RU2820701C1 - Nutrient medium for cultivation of sulphate-reducing bacteria of genus desulfovibrio species and method for production thereof - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention can be used for nutrient media for enrichment and accelerated cultivation of sulphate-reducing bacteria of the genus Desulfovibrio spp. There are disclosed qualitative and quantitative composition of a culture medium for cultivation of sulphate-reducing bacteria of the genus Desulfovibrio spp., as well as a method for production thereof.

EFFECT: sufficient growth of bacteria present in the biomaterial, their accumulation for subsequent seeding with the possibility of further study.

5 cl, 5 tbl, 4 ex

Description

The invention relates to the field of microbiology and can be used in practical and research work for the diagnosis and study of opportunistic microorganisms of the genus Desulfovibrio, namely, nutrient media for the enrichment and accelerated cultivation of sulfate-reducing bacteria.

Bacteria of the genus Desulfovibrio are anaerobic gram-negative spiral or curved motile rods that produce hydrogen sulfide (H ₂ S) and desulfoviridine during their life, which, under UV radiation, fluoresces red at an alkaline pH and green at an acidic pH [Machaca M, Bodean ML, Montana S, Garcia SD, Stecher D, Vay CA, Almuzara MN. Desulfovibrio desulfuricans [Description of a case of abdominal sepsis due to Desulfovibrio desulfuricans]. Rev Argent Microbiol. 2022 Jun 7:S0325-7541(22)00028-1. Spanish, doi: 10.1016/j.ram.2022.05.002. Epub ahead of print. PMID: 35688718; HagiyaH, KimuraK, Nishi I, Yamamoto N, Yoshida H, Akeda Y, Tomono K. Desulfovibrio desulfuricans bacteremia: A case report and literature review. Anaerobe. 2018 Feb;49:112-115. doi: 10.1016/j.anaerobe.2017.12.013. Epub 2018 Jan 4. PMID: 29305996; Goldstein EJ, Citron DM, Peraino VA, Cross SA. Desulfovibrio desulfuricans bacteremia and review of human Desulfovibrio infections. J Clin Microbiol. 2003 Jun;41(6):2752-4. doi: 10.1128/JCM.41.6.2752-2754.2003. PMID: 12791922; PMCID: РМС156571].

According to published data, sulfate-reducing bacteria (SRB) colonize bottom sediments of brackish lakes, as well as the gastrointestinal tract (GIT) of mammals, including humans [Marquis TJ, Williams VJ, Banach DB. Septic arthritis caused by Desulfovibrio desulfuricans: A case report and review of the literature. Anaerobe. 2021 Aug;70:102407. doi: 10.1016/j.anaerobe.2021.102407. Epub 2021 Jun 18. PMID: 34153468; Hagiya H, Kimura K, Nishi I, Yamamoto N, Yoshida H, Akeda Y, Tomono K. Desulfovibrio desulfuricans bacteremia: A case report and literature review. Anaerobe. 2018 Feb;49:112-115. doi: 10.1016/j.anaerobe.2017.12.013. Epub 2018 Jan 4. PMID: 29305996; Carli T, Diker KS, Eyigor A. Sulphate-reducing bacteria in bovine faeces. Lett Appl Microbiol. 1995 Oct;21(4):228-9. doi: 10.1111/j.l472-765x.l995.tb01047.x. PMID: 7576512; Nasreddine R, Argudin MA, Herpol M, Miendje Deyi VY, Dauby N. First case of Desulfovibrio desulfuricans bacteraemia successfully identified using MALDI-TOF MS. New Microbes New Infect. 2019 Oct 23;32:100614. doi: 10.1016/j.nmni.2019.100614. PMID: 31763046; PMCID: РМС6859274].

CRP is present in the body asymptomatically and can be opportunistic microorganisms leading to intra-abdominal infections, bacteremia, and also cause exacerbation of inflammatory diseases of the gastrointestinal tract (Crohn's disease, ulcerative colitis, irritable bowel syndrome), mental and cognitive diseases (Parkinson's disease, autism spectrum disorder (ASD), etc.). According to a model experiment on rodents, bacteria of the genus Desulfovibrio can be one of the etiological factors in the development of iron deficiency anemia (IDA).

The pathogenetic effects of bacteria of the genus Desulfovibrio are currently poorly studied. The reasons for this, among other things, are difficulties in cultivating and identifying cultures, as well as a slow growth rate, which complicates the routine examination of clinical biomaterial in laboratory diagnostics.

According to known data, the life activity of bacteria of the genus Desulfovibrio requires 3 groups of components: electron donors and acceptors, and growth factors. Electron donors include starch, sodium succinate, maltose, mannitol and sodium acetate. The group of electron acceptors includes manganese sulfate, ferrous sulfate and sodium thioglyconate. Growth factors are considered to be blood (5%), hemin, Tween 80 and vitamins B6, B12 and C [Chen YR, Zhou LZ, Fang ST, Long HY, Chen JY, Zhang GX. Isolation of Desulfovibrio spp.from human gut microbiota using a next-generation sequencing directed culture method. Lett Appl Microbiol. 2019 Jun; 68(6):553-561. doi: 10.1111/lam.l3149. Epub 2019 Apr 3. PMID: 30835854; Liu F, Li J, Wu F, Zheng H, Peng Q, Zhou H. Altered composition and function of intestinal microbiota in autism spectrum disorders: a systematic review. Transl Psychiatry. 2019 Jan 29;9(1):43. doi:10.1038/s41398-019-0389-6. PMID: 30696816; PMCID: РМС6351640].

Known general purpose media used for growing SRB:

Sulphate API Agar w/o Sodium Lactate (API) medium [https://exodocientifica.com.br/_technical-data/M309.pdf], the following composition:

yeast extract 1,000 g/l magnesium sulfate 0.200 g/l ascorbic acid 0.100 g/l dipotassium phosphate 0.010 g/l iron ammonium sulfate 0.100 g/l sodium chloride 10,000 g/l agar 14,000 g/l final pH 7.4±0.2,

where it is necessary to dissolve 25.41 mixtures in 1000 ml of distilled water, add 4 ml of sodium lactate, then heat until the ingredients are completely dissolved, without boiling, pour the medium into test tubes and sterilize.

A known nutrient medium for Desulfovibrio with 1% NaCl [Institute of Biochemistry and Physiology of Microorganisms named after. G.K. Scriabin of the Russian Academy of Sciences - A separate division of the Federal State Budgetary Institution of Science “Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”; All-Russian collection of microorganisms - VKM; Additional Information; Nutrient media; No. 54; https://www.vkm.ru/rus/Catalogue.htm; Nutrient media description file] containing:

K ₂ NPO ₄ - 0.01 g;

NaCl - 10.0 g;

MgSO ₄ - 0.2 g;

Na lactate (40%) - 4.0 ml;

Mohr's salt solution - 1.0 ml;

yeast extract - 1.0 g;

ascorbic acid - 0.1 g;

agar - 6.0 g;

distilled water - 1000.0 ml,

where Mohr's salt solution contains:

Fe(NH ₄ )2(SO ₄ )2×6 H ₂ O - 1.0 g

distilled water - 5.0 ml.

The solutions are sterilized separately by autoclaving at 121°C for 15 minutes.

A nutrient medium for Desulfovibrio with lactate is also known [Institute of Biochemistry and Physiology of Microorganisms named after. G.K. Scriabin of the Russian Academy of Sciences - A separate division of the Federal State Budgetary Institution of Science “Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”; All-Russian collection of microorganisms VKM; Additional Information; Nutrient media; No. 92; https://www.vkm.ru/rus/Catalogue.htm; Nutrient media description file] containing:

Solution 1:

K ₂ NRO ₄ - 0.5 g;

NH ₄ C1 - 1.0 g;

CaCl ₂ ×6H ₂ O - 0.1 g;

MgSO ₄ × 7 H ₂ 0 - 2.0 g;

Na ₂ SO ₄ - 1.0 g;

Na lactate - 5.0 g;

yeast extract - 1.0 g;

resazurin - 0.0 1 g;

cysteine - 0.5 g;

distilled water - 950.0 ml.

Solution 2:

NaHCO ₃ - 4.0 g;

distilled water - 40.0 ml.

Solution 3:

Na ₂ S×9H ₂ O - 300.0 mg;

distilled water - 6.0 ml.

Solution 4:

FeSO ₄ × 7H ₂ O 0.4 g;

distilled water - 10.0 ml.

pH - 6.8

To prepare the medium, all solutions are sterilized separately by autoclaving at 121°C for 15 minutes. Before sterilization, solution 1 is brought to a boil, purged with a gas mixture of 97% N ₂ and 3% H ₂ and sterilized in the atmosphere of this gas mixture. Solution 3 is sterilized in an N ₂ atmosphere.

To obtain an enrichment culture of sulfate-reducing bacteria, Towson or Postgate media are used [https://www.bibliotekar.ru/2-7-78-biologiya-pochv/65.htm], having the following composition:

Towson's medium (g/l): (NH4)2SO4 - 4.0, K2HPO4 - 0.5, MgSO4X X7H2O - 1.0, Mohr's salt - 0.5, calcium lactate - 5.0. Sometimes yeast water is added to the medium (1 ml/100 ml of medium).

Postgate medium (g/l): KH2PO4 - 0.5, NH4C1 - 1.0, CaSO4-2H2O - 1.0, MgSO4-7H2O - 2.0, sodium lactate - 3.5, yeast extract - 1.0, FeS04-7H20 - 0.5, sodium thioglycolate - 1.0, or ascorbic acid - 1.0.

Preparation of VKM culture media and Postgate media involves the separate preparation of several media solutions and their subsequent mixing. Also, the above-mentioned media have a common disadvantage, which is the slow growth rate of the target bacterium, which complicates their use in routine work.

In view of the fact that the cultivation of microorganisms on nutrient media is one of the main methods for studying bacteria in microbiology, and the existing media for cultivating bacteria of the genus Desulfovibrio are difficult to prepare, there is an urgent need to obtain a nutrient medium that is the least labor-intensive in preparation, containing all the necessary components for the life activity of the target cultures and facilitating rapid detection of the target microorganism in the biomaterial being studied.

The purpose of the proposed solution is to obtain a nutrient medium for the cultivation of sulfate-reducing bacteria of the genus Desulfovibrio spp., ensuring sufficient growth of bacteria present in the biomaterial, capable of their accumulation for subsequent sowing and the possibility of further study, as well as to develop the least labor- and resource-intensive method for preparing such a medium .

The goal is achieved due to the qualitative composition and ratio of the components of the nutrient medium indicated in Table 1.

In addition, to obtain a nutrient medium for the cultivation of sulfate-reducing bacteria of the genus Desulfovibrio spp. in the solid phase (agar nutrient medium), agar in an amount of 9.0 g/l is additionally added to the components indicated in Table 1.

The inventive nutrient medium is obtained in the following way. To prepare the nutrient medium, weigh all components on an analytical balance according to Table 1. Mix the following components: sodium lactate, casein hydrolyzate, corn starch, dehydrated beef broth, vitamin B1, vitamin B2, vitamin B6, vitamin B3, vitamin B9, vitamin B5, vitamin B7, vitamin B12, magnesium sulfate, glucose, sodium chloride, potassium hydrogen phosphate, ammonium chloride, sodium pyrosulfite. To obtain an agar nutrient medium, agar in an amount of 9.0 g/l is also added to the mentioned components.

Then add 1000 ml of distilled water, adjust the pH to 7.3 ± 0.2 by adding 0.1 N hydrochloric acid solution or 0.1 N sodium hydroxide solution, and dissolve all components, bringing the resulting mixture to a boil. Ascorbic acid and ferrous sulfate are added to the cooled mixture and mixed. The resulting solution is then poured into sterile glass bottles for autoclaving. Sterilization is carried out by autoclaving using high temperature under a pressure of 1.0 atm. at a temperature of 120.6°C for 15-30 minutes.

The finished nutrient medium is yellowish in color, transparent or slightly opalescent.

The prepared nutrient medium can be stored in the refrigerator at a temperature of +4-8°C for up to 30 days and used as needed.

The declared decision was received at the Federal Budgetary Institution Central Research Institute of Epidemiology of Rospotrebnadzor as part of the research topic “Clinico-pathogenetic substantiation and improvement of therapeutic programs for the preservation and restoration of the human microbiome in widespread infectious diseases.”

The essence of the proposed solution is illustrated by the following examples:

Example No. 1. Selection of the composition of a nutrient medium for the cultivation of sulfate-reducing bacteria of the genus Desulfovibrio spp.

Initially, casein hydrolyzate, starch and beef broth, which were part of the medium, were used as nutritional components to determine the sensitivity of bacteria to antibiotics in Mueller-Hinton broth.

Taking into account the need for the growth of bacteria of the genus Desulfovibrio spp. B vitamins and iron (II) ions, the composition of the medium was selected, presented in Table 2. Table 2. Composition of the nutrient medium, option 1.

Preparation of the nutrient medium according to option 1 included the following steps:

1. Weighing the dry components of the medium on an analytical balance, mixing all components except ferrous sulfate and ascorbic acid;

2. Dissolving the resulting sample in 500 ml of distilled water at room temperature 25°C;

3. After dissolving the components of the medium, add sodium lactate (4 ml).

4. Combine the resulting solution with 500 ml of distilled water, preheated to 70°C;

5. Bringing the pH of the medium to 7.3±0.2 by adding 0.1 N. hydrochloric acid solution or 0.1 N. sodium hydroxide solution;

6. Bring the resulting medium to a boil (101°C) and boil for 1 minute;

7. Add iron sulfate (0.5 g) and ascorbic acid (0.1 g) to the medium cooled to 55°C;

8. Sterilization of the medium by autoclaving under a pressure of 1.0 atm. (120.6°C) for 15-30 minutes.

To determine the growth properties of the medium, we used the prepared medium at room temperature (25°C), poured into 4 ml test tubes. The growth properties of the medium were checked by inoculating 100 μl of a collection culture of Desulfovibrio desulfuricans VKM B-1799 t with a density of 1 McFarland turbidity standard.

The culture was grown under anaerobic conditions at 37°C. After 14 days, no growth of turbidity or blackening of the medium was observed.

Due to the ineffectiveness of the nutrient medium according to option 1, a medium composition was proposed with the addition of glucose as an electron donor and components of the prototype medium - sodium chloride, magnesium sulfate and potassium hydrogen phosphate (Table 3).

The preparation of the nutrient medium was similar to the preparation of the medium of option 1. Determination of the growth properties of the nutrient medium was checked by inoculating 100 μl of a collection culture of Desulfovibrio desulfuricans VKM B-1799 ^t with a density of 1 McFarland turbidity standard.

The culture was grown under anaerobic conditions at 37°C. After 14 days, no growth of turbidity or blackening of the medium was observed.

Due to the ineffectiveness of the nutrient medium according to option 2, the composition according to option 3 was proposed, including an increased amount of B vitamins and the addition of vitamins B7 and B12 (Table 4).

The preparation of the nutrient medium was similar to the preparation of the medium of option 1. Determination of the growth properties of the medium was checked by inoculating 100 μl of a collection culture of Desulfovibrio desulfuricans VKM B-1799 t with a density of 1 McFarland turbidity standard.

The culture was grown under anaerobic conditions at 37°C. After 14 days, no growth of turbidity or blackening of the medium was observed.

To cultivate fastidious microorganisms, vitamin K was added to the medium of option 4 as an organic growth factor (Table 5).

The preparation of the nutrient medium was similar to the preparation of the medium according to option 1. Determination of the growth properties of the medium was checked by inoculating 100 μl of a collection culture of Desulfovibrio desulfuricans VKM B-1799 t with a density of 1 McFarland turbidity standard.

The culture was grown under anaerobic conditions at 37°C. After 14 days, no growth of turbidity or blackening of the medium was observed.

Due to the fact that the developed media of options 1-4 included all the components necessary for the growth of Desulfovibrio spp. cultures and taking into account the retardation of bacterial growth with an excess of vitamins, a medium composition containing a smaller amount of B vitamins and lactate was proposed. In addition, the proposed medium contained iron (II) and magnesium ions as inorganic growth factors; B vitamins acted as organic growth factors. Lactate and glucose acted as electron donors; as reducing agents - ascorbic acid, sodium sulfite and iron (II) ions. The composition and quantitative indicators of the components of the resulting nutrient medium are presented in Table 1. The nutrient medium was prepared according to the claimed method.

In the resulting nutrient medium, the culture was grown under anaerobic conditions at 37°C. After 3 days of growth, blackening of the medium was observed. Identification of the grown culture showed the presence of curved and spiral gram-negative bacteria in the smear, as well as red fluorescence when the pH of the environment changed to alkaline. The results obtained allowed us to identify the culture as Desulfovibrio spp.

Example 2. Preparation of a nutrient medium for the cultivation of sulfate-reducing bacteria of the genus Desulfovibrio spp. in the liquid phase.

To prepare the claimed nutrient medium, dry components were weighed on an analytical balance in the quantities indicated in Table 1.

Then sodium lactate, casein hydrolyzate, corn starch, dehydrated beef broth, vitamin B1, vitamin B2, vitamin B6, vitamin B3, vitamin B9, vitamin B5, vitamin B7, vitamin B12, magnesium sulfate, glucose, sodium chloride, potassium hydrogen phosphate were mixed , ammonium chloride, sodium pyrosulfite. Then 1000 ml of distilled water was added and the pH was adjusted to 7.3 by adding 0.1 N hydrochloric acid solution.

The components of the resulting mixture were dissolved, bringing it to a boil, after which ascorbic acid and ferrous sulfate were added to the cooled mixture and mixed.

Then the resulting solution was poured into sterile glass bottles for sterilization and autoclaving was carried out under a pressure of 1.0 atm. at a temperature of 120.6°C for 15 minutes. The nutrient medium obtained in this way was transparent and yellowish in color. The prepared nutrient medium was stored in the refrigerator at a temperature of +4-8°C for 30 days and used as needed.

Example 3. Preparation of a nutrient medium for the cultivation of sulfate-reducing bacteria of the genus Desulfovibrio spp. in the solid phase (agar nutrient medium).

To prepare the claimed nutrient medium, dry components were weighed on an analytical balance in the quantities indicated in Table 1.

Then sodium lactate, casein hydrolyzate, corn starch, dehydrated beef broth, vitamin B1, vitamin B2, vitamin B6, vitamin B3, vitamin B9, vitamin B5, vitamin B7, vitamin B12, magnesium sulfate, glucose, sodium chloride, potassium hydrogen phosphate were mixed , ammonium chloride, sodium pyrosulfite. Agar was added to the resulting mixture - 9.0 g/l. Then 1000 ml of distilled water was added, the pH was adjusted to 7.4 by adding 0.1 N. sodium hydroxide solution.

The components of the resulting mixture were dissolved, bringing it to a boil, after which ascorbic acid and ferrous sulfate were added to the cooled medium and mixed.

Then the resulting solution was poured into sterile glass bottles for sterilization and autoclaving was carried out under a pressure of 1.0 atm. at a temperature of 120.6°C for 30 minutes. The nutrient medium obtained in this way had a yellowish color and was slightly opalescent. The finished nutrient medium was stored in the refrigerator at a temperature of +4-8°C for 30 days and was used as needed.

Example 4. Assessment of the growth of the target bacterium in a nutrient medium for the cultivation of sulfate-reducing bacteria of the genus Desulfovibrio spp.

The growth properties of the nutrient medium for the cultivation of sulfate-reducing bacteria of the genus Desulfovibrio spp, prepared according to the claimed method, were determined on a collection strain, which was the type strain of the genus Desulfovibrio sp. from the All-Russian Collection of Microorganisms (Pushchino): Desulfovibrio desulfuricans sp.desulfuricans VKM B-1799 T.

The inventive nutrient medium, obtained by the methods described in examples 2 and 3, was inoculated with the collection strain Desulfovibrio desulfuricans VKM B-1799 t, anaerobic conditions were created using an anaerostat, Gaspack-type bags manufactured by Oxoid (Thermo Fisher Scientific, USA) and placed in a thermostat at 37°C (CO ₂ 6%). The growth of bacteria of the genus Desulfovibrio sp. found in the studied material was obtained after 48-72 hours of growth.

The growth of the culture was assessed by the blackening of the medium - a black precipitate or the growth of colonies in the thickness of the agar. Bacteria were identified using the following methods:

- samples were illuminated under UV, bringing the pH of the medium to alkaline or acidic;

- smears were prepared and stained with Gram, followed by the use of light microscopy.

The results obtained allowed us to identify the culture as Desulfovibrio spp.

Thus, the claimed nutrient medium obtained according to the proposed method provides rapid and sufficient for cultivation growth of sulfate-reducing bacteria of the genus Desulfovibrio spp., and also has the ability to accumulate them for subsequent study.

Claims (12)

1. Nutrient medium for the cultivation of sulfate-reducing bacteria of the genus Desulfovibrio spp., containing distilled water, sodium lactate, casein hydrolyzate, corn starch, dehydrated beef broth, ferrous sulfate, vitamin B1, vitamin B2, vitamin B6, vitamin B3, vitamin B9, vitamin B5, vitamin B7, vitamin B12, ascorbic acid, magnesium sulfate, glucose, sodium chloride, potassium hydrogen phosphate, ammonium chloride, sodium pyrosulfite, with the following content of components: distilled water 1000 ml/l sodium lactate 3.00 ml/l casein hydrolysate 0.01 g/l corn starch 0.01 g/l dehydrated beef broth 0.15 g/l iron sulfate ferrous 0.50 g/l vitamin B1 0.10 g/l vitamin B2 0.15 g/l vitamin B6 0.30 g/l vitamin B3 0.75 g/l vitamin B9 0.05 g/l vitamin B5 0.35 g/l vitamin B7 0.19 g/l vitamin B12 0.10 g/l ascorbic acid 0.10 g/l magnesium sulfate 1.00 g/l glucose 1.00 g/l sodium chloride 0.10 g/l potassium hydrogen phosphate 0.50 g/l ammonium chloride 1.00 g/l sodium pyrosulfite 0.50 g/l 2. Nutrient medium for the cultivation of sulfate-reducing bacteria of the genus Desulfovibrio spp. according to claim 1, characterized in that it additionally contains agar - 9.0 g/l. 3. Nutrient medium for the cultivation of sulfate-reducing bacteria of the genus Desulfovibrio spp. according to claims 1 and 2, characterized in that it additionally contains 0.1 n. hydrochloric acid solution or 0.1 N. sodium hydroxide solution until the pH is 7.3±0.2. 4. Method for obtaining a nutrient medium for the cultivation of sulfate-reducing bacteria of the genus Desulfovibrio spp.according to claim 1, including the sequential implementation of the following stages: (i) mixing sodium lactate - 3.00 ml/l; casein hydrolyzate - 0.01 g/l; corn starch - 0.01 g/l; dehydrated beef broth - 0.15 g/l; vitamin B1 - 0.10 g/l; vitamin B2 - 0.15 g/l; vitamin B6 - 0.30 g/l; vitamin B3 - 0.75 g/l; vitamin B9 - 0.05 g/l; vitamin B5 - 0.35 g/l; vitamin B7 - 0.19 g/l; vitamin B12 - 0.10 g/l; magnesium sulfate - 1.00 g/l; glucose - 1.00 g/l; sodium chloride - 0.10 g/l; potassium hydrogen phosphate - 0.50 g/l; ammonium chloride - 1.00 g/l; sodium pyrosulfite - 0.50 g/l; (ii) bringing the total volume of the mixture obtained in step (i) to 1000 ml by adding distilled water; (iii) adjusting the pH to 7.3 ± 0.2 by adding 0.1 N to the mixture obtained in step (ii). hydrochloric acid solution or 0.1 N. sodium hydroxide solution; (iv) dissolving the components of the mixture obtained in step (iii) by bringing it to a boil; (v) mixing the cooled mixture obtained at stage (iv) with ascorbic acid - 0.10 g/l and ferrous sulfate - 0.50 g/l; (vi) sterilization of the mixture obtained in step (v) in sterilized glass bottles by autoclaving under a pressure of 1.0 atm. at a temperature of 120.6°C for 15-30 minutes. 5. Method for obtaining a nutrient medium for the cultivation of sulfate-reducing bacteria of the genus Desulfovibrio spp. according to claim 4, characterized in that at stage (i) agar is added - 9.0 g/l.