RO135526A0 - Microbiological box for providing the microclimate for bacterial films growth and development - Google Patents

Abstract

The invention relates to a microbiological box which provides the microclimate for the growth and development of biofilm-producing bacteria in various incubators or thermostats, the box having applicability to the field of human and veterinary medicine, microbiology and hygiene. According to the invention,the microbiological box consists of two hollow bodies (1 and 2), preferably in the form of parallelepipedal boxes assembled together by clipping, having positioned between them a grate (3), perimetrally provided with a silicone gasket (10), made of plastic or metal autoclavable at 120°C, which delimits two spaces: an upper space in which the microplates (11) with culture media and bacteria are placed, which is isolated from the outside by a window provided with a filter (4), the body ( 1) being provided with a lateral hole (7) with a gasket, in which there is placed a dry thermometer (5) which controls the ambient temperature in the box, and a lower space containing a quantity of water (9) sufficient to ensure humidity and thermal inertia of the microclimate parameters necessary for the correct growth and development of bacterial biofilms, the temperature of water (9) being measured by means of a wet thermometer ( 6) with a wick placed laterally on the body (2) through a hole (7) provided with a gasket.

Description

RO 135526 AO

STATE OFFICE FOR INVENTIONS and more Patent application for the invention.

Denied date

MICROBIOLOGICAL BOX TO ENSURE THE MICROCLIMATE FOR THE GROWTH AND DEVELOPMENT OF BACTERIAL BIOFILMS

The invention relates to a simple device for ensuring the microclimate for the growth of biofilm-producing bacteria in various incubators or thermostats. The specific field of application is that of human and veterinary medicine, microbiology and hygiene.

Biofilms are bacterial populations/communities that produce an extracellular matrix, through which they attach to various surfaces (Donlan 2012). They generate numerous problems both in the medical system ("device-related infections") and in other sectors of activity (food industry, drinking water distribution systems, etc. - Fia 2005, Imre 2020, Flemming 2002).

Biofilm-producing bacteria are responsible for most nosocomial and recurrent infections. The biofilm, consisting of an adherent exopolysaccharide matrix, is the main virulence factor underlying these infections. The attachment of bacteria to biotic (host cells) or abiotic (instrument, medical equipment) surfaces is the first step in biofilm formation, followed by aggregation, through intimate attachment of bacterial cells to each other. Aggregation continues as the biofilm matures. This stage is followed by dispersal (initiated under certain conditions, such as the presence of phenol-soluble modulins), at which point the sessile bacterial cells revert to the planktonic form. in the sessile form, embedded in the biofilm, the bacteria are much more resistant to antimicrobial agents than in the planktonic form (Kirmusaoglu 2019).

Over time, numerous in vitro models have been developed to study the formation and development of bacterial biofilms (McLean 2004). in the way

RO 135526 Current AO, for the phenotypic identification of biofilm-producing bacteria, two methods are used: seeding on culture media containing Congo red dye (Freeman 1989) and the technique in microtitration plates (Christensen 1985). while the first method allows the identification of mainly biofilm-producing staphylococci, the second one addresses any bacterial species of interest (Flemming 2002).

The microtitre plate technique has successfully replaced the first method of macroscopic evaluation of bacterial biofilms, which involved the use of plastic test tubes/De Castro Melo 2013). This method allows the quantitative assessment of the matrix, together with viable and non-viable cells (biofilm and biomass assays), viable bacterial cells (viability assays) and extracellular matrix composition (biomass), based on specific stainings (assays for the quantification of matrix) (Peeters 2008).

The crystal violet (CV) microtiter plate technique is an indirect method of quantifying biofilm and biomass, which forms on the entire surface of the wells (on the walls and at the base) (Christensen 1985, Stepanovic 2000). Crystal violet is a basic dye that attaches to negatively charged molecules and polysaccharides in the extracellular matrix, as well as to the wall of viable and non-viable bacterial cells embedded in biofilm (Li 2003, Pitts 2003). Although modifications have been made to it over time to increase its accuracy (Stepanovic 2000), this method still suffers from the disadvantage of the "edge/marginal effect". The "marginal effect" translates into obtaining higher optical densities in the marginal wells of the microtiter plate compared to the center of the plate. This phenomenon occurs primarily because marginal wells are better ventilated, thus providing more oxygen to support bacterial growth. On the other hand, when the plates are incubated for more than 24 hours, the liquid evaporates from these marginal wells, a phenomenon that causes planktonic cells to attach to the walls of the wells and obtain false positive results and

RO 135526 AO inhomogeneous. since it is difficult to intervene on the oxygenation level in the center of the plate, without raising contamination problems, most attempts to reduce the "edge effect" have been based on ensuring a relative humidity in the plate or in the environment in which it is incubated high, to minimize evaporation (Shukla 2017).

Shukla et al. (2017) managed to obtain more homogeneous results by depositing the maximum amount of sterile distilled water in the marginal wells. Although effective, this method improvement has the disadvantage of significantly reducing the usable area of the microtiter plate.

Incubation in the climatic chamber, with the provision of a relative humidity above 85-90%, is another attempt to prevent excessive evaporation from marginal wells (Shukla 2017). However, these equipments are not a "must have" in any laboratory, on the one hand, and on the other hand, they present the disadvantage of large temperature fluctuations, in order to maintain the humidity relative to the set values. in a recent synthesis of the specialized literature, regarding the environmental factors that influence the formation of bacterial biofilms, Abdallah et al. 2014 emphasize the importance of temperature, relative humidity, nutrient availability, oxygen concentration, environmental pH, and surface type for biofilm formation by pathogenic bacteria, such as S. aureus (Abdallah 2014). regarding the effects of temperature variation on biofilm formation in S. aureus, these remain uncertain, with discrepancies between studies undertaken in this regard. Some authors claim that, on the surface of polystyrene, biofilm-biomass production was more increased at 37°C compared to 25°C (Choi 2013, Vazquez-Sanchez 2013). Others, on the other hand, report an increased number of viable cells adherent to the biofilm on the stainless steel surface at 25°C compared to 37°C (Pagedar 2010). Da Silva Meira et al. (2012) claim, however, that there is no definite effect of incubation temperature (7 and 28°C) on biofilm formation in the case of S. aureus (Da Silva Meira 2012). XÎî?

RO 135526 AO

The invention US20060166358A1 is known which presents a way of incubation for microorganisms to form a biofilm in the wells of plates by ensuring the flow of a flow of growth medium and by repeatedly changing the direction of the liquid flow and a test made from the resulting biofilm. Microorganisms are incubated to form a biofilm on well plates arranged in rows, each row with several wells, while ensuring a flow of liquid growth medium through each row of wells and an analysis made of the resulting biofilm. The sensitivity of the biofilm to the antimicrobial preparation can be determined by treating rows of wells with antimicrobial reagent before performing the test, by treating each row of wells with a different antimicrobial preparation, and by treating each well in the row with a different concentration of antimicrobial preparation.

The invention WO2012024897A1 is known which provides a method of preparing a biofilm, which comprises: inoculating a sample of source bacteria on a substrate, by directly dripping the sample of source bacteria onto the substrate, and cultivating the sample of source bacteria in a medium flow of non-cyclic culture to form a biofilm on the substrate. The invention also provides a device for the formation of a biofilm and uses of the biofilm in the testing and screening of antimicrobial preparations. The device and method of the present invention saves time for growing the culture, reduces contamination, so it can be used to produce biofilms without the need for anaerobic medium or pH adjustment in the culture medium.

WO2016076842A1 - in vitro model of oral biofilm in interdental spaces and use thereof discloses a biofilm model including: a support and a plurality of substrate pairs attached to a support, wherein an oral biofilm is formed on the substrate pairs , in which the pairs of

RO 135526 AO ⁵ substrates each include a first element and a second element and wherein the first element and the second element of each substrate pair are arranged to form a space between the two elements. Methods for evaluating oral biofilm formation using the in vitro oral biofilm model and methods for testing agents, such as oral compositions, for biofilm reduction using the oral biofilm model are also provided.

The examples presented in the state of the art ensure the development of different types of biofilms, but do not ensure the conditions of the microbiological unit nor the control of humidity in the micro-environment of the microbiological unit and are rather complex and expensive technical means.

The technical problem of the invention is the realization of a product of the type of a box that is constituted as a microbiological unit that ensures favorable conditions (temperature, humidity and air pressure) for growth and development, without the contamination of bacterial biofilms.

The microbiological box for ensuring the microclimate for the growth and development of bacterial biofilms, which contains plates with bacteria in a space with a controlled microclimate, is made up of hollow bodies, preferably of parallelepipedal shape, assembled together by clipping on the gasket of a grill. The grill delimits two spaces - an upper one, in which the plates with media and bacteria are placed during incubation and which is isolated from the outside environment by means of a window with a filter, and a lower space in which a sufficient amount of water is arranged to ensure humidity and thermal inertia in order to maintain the microclimate parameters necessary for the growth and correct development of bacterial biofilms. in the upper space the temperature of the environment in the box is measured by a thermometer

<· /

RO 135526 AO dry, placed laterally on the body through a hole with a gasket, and in the lower space the water temperature is measured by a wet thermometer with a wick, placed laterally on the body through a hole provided with a gasket.

The microbiological box for ensuring the microclimate for the growth and development of bacterial biofilms has a grill that delimits the two spaces of the upper and lower bodies. It ensures the communication between them, being made of autoclavable plastic or metallic material equipped around the perimeter with a gasket that allows the upper and lower bodies of the microbiological box to be tightly closed.

The microbiological box for ensuring the microclimate for the growth and development of bacterial biofilms according to the invention has the following advantages:

- allows the creation of independent microbiological units, reducing the risk of contamination of samples (different bacteria) that are simultaneously in the incubation equipment / thermostat;

- improves the microclimate in terms of temperature variations that may occur when the incubator / thermostat is opened as a result of the creation of a micro-environment and the thermal inertia of the water in the tank;

- maintains humidity at a constant level throughout the formation of the bacterial biofilm;

- allows the growth and development of bacteria in aerobiosis / anaerobiosis conditions (if the filter is replaced with a sealed membrane).

An example of an embodiment of the invention is given below in connection with the figures which represent:

Fig Box diagram defining the microbiological unit for the growth of bacterial biofilms.

Fig. 2. Schematic of the grid separating the box bodies.

RO 135526 AO

Fig. 3. Schematic of the sandwich filter.

Fig. 4. The overall diagram of the unfolded representation of the component elements of the ventilation window.

The purpose of the present invention is to effectively ensure the humidity and temperature necessary for the growth of bacteria in the appropriate culture environment, avoiding contamination that may occur in incubators/thermostats.

The microbiological box for ensuring the microclimate for the growth and development of bacterial biofilms (fig. 1) consists of a set of two cave-shaped bodies, an upper one 1 and a lower one 2, which are tightly closed to each other by clipping on a grill 3 provided around the perimeter with a silicone gasket 10 (Fig 2). The box, as a microbiological unit, in which the upper body is delimited from the lower body by the grid 3, forms two chambers, one upper, in which the plates with wells containing the culture media, control media and biofilm-forming bacteria are located, and one lower, in which there is water in sufficient quantity to maintain humidity in the microbiological unit and to ensure thermal inertia in the box.

Temperature and humidity are controlled by means of two thermometers. The dry thermometer 5 is inserted through a hole provided with a silicone gasket 7 in the upper chamber, in which the plates with culture media and bacteria are placed, and the wet thermometer is inserted through a hole provided with a silicone gasket in the lower chamber, in which is water to maintain humidity; the thermometer sensor is wetted by a wick that remains in contact with the water in the lower body (microbiological unit tank). Determining the humidity value can be done easily, with the help of a diagram that considers the temperatures recorded on the dry bulb and the wet bulb. To ensure the biological barrier functions and to maintain the aerobic environment, filter 4 is used, with a surface of at least 5% of the area of the upper face of

RO 135526 AO of the box, arranged in a sandwich between two grates on the upper body of the box (Fig.3).

The microbiological box for ensuring the microclimate for the growth and development of bacterial biofilms is preferably made of transparent and autoclavable materials and has two bodies that are hermetically closed, but which benefit from air through vents equipped with filters. The two bodies of the box are assembled by tightening on a gasket in the form of a microbiological unit consisting of:

- an upper body, usually parallelepiped (with length and width appropriate to the size and number of inserted plates - preferably a multiple of L and / of the microplate used and h at least three times the height of the microplate), which forms a chamber, in which they can be placed different types of plates in which the media and bacteria used for the formation of the biofilm are located, which in the upper part has a hole covered by a filter and, on the side, a hole provided with a seal for the dry thermometer;

- an autoclavable plastic or metal grill that allows the upper body chamber to be separated from the lower body chamber (the water tank), equipped around the perimeter with a seal that allows the two bodies of the microbiological unit to be hermetically closed - above the grill they are placed in a single layer one or more rows (depending on the size of the microbiological unit) of microplates, recommended with the same bacteria;

- a lower body, usually parallelepiped and of the same shape as the upper body, which is constituted in the water reservoir of the microbiological unit, which ensures the humidity and thermal inertia necessary for the development of the bacterial film, which in the upper part has a hole provided with a gasket for wet thermometer;

- a filter fixed on the upper part of the upper body by a sandwich type assembly formed between a discontinuous upper element 401,

RO 135526 AO the filter paper 402, the filter housing of the upper body 403 which is formed by inserting some feet 405 through the holes of the filter (404) into the holes provided at the level of the upper body 406 and sliding the part 401 with the filter 402 over the housing 403, until when the head of the nails 405 fixes the elements in a sandwich type assembly;

- a dry thermometer that attaches to the upper chamber;

- a wet thermometer fitted with a wick that reaches the lower body reservoir (microbiological unit reservoir).

For example, the results obtained by the growth and formation of biofilm in the first 36 hours by a bacterial strain S. aureus subjected to incubation in the microbiological box (microbiological unit - UM), in the thermostat (T), but with the elimination of marginal influences (edge effect= acronym EE) and climate chamber (CC) provide optical densities (measured using TECAN NanoQuant Microplate® equipment) that support the advantages of using the invention. The statistical analysis (ANOVA - one way between subjects) run after the independent variable - the type of incubation UM, T, EE and CC - and the optical density measured after the first 36 hours, as the dependent variable, reveals the importance of the type of incubation - F (3, 236) = 140.82 to/? = 0.000. The Tukey test points out that the optical density in UM (l.17±0.33) is higher than the optical densities in T (0.57±0.21 la/2=0.000), CC (0.45±0.10 la/2=0.000) and EE (l.00±0.21 la/2=0.018).

The same statistical analysis (ANOVA - one way between subjects) performed after 72 hours reveals the importance of the type of incubation - F (3, 236) = 358.82 la/2 = 0.000. The Tukey test emphasizes that the optical density at 72 hours in UM (0.28±0.24) is higher than the optical densities in T (l.33±0.38 la/2=0.000), CC (0.45± 0.04 la/2=0.000) and EE (l.26±0.22 la/2=0.000).

Repeated-measures ANOVA (AKA within-subjects effects) statistical analysis after 36, 48, and 72 hours of incubation, performed by type of incubation, as

RO 135526 AO independent variable and optical densities, as dependent variable, confirmed the significant differences in optical densities generated by the type of incubation (CC, T, EE and UM) - F (1,831, 432,225) = 197,629, la/? = 0.000.

The exemplified experiments allow the rejection of the null hypothesis that there are no significant differences in optical density as a result of the type of incubation. According to the results obtained, the best growth (expressed by the highest optical density) occurred in the microbiological unit of bacterial biofilm production (MU).

The system according to the invention can be used for any type of bacteria, any type of plates and devices for the production of bacterial biofilm and allows the simultaneous use of the incubator/thermostat with different bacterial strains, each in its own microbiological unit, facilitating the formation of the bacterial biofilm in humid conditions constant and with minimal variations of 5 S 5 temperature. The system according to the invention is particularly recommended for the study of resistance to antimicrobial preparations of different bacterial strains producing biofilms.

Bibliography:

1. Donlan RM, Costerton JW: Biofilms: survival mechanisms of clinically relevant microorganisms. Clin Microbiol Rev 2002; 15:167-193;

2. Fux CA, Costerton JW, Stewart PS, Stoodley P: Survival strategies of infectious biofilms. Trends Microbiol 2005; 13:34-40; Imre K, Herman V, Morar A: Scientific Achievements in the Study of the Occurrence and Antimicrobial Susceptibility Profile of Major Foodbome Pathogenic Bacteria in Foods and Food Processing Environments in Romania: Review of the Last Decade. BioMed Research International 2020; Flemming HC: Biofouling in water Systems — cases, causes and countermeasures. Appl Microbiol Biotechnol 2002; 59:629-640

RO 135526 AO

3. Kirmusaoglu, Sahra. (2019). The Methods for Detection of Biofilm and Screening Antibiofilm Activity of Agents. 10.5772/intechopen.84411.

4. McLean RJC, Bates CL, Bames MB, McGowin CL, Aron GM 2004: Methods of studying biofilms. Microbial Biofilms. ASM Press.

5. Freeman DJ, Falkiner FR, Keane CT: New method for detecting slime production by coagulase negative staphylococci. J Clin Pathol 1989; 42:872-874.

6. Christensen GD, Simpsonv WA, Yonger JJ, Baddor LM, Barrett FF, Melton DM, Beachey EH: Adherence of coagulase-negative Staphylococci to plastic tissue culture plates: a quantitative model for the adherence of Staphylococci to medical devices. J Clin Microbiol 1985; 22:996-1006;

7. de Castro Melo P, Ferreira LM, Filho AN, Zafalon LF, Vicente HI, de Souza V: Comparison of methods for the detection of biofilm formation by Staphylococcus aureus isolated from bovine subclinical mastitis. Braz J Microbiol 2013; 44(1): 119-24. two: 10.1590/S151783822013005000031;

8. Peeters E, Nelis HJ, Coenye T: Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates. J Microbiol Methods 2008; 72(2):157-65. two: 10.1016/j.mimet.2007.11.010;

9. Stepanovic S, Vukovic D, Dakic I, Savic B, Svabic-Vlahovic M: A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods 2000; 40:175-179.

10. Li X, Yan Z, Xu J: Quantitative variation of biofilms among strains in natural populations of Candida albicans. Microbiology 2003; 149:353362.

RO 135526 AO .Pitts B, Hamilton MA, Zelver N, Stewart PS: A microtiter-plate screening method for biofilm disinfection and removal. J Microbiol Methods 2003; 54:269-276.

12. Shukla S, Toleti SR. An Improved Crystal Violet Assay for Biofilm Quantification in 96-Well Micro-Titre Plates. 2017; 10.1101/100214 .Abdallah, M., Benoliel, C., Drider, D. et al: Biofilm formation and persistence on abiotic surfaces in the context of food and medical environments. Arch Microbiol 2014; 196:453-472.

https://d0i.0rg/l 0.1007/s00203-014-0983-1 .Choi N-Y, Kim B-R, Bae Y-M, Lee S-Y: Biofilm formation, attachment, and cell hydrophobicity of foodbome pathogens under varied environmental conditions. J Korean Soc Appl Biol Chem 2013; 56:207220. doi:10.1007/sl3765-012-3253-4. Vazquez-Sanchez D, Habimana O, Holck A: Impact of food-related environmental factors on the adherence and biofilm formation of natural Staphylococcus aureus isolates. Curr Microbiol 2013; 66:1 ΙΟΙ 21. doi: 10.1 007/s00284-012-0247-8 .Pagedar A, Singh J, Batish VK. Surface hydrophobicity, nutritional contents affect Staphylococcus aureus biofilms and temperature influences its survival in preformed biofilms. J Basic Microbiol. 2010 Dec;50 Suppl LS98-106. two: 10.1002/jobm.201000034. PMID: 20586075.

.da Silva Meira QG, de Medeiros Barbosa I, Alves Aguiar Athayde AJ, de Siqueira-Jiinior JP, de Souza EL: Influence of temperature and surface kind on biofilm formation by Staphylococcus aureus from food-contact surfaces and sensitivity to sanitizers. Food Control 2012; 25:469-475.

two: 10.1016/j .foodcont.2011.11.030

Claims (5)

RO 135526 AO demand 1. The microbiological box for ensuring the microclimate for the growth and development of bacterial biofilms containing some plates with bacteria (11) in a space with a controlled microclimate characterized by the fact that it is constituted by some bodies (1 and 2) of caves, preferably of the shape of parallelepiped boxes assembled together by clipping, a grid (3) through which two spaces are delimited - an upper one, in which microplates with culture media and bacteria (11) are placed during incubation and which is isolated from the environment of the incubator by means of a window with a filter (4) and a lower space, in which there is a quantity of water (9) sufficient to ensure humidity and thermal inertia in maintaining the microclimate parameters necessary for the growth and correct development of bacterial biofilms, in the upper space the temperature of the medium in the box being controlled by a dry thermometer (5) placed laterally on the body (1) through a hole with gasket (7), and in the lower space the temperature of the water (9) being measured by a wet thermometer (6) with a wick, placed laterally on the body (2) through a hole with gasket (7); 2. The microbiological box for ensuring the microclimate for the growth and development of bacterial biofilms according to claim 1, characterized in that the grill (3) through which communication is ensured but also the delimitation of the two spaces of the bodies (1 and 2) is made of autoclavable plastic material or metallic and is equipped around the perimeter with a gasket (10) that allows the bodies (1 and 2) of the microbiological box to be closed tightly; 3. The microbiological box for ensuring the microclimate for the growth and development of bacterial biofilms according to claim 1 characterized in that the filter window (4) located on the upper part of the body (1) is constituted as a sandwich-type assembly in which an upper frame ( 405), RO 135526 AO formed between a discontinuous upper element (401), a filter paper (402), a filter housing (403) of the upper body (1) is formed with the introduction of legs (405) through the holes of a filter (404) ) in the holes provided at the level of the upper body (1) and sliding the discontinuous upper element (401) with the filter paper (402) over the recess (403), until the head of the legs (405) tightens and fixes the elements of the sandwich assembly; the filter (402) ensures aerobic conditions and contributes to reducing the risk of plate contamination in the microbiological unit. 4. The microbiological box for ensuring the microclimate for the growth and development of bacterial biofilms according to claim 1 characterized in that at the level of the two bodies (1 and 2) it is allowed to insert one thermometer (5 and 6), one dry in the upper body ( 1) and one moistened by means of a wick, in the lower body (2), for tracking the temperature parameter and calculating the humidity parameter based on a diagram using the temperatures from the two thermometers. 5. The microbiological box for ensuring the microclimate for the growth and development of bacterial biofilms according to claim 1, characterized in that the entire assembly is made up of autoclavable components at a temperature of 120 degrees Celsius for at least 30 minutes.