[Show abstract][Hide abstract] ABSTRACT: Effect of human inherent immunity factors, a gene-encoded antibacterial peptide indolicidin (Ind) and a cytokine interleukin 1 (IL-1), 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 lagor exponentialphase 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. IL-1, 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 wall 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.
[Show abstract][Hide abstract] ABSTRACT: Currently, limited information is available on the enzymatic activities in ancient paleosols and permafrost, which are recognized as unique depositories of extant microorganisms. In this work, samples from buried and modern top Kastanozems (Haplic Calcisol), modern tundra (Gleysol) soils, and permafrost were tested for amylase activity based on hydrolysis of starch to low-molecular sugars that are important energy sources for microorganisms. Our study demonstrated the preservation of amylase in buried paleosols (300 and 2000 years old) and permafrost (15,000-60,000years old). The enzyme activity in buried soils was 5-7 times lower than in top soils and did not significantly (P>0.05) differ from permafrost and tundra soils. Exogenously added alkylresorcinols, valine, and glycine influenced amylolytic activity in soil and permafrost samples in the dose-dependent manner. The amylase response to stimulating concentrations of alkylresorcinols and amino acids was greater in buried paleosols (4.3-11.5-fold) than in top soils (1.9-3.8-fold) over the enzymatic activities in the controls. Positive response of soil amylase even to little changes in concentrations of the low-molecular-weight compounds may be important during recovery of biological activity of microorganisms. Results of this study contribute to a more complete evaluation of hidden biological potential of ancient soils and permafrost and are relevant to paleomicrobiology and soil biology.
No preview · Article · Oct 2015 · European Journal of Soil Biology
[Show abstract][Hide abstract] ABSTRACT: 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 Sm) 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.
[Show abstract][Hide abstract] ABSTRACT: The heterotrophic mesophilic microbial 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. The 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 from subglacial sediment samples. Our previous investigations demonstrated the necessity for special procedures for efficient reactivation of the cells from microbial communities of replace with buried soil and permafrost samples collected in the Arctic zone. The differing responses 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.
[Show abstract][Hide abstract] ABSTRACT: The structure and specific characteristics of the hydrolytic microbial complexes from chestnut paleosols buried under the barrows of different ages (similar to 4500 and similar to 3500 years) was compared with their modern analogue in microcosm experiments. Potential activity of the hydrolytic complex of the microbial community of the barrow paleosols was found to be higher than in the modern soil complex. The share of metabolically active cells revealed by FISH after the introduction of a growth-stimulating polysaccharide into the paleosol microcosm was 50% of the whole prokaryotic cell number. The paleosol community exhibited a more pronounced response to addition of the substrate than the modern soil community. The differences in the phylogenetic taxonomic structure of the prokaryotic metabolically active hydrolytic complex in the buried and modern soils were revealed. The hydrolytic complex of modern soil was more diverse, while the dominant hydrolytic organisms revealed in paleosols were unicellular and mycelial Actinobacteria, as well as Proteobacteria.
[Show abstract][Hide abstract] ABSTRACT: A comparative study was conducted on the adaptive mechanisms of the strains Arthrobacter oxydans K14 and Acinetobacter lwoffii EK30A isolated from permafrost subsoil sediments and of those of the analogous collection strains (Ac-1114 Type and BSW-27, respectively). In each pair of the strains compared, the strains differed in terms of (i) growth-related, physiological, and biochemical properties; (ii) resistance to stress factors; (iii) capacity for generation of dormant forms (DFs) under growth arrest conditions, and (iv) intrapopulation production of phase variants. The strains isolated from permafrost displayed a lower growth rate but were more resistant to repeated freezing-thawing treatment than the collection strains. Under the same growth conditions, the permafrost strains formed larger numbers of cystlike anabiotic DFs, extraordinarily small cells, and forms that became nonculturable during long-term storage. Resuscitation of the nonculturable forms resulted in a 2- to-7-fold increase in the percentage of FISH-detectable metabolically active cells. The permafrost strains were also distinguished by increased genome lability. This facilitated their dissociation into intrapopulation variants with phenotypically distinct colonial and morphological properties and different antibiotic resistance. The phenotypic variability was more prominent in Arthrobacter (for which it was not reported previously) than in Acinetobacter. In the populations produced by plating the dormant bacterial forms, the qualitative and quantitative characteristics of the phase variant spectra varied depending on the formation conditions and the composition of the solid media used for the plating. Thus, the permafrost isolates of A. oxydans and Ac. lwoffii were distinguished from their collection analogs by a more manifest adaptive potential including stress resistance, the intensity of DF generation under growth arrest conditions, and increased intrapopulation variability.
[Show abstract][Hide abstract] ABSTRACT: Methods of reactivating the dormant forms (DFs) and nonculturable cells (NCs) of the bacterial communities of buried paleosoils and subsoil permafrost stored for long periods of time (thousands to millions of years), including completely sterile samples (CFU = 0), were developed. They were based on washing the DFs and NCs to remove anabiosis autoinducers (spore germination autoinhibitors) and introducing low molecular weight extracellular growth regulators of microbial or plant origin, such as alkylhydroxybenzenes of the alkylresorcinol subtype, indoleacetic acid, and wheat germ agglutinin. It was revealed that the dormant communities of permafrost and buried soils differed in their sensitivity to reactivating factors, probably due to different natural storage conditions of the tested soil substrates and the heterogeneity of dormant populations. The latter was confirmed by FISH (fluorescent in situ hybridization): applying the reactivation methods to the cells of the dormant permafrost community resulted in an increase in the number of metabolically active cells from 5 to 77% of their total number. In contrast, the addition of microbial anabiosis autoinducers (C12-AHB) to background surface soil and permafrost samples caused the transition of bacterial cells to the dormant or the nonculturable state, depending on the C12-AHB concentration and the sensitivity of the cells from the control soil or permafrost’ to it. The data obtained contribute to our knowledge concerning the role of intercellular communication factors and the survival of microorganisms under extreme environmental conditions.
[Show abstract][Hide abstract] ABSTRACT: Skin secretory amphibian antimicrobial peptides are the part of their immune defense. The present work is devoted to the study of the influence of “water environment stress” and additional bacterial impact on the composition of the skin secretion of the Common frog (Rana temporaria) by means of high-performance liquid chromatography (HPLC) and matrix assisted laser desorption/ionization (MALDI) mass spectrometry. It was shown that the contact of the amphibian species with Micrococcus luteus and Staphylococcus aureus stimulates the release of antimicrobial peptides, maintains the high bradykinin and related peptides levels in the skin secretion and influences the processing of the latter ones. The possibilities of mass spectrometric profiling by using HPLC and MALDI were demonstrated. This feature allows the detection of potentially bioactive peptides for their future direct testing, as has been shown for temporin M and brevinin 1Tb in the present study.
Full-text · Article · Dec 2011 · Journal of Analytical Chemistry
[Show abstract][Hide abstract] ABSTRACT: The ability of the symbiotrophic rhizobium Sinorhizobium meliloti P221 to produce cells having all the properties of resting forms (RFs) during the development cycles of the culture or after
addition of the threshold concentrations of anabiosis autoinducers was demonstrated. The numbers, properties, and ultra-structure
of S. meliloti resting forms depended on the conditions of growth and poststationary-phase incubation. In the four-month poststationary-phase,
cultures grown in media deficient in some nutrient elements and energy sources (nitrogen, phosphorus, or oxygen), numerous
cells (24–76% of the number of CFUs in the stationary-phase cultures) exhibiting a high degree of heat resistance and reversibly
inhibited metabolic activity (the absence of endogenous respiration) were detected. According to their ultrastructure, all
the resting forms detected in starving cultures were divided into three groups: (1) cystlike resting cells (CRCs) with thick
cell envelopes and compacted nucleoids, (2) CRCs containing numerous (up to three-quarters of their volumes) polyhydroxyalkanoate
inclusions, and (3) RFs similar to Azotobacter cysts. The resting forms obtained in the culture grown at high concentrations (5 × 10−5 M) of C12-AHB, a chemical analogue of microbial anabiosis autoregulators, were incapable of endogenous respiration and retained the
colony-forming ability. The CFU number after plating of these resting forms was twice as high as in the control culture; the
heat resistance of these cells (55°C, 10 min) was an order of magnitude higher. The bacterial cells obtained from the resting
forms either had a mixed (Swa+Gri+) type of motility in semisolid agar, typical of the dominant phenotype of the parent cells, or switched to the Gri+ type. Emergence of different motility phenotypes depended on the conditions of RF formation. More severe stress conditions
of RF formation induced the emergence of the Gri+ type of cell motility. The results obtained can be used for development of a new generation of bacterial preparations based
on bacterial CRCs which are able to preserve their viability for a long time and are highly resistant to stress impacts.
[Show abstract][Hide abstract] ABSTRACT: The colony-forming ability of long (3–9 months) incubated cystlike resting cells (CRC) of the nonspore-forming gram-positive
bacteria Micrococcus luteus and Arthrobacter globiformis was studied in this work. The preservation of the CRC proliferative potential as assayed by plating on standard LB agar was
shown to depend on the conditions of the formation of the dormant cells. In aged post-stationary cultures of micrococci and
arthrobacters grown under carbon and phosphorus limitation the number of colony-forming units (CFU/ml) of CRC decreased in
the course of 3–9 month incubation to the level of 106–107 CFU/ml. However, M. luteus CRC obtained under carbon and nitrogen limitation and A. globiformis CRC obtained under nitrogen limitation and starvation completely lost their ability to form colonies on standard solid medium
after 4–6 months of incubation and turned into a ‘non-culturable’ (non-platable) state. In this case, the ratio of live cells
in the population of M. luteus and A. globiformis ‘non-culturable’ CRCs (determined by the Live/Dead staining test) was 10–44% of the total cell number. To study the possible
preservation of proliferative potential in non-platable CRCs, various methods of their reactivation were applied. Although
preincubation of CRC suspensions in a buffer solution of 0.1 M K2HPO4 (pH 7.4) or in the presence of lysozyme (1 or 10 μg/ml) resulted in increased numbers of live cells (determined by the Live/Dead
test) or in disruption of the cell conglomerates, it did not increase considerably the CFU titer on LB medium. Variations
in the medium composition, such as addition of sodium pyruvate as an antioxidant or dilution of the medium, promoted the formation
of macrocolonies by a small portion of nonplateable CRC of M. luteus (50−80 CFU/ml), whereas the number of the cells capable of microcolony formation (mCFU) was 1.8–6.8 × 105 mCFU/ml, exceeding the CFU titers by four orders of magnitude. The application of semisolid agar and the most probable number
(MPN) method was the most efficient for determination of the mCFU titer, and an almost complete reversion of ‘non-culturable’
micrococcal CRCs to microcolony formation was observed (up to 2.3 × 107 mCFU/ml). The usefulness of diluted complete media for the restoration of the colony-forming ability of the dormant forms
was confirmed in experiments with ‘nonculturable’ CRCs of A. globiformis. The development of special procedures and methods for determining actively proliferating cells not detected by ordinary
methods is of great importance for advanced monitoring studies.
[Show abstract][Hide abstract] ABSTRACT: Chestnut paleosols buried under steppe kurgans about 4800, 4000, and 2000 years ago and their background analogues were studied
in the dry steppe zone on the Volga-Don interfluve. Morphological, chemical, microbiological, biochemical, and radiocarbon
studies were performed. Paleoclimatic conditions in the region were reconstructed on the basis of paleosol data. The ages
of microbial fractions isolated from the buried and surface soils were determined using the method of 14C atomic mass-spectrometry. It reached 2100 years in the A1 horizon of the buried paleosol, which corresponded to the archaeological
age of the kurgan (1st century AD). The ages of microbial biomass isolated from the B2 horizons of the buried paleosol and
the background surface soil comprised 3680 ± 35 and 3300 ± 30 years, respectively. The obtained data confirmed our assumption
about preservation of microorganisms of the past epochs in the paleosols buried under archaeological monuments. It is ensured
by various mechanisms of adaptation of soil microbial communities to unfavorable environmental conditions (anabiosis, transformation
of bacteria into nanoforms, etc.). The possibility to stimulate germination of the ancient dormant microbial pool isolated
from the buried paleosols by 2–3 orders of magnitude with the use of β-indolyl-3-acetic acid as a signal substance was demonstrated.
No preview · Article · Dec 2008 · Eurasian Soil Science
[Show abstract][Hide abstract] ABSTRACT: The structure of individual cells in microbial populations in situ of the Arctic and Antarctic permafrost was studied by scanning and transmission electron microscopy methods and compared with that of cyst-like resting forms generated under special conditions by the non-spore-forming bacteria Arthrobacter and Micrococcus isolated from the permafrost. Electron microscopy examination of microorganisms in situ revealed several types of bacterial cells having no signs of damage, including "dwarf" curved forms similar to nanoforms. Intact bacterial cells in situ and frozen cultures of the permafrost isolates differed from vegetative cells by thickened cell walls, the altered structure of cytoplasm, and the compact nucleoid, and were similar in these features to cyst-like resting forms of non-spore-forming "permafrost" bacterial strains of Arthrobacter and Micrococcus spp. Cyst-like cells, being resistant to adverse external factors, are regarded as being responsible for survival of the non-spore-formers under prolonged exposure to subzero temperatures and can be a target to search for living microorganisms in natural environments both on the Earth and on extraterrestrial bodies.
[Show abstract][Hide abstract] ABSTRACT: Non-spore-forming bacteria of the genera Micrococcus and Arthrobacter, including the isolates from permafrost sediments, were found to be able to form cystlike cells under special conditions. Cystlike cells maintained the viability during long-term storage (for up to several years), had undetectable respiratory activity and the elevated resistance to heating and other unfavorable conditions, possessed the specific fine structure and morphology, and were formed in the life cycles of the microorganism. These properties allow cystlike cells to be attributed to a new type of resting microbial forms. Furthermore, the distinctive feature of resting cystlike cells was their low P/S ratios and high Ca/K ratios in comparison to vegetative cells as shown by X-ray microanalysis. The experimentally obtained bacterial cystlike cells with thickened and laminated cell walls and altered texture of the cytoplasm were similar to the cells abundant in native microbial populations isolated from permafrost sediments and ancient soils of the Kolyma lowland (Siberia, Russia). Due to the inherent elevated resistance to adverse conditions and maintenance of viability for prolonged periods, resting cystlike cells are likely to ensure long-term survival of non-spore-forming bacteria in cold environments.
No preview · Article · Jan 2003 · Proceedings of SPIE - The International Society for Optical Engineering
[Show abstract][Hide abstract] ABSTRACT: Non-spore-forming bacteria of the genera Arthrobacterand Micrococcus, isolated from permafrost subsoil, were found to produce greater amounts of the d
1extracellular 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 d
1preparation isolated from the culture liquid of the permafrost isolate Arthrobacter globiformis245 produced an effect on the collection strain Arthrobacter globiformisB-1112 and also on Micrococcus luteusand Bacillus cereus.The d
1preparation from the permafrost isolate of Arthrobacterdiffered from the chemical analogue of this factor, 4-n-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 d
1preparation. 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 d
1anabiosis-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 long-term cooling.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.