RU2265057C2 - Method for indication of biocide activity of peroxide disinfecting solutions to botulinic neurotoxin - Google Patents

Abstract

FIELD: biotechnology, toxins.

SUBSTANCE: invention relates to methods for indication of biocide activity of peroxide disinfecting solutions to toxins of bacterial origin. Method involves preparing preparations of globular protein complexes of class C initiating ice formation of supercooled water that are produced by microorganisms of genus Erwinia and Pseudomonas and effect of the disinfecting composition on prepared preparation and exposition. Effectiveness of disinfectant is evaluated by retaining specific properties of proteins after disinfecting treatment by the instrumental cryoscopic microdrop method. Method provides carrying out the safety and rapid evaluation of biocide activity of peroxide disinfecting solutions with respect to botulinic neurotoxin. Invention can be used in indication of biocide activity of peroxide disinfecting solutions to botulinic neurotoxin.

EFFECT: improved method for indication.

4 tbl, 3 ex

Description

The invention relates to methods for determining the disinfecting activity of peroxide solutions against microbial toxins of protein nature and can be used in biotechnology, sanitary, medical and applied microbiology.

Determination of the biocidal activity of disinfectants involves their exposure to the test object, followed by an assessment of its biological activity before and after exposure. In relation to botulinum neurotoxin, the determination of biological activity consists in the quantitative determination of a protein by its toxic properties.

There are a large number of methods for determining the activity of botulinum toxins, among which it is necessary to note the indication of botulinum toxins by the reaction of delayed phagocytosis and its restoration in the presence of specific sera (Matveev K.I. Botulism. - M., 1959, - 407 p.). However, the method did not find wide recognition due to the orientation of the results. Conventional serological methods based on the reaction between the toxin and the corresponding antibodies remain uncommon, due to insufficient sensitivity. Currently, numerous variants of enzyme-linked immunosorbent enzyme-linked solid or homogeneous methods of analysis have been developed. They are based on the specific fixation of certain antibodies or antigens on an insoluble basis, followed by testing the associated component using an indicator system - an antibody (or antigen) covalently bound to the enzyme (Egorov A.M., Osipov A.P., Dzantiev B.B. Gavrilova EM Theory and practice of enzyme immunoassay. - M., 1991, - 286 p.). However, the implementation of the enzyme immunoassay requires individual specificity of the conjugates for each type of antigens and a high degree of purification, which reduces the possibility of their use in wide practice.

There is also a method of determining the activity of toxins to reduce the luminescence of bacteria after exposure to toxins. The method is informative, to some extent instrumental, but is not sufficiently accurate due to the use of a biological component (Method for determining the activity of bacterial toxins, patent RU 2075081. Polyakov VM, Tekutiev SI, Andrusenko IT, etc., 1997) .

As the closest analogue to the determination of the biocidal activity of peroxide disinfectant solutions to toxins of microbial origin, the suspension method was selected, including the "Method" for determining the effectiveness of disinfectants in relation to toxins by titration in laboratory animals "(Labinskaya AS Microbiology with the technique of microbiological research. - M .: Medicine, 1978, S.258-260).

The toxicity of the test sample is determined by titration on white outbred mice weighing 18-20 g without a banal pathology. For this, the sample is diluted with a phosphate-nutrient buffer solution with a pH of 6.5 ... 6.7 in ten-fold increments. Each dilution was administered intraperitoneally to 4 white mice, 0.5 ml each. The control is a test sample warmed up for 30 minutes at 100 ° C in similar dilutions. Infected and control animals are monitored for 4 days, daily recording their death and noting the appearance of symptoms characteristic of intoxication with botulinum neurotoxin (rapid breathing, complete muscle relaxation, retraction of the muscles of the abdominal wall - wasp waist, paralysis of the extremities). After obtaining the results of a preliminary study, toxic activity is determined in the same way, but with a two-step sample dilution. At the same time, the protein concentration in the sample is determined by the method of Lowry (Loury O.N. 3 Farr AR et al. Protein measurement with the Folin phenol reagent // J. Biol. Chem. - 1951. - Vol.19. - P.265-275 ) The activity of the sample is expressed by the value of LD ₅₀ in ml ^-1 with the definition of standard deviation. The specific activity of the LD ₅₀ sample in ml ^{-1 of} protein is also calculated. Sample activity (LD ₅₀ ) is calculated according to the Kerber method in the modification of Ashmarin (Ashmarin I.P., Vorobyov A.A. Statistical methods in microbiological research - L .: Medgiz, 1962). The duration of the analysis of a biological sample in laboratory animals is from 7 to 8 days.

In common with the claimed method is the stage of preparation of the studied protein preparation of preliminary dilutions of the required multiplicity, modes and methods of disinfection, sampling for analysis.

The disadvantages of this method include the complexity and complexity of determining the biocidal activity of disinfecting peroxide solutions from a biological sample in laboratory animals, the duration and low accuracy of determination due to the use of laboratory animals that are not standardized for sensitivity.

The objective of the invention is to develop a method that provides easy, rapid determination of the effectiveness of disinfectants against botulinum neurotoxin, which is a globular protein complex formed from several subunits, at the stages of development of prescriptions of disinfectant compositions, modes and technical means for their use.

The problem is solved due to the fact that the method of indicating the biocidal activity of peroxide disinfectant solutions provides for the use of:

as an object of test exposure, preparations of bacterial proteins initiating ice formation (BIL), the biological activity of which is recorded by the presence in the sample of nucleation centers (CNs) of supercooled water formed by proteins of this type;

drip freezing techniques for quantitative detection of CN in samples, the essence of which is to calculate the relative number of crystallizable microdrops of serial dilutions of the studied samples at a certain supercooling temperature and use the known dependence of the concentration of CN on the proportion of freezing microdrops that obeys the Poisson distribution;

rational analysis temperature, which is in the range from minus 7 ° C to minus 11 ° C, because at this temperature, pure water and dilute solutions of salts that do not contain heterogeneous nuclei of nucleation in microdrops remain in a supercooled state for a long time;

temperature-controlled mini-criostat (manufactured by Bio-Mashpribor OJSC, Yoshkar-Ola), constructed on the basis of Peltier elements that provide cooling and thermal stabilization of the working surface with an accuracy of 0.2 ° C in the temperature range from minus 1 ° C to minus 13 ° C;

active producer strains of ice formation initiating proteins (BIL) - microorganisms Erwinia herbicola, strains No. 1211 and No. 1123 (it is also possible to use Pseudomonas syringae, strain 1567, Pseudomonas fluorescens strain 1475) from the All-Russian collection of microorganisms that produce significant amounts of protein complexes initiating crystallization of supercooled water at a temperature of minus 7 ° С and below, representing globular formations of several subunits, having a molecular weight of about 150 kDt and an isoelectric point in acidic not pH, which is similar to botulinum neurotoxin (Vinogradova I.D., Uvarova R.N., Ivanov K.K. et al. Obtaining neurotoxin and hemaglutinin Clostridium botulinum type A and neurotoxin characterization // Biochemistry. - 1983. - T.4 , issue 5 - С: 788-795; Burke MJ, Lindow SE Surface properties and size of the ice nucleation site in ice nucleattion active bacteria: Theoretical considerations // Cryobiology. - 1990 - Vol.- 27, No. 1. - P.80-84). Another evidence of the similarity between ice-forming proteins and botulinum neurotoxin proteins is comparable levels of activation energy (E _act ) and preexponential factor (A) in the Arrhenius equation (k = A _exp (-E _act / RT), which characterizes the thermal stability of microorganisms. So, the activation energy and preexponential factor for Clostridium botulinum type A are 72 kcal × mol ^-1 and 2 × 10 ³⁹ s ^-1 , and for Pseudomonas fluorescens 69 kcal × mol ^-1 and 3 × 10 ⁴³ s ^-1 respectively (Munblit V.Ya., Talroze V. L., Trofimov V.I. Thermal inactivation of microorganisms. - M .: Nauka, 1985, - 247 p.).

The method includes the following steps:

preparation of a test object from a protein preparation;

inactivation of the drug with peroxide disinfectants;

determination of the content of CN in the test object before and after exposure to a disinfectant solution using the cryoscopic microdrop method;

processing the results.

The method is as follows.

To obtain a test drug, a producer culture is grown from which BIL is isolated.

The microbes of producers are grown on the surface of a dense nutrient medium, based on an enzymatic meat hydrolyzate containing amine nitrogen in an amount of (110 ± 10) mg%, having a concentration of hydrogen ions (7.2 ± 0.2) units. pH Microbial cultures are obtained at a temperature of (24 ± 4) ° C for 3 days, followed by washing the colonies with physiological sodium chloride solution.

Isolation of BIL is carried out by destroying the cells of a suspension of microorganisms by shaking with glass beads or sonication. BIL is isolated from the culture fluid by salt precipitation in the presence of ammonium sulfate at a pH of 3.5 ... 5.5 units. pH followed by centrifugation. Based on the obtained protein paste, a test preparation is prepared corresponding to a solution of botulinum neurotoxin in terms of protein, mineral salts and acidity.

Inactivation of the protein preparation by the investigated disinfectant solutions is carried out by the suspension method with a ratio of the drug and the disinfectant 1: 9 and exposure from 5 to 30 minutes with periodic shaking.

To register CN in samples, the following operations are carried out:

according to the description attached to the cryostat, prepare the device for operation. Set the operating temperature to minus 7 ° C.

A diluting liquid, a phosphate buffer, is prepared on the basis of solutions of salts of monosubstituted and disubstituted potassium phosphate and distilled water. The concentration of hydrogen ions (pH) should be in the range of 7.3 ... 7.6 units. pH The prepared buffer is sterilized at 120 ° C for 30 minutes. The sterile buffer is checked for abiogenic nucleation sites. For this, the buffer is poured into sterile tubes of 4 ... 10 ml and frozen in a cryostat at a temperature of minus (7 ... 9) ° С for 5 minutes. Tubes frozen at this temperature are discarded.

The preparation of waxed cuvettes is carried out from aluminum foil and molten paraffin dissolved in xylene or chloroform at a concentration of 3%. Ditches are prepared in the form of a circle with a diameter of 8 ... 10 cm with curved edges and a side height of 5 ... 10 mm. The paraffin solution is applied to the surface of the cuvette in excess. Excess solution is drained. The cuvette is dried for 2 ... 3 minutes above the flame of an alcohol lamp to evaporate the solvent.

Sterile volumetric tubes in the amount necessary to perform the required ten-fold dilutions are filled with 4.5 cm ^{3 of} phosphate buffer. The test sample is thoroughly mixed and 0.5 cm ^{3 is} introduced into the first tube with phosphate buffer. Further dilutions are carried out in ten-fold increments. The initial sample and all 5 ... 8 ten-fold dilutions are subjected to analysis.

A micropipette is applied to the surface of an aluminum waxed cuvette with 20 ... 40 drops (0.01 cm ³ volume) (controls of a phosphate buffer and a test disinfectant solution) and each analyzed dilution. The cuvettes are installed in a cryostat and visually determine the number of frozen drops in each cuvette three minutes after the start of crystallization. Freezing drops recorded by their turbidity. Change the temperature of the cooling surface of the cryostat and after 3 min repeat the count of the number of frozen microdrops.

When calculating the concentration of nucleation centers, cuvettes are used in which a part of the applied droplets is frozen. The analysis is considered valid if in the controls (phosphate buffer and the test disinfectant solution), the CNs at the selected temperature are not recorded.

The calculation of the concentration of CN in the samples is carried out in the following order.

For each dilution of the sample (i), the fraction of frozen droplets is calculated using formulas 1 and 2 — the crystallization effect (f _i ) and the concentration of nucleation centers (C _fi ) corresponding to this effect.

where: f _i - crystallization effect, fraction;

С _fi - concentration of nucleation centers in the test fluid, c.n. cm ^-3 ;

N _oi is the total number of test drops of the test dilution on this cuvette;

N _3i - the number of frozen drops of the investigated dilution on this cuvette;

d is the degree of dilution;

V is the droplet volume on the cuvette (0.01 cm ³ ).

On the basis of the obtained values by the least squares method, the concentration of nucleation centers corresponding to the 50% crystallization effect is calculated.

Statistical processing of the results of parallel determinations is performed in accordance with generally accepted standards.

The presence of a causal relationship between the totality of the essential features of the claimed object and the achieved technical results are shown in table 1.

Table 1
Information about the causal relationship between the totality of the essential features of the claimed object and the achieved technical results Types of technical result and their sizes Actual or Estimated Indicators The set of distinctive features that improve the performance of the claimed object prototype the claimed object 1 2 3 4 The average duration of the determination of the biocidal effect of the disinfectant solution in 10 samples, hour 96 2 Using the method of drip freezing of microbial proteins imitating botulinum neurotoxin, determined by the instrumental method, which provides an express determination of the biocidal activity of peroxide disinfectants in comparison with the prototype, where the disinfecting effect is recorded on a biological sample in laboratory animals. 1 2 3 4 Ensuring the safety of working personnel when performing work on disinfection and recording its effectiveness (required / not required) required not required The safety of working personnel when working with active preparations of botulinum neurotoxin is ensured by the relevant standards and rules for working with pathogenic materials of 1-2 pathogenicity groups in accordance with SP 1.2.011-94, work in laboratory facilities equipped with special biosafety engineering systems, and staff qualifications , which is excluded when implementing the proposed method of indication. Error in determining the effectiveness of a disinfection measure, percentage > 25 <10 The use of non-standardized laboratory animals (white mice) does not provide an accuracy of recording the effect better than 25% (permissible biological error), with 10% accuracy of the instrumental method in the proposed method of indication. 1 2 3 4 The average number of tests to determine the biocidal effect by two laboratory assistants per shift, samples 10 40 The productivity of the tests of the biocidal effect of the prototype is lower due to the need to perform them in zoned laboratories and personal protective equipment, as well as the duration (96 hours) of observation of laboratory animals to record the results, which excludes the proposed method.

The proposed method for indicating the biocidal activity of peroxide disinfectant solutions against microbial toxins has the following advantages over the prototype:

the duration of the registration of the biocidal effect of the disinfectant on ice-forming proteins, which are a biological simulator of botulinum neurotoxin, is reduced;

danger to personnel performing disinfection measures and recording results is eliminated;

the accuracy of determining the effectiveness of disinfection is improved by eliminating another biological factor - the use of laboratory animals to determine residual toxicity and completeness of disinfection measures;

research costs are significantly reduced due to the exclusion of work requiring special protective measures (zoned laboratories and vivariums, PPE of the skin and respiratory organs, special training of personnel, etc.);

productivity increases when using the instrumental method of recording the biocidal effect of peroxide disinfectants by the activity of biological nucleation centers.

The possibility of implementing the proposed method is shown in the following examples.

Example 1. The evaluation of the destructive effect of a 5% hydrogen peroxide solution on the BIL of Erwinia herbicola bacteria was carried out in dynamics at the temperatures of recording the crystallization effect of minus 7 ° C and minus 11 ° C by the suspension method.

table 2
The destructive effect of hydrogen peroxide on BIL bacteria Erwinia herbicola strain No. 1211 Disinfectant exposure time, min The concentration of nucleation centers at a temperature of minus ... ° C, tsn · cm ^-3 7 eleven 0 4.7 · 10 ⁴ 5,410 ⁵ 5 3.110 ² 5.1 · 10 ⁴ fifteen 0.110 ¹ 2.6 · 10 ³ thirty 0 1.5 · 10 ²

From the data presented it follows that a simple solution of hydrogen peroxide has some destructive activity in relation to the studied proteins. Larger and more labile protein complexes, recorded at a temperature of minus 7 ° С, are inactivated in 30 minutes. Stable protein complexes recorded at a temperature of minus 11 ° C are not completely inactivated. Their residual concentration after a 30-minute exposure indicates that complete destruction of the proteins does not occur.

Example 2. Evaluation of the destructive effect of activated formic acid solution of hydrogen peroxide on proteins Erwinia herbicola strain No. 1211 under the same conditions as in example 1.

Table 3
The destructive effect of the activated hydrogen peroxide solution on the BIL of bacteria Erwinia herbicola strain No. 1211 Disinfectant exposure time, min The concentration of nucleation centers at a temperature of minus ... ° C, tsn · cm ^-3 7 eleven 0 5.110 ³ 5.8 · 10 ⁵ 5 2.0 · 10 ¹ 3.110 ³ fifteen 0 1.4 · 10 ¹ thirty 0 0

Note. The composition of the disinfecting solution: hydrogen peroxide 5%, formic acid 1%, sulfonol 0.3%.

The results obtained indicate that the destructive activity of the activated hydrogen peroxide solution is significantly higher than that of its simple solution. However, the achievement of complete destruction of proteins is achieved only after a 15-minute exposure, which indicates some unreliability of the disinfectant used for these purposes.

Example 3. Evaluation of the destructive effect of a 10% solution of hydrogen peroxide with a 35% solution of ethyl alcohol on Erwinia sp. No. 16. The conditions of the experiment are similar to example No. 1.

The effect of the alcohol-peroxide solution of the tested concentration causes the complete destruction of the proteins of Erwinia herbicola strain No. 1211 after a 5-minute exposure, which indicates a high biocidal effect of the action of the tested solution.

Table 4
The destructive effect of alcohol-peroxide solution on the BIL of bacteria Erwinia herbicola strain No. 1211 Time
disinfectant exposure, min The concentration of nucleation centers at a temperature of minus ... ° C, tsn · cm ^-3 7 eleven 0 5.510 ³ 6.310 ⁵ 5 0 0 fifteen 0 0 thirty 0 0

The adequacy of the use of proteins initiating ice formation as a model of botulinum neurotoxin for the rapid determination of the effectiveness of disinfectants is due to the results of experimental studies of their effect on neurotoxin. Studies of the effectiveness of disinfectants based on hydrogen peroxide have shown that complete inactivation of botulinum toxin is observed in the range from 5 to 30 minutes. It was found that the maximum biocide is shown by a 10% hydrogen peroxide solution with a 35% solution of ethyl alcohol, the minimum by a hydrogen peroxide solution, and a hydrogen peroxide solution activated by formic acid with the addition of sulfonol occupies an intermediate position. These results are completely consistent with experimental data obtained using the ice-forming proteins of bacteria Erwinia herbicola strain No. 1211 (tables 2, 3 and 4).

It should also be noted that the current regulatory documentation (Sanitary and epidemiological rules. Safety of working with microorganisms of 1-11 pathogenicity (danger) groups - SP - 1.3.1285 - 03) requires the use of disinfectant solutions based on hydrogen peroxide for inactivation of microbial toxins.

Claims (1)

A method for indicating the biocidal activity of peroxide disinfectant solutions to botulinum neurotoxin, which consists in preparing preparations of globular proteins of a bacterial nature, the effect of the disinfecting composition on prepared preparations, exposure and evaluating the effectiveness of the disinfectant in preserving the specific properties of proteins after disinfection, characterized in that it is a globular protein preparation class C protein complexes are used that initiate ice formation supercooled water produced by microorganisms of the Erwinia and Pseudomonas genera, and the effectiveness of disinfecting solutions is assessed by instrumental cryoscopic microdrop method.