Quorum sensing strain-based device and method for in-situ evaluation of disinfection

Abstract

A quorum sensing strain-based device and method for in-situ evaluation of disinfection. The device comprises a microfluidic reactor; two identical independent culture spaces are provided at the position, corresponding to a chip, in the microfluidic reactor; two ends of each culture space are further connected to an inflow channel and an outflow channel; the inflow channel is suitable for a bacterial liquid having a different quorum sensing capability, a culture medium or a disinfectant to enter the culture space by means of the inflow channel, to culture a biofilm or evaluate the disinfection.

Description

In-situ evaluation and disinfection device and method based on quorum sensing strains Technical Field

[0001] The present invention relates to the field of microbial experimental technology, and in particular to a quorum sensing strain-based in-situ evaluation and disinfection device and method. Background Art

[0002] Disinfection is a crucial technology for ensuring drinking water safety and wastewater treatment and reuse. Especially during the COVID-19 pandemic, sewage treatment plants and waterworks have implemented specialized disinfection procedures to disinfect target water bodies. This can block the growth and spread of pathogenic microorganisms in water and is crucial for preventing waterborne diseases. The spread of drug-resistant bacteria and the formation of drug-resistant biofilms have become a major public health concern worldwide. Many bacteria adopt biofilms as their default growth mode and are ubiquitous in water supply systems, healthcare systems, and natural water bodies. However, in many cases, biofilm growth poses a serious threat to human health and the sustainable development of environmental systems, negatively impacting the environment, industry, and human health. Bacteria easily form aggregates by combining with suspended matter in wastewater, which can lead to increased drug resistance and disinfection resistance. Quorum sensing, in which bacteria sense the number of cells in their surroundings through the secretion of signaling molecules, is a crucial regulatory system that influences bacterial growth and metabolism. Quorum sensing is a key factor influencing disinfection effectiveness. Therefore, inhibiting and removing biofilms with varying quorum sensing capabilities in water systems is particularly important. However, it is still unclear whether different disinfection technologies affect the formation of biofilms of different quorum-sensing bacterial strains during wastewater treatment. Therefore, detailed studies on the growth of biofilms of different quorum-sensing bacteria under disinfection stimuli are needed to fully understand their stress response mechanisms. Summary of the Invention

[0003] The problem solved by the present invention is to solve the problem that the prior art cannot obtain detailed information on the growth conditions and stress response mechanisms of different quorum sensing bacterial biofilms under disinfection stimulation.

[0004] To solve the above problems, the present invention provides an in situ evaluation and disinfection device based on quorum sensing strains, including a microfluidic reactor, wherein two identical independent culture spaces are provided inside the microfluidic reactor corresponding to the chip position, and each of the culture spaces is also connected to an inflow channel and an outflow channel at both ends. The inflow channel is suitable for bacterial liquid, culture medium or disinfectant with different quorum sensing capabilities to enter the culture space through the inflow channel to cultivate biofilm or evaluate the disinfection situation.

[0005] Furthermore, it also includes a connected syringe and a micro-injection pump, the end of the syringe is connected to one end of the inflow channel, and the micro-injection pump is suitable for controlling the injection volume and injection time of the syringe into the inflow channel.

[0006] Furthermore, the number of chips inside the microfluidic reactor ranges from 1 to 4.

[0007] Furthermore, the inflow channel and the outflow channel are both plastic hoses; a steel needle is provided at the end of the syringe, and the steel needle is connected to one end of the inflow channel.

[0008] Furthermore, the microfluidic reactor is formed by bonding polydimethylsiloxane and glass, and the glass serves as a result substrate.

[0009] Furthermore, it further comprises a collecting container, which is connected to the outflow end of the outflow channel and is suitable for collecting the solution flowing out of the culture space through the outflow channel.

[0010] The advantage of the in situ evaluation and disinfection device based on quorum sensing strains described in the present invention over the existing technology is that the present invention can realize the observation and comparison of biofilm formation of bacterial liquids with different quorum sensing capabilities through independent culture spaces in microfluidic reactors, and conduct detailed research on the growth of biofilms of different quorum sensing bacteria under disinfection stimulation to fully understand their stress response mechanism.

[0011] To solve the above problems, the present invention also provides a method for in-situ evaluation and disinfection based on quorum sensing strains, based on the in-situ evaluation and disinfection device based on quorum sensing strains, comprising the following steps:

[0012] Step S1: UV sterilization of the quorum sensing strain-based in situ evaluation disinfection device;

[0013] Step S2: preparing a culture medium and a disinfectant, and preparing bacterial solutions with different quorum sensing abilities, and using different syringes to draw the culture medium, the disinfectant, and the bacterial solution respectively;

[0014] Step S3: turning on the microinjection pump switch, setting corresponding flow rates, and injecting the culture medium and the bacterial solution into different culture spaces respectively by controlling the syringes, and then injecting the disinfectant into the culture spaces respectively, so that the biofilm grows or is inactivated in the culture spaces of the microfluidic reactor;

[0015] Step S4: After culturing for different experimental times, the microfluidic reactor was taken out and placed under a laser confocal microscope for staining and observation.

[0016] Furthermore, the bacterial solution in step S2 includes one of Pseudomonas aeruginosa, Gram-positive bacteria and hydrogen-producing bacteria, and the bacterial solution contains self-fluorescent protein.

[0017] Furthermore, in step S3, the disinfectant includes ozone or a chlorine-containing culture medium.

[0018] Furthermore, the operations of step S2, step S3 and step S4 are suitable for being performed at room temperature.

[0019] The advantage of the in situ evaluation disinfection method based on quorum sensing strains described in the present invention over the existing technology is that the method described in the present invention can be applied to the detection of a variety of strains with different quorum sensing capabilities and quickly obtain detection data. It can also evaluate the efficiency of different disinfection technologies in situ, and explore the inactivation effects of various disinfectants including sodium hypochlorite and ozone disinfection on strains with different quorum sensing capabilities. In this way, the response changes of signal molecules to disinfection are revealed from the perspective of quorum sensing, providing guidance for the removal of opportunistic pathogens in sewage. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 is a schematic structural diagram of an in-situ evaluation and disinfection device based on quorum sensing strains according to an embodiment of the present invention;

[0021] FIG2 is a schematic diagram of a biofilm staining microscopy according to a specific embodiment of the present invention;

[0022] FIG3 is a scatter plot 1 of thickness changes of four biofilms with different quorum sensing capabilities according to a specific embodiment of the present invention;

[0023] FIG4 is a second scatter plot of thickness changes of four biofilms with different quorum sensing capabilities according to a specific embodiment of the present invention;

[0024] FIG5 is a third scatter plot of thickness changes of four biofilms with different quorum sensing capabilities according to a specific embodiment of the present invention.

[0025] Reference numerals:

[0026] 1-microinjection pump; 2-syringe; 3-steel needle; 4-inflow channel; 5-microfluidic reactor; 6-outflow channel; 7-collection container. Implementation Method

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0028] As shown in Figure 1, an embodiment of the present invention provides an in situ evaluation and disinfection device based on quorum sensing strains, including a microfluidic reactor 5, wherein two identical independent culture spaces are provided inside the microfluidic reactor 5 corresponding to the chip position, and each of the culture spaces is also connected to an inflow channel 4 and an outflow channel 6 at both ends. The inflow channel 4 is suitable for bacterial liquid, culture medium or disinfectant with different quorum sensing capabilities to enter the culture space through the inflow channel 4 to cultivate biofilm or evaluate the disinfection situation.

[0029] The embodiment of the present invention can realize the observation and comparison of biofilm formation of bacterial liquids with different quorum sensing capabilities through the independent culture space in the microfluidic reactor 5, and conduct detailed research on the growth of biofilms of different quorum sensing bacteria under disinfection stimulation to fully understand their stress response mechanism.

[0030] In some preferred embodiments, the apparatus further comprises a syringe 2 and a microinjection pump 1 connected thereto, wherein the end of the syringe 2 is connected to one end of the inflow channel 4, and the microinjection pump 1 is adapted to control the injection volume and injection time of the syringe 2 into the inflow channel 4. Thus, the injection flow rate is precisely controlled, thereby improving the accuracy.

[0031] In some preferred embodiments, the number of chips inside the microfluidic reactor 5 ranges from 1 to 4.

[0032] In some preferred embodiments, the inflow channel 4 and the outflow channel 6 are both plastic hoses; a steel needle 3 is provided at the end of the syringe 2 , and the steel needle 3 is connected to one end of the inflow channel 4 .

[0033] In some preferred embodiments, the microfluidic reactor 5 is formed by bonding polydimethylsiloxane and glass, with the glass serving as the substrate, thereby simplifying the structure and facilitating cleaning and experimental observation.

[0034] In some preferred embodiments, a collecting container 7 is further included, which is connected to the outflow end of the outflow channel 6 and is suitable for collecting the solution flowing out of the culture space through the outflow channel 6.

[0035] The present invention also provides a method for in-situ disinfection based on quorum sensing strains, which comprises the following steps:

[0036] Step S1: UV sterilization of the quorum sensing strain-based in situ evaluation disinfection device;

[0037] Step S2: preparing a culture medium and a disinfectant, and preparing bacterial solutions with different quorum sensing capabilities, and using different syringes 2 to draw the culture medium, the disinfectant, and the bacterial solution respectively;

[0038] Step S3: turning on the microinjection pump 1, setting corresponding flow rates, and injecting the culture medium and the bacterial solution into different culture spaces respectively by controlling the syringe 2, and then injecting the disinfectant into the culture spaces respectively, so that the biofilm grows or is inactivated in the culture spaces of the microfluidic reactor 5;

[0039] Step S4: After culturing for different experimental times, the microfluidic reactor 5 is taken out and placed under a laser confocal microscope for staining and observation.

[0040] The method described in the embodiments of the present invention can be applied to the detection of various strains with different quorum sensing capabilities and quickly obtain detection data. It can also evaluate the efficiency of different disinfection technologies in situ, and explore the inactivation effects of various disinfectants, including sodium hypochlorite and ozone disinfection, on strains with different quorum sensing capabilities. In this way, the response changes of signal molecules to disinfection are revealed from the perspective of quorum sensing, providing guidance for the removal of opportunistic pathogens in sewage.

[0041] In some preferred embodiments, the bacterial solution in step S2 includes one of Pseudomonas aeruginosa, Gram-positive bacteria, and hydrogen-producing bacteria, and the bacterial solution contains self-fluorescent proteins, thereby being suitable for collecting observation data later.

[0042] In some preferred embodiments, in step S3, the disinfectant includes ozone or a chlorine-containing culture medium. Thus, the disinfectant is easily available and the experimental effect is good.

[0043] In some preferred embodiments, the operations of step S2, step S3 and step S4 are suitable for being performed at room temperature. DETAILED DESCRIPTION

[0044] A quorum sensing-based in situ bacterial strain assessment and disinfection device comprises a microinjection pump 1, a syringe 2, a steel needle 3, an inflow channel 4, a microfluidic reactor 5, an outflow channel 6, and a collection container 7. The syringe 2, inflow channel 4, and outflow channel 6 are all disposable plastic products, and the microfluidic reactor 5 is made of PDMS bonded to glass. The microfluidic reactor 5 can be composed of up to four chips, each with two independent culture chambers. Each culture chamber is 20 mm long, 2 mm wide, and 0.2 mm deep. The interior of the chip is used to simulate microbial biofilm formation. The collection container 7 is used to collect exudate culture medium and bacterial liquid. The microinjection pump 1 controls the flow rate of the solution by controlling the syringe 2. DETAILED DESCRIPTION

[0045] This specific embodiment is based on the quorum sensing strain in situ evaluation disinfection device provided in specific embodiment 1. It performs sodium hypochlorite and ozone simulated disinfection on the wild-type strain of Pseudomonas aeruginosa carrying green fluorescent protein and mutants 1, 2 and 3 to evaluate the efficiency of different disinfection technologies in situ. The specific steps are as follows:

[0046] Step S1: All components of the quorum sensing strain-based in situ assessment and disinfection device were sterilized by ultraviolet light in a clean bench for 20 min. The entire pathway of the in situ assessment and disinfection device was cleaned using syringe 2 filled with anhydrous ethanol. The pathway was then flushed three times in the clean bench using syringe 2 filled with sterile water. The microinjection pump 1 parameter was set to 200 µl / min.

[0047] Step S2: Prepare the culture medium and disinfectant: The culture medium is prepared by adding 5 g of yeast powder, 10 g of tryptone, and 10 g of NaCl. The pH is adjusted to 7.2-7.4 and the volume is then fixed to 1 L. The prepared culture medium is then filtered through a membrane using a vacuum filtration pump and dispensed into several 100 mL conical flasks. The culture medium is then sterilized under high pressure (121°C, 20 min). The culture medium is cooled to room temperature and stored for later use. The disinfectant is prepared by taking a certain volume of sodium hypochlorite solution so that the chlorine concentration in the culture medium reaches 2 mg / L. The ozone generator is turned on to a concentration of 2 mg / L, and ozone is introduced into the liquid culture medium through a rubber tube to prepare an aqueous solution exposed to ozone gas.

[0048] Prepare bacterial suspension: Add 1% inoculum of wild-type Pseudomonas aeruginosa and three signal molecule mutants taken from a 4°C refrigerator. Cultivate in a 37°C shaker until the logarithmic phase is activated. Aspirate with a sterile syringe 2 and place on a microinjection pump 1.

[0049] Step S3: One of the culture chambers served as a blank control, flowing only LB medium at a flow rate of 200 µl / min. The other culture chambers were each fed with suspensions of the wild-type strain and three different mutants. Microinjection pump 1 was set to 60 µl / min and allowed to stand for 2 hours to allow biofilm formation on the chip. Finally, LB medium was continuously flowed at a flow rate of 200 µl / min to allow continued biofilm growth. Simultaneously, the experimental culture chambers were disinfected by sequentially passing medium containing sodium hypochlorite and medium exposed to ozone.

[0050] Step S4: Biofilm microfluidic reactors 5 were removed from the control and experimental groups after 2, 24, 36, and 48 h of incubation, and incubated with 3 μM PI dye at room temperature in the dark for 15 minutes. After rinsing with sterile water, the biofilms were immediately observed and imaged under a confocal laser scanning osmosis (CLSM) to monitor the biofilm morphology and thickness on each chip. Three independent fields of view were randomly selected for image acquisition at 10x, 20x, and 40x magnification, and fluorescence intensity was analyzed using Zeiss Zen software.

[0051] Under CLSM, red fluorescence indicates dead bacteria in the biofilm, while green fluorescence indicates live bacteria. As shown in Figure 2, after 48 hours of incubation, the signal molecule-deficient strains (mutants 1 and 3) showed higher levels of dead bacteria in the biofilm after disinfection, while the signal molecule-oversecreting strain (mutant 2) showed higher levels of live bacteria in the biofilm after disinfection. 3D scanning was used to monitor changes in biofilm thickness. Based on the ZY top view, distance distance was used to measure changes in biofilm thickness over time. As shown in Figure 3, the samples were observed under CLSM after 2, 24, 36, and 48 hours of incubation. Over time, the average biofilm thickness of the wild-type strain increased to 29.37 µm, 32.50 µm, 46.88 µm, and 50.60 µm, respectively. The average biofilm thickness of mutant 1 increased to 17.54 µm, 30.34 µm, 45.15 µm, and 45.37 µm, respectively. The average biofilm thickness of mutant 2 increased to 27.78 µm, 32.07 µm, 40.80 µm, and 51.30 µm, respectively. The average biofilm thickness of mutant 3 increased to 26.28 µm, 30.37 µm, 40.75 µm, and 45.85 µm, respectively. After sodium hypochlorite disinfection, biofilm formation of all four strains was inhibited, as shown in Figure 4. After ozone disinfection, ozone had the strongest inhibitory effect on biofilm formation, as shown in Figure 5. In summary, after 48 hours of real-time monitoring, the following two conclusions can be drawn: First, in terms of biofilm thickness, for P. aeruginosa strains, signal molecule mutant 2 was able to form a higher average biofilm thickness, while mutant 3 formed the lowest average biofilm thickness after 48 hours. In the initial stage of biofilm formation, signal molecule mutant 1 formed the lowest biofilm thickness. This suggests that the secretion of signal molecules promotes biofilm formation and plays a certain protective role against damage by disinfectants.

[0052] Although the present invention is disclosed as above, the scope of protection disclosed by the present invention is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention, and these changes and modifications will fall within the scope of protection of the present invention.

Claims

1. A quorum sensing strain in-situ evaluation and disinfection device, characterized in that: It comprises a microfluidic reactor, wherein two identical independent culture spaces are arranged inside the microfluidic reactor corresponding to the chip position, and each culture space is connected with an inflow channel and an outflow channel at both ends. The inflow channel is suitable for bacterial liquid, culture medium or disinfectant with different group sensing capabilities to enter the culture space through the inflow channel to cultivate biofilm or evaluate the disinfection condition.

2. The quorum sensing strain in-situ evaluation and disinfection device according to claim 1 is characterized in that: It also includes a connected syringe and a micro-injection pump, wherein the end of the syringe is connected to one end of the inflow channel, and the micro-injection pump is suitable for controlling the injection volume and injection time of the syringe injected into the inflow channel.

3. The quorum sensing strain in-situ evaluation and disinfection device according to claim 1 is characterized in that: The number of chips inside the microfluidic reactor ranges from 1 to 4.

4. The quorum sensing strain in-situ evaluation and disinfection device according to claim 2 is characterized in that: The inflow channel and the outflow channel are both plastic hoses; a steel needle is provided at the end of the syringe, and the steel needle is connected to one end of the inflow channel.

5. The quorum sensing strain in-situ evaluation and disinfection device according to claim 1, characterized in that: The microfluidic reactor is formed by bonding polydimethylsiloxane and glass, and the glass is a result substrate.

6. The quorum sensing strain in-situ evaluation and disinfection device according to claim 1, characterized in that: It also includes a collecting container, which is connected to the outflow end of the outflow channel and is suitable for collecting the solution flowing out of the culture space through the outflow channel.

7. A method for in-situ evaluation and disinfection based on quorum sensing strains, characterized in that: The quorum sensing strain in-situ evaluation and disinfection device according to any one of claims 2 to 6 comprises the following steps: Step S1: UV sterilization of the quorum sensing strain-based in-situ evaluation disinfection device; Step S2: preparing a culture medium and a disinfectant, and preparing bacterial solutions with different quorum sensing capabilities, and using different syringes to draw the culture medium, the disinfectant, and the bacterial solution respectively; Step S3: turning on the microinjection pump switch, setting corresponding flow rates respectively, injecting the culture medium and the bacterial solution into different culture spaces respectively by controlling the syringe, and then injecting the disinfectant into the culture spaces respectively, so that the biofilm grows or is inactivated in the culture space of the microfluidic reactor; Step S4: After culturing for different experimental times, the microfluidic reactor is taken out and placed under a laser confocal microscope for staining observation.

8. The method for in-situ evaluation and disinfection based on quorum sensing strains according to claim 7, characterized in that: The bacterial solution in step S2 includes one of Pseudomonas aeruginosa, Gram-positive bacteria and hydrogen-producing bacteria, and the bacterial solution contains self-fluorescent protein.

9. The method for in-situ evaluation and disinfection based on quorum sensing strains according to claim 7, characterized in that: In step S3, the disinfectant includes ozone or a chlorine-containing culture medium.

10. The method for in-situ evaluation and disinfection based on quorum sensing strains according to claim 7, characterized in that: The operations of step S2, step S3 and step S4 are suitable to be performed at room temperature.

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