 9.- shows in graphs A to C the efficacy of the heating system of the invention against two strains of P. aeruginosa (Pa1016 and Pa46) and a strain of K. pneumoniae (Kp16) that grow on silicone discs, determined by quantitative culture.

Preferred embodiment of the invention

Figure 1 shows a perspective view of the biofilm formation prevention system (1) for respiratory medical devices according to a preferred implementation of the invention, in which the system comprises a thermal radiator (2) and a cover (3) of thermal isolation. The thermal radiator (2) is an elongated metallic body that has first and second ends (2a, 2b) and a plurality of channels (4), which extend longitudinally along the thermal radiator and establish fluid communication between the ends. (2a, 2b) first and second for the passage and heating of the ventilation air through them.

In this preferred example, there are nine channels (4) each with a diameter of 3mm, so the cross-sectional area of the holes is 63.617mm2, which matches with the area of a conventional endotracheal tube, so the thermal radiator ensures homogeneous air flow through an endotracheal tube.

The thermal radiator (2) can be manufactured as a unitary body composed, for example, of stainless steel or another suitable metal or metal alloy.

The thermal radiator (2) of this embodiment is specially configured to be coupled to an endotracheal tube (24) and to forced ventilation equipment (22). For this, a first end (2a) of the radiator has a male configuration and a second end (2b) has a female configuration.

The thermal radiator (2) could also be attached to other respiratory medical devices, such as a breathing mask.

The system also includes an electrical resistance consisting of a heating wire (21) that is wound around the thermal radiator (2) to heat it when an electrical current flows through it. The resistor is isolated by a Kapton layer.

The entire extension of the channels (4) is surrounded by the heating wire (21), so that the heating wire (21) provides a uniform and appropriate heat transmission to the radiator (2), which in turn can raise the temperature of the air flow entering the respiratory medical device.

central. Preferiblemente, los canales (4) son paralelos al eje (x). Con esta distribución de canales, una parte principal del flujo de aire circula cerca de la superficie (5) externa en la que está enrollado el hilo (21) de calentamiento, es decir, más cerca de la fuente de calor.As shown in figure 2B, the thermal radiator (2) in this example is a cylindrical body, and a central channel (4 ') is arranged along the axis (x) of the thermal radiator, while the other channels (4) are circumferentially distributed around the canal

central. Preferably, the channels (4) are parallel to the axis (x). With this channel arrangement, a main part of the air flow circulates close to the external surface (5) on which the heating wire (21) is wound, that is, closer to the heat source.

As shown in figure 2A, the inlet and outlet (9a, 9b) of the channels (4 ', 4) have a concave shape, preferably a cone shape, which serves to allow air to enter and exit the radiator ( 2) in a more homogeneous way.

The thermal insulation cover (3) is a cylindrical body, dimensioned so that the thermal radiator (2) with the heating wire (21) can be housed inside the cover (3) as shown in figure 3. From In this way, the radiator and the wire are thermally insulated from the outside, and the temperature is kept continuous inside the cover, and at the same time, accidental burns to users are avoided. The insulation cover (3) can be obtained, for example, from polyoxymethylene (POM).

Furthermore, the thermal insulation cover (3) has several annular grooves (6), which allow better insulation and ventilation of the radiator.

Respectively at each end (2a, 2b), the thermal radiator (2) has annular extensions (8a, 8b), which serve as contact areas between the radiator (2) and an internal surface of the cover (3) and to prevent the contact between the wire (21) and the cover (3).

The heating wire (21) is formed by two filaments connected in parallel; one of them being composed of nickel-chromium (preferably an 80/20 nickel-chromium alloy) and the other one of nickel-aluminum. The heating wire (21) has a resistance of 10 Q, so by applying 12 V, it produces a power of 14.4 W.

The heating system preferably operates on alternating current, but could also operate on direct current. Each filament is covered with a Teflon sleeve, and both are covered with a 0.8mm Teflon tube to provide insulation.

The system incorporates a temperature sensor (10) thermally coupled with the thermal radiator (2) to measure its temperature while it is heating up. The temperature sensor (10) is thermally coupled with an outer surface of the thermal radiator (2) and has high precision (class A) with 0.3 ° C tolerance, and allows to block the heating system in case of damage or crossover accidental. Preferably, the temperature sensor (10) is implemented as a Pt100 sensor.

The system also comprises a temperature controller (11), so that the thermal radiator (2) can be connected to the controller (11) by means of a cable (7) that includes four wires, specifically: two wires to connect the wire (21) heating and two wires to connect the temperature sensor (10) with the controller (11). For this end, The cable (7) has a four-pole connector (20) that can be plugged into a complementary connector provided on the controller (11).

In this way, the temperature controller (11) can operatively communicate with the temperature sensor (10) and with the heating wire (21), which is adapted to maintain the temperature of the radiator (2) within a temperature range. wanted. Preferably, the temperature controller (11) is further adapted to maintain the temperature within the endotracheal tube within the range of 37-50 ° C, and more preferably around 42 ° C, in order to prevent biofilm formation within the tube. tube.

Figure 4 shows in a perspective view the heating system (1) in use, in which the thermal radiator (2) is shown connected to a ventilation equipment (22) and also connected to an endotracheal tube (24). In particular, the first end (2a) of the radiator (2) with male configuration is connected to the ventilation equipment (22) by means of flexible breathing tubes (23) and the second end (2b) with female configuration is connected to a tube (24) endotracheal. As illustrated in the figure, the radiator (2) can be easily attached by simply inserting the ends (2a, 2b) of the radiator respectively into the endotracheal tube and the ventilation equipment.

Figure 5 shows the electrical diagram of the temperature controller (11), which comprises: a digital temperature regulator (12) with a proportional-integral-derivative (PID) control system, which allows stopping the thermal inertia when necessary. Furthermore, this control is improved by a FUZZI procedure based on an artificial intelligence program. It has a version to activate the heating and another to deactivate it when the temperature is exceeded. If a second control system were required, it could be incorporated as a second lock into the system.

The controller (11) further includes a cyclical timer (13) that communicates with the temperature regulator (12). The cyclical timer (13) allows the activation of the system during a determined period of time, and stops the operation automatically when the temperature rises above a predefined value.

The controller (11) also includes a solid state relay (14) operated by the cyclic timer (13) and a transformer (15) connected to the output of the relay (14), so that the AC input (16) is applies to the primary side of the transformer. The thread (21) The heating element is connected to the output of the transformer (15) by means of the cable (7), which in this case provides an output of 12 volts.

The digital temperature regulator (12) is connected with the temperature sensor (10) connected to the radiator (2), in order to regulate the temperature based on the temperature of the radiator (2).

The temperature controller (11) is fed from the low voltage AC source (16) through a fuse (17) and a selector (18) with three positions (central, disabled; position D, direct action and position C, cyclic interval). The controller also includes a safety relay (19) operated by the digital temperature regulator (12) and connected to interrupt the solid state relay (14), to stop heating in case of reaching a temperature higher than that determined by the thermostat.

1. In vitro efficacy studies

1.1 Strains

For biofilm formation, Gram negative XDR isolates were studied. Three Acinetobacterbaumannii strains were tested (AbI1; isolate harboring NDM-2 and OXA-51, only sensitive to colistin and tigecycline (sequence typing (ST) -103), AbI4; isolate harboring OXA-51, sensitive only to colistin, amikacin and tigecycline (ST-2) and Ab60; isolate that harbors OXA-51 and AmpC of hyperproduction, resistant to gentamicin (ST-38)), two strains of Klebsiella pneumoniae (Kp6; that harbors a beta-lactamase of extended spectrum (ESBL) only sensitive to colistin, fosfomycin and imipenem and Kp16; hyperproduction of cephalosporinase, only sensitive to colistin, clavulanic acid and gentamicin) and three strains of Pseudomonas aeruginosa (Pa3; which harbors a VIM-2 carbapenemase, only sensitive to colistin and a worldwide disseminated isolate (ST-235), Pa46; harboring a VIM-2 carbapenemase, only sensitive to colistin and amikacin (ST-111) and Pa1016; harboring hyperproducing AmpC, OprD inactivation (Q142X ), only sensitive to colistin and amikacin and an isolate dis issued in Spanish hospitals (ST-175)). A methicillin- resistant Staphylococcus aureus (MRSA15) strain was also studied. All of them were isolated from patients at the Vall d'Hebron University Hospital (VHUH).

All strains were stored in skim milk at -80 ° C in cryovial storage containers. Before each experiment, the strains were plated on trypticase soy agar (TSA, BioMérieux® SA, Marcy l'Etoile, France) and incubated at 37 ° C for 24 h.

1.2 Substrates used in biofilm formation

Two types of substrates were used for biofilm formation: silicone discs (15 mm diameter and 0.5 mm thick, Merefsa, Barcelona, Spain) and polyvinyl chloride discs (PVC; 15 mm diameter and 0.5 mm thick; Servicio Estación SA, Barcelona, Spain). In order to improve biofilm formation, K. pneumoniae strains were grown in thicker PVC discs (15 mm diameter and 1 mm thick; Servicio Estación SA, Barcelona, Spain).

1.3 Formation of biofilm on silicone or PVC discs

For the formation of biofilm on the surface of the discs, the protocol described by Chandra et al.28 was followed, with some modifications. First, the biofilm producing strains were grown overnight in trypticase soy broth (TSB; Becton Dickinson and Company, Le Pont de Claix, France) at 37 ° C and 60 rpm. After centrifuging and washing the cell suspension three times with sterile phosphate buffered saline, pH 7.2 (PBS; Merck, Germany), an inoculum of 1107 colony forming units (CFU) / ml was prepared with PBS, pH 7 ,2. Second, 4 ml of the inoculum and silicone or PVC discs were added to each well of a 12-well plate (Sarstedt AG & Co, Nümbrecht, Germany). The plates were incubated for 30 or 90 min at 37 ° C (adhesion step) and then placed in a new plate containing 4 ml of fresh TSB. The plate was then incubated for 24 h at 37 ° C with shaking at 60 rpm (growth stage).

1.4 Application of the treatment of the system of the invention

1.4.1 Treatment after accession

After the adhesion step of the biofilm formation on the surface of the discs, the discs were placed in a new 12-well plate. Three of them were incubated at 37 ° C (control group), while the other three were placed on a heating plate (Sanara, Barcelona, Spain). 1, 2 and 3 shocks of 42 ° C, 45 ° C or 50 ° C were applied for 15 min. Between shocks, the discs were incubated at 37 ° C for 30 min. A thermometer (Omega Instruments, Manchester, UK) and a probe (Sanara, Barcelona, Spain) were used to check the temperature on a control disk throughout the experiment.

After each shock, the discs were placed in a new plate containing 4 ml of TSB to follow the growth stage. The plate was incubated at 37 ° C for 24 h with shaking at 60 rpm. Then, the effectiveness of the system of the invention was evaluated.

1.4.2 Treatment after growth

The protocol used was similar to that of the treatment after adherence, with some modifications.

After the growth stage of biofilm formation on the surface of the discs, the discs were placed on a new plate. Three of them were incubated at 37 ° C (control group), while the other three were placed on a heating plate (Sanara, Barcelona, Spain). 1,2, 3, 4 and 5 shocks of 50 ° C were applied for 15 min. Between shocks, the discs were incubated at 37 ° C for 30 min. A thermometer (Omega Instruments, Manchester, UK) and a probe (Sanara, Barcelona, Spain) were used to check the temperature on a control disk throughout the experiment.

1.5 Evaluation of the effectiveness of the system of the invention

1.5.1 Quantitative culture

Disks were placed in a new 12-well plate containing 1 ml of TSB in each well. The biofilm was scraped (cell scraper, Sarstedt. AG & Co, Nümbrecht, Germany), seeded in TSA and incubated at 37 ° C for 24 h. The cells were then quantified and expressed as Log ¹⁰ CFU / ml.

1.5.2 Confocal laser scanning microscopy

The efficiency of the system of the invention was visualized using confocal laser scanning microscopy (CLSM) with an Olympus FV1000 device with excitation wavelengths of 488 and 568 nm and a magnification of 60x. Biofilms were stained using a LIVE / DEAD® BacLight ™ viability kit (Molecular Probes, Invitrogen, Leiden, The Netherlands) following the manufacturer's instructions, which consisted of staining with a mixture of SYTO 9 (3.34 mM solution in DMSO ) and propidium iodide (20 mM solution in DMSO) and incubate at room temperature in the dark for 30 min. Three zones of the biofilm of each silicone disc were explored with a step size of 2 ^ .m. Simultaneous dual channel imaging was used to visualize green (live cells) and red (dead cells) fluorescence. IMARIS 8 software (Bitplane, Belfast, UK) was used to create a projection view of the formed biofilms and ImageJ 1.45s software package was used to calculate the value of live pixels (green).

The results were expressed as the percentage of cell viability of the discharges compared to the control group.

2. In vivo safety studies

2.1 Endotracheal intubation model

2.1.1 Animals

For the study, male New Zealand rabbits weighing between 2 kg-2.3 kg (Granja Cunícola San Bernardo, Navarra, Spain) were individually housed in regulation cages, they were provided with water and food ad libitum throughout the experiments. and housed in a reverse 12hr / 12hr light / dark cycle.

2.1.2 Ethics

This study was conducted in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals and the guidelines of Animal Research: Reporting of In Vivo Experiments (Animal Research: Reports in experiments vivo) (ARRIVE). All the experimental procedures were carried out according to the Catalan, Spanish and European laws and regulations on the protection of animals used for experimental and other scientific purposes.

The experimental protocol was approved by the Institution's Experimental Animal Ethics Committee (registration number 69/16 CEEA).

2.1.3 Anesthesia

First, the animals were anesthetized by an intramuscular (i.m.) injection of ketamine 100 mg / kg (Pfizer, Madrid, Spain) plus xylazine 20 mg / kg (Laboratorios Calier S.A., Barcelona, Spain). When the animal did not have a corneal reflex, a line was placed in the peripheral vein of the ear for anesthetic maintenance with a continuous infusion of fentanyl 1 ^ .g / kg / h and midazolam 0.1 mg / kg / h at a rate of infusion of 1 ml / h. If the animal was observed to be awake during the experiment, a bolus injection of 0.5 ml vecuronium 0.1 mg / kg / h was also given. He was also maintained with Ringer's lactate fluid therapy (B. Braun, Barcelona, Spain) at a rate of 1-2 ml / kg / h.

2.1.3.1 Endotracheal intubation

For endotracheal intubation, the protocol described by Thompson et al.29 was followed, with some modifications.

When the rabbit reached the anesthetic plane, it was placed on the preparation table with its head slightly extended over the edge of the table and in straight alignment with the spine. After checking for complete relaxation of the jaw by lifting the head, gauze was used to pull the rabbit's tongue up to the lower right incisors, taking care to avoid trauma to the incisors. Lidocaine was nebulized 2% (Inibsa, Barcelona, Spain) in the larynx of the rabbit to locally anesthetize the vocal cords and to avoid the risk of laryngospasm. Then, while the rabbit's head was extended back and the neck of the rabbit forward to maintain an open airway and view the larynx, the endotracheal tube (3.5 mm ID, Covidien, Mansfield, USA) was slowly inserted. ) from the right side of the rabbit until some resistance was noted (confirmation that the larynx had been reached). To prevent oxygen desaturation, it was important to keep the neck extended as described to maintain an open airway during intubation. The successful introduction of the tube was done by observing the fogging of a glass / mirror at the end of the tube and listening to the air flow. Consequently, the tube was gradually introduced into the desired position. To secure the endotracheal tube, the rabbit was placed on its side and an umbilical tape was used around the tube.

2.1.4 Ventilation parameters

The animals were ventilated using a mechanical respirator and a humidifier (Serve Ventilator 300, Siemens, Germany). The mechanical ventilator was used in neonatal mode and with pressure control, with a peak airway pressure of 15 cmH ² O and a PEEP of 5, a respiration rate of 44 breaths / min, an inspiration period of 0 , 35 s, an inspiration tidal volume of 40 ml and an O ² concentration of 50%. Inspiratory gases were conditioned through the heated humidifier.

2.1.5 Application of the treatment of the system of the invention

After intubation of the animal, an electrical resistance (Forbac 100 CE, Sanara, Barcelona, Spain) was placed between the endotracheal tube and the respirator tube and was plugged into a control box (Gavalbac 100, Sanara, Barcelona, Spain) . The temperature of the control box was varied in order to maintain the temperature within the endotracheal tube at 42 ° C. 3 shocks of 42 ° C were applied for 15 min leaving 30 minutes without heat between shocks. The temperature within the endotracheal tube was verified throughout the experiment using a thermometer (Omega Instruments, Manchester, UK) and a probe (Sanara, Barcelona, Spain).

2.1.6 Evaluation of the safety of the system of the invention Immediately after the application of the system of the invention or 24 h later, the animals were sacrificed by an intraperitoneal injection of pentobarbital 200 mg / kg. The lungs were then aseptically removed and kept in 10% neutral buffered formalin (Diapath SpA, Martinengo, Italy). System security The invention was evaluated by analyzing the lungs microscopically and macroscopically by a VHUH pathological anatomy laboratory.

RESULTS

1. In vitro sensitivity studies

1.1 Efficacy of the system of the invention applied after the adhesion step a) In silicone discs

By applying the system of the invention after the adhesion step, a discharge of 50 ° C for 15 min was necessary to prevent the formation of the biofilm of the strains Pa1016 and Kp16 on the surface of the silicone discs (see the figure 6).

In addition, the effect of reducing the temperature to 45 ° C was also tested. In this case, after 90 and 30 min of adhesion, the eradication of the growth of the strain Pa1016 on silicone discs was achieved by applying 3 shocks of 45 ° C for 15 min (see Figure 7).

Figure 6. Efficacy of the system of the invention against a strain of P. aeruginosa (Pa1016) and a strain of K. pneumoniae (Kp16) growing on silicone discs, determined by quantitative culture. The treatment consisted of applying from 1 to 5 shocks of 50 ° C for 15 min and was applied after 90 min of adhesion of the biofilm.

Figure 7. Efficacy of the system of the invention against a Pa1016 strain that grows on silicone discs by applying 1, 2 or 3 shocks of 45 ° C for 15 min. The treatment was applied after 90 min (A) or 30 min (B) of the adhesion stage of the biofilm formation. Cells were stained with the LIVE / DEAD® Viability Kit and visualized using confocal laser scanning microscopy (60x magnification). ImageJ software was used to calculate the number of green pixels (viable cells). The results were expressed as the percentage of viable cells after unloading compared to the control group.

b) On PVC discs

The results of the efficacy of the system of the invention applied after 30 minutes of adhesion on PVC discs are shown in figure 8. The application of the system of the invention successfully prevented the formation of biofilm on PVC discs by 100% (8 of 8) of the strains tested: 2 shocks of 42 ° C were necessary for 15 min to prevent biofilm formation from 2 of the 8 strains tested (AbI1 and MRSA15). In addition, the application of 3 shocks of 42 ° C was required during 15 min to avoid the biofilm formation of the rest of the strains tested: two strains of P. aeruginosa (Pa1016 and Pa3), two strains of A. baumannii (AbI4 and Ab60) and two strains of K. pneumoniae (Kp6 and Kp16).

Figure 8. Efficacy of the system of the invention against different strains that grow in PVC discs by applying 1, 2 or 3 discharges of 42 ° C for 15 min. The treatment was applied after 30 min of the adhesion stage of the biofilm formation. Cells were stained with the LIVE / DEAD® Viability Kit and visualized using confocal laser scanning microscopy (60x magnification). ImageJ software was used to calculate the number of green pixels (viable cells). The results were expressed as the percentage of viable cells after unloading compared to the control group.

1.2 Efficacy of the system of the invention applied after the growth stage a) In silicone discs

The results of the efficacy of the system of the invention applied after the formation of biofilm on the surface of the silicone discs are shown in Figure 9. The application of 4 shocks of 50 ° C for 15 min against two strains of P. aeruginosa (Pa1016 and Pa46) and a K. pneumoniae strain (Kp16) allowed the complete eradication of an 8 log ¹⁰ CFU / ml biofilm. However, in the case of Kp16, with only 1 discharge of 50 ° C for 15 min, the reduction was 50%.

Finally, a reduction of more than 70% of the biofilm by Pa46 was achieved by applying 2 shocks of 50 ° C for 15 min, while the total eradication of this strain was obtained by applying only 2 shocks of 50 ° C for 15 minutes. min.

Figure 9. Efficacy of the system of the invention against two strains of P. aeruginosa (Pa1016 and Pa46) and one strain of K. pneumoniae (Kp16) growing on silicone discs, determined by quantitative culture. The treatment consisted of applying from 1 to 5 discharges of 50 ° C for 15 min and was applied after the growth stage of the biofilm formation.

2. In vivo safety studies

The microscopic and macroscopic analyzes of the lungs of the animals showed that the application of the system of the invention did not damage the anatomical structures.

Claims (17)

1.

- Biofilm formation prevention system for respiratory medical devices, the system comprising: a thermal radiator (2) adapted to engage in fluid communication with a respiratory medical device, to raise the temperature of the air flow entering the respiratory medical device, in which the thermal radiator (2) is an elongated metallic body having first and second ends (2a, 2b), and a plurality of channels (4 ', 4) extending longitudinally along the interior of the body and which establish a fluid communication between the first and second ends (2a, 2b) for the passage of ventilation air from one end to the other end, the system further comprising at least one heating wire (21) wound around the thermal radiator (2) to heat the air flowing through the channels (4). 2.

- System according to claim 1, in which one end (2a) of the thermal radiator (2) is configured as a male connection tube and the other end (2b) is configured as a female connection tube. 3.

- Inhalation air heating system according to claim 1 or 2, in which one end (2a) is configured to engage with a source of ventilation air and the other end (2b) is configured to engage in fluid communication with a Endotracheal tube. Four.

- System according to any of the preceding claims, in which the heating wire (21) includes a first and a second filament connected in parallel, and in which the first and second filaments are obtained from different metals or metal alloys . 5.

- System according to claim 4, in which the first filament is a nickel-chromium alloy and the second filament is a nickel-aluminum alloy. 6. - System according to claim 5, in which the first filament is a nickel-chromium 80/20 alloy. 7.

- System according to any of the preceding claims, further comprising at least one temperature sensor (10) thermally coupled with the thermal radiator (2). 8.

- System according to any of the preceding claims, further comprising a temperature controller (11) operatively communicated with the temperature sensor (10) and with the heating wire (21), and adapted to maintain the temperature of the radiator (2) within a desired temperature range. 9.

- System according to claim 8, in which the temperature is within the range of 80 - 90 ° C. 10.

- System according to claim 9, in which the temperature controller (11) is further adapted to maintain a temperature within an endotracheal tube connected to the radiator (2) within the range of 37-50 ° C, and preferably approximately 42 ° C, in order to prevent the formation of biofilm inside the tube. eleven.

- System according to claim 8 or 9, in which the temperature controller (11) includes a timing device adapted to activate the heating wire (21) for predefined periods of time and at predefined time intervals. 12.

- System according to claim 10, wherein the time period is within the range of 10 to 20 minutes, and preferably approximately 15 minutes, and wherein the time range is within the range of 20 to 40 minutes, and preferably 30 minutes. 13.

- System according to any of the preceding claims, in which the thermal radiator (2) has a central channel arranged axially, and a set of channels arranged circumferentially around the central channel and which also extend longitudinally within the radiator, so that the Circumferentially arranged channels are parallel to the central channel. 14. - System according to any of the preceding claims, in which the thermal radiator (2) has a cylindrical configuration. fifteen.

- System according to any of the preceding claims, further comprising a thermal insulation cover (3), so that the thermal radiator (2) and the heating wire (21) are enclosed within the insulation cover (3). 16.

- System according to any of the preceding claims, further comprising electrically insulating sleeves, which respectively electrically isolate the first and second filaments of the heating wire (21). 17.

- System according to any of the preceding claims, in which the inlet and outlet (9a, 9b) of the channels (4 ', 4) have a concave shape, preferably a shape of cone.