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.

Glucose Catabolite Repression Participates in the Regulation of Sialidase Biosynthesis by Antarctic Strain Penicillium griseofulvum P29.

Sialidases (neuraminidases) catalyze the removal of terminal sialic acid residues from glycoproteins. Novel enzymes from non-clinical isolates are of increasing interest regarding their application in the food and pharmaceutical industry. The present study aimed to evaluate the participation of carbon catabolite repression (CCR) in the regulation of cold-active sialidase biosynthesis by the psychrotolerant fungal strain Penicillium griseofulvum P29, isolated from Antarctica. The presence of glucose inhibited sialidase activity in growing and non-growing fungal mycelia in a dose- and time-dependent manner. The same response was demonstrated with maltose and sucrose. The replacement of glucose with glucose-6-phosphate also exerted CCR. The addition of cAMP resulted in the partial de-repression of sialidase synthesis. The CCR in the psychrotolerant strain P. griseofulvum P29 did not depend on temperature. Sialidase might be subject to glucose repression by both at 10 and 25 °C. The fluorescent assay using 4MU-Neu5Ac for enzyme activity determination under increasing glucose concentrations evidenced that CCR may have a regulatory role in sialidase production. The real-time RT-PCR experiments revealed that the sialidase gene was subject to glucose repression. To our knowledge, this is the first report that has studied the effect of CCR on cold-active sialidase, produced by an Antarctic strain.

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.

Structural and functional characterization of cold-active sialidase isolated from Antarctic fungus Penicillium griseofulvum P29.

The fungal strain, Penicillium griseofulvum P29, isolated from a soil sample taken from Terra Nova Bay, Antarctica, was found to be a good producer of sialidase (P29). The present study was focused on the purification and structural characterization of the enzyme. P29 enzyme was purified using a Q-Sepharose column and fast performance liquid chromatography separation on a Mono Q column. The determined molecular mass of the purified enzyme of 40 kDa by SDS-PAGE and 39924.40 Da by matrix desorption/ionization mass spectrometry (MALDI-TOF/MS) analysis correlated well with the calculated mass (39903.75 kDa) from the amino acid sequence of the enzyme. P29 sialidase shows a temperature optimum of 37 °C and low-temperature stability, confirming its cold-active nature. The enzyme is more active towards α(2 â†’ 3) sialyl linkages than those containing α(2 â†’ 6) linkages. Based on the determined amino acid sequence and 3D structural modeling, a 3D model of P29 sialidase was presented, and the properties of the enzyme were explained. The conformational stability of the enzyme was followed by fluorescence spectroscopy, and the new enzyme was found to be conformationally stable in the neutral pH range of pH 6 to pH 9. In addition, the enzyme was more stable in an alkaline environment than in an acidic environment. The purified cold-active enzyme is the only sialidase produced and characterized from Antarctic fungi to date.

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.

Distribution of a novel enzyme of sialidase family among native filamentous fungi.

Sialidases (neuraminidases, EC 3.2.1.18) are widely distributed in biological systems but there are only scarce data on its production by filamentous fungi. The aim of this study was to obtain information about sialidase distribution in filamentous fungi from non-clinical isolates, to determine availability of sialidase gene, and to select a perspective producer. A total of 113 fungal strains belonging to Ascomycota and Zygomycota compassing 21 genera and 51 species were screened. Among them, 77 strains (11 orders, 14 families and 16 genera) were able to synthesize sialidase. Present data showed a habitat-dependent variation of sialidase activity between species and within species, depending on location. Sialidase gene was identified in sialidase-positive and sialidase-negative strains. . Among three perspective strains, the best producer was chosen based on their sialidase production depending on type of cultivation, medium composition, and growth temperature. The selected P. griseofulvum Р29 was cultivated in 3L bioreactor at 20 °C on medium supplemented with 0.5% milk whey. The results demonstrated better growth and 2.3-fold higher maximum enzyme activity compared to the shaken flask cultures. Moreover, the early occurring maximum (48 h) is an important prerequisite for future up scaling of the process.

High Production of Neuraminidase by a Vibrio cholerae Non-O1 Strain--the First Possible Alternative to Toxigenic Producers.

Vibrio cholerae neuraminidase (VCNA) is widely used in biochemical and medical research, in processes for preparing homogenous sialoconjugates, and in the pharmaceutical industry. Its production by non-toxigenic strains is quite desirable, in order to avoid the expensive safety measures. Here, we report the first method for highly effective production of a novel, purified V. cholerae extracellular neuraminidase from a non-toxigenic strain. The enzyme is highly active, and its properties, as well as the responsible gene nanH, are practically identical with those of the toxigenic strains. It cleaves α,2 → 3 and α,2 → 6 glycosidic bonds with highest affinity (K M 1.7 × 10(-5) µM) for human transferrin. The deduced amino acid sequence of the enzyme reveals three binding sites for Ca(2+) and one for sialic acid. The sequence analysis of the nanH gene, being the first for a V. cholerae non-O1 strain, shows 99% identity with a new nanH allele of an O1 Vibrio strain. The simple laboratory technology for efficient production of the new VCNA is based on the use of common and cheap nutrient media and easily available inducer--glycomacropeptide. The rapid purification consists of salting-out and diethylaminoethanol (DEAE) and Q-Sepharose chromatography steps. Purified preparation contains no aldolase and protease, which gives the production scheme a great potential for industrial application.

Biochemical studies on the production of neuraminidase by environmental isolates of Vibrio cholerae non-O1 from Bulgaria.

Neuraminidase is a key factor in the infectious process of many viruses and pathogenic bacteria. The neuraminidase enzyme secreted by the etiological agent of cholera - Vibrio cholerae О1 - is well studied in contrast with the one produced by non-O1/non-O139 V. cholerae. Environmental non-O1/non-O139 V. cholerae isolates from Bulgaria were screened for production of neuraminidase. The presence of the neuraminidase gene nanH was detected in 18.5% of the strains. Тhe strain showing highest activity (30 U/mL), V. cholerae non-O1/13, was used to investigate the enzyme production in several media and at different aeration conditions. The highest production of extracellular neuraminidase was observed under microaerophilic conditions, which is possibly related to its role in the infection of intestine epithelium, where the oxygen content is low. On the other hand, this is another advantage of the microbe in such microaerophilic environments as sediments and lake mud. The highest production of intracellular neuraminidase was observed at anaerobic conditions. The ratio of extracellular to intracellular neuraminidase production in V. cholerae was investigated. The temperature optimum of the enzyme was determined to be 50 °C and the pH optimum to be 5.6-5.8.