RU2435845C1 - METHOD OF Shewanella BACTERIA RECOVERY AND IDENTIFICATION - Google Patents

Abstract

FIELD: medicine. ^ SUBSTANCE: method provides test inoculation of a nutrient medium containing pancreatic fish flour hydolyzate, fermentative meat peptone, NaCl, Tween-80, CaCl2, sodium thiosulphate (Na2S2O3 ù 5H2O), ferrous ammonium sulphate ((NH4)2SO4 ù FeSO4 ù 6H2O), sorbite, bromthymol blue, irgasan (DP-300), rifampicin, NaOH, agar and distilled water in the preset proportions. Pancreatic fish flour hydolyzate, peptone, NaCl, agar are dissolved with heating, sterilised at 121C for 20 min and thereafter added in a hot medium of the other components specified above. The inoculations are incubated on the nutrient medium in aerobic conditions at temperature 37C and/or 28C for 24-48 h and assessed by the presence of black-centre green or dark grey colonies surrounded by a cloudy precipitate zone in the nutrient medium. ^ EFFECT: invention allows simplifying and providing higher specificity of Shewanella bacteria recovery and identification. ^ 2 ex

Description

The invention relates to the field of microbiology. It can be used in bacteriological studies on the isolation and identification of bacteria of the genus Shewanella and the production of culture media for these studies.

A known method of isolation and identification of bacteria of the genus Shewanella directly from the source material on marine 2216 agar (Difco Lab.) Or nutrient agar (Oxoid) without prior enrichment. Colonies of Shewanella bacteria are often recognized by their pale or pink (salmon-colored) coloration (Bowman JP Genus XIII. Shewanella Mac Donell and Colwell 1986,355 (Effective publication: Mac Donell and Colwell 1985,180), in Bergey's Manual of Systematic Bacteriology, 2 ^nd ed., Garriti GM, Brenner DJ, Krieg NR, and Staley JT, Eds., New York: Springer, 2005, Vol.2, part B, pp. 480-491).

There is also known a method for isolation and identification of bacteria of the genus Shewanella on traditional nutrient media, including MacConkey agar, on which they grow well, forming yellow-brown colonies with a diameter of 1-2 mm after 18-24 hours of incubation. Further, isolates from tan colonies are identified by labor tests (O / F test with glucose for the absence of fermentation, test for the presence of oxidase, test for the production of hydrogen sulfide on Kligler medium). Tests are then used to identify species of S. putrefaciens, S. algae. Both species hydrolyze gelatin, have nitrate reductase, ornithine decarboxylase; unlike S. putrefaciens, S. algae has beta hemolysis on blood agar with sheep blood, grows at 42 ° C and in a nutrient medium with 6% NaCl. Automated identification systems do not distinguish between the species S. putrefaciens, S. algae, since in the program interfaces of the 20E, 20 NE, ID 32GN, Vitek systems, databases include only the S.putrefaciens type (N.M. Holt, B. Gahrn-Hansen, B. Brunn Shewanella algae and Shewanella putrefaciens: clinical and microbiological characteristics. Clin. Microbiol. Infect. 2005; Vol. 11, No. 5: p. 347-352).

A known method for the isolation and identification of Shewanella bacteria by preliminary accumulation under aerobic conditions in a liquid medium containing 1% peptone, 1% sodium chloride and 0.1% bile salts (Difco). After subsequent reseeding on DHL Agar (deoxycholate-hydrogen sulfide-lactose agar) and incubation under aerobic conditions, colonies of non-fermenting bacteria producing hydrogen sulfide are selected. Isolates are finally identified as Shewanella and various species (S. putrefaciens, S. algae) by additional taxonomic tests: determination of oxidase, fermentation and oxidation of carbohydrates on O / F medium, lipases, gelatinases, ureases, arginine dihydrolases, lysine decarboxylases, ornithine decarboxylases, and trihydrogen sulfide growth on SS-arape and Simmons citrate medium; growth on nutrient agar with 1.5% sodium chloride at 37 ° C and 42 ° C for 7 days and 14 days at 4 ° C; tolerance to 6% sodium chloride at 30 ° C for 48 hours; hemolysis on blood agar with ram blood at 30 ° C for 48 h (N.Nozue, T. Hayashi, Y. Hashimoto, T. Ezaki, K. Hamasaki, K. Ohwada, Y. Terawaki. Isolation and Characterization of Shewanella alga from Human Clinical Specimens and Emendation of the Discription of S.alga Simidu et aL, 1990, 335. Intern. J. System. Bacteriology, Oct. 1992, Vol. 42, No. 4, p. 628-634). Note to the method. Composition and use of the DHL Agar acc. to Sakasaki (Merck Microbiology Manual 12 ^th Edition, 2005, p. 262-263). Composition, g / l: peptone from casein 10.0; meat peptone 10.0; meat extract 3.0; lactose 10.0; sucrose 10.0; L-cysteine chloride 0.2; sodium citrate 1.0; sodium deoxycholate 1.5; sodium thiosulfate 2.0; ammonium iron (III) citrate; neutral red 0.03; agar 15.0; distilled water 1 l; pH 7.2 ± 0.2. Preparation of the medium: all components are suspended in water, dissolved by heating (do not sterilize in a steam sterilizer), poured into Petri dishes. Crops are incubated aerobically at 35 ° C for 24-48 hours. Colonies of bacteria fermenting lactose or sucrose are pink or red; colonies of bacteria that do not ferment them are colorless; colonies of bacteria producing hydrogen sulfide become black. Type of colony: Salmonella — colorless with a black center; Citrobacter is colorless. sometimes with a black center; Proteus - colorless, surrounded by a dark brown zone, the center has a weak black color; Escherichia coli - red; Shewanella - colorless with a black center.

The prototype of the proposed method, we have selected the method according to H. Nozue, T. Hayashi et al using DHL Agar nutrient medium, since in both methods the isolation and identification of Shewanella bacteria is carried out on solid nutrient media based on the production of hydrogen sulfide.

The disadvantages of the prototype method and analogues are:

- insufficient specificity of identification of colonies of Shewanella bacteria on the primary culture medium due to the possible presence of colonies of other bacterial genera with the same differentiating characteristics (the presence of colorless colonies with a black center on the hydrogen producing hydrogen sulfide Salmonella, Citrobacter, Proteus on DHL Agar medium; the presence on nutrient agar (Oxoid ) pink-colored colonies of bacteria Pseudomonas stutzeri, Alcaligenes faecalis, Roseomonas spp .; the presence of yellow-brown bacteria colonies Pseudomonas spp., Roseomonas spp. on the McConkey medium;

- the complexity of the study on the identification of bacteria of the genus Shewanella due to the need to use additional tests to study isolated colonies (determination of oxidase, fermentation and oxidation of carbohydrates by O / F test, production of hydrogen sulfide).

The aim of the invention is to increase the specificity and simplification of the study on the isolation and identification of bacteria of the genus Shewanella.

In accordance with the invention, the goal is achieved by the fact that the test material is seeded on a solid nutrient medium containing irgazan (0.14-0.2 g / l) and rifampicin (0.0005-0.001 g / l) as an inhibitor of extraneous microflora; sodium thiosulfate (0.35 g / l), Mora salt, sorbitol, bromothymol blue, tween-80, calcium chloride, sodium chloride, sodium hydroxide in the following ingredients, g / l: pancreatic hydrolyzate of fish meal - 12.0; enzymatic peptone from meat - 12.0; NaCl - 6.0; tween-80 - 5.0; CaCl ₂ 0.2; Na ₂ S ₂ O ₃ · 5H ₂ 0 - 0.35; (NH ₄ ) ₂ SO ₄ · FeSO ₄ · 6H ₂ O — 0.25; sorbitol - 13.0; bromothymol blue - 0.08; irgazan (DP-300) - 0.14-0.2; rifampicin - 0.0005-0.001; NaOH - 0.8; agar - 12.0; distilled water - 1 l; pH 7.2 ± 0.2; while the hydrolyzate of fish meal, peptone, sodium chloride, agar are dissolved by heating, sterilized for 20 min at 121 ° C, after which the remaining ingredients are added to the hot medium, poured into sterile Petri dishes; crops are incubated at 37 ° C and / or 28 ° C under aerobic conditions for 24-48 hours; determine the belonging of the isolated bacteria to the genus Shewanella by the presence of green colonies with a black center or dark gray, surrounded by a zone of turbid precipitate in the nutrient medium.

A distinctive essential feature of the method is the use of irgazan (0.14-0.2 g / l) and rifampicin (0.0005-0.001 g / l) as an inhibitor of extraneous microflora in the nutrient medium. A distinctive essential feature was not used in the prototype and analogues, and is also not known from the prior art. We first established on the isolated strains a high level of resistance of bacteria of the genus Shewanella (S.algae, S. putrefaciens) to irgazan: 128-256 mg / l. This allowed the use of irgazan (DP-300) in the nutrient medium of the proposed method in the range of such high concentrations (0.14-0.2 g / l) that are not used in known selective media with irgazan, for example, in a nutrient medium for the isolation of Yersinia CIN -Agar (Hi-Media) content of irgazan 0.004 g / l; in a nutrient medium for isolation of pseudomonas Pseudomonas Isolation Agar (Hi-Media, Difco) 0.025 g / l of irgazan. The concentration of rifampicin in the nutrient medium was determined experimentally: only in the range of 0.0005-0.001 g / l rifampicin does not inhibit the growth of shevanelles and their production of hydrogen sulfide with effective suppression of the growth of gram-positive microflora. Moreover, the nutrient medium of the proposed method has a high analytical sensitivity: 1-2 CFU / ml ^-1 Shewanella bacteria (S.algae, S. putrefaciens).

A distinctive essential feature of the method directly determines the achievement of the goal - increasing the specificity of the isolation and identification of bacteria of the genus Shewanella. When studying 80 strains of 30 species of 24 bacterial genera, it was found that irgazan and rifampicin in the medium of the proposed method completely inhibit the growth of bacteria of the genus Salmonella (S.enterica serovar Enteritidis, S.enterica serovar Typhimurium) and the genus Proteus (P.vulgaris, P. mirabilis, P.penneri) producing hydrogen sulfide, but does not inhibit the growth of bacteria of the genus Citrobacter (complex C. freundii producing hydrogen sulfide). Thus, the number of species of bacteria is sharply reduced, the colonies of which must be distinguished from the colonies of Shevanella on the basis of hydrogen sulfide formation. In addition, these inhibitors completely inhibit the growth of bacteria of the genera Escherichia, Shigella, Enterobacter, Klebsiella, Hafnia, Staphylococcus, Bacillus (B. subtilis), but do not inhibit the growth of bacteria of the genera Serratia, Morganella, Providencia (P.rettgeri), Aeromonas, Vibrio, Pseudomonas, Stenotrophomonas, Acinetobacter, Achromobacter, Enterococcus.

The second distinctive essential feature of the method is the use of sodium thiosulfate complex (0.35 g / l), Mohr's salt, sorbitol to detect hydrogen sulfide production by Shewanella bacteria and to suppress the formation of hydrogen sulfide by Citrobacter bacteria. This distinctive essential feature was not used in analogues and only one of its component (sodium thiosulfate) is used in the prototype. A distinctive essential feature does not follow explicitly from the prior art. We experimentally found that only a significantly reduced concentration of sodium thiosulfate (0.35 g / l), in contrast to its concentration in the prototype (2.0 g / l), provides in the presence of Mohr's salt and sorbitol a complete suppression of the formation of hydrogen sulfide by the bacteria Citrobacter when copious formation of hydrogen sulfide in colonies of bacteria Shewanella. A distinctive essential feature provides the direct achievement of the goal - increasing the specificity of isolation and identification of Shevanella bacteria, since on the nutrient medium of the proposed method, all colonies with a black color (producing hydrogen sulfide) will contain only Shevanella bacteria. Bromothymol blue and sodium hydroxide in a nutrient medium provide an optimal pH of the medium and additionally differentiate the bacteria, cytrobacter (yellow) and shevanella (green).

The third distinctive essential feature of the method is the use of a complex of tween-80 and calcium chloride to detect bacterial lipase. This feature was not used in the prototype and analogues, but is known from the prior art. It directly ensures the achievement of the goal of increasing the specificity of the isolation and identification of Shevanella, since it further differentiates colonies of Shevanella having a lipase (turbid precipitate zone around the colonies) from bacterial colonies without lipase (there is no precipitate zone).

The combination of these distinctive essential features determines the simplification of the isolation and identification of bacteria of the genus Shewanella, since the identification of Shewanella colonies by the sign of hydrogen sulfide formation, as well as by the presence of lipase and the absence of sorbitol fermentation reliably provides direct identification of these bacteria on the nutrient medium of the proposed method, which eliminates the need for additional taxonomic tests of generic identification.

The essential features of the method are some components of the nutrient medium that provide the nutritional needs of Shewanella bacteria, g / l: pancreatic hydrolyzate of fish meal 12.0; enzymatic peptone from meat 12.0; sodium chloride 6.0; agar 12.0. These components correspond to the composition of the commercial nutrient medium "nutrient agar for the cultivation of dry microorganisms (timing agar)", which is convenient for preparing the medium of the proposed method. Cultivation of crops should be carried out at 37 ° C and / or 28 ° C, as some species of bacteria of the genus Shewanella (S.baltica) do not grow at 37 ° C.

Examples confirming the possibility of implementing the method.

Example 1. The studied material is exudate from the chest cavity of a patient P. is inoculated with a loop on the nutrient medium of the proposed method in a Petri dish, the composition and preparation of which are indicated in the note; the crops are incubated at 37 ° C for 24 h under aerobic conditions and the result is taken into account: the presence on the medium of colonies with a diameter of 2-3 mm in green with a black center surrounded by a cloudy precipitate zone in a nutrient medium indicates their belonging to the genus Shewanella; accompanying colonies with a diameter of 3 mm in yellow color without blackening, surrounded by a zone of turbid precipitate, belong to a different genus of bacteria. The control identification of the isolated isolates by additional species identification tests showed that bacteria isolated from green colonies with a black center surrounded by a precipitation zone belong to the species Shewanella algae; bacteria from yellow colonies without blackening, surrounded by a precipitate zone, belong to the species Serratia marcescens.

Note to example 1. The method of preparation of the nutrient medium of the proposed method. In 1 l of distilled water contribute, g / l: pancreatic hydrolyzate of fish meal - 12.0; enzymatic peptone from meat - 12.0; NaCl - 6.0; agar - 12.0; dissolved by heating, sterilized in a steam sterilizer at 121 ° C for 20 minutes; then in a hot medium add tween-80 (sterile) - 5 ml; CaCl ₂ - 0.2 (2 ml of a sterile 10% solution of CaCl ₂ ); sodium thiosulfate - 0.35; Mohr's salt - 0.25; sorbitol 13.0; bromothymol blue - 0.08 (5 ml of a 1.6% aqueous solution); irgazan (DP-300) - 0.14 (7 ml of a 2% solution in a 1 N NaOH solution); rifampicin - 0.0005 (0.5 ml of a 0.1% solution on dimethyl sulfoxide); NaOH - 0.8 (20 ml of 1 N NaOH solution); pH 7.2 ± 0.2; poured into sterile Petri dishes. The medium has a green color, transparent, suitable for use for 14 days when stored from + 4 ° C to + 8 ° C. The control of the nutrient medium is the presence of colonies with a diameter of 2-3 mm green with a black center, surrounded by a zone of turbid precipitate when using the control strain Shewanella algae 68 according to the method of example 1.

Example 2. The test material - water from the Neva River, 0.1 ml is triturated with a spatula on the nutrient medium of the proposed method in Petri dishes containing irgazan 0.2 g / l and rifampicin 0.001 g / l (the rest of the composition and preparation of the medium according to the note for example one). Crops are incubated at 28 ° C for 48 h, after which the result is taken into account - the presence on the medium of two colonies with a diameter of 3-4 mm of green color with a wide black zone in the center, surrounded by a muddy precipitate zone in the nutrient medium, indicates their belonging to the genus Shewanella ; colonies of other genera of bacteria were also detected - yellow in diameter with a diameter of 3 mm without a precipitation zone; 2 mm in diameter blue with a precipitation zone; 3 mm blue with a wide precipitation zone. The control identification of the isolated isolates showed that the bacteria from one green colony with a black center and a precipitation zone belong to the species Shewanella putrefaciens; bacteria from the second colony belong to the species Shewanella baltica; bacteria from yellow colonies with a diameter of 3 mm without a precipitation zone are Citrobacter freundii; bacteria from blue colonies with a diameter of 2 mm with a precipitation zone - Stenotrophomonas maltophilia; bacteria from blue colonies with a diameter of 3 mm with a wide precipitation zone - Aeromonas caviae.

Thus, at all limiting concentrations of the main components of the nutrient medium of the proposed method (irgazan, rifampicin), temperature conditions (37 ° C and 28 ° C) and exposure time (24-48 hours), clear results of the identification and identification of bacteria of the genus Shewanella were obtained.

When studying the nutrient medium of the proposed method for sowing 30 strains of bacteria producing hydrogen sulfide (5 strains of Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, Proteus vulgaris, Proteus mirabilis, Citrobacter freuindii, Citrobacter braakii) and 48 strains of other 26 species of bacteria (except ) indicated above, not a single false-positive result of their identification as Shewanella, that is, the claimed method has a high analytical specificity. Moreover, the nutrient medium of the proposed method has a high analytical sensitivity for Shewanella bacteria - 1-2 CFU / ml ^-1 .

The inventive method for the isolation and identification of bacteria of the genus Shewanella simplifies this study, since on the nutrient medium of the proposed method, direct generic identification of Shewanella by colony type is achieved without additional studies.

Claims (1)

A method for the isolation and identification of bacteria of the genus Shewanella, which provides for the inoculation of the test material on a nutrient medium containing sources of nitrogen, carbon, sulfur, carbohydrates, inhibitors of extraneous microflora, pH and hydrogen sulfide formation indicators, agar and distilled water; incubation of crops at 37 ° C and / or 28 ° C under aerobic conditions for 24-48 hours; determination of the belonging of the isolated bacteria to the genus Shewanella by the presence of green colonies with a black center or dark gray, surrounded by a cloudy precipitate zone in a culture medium, characterized in that the culture medium contains irgazan as an inhibitor of extraneous microflora (0.14-0.2 g / l) and rifampicin (0.0005-0.001 g / l); sodium thiosulfate (0.35 g / l); Mora salt, sorbitol, bromothymol blue, tween-80, calcium chloride, sodium chloride, sodium hydroxide with the following ingredients, g / l:
pancreatic hydrolyzate of fish meal 12.0 meat peptone 12.0 NaCl 6.0 twin 80 5,0 CaCl ₂ 0.2 Na ₂ S ₂ O ₃ · 5H ₂ O 0.35 (NH ₄ ) ₂ SO ₄ · FeSO ₄ · 6H ₂ O 0.25 sorbitol 13.0 bromothymol blue 0.08 Irgazan (DP-300) 0.14-0.2 rifampicin 0.0005-0.001 NaOH 0.8 agar 12.0 distilled water 1 liter
pH 7.2 ± 0.2; while the hydrolyzate of fish meal, peptone, sodium chloride and agar are dissolved by heating, sterilized for 20 min at 121 ° C, after which the remaining ingredients are added to the hot medium.