[Characterization of the Structure of the Prokaryotic Complex of Antarctic Permafrost by Molecular Genetic Techniques].

A prokaryotic mesophilic organotrophic community responsible for 10% of the total microbial number determined by epifluorescence microscopy was reactivated in the samples ofAntarctic permafrost retrieved from the environment favoring long-term preservation of microbial communities (7500 years). No culturable forms were obtained without resuscitation procedures (CFU = 0). Proteobacteria, Actinobacteria, and Firmicutes were the dominant microbial groups in the complex. Initiation of the reactivated microbial complex by addition of chitin (0.1% wt/vol) resulted in an increased share of metabolically active biomass (up to 50%) due to the functional domination of chitinolytics caused by the target resource. Thus, sequential application of resuscitation procedures and initiation of a specific physiological group (in this case, chitinolytics) to a permafrost-preserved microbial community made it possible to reveal a prokaryotic complex capable of reversion of metabolic activity (FISH data), to determine its phylogenetic structure by metagenomic anal-ysis, and to isolate a pure culture of the dominant microorganism with high chitinolytic activity.

[Applicability of MALDI Mass Spectrometry for Diagnostics of Phase Variants in Bacterial Populations].

Efficiency of MALDI mass spectrometry for differentiation between phenotypic phase variants (in colony morphology and virulence/avirulence) was investigated.for saprotrophic and opportunistically pathogenic bacteria of five genera (Acinetobacter, Arthrobacter, Rhodococcus, Corynebacterium, and Escherichia). Analysis of MALDI spectra (on the SA and HCCA matrices) included: (1) determination of similarity of the protein spectra as a percentage of the common protein peaks to the total amount of proteins, which reflects the phylogenetic relationships of the objects and has been recommended for identification of closely related species; (2) comparison of intensities of the common peaks; and (3) the presence of specific peaks as determinative characteristics of the variants. Under the standard analytical conditions the similarity between the MALDI profiles was shown to increase in the row: genus-species-strain-variant. Assessment of intensities of the common peaks was most applicable for differentiation between phase variants, especially in the case of high similarity of their profiles. Phase variants (A. oxydans strain K14) with similar colony morphotypes (S, R, M, and S(m)) grown on different media (LB agar, TSA, and TGYg) exhibited differences in their protein profiles reflecting the differences in their physiological characteristics. This finding is in agreement with our previous results on screening of the R. opacus with similar colony morphology and different substrate specificity in decomposition of chlorinated phenols. Analysis of MALDI spectra is probably the only efficient method for detection of such variants.

[Effect of Inherent Immunity Factors of Development of Antibiotic Tolerance and Survival of Bacterial Populations under Antibiotic Attack].

Effect of human inherent immunity factors of, a gene-encoded antibacterial peptide indolicidin (Ind) and a cytokine interleukin 1 (IL1) on formation of antibiotic-tolerant persister cells surviving in the presence of ciprofloxacin (Cpf, 100 µg/mL) and ampicillin (Amp, 100 µg/mL) in submerged bacterial cultures (Staphylococcus aureus FGA 209P, Escherichia coli K12, and Pseudomonas aeruginosa PAO1) was studied. While Ind in physiological concentrations (0.3 and 3.0 µg/mL) introduced to the lag- or exponential-phase cultures of test organisms exhibited no reliable effect on population growth, the number of persisters increased at 3.0 µg/mL. Bactericidal Ind concentrations (9 µg/mL) suppressed S. aureus growth (-0.1% of surviving cells) with subsequent recovery due to development of the more antibiotic-tolerant white variant. Treatment with Cpf after Ind addition resulted in mutual potentiation of their antimicrobial activity, with the number of S. aureus persisters 2 to 3 orders of magnitude lower than in the case of the antibiotic alone. IL1, another immunity factor, when introduced (0.1-1 ng/mL) to the exponentially growing S. aureus culture (but not to the lag phase culture) had a temporary growth-static effect, with the number of persisters surviving Cpf treatment (100 µg/mL) increasing by 1 to 2 orders of magnitude. Electron microscopy revealed significant alterations in the outer cell envelope layer of surviving S. aureus cells, which should be associated with their changed antigenic properties. Thus, the factors of human inherent immunity have a dose-dependent effect on the growth of bacterial populations. In combination with antibiotics, they exhibit synergism of antimicrobial action (indolicidin) and minimize (indolicidin) or increase (interleukin 1) the frequency of formation of persister cells responsible for survival of a population subjected to an antibiotic attack.

[Prokaryotic community of subglacial bottom sediments of Antarctic Lake Untersee: detection by cultural and direct microscopic techniques].

The heterotrophic mesophilic component was studied in microbial communities of the samples of frozen regolith collected from the glacier near Lake Untersee collected in 2011 during the joint Russian-American expedition to central Dronning Maud Land (Eastern Antarctica). Cultural techniques revealed high bacterial numbers in the samples. For enumeration of viable cells, the most probable numbers (MPN) method proved more efficient than plating on agar media. Fluorescent in situ hybridization with the relevant oligonucleotide probes revealed members of the groups Eubacteria (Actinobacteria, Firmicutes) and Archaea. Application of the methods of cell resuscitation, such as the use of diluted media and prevention of oxidative stress, did not result in a significant increase in the numbers of viable cells retrieved form subglacial sediment samples. Our previous investigations demonstrated the necessity for special procedures for efficient reactivation of the cells from microbial communities of preserved fossil soil and permafrost samples collected in the Arctic zone. The differences in response to the special resuscitation procedures may reflect the differences in the physiological and morphological state of bacterial cells in microbial communities subject to continuous or periodic low temperatures and dehydration.

[Synthesis of anabiosis autoinducers in non-spore-forming bacteria as a mechanism regulating their activity in soil and subsoil sedimentary rocks]. / Sintez autoinduktorov anabioza u nesporoobrazuiushchikh bakterii kak mekhanizm reguliatsii ikh aktivnosti v pochve i podpochvennykh osadochnykh porodakh.

Non-spore-forming bacteria of the genera Arthrobacter and Micrococcus, isolated from permafrost subsoil, were found to produce greater amounts of the d1 extracellular factor than closely related collection strains isolated from soil. The effect of this factor, responsible for cell transition to anabiosis, was not species-specific. Thus, the d1 crude preparation isolated from the culture liquid of the permafrost isolate Arthrobacter globiformis 245 produced an effect on the collection strain Arthrobacter globiformis B-1112 and also on Micrococcus luteus and Bacillus cereus. The crude d1 preparation from the permafrost isolate of Arthrobacter differed from the chemical analogue of this factor, 4n-hexylresorcinol, in the level of the induced cell response, which may have resulted from different cell sensitivity to various homologs of alkylhydroxybenzenes contained in the d1 preparation. Thus, additional evidence was obtained indicating that autoregulation of bacterial growth and development is implemented at the level of intercellular interactions in microbial communities. Abundant production of the d1 anabiosis-inducing factors by bacteria isolated from permafrost subsoil is probably a result of special antistress mechanisms responsible for the survival of these bacteria under extreme conditions of natural deep cooling.

[Reproductive resting forms of Arthrobacter globiformis]. / Reproduktivnye pokoiashchiesia formy Arthrobacter globiformis.

Submerged cultures of Arthrobacter globiformis grown in media unbalanced with respect to carbon and nitrogen sources were found to contain cells exhibiting features typical of resting forms: long-term viability, specific ultrastructure, dormant metabolism, and thermoresistance. Such cells were produced not only in the collection strain VKM B-1112, but also in the A. globiformis strains isolated from 2- to 3-million-year-old permafrost sediments.

[Formation of resting forms of Arthrobacter globiformis in autolysing cell suspensions]. / Obrazovanie pokoiashchikhsia form Arthrobacter globiformis v avtoliziruiushchikhsia suspenziiakh.

Under conditions of spontaneous or induced autolysis of thick cell suspensions, Arthrobacter globiformis strains produced cells exhibiting features typical of resting microbial forms. The number of viable resting cells was greater under conditions of induced rather than spontaneous autolysis. The thermoresistance of the resting cells of A. globiformis strains isolated from 2- to 3 million-year-old permafrost was higher than that of the collection A. globiformis strain.