RU2101351C1 - Method of differentiation of anthrax microbe strains by virulence in vitro - Google Patents

Abstract

FIELD: medicinal microbiology. SUBSTANCE: invention relates to intraspecies taxonomy of Bacillus anthracis. Method involves the use capsule-formation in vitro and additionally toxin production. Both parameters were determined simultaneously on agar for immunological investigation. This ensures to differentiate strains for the following 5 groups: high virulent, moderate virulent, weak virulent, avirulent and apathogenic while the known method ensures to detect only two groups: virulent and avirulent. EFFECT: improved method of differentiation. 3 tbl, 3 dwg

Description

 The invention relates to medical microbiology, and in particular, to the determination of virulence and differentiation of strains of Bacillus anthracis on this basis in vitro.

 The pathogenicity of the anthrax microbe is determined by two factors - the capsule and exotoxin. These two main pathogenicity factors are produced by the microorganism in vivo during the pathological infectious process in the body of an animal or person sensitive to anthrax, as well as in vitro when cultured on artificial nutrient media under conditions that simulate the internal environment of the macroorganism. The strains of the anthrax microbe differ in the production of these factors, which as a result determines their different virulence. Therefore, the virulence of the strains can be estimated in vivo by the dose of B.anthracis cells, causing the death of half in all animals taken in the experiment, or in vitro by the presence and nature of the capsule and exotoxin production.

There is a method of differentiating strains of anthrax microbe by virulence in vivo [1] consisting in preparing a suspension of spores of anthrax microbe with a concentration of 1, 10, 100, 1000 and 10,000 spores in an infectious dose, each of which infects 6 outbred mice or 3 rabbits or guinea pig of each of the last three doses. Then, the death of animals from anthrax is recorded within 10 days after infection, DCL for rabbits or guinea pigs, or LD ₅₀ for white mice is calculated. Strains are virulent if DCL for guinea pigs or rabbits or LD ₅₀ for white mice does not exceed 10,000 spores.

 The disadvantages of this method are the use of a large number of laboratory animals (30 white mice or 9 guinea pigs or rabbits), the dependence of the results on the physiological state of animals, the ambiguity of determining virulence in different types of laboratory animals, the low degree of differentiation of strains with the release of only two groups of virulent and avirulent .

Closest to the proposed is a method for differentiating strains of anthrax microbe by virulence in vitro [2] which involves the parallel seeding of strains on nutrient agar and capsule formation medium, cultivation of crops on nutrient agar in an air atmosphere, and capsule formation in a CO ₂ atmosphere followed by capsule formation and differentiation of strains into 3 groups depending on the ability to capsule formation and its nature. I forming a capsule in an atmosphere of CO ₂ , II forming a capsule in an atmosphere of CO ₂ and in air, III not forming a capsule, while group I are virulent strains, and II and III are avirulent.

 The disadvantage of this method is the inability to differentiate strains of groups I and III by virulence, although in experiments on white mice in vivo strains of group II are virulent and group III avirulent. This disadvantage is determined by the inability to take into account the toxin formation of the strains.

It was experimentally shown that while simultaneously determining in vitro capsule production in a CO ₂ atmosphere and in air and toxin production in a CO ₂ atmosphere, all strains can be divided into 5 groups having different virulence determined in an animal experiment.

 The aim of the invention is to improve the accuracy of the method of differentiation of strains by virulence in vitro.

The goal is achieved in that the method of differentiating strains of the anthrax microbe by virulence in vitro involves the parallel plating of the cells of the tested strains on the surface of nutrient agar and a dense capsule-forming medium, cultivation of crops on nutrient agar in an air atmosphere, and on the capsule-forming medium in an atmosphere of CO ₂ , followed by the formation of CO ₂ capsules, moreover, Hottinger agar is used as nutrient agar, and agar for immunological research containing antitoxic antibodies (RYE), crops thereon cultured for 40-48 hours at 35-37 ^o C, then kept at 4-10 ^o C for 48-72 hours and further keeping carried formation toxin strains.

In relation to the prototype of the proposed method has distinctive features using Hottinger as nutrient agar, as an RYE capsule formation medium, culturing on RYE in a CO ₂ atmosphere for 40-48 hours at 35-37 ^o C, followed by aging at 4-10 ^o C for 48-72 hours and accounting for the formation of toxin.

 This achieves the goal of increasing the accuracy of differentiation of strains by virulence in vitro.

The proposed method allows to select instead of 2 degrees 5 degrees of virulence of strains with the corresponding phenotype:
1. Highly virulent, forming a capsule and toxin in an atmosphere of CO ₂ and not forming a capsule in air.

2. Moderately virulent, forming a capsule and not forming a toxin in the atmosphere of CO ₂ and not forming a capsule in the air.

3. Weakly virulent, forming a capsule in an atmosphere of CO ₂ and in air and not forming a toxin in an atmosphere of CO ₂ .

4. Avirulent not forming a capsule in any conditions and forming a toxin in an atmosphere of CO ₂ ;
5. Apatogenic not forming a capsule and toxin. In addition to higher accuracy of differentiation, the method also has an advantage over the known method of differentiation of strains by virulence in vivo, since it does not require the use of laboratory animals, reduces the study time (from 10 to 6 days) and material costs, allowing the use of 50 strains instead of 300 rabbits for differentiation or guinea pigs or 1,500 white mice 4 cups with culture media.

The invention is as follows. RYE [3] is prepared by dissolving in 358 ml of distilled water the ingredients, which are taken in the ratio, g:
1-Arginine • HCl 0.125
1-Aspartic acid 0.184
1-Valine 0.137
1-Histidine • HCl 0.055
Glycine 0.065
1-monosodium glutamate 0.612
1-Isoleucine 0.170
1-Leucine 0.230
1-Lysine 0.230
1-Methionine 0.073
1-Proline 0.043
1-Serine 0.235
1-Threonine 0.120
1-tyrosine 0.144
1-Tryptophan 0.035
1-Phenylalanine 0.125
1-Cystine 0.025
Adenine 0.0021
Uracil 0.0014
Thiamine • HCl 0.001
CaCl ₂ • 2H ₂ O 0.012
MgSO ₄ • H ₂ O 0.0099
MnSO ₄ • H ₂ O 0,0009
K ₂ HPO ₄ 3,000
NaHCO ₃ 8,000
d (+) glucose 2,500
Yeast Extract 8,000
The pH of the medium is monitored and, if necessary, adjusted to 8.0. The resulting solution of the nutrient base of the medium is sterilized by filtration through a membrane filter with a pore diameter of 0.22-0.45 μm. A portion of agarose 15 g is melted in 500 ml of distilled water and sterilized by autoclaving at 121 ^o C for 20 minutes A 358 ml sterile nutrient base solution and lamb antiserum against spores of anthrax vaccine strain 34F ₂ in a 142 ml volume are added to molten and cooled to 50 ^° C agarose, mixed and poured into 10-12 ml Petri dishes. Prepare suspensions of spores of the studied strains with an optical density corresponding to a CCA turbidity GISK 10 units. Inoculum is applied by loop with plaques with a diameter of 3-4 mm or a replicator of 5 to 25 inoculum in parallel on one cup with RYE and one cup with Hottinger agar. To control the quality of the medium, a culture of a known virulent strain is sown. RYE plates are placed in a microaerostat or CO ₂ incubator. 25% of the air is pumped out of the microaerostat by a vacuum pump, focusing on the readings of the manovacuum meter, and replaced with carbon dioxide. In a CO ₂ incubator, plates are cultured with 10% carbon dioxide. RYE crops were grown at 35-37 ^° C for 40-48 hours, then transferred to a refrigerator from a microaerostat or a CO ₂ incubator, and kept there for 48-72 hours at 4-10 ^° C. Cups with Hottinger agar were incubated at 37 ^o C in a normal air atmosphere for 24 hours

At the end of the indicated incubation periods, the plates are scanned, recording the colony morphology. The capsule producing culture form colonies in the SM form rounded, smooth, shiny, with a mucous capsule substance on the surface. Non-capsule-producing cultures grow in the R-form in the form of dull, dull, dry, rough colonies with a fibrous edge. To confirm the formation of capsules from the colonies, smears for microscopy are prepared. Smears are made on fat-free glass slides, dried in air and fixed in 96 ^° ethanol with 6% hydrogen peroxide for 20 minutes. Smears are stained with a Rebiger solution for 15-20 s, washed and dried. Microscopy smears using a lens 90 with oil immersion. Capsules turn pink or remain unpainted, bacteria in dark purple.

 Toxin formation is detected by the method [3]. For this, RYE cups are viewed under natural light on a black background using a sheet of black paper. Around the toxin-producing colonies in the medium, concentric halos of immunoprecipitation are formed in the form of white thin rings. The toxin producing colonies lack such halos.

Cultures relate to:
1) highly virulent if they produce a capsule and toxin in a CO ₂ atmosphere and do not produce a capsule in air;
2) moderately virulent, if they do not produce a toxin, produce a capsule in an atmosphere of CO ₂ and do not produce it in air;
3) weakly virulent if they do not produce a toxin and produce a capsule in an atmosphere of CO ₂ and in air;
4) avirulent if they produce a toxin but do not produce a capsule;
5) pathogenic if they do not produce either a capsule or a toxin.

 The results of experimental tests of the invention are illustrated by comparative tables and graphic materials.

 Table 1 presents the comparative results of the differentiation of B. anthracis strains by the proposed method and the analogue method; table 2 - comparative results of the differentiation of strains of B.anthracis in vitro by the proposed method and the prototype method; in table 3 the results of differentiation of B.anthracis strains by virulence in vitro by the proposed method using a replicator.

 Figure 1 presents the drawings of colonies on Petri dishes for differentiation of B. anthracis strains by virulence by the proposed method and prototype method, where A is a cup with RYE, B is a cup with Hottinger agar, C is a cup with bicarbonate agar. Sector numbers correspond to the sequence numbers of the strains given in table. 2. On Petri dishes A and B, accounting for the growth of colonies of the studied strains by the proposed method. On plates B and C, accounting for the growth of colonies of the same strains by the prototype method.

 In FIG. 2 presents drawings of strain colonies on Petri dishes, differentiated by the proposed method using a replicator, where A is a cup with RYE, B is a cup with Hottinger agar, C is a diagram of the locations of strain colonies on plates A and B. The colony numbers in Figure C correspond to the sequence numbers of the strains, given in table 3.

In FIG. 3 presents drawings of colony strains on Petri dishes with RYE, differentiated by the proposed method, with different cultivation times in an atmosphere of CO ₂ at 37 ^° C, where A is the cultivation time 24 hours, B is the cultivation time 40 hours, C the cultivation time 48 hours, D the cultivation time is 72 hours. The sector numbers on the plates correspond to the serial numbers of the strains shown in Table 2.

 The possibility of practical implementation of the invention using the full totality of the claimed features is shown in specific examples.

Example 1. Prepared in accordance with the prescription, one cup of RYE and one cup of Hottinger agar. Prepared suspensions of spores with optical density corresponding to CCO GISK 10 units. the following strains of anthrax microbe: 81/1, 228, 140P, STI-1, 12 / 16-1. On the surface of one cup with each of the media, having previously drawn the reverse side of the bottom of the cup into sectors or squares, a plaque of a bacteriological loop was inoculated into the corresponding sectors of the spore suspension of the listed strains. A Cup with Hottinger agar was placed in a thermostat with a temperature of 37 ^o C and a normal air atmosphere, kept for 24 hours, after which capsule formation was taken into account. The RYE cup was placed in a microaerostat, from which 25% of the air was pumped out and replaced with carbon dioxide, kept at 37 ^° C for 48 hours, then capsule formation was taken into account, and then transferred to a refrigerator and kept at 4 ^° C for 72 hours. at the end of this period, toxin formation at RYE was taken into account and differentiation of the strains was carried out in accordance with the given scheme.

Spore suspensions of the same strains were prepared so that 0.2 ml contained 1, 10, 100, 1000 and 10000 spores for each strain. White doses of outbred mice (6 animals per dose) were infected with these doses, and 3 rabbits were infected with doses of 100, 1000 and 10,000 spores. The death of animals from anthrax within 10 days after infection was recorded and LD ₅₀ for white mice and DCL for rabbits were determined in accordance with [1] If LD ₅₀ for white mice or DCL for rabbits did not exceed 10,000 spores, the strains were virulent if they were above 10,000 considered avirulent.

Example 2. Prepared in accordance with the prescriptions for one cup of culture media: 1) RYE, 2) 1% bicarbonate agar, 3) Hottinger agar. To prepare 1% bicarbonate agar, 10 g of baking soda was dissolved in 100 ml of sterile distilled water with light heating on a flame of an alcohol lamp, then 10 ml of the obtained 10% soda solution was added to 100 ml of Hottinger agar melted and cooled to 50 ^° C and poured into Petri dishes. Prepared suspensions of spores with optical density corresponding to CCO GISK 10 units. the following strains of anthrax microbe: 81/1, 228, 140P, STI-1, 12 / 16-1. On the surface of one cup with each of the media, having previously drawn the reverse side of the bottom of the cup into sectors or squares, a plaque of a bacteriological loop was inoculated into the corresponding sectors of the spore suspension of the listed strains. A Cup with Hottinger agar was placed in a thermostat with a temperature of 37 ^o C and a normal air atmosphere, kept for 24 hours, after which capsule formation was taken into account. RYE cups and bicarbonate agar were placed in a microaerostat, from which 25% of the air was pumped out and replaced with carbon dioxide, kept at 35 ^° C for 48 hours, then capsule formation was taken into account, and then the RYE cup was transferred to a refrigerator and kept at 10 ^° C within 48 hours. At the end of this period, toxin formation at RYE was taken into account. Differentiation of the strains in accordance with the proposed method was carried out, taking into account the growth results on RYE and Hottinger agar, and according to the prototype method, considering the growth on bicarbonate agar and Hottinger agar.

 Example 3. A cup was prepared with RYE according to the recipe and technology described above, as well as a cup with Hottinger agar. Analogously to example 1, suspension of spores of 25 strains of anthrax microbe was prepared. 25 strains were sown in parallel on a pair of Hottinger-RYE agar plates using a 25-pin replicator, for which 0.1 ml of the spore suspension of one of the strains was added to each well of the replicator matrix, then the replicator was lowered into the matrix and transferred to a dense medium RYE. The same operation was repeated with a cup of Hottinger agar. The plates were incubated and the results taken into account as described in the previous example.

 Example 4. Prepared, as described above, 4 cups of RYE and one cup of Hottinger agar. The same strains were seeded onto the sectors of the cups with loop plaques as in Example 1. One RYE cup was grown in an anaerostat for 24 hours, the second 40 hours, the third 48 hours and the fourth 72 hours. At the end of these periods, capsule formation was taken into account, and after holding the cups for 72 hours in the refrigerator - toxin formation. Differentiation of the strains was performed as described in the previous examples.

As can be seen from table 1, the differentiation of the proposed method allows you to select 5 groups of strains instead of 2, allocated during differentiation according to the similar method. Differences in the virulence of the first three groups, identified using the proposed method, are confirmed by quantitative differences in the LD ₅₀ value for white mice. The proposed method eliminates the ambiguity in the interpretation of the virulence of group 3 strains, which is characteristic of the analogous method using different laboratory animals, in which strain 140P can be regarded as virulent (for white mice) and avirulent (for rabbits). Thus, the proposed method improves the accuracy of determination and objectivity of the results compared with the analogue method.

 Comparative results of differentiation by the proposed method and the prototype method, shown in table 2 and figure 1, show that the prototype method (B and C, figure 1) does not take into account the production of toxin by strains, and differentiation is only based on differences in capsule production. Capsule-producing strains (1, 2, and 3) form on RYE (C) and Hottinger agar (B) rounded mucous shiny colonies coated with a capsule layer. In contrast to the prototype method in the proposed method (A and B, Fig. 1) on RYE (A), in addition to capsule formation in strains 1, 2 and 3, toxin formation in strains 1 and 4 is also recorded as clearly visible on a dark background white rings of immunoprecipitation around colonies in medium in a Petri dish. Accounting for toxin formation allows to select 5 groups instead of 2 virulence groups in the prototype method (Table 2) and thereby increase the accuracy of differentiation of strains in vitro.

 Using a replicator with 25 pins allows the simultaneous differentiation of up to 25 strains on one Petri dish with RYE and one with Hottinger agar. Figure 2 shows that the nearby colonies producing toxin are surrounded by merged halos of immunoprecipitation, which allows you to immediately distinguish them from the rest of the colonies. Accounting for capsule and toxin formation allows us to simultaneously differentiate all 25 strains with their division into 5 groups, which is reflected in Table 3.

The results of the experiments showed that the claimed time interval of cultivation at RYE prt 37 ^o C in an atmosphere of CO _{2 is} necessary and sufficient to obtain the desired result. Figure 3 shows that cultivation for 24 hours is not enough, since the colonies are small, and immunoprecipitation is not observed (A). Cultivation for 40 (B) and 48 (C) h provides toxin formation (1 and 4) and capsule formation (1,2 and 3) that are clearly visible to the eye. When cultured for 72 hours, the colonies “outgrow” and toxin formation becomes impossible (D).

The results of the experiments showed that the claimed culture temperature ranges of 35 ^o C (example 2) - 37 ^o C (example 1) and keeping in the cold cups with crops on RYE 4 ^o C (example 1) - 10 ^o C (example 2), and also the time of keeping them in the cold 48 hours (example 1) - 72 hours (example 2) are necessary and sufficient to obtain the necessary positive result.

 Thus, the proposed method for differentiation of B.anthracis strains by virulence in vitro increases the accuracy of differentiation. Unlike the known methods, it allows you to select instead of 2 5 groups of strains with different virulence. The method requires significantly less time and money, is not associated with the use of laboratory animals and therefore is devoid of the disadvantages of the in vivo method, determined by differences in sensitivity to infection in different types of laboratory animals and the physiological state of animals of the same species.

Sources of information
1. Burgasov P.N. Rozhkov G.I. Siberian ulcer infection. M. Medicine, 1984. -208 p.

 2. Thorne C. B. Biochemical properties of virulent and avirulent strains of Bacillus anthracis // Ann. Met.N.-Y. Acad.Sci.-1960.-Vol.88.-N 6.-P.1024-1033.

 3. Ivins B. E. Welkos S.L. Cloning and expression of Bacillus anthracis protective antigen gene in Bacillus subtilis // Infect.and Immun.-1986.-Vol. 54.-N 2.-P.537-542.

Claims (1)

A method for differentiating strains of the anthrax microbe by in vitro virulence, including the parallel seeding of cells of the tested strains on the surface of nutrient agar and a dense capsule-forming medium, culturing crops on nutrient agar in an air atmosphere, and on capsule-forming medium in a CO ₂ atmosphere, followed by capsule formation, characterized in that that Hottinger agar is used as nutrient agar, and an anti-Agar agar is used as a capsule formation medium for immunological studies classically antibody crops cultivated thereon at 48 for 40 hours at 35 37 ^o C, then held at April 10 ^o C for 48 72 hours and further keeping carried toxin formation.

Images