OSMAC Strategy Integrated with Molecular Networking for Accessing Griseofulvin Derivatives from Endophytic Fungi of Moquiniastrum polymorphum (Asteraceae)

Three endophytic fungi isolated from Moquiniastrum polymorphum (Less.) G. Sancho (Asteraceae) were cultivated using the one strain many compounds (OSMAC) strategy to evaluate the production of griseofulvin derivatives. Extracts obtained were analyzed by HPLC–MS/MS and the chromatographic and spectrometric data used to elaborate a feature-based molecular network (FBMN) through the GNPS platform. This approach allowed the observation of differences such as medium-specific and strain-specific production of griseofulvin derivatives and variations of cytotoxic activity in most extracts. To evaluate the efficiency of the OSMAC approach allied with FBMN analysis in the prospection of compounds of biotechnological interest, griseofulvin and 7-dechlorogriseofulvin were isolated, and the relative concentrations were estimated in all culture media using HPLC–UV, allowing for the inference of the best strain–medium combinations to maximize its production. Malt extract-peptone broth and Wickerham broth media produced the highest concentrations of both secondary metabolites.

Prevalence and Types of Bacteria Associated With Ocular Infections of Patients Visiting the Optometry Clinic of Federal University of Technology Owerri

The prevalence and types of bacteria associated with ocular infections were studied using swab samples from ocular infected patients attending the Department of Optometry, Federal University of Technology, Owerri clinic. A total of fifty specimens were collected from patients comprising fourteen males and thirty-six females with ocular infections and analyzed aseptically in the Biotechnology laboratory within thirty minutes of collection. The samples were maintained on peptone broth in test tubes and about 1 ml of the overnight peptone broth culture was transferred into sterile petri dishes containing the culture media (nutrient, blood and macConkey agar). Standard microbiological and biochemical protocols were used for isolation, characterization and identification of the bacterial isolates. All specimens had bacterial growth. Fifty-seven bacterial isolates; 35 Gram positive and 22 Gram negative bacteria were identified. These fell into twelve species; Bacillus sp., Corynebacterium sp., Pseudomonas aeruginosa, Haemophilus sp., Staphylococcus aureus, Lactobacillus sp., Klebsiella sp., Citrobacter sp., Proteus mirabilis, Streptococcus pneumoniae, Listeria sp. and Neisseria sp. The predominant bacterial species isolated was Bacillus sp. 17 (29.8%) while Streptococcus sp., Listeria sp., and Neisseria sp. were the least with 1 (1.8%) each. The prevalence rate of bacteria was higher among the female gender within the age group 21 -30 years. The burden of bacterial infections of the eyes is high. The prevalence and types of bacteria may not be exactly the same in every part of the world. To mitigate the burden of ocular infections, physicians need to comply with etiologic approach of diagnosis and treatment regimen.

Halotolerance of pseudomonads isolated from aquatic environment and fish (Sander lucioperca) in the Volga River delta

Introduction. The material shows halophiles bacteria R. Psendomonas the water’s microbiota and the pikeperch in spring and autumn. The attachment of Pseudomonas isolated from these habitats, certain Delta areas has not been identified. Materials and methods. 190 “water” strains and 720 ones isolated from fish were experimentally tested. In meat-peptone broth (MPB) with 3.0, 7.0, and 10.0% NaCl content, daily cultures of analyzed bacteria were sown, incubation of which was carried out at 37 0 C, and the results were taken into account after 24 and 48 hours. Results. There were no significant differences in halophilicity in the analyzed strains, except for the autumn season, especially in 10.0% salt solution. Among the isolated pseudomonads, the maximum halotolerance in both biotopes was observed in P. fluorescens and P. alcaligenes in P. putida. Halophiles strains of Pseudomonas that infect the water and fish had seasonal cycles. A slight decrease in the halophilicity of pseudomonades persistent in water and fish only at concentrations of 3.0 and 10.0 mg/l from spring to summer (1.1-1.2 times), and their significant rise in autumn (1.5 and 1.4 times) in the salt concentration of 3.0 mg/l. In the tested strains in spring and autumn, increased salt tolerance values were noted, which was determined by the hydrological and hydrochemical features of Delta waters and the “transfer” of bacteria in the body of walleye during its migration from the sea to the river. Conclusion. Analysis of long-term material showed high halophiles studied strains of pseudomonad, indicating that their sanitary and epidemiologic danger, and the ability to remain viable in salted fish products

Efficacy of Peptone Glycerol Broth in Long-term Storage of the Bacterial and Yeast Cultures

Introduction: Bacterial isolates and control strains stocking is an integral part of clinical microbiology laboratories. This is an essential step in maintaining quality. Preserving the strains without altering the character is an essentiality. There are different stock culture preparations studied in past showing varied level of performance. Aim: To evaluate the performance in terms of longevity and phenotypic character preservation of Peptone Glycerol Broth (PGB) in comparison to Brucella Glycerol Broth (BGB) and Skim Milk (SKM). Materials and Methods: The present study was a prospective analytical study. Three quality control strains and seven clinical isolates with different types of resistance were stocked in triplicates with cryobead based peptone broth with 15% glycerol, Brucella Broth (BB) with 15% glycerol and 10% SKM and stored at -80°C. Isolates were revived in monthly pattern, quarterly pattern and once after 10 months to assess the variations in viability and loss of phenotypic properties arising out of repeated freeze thaw and contaminations. Viability was assessed by time taken to produce observable confluent growth on revival. Metabolic characters and antibiotic susceptibility testing were compared before and after stock revival at intervals. Results: The phenotypic characters like metabolic features and antibiotic susceptibility were preserved with all three preparations both with repeated freeze thaw and single freeze thaw at the end of 10 months. PGB and BGB had a 100% revival rate of stored isolates with a confluent growth at 24 hours in comparison to 93.56% with SKM. Conclusion: Cryobead preparation of peptone broth-15% glycerol can be used as an effective preparation for stock culture maintenance of non-fastidious bacteria and yeast.

DEVELOPMENT OF SELECTIVE MEDIUM FOR BACTERIA Y .RUCKERI

In this work the research results on development of selective biphase medium for detachment and bacterial identification of microorganism Y. ruckeri are shown. The selective medium consists of 2 phases: 1)liquid, on the basis of meat-peptone broth, sodium azide (NaN₃) and sodium dodecyl sulphate; 2) dense, including agar, maltose, trypton and bromthymol blue. It was established that substrate on the basis of sodium azide and dodecyl sulphate admit growth of Y. ruckeri on dense phase only under condition of weakening NaN₃ with 0,08%, based on its molecule migration into agar. During the test on specificity, engineered biphase medium showed presence of adequate stability of microorganisms of Yersinia species to inhibitory environmental components. Bacteria Y. ruckeri , Y. enterocolitica, Y. pseudotuberculosis species showed good growth even in weak concentrations, but it was not showed by used I experiments bacteria of grampositive and gram-negative groups, such as Staphylococcus aureus, Bacillus cereus, Escherichia coli, Flavobacterium psychrophilum, Аeromonas hydrophila. So the use of this selective medium is entirely sufficient for primary differentiation carrying out of bacteria Yersinia species from other gram-positive and gram-negative microorganisms found in fish water area. For further differentiation of Yersinia ruckeri, cultures it is enough to cultivate cultures, obtained on experimental medium, on Giss medium with arabinose 48 hours at the temperature of 260С. Bacteria Y. species doesn’t ferment arabinose, in contrast to other bacteria Yersinia , which allows to make clear specific belonging of studied cultures.

Quantifying the Influence of Relative Humidity, Temperature, and Diluent on the Survival and Growth of Enterobacter aerogenes

ABSTRACT Survival of bacteria on surfaces plays an important role in the cross-contamination of food. Temperature, relative humidity (RH), surface type, and inoculum diluent can affect bacterial survival. This study was conducted to examine how temperature, RH, and diluent affect the survival of Enterobacter aerogenes on stainless steel, polyvinyl chloride, and ceramic tile. Although surface type had little effect on survival, temperature had a clear effect. E. aerogenes survival was highest at 7°C and 15 and 50% RH on all surfaces. Some diluents allowed growth under high RH conditions. Cell populations in distilled water inoculated onto each surface decreased initially compared with populations in 1% phosphate-buffered saline (PBS) and 0.1% peptone broth. At 15 and 50% RH, cell populations in 1% PBS declined more sharply after 120 h than did those 0.1% peptone, but populations in both diluents had similar declines up to 3 weeks. Cell populations in 0.1% peptone had the greatest growth and reached the highest population density (∼8 log CFU/mL). Cell populations in PBS and distilled water increased by ∼2 log CFU/mL. When cells in 0.1% peptone were inoculated onto stainless steel at 100% RH, populations increased to ∼7 log CFU per coupon, whereas cells in 1% PBS increased to ∼5 log CFU per coupon followed by a decline over 3 weeks. DMFit and GInaFiT software modeled inactivation on surfaces at all conditions other than 100% RH at 21°C. These findings have important implications for experiments in which microorganisms are inoculated onto foods or food contact surfaces because the growth observed may be affected more by the inoculum diluent at high or uncontrolled RH than by the type of inoculated surface. HIGHLIGHTS

Changes in growth characteristics and biofilm-forming activity of Escherichia coli with cholesterol available

Aim.To assess the growth properties and formation of E. сoli biofilms with different concentrations of cholesterol available. Materials and methods. E. coli strains were cultivated in the meat-peptone broth with cholesterol added in the concentration of 3, 5, 7 and 9 mmol/l as well as in the nutrient medium without cholesterol. The growth parameters and cholesterol concentration in the samples prior to and after cultivating microorganisms and the effect of cholesterol on biofilm-forming activity of E. coli was determined. Results. Cholesterol was shown to have no bactericidal effect on E. coli, however, it is able to change the duration of growth phases of the strains that is, possibly, caused by adaptation to the presence of cholesterol and switching of metabolic pathways. Cholesterol has a stimulating effect on accumulation of biomass by the test-strain and its biofilm-forming activity. Conclusions. In the study performed, the ability of E. coli to metabolize human cholesterol was shown.

HALOTOLERANCE AEROMONADS ISOLATED FROM WATER AND PERCH (SANDER LUCIOPERCA) IN THE DELTA OF THE VOLGA RIVER

The article presents the results of a study of halotolerance in aeromonads isolated from 447 specimens of perch (Sander lucioperca) and 375 water samples in areas of its habitat in the delta of the Volga River. They were subdominant in the microbial landscape of these biotopes. There were no significant differences inoculation aeromonads found in various parts of the delta. Their halotolerance was studied by means of inoculation of daily pure cultures of meat--peptone broth with 3, 7 and 10% of sodium chloride content and incubation at 37◦C. All the studied microflora of this spieces was established to have significant indices of halotolerance with a predominance in water isolates. Whereby in cases of 3.0 and 7.0% NaCl concentrations were 2.2 times more and in the 10.0% NaCl solution with water and fish strains had similar indices, showing them to be of “marine origin”. Among isolated aeromonads, shattering the water and fish most halophilic strains of A. hydrophila and A. sobria, and halophobic strains were presented by A. caviae. As a rule, water strains had stability indices above in the average of 1.3 times higher than fish ones. Epidemiologically important strains of A. sobria were isolated from water more frequently than from fish, whereas A. hydrophila was isolated as in water as in fish at the same level. Halotolerance of isolated aeromonads in hydroecosystems of the delta of the Volga River had seasonal specificity and dynamics. The gain in halotolerance in aquatic strains of aeromonads in spring and autumn was caused by natural and climatic processes and the elevation in the salinity of delta waters. Enhanced halophilic strains of fish in these seasons is determined by their migration with fish, because in seasons pike migrates from the sea to the river ecosystem.

Features of formation of Yersinia enterocolitica biofilms

Aim: The work aimed to study the morphology of colonies and their comparison by features of the formation of Yersinia enterocolitica biofilms. Materials and Methods: Bacteria were cultured on a Yersinia Selective Agar medium ("CIN-agar") at 28°C for 24 h. The microorganisms were grown in meat-peptone broth with 1.0% glucose to measure the absolute values of the optical density of the culture. The optical density of the liquid was determined in a microplate photometric analyzer Immunochem-2100 (HTI, USA) at a wavelength of 490 nm. For the study of biofilms, the specimens were fixed for 3-5 h in pairs of 25.0% solution of glutaraldehyde (according to DV), and pairs of a 1.0% aqueous solution of osmic acid (OSO4) were used for contrasting for 2-3 min. The specimens were examined with stereoscopic microscopy "BIOMED MS-1 Stereo" (Russia) and scanning electron microscope "TM 3030 plus" (Holland). Results: With stereoscopic microscopy of the colonies of Y. enterocolitica, the S-forms had an elevated intensely colored center, radial striation along the periphery, smooth edges, d ≤ 1.0 mm. R-form colonies had a dark color and a dry surface, were tuberous and had a dense center with a peripheral ridge, rugged edges, d ≥ 1.5 mm. The optical density of the Y. enterocolitica S-form showed that this type of microorganism belongs to the moderate producers of biofilms since the optical density of the sample (density of the sample - Ds) exceeded the optical density of control (density of the control - Dc) by 3 times. In Y. enterocolitica R-form (D ≤ 0.197) weakly produced biofilms, the optical density of the sample exceeded the optical density of the control by <2 times. Conclusion: The ability to form biofilms, the variability of phenotypic features, and the multiplicity of virulence factors of bacteria significantly reduce the effectiveness of diagnostic studies. The development of accelerated methods of detection and differentiation of the virulent properties of pathogenic bacteria will allow scientifically to substantiate and develop a set of measures aimed at preventing animal diseases and obtaining safe livestock products to prevent human diseases. Thus, we need to pay attention to which forms of colonies do Y. enterocolitica form on solid nutrient media: S- or R-forms. Through this study, we know that bacteria-forming S-shaped colonies are more capable of forming biofilms than R-forms. It means that they are more pathogenic and can cause persistent infections due to adhesion and biofilm formation.

MONITORING OF FORMATION OF BIOFILMS BY OPPORTUNISTIC AND PATHOGENIC BACTERIA

The aim of the work is monitoring the formation of biofilms by opportunistic and pathogenic microorganisms. Materials and methods. The cultures of the genera Salmonella, Escherichia, Yersinia, Proteus, Citrobacter, Enterobacter, Prtovidenzia, Morganella, Klebsiella, Cronobacter, Pseudomonas, Bacillus, Staphylococcus were used in the work. The studied microorganisms were cultured in polystyrene 96-well plates. For this purpose, a daily culture of microorganisms was introduced into the wells with meat-peptone broth, having previously established a concentration of 104 mc / ml, and incubated for 24...96 hours at temperature of 37 °C. Then the medium with plankton cells was removed from the wells. 200 μl of filtered 0,1% crystal violet solution was poured into the wells of the plate and the plates were kept for 10...15 min at room temperature. Then dye was removed from the wells. Unbound dye was thoroughly washed with saline or distilled water. The plates were turned over on filter paper and dried. The presence and density of biomatrix (biofilm) was determined visually by the intensity of staining the surfaces of plates. Then, for the extraction of paint from the film, 200 μl of 96% ethanol was added to the wells and the optical density was measured on KFK-3KM spectrophotometer at a wavelength of 590 nm. Results of research. The results of the experiments allowed us to assert that within 48 hours of cultivation microorganisms form a mature biofilm, which can serve as a model for studying the process of biofilm formation. Biofilm of microorganisms of different taxonomic groups differs in density. In addition, even bacteria belonging to the same genus, under the same conditions, can form a biofilm, the density of which differs by 30...60%.