Meat-peptone Broth Research Articles

Comparative analysis of bactericidal properties of synthetic peptides from the active center of GM-CSF - ZP2 against different gram-negative bacteria

Objective of this study was to carry out comparative analysis of bactericidal activity of synthetic peptide ZP2 (SP ZP2) against museum strains and clinical isolates of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. Materials and methods. We used museum strains of E. coli (ATCC 25922) and P. aeruginosa ATCC 27853, as well as 104 clinical isolates of Enterobacteriaceae, including E. coli (n = 22) and K. pneumoniae (n = 82), and 98 clinical isolates of non-fermenting Gramnegative bacteria, including P. aeruginosa (n = 43) and A. baumannii (n = 55), isolated from patients with various gynecological and surgical infections. Bactericidal activity of SP ZP2 (final concentration 10 μg/ml) against the microorganisms was assessed by difference in optical density (OD) for experimental and control broth cultures after 20 min of contact of bacterial suspensions (5 x 108 CFU/ml) with SP ZP2 (in control — with distilled water), adding meat-peptone broth and 4-hour incubation at 37 °C. The effect of SP ZP2 was expressed by the Bactericidal Activity Index (BAI, %). Results. Using the in vitro assays, we have found that SP ZP2 had a pronounced bactericidal effect on both the reference strains of E. coli and P. aeruginosa, and majority (95.5-98.2%) of the studied clinical isolates of Gram-negative bacteria, regardless of their species. With regard of the average BAI values, the tested bacterial species may be ranked by increasing their sensitivity to the synthetic ZP2 peptide as follows: P. aeruginosa (74.0±2.3%) — E. coli (77.6±3.5%) — K. pneumoniae (82.8±1.6%) — A. baumannii (84.3±1.7%). In addition, significant intraspecific variability of clinical isolates of Gram-negative bacteria was revealed for their sensitivity to bactericidal effect of SP ZP2. Conclusion. The synthetic ZP2 peptide is able to inhibit growth of Gram-negative bacteria, as well as exerts a bactericidal effect, thus considering it as a promising candidate for development of new effective drugs with combined immunobiological properties for combatting infectious and inflammatory conditions caused by the indicated microorganisms which show common resistance to a wide range of antimicrobial drugs used in clinical practice.

A STUDY OF THE EFFICIENCY OF CULTURE MEDIA FOR THE RECOVERY OF LYOPHILIZED PATHOGENIC BACTERIA

Today, the most appropriate way of long-term storage of most microorganism strains in collections is their lyophilization. The rate and quality of the recovery of biological properties in lyophilized bacterial cultures depend on the quality of culture media used for this purpose. The purpose of this study was to investigate the effectiveness of culture media for the recovery of lyophilized pathogenic bacteria. The strains of Salmonella typhimurium, Pasteurella multocida, Yersinia pseudotuberculosis, and Staphylococcus aureus from the collection of the Ukrainian Laboratory of Quality and Safety of Agricultural Products of the National University of Life and Environmental Sciences of Ukraine were used in the study. In order to restore lyophilized bacterial cultures, several variants of culture media were used: meat-peptone broth (Pharmactive, Ukraine) with the addition of 5% equine serum and 0.5% glucose; meat-peptone broth (Pharmactive, Ukraine) with the addition of 5% bovine serum and 0.5% glucose; meat-peptone broth (Pharmactive, Ukraine) with the addition of 5% bovine serum and 0.5% glucose; brain-heart infusion broth (HiMedia, India) and tryptone soya broth (HiMedia, India). Faibich protective medium was used as a cryoprotectant. It was found that in the process of sublimation and cryopreservation of cultures of Salmonella typhimurium, Pasteurella multocida, Yersinia pseudotuberculosis, and Staphylococcus aureus, the loss of viable microbial cells was 99.91–99.987%. The concentration of viable microbial cells in lyophilized cultures of Salmonella typhimurium, Pasteurella multocida, Yersinia pseudotuberculosis, Staphylococcus aureus was recorded in the range of 1.4 × 10 4 –1.7 × 10 6 CFU/cm 3 . During a single passage on different culture media, obtained biomass of the studied cultures was in the initial titer x × 10 9 . The highest productivity in recovered cultures was obtained using brain-heart infusion broth manufactured by HiMedia, India (concentration of live microbial cells in experimental cultures was recorded in the range of 5.7–6.4 × 10 9 CFU/cm 3 ). Keywords : Salmonella, Pasteurella, Yersinia, Staphylococcus, nutrient media, lyophilization

ИЗЫСКАНИЕ ОПТИМАЛЬНОЙ ПИТАТЕЛЬНОЙ СРЕДЫ ДЛЯ ПОЛУЧЕНИЯ АНАТОКСИНОВ БАКТЕРИЙ CLOSTRIDIUM PERFRINGENS

The article presents the results of studies on the selection of the optimal nutrient medium for the cultivation of bacteria Cl. perfringens. Meat-peptone broth, Hottinger broth, thioglycolic medi-um, Kitta-Tarozzi medium, meat-liver-casein were tested for these purposes. It has been established that the meat-liver-casein medium is the most optimal nutrient medium for the cultivation of Cl. perfringens bacteria and the production of toxoids. To obtain toxoids, production strains of Cl. perfringens No. 28, 392, 213 were grown under anaerobic conditions of the bioreactor on meat-liver-casein medium for 6–9 h at a temperature of 37–38 °C. Inactivation of bacteria and their toxins was carried out by adding formalin to a culture of 0,7 % final concentration and maintaining at a temperature of 37-38 °C for 10 days. Inactivated biomass of bacteria Cl. perfringens and toxoids was precipitated with aluminum hydroxide. The antigenic properties of the obtained preparation were studied in rabbits by double subcutaneous administration of it in a dose of 2 cm3. It was estab-lished that inactivated bacteria Cl. perfringens and toxoids sorbed on aluminum hydroxide have high antigenic activity and induce the production of specific antibodies in blood serum in rabbits in titers up to 13,84 ± 0,22 log2.

Sensitivity of archival and clinical enterobacteria strains to synthetic GM-CSF active center ZP2 peptide

Objective: to study sensitivity of various enterobacterial species to the bactericidal action of the ZP2 synthetic GM-CSF active center peptide after long-term storage. In vitro experiments were carried out with archival archival test strains Escherichia coli K12 (GISK 240367) and E. coli (ATCC 25922), as well as 104 clinical isolates of E. coli (n = 22) and Klebsiella pneumoniae (n = 82) obtained from patients with surgical pathology. The bactericidal effect of the ZP2 peptide (final concentration 10 μg/ml) on microorganisms was assessed by measuring optical density (OD) difference between experimental and control broth cultures after 20 min exposure of bacterial suspensions (5 × 108 CFU/ml) with the ZP2 peptide (in control – with distilled water), added with meat-peptone broth and incubated at 37 °C for 4 hours. The ZP2 peptide effect was presented as Bactericidal Activity Index (BAI, %). It was experimentally found that the archival E. coli test strains as well as most of examined clinical strains of E. coli and K. pneumoniae (95.5 and 97.6%, respectively) showed sensitivity to the bactericidal effect of the synthetic ZP2 peptide (at a final concentration of 10 μg/ml ) with BAI averaging 77.6±3.5 and 82.8±1.6% and its relatively wide variation range 45.8 ... 98.9 and 42.1 ... 99.8%, respectively. The 5-year storage of synthetic ZP2 peptide at 8 °C exerted bactericidal effect on antibiotic-resistant enterobacterial strains. The data obtained can be used to develop ZP2 peptide-based drugs aimed at combating pyo-inflammatory processes caused by antibiotic-resistant E. coli and K. pneumoniae strains.

BIOLOGICAL ACTIVITY OF NICKEL NANOPARTICLES

Objective. Study the biological activity of nickel nanoparticles (Ni NPs) against pathogenic viruses and fungi, pathogens of farm animals and plants, investigate their bactericidal and growthregulating properties. Methods. Virological, microbiological and statistical. Virucidal activity of NiNPs was evaluated by the difference of virus titres in the control and under the action of NPs, bactericidal — by the difference of bacterial titres, fungicidal and fungistatic — by the difference of diameters of fungal colonies, growth-regulating — by the difference in leaf blade length and roots ofgerms of winter wheat. The antiviral activity of NPs was determined in the culture of versenalisedembryonic porcine kidney cells. Bactericidal activity was investigated in a liquid digest medium.Fungistatic activity was determined on wort agar. Wheat germs were germinated in Petri dishesaccording to generally accepted methods. The viral titre was calculated by Reed-Muench method.The bacterial titre was determined by seeding the suspensions on agar medium. Statistical processing was performed in Microsoft Office Excel and statsoft STATISTICA 12, using Student's t-test,post-hoc tests: Fisher’s least significant difference test and Duncan’s new multiple range test.Results. It was found that Ni NPs show high virucidal activity against Teschovirus A strainDniprovskyi-34, reducing the virus titre by 2.46 lg TCD50/cm3, and have a chemotherapeutic indexof 4. Ni NPs have low fungistatic activity against Acremonium cucurbitacearum 502, reducing theaverage diameter of colonies by 6.58 to 20.22 %. Regarding Acremonium strictum 048 and Fusarium sp. 072, Ni NPs have a slight stimulating activity, increasing the average diameter of the colonies by 10.18 to 12.44 % and 14.84 to 22.18 %, respectively. Under the action of Ni NPs, the titre ofBacillus subtilis ІМВ В-7023 and Pseudomonas chlororaphis subsp. aureofaciens UKM B-306(2687) upon the cultivation of bacteria in meat-peptone broth significantly (by 80.96 to 82.77 %and 15.67 to 36.39 %, respectively) increases. Ni NPs exhibits growth-promoting properties onwheat germs, increasing the average length of leaf blades and roots by 41.79 and 36.76 %, respectively. Conclusion. According to the study findings, Ni NPs cannot be recommended for the creation of antifungal preparations. Further research is needed to develop Ni NP-based antiviral preparations, virucidal and disinfecting agents, growth- regulating products for cultivated plants, and toincrease the titre of beneficial soil bacteria.

РАЗРАБОТКА СЕЛЕКТИВНОЙ СРЕДЫ ДЛЯ БАКТЕРИИ 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.

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.

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. enterocolitic a 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. enterocolitic a 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.

МОНИТОРИНГ ОБРАЗОВАНИЯ БИОПЛЕНОК УСЛОВНО-ПАТОГЕННЫМИ И ПАТОГЕННЫМИ БАКТЕРИЯМИ

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%.

Sensitivity of biofilm and planktonic forms of non-fermenting bacteria to the action of antiseptics

It is known that bacteria colonize endotracheal intubation tubes [EIT], form biofilms. Bacterial cells that are in the film matrix are protected from the effects of external factors and differ in high levels of resistance to the action of antibiotics, antiseptics, and disinfectants. The purpose of our study was to determine the sensitivity of the antiseptics of biofilm and planktont forms of non-fermentable gram-negative bacteria to the action of the antiseptics of decamethoxin, chlorohexidine bigluconate, povidone iodine, hydrogen peroxide. In order to create bacterial biofilms, sterile fragments of the endotracheal tubes were placed in test tubes with meat-peptone broth and the daily culture of the studied strain of microorganisms was blocked. To determine the influence of antiseptics on biofilms of bacteria, the method of two successive dilutions was used. The results of a comparative study of the activity of antiseptic agents (decamethoxin, bichluconate chlorhexidine, povidone iodine, hydrogen peroxide) in relation to planktonic and biofilm forms of pseudomonad and acinetobacter strains testify that in all investigated antiseptics and bacterial films pseudomonads forms are more resistant to drags preparations than in planktonic form. In bichluconate, chlorhexidine and decamethoxin MBcK for a bacterial film are 3,2 times greater than for a planktonic form. The smallest difference MBtsK for planktonic and biofilm forms of pseudomonad was in the drug povidone-iodine. MBcC bichluconate chlorhexidine for the biofilm form of acinetobacter, in comparison with planktonic form, was 3,3 times higher, for decamethoxin in 3.7 times, for povidone-iodine in 3,5 times. Biopharmaceutical forms of gram-negative nonfermenting bacteria have the ability to achieve high levels of resistance to antiseptics.

Features of resistance formation of gram-negative non-fermenting bacteria to antibiotics

The problem of the antibiotic resistance development of pathogenic microorganisms to the main groups of antibacterial drugs has evolved from medical to socio-economic. There is a resistance increase of P.aeruginosa, A.baumannii belonging to the group of gram-negative non-fermenting bacteria (GNB), to antibiotics that are used in medicine. The possibility of acquiring GNB resistance to the main groups of antibacterial drugs is related to the ability of bacteria to acquire new genetic information. The production of metal-beta-lactamase by GNB become a widespread problem of resistance to beta-lactam antibiotics. Detection of resistance genes to beta-lactam antibiotics blaVIM, blaOXA 23, blaOXA 40, blaOXA 69 and blaOXA 100 was performed using real time polymerase chain reaction (PCR-RT). Sensitivity of isolated strains of microorganisms to antibiotics was investigated using the standard disco-diffusion method (DDM). Influence of meropenem on formation of microorganism resistance was investigated in vitro by method of microorganisms passage on meat-peptone broth (MPB) with increasing concentrations of antibiotics.&#x0D; In the process of research, 14 clinical strains of P. aeruginosa and 30 strains of A.baumannii were isolated and identified, almost all strains of these types of bacteria, characterized by resistance to antibiotics-carbapenems and third and fourth generation of cephalosporins. At the same time, the three strain-carries of markers of beta-lactamase products exhibited resistance to carbapenems, and one of the strains (P.aeruginosa No. 68) was capable of producing MBL, according to DDM, was sensitive to carbapenems and MCC was 31.2 μg / ml for it. Among the investigated strains of A.baumannii 18 (60%) out of 30 isolated ones were potential producers of beta-lactamases, capable of inactivating carbapenem antibiotics. In this case, only 6 out of 18 strains showed resistance to carbapenems according to DDM. Sensitivity to carbapenems revealed all strains of the beta-lactamase producers OXA 69 and OXA 100 and three strains – OXA 23. In this work, modern ideas about the mechanisms of development of resistance of GNB were described, isolated from patients of medical institutions (Vinnytsa city), the most common are producers of beta-lactamase types of OXA 23, OXA 69, OXA 100 and VIM.

Synthesis and Characterization of a New PAMAM Metallodendrimer for Antimicrobial Modification of Cotton Fabric

The synthesis and spectral characterization of a new PAMAM metallodendrimer modified with four 4-hexylamino-1,8-naphthalimide units was described. The chemical structure of metallodendrimer has been analyzed with Fourier transform infrared spectroscopy, electronic, nuclear magnetic resonance spectroscopy, and scanning electron microscopy (SEM) analysis. The antimicrobial activity of PAMAM dendrimer and its Zn complex [Zn2(D)(NO3)4] was investigated in agar medium and in meat-peptone broth against several pathogenic bacteria and yeasts. The antimicrobial screening showed good inhibition activity of the compounds better expressed against the test Gram-positive bacteria and yeasts. The antimicrobial efficacy of cotton fabrics treated with each of the dendrimers was evaluated, and their anti-adhesive and biofilm inhibiting potential was analyzed by SEM. The complex [Zn2(D)(NO3)4] showed stronger antimicrobial and biofilm inhibiting abilities than those of the ligand. The cytotoxicity effect of the dendrimers was also investigated in vitro in model HEp-2 cell line.

K. OXYTOCA BACTERIOPHAGES ISOLATION METHODS IMPROVEMENT

The article presents the results of a study related to increasing the efficiency of phage isolation of bacteria of the species K. oxytoca, by developing the optimal composition of the medium used in the work. In scientific research, in almost all methods associated with the isolation of bacteriophages, meat-peptone broth and meat-peptone agar are used as the nutrient basis. The peculiarities of growth and cultivation of microorganisms create certain difficulties for the isolation of phages active against bacteria of the species K. oxytoca. The selection of components and the creation of an environment that would ensure the optimal growth of both the bacterial culture and the reproduction of the virus makes it possible to facilitate the isolation of bacteriophages. The number of bacterial strains used in the work was 7. All strains of cultures were obtained from the Museum of the Department of Microbiology, Virology, Epizootology and Veterinary and Sanitary Expertise of the Federal State Budget Educational Institution of Higher Education “Ulyanovsk State Agrarian University named after P.A. Stolypin”. The studies included 2 main stages. The first stage consisted in isolation of bacteriophages by the method of isolation from the external environment by the method of Adelson L.I., Lyashenko E.A. The material for the studies were samples: soil, sewage sample, fecal samples (2). Only 4 samples. According to the chosen method, the sowing of the putative phagolysate was carried out on meat-peptone agar (1.5%) and the agar for isolating bacteriophages (Aph) (1.5%). A positive result was the presence on the environment of negative colonies, clearly visible on the matt background of deep growth of bacteria. A negative result is a continuous growth (“lawn”) of bacterial culture. As a control, the culture of the microorganism studied was used for the media. In the course of the conducted studies for the first stage, 2 bacteriophages were isolated, active against the desired species of microorganism. The second stage consisted in a comparative analysis of the indices of cultivation of the isolated phages on standard meat-peptone agar and the proposed medium for isolation of bacteriophages. The main biological properties of the phages studied were also determined: morphology of negative colonies, lytic activity, specificity. The negative colonies formed on the compared media were classified as one type. The lytic activity of the studied phages on meat-peptone agar and Aph medium was the same. According to Grazia 2 × 107 BOE/ml for bacteriophage KO-3 UGSHA, (3–4) × 108BOE/ml for bacteriophage KO-8724 UGSHA. The isolated bacteriophages showed no lytic activity against strains of heterogeneous crops (Escherichia spp., Citrobacter spp., Serratia spp., Hafnia spp., Yersinia spp.).

МЕТОДОЛОГИЧЕСКИЕ ПОДХОДЫ К ИЗУЧЕНИЮ ФОРМИРОВАНИЯ БИОПЛЕНОК УСЛОВНО-ПАТОГЕННЫМИ И ПАТОГЕННЫМИ ЭНТЕРОБАКТЕРИЯМИ

To study the process of biofilm formation, microorganisms were cultured in 96-well plates, on meat-peptone broth, stained with a 0,1% solution of crystalline violet for 10...15 minutes, after which the unbound dye was washed off. The quantitative accounting of the bound dye was carried out by spectrophotometry at a wavelength of 490 nm. The technique for making bacterial preparations for light and scanning electron microscopy on dodged glasses immersed in Petri dishes with a liquid nutrient medium is proposed. A suspension of bacteria at a concentration of 105 m.k/ml in a volume of 5 ml was shaken on Vortex apparatus and introduced into Petri dishes with 20 ml of meat-peptone broth. Sterile non-greased cover glasses were placed on sterile object glasses and immersed in a liquid nutrient medium in Petri dishes. The material was incubated for 18...24 hours at 37 °C. Then the cover slips were removed with tweezers and some of them were stained with 1% aqueous solution of methylene blue (for light microscopy), and some were placed in Petri dishes with bottomed filters (for electron microscopy). The latter, in order to preserve natural architectonics, were fixed in vivo by pairs of 25% glutaraldehyde for 3...5 hours. Vapors of 2...4% osmic acid solution were used for 2...3-minutes to contrast the preparations. After treatment with vapors of osmic acid, biofilms with included bacteria acquired yellowish or brown color. The obtained preparations after dehydration with propylene oxide vapors and spraying with gold ions were examined in a scanning electron microscope (SEM). The technique allows us to study the phases of development of biofilms and obtain objective data on the morphology of populations of pathogenic and conditionally pathogenic bacteria without disturbing natural architectonics. It is shown that the intensity of biofilm formation by pathogenic microorganisms, such as salmonella, Yersinia, Staphylococcus aureus was slightly higher than that of non-pathogenic: Escherichia, Proteus, Citrobacter, Enterobacter.

Антимікробна активність нового хіміотерапевтичного препарату на основі флюмеквіну щодо Aeromonas Hydrophila

In the article presented results of study of the antimicrobial activity of the chemotherapeutic drug on the basis of the active substance – flumequine.The sensitivity of the microflora of the biomaterial to antibiotics was determined and the minimum inhibitory concentrations of flumequin, the active substance of the drug «Flyumek», were determined.For the study were selected 5 fish – carps affected by aeromonosis. The diagnosis for aeromonosis was based on epizootological data, clinical signs of the disease, pathological and anatomical changes and the results of bacteriological research. The fish were slaughtered and crops were made from the affected parts of the skin, liver and kidneys. Sowing was carried out on meat-peptone broth (MPB) and meat-peptone agar (MPA). The seeds were incubated in a thermostat at a temperature of 26 °C for 48 hours. The sensitivity of the microflora of the biomaterial to antibiotics was determined by agar diffusion using standard antibiotic disks and the Müller-Hinton medium. In order to isolate and identify the Aeromonas hydrophila bacterium, the primary culture of the pathological material was performed on the MPA, which was incubated in the thermostat at 26 °C for 24 hours. The next step was to isolate pure cultures of microorganisms and make smears that were stained with Gram in the Björk modification. The method of serial dilutions in a liquid nutrient medium was determined by the minimum inhibitory concentrations (MICs) of flumequine in the preparation of «Flyumek» for Aeromonas hydrophila isolates.The results of the test for the sensitivity of the microflora of the biomaterial from the patient to the aeromonosis of the fish showed high sensitivity of the microorganisms to flumequin, tetracycline, fluorophenicol and trimethoprim, moderate neomycin sensitivity and resistance to amoxicilline, tylosine, lincomycine and colistine. The diameter of the growth retardation zone around the disks with flumequin was – 23,2 mm in the crop sown from the skin, from the kidneys – 30,0 mm, from the liver – 30,4 mm.According to the obtained values of the minimum inhibitory concentrations of flumequine, all investigated isolates of Aeromonas hydrophila were sensitive to the drug «Flyumek» and amounted to 1.0–2.0 μg ml.The results of the conducted studies indicate that fluoroquinolone antibiotic flumequin has a high antimicrobial activity against the bacterium Aeromonas hydrophila, an aeromonosis agent of carp fish.

Аntimicrobial and anticancer activity of new poly(propyleneamine) metallodendrimers

The synthesis, EPR characterisation and biological evaluation of two new metallodendrimers, i.e. a poly(propyleneamine) dendrimer functionalized at the external surface with 4-bromo-1,8-naphthalimide and conjugated with Cu(II) and Zn(II), was performed with the aim to evaluate their antimicrobial and anticancer activity. The antimicrobial activity was investigated in meat-peptone broth against bacteria B. subtilis and P. aeruginosa, and the yeast C. lipolytica. The results showed that the compounds inhibited effectively the tested pathogens even after their deposition on a textile fabric. Anticancer activity was investigated against three human permanent cell lines from non-small cell lung cancer (A549), triple negative breast cancer (MDA-MB-231) and carcinoma of the uterine cervix (HeLa) in the c = 0.01–30 μM concentration range. The results suggest that these compounds are promising for application in biomedicine as anticancer drugs in the design of new effective preparations. The antimicrobial and anticancer activity may be related to the peculiar structural and dynamical properties revealed for the Cu(II) complexes, by a computer aided analysis of the electron paramagnetic resonance (EPR) spectra. This analysis indicated the formation, at the lowest Cu(II) concentrations, of a flexible rhombic Cu-N4 coordination with the internal amino groups of the dendrimer, which transformed into a Cu2-N4 coordination already at 0.25 equiv. of Cu(II).

Preparation, characterization, and antibacterial activity of composite material: Cotton fabric/hydrogel/silver nanoparticles

ABSTRACTA new composite cotton fabric with hydrogel containing silver nanoparticles (AgNPs) has been synthesized by two steps, and simultaneous in situ synthesis of AgNPs under visible light irradiation has been performed. The influence of silver nitrate concentration upon the hydrogel and AgNP properties was studied by colorimetric analysis, scanning electron microscopy, and transmission electron microscopy. The antibacterial activities of the composite materials have been investigated against Acinetobacter johnsonii and Escherichia coli in agar medium and meat-peptone broth. The results showed high inhibition activity toward both test cultures which were better expressed against A. johnsonii.

Low-Temperature Conservation of Collection Cholera Vibrio Strains

Objective of the study is to evaluate cholera vibrio resistance to freezing and freeze preservation at -70 °C in the presence of cryoprotectors. The study outlines the results of storage of seven different V. cholerae strains within three-years term in the protective media containing glycerin or lactose (10, 20, 30 % aquatic solutions of glycerin, meat-peptone broth with 10 % of glycerin, a medium with 15 % of lactose and 3 % of gelatin) as cryoprotectors. Demonstrated is the fact that cholera vibrio death occurs during freezing-refreezing procedure and preservation of samples. After three-year conservation the numbers of living cells decrease in all types of media, but to different extent. The highest survival rate in cholera vibrios after freezing-refreezing procedure and freeze preservation in accordance with applicable protocols is observed in lactose-gelatin medium. When using aquatic solution of glycerin as protective medium, optimum turns out to be the cryoprotector concentration equal to 20 %. After three-year preservation at -70 °C all the strains, irrespective of the protective medium composition, retain their basic diagnostically significant phenotypic characteristics. Thus, cholera vibrios can be conserved free from subcultures within three-year term (period of observation) without alterations to their basic diagnostic properties in all tested protective media but at -70 °C.

Influence of Some Food Industry Technologies on the Characteristics of Pseudomonas aeruginosa Strains

H r Ii z 0., Jindra L u k Ii S 0 v Ii: Influence of Some Food Industry Technologies on the Characteristics of Pseudomonas aeruginosa Strains. Acta vet. Brno, 57, 1988:169-181. An investigation was made into the effects of 2 and 3% con­ centration of NaCl,2.5% nitrite salting mixture, freezing and milk fermentation on the characteriStics. of 5 typical ~­ monas aeruginosa strains isolated from water (1), ·bull semen (2) and bovine mastitis (2). the results were as follows: 1. On examination of 75 re-isolated P. aeruginosa strains qualitative changes were found 12 times, being recorded in a total of 10 (13.3%) strains. they concerned only dubious to negative attempts at gelatin hydrolysis (9 times), pyover­ dine loss (twice) and absence of visible gas from KN02 (once). Another frequent finding was the loss of blue-green pigmentati<?n on King' s agar A, but the production of pyocyanin in Gessard s medium was unaffected. 2. A total of 55 minor qUllJ'ltitative changes were found in 4 strains of the foregoing group and in 41 (54.7% other strains. they concerned delayed growth at 41"C (35 times), production of visible gas from KNO only after adaptation passage in nitrate broth (16 times); delayed oxidase reaction (twice), decreased motility (once) and delayed nitrate reduction (once). they occurred mainly in cases where 2 or 3% NaCl or nitrite salting mixture was added. 3. In 16 (21.3%) strains, decreased mortality of mice inoculated i.p. with 0.2 ml of 18- to 24 h culture was recorded. Almost half of these strains came from meat-peptone broth containing 2% NaCl. Involved in this phenomenon were 12 (23.5%) out of 51 aberrant strains and 4 (16.6%) out of 24 typical strains.

A Study of the so-called Implantation of the Bacillus Bulgaricus

ing bacilli found in the fermented milk Yahourth were able to check the growth of putrefactive bacteria in the intestines, in all the articles published in attempted confirmation of his claims, the methods used to identify the bacillus are inadequately described. This work was undertaken with the purpose of determining, in the light of a better understanding of the cultural peculiarities of the bacillus bulgaricus, whether or not a true implantation took place. In the human intestine there occur normally bacilli that culturally and morphologically resemble this bacillus very closely. The group of organisms, which includes the bacillus bulgaricus, is distinguished from other groups of bacteria by the ability of its members to grow in media containing considerable amounts of acid; because of this characteristic, these bacilli are called aciduric or acid-tolerant organisms. In a previous paper1 the writer was able to show that, contrary to the usual statement, these bacteria grow luxuriantly in an ordinary laboratory medium, viz., unneutralized meat-peptone broth containing glucose or other suitable carbohydrate, and that agar prepared from this broth, with or without the addition of 0.2 percent of sodium oleate, is an excellent solid medium. It was also shown that while the bacillus bulgaricus, from its cultural and perhaps biochemical properties, also belongs to this group of sugar fermenting, acid-tolerating organisms, it differs from the bacteria of the type of the bacillus acidophilus in failing to ferment maltose. Some strains do not attack saccharose.