La eliminación del biofilm El uso de aerosoles de dióxido de carbono sin purga de nitrógeno

Resumen

Biofilms on surfaces can be effectively and rapidly removed by using a periodic jet of carbon dioxide aerosols without a nitrogen purge.

Resumen

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. Therefore, it is highly desirable to remove biofilms from surfaces. Many studies on CO₂ aerosol cleaning have employed nitrogen purges to increase biofilm removal efficiency by reducing the moisture condensation generated during the cleaning. However, in this study, periodic jets of CO₂ aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from polished stainless steel surfaces. CO₂ aerosols are mixtures of solid and gaseous CO₂ and are generated when high-pressure CO₂ gas is adiabatically expanded through a nozzle. These high-speed aerosols were applied to a biofilm that had been grown for 24 hr. The removal efficiency ranged from 90.36% to 98.29% and was evaluated by measuring the fluorescence intensity of the biofilm as the treatment time was varied from 16 sec to 88 sec. We also performed experiments to compare the removal efficiencies with and without nitrogen purges; the measured biofilm removal efficiencies were not significantly different from each other (t-test, p > 0.55). Therefore, this technique can be used to clean various bio-contaminated surfaces within one minute.

Introducción

Biofilms are complex bacterial community structures in which bacterial cells are embedded within self-produced matrices of extracellular polymeric substances, held together, and protected from the external environment. Biofilms can present a public health risk and cause economic losses because they can form on various surfaces, including medical implant materials and devices, food processing equipment, and heat exchangers. In fact, biofilms have been found to be associated with 65% of all bacterial infectious diseases in humans, according to the Centers for Disease Control and Prevention¹.

Autoclaves and disinfectants such as chlorine have generally been used for the inactivation of biofilms. However, the use of an autoclave is limited for surfaces that can neither withstand high temperature steam nor be placed into the autoclave. Disinfectants are not suitable for surfaces sensitive to chemical treatment or prone to collecting toxic oxidation products². In addition, the biofilm should not only be inactivated, but also removed in order to prevent the attachment of new cells onto the surface, thereby forming a new biofilm³. However, it is difficult to remove biofilms using methods based on viscous fluid shear because the flow velocity near the surface is almost zero and the shear force usually cannot overcome the adhesive forces of micron- and submicron-sized substances. Moreover, the biofilm matrix is known to act as a physical and chemical barrier¹.

Many physical and mechanical techniques have been developed to remove biofilms from surfaces, including ultrasonic vibration¹, electric currents⁴, laser irradiation⁵, and high-pressure water sprays⁶. Each technique has its own pros and cons. Ultrasonic vibration and electric current can be used to control biofilm formation; however, they require a particular configuration and conductive surfaces, respectively, requiring additional shear stress^{1, 4}. Laser irradiation can be applied to a limited area and to hard surfaces; however, some live and dead cells remain on the surface⁵. High-pressure water sprays effectively remove biofilms; however, their high momentum can cause damage to soft substrates⁶.

Biofilm removal using CO₂ aerosols has been previously proposed. It has shown promising results, with high removal efficiencies within a short time^7-11. CO₂ aerosols are generated by adiabatic expansion of a high-pressure CO₂ gas through a nozzle, and they are applied to the surfaces contaminated with a biofilm. This cleaning technique utilizes the momentum transfer of solid CO₂ particles and the solvent action of the melted CO₂ liquids, followed by the aerodynamic shear force of the CO₂ gas¹². Compared with high-pressure N₂ gas jets, CO₂ aerosol jets at the same stagnation pressure are much more effective in removing E. coli biofilms⁷. Moreover, although the momentum of the solid CO₂ that is delivered to the bacteria is considerably high, the momentum of the total aerosol jet applied to the solid surface is substantially lower than that of water jets. Therefore, damage-free cleaning is possible using this CO₂ aerosol technique.

In this protocol, periodic jets of CO₂ aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from a polished stainless steel surface. In fact, nitrogen purges have been used in many CO₂ aerosol studies to increase the removal efficiency by reducing the moisture condensation generated during the cleaning, even though heating with a hot plate or infrared lamps and employing dry boxes have also been adopted¹². The surface biofilm formation and the optimized cleaning procedures are described below. The removal efficiency was evaluated by measuring the fluorescence intensity of the biofilm on the surface.

Protocolo

1. Preparación de superficies para la formación de biofilm

1. Cortar 1 mm de grosor, 304 placas de acero inoxidable en chips (10 × 10 mm ²⁾ con un cortador mecánico.
2. Realizar la limpieza por ultrasonidos de los chips en acetona, metanol, y desionizada (DI) secuencialmente agua durante 10 min cada uno. Use un recipiente resistente a los disolventes, hecho de sustancias tales como vidrio, para eliminar la contaminación orgánica.
3. Enjuague los chips con un chorro de agua DI durante 3-5 seg.
4. Se secan las fichas utilizando el flujo de gas _N2 durante 3-5 seg.

2. Preparación del cultivo bacteriano

1. Tome una P. putida KT2440 (proporcionado amablemente por el Prof. Sung Kuk Lee, UNIST, Corea del Sur) stock almacenado en un -80 ° C congelador.
2. Descongele a temperatura ambiente después de 1 min, y la capa superior de la solución madre congelada se convierte en aguanieve. Sumergir un bucle en la capa derretida de la solución madre.
3. Utilice este bucle a la raya a las bacterias en un medio Luria-Bertani placa (LB) que contiene 1,5% de agar.
4. Incubar la placa durante la noche a 30 ° C para crecer las colonias bacterianas.
5. Elija una única colonia de la placa usando un bucle fresco.
6. Inocular 10 ml de caldo LB en un tubo cónico de 50 ml con el bucle que contiene la colonia bacteriana única.
7. Incubar el caldo en una incubadora de agitación durante 16 horas a 30 ° C y 160 rpm.

3. La formación de biopelículas en la Superficie

1. Recoger cada una de las fichas preparadas con unas pinzas y sumergirlos en un 70% de etanol 5 veces durante 1-2 segundos cada uno, para esterilizar la superficie de cada chip. Asegúrese de que las virutas se llevan a cabo con pinzas durante la inmersión.
2. Sumergir cada chip en agua DI autoclave y en LB caldo de forma secuencial, 5 veces durante 1-2 segundos cada uno, para eliminar el etanol restante.
3. Colocar estos chips en placas de cultivo de 6 pocillos con 2 chips y 5 ml de caldo LB por pocillo.
4. Diluir el cultivo bacteriano hasta que la concentración en los tramos caldo LB8 × 10 ⁸ células / ml (densidad óptica a la longitud de onda 600 nm: ~ 0,8).
5. Inocular cada pocillo con 50 l de cultivo bacteriano diluido.
6. Incubar las placas a 30 ° C sin agitación durante 24 horas para la formación de biopelículas.

4. Limpieza de CO ₂ en aerosol

1. Dip los chips de la biopelícula formada en tampón acetato de amonio 10 mM (volátiles) 5 veces para eliminar débilmente unidos y bacterias planctónicas.
2. Seque estos chips en una cabina de seguridad biológica, donde el aire fluye suavemente.
3. Inmediatamente después del secado, colocar un chip en el lugar de carga, que es de 20 mm de la boquilla de _CO2 a lo largo del eje del chorro. Incline el eje del chorro a un ángulo de 40 °.
4. Establecer las presiones de estancamiento de _CO2 y _N2 gaseoso a 5,3 MPa y 0,7 MPa, respectivamente, utilizando los reguladores de presión de gas.
5. Aplicar el chorro de aerosol en la parte central del chip. Aerosoles blancos incluyendo el _CO2 sólido debese visible. "Encender" la válvula de solenoide para el CO ₂ durante 5 segundos, y luego convertirlo en "off" durante 3 segundos (ciclo: 8 seg) periódicamente con un interruptor controlado manualmente. Si una purga de nitrógeno tiene que ser utilizado, a su vez en la válvula de solenoide para un suministro continuo de N _2.
6. Tratar los chips con CO ₂ aerosoles para 16, 40, y 88 seg con y sin purgas de nitrógeno. Conservar los chips sin tratamiento como controles negativos.

5. Análisis de eficacia de la eliminación

1. Preparar 1 M mancha de ácido nucleico de fluorescencia verde (longitud de onda de excitación / emisión: 480/500 nm) en agua DI para teñir las células bacterianas en el control y patatas fritas tratadas con aerosol.
2. Coloque las fichas en la solución de tinción.
3. Se incuban las fichas en una incubadora sin luz a 37 ° C durante 30 minutos.
4. Después de la incubación, lavar suavemente las virutas con un chorro de agua DI para eliminar el exceso de colorante fluorescente.
5. Se seca el wi fichasTH flujo de gas N _2.
6. Tome fluorescencia imágenes microscópicas de 5 campos aleatorios de vista (321 × 240 m ²⁾ para cada chip utilizando un microscopio de epifluorescencia, una lente objetivo de 40X, y una cámara CCD.
7. Obtener la intensidad de fluorescencia para cada imagen utilizando un software de procesamiento de imágenes tales como ImageJ. En ImageJ, utilizar la función de "Antecedentes Reste" en el menú "Acción", y seleccione "densidad integrado" en la ventana "Establecer medidas" en el menú "Analizar". Hacer el "medición" en el menú "Analizar" para obtener la intensidad de fluorescencia.
8. Calcular la eficiencia de eliminación de biofilm de acuerdo con la fórmula siguiente: 100% x (I de chips de control - I de virutas tratadas) / (I de chips de control), donde I es la intensidad de la fluorescencia calculada.
9. Obtener el promedio de las eficiencias de eliminación y las desviaciones estándar. Use por lomenos 4 chips para cada caso.

Resultados

CO ₂ aerosoles se utilizan para eliminar el P. putida biofilms de SUS304 superficies (Figura 1). La mayor parte de las superficies estaban cubiertas por el biofilm después de 24 horas de crecimiento. La mayor parte de la biopelícula se retiró usando el CO ₂ aerosoles (Figura 2). Como era de esperar, la Figura 3 muestra un aumento de la eficiencia de eliminación de biofilm como el tiempo de tratamiento aerosol CO ₂ aumentado....

Discusión

Previously, we conducted optimization studies on CO₂ gas pressure, jet angle, and distance to the solid surface in CO₂ aerosol cleaning⁷. Unlike our previous studies, in the present study, a nitrogen purge was not included in the aerosol (Figure 1). Moreover, 304 stainless steel was used in this protocol, since it is one of the most common stainless steels and is widely used in the food industry. The polished surface is beneficial for fluorescence analysis because of a un...

Materiales

Name	Company	Catalog Number	Comments
304 stainless steel	Steelni (South Korea)		Polished and diced ones
Ultrasonic cleaner	Branson (USA)	5510E-DTH
Luria-Bertani (LB)	Becton, Dickinson and Company (USA)	244620	500 g
Agar	Becton, Dickinson and Company (USA)	214010	500 g
6-well culture plate	SPL Life Sciences (South Korea)	32006
Ammonium acetate buffer	Sigma-Aldrich (USA)	667404	10 mM
Dual gas unit	Applied Surface Technologies (USA)	K6-10DG	One nozzle for CO2 gas & 8 nozzles for N2 gas
SYTO9	Thermo Fissher Scientific (USA)	Invitrogen	Excitaion: 480 nm Emission: 500 nm
Epifluorescence microscope	Nikon (Japan)	Eclipse 80i
40X objective lens	Nikon (Japan)	Plan Fluor	NA: 0.75
CCD camera	Photometrics (USA)	Cool SNAP HQ2	Monochrome