Microbiological study of wounds and large intestine of sturgeons using β-cyclodextrin complex with levofloxacin

Background. The research laboratory Progressive biotechnologies in aquaculture of the Saratov State University of Genetics, Biotechnology and Engineering named after N.I. Vavilov studied the effect of complexes of ß-cyclodextrin on the healing of skin wounds in hybrid sturgeon fish. The total microbial count of sturgeon cut wounds was determined using the culture method and the composition of the microflora of the fish colon using the qPCR method under the influence of the ß-cyclodextrin complex. This complex was ß- cyclodextrin with levofloxacin included in a chitosan shell. The complex was adsorbed on the surface of the feed that the sturgeons received daily. Changes in the total microbial count demonstrate the nature of the inflammatory process and the effectiveness of the use of therapeutic drugs. The study of the colon microbiota also demonstrates the result of using these complexes in fish. It was found that the use of antibacterial complexes of ß-cyclodextrins with chitosan on sturgeons leads to a significant decrease in the total microbial count of the wound surface. The highest antimicrobial activity was established for the chitosan-ß-cyclodextrin complex with 20 and 15% levofloxacin content. The presence of bacteroides, eubacteria, clostridia, peptostreptocci, enterobacteria, lactobacilli and staphylococci were determined in the composition of the normal flora. At the same time, by the end of the experiment, the microbiome of the large intestine of fish is characterized by the restoration of the number of lactic acid bacteria. The research results can be used in aquaculture in the process of fish cultivation in the treatment of injuries to the outer coverings received during transportation and sorting.

Purpose. Reproduction and cultivation of fish in aquaculture is inevitably associated with trauma to the skin of fish, infection with saprophytic, opportunistic and pathogenic microorganisms during transportation, sorting, dense planting, and transition to new feed. All these factors reduce the immune status of the fish organism and lead to the occurrence of various diseases that affect all metabolic processes. Currently, antibiotics of various groups with a wide spectrum of action are use to combat infectious diseases, as well as for prevention purposes. Due to the specific lifestyle of aquatic organisms, antibiotics are introduced into the fish organism in aquaculture using medicinal baths, injections, or orally with feed.

Materials and methods. The effect of the chitosan-ß-cyclodextrin complex on the fish organism under aquarium conditions was studied. This complex was synthesized and provided by the Department of Chemical Enzymology of the Lomonosov Moscow State University. The studied complex is a light yellow powder, slowly soluble in water due to the content of chitosan and cyclodextrin and added to fish feed. For the experiment, 5 groups of sturgeons with wounds in the form of dorsal cuts of the skin 2 cm long and 0.5 cm deep were formed using the pair-analogue method. Before the experiment, the fish were fed with compound feed with a peroxide value of 24.68±2.22 for 10 days to form a model digestive disorder and intestinal dysbiosis. The fish received feed with the preparation daily (3 times a day). Individuals of the 1st and 2nd control groups did not receive the studied complex, in addition, individuals of the 2nd control group continued to receive low-quality feed during the experiment. The experimental groups received high-quality feed with the complex in different dosages of levofloxacin (the first - a complex with 20%, the second - with 15% and the third - with 10% of the antibiotic, respectively) for 7 days.

Results. The study of sturgeon wound microflora showed that the studied chitosan-ß-cyclodextrin complex with an antibiotic reduces the TMC of cut wounds. In individuals of the control groups (K1 and K2), the inflammatory process in the wounds continued until the end of the experiment (day 8). On the 8th day, the greatest suppressive effect on the microflora of fish wounds was exerted by complexes with 15 and 20% antibiotic content: in both groups, the decrease in TMC relative to day 1 was 1000 times, below K1 by 1000 times, and K2 by 10000 times. The results of assessing the diversity of sturgeon large intestine microflora indicate that the control group of fish is characterized by normal microflora: bacteroides, eubacteria, clostridia, peptostreptocci, enterobacteria, lactobacilli and staphylococci. In the experimental groups a decrease in the number of lactobacilli, enterobacteria, fusobacteria, eubacteria and clostridia genomes was recorded. Lactate-utilizing bacteria were not detected. Gradual recovery of the fish organism is confirmed by the presence of lactobacilli on the 14th day in the same quantity as before injury and the use of the complex, as well as the absence of mycoplasmas, streptococci and Candida fungi.

Conclusion. A study of wound microflora using the culture method showed that chitosan-ß-cyclodextrin complexes with levofloxacin, used in the treatment of sturgeons, have a significant antimicrobial effect on the number of microorganisms compared to the control groups, with the best effect by the end of the experiment being provided by complexes with 15 and 20% levofloxacin.

A study of wound microflora using the culture method showed that chitosan-ß-cyclodextrin complexes with levofloxacin, used in the treatment of sturgeons, have a significant antimicrobial effect on the number of microorganisms compared to the control groups, with the best effect by the end of the experiment being provided by complexes with 15 and 20% levofloxacin. Molecular genetic research using the PCR method in real time determined the presence of bacteroids, eubacteria, clostridia, peptostreptocci, enterobacteria, lactobacilli and staphylococci in the composition of the normal flora. In the 14 days the microflora of the large intestine of fish is characterized by a confident restoration of the number of lactic acid bacteria that regulate immune processes.