Anatomical, pathological, and histological features of experimental respiratory infection of birds by biofilm-forming bacteria Staphylococcus aureus.

Background and Aim: The pathogenesis of staphylococcal infections is mediated by virulence factors, such as enzymes, toxins, and biofilms, which increase the resistance of microorganisms to host immune system evasion. Testing and searching for standardized multi-level algorithms for the indication and differentiation of biofilms at the early stages of diagnosis will contribute to the development of preventive measures to control the critical points of technology and manage dangerous risk factors for the spread of infectious diseases. This research aimed to study the main stages of Staphylococcus aureus biofilm formation in in vitro experiments and to analyze the dynamics of respiratory syndrome development in chickens infected with these bacteria. Materials and Methods: Experimental reproduction of the infectious process was performed using laboratory models: 10-day-old White Leghorn chickens (n = 20). Before the experiments, the birds were divided into two groups according to the principle of analogs: Group I (control, n = 10): the birds were intranasally inoculated with 0.5 cm3 of 0.9% NaCl solution; Group II (experiment, n = 10): the birds were intranasally inoculated with a suspension of S. aureus bacteria, 0.5 cm3, concentration 1 billion/cm3. Results: Colonization of individual areas of the substrate under study in vitro occurred gradually from the sedimentation and adhesion of single motile planktonic cells to the attachment stage of microcolony development. Staining preparations with gentian violet due to the "metachromosia" property of this dye are a quick and fairly simple way to differentiate cells and the intercellular matrix of biofilms. Fixation with vapors of glutaraldehyde and osmium tetroxide preserves the natural architecture of biofilms under optical and scanning electron microscopy. Pure cultures of S. aureus microorganisms were isolated from the blood, lungs, small intestine, liver, kidneys, and spleen after 5-10 days during experimental infection of chickens. Clinical signs of respiratory syndrome developed within 5-6 days after infection. Acute and subacute serous-fibrinous airsacculitis, characterized by edema and thickening of the membranes of the air sacs and the presence of turbid, watery, foamy contents in the cavity, was the most characteristic pathomorphological sign. The signs of acute congestive hyperemia and one-sided serous-fibrinous pneumonia developed with significant thickening of fibrinous deposits. In Garder's gland, there was an increase in the number of secretory sections, indicating hypersecretion of the glands. In the lymphoid follicles of Meckel's diverticulum, leukocytes, usually lymphocytes, and pseudoeosinophils were detected. Conclusions: Hydration and heteromorphism of the internal environment of biofilms determine the localization of differentiated cells in a three-dimensional matrix for protection against adverse factors. The most characteristic pathomorphological sign was the development of acute and subacute serous-fibrinous airsacculitis when reproducing the infectious process in susceptible models. There was a significant thickening of fibrinous deposits and signs of acute congestive hyperemia and one or two serous-fibrinous pneumonia developed.

Bird immunobiological parameters in the dissemination of the biofilm-forming bacteria Escherichia coli.

Background and Aim: With the development of industrial maintenance technology, a group of pathogens called avian pathogenic Escherichia coli (APEC) became very common. The initiation, development, and outcome of the infectious process mediated by virulent APEC strains occur through a decrease in the colonization resistance of the intestine, an immunobiological marker of homeostasis stability in susceptible species. This study focused on the pathogenetic features of colibacillosis and the morphological features of E. coli. Materials and Methods: Clinical, immunological, bacteriological, and histological studies were conducted on 15-day-old white Leghorn birds (n = 20). The birds were divided into two groups: Control group (Group I; n = 10) with birds intranasally inoculated with 0.5 mL of 0.9% NaCl solution and experimental group (Group II; n = 10) with birds intranasally inoculated with 0.5 mL of an E. coli suspension at 1 billion/mL. Results: During the biofilm formation, clusters of microcolonies were formed as a gel-like intercellular matrix that accumulated due to cell coagulation. The intercellular matrix "glues" heteromorphic cells together and forms a structure of densely packed heteromorphic cells arranged in an orderly manner and growing in different directions. During the experimental reproduction of E. coli, excessive growth was observed in material isolated from poultry. Pathogenic E. coli strains implementing virulence factors adhered to the receptors of erythrocytes, alveolocytes, and enterocytes. Multicellular heterogeneous biofilms, united by an intercellular matrix, were located at the apical poles of the respiratory tract alveolocytes and enterocytes of the terminal ileum villi. Many bacteria exudate containing desquamated epithelial cells with an admixture of mucus, and polymorphonuclear leukocytes were detected in the lumen of the birds' abdominal organs. Invasive bacteria damaged the epithelial layer, violated the endothelial layer of blood vessels, and developed inflammatory hyperemia of the lamina propria of the respiratory and digestive systems' mucous membrane. A correlative dependence of changes developed by the type of delayed hypersensitivity reaction was established. Signs of accidental transformation of the thymus, atrophy of the bursa of Fabricius, disseminated thrombosis, and septic spleen developed. Moreover, toxic cardiomyocyte dystrophy, signs of congestive vascular hyperemia, massive disintegration of lymphocytes, macrophage reactions, perivascular edema resulting from the release of plasma, and shaped blood elements were detected. Conclusion: The development and outcome of the infectious process in escherichiosis primarily depend on the homeostasis stability of susceptible species and virulence factors of the pathogenic microorganisms. One of the selected strains, E. coli O78:K80 displayed the highest ability to form biofilms. Its strong adhesion ability to bird erythrocytes was demonstrated. Deepening the scientific knowledge of the interaction between eukaryotes and prokaryotes will contribute to a better understanding of the pathogenetic aspects of avian escherichiosis and eventually find promising anti-adhesive drugs that could reduce primary bacterial contamination in vivo and in vitro.

Interaction of Cyprinus carpio Linnaeus with the biofilm-forming Aeromonas hydrophila.

Background and Aim: The resistance of susceptible fish populations and the adaptive potential of heterogeneous biofilms, which cause multiple antibacterial resistance and long-term persistence of microorganisms, mediate the development and outcome of the infectious process. The study of the fish immunological parameters in interaction with biofilm-forming bacteria is of practical importance for assessing the stability of the homeostasis of the fish. This study aimed to determine the immunobiological parameters of Cyprinus carpio Linnaeus when interacting with biofilm-forming bacteria Aeromonas hydrophila. Materials and Methods: Clinically healthy fish C. carpio L. (Linnaeus, 1758) of both sexes, aged 4 years, and weighing 1.0-1.5 kg (n = 10), were used in this study. The fish were taken from the pond of the VNIIR experimental base in the period of 2020-2022. The standard method was employed to determine the phagocytic activity of blood cells, the total redox activity of neutrophils, and the bactericidal activity of blood serum. Results: After 24-48 h of cultivation in nutrient broth, the implementation of the processes of intercellular communication of bacteria had common patterns of formation of the heterogeneous structure of biofilms. Moreover, analyzing the optical density indices (density, D), it was observed that A. hydrophila was a strong producer of biofilms, as the optical density of the sample (density of sample, Ds) exceeded the optical density of the control (density of control, Dc) by more than 4 times (D = 0.464 ± 0.07). The ratio of the average number of microorganisms attached to the surface of one erythrocyte (average adhesion index) and the percentage (%) of erythrocytes having bacteria on their surface (adhesion coefficient [AC]) was 14.05 ± 0.72, and the adhesion index, AI was ≥4.00, indicating A. hydrophila to be highly adhesive. In addition, the AC of erythrocytes having bacteria on the surface was 14.05% ± 0.72%. A direct correlation was established (R2 = 0.94) between the AC (14.05% ± 0.11%-13.29% ± 0.08%) and the phagocytic index (11.3% ± 0.29%-32.0% ± 0.8%). The indicators of spontaneous nitro blue tetrazolium were 103.20 ± 11.70 when estimating the total redox activity of neutrophils. The optical density increased to 182.10 ± 21.12 with the addition of 20.0 mL of A. hydrophila bacteria (1 billion/mL) and the activity of neutrophils also increased. Conclusion: Among the markers of homeostasis stability, immunological indicators most fully reflect the mechanisms of initiation, development, and outcome of the infectious process mediated by the interaction of adhesive molecules of multicellular eukaryotes and adhesives of infectious disease pathogens. The research will contribute to further understanding the potential mechanism of quorum-sensing molecules and the search for new anti-adhesive drugs that reduce the formation of biofilms.

Reduction in Pathogenicity in Yeast-like Fungi by Farnesol in Quail Model.

Candida albicans was the first eukaryotic microorganism to exhibit quorum-sensing through the secretion of the sesquiterpene E, farnesol. This molecule is generated by dephosphorylation of farnesyl pyrophosphate in the mevalonate biosynthetic pathway in mammalian and yeast cells. Exogenous farnesol inhibits yeast-to-hyphal formation in a concentration- and time-dependent manner at the earliest stage of hyphal development. Much research has been devoted to studying the role of farnesol as an inhibitor of hyphal morphogenesis; however, little research has been published regarding the in vivo impacts of farnesol on fungal virulence and the development of Candida infection. While other studies have examined the impact of multiple doses of farnesol in addition to antimycotics, we hypothesize that C. albicans treated with a single dose of this quorum-sensing molecule could reduce fungal virulence in a quail model.

Effects of farnesol and lyticase on the formation of Candida albicans biofilm.

BACKGROUND AND AIM: Candida albicans is a dimorphic fungus that has both yeast and filamentous forms. It is part of the normal flora in the oral and genital areas of mammals. One factor for the pathogenicity of C. albicans is its ability to switch from yeast to hyphae. The hyphal form adheres and penetrates tissues more readily than the yeast form and produces biofilms that are associated with chronic infection. Biofilms are protective niches that enable microorganisms to be more resistant to antibiotic treatment, thus allowing for persistent infection. The first stage in the transition from yeast to hyphae involves the formation of a germ tube, and this transition is triggered by interactions with host cells. Germ tube formation is dependent on serum, pH, temperature, and quorum-sensing molecules (QSMs). Farnesol, which is a QSM in C. albicans, can prevent yeast to hyphae conversion and inhibits the growth of fungal biofilm. Lyticase is a synergistic enzyme complex that catalyzes yeast cell lysis by b-1,3-glucanase and is a highly specific alkaline protease that produces protoplasts or spheroplasts. This study investigated the effect of farnesol and lyticase on the formation of C. albicans biofilms. MATERIALS AND METHODS: C. albicans ATCC 2091 was cultivated on liquid and solid Sabouraud media. The presence of C. albicans was confirmed using HiCrome Candida Agar chromogenic medium. Enzyme activities were assayed using a HiCandida Identification Kit. The morphology and densitometry parameters of C. albicans biofilms were considered in the presence of farnesol (Sigma-Aldrich, Germany), lyticase (from Arthrobacter luteus; Sigma-Aldrich, Germany), and farnesol-lyticase. RESULTS: This study shows that both farnesol and lyticase possess antifungal activity against C. albicans biofilms. A significant difference among treatment groups (p<0.05) was observed from strong biofilm production to medium and weak. CONCLUSION: Many studies have been devoted to the antimicrobial action of farnesol. Bacterial enzyme lyticase is also used to degrade fungal cell walls. Both molecules show substantial antifungal properties that are similar to the properties of modern antimycotics. The current study demonstrates that farnesol and lyticase can disrupt biofilm formation in C. albicans ATCC 2091, which is an effective biofilm producer.

Morphological and adhesive properties of Klebsiella pneumoniae biofilms.

BACKGROUND AND AIM: The study of biofilm-forming ability of Gram-negative microflora has great practical importance for assessing the effectiveness of antibiotic therapy and finding new ways to diagnose and inhibit the growth of biofilms. This is because poor penetration of antibacterial drugs into the biofilm can lead to the selection of resistant strains and has a consequence evident by the occurrence of relapse of infection in animals. This study aimed to evaluate morphological and densitometric indicators of biofilm formation as well as adhesive properties of Klebsiella pneumoniae. MATERIALS AND METHODS: K. pneumoniae was cultured at 37°C for 2-144 h in vitro. The specimens for optical microscopy were prepared by fixation with a 1:1 alcohol-ether mixture for 10 min and stained with a 0.5% solution of gentian violet for 2 min, and the optical density index was evaluated at a wavelength of 490 nm. Further, the adhesive properties of the microorganisms were determined at a concentration of 1 billion/ml and a suspension of ram erythrocytes at a concentration of 100 million/ml when cultured at 37°C for 24 h. Blood smears were prepared and stained with 0.5% gentian violet. RESULTS: K. pneumonia cultured at 37°C after 24 h on the meat peptone agar formed large, convex, mucous, and white colonies (d=3.0-6.0 mm). With the growth in the meat and peptone broth, uniform turbidity of the medium was observed. Analyzing the optical density indices (density, D), it was found that K. pneumoniae were good producers of biofilms (D=0.528±0.31). Data for indicators of adhesive properties of K. pneumoniae were as follows: Average adhesion index, 4.56±0.14; adhesion coefficient, 1.07±0.52; and adhesion index, 4.26±0.07. The studied bacteria had high adhesive activity. A direct correlation dependence (R=0.94) of the optical density of biofilms (D≥0.514-0.551) and AAI (4.15±0.28-4.76±0.75) was established. CONCLUSION: This study has demonstrated that K. pneumoniae had high adhesive activity, was strong producer of biofilms, and the optical density of the sample exceeded the optical density of the control by more than 4 times.