Pathogenic Potential of Opportunistic Gram-Negative Bacteria Isolated from the Cloacal Microbiota of Free-Living Reptile Hosts Originating from Bulgaria.

Reptiles are known to be asymptomatic carriers of various zoonotic pathogens. A number of Gram-negative opportunistic commensals are causative agents of bacterial infections in immunocompromised or stressed hosts and are disseminated by reptiles, whose epidemiological role should not be neglected. Since most studies have focused on exotic species, in captivity or as pet animals, the role of wild populations as a potential source of pathogens still remains understudied. In the present study, we isolated a variety of Gram-negative bacteria from the cloacal microbiota of free-living lizard and tortoise hosts (Reptilia: Sauria and Testudines) from the Bulgarian herpetofauna. We evaluated their pathogenic potential according to their antibiotic susceptibility patterns, biofilm-forming capacity, and extracellular production of some enzymes considered to play roles as virulence factors. To our knowledge, the phenotypic manifestation of virulence factors/enzymatic activity and biofilm formation in wild reptile microbiota has not yet been widely investigated. All isolates were found to be capable of forming biofilms to some extent and 29.6% of them could be categorized as strong producers. Two strains proved to be excellent producers. The majority of the isolated strains showed extracellular production of at least one exoenzyme. The most pronounced pathogenicity could be attributed to the newly isolated Pseudomonas aeruginosa strain due to its multiresistance, excellent biofilm formation, and expression of exoenzymes.

Functional Hydrogels for Delivery of the Proteolytic Enzyme Serratiopeptidase.

Hydrogels are superior wound dressings because they can provide protection and hydration of the wound, as well as the controlled release of therapeutic substances to aid tissue regeneration and the healing process. Hydrogels obtained from natural precursors are preferred because of their low cost, biocompatibility, and biodegradability. We describe the synthesis of novel functional hydrogels based on two natural products-citric acid (CA) and pentane-1,2,5-triol (PT, a product from lignocellulose processing) and poly(ethylene glycol) (PEG-600)-via an environment friendly approach. The hydrogels were prepared via monomer crosslinking through a polycondensation reaction at an elevated temperature in the absence of any solvent. The reagents were blended at three different compositions with molar ratios of hydroxyl (from PT and PEG) to carboxyl (from CA) groups of 1:1, 1:1.4, and 1.4:1, respectively. The effect of the composition on the physicomechanical properties of materials was investigated. All hydrogels exhibited pH-sensitive behavior, while the swelling degree and elastic modulus were dependent on the composition of the polymer network. The proteolytic enzyme serratiopeptidase (SER) was loaded into a hydrogel via physical absorption as a model drug. The release profile of SER and the effects of the enzyme on healthy skin cells were assessed. The results showed that the hydrogel carrier could provide the complete release of the loaded enzyme.

Novel sialidase from non-pathogenic bacterium Oerskovia paurometabola strain O129.

Bacterial sialidases are enzymes that are involved in a number of vital processes in microorganisms and in their interaction with the host or the environment. Their wide application for scientific and applied purposes requires the search for highly effective and non-pathogenic producers. Here, we report the first description of sialidase from Oerskovia paurometabola. The extracellular enzyme preparation was partially purified. The presence of sialidase was confirmed in native PAGE treated with the fluorogenic substrate 4MU-Neu5Ac. Maximum enzyme activity was registered at 37 °C and in the pH range of 4.0-5.5. The influence of metal ions and EDTA was examined. It was demonstrated that EDTA, Mn2+ and Ba2+ ions inhibit the sialidase activity to different extent, while Cd2+, Fe2+ and Fe3+ have stimulating effect on it. These features are studied for the first time concerning sialidase of Oerskovia representative. Cell bound sialidase and sialate aldolase were also established.

Safe Sialidase Production by the Saprophyte Oerskovia paurometabola: Gene Sequence and Enzyme Purification.

Sialidase preparations are applied in structural and functional studies on sialoglycans, in the production of sialylated therapeutic proteins and synthetic substrates for use in biochemical research, etc. They are obtained mainly from pathogenic microorganisms; therefore, the demand for apathogenic producers of sialidase is of exceptional importance for the safe production of this enzyme. Here, we report for the first time the presence of a sialidase gene and enzyme in the saprophytic actinomycete Oerskovia paurometabola strain O129. An electrophoretically pure, glycosylated enzyme with a molecular weight of 70 kDa was obtained after a two-step chromatographic procedure using DEAE cellulose and Q-sepharose. The biochemical characterization showed that the enzyme is extracellular, inductive, and able to cleave α(2→3,6,8) linked sialic acids with preference for α(2→3) bonds. The enzyme production was strongly induced by glycomacropeptide (GMP) from milk whey, as well as by sialic acid. Investigation of the deduced amino acid sequence revealed that the protein molecule has the typical six-bladed ß-propeller structure and contains all features of bacterial sialidases, i.e., an YRIP motif, five Asp-boxes, and the conserved amino acids in the active site. The presence of an unusual signal peptide of 40 amino acids was predicted. The sialidase-producing O. paurometabola O129 showed high and constant enzyme production. Together with its saprophytic nature, this makes it a reliable producer with high potential for industrial application.