Lethal activity of the museum and clinical strains of C. Difficile

Abstract

Introduction. At present С. difficile infection (CDI) is considered to be nosocomial worldwide, this problem is studied in depth and currently there are large-scale monitoring studies being carried out. There is a considerable quantity of different tests for CDI diagnosis. These tests include the cytopathogenic test and the toxin neutralization reaction (determination of toxin B), latex agglutination (determination of the glutamatedehydrogenase), ELISA test (determination of the A or B toxins, or determination of the A and B toxins simultaneously), PCR. At the same time, it is known that the isolation of the agents and its identification without determination of the ability of the latter to produce toxins is insufficient, as the non-toxicogenic strains do not play any role in the human pathology. The modern diagnostic methods present in Ukraine (PCR, ELISA, etc.)  have not become widespread in the laboratories of the state health establishments because of their complexity and cost, moreover, they cannot provide for all aspects required for the solution of this problem, such as the study of the properties and variability of the agent itself.  The toxins A and B have powerful cytotoxic and pro-inflammatory properties. These toxins are glycosyltransferase enzymes that catalyze the inactivation of Rho proteins responsible for actin cytoskeleton organization and epithelial barrier function. The destruction of the epithelial cells leads to the disruption of the water-electrolyte exchange, which facilitates the secretion of the fluid into the intestinal space. The modern studies confirm the important role of both toxins A and B, and the distribution of the clinical symptoms beyond the intestine to other organs (heart, kidneys, brain), which points to the presence of the systemic toxemia and plays the main role in the prognosis of the CDI development.  In the laboratory of the anaerobic infections of the SI “IMI NAMS” in the 2017-2019 years, studies devoted to the identification and study of the circulating clinical C. difficile strains were carried out. Aim of the study. To verify the toxin-producing properties of the clinical and museum strains of C. difficile in different nutrient media. Materials&methods. The toxin producing properties were verified in 7 isolated clinical strains and 2 museum strains (C. difficile №258, №281 А), obtained from the L. V. Tarasevich SISK in 1988. In order to isolate the toxin, the microorganisms were cultivated on the liver and heart-brain broth (HBB) in anaerobic conditions. After 72 hours of cultivation, bacterial cells were isolated with the help of centrifugation for 30 minutes at 8 000 rpm with the subsequent filtration of the supernatant through 0, 45 μm membranous filters (Sartorius) [2, 18]. Lethal activity was determined in the experiments on white mice with the help of the LD50/ml determination method [18, 19]. The animal experiments were carried out according to the requirements of the European Convention of the Protection of Vertebrate animals that are used for research and other scientific purposes and practical scientific recommendations [20-21]. Results&discussion. The carried out experimental studies have shown tha the museum strains of C. difficile produced toxins with different lethal activity. In the cultural filtrate of the museum strain № 258, obtained on the liver broth, the lethal activity was 7,9 LD50/ml  (LD50 max = 1:5,4 ml; LD50 mіn = 1:9,5 ml, р = 0,05), on the heart-brain broth – 10,0 LD50/ml (LD50 max = 1:8,3 ml; LD50 mіn = 1:12,5 ml, р = 0,05).  Museum strain № 281 А produced a more active toxin. Its lethal activity was correspondingly 12,5 LD50/ml (LD50 max = 1:10,0 ml; LD50 mіn = 1:14,8 ml, р = 0,05), and – 19,9 LD50/ml (LD50max = 1:17,8 ml; LD50 mіn = 1:20,5 ml, р = 0,05). Conclusion. Filtrates obtained from clinical strains of C. difficile have not demonstrated lethal activity after an intraperitoneal injection into mice, which allows us to consider them non-toxicogenic in advance and as such as those that do not have epidemic significance. The studies museum strains of C. difficile № 258 and 281 А produced lethal toxins of different activity. The toxin-producing activity of the C. difficile 281 А strain was reliably (р≤0,05) higher than that of the C. difficile № 258 strain. The toxin-producing activity of both strains was reliably higher on the heart-brain broth compared to the data obtain on the liver broth.