RU2372406C1 - Method of revealing contamination of objects in external environment by gram-negative bacteria pseudomonas and acinetobacter - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method deals with analysis of washout samples. Washout samples are filtered through bacterial filters with a pore diametre of 0.22 mcm that concentrate bacteria that are in small numbers and/or dormant forms followed by a PCR-analysis of the eluate total DNA using a genus-specific and species-specific primers for these bacteria.

EFFECT: invention enables to improve effectiveness of revealing contamination of objects of the external environment by bacteria.

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Description

The invention relates to the field of medicine, namely epidemiology and sanitary microbiology.

The effectiveness of microbiological monitoring of the sanitary condition in hospitals of various profiles largely depends on the diagnostic tests used. The solution to this problem is complicated in some cases by the ineffectiveness of bacteriological diagnostics, since in modern conditions the circulation of hospital strains can be accompanied by the formation of uncultivated forms of bacteria (A.V. Sokolenko, T.N. Mukhina, 2006) that are not detected by traditional cultural methods.

A known method of planned bacteriological control in hospitals, based on the determination of the total microbial number and the presence of sanitary-indicative microorganisms (including gram-negative bacteria of the group of Escherichia coli, etc.) in accordance with the order of the Ministry of Health of the USSR No. 720 of 07/31/1978, Appendix No. 2 and MU for the epidemiological surveillance of nosocomial infections. Ministry of Health of the USSR No. 28-6 / 34 dated 02.12.87.

The disadvantages of the prototype: time-consuming, time-consuming, does not reveal resting forms.

Effect: shortening the study time, the ability to identify resting forms of gram-negative bacteria.

The solution of this problem is carried out as follows: during sanitary control in medical institutions, washes from environmental objects are filtered through bacterial filters, the pore sizes of which ensure the concentration of all forms of bacteria, including those that are at rest, but exclude filter contamination by random DNA fragments. Then the filters are placed in a test tube with 1 ml of ultrapure water, boiled, centrifuged, the supernatant is separated, total DNA is precipitated from it with isopropyl alcohol and subjected to PCR analysis using species-specific and genus-specific primers of gram-negative bacteria.

The method is as follows: washings are obtained with a sterile cotton swab or gauze napkins of size 5 × 5 cm, for the moistening of which a sterile saline solution is used. When controlling small objects, washings are taken from the surface of the entire object. When controlling objects with a large surface, washings are carried out in several places of the studied object with an area of 100-200 cm ² . Tampons and napkins are placed in a bottle with 200 ml of physiological sterile solution.

In the conditions of a laminar box with strict adherence to aseptic and antiseptic rules, bacteria are deposited on sterile nitrocellulose bacterial filters with a filter surface of 25 mm and a pore diameter of 0.22 μm using a filter unit with a vacuum of 150 mm Hg. Each swab sample (200 ml) is filtered through one filter. The number of filters is determined by the number of samples taken for analysis. Next, the filters are sterilely transferred to test tubes of the Eppendorf type with 1 ml of ultrapure water, previously crushed with a sterile scalpel. Isolation of total DNA for the polymerase chain reaction is carried out according to the method described by G.G. Stone (1994). Eppendorfs with filters are heated for 10 minutes at a temperature of 97 ° C in a solid-state thermostat "Termite", centrifuged for 10 minutes at 10,000 rpm, the supernatant is transferred to another tube. For concentration, DNA is precipitated with isopropanol (1: 1), centrifuged for 10 minutes at 13000 rpm, the supernatant is removed, dried, the precipitate is dissolved in 25 μl of TE (10 mM Tris-HCl, 1 mM EDTA) and used for genetic studies. Storage of samples at a temperature of -18 ° C.

Molecular genetic studies are carried out by PCR (specific PCR) using primers (table), complementary to a specific DNA sequence characteristic of a strictly defined type of microorganism (Pseudomonas aeruginosa, Acinetobacter baumanii, etc.) and universal primers that allow amplification of fragments of genes present in all microorganisms of a particular taxonomic group (Pseudomonas, Klebsiella, etc.).

Primers for PCR analysis of 16S RNA genes of the most significant representatives of gram-negative bacteria - causative agents of nosocomial infections (F. Widmer et.al., 1998; T. Spilker, T. Coenye, 2004; J.F. Turton et.al., 2005). Microorganism Primer Sequence Fragment, bp Pseudomonas spp. Ps-for ggtctgagaggatgatcagt 969 Ps-rev ttagctccacctcgcggc P. aeruginosa PA-SS-F gggggatcttcggacctca 956 PA-SS-R tccttagagtgcccacccg A. baumannii Acinetf gaaggtagcttgctac 410 Acinetr actatctctaggtattaactaaagt

The amplification mode for AcinetF / AcinetR primers is the initial denaturation cycle for 2 minutes at 94 ° C, the following 35 cycles: 94 ° C for 30 s; 50 ° C - 60 s; 72 ° С - 90 s and the final cycle - 5 min at 72 ° С; for Ps-for / Ps-rev primers, the initial denaturation cycle is 5 minutes at 95 ° C, the following 40 cycles: 94 ° C - 30 s; 65 ° C - 60 s; 72 ° C - 70 s, the final cycle - 10 min at 72 ° C; for primers PA-SS-F / PA-SS-R, the initial denaturation cycle is 2 min at 95 ° C, then 35 cycles: 94 ° C - 20 sec; 58 ° C - 20 sec; 72 ° C - 40 sec. The final cycle of 1 min at 72 ° C.

Electrophoretic separation of the reaction products was carried out on a 1.2% agarose gel in Tris-borate buffer at an electric field strength of 6 V / cm. The bands were visualized and the data were documented after staining the gel with ethidium bromide using the BioDocAnalyze gel documentation system (Biometra, Germany).

To confirm the effectiveness of the proposed method for genetic control of microbial contamination of environmental objects in medical institutions, 10 studies were conducted in two large clinics of Perm.

Examples of practical application:

Example 1

The contamination of environmental objects by gram-negative non-fermenting bacteria was monitored in the general department of the Perm Regional Infectious Diseases Hospital. Samples were taken from the following facilities: No. 1 - a dining table and a bed in the ward; No. 2 - a table for collecting tests in a common corridor, No. 3 - a table in the treatment room.

PCR analysis of the DNA obtained from the samples as described above with the genus and species-specific primers for the 16S RNA genes of Pseudomonas spp. and A. baumannii.

By amplification with Ps-for / Ps-rev primers, fragments corresponding to the size of the 16S RNA gene of bacteria of the genus Pseudomonas were detected (Fig. 1). At the same time, bacteriological control was carried out in accordance with Appendix No. 2 to the Order of the Ministry of Health No. 720. To isolate staphylococci, inoculation was made directly on a Petri dish with yolk-salt agar of bacteria of the group of Escherichia coli - in 10-20% gall broth, followed by transfer to Endo medium. To identify Pseudomonas aeruginosa - direct culture on blood agar and Endo medium. Bacteriological research on Pseudomonas aeruginosa negative. Thus, using molecular genetic analysis, it was established that bacteria taken from the surface of the table in the treatment room of the department contain bacteria of the genus Pseudomonas.

Example 2

The contamination of environmental objects was carried out in the surgical department of the regional regional hospital in Perm. Samples were taken from the following objects: No. 1 - endotracheal tube of a ventilator; No. 2 - operating table, No. 3 - bed in the intensive care unit.

Amplification with primers Ps-for / Ps-rev and PA-SS-F / PA-SS-R gave a positive result with material from sample No. 1, which was obtained within one working day (8 hours). A bacteriological study confirmed the presence of P. aeruginosa bacteria in the flush, but it took 3 days (72 hours) to conduct it.

Example 3. The control of contamination by gram-negative non-enzymatic bacteria of environmental objects in the intensive care unit of CIB No. 1 in Perm. Samples were taken from the following facilities: No. 1 - bed; No. 2 - a bedside table, No. 3 - a window sill, No. 4 - a washstand; No. 5 - a ship.

Genetic research was carried out in a similar way with three types of primers. Bacteriological research on Pseudomonas aeruginosa and acinetobacteria - negative. In samples No. 4 and No. 5, DNA fragments were found encoding the A. baumannii 16S RNA gene.

The positive effect of the proposed method is a better control of bacterial contamination of environmental objects in medical institutions, taking into account uncultivated forms of microorganisms, which under certain conditions can cause nosocomial infection, but are not detected using the existing bacteriological method.

Claims (1)

A method for detecting the contamination of environmental objects by gram-negative bacteria of the genus Pseudomonas and Acinetobacter by studying swabs, characterized in that the swabs are filtered through bacterial filters with a pore diameter of 0.22 μm, on which bacteria contained in minimal quantities and / or resting forms are concentrated, with subsequent PCR analysis of eluted total DNA using genus-and species-specific primers for these bacteria.

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