Characterization of virulent Escherichia coli strains isolated from patients with urological infection

Objective. Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually.Purpose: Characteristics of non-hospital strains of UPEC isolated from patients with UTI in Yaroslavl in 2016– 2017.Materials and methods. Susceptibility of UPEC strains (n = 20) to antibacterials was measured by the serial dilution method; the antibiotic resistance and virulence genes, phylogroups, O-serogroups and sequence types were identified by PCR and whole genome sequencing. The virulence of the strains was studied using the model of Galleria mellonella larvae.Results. UPEC strains were classified as resistant (n = 11) and multi-drug resistant (n = 9) pathogens. Betalactamase genes blaTEM (n = 10), blaCTX-M (n = 6), class 1 integrons (n = 8), and gene cassettes dfrA17-aadA5 (n = 2), dfrA1 (n = 1) and aacA4-cmlA1 (n = 1) were identified. UPEC-virulence genetic determinants coding adhesins fimH, papG, sfaS, focG, afa/draBC, csgA, siderophores iroN, fyuA, iutA, counteracting factors of host immunity ompT, traT, toxins hlyA, cnf1, usp, capsule transporter kpsMTII, colicin cvaC, and pathogenicity islands I536, II536, III536, IV536, IIJ96 и IICFT073 were detected. Highly virulent and slightly virulent for G. mellonella larvae UPEC strains were obtained with LD50 104–105 and 106–107 CFU, respectively. The phylogroups A, B1, B2, E and F, serogroups О2, О4, О6, O9, O11, О15, О18, О25, О75 and O89, known sequence types ST14, ST58, ST69, ST73, ST93, ST127, ST131, ST-141, ST165, ST297, ST457, ST537, ST744, ST1434 and novel ST9239 and ST10102 were revealed.Conclusions. The identified genetic diversity of non-hospital UPEC strains is consistent with the observed global trend in the spread of human pathogens, which are characterized with both high virulence and multiple drug resistance. This makes possible to assess prospectively the current epidemiological situation, give a forecast for its development in the future, as well as determine the optimal therapeutic options.

Method for the estimation of antibacterial activity of the polyfunctional protein from transferrin family in the experimental model of the kinetics of Staphylococcus aureus development

Introduction. Lactoferrin is a cationic monomeric glycoprotein produced by acinar cells and glands, present in different places of the mucous membrane in different concentrations. In connection with the development of various variants of hygienic and medicinal products for the treatment of inflammatory diseases of the oral cavity based on lactoferrin, there was a need for an objective assessment of its antibacterial and antibiofilms properties, followed by an analysis of the preservation of activity in various variants of the isolation of this protein from the substrate and storage.Aim - to improve the effectiveness of evaluating the antibacterial activity of lactoferrin and the duration of its preservation in various biological substrates containing the active substance and individual experimental batches of the manufactured drug using automatic cultivation.Materials and methods. As part of the experiment, a microbiological diagnostic technique employing a system for the automatic cultivation of microbial populations was used. A pre-prepared bacterial suspension was inoculated into the nutrient broth and the studied lactoferrin samples were added, followed by cultivation and analysis of the possible antibacterial effects of transferrin protein. To determine the sensitivity of the isolated strains, we used our own modification of the serial dilution method developed at the Department of microbiology, virology, immunology of the A.I. Yevdokimov Moscow State University of Medicine and Dentistry. The experiment was based on the programmed automatic cultivation using the RTS-1 bioreactor. The interpretation of the results was carried out by changing the optical density at a wavelength of λ = 850 nm. The study of the growth dynamics of microorganisms was carried out in several repetitions, which was reflected in the graphs of the development of bacterial populations. The assessment of the growth control of the corresponding bacterial species was reflected in the change in the optical density values, on the basis of which the curve was built. Results and discussion. According to the results of an experimental study of the growth curves of bacterial populations, statistically significant differences in the number of viable cells in different phases of the growth curves were noted, when using different lactoferrin samples. Higher activity of human recombinant lactoferrin samples was established. An analysis of growth dynamics revealed differences in the onset of the maximum reproduction and its inhibition under the influence of various aggravating factors during cultivation. The bacteriostatic effect of lactoferrin is realized through the binding of iron ions, depriving the bacteria of this microelement, causing inhibition of their development. Along with this, lactoferrin is active against certain virulence factors of microorganisms, splitting them like serine proteases, and thus prevents their penetration into human cells.Conclusion. The method used for automatic cultivation of microorganisms in the bioreactor used allows one to obtain reproducible results, is available for wide use, and can be recommended for obtaining objective, comparable, reliable information about the antimicrobial properties of various samples of the bactericidal protein lactoferrin produced by the domestic pharmaceutical industry. The studied substrate containing recombinant human lactoferrin of Russian production is characterized by high antibacterial activity that persists for 3 years as minimum.

Poly (ε-caprolactone)/Poly (lactic acid) fibers produced by rotary jet spinning for skin dressing with antimicrobial activity

The rotary jet spinning technique permits the production of biomaterials that can be used as devices that come into contact with biological systems (including biological fluids) for diagnostic or surgical applications. These materials are composed of synthetic or natural compounds and allow the incorporation of drugs for therapeutic purposes. Two solutions containing 50% poly(lactic acid) (PLA) and 50% poly(ε-caprolactone) (PCL) diluted in three different solvents were prepared for rotary jet spinning (RJS) process. Vancomycin, an antibiotic indicated for the treatment of severe staphylococcal infections in patients with penicillin allergy, was added in the polymer solutions, to obtain drug-loaded fibrous mats. Morphological surface characterization by scanning electron microscopy revealed heterogeneous pores in the microfibers. Vancomycin loading interfered with the morphology of all samples in terms of fiber size, leading to smaller diameter fibers. Attenuated total reflectance/Fourier transform infrared spectroscopy was used for identification of the samples. The vibrational characteristics of PCL/PLA and vancomycin were consistent with expectations. Vero cell culture assays by the extract dilution and direct contact methods revealed the absence of cytotoxicity, except for the sample prepared with 50% of PCL and of a 9/2 (V/V) vancomycin content, with the growth of confluent and evenly spread cells on the fibrous mats surface. Microbiological analysis, performed on Staphylococcus aureus by the halo inhibition test and by the broth dilution method, showed that the antibacterial activity of vancomycin was maintained by the loading process in the polymer fibers. The results showed that rotary jet spinning produces satisfactory amounts of Vancomycin-loaded fibers, as potential web dressing for wound repair

Weight-loss in obese dogs promotes important shifts in fecal microbiota profile to the extent of resembling microbiota of lean dogs

Background Among the undesirable changes associated with obesity, one possibility recently raised is dysbiosis of the intestinal microbiota. Studies have shown changes in microbiota in obese rats and humans, but there are still few studies that characterize and compare the fecal microbiota of lean, obese and dogs after weight loss. Thus, this study aimed to evaluate the effects of a weight loss program (WLP) in fecal microbiota of dogs in addition to comparing them with those of lean dogs. Twenty female dogs of different breeds, aged between 1 and 9 years were selected. They were equally divided into two groups: Obese group (OG), with body condition score (BCS) 8 or 9/9, and body fat percentage greater than 30%, determined by the deuterium isotope dilution method, and lean group (LG) with BCS 5/9, and maximum body fat of 15%. Weight loss group (WLG) was composed by OG after loss of 20% of their current body weight. Fecal samples were collected from the three experimental groups. Total DNA was extracted from the feces and these were sequenced by the Illumina methodology. The observed abundances were evaluated using a generalized linear model, considering binomial distribution and using the logit link function in SAS (p < 0.05). Results The WLP modulated the microorganisms of the gastrointestinal tract, so that, WLG and LG had microbial composition with greater biodiversity than OG, and intestinal uniformity of the microbiota (Pielou’s evenness index) was higher in OG than WLG dogs (P = 0.0493) and LG (P = 0.0101). In addition, WLG had values of relative frequency more similar to LG than to OG. Conclusion The fecal microbiota of the studied groups differs from each other. The weight loss program can help to reverse the changes observed in obese dogs.

Design, Molecular Docking, Synthesis, Characterization and Biological Activities of Novel Thiazole Derivatives

Aim: The study aims to design & synthesize novel thiazole derivatives as potent antitubercular agents with minimal side effects. Background: The emergence and rapid spread of multi-drug resistant infectious microbial flora embracing a variety of bacterial as well as mycobacterium strains are causing a threat to public health worldwide. Objective: Owing to the importance, we designed compounds with thiazole functionality coupled with Schiff base and thiosemicarbazide, predicted the molecular properties and antitubercular potency of designed compounds by the in-silico method, and synthesized fifteen novel thiazole analogs, characterized and tested in vivo antitubercular, antibacterial and antioxidant potencies. Methods: Molinspiration online tool was used to predict the molecular properties and molecular docking was used to predict the antitubercular potency. FT-IR, 1H-NMR, 13C-NMR, Mass spectroscopy and bases of elemental analysis are employed to confirm the structure of compounds. 10-Fold serial dilution method, agar streak dilution test and DPPH radical scavenging methods are used to estimate antitubercular, antibacterial and antioxidant potency of title analogs, respectively. Results: Multi-step synthesis was used to synthesize a variety of novel thiazole derivatives coupled with Schiff base and thiosemicarbazide. Synthesized title compounds displayed a varying degree of antitubercular, antibacterial and antioxidant activities (mild to good). The title compounds possessing deactivating group exhibited superior activities than activating group, while unsubstituted analogs displayed intermediate activities. In addition, para-substituted analogs showed slightly higher activity than the corresponding meta substituted analogs. Conclusion: Among fifteen tested title compounds, the potent compound of this series was found to be 1-(4-nitrobenzylidene)-4-(4-(4-methoxyphenyl)thiazol-2-yl)thiosemicarbazide (BTS14), which might be extended as a novel class of antitubercular and antibacterial agents.

Mutations in Sdh Gene Subunits Confer Different Cross Resistance Patterns to SDHI Fungicides in Alternaria alternata Causing Alternaria Leaf Spot of Almond in California

Alternaria leaf spot caused by Alternaria alternata and A. arborescens is a common disease of almond in California. Succinate dehydrogenase inhibitors (SDHIs) are widely used for its management, however, we observed reduced performance of SDHI fungicides at some field sites. Thus, we evaluated the sensitivity of 520 isolates of the main pathogen A. alternata from major production areas collected between 2006 and 2019 to boscalid and of a subset of 204 isolates to six members of the SDHIs belonging to six sub-groups. Additionally, 97 isolates (14 sensitive and 83 with reduced sensitivity) of the 204 were used to determine the molecular mechanisms of resistance. A wide range of in vitro concentrations to effectively inhibit mycelial growth by 50% (EC50 values) was determined for each fungicide using the spiral gradient dilution method. Some isolates were highly resistant (EC50 values >10 μg/ml) to boscalid (a pyridine-carboxamide), pyraziflumid (a pyrazine-carboxamide), and fluxapyroxad (a pyrazole-4-carboxamide), but not to fluopyram (a pyridinyl-ethyl-benzamide), isofetamid (a phenyl-oxo-ethyl thiophene amide), and pydiflumetofen (a N-methoxy-(phenyl-ethyl)-pyrazole-carboxamide). There was no strong cross resistance among the fungicides tested, including for the two pyrazole-4-carboxamides fluxapyroxad and penthiopyrad (tested for 33 of the 204 isolates). The comparison of EC50 values for fluopyram and isofetamid resulted in the highest coefficient of determination (R2 = 0.582) among ten pairwise comparisons between sub-groups. Sequence analyses of the 97 isolates revealed five mutations in SdhB, SdhC, or SdhD subunits of the Sdh target gene among 73 isolates with reduced sensitivity to at least one SDHI. No mutations were detected in the 14 sensitive isolates and in 10 of the 83 isolates with reduced sensitivity. The most common mutation (59 isolates) was H134R in SdhC. Other mutations included H277Y (8 isolates) and H277L (2 isolates) in SdhB, as well as G79R (2 isolates) and S135R (2 isolates) in SdhC. Mutations H277Y in SdhB and S135R in SdhC were only present in isolates collected in 2012 or earlier. Both conferred mostly high levels of resistance to boscalid and also reduced sensitivity to pyraziflumid, fluxapyroxad, and isofetamid with intermediate EC50 levels. Mutations H277L in SdhB, as well as H134R and G79R in SdhC, that were found in isolates obtained after 2012 had very similar resistance phenotypes with different levels of resistance to boscalid, pyraziflumid, and fluxapyroxad, whereas sensitivity to fluopyram, isofetamid, and pydiflumetofen was mostly less affected. Our data for SDHI fungicides do not support the classical concept of positive cross resistance within a single mode of action. Because some mutations conferred resistance to multiple SDHI sub-groups, however, resistance management needs to consider all SDHIs as a homogenous group that should be mixed or rotated with other modes of action prior to resistance development to either mode of action.

Development of Inorganic Impregnated Phase Change Material(PCM) in the Pores of Activated Carbon

Objectives : Recently, energy-related research has shifted from developing alternative energy to the efficient management technology of the produced energy. As an alternative, research on phase change materials (PCMs) capable of absorbing and releasing heat as an energy medium has been conducted. This study developed a more efficient heat storage medium using activated carbon as a medium for the phase change material. At the same time, we developed a method for efficiently impregnating the phase change material into the activated carbon pores.Methods : The activated carbon used in this experiment was charcoal powder activated carbon (250-350 mesh) and granular activated carbon. The inorganic phase change materials used in the experiment was manganese nitrate hexahydrate. The method for impregnating the phase change material was pressurization method and dilution method. The heat absorption / emission capacity of the developed material was examined within the range of 10℃ to 50℃.Results and Discussion : The Scanning electron microscope (SEM) and Transmission electron microscopy energy-dispersive X-ray spectroscopy (TEM-EDX) analysis showed that the phase change material was filled in the pore of activated carbon. When the phase change material is filled by the pressurized method, the material properties of manganese nitrate hexahydrate are reflected, resulting in absorption and release of heat at each phase change temperature. As a result of experiments for the selection of the optimum solvent in the phase change material filling study using the dilution method, when ethanol was used as the solvent, the heat absorption was clearly observed even after the phase change material was loaded. As a result of selecting the optimal dilution ratio, the ratio of ethanol was determined to be 1:1 as the dilution ratio with the lowest amount of floating activated carbon. The optimal solvent removal method experimental results show that the heat absorption/release section occurred when the ethanol was removed by evaporation at 85℃ temperature.Conclusions : 1) Both the pressurization method and the dilution method are filling methods in which inorganic phase change materials can be immobilized inside activated carbon, and heat absorption and release characteristics are maintained even after loading. 2) The heat absorption release was maintained for ethanol and the optimal dilution ratio was 1:1. 3) In case of the dilute solvent removal method, the heat absorption/release capacity was maintained when the solvent was removed using only the vaporization method.

Isolation of MDCK cells with low expression of mdr1 gene and their use in membrane permeability screening

The Madin-Darby canine kidney (MDCK) cell line is frequently used for permeability screening in drug discovery. It contains endogenous transporters, most prominently canine multidrug resistance P-glycoprotein (Mdr1), which can interfere with studies of P-glycoprotein substrate assessment and permeability measurements. Because MDCK wild type (WT) is genetically heterogeneous, an isolation procedure was investigated in this study to obtain the subclonal line with low P-glycoprotein expression. The best clone obtained had up to 3-fold lower amprenavir efflux and P-glycoprotein expression in comparison to WT. Of 12 standard compounds tested that exhibited active efflux in WT cells, 11 showed a decrease in efflux in the isolated clone. However, the decrease was not below the cut-off value of 2, indicating residual P--glycoprotein activity. Clone isolation via the limiting dilution method, combined with bidirectional amprenavir permeability for clone selection, successfully identified MDCK clones with substantially lower P-glycoprotein efflux and has been demonstrated as a useful tool for assessing passive permeability in early drug discovery.

Synthesis, Characterization and Evaluation of Biological Activities of Sn(II) Complexes of Schiff Base Incorporating Sulpha Drugs

In this study, we report synthesis, characterization and biological activities of four sulpha drug based Schiff base ligands and their Sn(II) complexes. The Schiff bases and their Sn(II) complexes have been synthesized by traditional methods and characterized by the spectral techniques IR, NMR (1H and 13C), mass and TGA-DTA. Newly synthesized Schiff bases (L1-L4) and their Sn(II) complexes (C-1 to C-4) have been screened for antibacterial activity against bacterial strains S. aureus, S. pyogenus, E. coli, P. aeruginosa and antifungal activity against fungal strains C. albicans, A. niger, A. clavatus using broth micro dilution method. Best antimicrobial activity was shown by C-3 complex against E. coli (MIC, 50.0 µg/mL) and A. niger microbial strains (MIC, 100 µg/mL). Moreover, antimalarial activity against plasmodium falciparum was also studied. Complex C-3 was found to be more active against parasite P. falciparum (IC50, 0.04 µg/mL). Results showed that dichloride tin complexes are more active with respect to their corresponding Schiff base ligands.