CN110583697B - Efficient chemical biological agent for removing pseudomonas aeruginosa biofilm and application thereof - Google Patents

Efficient chemical biological agent for removing pseudomonas aeruginosa biofilm and application thereof Download PDF

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CN110583697B
CN110583697B CN201910891646.6A CN201910891646A CN110583697B CN 110583697 B CN110583697 B CN 110583697B CN 201910891646 A CN201910891646 A CN 201910891646A CN 110583697 B CN110583697 B CN 110583697B
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pseudomonas aeruginosa
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汪美贞
肖俊炜
孙峰
李张强
单玉菊
冯华军
沈东升
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Zhejiang Gongshang University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates

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Abstract

The invention discloses a high-efficiency chemical biological agent for removing a pseudomonas aeruginosa biomembrane, which comprises the following components by the total mass of 100 percent: 60-75% of microbial preparation and 25-40% of active oxygen radical scavenger; the microbial preparation is a delta LasR strain and/or a delta LasI strain. The efficient chemical biological agent for removing the pseudomonas aeruginosa biomembrane has the advantages of greenness, economy, universality, durability and the like, is simple in formula, low in price of used components, wide in source and low in cost, and has a good effect of inhibiting the growth of the pseudomonas aeruginosa and the formation of the pseudomonas aeruginosa biomembrane.

Description

Efficient chemical biological agent for removing pseudomonas aeruginosa biofilm and application thereof
Technical Field
The invention relates to the field of membrane pollution removal, and in particular relates to a high-efficiency chemical biological agent for removing a pseudomonas aeruginosa biomembrane and application thereof.
Background
Biofilms, also known as biofilms, are organized aggregates of microorganisms. The biological membrane is composed of lipid, protein and saccharide, and has strong physical resistance to antibiotics and can promote the destruction of host immune defense mechanism. Biofilm formation on living pipelines, industrial production, medical instruments, causes erosion and blockage of pipelines, contamination of products, reduction of heat conduction, and causes bacterial infection, etc., which causes many problems in production and life.
Pseudomonas aeruginosa (P.a) is widely distributed in nature and exists in various humid environments, such as water, air, human skin, respiratory tract and intestinal tract. In many practical production operations, the problem of film formation of pseudomonas aeruginosa is difficult to eliminate, and the production efficiency is seriously influenced. This bacterium is one of the most common infectious bacteria that aggravate diseases such as cystic fibrosis, chronic bronchitis, etc. in hospitals. The reason for this is that the Pseudomonas aeruginosa strain has a strong biofilm-forming ability, making it difficult to kill the bacterium.
Quorum Sensing (QS) is a mechanism of information exchange between bacteria. Quorum sensing is a process in which a wild-type strain (WT) secretes a public substance such as elastase to stabilize a population, and quorum sensing is called quorum sensing cooperation. Quorum sensing spoofing refers to a common substance that is not secreted by an individual spoofer (Δ LasR/Δ LasI) but can be generated by other surrounding cooperative individuals. This action can cause a burden on the entire population, and if a large number of rogue seeds are populated in the population, the population may collapse. When a spoof is present, quorum sensing partners may secrete hydrogen cyanide blocking their electronic respiratory chain to inhibit the massive growth of the spoof. The formation of the biomembrane is a cooperative behavior, and the introduced cheater is introduced to destroy the quorum sensing of the pseudomonas aeruginosa and simultaneously eliminate Reactive Oxygen Species (ROS) so as to lead the introduced cheater to accelerate the destruction of the quorum sensing of the pseudomonas aeruginosa, thereby providing a new strategy for removing the biomembrane.
Existing methods of removing and inhibiting biofilm formation: chinese patent with publication No. CN105613584B discloses a method for improving the removal and sterilization efficiency of silver ions on a biological membrane, and the removal and sterilization efficiency of inorganic silver ions on the biological membrane is improved by coupling a small molecular substance bis (3-aminopropyl) amine with dissociation effect on the biological membrane with silver ions, but the raw materials of the method comprise high-cost materials such as inorganic silver ions, bis (3-aminopropyl) amine and the like, and are not suitable for commercial production; the Chinese patent with publication No. CN1901801B discloses that gallium inhibits the formation of biofilm, and the equipment or equipment surface is coated with gallium-containing composition with concentration enough to inhibit the growth and formation of biofilm.
Disclosure of Invention
Aiming at the defects in the field, the invention provides the high-efficiency chemical biological preparation for removing the pseudomonas aeruginosa biomembrane, which has low cost and wide component sources, can obviously inhibit the growth and the membrane formation of the pseudomonas aeruginosa and can efficiently remove the pseudomonas aeruginosa biomembrane.
The high-efficiency chemical biological agent for removing the pseudomonas aeruginosa biofilm comprises the following components by the total mass of 100 percent:
60 to 75 percent of microbial preparation,
25 to 40 percent of active oxygen free radical scavenger;
the microbial preparation is a delta LasR strain and/or a delta LasI strain.
The invention removes the pseudomonas aeruginosa biomembrane by interfering the quorum sensing of microorganisms. Quorum sensing refers to the continuous production of some self-induced small-molecule compounds (autoinducers) during the growth of bacteria, and bacteria judge the surrounding environment by sensing the concentration of the autoinducers. When these auto-inducing substances reach a certain threshold, the expression of some related genes is regulated within the population to adapt to the change of environment.
The invention selects the wild type strain corresponding to the microbial preparation according to the type of the pseudomonas aeruginosa in the pseudomonas aeruginosa biomembrane to be removed, and can adopt various commercially available strains, such as various pseudomonas aeruginosa sold by the China general microbiological culture collection center.
The delta LasR is a mutant strain obtained by knocking out a lasR gene of pseudomonas aeruginosa, and the mutant strain can not receive a signal sent by a signal molecule synthetase LasI, namely, some responses and functions to wild pseudomonas aeruginosa can be deleted. The mutant strain can well remove the biological membrane when used in the invention.
The lasI gene controls the synthesis of a signal molecule at the top of the quorum sensing of the pseudomonas aeruginosa, and the delta LasI mutant strain with the lasI gene knocked out cannot synthesize an intermediate substance for exchanging bacteria, so that the quorum sensing cannot be started by the quorum sensing, namely, some corresponding functions of wild pseudomonas aeruginosa are also deleted. The mutant strain used in the present invention can also remove biofilm well.
The invention mainly uses quorum sensing, introduces deceptive delta LasR strains and/or signal molecule synthetase mutant delta LasI, greatly utilizes public substances of wild strains to ensure that the public substance supply of the population is insufficient and active oxygen radical scavengers are used to inhibit or even kill bacteria, thereby economically and efficiently accelerating the removal of the pseudomonas aeruginosa biomembrane.
Preferably, the high-efficiency chemical biological agent for removing the pseudomonas aeruginosa biofilm comprises the following components in percentage by mass of 100 percent:
60 to 70 percent of microbial preparation,
30 to 40 percent of active oxygen free radical scavenger.
Further preferably, the high-efficiency chemical biological agent for removing the pseudomonas aeruginosa biofilm comprises the following components by the total mass of 100 percent:
65 to 70 percent of microbial preparation,
30 to 35 percent of active oxygen free radical scavenger.
Preferably, the microbial preparation is a Δ LasR strain and/or a Δ LasI strain grown to logarithmic growth phase in LB medium.
Preferably, the microbial preparation is a Δ LasR strain and a Δ LasI strain. The colony induction cooperation is influenced by increasing the number of deceptive delta LasR and/or delta LasI strains in the bacterial population, cyanide secreted by the population cannot play a role through an active oxygen free radical remover, the colony induction supervision effect is reduced, the stability of colony induction is damaged, the population is collapsed, and the aim of accelerating the removal of the pseudomonas aeruginosa biomembrane is fulfilled.
Further preferably, the microbial preparation comprises the following components by the total mass of 100 percent:
80 to 90 percent of delta LasR strain,
10 to 20 percent of delta LasI bacterial strain.
Still further preferably, the microbial preparation comprises the following components by weight percent based on 100 percent of the total mass:
80 to 85 percent of delta LasR strain,
15-20% of delta LasI strain.
Preferably, the active oxygen radical scavenger is at least one of N-Acetyl-L-cysteine (NAC), Catalase (CAT), and superoxide dismutase (SOD). NAC is a thiol-containing antioxidant that increases cellular free radical capture. CAT is called catalase and has the main function of eliminating H2O2And H is2O2The higher the concentration the faster the reaction rate. Both can effectively eliminate active oxygen free radicals in the bacterial body, accelerate collapse of pseudomonas aeruginosa population and remove biological membranes.
Further preferably, the active oxygen radical scavenger is N-acetyl-L-cysteine and catalase. Mixing N-acetyl-L-cysteine and catalase uniformly to obtain the composite active oxygen radical scavenger. The composite active oxygen free radical scavenger has better effect than the active oxygen free radical scavenger with single component, wherein the N-acetyl-L-cysteine and the catalase can play a synergistic role, the inhibition effect is enhanced, the better stability is kept, and the inhibition efficiency is further improved.
Still further preferably, the active oxygen radical scavenger comprises the following components by weight percentage of 100 percent in total mass:
70 to 80 percent of N-acetyl-L-cysteine,
20 to 30 percent of catalase.
Still more preferably, the active oxygen radical scavenger comprises the following components by weight percentage of 100 percent in total mass:
75 to 80 percent of N-acetyl-L-cysteine,
20 to 25 percent of catalase.
The invention also provides application of the high-efficiency chemical biological agent for removing the pseudomonas aeruginosa biomembrane in removing the pseudomonas aeruginosa biomembrane.
In the high-efficiency chemical biological preparation for removing the pseudomonas aeruginosa biofilm, the microbial preparation needs to be frozen and stored, the frozen and stored microbial preparation is restored to activity when in use, the microbial preparation is cultured in an LB culture medium to a logarithmic phase and then is added for use, and the adding amount and the treatment time of the microbial preparation are determined according to the thickness and the form of the biofilm. The active oxygen free radical scavenger is fully dissolved in water and then added for use.
Preferably, the application comprises: and (3) adding a microbial preparation into the pseudomonas aeruginosa biomembrane, and adding an active oxygen free radical scavenger after 1-5 h interval. The addition mode can accelerate the destruction of the stability of the pseudomonas aeruginosa population, can effectively accelerate the removal of the biological membrane, and the pseudomonas aeruginosa population can be rapidly collapsed within 7-8 days. The results, combined with confocal laser microscopy, show that the amount of biofilm and the thickness of biofilm were significantly reduced after the formulation of the present invention was added.
For some daily biofilm infections, the interval time is preferably 1-1.5 h; for some engineering biological membrane blockage such as membrane fouling of a Membrane Bioreactor (MBR), the interval time is preferably 2.5-3.5 h.
Compared with the prior art, the invention has the main advantages that: the efficient chemical biological preparation for removing the pseudomonas aeruginosa biomembrane utilizes quorum sensing deception and interference quorum sensing supervision, can collapse the pseudomonas aeruginosa population within seven days, has the advantages of being green, economic, universal, durable and the like, is simple in formula, low in price of used components, wide in source, low in cost, good in effect, capable of inhibiting the growth of the pseudomonas aeruginosa and the formation of the pseudomonas aeruginosa membrane, and worthy of popularization and application.
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FIG. 1 is a graph of population density of each group over time in application example 1;
FIG. 2 is a graph showing the results of biofilm detection by the crystal violet staining method in application example 3.
Detailed Description
The invention is further described with reference to the following drawings and specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are conducted under conditions not specified, usually according to conventional conditions, or according to conditions recommended by the manufacturer.
Example 1
The high-efficiency chemical biological agent for removing the pseudomonas aeruginosa biofilm comprises the following components in percentage by mass of 100 percent:
70 percent of delta LasR strain and delta LasI strain,
24 percent of N-acetyl-L-cysteine,
6% of catalase.
Wherein the mass ratio of the DeltaLasR strain to the DeltaLasI strain is 90: 10.
Example 2
The high-efficiency chemical biological agent for removing the pseudomonas aeruginosa biofilm comprises the following components in percentage by mass of 100 percent:
delta LasR strain + Delta LasI strain 65%,
26 percent of N-acetyl-L-cysteine,
and 9% of catalase.
Wherein the mass ratio of the DeltaLasR strain to the DeltaLasI strain is 85: 15.
Example 3
The high-efficiency chemical biological agent for removing the pseudomonas aeruginosa biofilm comprises the following components in percentage by mass of 100 percent:
70 percent of delta LasR strain,
24 percent of N-acetyl-L-cysteine,
6% of catalase.
Example 4
The high-efficiency chemical biological agent for removing the pseudomonas aeruginosa biofilm comprises the following components in percentage by mass of 100 percent:
70 percent of delta LasI bacterial strain,
24 percent of N-acetyl-L-cysteine,
6% of catalase.
Example 5
The high-efficiency chemical biological agent for removing the pseudomonas aeruginosa biofilm comprises the following components in percentage by mass of 100 percent:
delta LasR strain + Delta LasI strain 65%,
28 percent of N-acetyl-L-cysteine,
and 7% of catalase.
Wherein the mass ratio of the DeltaLasR strain to the DeltaLasI strain is 80: 20.
Comparative example 1
The chemical biological preparation for removing the pseudomonas aeruginosa biofilm comprises the following components in percentage by mass of 100 percent:
70 percent of pseudomonas aeruginosa wild type strain (WT),
15 percent of delta LasR strain,
Δ LasI strain 15%.
Comparative example 2
The chemical biological preparation for removing the pseudomonas aeruginosa biofilm comprises the following components in percentage by mass of 100 percent:
WT 100％。
application example 1
The chemical biological agents of examples 1 to 5 and comparative examples 1 to 2 were fully dissolved in deionized water for use. Inoculation of starting OD in sterile Environment600When inoculating pseudomonas aeruginosa of 0.05 and 4mL casein culture medium, firstly adding 200 mu L of microbial preparation into the culture medium, adding 100 mu L of active oxygen free radical scavenger into a shaking table for 3 hours, then culturing for 24 hours in the shaking table environment of 36 ℃ and 250rpm, and detecting. Each set of examples or comparative examples was set to 3 biological replicates. Population density was measured every two days as shown in table 1, figure 1.
The pseudomonas aeruginosa wild strain of the application example adopts pseudomonas aeruginosa with the China general microbiological culture Collection center number of CGMCC 1.2421, and the delta LasR strain and the delta LasI strain are mutant strains obtained by homologous recombination or CRISPR gene knockout.
LB medium of this application: dissolving 1% TRYPTONE (10 g/L), 0.5% NaCl (5g/L), and 0.5% YEAST EXTRACT (5g/L) in deionized water, and sterilizing with autoclave at 120 deg.C.
The casein culture medium of the application example: and (4) after the PM culture medium is sterilized at high temperature, shaking up, and standing to a clear state. Casein was added at 10g/L and stirred until completely dissolved. Filter sterilized with a 0.22 μm filter system for use.
PM culture medium: 1g/L (NH)4)2SO4，1.7g/L KH2PO4，1.775g/L NaHPO4，0.0025g/L EDTA，0.011g/L ZnSO4·7H2O，0.00154g/L MnSO4·H2O，0.000392g/L CuSO4·5H2O，0.00025g/L Co(NO3)2·6H2O，0.000177g/L Na2B4O7·10H2O，0.0667g/LCaCl2·2H2O，0.289g/L MgSO4，0.000185g/L(NH4)6Mo7O244H2O, 0.146g/L KOH, 0.2g/L nitrilotriacetic acid.
TABLE 1 dilution 10-6Number of CFU in time
Group of Day 1 CFU Day 3 CFU Day 5 CFU Day 7 CFU
Example 1 82±2 79±3 3±1 4±1
Example 2 96±3 91±2 58±1 3±2
Example 3 105±2 100±1 75±2 4±1
Example 4 102±1 99±2 88±3 3±1
Example 5 90±2 91±4 80±2 67±4
Comparative example 1 99±4 103±3 97±3 114±3
Comparative example 2 93±3 122±2 100±4 112±2
As can be seen from the results in Table 1 and FIG. 1, the Pseudomonas aeruginosa populations of examples 1-5 collapsed within seven days, while the groups of comparative examples 1-2 did not collapse and had good growth conditions, indicating that the preparation of the present invention can inhibit or even kill Pseudomonas aeruginosa, i.e., accelerate the removal of biofilm.
Application example 2
The chemical biological agents of examples 1 to 5 and comparative examples 1 to 2 were fully dissolved in deionized water, filtered with a 0.22 μm filter membrane, and then placed in a closed container for use. Inoculating the starting OD in a culture dish special for a laser confocal microscope in an aseptic environment600When inoculating pseudomonas aeruginosa of 0.05 and 2mL LB culture medium, firstly adding 100 mu L of microbial preparation into the culture medium, adding 50 mu L of active oxygen free radical scavenger at an interval of 3 hours, and then culturing for 12 hours in a constant temperature incubator at 36 ℃. Each set of examples or comparative examples was set to 3 biological replicates. And observing the ratio of the pseudomonas aeruginosa wild strain to the delta LasR strain and the form of the biological membrane by using a laser confocal microscope. The pseudomonas aeruginosa wild type strain was mCherry-labeled (red under a laser confocal microscope) and the Δ LasR strain was GFP-labeled (green under a laser confocal microscope).
The pseudomonas aeruginosa wild strain of the application example adopts pseudomonas aeruginosa with the China general microbiological culture Collection center number of CGMCC 1.2421, the delta LasR strain is a mutant strain obtained by homologous recombination or CRISPR gene knockout, and the fluorescent marker strain is obtained by plasmid introduction.
As a result, the thickness of the biological membrane of the pseudomonas aeruginosa population corresponding to the examples 1-5 is 30-40 μm, while the thickness of the biological membrane of the groups corresponding to the comparative examples 1-2 is 60-80 μm, which shows that the preparation of the invention can effectively reduce the formation of the biological membrane of the pseudomonas aeruginosa.
Application example 3
The chemical biological agents of examples 1 to 5 and comparative examples 1 to 2 were fully dissolved in deionized water, filtered with a 0.22 μm filter membrane, and then placed in a closed container for use. Adding 100 mu L of LB culture medium into each hole of a 96-hole polystyrene micropore culture plate, inoculating 10 mu L of overnight-cultured pseudomonas aeruginosa, adding 5 mu L of microbial preparation into each hole, adding 2.5 mu L of active oxygen radical scavenger at intervals of 3 hours, and standing and incubating for 24 hours at 36 ℃. The medium was carefully aspirated off and the wells were washed 3 times with 200 μ L sterile PBS buffer per well. Add 100. mu.L of methanol to each well and fix for 15min, and carefully aspirate until the methanol is completely air dried. Adding 200 μ L of 1% crystal violet solution into each well, dyeing at room temperature for 30min, sucking out the dye, washing the residual dye with distilled water, drying, adding 200 μ L of 95% ethanol, dissolving, and detecting the absorbance at 570nm with an enzyme-labeling instrument. Each set of examples or comparative examples was set to 3 biological replicates and the results are shown in figure 2.
The pseudomonas aeruginosa wild strain of the application example adopts pseudomonas aeruginosa with the China general microbiological culture Collection center number of CGMCC 1.2421, and the delta LasR strain and the delta LasI strain are mutant strains obtained by homologous recombination or CRISPR gene knockout.
As a result, it was found that the biofilms were detected by the crystal violet staining method, and the Pseudomonas aeruginosa populations OD according to examples 1 to 55701-2, and OD corresponding to comparative examples 1-2570And 4-5, which shows that the preparation can effectively reduce the formation of the pseudomonas aeruginosa biofilm.
Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the above description of the present invention, and equivalents also fall within the scope of the invention as defined by the appended claims.

Claims (9)

1. The high-efficiency chemical biological agent for removing the pseudomonas aeruginosa biofilm is characterized by comprising the following components in percentage by mass of 100 percent:
60 to 75 percent of microbial preparation,
25 to 40 percent of active oxygen free radical scavenger;
the microbial preparation is a delta LasR strain and/or a delta LasI strain;
the delta LasR strain is a mutant strain obtained by knocking out a lasR gene from pseudomonas aeruginosa;
the delta LasI strain is a mutant strain obtained by knocking out a lasI gene of pseudomonas aeruginosa;
the active oxygen free radical scavenger is at least one of N-acetyl-L-cysteine, catalase and superoxide dismutase.
2. The efficient chemical biological agent for removing the pseudomonas aeruginosa biofilm according to claim 1, which comprises the following components by the total mass of 100%:
60 to 70 percent of microbial preparation,
30 to 40 percent of active oxygen free radical scavenger.
3. The efficient chemical biological agent for removing the pseudomonas aeruginosa biofilm according to claim 2, which comprises the following components by the total mass of 100%:
65 to 70 percent of microbial preparation,
30 to 35 percent of active oxygen free radical scavenger.
4. The efficient chemical biological agent for removing the pseudomonas aeruginosa biofilm according to claim 1, wherein the microbial agent is a Δ LasR strain and/or a Δ LasI strain grown to logarithmic growth phase in LB medium.
5. The high-efficiency chemical biological preparation for removing the pseudomonas aeruginosa biofilm according to claim 1 or 4, wherein the microbial preparation is a Δ LasR strain and a Δ LasI strain;
the microbial preparation comprises the following components in percentage by mass of 100 percent:
80 to 90 percent of delta LasR strain,
10 to 20 percent of delta LasI bacterial strain.
6. The efficient chemical biological agent for removing the pseudomonas aeruginosa biofilm according to claim 1, wherein the active oxygen radical scavenger is N-acetyl-L-cysteine and catalase.
7. The efficient chemical biological agent for removing the pseudomonas aeruginosa biofilm according to claim 6, wherein the active oxygen radical scavenger comprises the following components by the total mass of 100%:
70 to 80 percent of N-acetyl-L-cysteine,
20 to 30 percent of catalase.
8. The use of the high efficiency chemical biological agent for removing the pseudomonas aeruginosa biofilm according to any one of claims 1-7 in the removal of the pseudomonas aeruginosa biofilm.
9. The use according to claim 8, comprising: and (3) adding a microbial preparation into the pseudomonas aeruginosa biomembrane, and adding an active oxygen free radical scavenger after 1-5 h interval.
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