RU2619220C1 - Method for mycobacteria detection from surfaces - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for mycobacteria detection from surfaces involves material collection from the surface using a swab moistened with saline and processing thereof by means of "Septustin" disinfectant in a 1% aqueous solution. At that, "Septustin" is mixed with a suspension of the studied material in a ratio of 1:4 for 30 minutes. Moreover, overall exposure incudes 15 minutes of centrifugation at 3000 rpm. The studied material is centrifuged for 15 min at 3000 rpm and the residue is washed with physiological saline twice by centrifugation under the same regime. Plating from the residue is carried out on a solid growth medium at the optimum temperature and time with subsequent colonies isolation and identification.

EFFECT: invention improves the accuracy of laboratory research and reduces research time.

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Description

A method for detecting mycobacteria from surfaces

The invention relates to microbiology and can be used for epidemiological purposes to detect massive contamination of surfaces with mycobacteria and quality control of disinfection measures.

Tuberculosis is a separate group of diseases in which environmental objects are of great importance in the occurrence and spread of this infection. The development of the infectious and the nature of the epizootic process are determined by a whole range of factors, including the mechanism of transmission of infection. The main ways of contracting tuberculosis are aerogenic and alimentary. Given that with tuberculosis, the pathogen is localized throughout the body, its excretion occurs multiple times (with sputum when coughing, feces, milk), and mycobacteria isolated from the body of a sick animal retain their virulence and viability: in manure - up to 7 months ; in corpses - up to a year; in the soil - more than two years; in meat - up to a year / 1 /, due to the structure of the cell wall and its high content of lipids and waxes. Therefore, contaminated environmental objects - air, soil, water, feed and animal products - play an important role in the emergence of new foci of tuberculosis. Mycobacteria with lost virulence in the presence of susceptible animals lead to infection / 2, 3 /. Atypical mycobacteria reproduce freely in the environment (soil, water), they are isolated from the soil of meadows, pastures, cattle-breeding lands, samples of straw, silage, hay and haylage, compound feed, from vegetables, from samples of peat, trees, as well as reservoirs, wells and tap water. Under certain conditions, atypical mycobacteria, entering animals, can take root in it, causing sensitization to tuberculin, which is explained by their antigenic relationship with the causative agent of tuberculosis, thereby giving a cross-reaction with tuberculin, and is confirmed by their isolation from pathological material from dead animals that react on tuberculin / 4, 5 /. Under natural conditions, cattle sensitization occurs with atypical mycobacteria / 6 /. In some cases, some types of atypical mycobacteria can cause classic tuberculosis lesions (MAIS).

All known methods for detecting mycobacteria from surfaces are based on taking the wash off from the surface with a moistened swab, processing the test material before sowing to eliminate extraneous microflora, and sowing on nutrient media. The presence of mycobacteria and their number on the studied surfaces is judged on the basis of their growth on a nutrient medium / 7 /.

The detection of mycobacteria from environmental objects is complicated by the extreme degree of contamination of samples from livestock buildings with mechanical impurities and commonplace microflora. To suppress the growth of extraneous microflora and isolate pure cultures of tuberculosis pathogens, various methods of treating it with acid, alkali or other chemicals of various concentrations at different exposures are used depending on the degree of contamination.

In the practice of microbiological laboratories, caustic soda solutions are currently used (flotation method by O.V. Martov, 1971), calcium hypochlorite and other solutions, sulfuric solutions (A.F. Korzhinskaya, 1926; K.K. Kresling, 1929; A. N. Makkaveyskaya, 1956), hydrochloric acid (Yu.A. Yudenich, 1928), oxalic acid (the Gon-Levenshtein-Sumiosi method, which we consider as an analogue, provides for the use of solutions of sulfuric acid with a concentration of 3-5 or 6% or solution of oxalic acid concentration of 5 or 10% in the ratio: per 1 volume part of the material, 4 parts of acid, centrifugation for 10-15 minutes with a rotation speed of 3000 min, washing the precipitate 2-3 times with physiological saline using centrifugation and subsequent cultivation in a solid nutrient medium / 8 /.

The disadvantages of these methods include the destructive, to some extent, the effect of acids and alkalis on mycobacteria. However, the percentage of pollution remains high, especially when processing samples taken from environmental objects. Solutions of acids in low concentrations for processing the material studied for the presence of mycobacteria do not ensure its release from the accompanying microflora. So, the use of 3% solutions of sulfuric and hydrochloric acids and 5% oxalic acid solution at an exposure of 20-40 minutes does not ensure sterility of crops in 56% of cases, the use of 2-4% caustic soda solutions is ineffective due to the small percentage of mycobacteria sown. Detergents used in medical practice are unsuitable for processing, since in 70-87.5% of cases they do not suppress the growth of concomitant microflora in samples taken from environmental objects and in homogenates of biomaterial obtained from an animal / 9 /. When processing the material with a 5% solution of oxalic acid without subsequent washing with physiological saline, as recommended by Bannikova B.N. (1980), the growth of mycobacteria on Levenshtein-Jensen, Finn II media does not occur, since the pH of the medium in it varies below 6, 8/10 /.

The purpose of the invention is to increase the accuracy of laboratory diagnostics by reducing quantitative losses and maintaining the viability of tuberculosis mycobacteria in the test material while reducing the time of laboratory tests.

The goal is achieved by optimizing the conditions of pre-sowing treatment using the disinfectant "Septustine".

Septutin disinfectant (manufacturer of Uralstinol BIO LLC, Russia) is recommended for disinfection of veterinary surveillance facilities. This preparation from the group of cationic surfactants contains catamine AB as a disinfectant, as well as isopropyl alcohol, nonionic surfactant, sodium bicarbonate and bromphenol blue, has a wide spectrum of action against pathogens of infectious bacterial diseases (including tuberculosis), viral and fungal etiology / 11 / .

The method is as follows.

Wash samples from the surfaces of environmental objects are treated with Septutin disinfectant in the form of an aqueous solution of 1.0% concentration, which is mixed with a suspension of the test material in a ratio of 1: 4 for 30 minutes, and the total exposure includes 15 minutes of centrifugation with a frequency rotation of 3000 rpm, followed by twofold washing with physiological saline by centrifugation under the same mode.

The parameters of the method were tested in the course of preliminary studies to determine the effect of aqueous solutions of the Septutin disinfectant of various concentrations on the survival and sowing of mycobacteria from environmental objects, as well as on the commonplace microflora (tables 1, 2).

The feasibility and effectiveness of the claimed method was determined on 5 samples of swabs from various surfaces of the livestock building (table 3).

The essence of the method is illustrated by examples.

Example 1. From surfaces (1 sq. M) of the wall of the livestock building with a sterile cotton-gauze swab moistened with physiological saline, 5 wash-out samples were taken, which were combined into one sample.

The combined sample was divided into 5 samples of 5 ml of suspension. The test samples in a volume of 5 ml were centrifuged for 25 minutes with a rotation speed of 3000 rpm, after centrifugation, the supernatant was drained, the centrifugate was adjusted with sterile saline to 1 ml (suspension). The test material was treated with Septutin disinfectant in the form of an aqueous solution of 1.0% concentration, which was mixed with a suspension of the test material in a ratio of 1: 4 for 30 minutes, then the test material was centrifuged for 15 minutes at a speed of 3000 rpm (the centrifugation time was included in the total exposure to the suspension of a 1.0% Septupine aqueous solution), after centrifugation, the supernatant was decanted, the precipitate was washed 2 times with sterile saline solution using centrifugation in the same mode. From the sediment, crops were sown on Wednesday of Levenshtein-Jensen and Finn-2. Crops were incubated in an incubator at 37 ° C and examined for 90 days for the growth of colonies of mycobacteria. Throughout the entire period of the study, as colonies appeared, smears were prepared, followed by Gram and Tsil-Nielsen staining. Identification of the selected cultures was carried out by gas-liquid chromatography (GLC) on a Chromos GC 1000 instrument. The timing of the appearance of growth and the number of selected cultures of mycobacteria were taken into account. Control is the Gon-Levenshtein-Sumiosi method. The results are presented in table 4.

As can be seen from table 4, in the experimental samples on the 10th day, and in the control sample on the 12th day, yellow colonies appeared. When staining smears according to Ziehl-Nielsen, thin sticks of pale pink color were found, which were identified as M. gordonae, which was confirmed by gas-liquid chromatography (absence of tuberculostearic acid (C _{19 development} ), peak C ₁₄ and C _{14 development} - double, completely not branched). Growth over the entire surface was noted in the fourth sample processed by the claimed method.

Example 2. The identification of mycobacteria from the floor surface of the livestock building was carried out as in example 1. The results are presented in table 5.

As can be seen from table 5, in the third sample on day 10, yellow shiny colonies appeared. Thin sticks of pale pink color were found in smears stained according to Ziehl-Nielsen. The culture was identified by gas chromatography as M. scrofulacium (the presence of higher mycolic acids with the number of carbon atoms is greater than 20, the peak C _{24: 0 is} greater than _{22: 0} , the peak C _{26: 0} is absent).

Example 3. The detection of mycobacteria from the surface of the pipe of the livestock building was carried out as in example 1. The results are presented in table 6.

As can be seen from table 6, in the third and fourth experimental samples, mucous shiny yellow colonies grew on day 8. According to Tsil-Nielsen staining and morphological characteristics, they were identified as mycobacteria, which is confirmed by the study of the fatty acid spectrum of cells by gas-liquid chromatography. According to chemotaxonomic characteristics (peaks of acids with 17 and 19 carbon atoms and unsaturated fatty acids with more than 20 carbon atoms are clearly pronounced, behenic acid C _{22: 0} prevailed, C _{26: 0} peak was absent), mycobacteria were identified as M. vaccae. In all samples, yellow colonies appeared on day 12. When staining smears according to Ziehl-Nielsen, thin sticks of pale pink color were found, which were identified as M. gordonae, which was confirmed by gas-liquid chromatography (absence of tuberculostearic acid (C _{19 development} ), peak C ₁₄ and C _{14 development} - double, completely not branched).

Example 4. The detection of mycobacteria from the surface of the dung passage of the livestock building was carried out as in example 1. The results are presented in table 7.

As can be seen from table 7, on day 10 in the third sample appeared yellow shiny colonies. Smears stained according to Ziehl-Nielsen showed thin rods of pale pink color. The culture was identified by gas chromatography as M. scrofulacium (the presence of higher mycolic acids with more than 20 carbon atoms, C _{24: 0} peak, C _{22: 0} , C _{26: 0} peak, is absent). Mycobacteria were not detected in other samples.

Example 5. The detection of mycobacteria from the surface of the feeder of the livestock building was carried out as in example 1. The results are presented in table 8.

Example 6. The detection of mycobacteria from the surface of the drinking bowl of the livestock building was carried out as in example 1. The results are presented in table 9.

As can be seen from table 9, in all samples of swabs from the surface of the drinking bowl, colonies with a pale yellow pigment appeared. In smears when stained according to Ziehl-Nielsen, small, thin, acid-resistant bacilli and cocci were found. Chromatograms of C _{26: 0} peaks were absent, C _{24: 0} peak height was significantly higher than C _{22: 0} , C _{19: 0} peak in a significant amount, mycobacterium was identified as M. avium.

The research results are summarized in tables 10 and 11.

Example 7. Conducted the identification of mycobacteria in the economy, dysfunctional for tuberculosis. Samples were taken from the surfaces of environmental objects (feeders, walls, drinking bowls). The results are presented in table 12.

Studies have confirmed that a 1.0% aqueous solution of Septutin disinfectant at a 30-minute exposure restrained the development of concomitant microflora in materials from the surfaces of environmental objects. The claimed method improves the reliability of the results by increasing the information content of studies to identify mycobacteria, meets modern requirements when working with infectious material, safe and effective when working.

Information sources

1. Kuzin A.I. Tuberculosis of farm animals and its prevention. - M.: Rosagropromizdat, 1992 .-- 189 p.

2. Dekanosidze T.V. The resistance of mycobacterium tuberculosis in cattle to aerosols of disinfectants and the modes of their use: abstract. dis. ... cand. vet. Sciences: 16.00.03 / Dekanosidze Tamara Vladimirovna. - M., 1989 .-- 24 p.

3. Dudnitsky, I.A. Disinfection on farms, dysfunctional for brucellosis and tuberculosis / I.A. Dudnitsky, V.F. Brichko, I.D. Belyaev et al. // Veterinary medicine. - 1989. - No. 6. - S. 7-11.

4. Tupota S.G. A comprehensive comparative assessment of the epizootic situation of animal tuberculosis and methods for its diagnosis in agricultural enterprises and personal subsidiary plots: dis. ... cand. vet. Sciences: 06.02.02 / Tupota Sergey Grigorevich. IEVSiDV. - Novosibirsk, 2010 - 149 s.

5. Sharov A.N. The effectiveness of methods for the intravital diagnosis of tuberculosis / A.N. Sharov, L.A. Eroshenko, I.P. Sukhanov et al. // Veterinary medicine. - 2002. - No. 2. - S. 16-18.

6. Schurevsky V.E. Fast-growing atypical mycobacteria and their importance in the pathology of cattle / V.E. Schurevsky, N.P. Ovdienko, A.M. Kadochkin, V.N. Kudyakov // Veterinary medicine. - 1984. - No. 9. - S. 29-30.

7. Order of the Ministry of Health of the Russian Federation 109 dated March 21, 2003 “On the improvement of anti-TB measures in the Russian Federation”, Appendix 11 “Instructions for unified microbiological studies in the detection, diagnosis and treatment of tuberculosis” p. 205-206.

8. GOST 26072-89 (ST SEV 3457-81) Agricultural animals and poultry. Laboratory methods for the diagnosis of tuberculosis.

9. Schurevsky V.E., Kosenko V.I., Tazhgaliev N.M. The use of various chemicals to treat pathological material and their effect on the sowing rate of mycobacteria. / Bulletin VIEV. - 1987. - V. 64. - S. 15-19.

10. Kolychev N. characterization of mycobacteria isolated from the objects of animal and livestock farms. / Sat nt Sib. NIVI. - 1976 .-- S. 47-55.

11. Manual on the use of the drug “Septustine” for the disinfection of objects of veterinary surveillance. Uralstinol. - BIO. - 2002.

Claims (1)

A method for detecting mycobacteria from surfaces, including collecting the test material from the surface with a swab moistened with physiological saline, processing the test material before sowing to eliminate extraneous microflora, sowing on a solid nutrient medium, followed by cultivation at a temperature and temporary optimum, characterized in that as a means, inhibiting the development of extraneous microflora, they use the disinfectant Septupin in the form of an aqueous solution of 1.0% concentration, which I mix with a suspension of the material in a ratio of 1: 4 for 30 minutes, and the overall exposure consists of 15 minutes of centrifugation at 3000 rev / min, followed by washing twice with physiological saline by centrifugation under the same regime.