RU2745159C1 - Selective nutritional medium for isolation of dermatophytosi causes - Google Patents

Abstract

FIELD: veterinaty medicine.

SUBSTANCE: invention relates to veterinary medicine and veterinary microbiology and can be used for the diagnosis of animal dermatophytosis caused by filamentous fungi of the species Microsporum canis and Trichophyton mentagrophytes. Selective nutrient medium for isolation of pathogens of dermatophytosis includes glucose, mannitol, meat peptone, phenol red, agar-agar, cycloheximide, enrofloxacin and distilled water at a given content of components.

EFFECT: invention makes it possible to shorten the time of diagnosis of dermatophytosis in animals, to increase the reliability of the results of differentiation of fungi of dermatophytes from non-dermatophytes using routine microbiological methods and to expand the arsenal of nutrient media for isolation of pathogens of dermatophytosis.

1 cl, 2 tbl, 5 ex

Description

The invention relates to veterinary medicine and veterinary microbiology, and can be used for the diagnosis of animal dermatophytosis caused by filamentous fungi of the species Microsporum canis and Trichophyton mentagrophytes. The invention is an improved differential diagnostic environment for the detection of dermatophyte fungi from clinical material selected from animals with dermatological pathologies and / or suspicions of them.

Dermatophytoses of animals caused by mycelial species of fungi Microsporum canis and Trichophyton mentagrophytes are widespread in Russia and the world as a whole [Savinov, V.A. The prevalence of dermatophytosis in small domestic animals / V.A. Savinov // Advances in medical mycology. 2018.Vol. 19.P. 373-375]. In many regions of Russia, the disease occupies a leading place among dermatological pathologies. To prescribe and conduct effective treatment, veterinarians first need to determine the etiology of the disease. So, at present, the most common method for diagnosing dermatophytosis is a luminescent study of samples using a lamp with a Wood filter. In addition, microscopy of the affected coat and hair and the search for arthroconidia, hyphae, and mycelium of the fungus is a common diagnostic approach. For a number of reasons, the effectiveness of the above methods is low, despite their simplicity and low cost in implementation. The "gold standard" for the diagnosis of dermatophytosis is still the cultural method of research [Ovchinnikov, R.S. Mycological screening of domestic animals is an important way to prevent human dermatophytosis / R.S. Ovchinnikov, P.P. Ershov, A.V. Kapustin, V.A. Savinov, A.G. Gainullina // Advances in medical mycology. 2019. T. 20. S. 712-716]. Cultural methods make it possible not only to differentiate the causative agents of dermatophytosis, but also to determine their sensitivity to antimycotic drugs, which in turn contributes to the prescription of the correct therapy for the disease. However, the implementation of cultural diagnostic methods, in contrast to the luminescent method and the microscopy method, is possible only by highly qualified employees with certain training and in laboratory conditions. In turn, cultural methods using known nutrient media have their disadvantage, namely the prolonged growth of dermatophytes, which allows a final conclusion to be made no earlier than 14-16 days, which significantly affects the course of treatment of a sick animal [Savinov, V.A. Rapid diagnostics of animal dermatophytosis / V.A. Savinov, R.S. Ovchinnikov, A.V. Kapustin, A.A. Gainullina // Agricultural science. 2019. No. 10. S. 20-24].

State of the art

Currently, Sabouraud's medium is most widely used to isolate filamentous fungi from pathological and clinical material. This environment allows the growth of most micromycete fungi. The disadvantage of this environment, in the case of diagnosing dermatomycosis, is its low specificity. In particular, due to the long period of dermatophyte cultivation, fast-growing saprophytic molds from the test material (contaminating fungi) begin to grow on the medium, which subsequently complicates or completely eliminates the growth of dermatophytes. In addition to the low specificity, the use of this nutrient medium requires a long cultivation period. So, the formation of macroconidia occurs by 14-16 days, which significantly slows down the timing of the final diagnosis and the appointment of the necessary therapy.

Known nutrient medium for the cultivation of Microsporum canis [patent RU 2695675 C1 publ. 2019.03.05]. The disadvantage of this environment is the limited range of applications.

A known method for the diagnosis of dermatomycosis and a nutrient medium for dermatophytes [patent RU 2275631 C1 publ. 2006.04.14]. A significant drawback of this medium is its non-specificity, and the absence of indicator changes during cultivation, indicating the presence of dermatophyte growth.

S. Mayrhofer, U. Zitz, C. Hofacre, K.J. Domig // Poult Sci. 2019 Apr; 98(4): 1791-1804].Known environment DTM (Dermatophyte Test Medium) [Taplin, D. Isolation and recognition of dermatophytes on a new medium (DTM). / D. Taplin, N. Zaias, G. Rebell, H. Blank. // Arch Dermatol 1969 99: 203-209]. The medium contains papain digest of soy flour (10.0 grams), glucose (10.0 grams), cycloheximide (0.5 grams), phenol red indicator (0.2 grams), chlortetracycline (0.1 grams), gentamicin (0.1 grams). The principle of using the nutrient medium is based on changing the level of hydrogen ions (pH). So, in the process of growth, dermatophyte fungi absorb protein peptides, produce alkali and increase the pH value, as a result of which the indicator changes the color of the medium from yellow to red. The advantage of this environment is that sowing does not require special skills and can be carried out in a clinic, while the incubation of sowing can be carried out at room temperature, i.e. in the absence of special laboratory equipment. The results of the study using the designated medium can be obtained on the 7-9 day of the study. However, the significant disadvantages of this environment are the recorded cases of the occurrence of a false-positive reaction to the growth of fungi that do not belong to the group of dermatophytes [Savinov, V.A. Development of a differential diagnostic nutrient medium for the express diagnosis of animal dermatophytosis. / VA Savinov, RS Ovchinnikov, AV Kapustin, AI Laishevtcev, AM Gulykin // In IOP Conference Series: Earth and Environmental Science 2019, August, Vol. 315, No. 2, p. 022071. IOP Publishing.]. In addition, resistance to antibiotics that are part of the nutrient medium is widespread, which in turn reduces the diagnostic value of the medium. This phenomenon is due to the fact that the growth of bacteria acidifies the nutrient medium, thereby preventing the medium from changing color to red [Kaufer, NF Cycloheximide resistance in yeast: the gene and its protein. / NF Kaufer, HM Fried, WF Schwindinger, M. Jasin, JR Warner // Nucleic Acids Res. 1983 May 25; 11 (10): 3123-3135; Roth, N. The application of antibiotics in broiler production and the resulting antibiotic resistance in Escherichia coli: A global overview. / N. Roth,

S. Mayrhofer, U. Zitz, C. Hofacre, KJ Domig // Poult Sci. 2019 Apr; 98 (4): 1791-1804].

According to the above characteristics for existing and / or previously used nutrient media for the diagnosis of dermatomycosis, Dermatophyte Test Medium is the closest analogue that we take as a prototype.

The technical problem to be solved by this invention is the need to create a selective nutrient medium that allows, with minimal time costs and maximum diagnostic accuracy, to cultivate and differentiate the causative agents of animal dermatophytosis, namely Microsporum canis and Trichophyton mentagrophytes.

Disclosure of the essence of the invention

The technical result of the invention is to reduce the timing of diagnosing dermatophytosis in animals and increase the reliability of the results of differentiation of fungi of dermatophytes from non-dermatophytes using routine microbiological methods, expanding the arsenal of nutrient media for isolating pathogens of dermatophytosis.

The composition and preparation of the nutrient medium are as follows: 10.0 ± 1.0 g / L glucose, 10.0 ± 1.0 g / L mannitol, 10.0 ± 1.0 g / L meat peptone, 0.2 ± 0 , 02 g / l phenol red, 15.0-20.0 ± 1.5 g / l agar agar, are evenly mixed in 949.8-954.8 ml of distilled water (respectively). Using hydrochloric acid (HCl), the pH is adjusted to 5.5 ± 0.1. The mixture is autoclaved at 121 ° C for 15 minutes. After the medium has cooled down to 55 ± 5.0 ° C, cycloheximide 0.5 ± 0.05 g / l, enrofloxacin 0.1 ± 0.01 g / l are added aseptically, then poured into Petri dishes 22 ± 2, 5 ml or in sterile polymer containers of 10 ml. The medium is suitable for use after solidification and in the absence of condensation. The proposed diagnostic tool differs from its early counterparts in improved growth and selective properties, which make it possible to shorten the diagnostic time and increase the sensitivity in detecting the causative agents of animal dermatophytosis. The indicator component, which is part of the nutrient medium, makes it possible to detect the growth of dermatophytes already at the early stages of diagnosis, without resorting to subsequent microscopy of the culture, which, in turn, is necessary in classical diagnostics to confirm the belonging of cultures to dermatophytes.

The differences between the inventive medium and the prototype are in the composition, namely in the modified peptone to increase growth properties and accelerate the growth itself, in the addition of an additional carbohydrate that stimulates growth energy, in the set of antibacterial drugs necessary to reduce the secondary growth of bacterial cultures-contaminants.

On the proposed medium, the growth of the culture of Microsporum canis demonstrates a white loose-fluffy colony characteristic of the species with a moderately developed aerial mycelium. The growing margin is smooth, ciliate. The beginning of growth is noted starting from the 3rd day, and the color change starting from the 5th day.

The growth of the culture of Trichophyton mentagrophytes demonstrates the characteristic of the species, a convex, fluffy white colony with a well-developed aerial mycelium. The growing margin is smooth, ciliate. The beginning of growth on the medium is noted starting from 2 days, and the color change starting from 3 days.

Implementation of the invention

The examples below are illustrative and thus do not limit the scope of the present invention.

Example # 1. When studying the effect of peptones of various origins on the growth rate of dermatophytes, we used the classical formulation of Sabouraud's medium; in each experimental series of the medium, the peptone variety was changed. So, in the study, 4 types of peptones were used - casein (Casein peptone pancreatic digested), soy (Soyabean pepton), dry fermentation pepton, and meat (Meat Pepton). To study the growth energy on different variants of the medium, cultures of dermatophyte strains were inoculated with an injection in the center of the dish. The results obtained are shown in Table 1.

When studying the growth properties, the growth was recorded daily, starting from the second day after sowing. The diameter of the colonies and the intensity of air mycelium formation were measured. The degree of development of aerial mycelium was evaluated in points:

1 - aerial mycelium is poorly developed, translucent, cobweb

2 - aerial mycelium is moderately developed, velvety, powdery

3 - aerial mycelium is well developed, woolly, fluffy.

As can be seen from the results obtained, the medium with the addition of meat peptone showed the best growth qualities for all strains. Also on this medium, aerial mycelium is well formed.

Example # 2. The study of the effect of carbohydrates on the growth of dermatophytes was similar to the experiment with peptones, reflected in example No. 1. The experiment involved 14 different carbohydrates, namely: mannose, inositol, mannitol, lactose, arabinose, adonit, sorbitol, dulcit, xylose, rhamnose, raffinose, salicin, maltose, glucose. The best growth of dermatophytes was observed on a medium supplemented with mannitol. So, on the 7th day, a colony of M canis # 71-19 growing on a medium supplemented with mannitol had a diameter of 32 mm, while on media with other carbohydrates, the diameter of the colonies reached an average of 29-30 m. Colony Trichophyton mentagrophytes # 135 on media supplemented with mannitol it had a diameter of 35 mm, on media with other carbohydrates the average diameter was 32 mm.

Example No. 3. Evaluation of the inhibitory properties of the proposed environment in relation to bacteria was carried out using bacterial strains Pseudomonads aeruginosa, Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae, Staphylococcus pseudintermedius, Bacillus subtilis. Served as control medium Saburo. It was found that on the proposed medium, all studied bacteria are completely inhibited, except for Pseudomonas aeruginosa - during sowing, weak growth was observed, which did not affect the diagnostic properties of the medium in relation to dermatophytes. A similar increase in Ps was observed on Sabouraud's medium. aeruginosa.

Example No. 4. Influence of the quantitative composition of the nutrient medium on its growth properties.

When studying the influence of the quantitative composition of the nutrient medium on its growth properties, two batches of the nutrient medium were produced with the minimum and maximum amount of components specified in the recipe. Thus, the first batch of the medium had the following composition: 9 g / L glucose, 9 g / L mannitol, 9 g / L meat peptone, 0.18 g / L phenol red, 13.5 g / L agar-agar, 0, 45 g / L cycloheximide, 0.09 g / L enrofloxacin. The second batch of the medium included 11 g / L glucose, 11 g / L mannitol, 11 g / L meat peptone, 0.22 g / L phenol red, 21.5 g / L agar-agar, 0.55 g / L cycloheximide , 0.11 g / L enrofloxacin. When assessing the growth properties of the two series of the medium, no significant differences were found. Thus, in both series of the medium, the growth of the culture of Microsporum canis demonstrated a white loose-fluffy colony characteristic of the species with a moderately developed aerial mycelium. The growing margin is smooth, ciliate. The beginning of growth is noted from 3 days, and the color change from 5 days. The culture of Trichophyton mentagrophytes showed, on both medium options, a characteristic for the species a convex, fluffy white colony with a well-developed aerial mycelium. The growing margin is smooth, ciliate. The beginning of growth on the medium is noted from 2 days, and a color change occurs from 3 days.

Example No. 5. For a comparative assessment of the specificity and sensitivity of the proposed medium, the commercial DTM medium, and the Sabouraud medium, 40 samples of clinical material were inoculated on them. The seeding technique did not differ. The percentage of positive discharge of dermatophytes using the newly proposed medium was 31.0%, for the imported medium 32.5%, and for Sabouraud agar 22.5%, respectively. The visible growth of dermatophytes appeared most quickly on the Sabouraud medium, and almost simultaneously on the experimental medium and DTM of foreign production with a difference of 1-2 days. The reddening of the medium during the growth of dermatophytes on the experimental and imported media also occurred almost simultaneously - on 6-9 days, and became pronounced on the 10-11 days, but at the same time, on the proposed medium, the growth was recorded earlier than on the well-known DTM medium on 1-2 days. Active growth of contaminant molds, which prevents the growth of dermatophytes, was found in 27% of crops on Sabouraud's medium, which is obviously associated with the lowest frequency of isolation of dermatophytes on this medium. Molds were represented by the genera Aspergillus, Penicillium, Mucor, Alternaria, Cladosporium. On the proposed environment, mold growth was observed in only 15% of the samples, while 23.5% of the crops were contaminated with the imported DTM. It is important to note that on the imported DTM medium, reddening was observed in 13.2% of the crops during the growth of molds, i.e. received a false positive result, which leads to diagnostic errors in real practice. On the developed DTM medium, false redness caused by molds was not observed.

Claims (2)

Selective nutrient medium for isolation of pathogens of dermatophytosis, including glucose, agar-agar, protein component, cycloheximide and additional antibacterial components of the medium, phenol red, characterized in that it additionally contains mannitol, meat peptone as a protein component, and as an additional antibacterial component of the medium - enrofloxacin with the following content of components: glucose 10 ± 1.0 g manniol 10 ± 1.0 g meat peptone 10 ± 1.0 g phenol red 0.2 ± 0.02 g agar-agar 20.0 ± 1.5 g cyclohexamide 0.5 ± 0.05 g enrofloxacin 0.1 ± 0.01 g distilled water 49.8-954.8 ml