Drug sensitivity of low growing non-­­tuberculous mycobactria

Objective — analysis of drug resistance (DR) of some types of low growing non-­tuberculous mycobactria (NTMB) by the method of double serial microdilutions of drugs of different groups to determine the minimum inhibitory concentra­tions (MIC) in a liquid culture medium using the TREK Diagnostic Systems test system, Thermo Scientific SLOWMYCO for low growing mycobacteria. Materials and methods. Investigated 122 strains of low growing NTMB (M. avium, M. intracellulare, M. gordonae, M. kansasii, M. xenopi, M. malmoense and M. simiae), which were isolated in a liquid nutrient medium during sputum inoculation. DR was determined by the culture method using the TREK Diagnos­tic Systems kits (panel for low growing NTMB, which allows to determine the MIC of 13 drugs). The results were interpreted using an automatic bacteriological analyzer Sensititre Vizion System TREK Diagnostic Systems (USA). Results and discussion. Was found that for most M. avium strains the MIC of amikacin was 16.0—32.0 µg/ml, for ciprofloxacin — 16.0 µg/ml and higher, clarithromycin — 2.0—4.0 µg/ml, doxycycline — 16.0 µg/ml and above, ethambutol — 8.0—16.0 µg/ml. The MIC spectrum of ethionamide was distributed in the range from 1.2 to more than 20.0 µg/ml. MIC of isoniazid for most strains of M. avium was more than 8.0 µg/ml, linezolid — 16.0—32.0 µg/ml, moxifloxacin — 2.0—4.0 µg/ml, rifabutin — 0.25 µg/ml, rifampicin 4.0 µg/ml and above, streptomycin 64 µg/ml and above, and trimethoprim/sulfamethoxazole — more than 8.0/152 µg/ml. For M. intracellulare strains, in general, a similar situation with the M. avium strains with the MIC spectrum was observed. The work also provides MICs for the strains M. gordonae, M. kansasii, M. xenopi, M. simiae and M. malmoense. Based on the determination of the DR, the MIC50 and MIC90 values of each preparation of the SLOWMYCO panel were calculated for the studied species of low growing NTMB. Comparison of the MIC50 and MIC90 values with the limiting drug concentrations made it possible to determine the drugs effective against the studied types of NTMs. Conclusions. Determination of DR by the micromethod of serial dilutions in a liquid nutrient medium showed that most of the studied strains of low growing NTMPs are sensitive to clarithromycin and rifabutin. Amikacin, linezolid, and moxifloxacin were also quite effective. At the same time, drugs such as ethambutol, isoniazid, streptomycin, trimethoprim/sulfamethoxazole, suppressed the growth of the studied strains mainly in high concentrations, significantly exceeding the critical one.Until now, there is no single criterion for determining the DR NTM, and for the method used in this work, recommended by the Institute for Clinical and Laboratory Standards (USA), there are limitations in the interpretation of the results due to the untreated limit concentrations of drugs for various types of NTM. This is especially true for MAC, which play a major role in the development of mycobacteriosis. It was shown that the resistance profile of M. avium strains included the largest number of SLOWMYCO panel preparations compared to other types of NTMB, which dictates the need for studies aimed at comparing the results of in vitro DST with the effectiveness of therapy.

Secondary metabolite profiles and biological activity of extracts from various isolates fungi Alternaria sonchi depending on the composition of the liquid nutrient medium

Phytopathogenic fungus A. sonchi is able to produce metabolites with insecticidal properties. The composition of the culture media affected the metabolite profiles of the extracts. The results of the assessment of biological activity allowed to divide the working isolates with phytotoxic and insecticidal activity.

Cultivation of Rhizobium leguminosarum to produce exopolysaccharide

While studying the bacteria Rhizobium leguminosarum from different sources, a strain was isolated. Its growth on a liquid nutrient medium is accompanied by the accumulation of a significant amount of exopolysaccharide substance.

METHODOLOGICAL APPROACHES TO THE STUDY OF FORMATION OF BIOFILMS BY CONDITIONALLY PATHOGENIC AND PATHOGENIC ENTEROSBACTERIES

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.