Search

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium and also an opportunistic pathogen for both humans and animals, causing acute or chronic infections. It has been frequently detected in healthy and diseased reptiles, more commonly in captive ones. Since most studies are primarily on clinical isolates, the pathogenic potential of strains originating from wild animals is poorly explored. We isolated the strain P. aeruginosa PM1012 from the cloacal microbiota of a common wall lizard (Podarcis muralis Laurenti, 1768) from a free-living population. The effect of temperature, pH and salinity on its growth was evaluated. Antibiotic resistance, the expression of several virulence factors as some extracellular enzymes, pyocyanin production and biofilm formation were also assessed. Apart from intrinsic resistance, the newly isolated strain P. aeruginosa PM1012 presented an antibiotic susceptibility profile with a low resistance rate limited to meropenem and intermediate to ceftazidime and aztreonam. Protease, lipase and gelatinase secretion was detected. Strong pyocyanin production was observed in the optimal range of growth conditions. An excellent biofilm-forming capacity was manifested.

Studies on the gut microbiome of free-living reptiles in Europe are generally fragmentary and still missing in Bulgaria. We aimed to identify and compare the fecal microbiota profiles of five syntopic lizard species from three families: the European green lizard (Lacerta viridis), the common wall lizard (Podarcis muralis), the meadow lizard (Darevskia praticola) (Lacertidae), the European snake-eyed skink (Ablepharus kitaibelii) (Scincidae), and the European slow worm (Anguis fragilis) (Anguidae), which coinhabit a low mountainous area in the western part of the country. A high-throughput sequencing of the hypervariable V3-V4 region of the 16S rRNA gene, performed on the Illumina HiSeq2500 platform, was used. The core microbiota of lizard hosts seems to be species-specific. A dynamic phyla proportion between hosts was found. The richest alpha diversity was observed in D. praticola, and the lowest alpha diversity was observed in P. muralis and A. fragilis. Within the three lacertids, the microbiota of D. praticola and L. viridis were more closely related to each other than they were to those of P. muralis. Sharing a largely common trophic resource (all species except A. fragilis are mainly insectivorous) was not an indication of similarity in their gut microbial communities.

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 → 3) sialyl linkages than those containing α(2 → 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 fibrous cellulosic substrates impregnated with meta-polybenzimidazole (PBI)-stabilized carbon nanotubes/zinc oxide with different weight content of ZnO and with the use of dimethylacetamide as dispersant media. The pristine ZnO nanoparticle powder was prepared by plant extract-mediated synthesis using Vaccinium vitis-idaea L. The green synthesized ZnO possesses an average crystallite size of 15 nm. The formation of agglomerates from ZnO NPs with size 250 nm–350 nm in the m-PBI@CNTs/ZnO was determined. The prepared materials were investigated by PXRD analysis, XPS, SEM, EDS, AFM, and TEM in order to establish the phase and surface composition, structure, and morphology of the hybrids. The potential of the synthesized hybrid composites to degrade methylene blue (MB) dye as a model contaminant in aqueous solutions under UV illumination was studied. The photocatalytic results show that in the course of the photocatalytic reaction, the m-PBI@CNTs/ZnO 1:3 photocatalyst leads to the highest degree of degradation of the methylene blue dye (67%) in comparison with the other two studied m-PBI@CNTs/ZnO 1:1 and 1:2 composites (48% and 41%). The antibacterial activity of ZnO nanoparticles and the hybrid CNT materials was evaluated by the RMDA and the dynamic contact method, respectively. The profound antibacterial effect of the m-PBI@CNTs/ZnO hybrids was monitored for 120 h of exposition in dark and UV illumination regimes. The photocatalytic property of ZnO nanoparticles significantly shortens the time for bactericidal action of the composites in both regimes. The m-PBI@CNTs/ZnO 1:2 combination achieved complete elimination of 5.105 CFU/mL E. coli cells after 10 min of UV irradiation.

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.

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.

Pseudomonas aeruginosa is a ubiquitous environmental Gram-negative bacterium and also an opportunistic pathogen for both humans and animals, causing acute or chronic infections. It has been frequently detected in healthy and diseased reptiles, more commonly in captive ones. Since most studies are primarily on clinical isolates, the pathogenic potential of strains originating from wild animals is poorly explored. We isolated the strain P. aeruginosa PM1012 from the cloacal microbiota of a common wall lizard (Podarcis muralis Laurenti, 1768) from a free-living population. The effect of temperature, pH and salinity on its growth was evaluated. Antibiotic resistance, the expression of several virulence factors as some extracellular enzymes, pyocyanin production and biofilm formation were also assessed. Apart from intrinsic resistance, the newly isolated strain P. aeruginosa PM1012 presented an antibiotic susceptibility profile with a low resistance rate limited to meropenem and intermediate to ceftazidime and aztreonam. Protease, lipase and gelatinase secretion was detected. Strong pyocyanin production was observed in the optimal range of growth conditions. An excellent biofilm-forming capacity was manifested. [ABSTRACT FROM AUTHOR]

As a pathogenicity factor in some microorganisms, sialidase is a key target for inactivation, as this would have curative and preventive effects on various diseases. Significant results are already achieved with viral sialidase inhibitors, while such studies on bacterial enzymes are scarce. Pure natural compounds representing phenols and flavonoids, were tested for their inhibitory effect on sialidases from Vibrio cholerae non-O1, Arthrobacter nicotianae and Oerskovia paurometabola. All three enzymes were isolated, purified beforehand and stored under suitable conditions. Quinic and gallic acids showed the highest inhibitory activity - 76 to 100% against the three sialidases. Fisetin had a significant inhibitory activity on two of the enzymes. The structurally related thymol and thymoquinone exerted from 80 to 100% inhibition on at least one of the enzymes. Catechin and rutin had significant inhibitory activity, varying from 49 to 100%, on some of the enzymes. Quercetin, known for its inhibitory effect on viral sialidases, had a lesser impact on the studied enzymes. The suppressive effect of quinic acid, rutin and fisetin on bacterial sialidases is observed for the first time. [ABSTRACT FROM AUTHOR]

Novel fibrous cellulosic substrates impregnated with meta-polybenzimidazole (PBI)-stabilized carbon nanotubes/zinc oxide with different weight content of ZnO and with the use of dimethylacetamide as dispersant media. The pristine ZnO nanoparticle powder was prepared by plant extract-mediated synthesis using Vaccinium vitis-idaea L. The green synthesized ZnO possesses an average crystallite size of 15 nm. The formation of agglomerates from ZnO NPs with size 250 nm–350 nm in the m-PBI@CNTs/ZnO was determined. The prepared materials were investigated by PXRD analysis, XPS, SEM, EDS, AFM, and TEM in order to establish the phase and surface composition, structure, and morphology of the hybrids. The potential of the synthesized hybrid composites to degrade methylene blue (MB) dye as a model contaminant in aqueous solutions under UV illumination was studied. The photocatalytic results show that in the course of the photocatalytic reaction, the m-PBI@CNTs/ZnO 1:3 photocatalyst leads to the highest degree of degradation of the methylene blue dye (67%) in comparison with the other two studied m-PBI@CNTs/ZnO 1:1 and 1:2 composites (48% and 41%). The antibacterial activity of ZnO nanoparticles and the hybrid CNT materials was evaluated by the RMDA and the dynamic contact method, respectively. The profound antibacterial effect of the m-PBI@CNTs/ZnO hybrids was monitored for 120 h of exposition in dark and UV illumination regimes. The photocatalytic property of ZnO nanoparticles significantly shortens the time for bactericidal action of the composites in both regimes. The m-PBI@CNTs/ZnO 1:2 combination achieved complete elimination of 5.105 CFU/mL E. coli cells after 10 min of UV irradiation. [ABSTRACT FROM AUTHOR]

Sialic acids (Sia) represent a set of derivatives of nine-carbon sugar neuraminic acid that occupy a terminal position in oligosaccharide chains located on the surface of cells. They are very important for several physiological and pathological processes, including bacterial and viral infections. Until recently, Sia had been described mostly in animals, viruses, bacteria and protozoan species. In the last years, information on sialic acids existence in filamentous fungi has been published, but these data are very scarce. The same is valid for the enzymes that are involved in the synthesis, transport and degradation of Sia in fungi. So far, most data known regarding fungal sialidases are a result of published complete fungal genomes. However, based on the accumulated data, it appears that the kingdom of fungi probably harbours hitherto unknown mechanisms and enzymes of sialic acid metabolism. This review attempts to present the state of knowledge on sialic acids in fungi with respect to their structure, diversity and biological role, as well as Sia metabolism and enzymes involved in their conversion. The summary is presented at the background of the respective metabolic mechanisms possessed by eukaryotes and prokaryotes. The possible role of sialome and sialometabolism in fungi are also discussed.

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 β-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.

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. [ABSTRACT FROM AUTHOR]

Non-enzymatic cleavage of nucleic acids (DNA or RNA) is essential for developing novel antibiotics and chemotherapeutics, as well as new gene manipulation techniques. In this study, we investigated for deoxyribonuclease activity the allenic acids 2-methyl-4-phenylhexa-2,3-dienoic acid (racemic mixture, AK-1), S-2-methyl-4-phenylbuta-2,3-dienoic acid (AK-2) and R-2-methyl-4-phenylbuta-2,3-dienoic acid (AK-3) using supercoiled plasmid pUC18 DNA as a substrate. All three allenic acids demonstrated single strand deoxyribonuclease activity in the order AK2 = AK3 > AK1. Increasing concentrations of potassium-phosphate buffer (pH 7.5) in the reaction mixture completely inhibited the relaxation of the supercoiled pUC18 DNA, thus implying that the phosphodiester bond in DNA is the vulnerable site of attack by the allenic acids. When plasmid pUC18 DNA nicked with allenic acids was treated with T4 DNA ligase, it failed to regain its covalently closed circular form. This indicates that the allenic acids act as either hydrolytic agents producing 3'-PO4 and 5'-OH termini, or oxidizers, damaging DNA by free radical mechanism. [ABSTRACT FROM AUTHOR]

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 - 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. [ABSTRACT FROM AUTHOR]

Abstract  A moderately thermophilic Gram-positive, sporulating, rod-shaped strain of Bacillus with nitrile-degrading activity was isolated from polluted industrial waters. Whole cells and cell-free extracts from the end of exponential growth phase expressed 7.6 nkat mg−1 and 2.0 nkat mg−1 benzonitrile-degrading activity, respectively, after cultivation in a fermentor with complex medium containing benzonitrile as an inducer. The benzonitrile degradation took place via the nitrilase pathway directly to benzoic acid without intermediate formation of benzamide. Samples with benzonitrilase activity of 7.6 nkat mg−1 converted 3 mg benzonitrile in 1 h at 45°C. The half-life of benzonitrilase activity for a whole cell suspension and for cells immobilized in 2% agar was 4.5 min and 6 min at 70°C without substrate and 3 min at 90°C with substrate, respectively. The nitrilase had a broad substrate spectrum. The active biocatalyst obtained by immobilization was used in a continuous process and total biodegradation of 14.1 mM benzonitrile and 37.2 mM 4-cyanopyridine in a column bioreactor at 50°C for 5 h was achieved. [ABSTRACT FROM AUTHOR]