Lactic acid bacteria (LAB) play an essential role in biotechnology and biomedicine. However, their main disadvantage is the rapid death of their cultures and preparations during storage. Research on techniques of prolonging the survival time of lactic-acid bacteria under varied conditions is an important task that constituted the goal of the present work. The research subject was the lactic acid bacterium Enterococcus faecium. It was revealed that bacteria in aging planktonic cultures were rapidly losing viability (the viable cell number decreased by 2 to 4 orders of magnitude within 1 month). Under these conditions, the development cycle of the E. faecium population led to formation of cyst-like dormant cells of two different types, L forms and hypometabolic cells. Applying chemical stabilizers such as humic substances increased the viable cell number 2–3-fold. Surface immobilization (adsorption) on organosilanol or inorganic carriers such as silicon dioxide enabled elevating the number of starvation-surviving cells 1.25- to 3-fold. The most efficient approach was cell immobilization in silanol–humate gels that resulted in increasing the surviving cell number up to 35 times compared to the control. The data obtained provide an insight into the mechanisms of LAB survival and the forms of survivors under natural conditions, including the hypometabolic state and the presence of specialized dormant forms. These data can be utilized for developing techniques of long-term storage of LAB biopreparations.

We studied the possibility of using 4-hexylresorcinol to increase the efficiency of anti-mycobacterial chemotherapy. In an in vitro experiment, 4-hexylresorcinol increased the efficiency of rifampicin, kanamycin, and isoniazid against Mycobacterium smegmatis by 3-5 times. Experiments in sanitation of BALB/c mice infected with M. smegmatis showed the best efficacy of the isoniazid and 4-hexylresorcinol combination in comparison with isoniazid monotherapy. The growth-inhibiting activity of the combination of antibiotic rifabutin with 4-hexylresorcinol was shown on 6 strains of M. tuberculosis. A 2-fold decrease in the minimum inhibitory concentration of this antibiotic in the presence of half-minimum inhibitory concentration of 4-hexylresorcinol was demonstrated for monoresistant strain M. tuberculosis 5360/42Hr. On the mouse model of experimental tuberculosis caused by M. tuberculosis H37Rv, a 5-fold decrease in lung contamination and more rapid complete cure were achieved in animals treated with the combination of rifabutin and 4-hexylresorcinol in comparison with rifabutin monotherapy.

The survival of the microbial population in constantly changing environmental conditions, including those unfavorable for growth, is ensured by: (1) formation of a subpopulation of persister cells (Ps), maturing into ametabolic dormant forms (DFs); (2) protection of chromosomal DNA of stationary cells using the physicochemical mechanism of its co-crystallization with the nucleoid-associated protein Dps and the formation of a biocrystalline nucleoid (BN); and (3) the ability of DFs to germinate in a fresh environment, yielding a mixed population of phenotypically different variants, one of which will be the most adaptive to it. This study addressed two questions: (1) how BN is structurally organized in prokaryotic DFs, and (2) how nucleoid biocrystallization is related to the phenotypic heterogeneity of populations growing from DFs. The present work proposes a new model of BN decrystallization/recrystallization during heating/cooling of DFs at sublethal temperatures in a non-growth environment, which reproduces the dynamics of BN formation in the model of nucleoid organization as a folded globule. Electron microscopic analysis of structural changes in BN in heated/cooled DFs, together with determination of the dissociative spectra of the populations growing from them, allowed us to obtain the following new information. Biocrystallization of the nucleoid occurs in the following sequence: (1) incipient co-crystallization of DNA−Dps is accompanied by the division of the nucleoid volume with formation of a compacted nucleoid from superfolded DNA in the central region of the cell and loops of superfolded linear DNA extending from it; (2) co-crystallization of looped DNA−Dps is accompanied by its diverse geometric arrangement—toroidal, lamellar, etc.; and (3) crystallization of Dps-Dps, repeating the template folding of looped DNA−Dps and the formation of a multilayer structure of the Dps−Dps crystalline array. It was found that the actual heating of the DF (45‒70°C, 15 min), leading to decrystallization of looped DNA−Dps while maintaining the structure of the compacted nucleoid, did not affect the phase variation (colonial-morphological) spectrum of the population growing from the DFs. The change in its dissociative spectrum is influenced by the process of DNA−Dps recrystallization, during which, apparently, Dps binds not only to the former, but also to other DNA sites having affinity for Dps and, possibly, partially occupied by other nucleoid-associated proteins. This influences changes in DNA topology and its transcription.

Dormant forms of causative agents of healthcare-acquired infections Moraxella catarrhalis and Kocuria rhizophila have been obtained. Dormant forms cells retained viability during long-term storage (≈107 CFU/ml after 2 months) under provocative conditions (lack of nutrient sources; temperature 20°C, oxygen access) were characterized by heat resistance, and acquired special ultrastructural organization typical of dormant forms (compacted nucleoid, thickened cell wall). They were also capable of forming alternative phenotypes (dominant and small colony variants) in a new cycle of germination in a fresh medium. These results demonstrate that the dormant forms can be responsible both for survival in the environment and persistence in the host organism.

— The transcriptome of Esherichia coli cystlike dormant cells was investigated. RNA content in a single dormant cell was 0.26 fg, i.e., 13.5 times less than in a cell of a growing culture. The presence of mRNA in E. coli dormant cells has not been reported previously. The pools of gene reads for the dormant and growing cells were characterized, as well as the differential expression of all genes, calculated according to the special algorithm considering the average mRNA amount in a single cell. The notion of the genes active in every cell of the population was introduced. In each cell of the dormant and growing E. coli population, such genes were represented by the transcripts of 21 and 16% of the genes, respectively. The revealed cell heterogeneity regarding set of active genes is one of the reasons (and forms) of heterogeneity of bacterial populations. Sixty genes were revealed, for which activity increased twice or more during formation of E. coli dormant cells. These were the genes responsible for genome activity, structure and properties of the cell envelope, cell proliferation, stress adaptation, biofilm formation and functioning, and collective behavior, as well as the genes providing for survival of the cell population during germination of the dormant cells.

The culture liquid of lactic-acid bacteria (LABs) and bifidobacteria contained secondary metabolites fulfilling signal functions, such as guaiacol and γ-butyrolactone. In addition, 4-hexylresorcinol was detected in the cell extract of Bifidobacterium bifidum 791. No homoserine lactones were produced by the tested microorganisms. The presence of metabolites with signal functions in conjunction with the available data on the synthesis of neuroactive amines, amino acids, and prebiotics by LABs and bifidobacteria indicates that these bacteria can strongly influence the functioning of the whole human symbiotic microbiota as well as the human host’s health state.

Localization and structural organization of microbial biofilms developing in anthropogenic ecological niches of meat-processing plants using different raw materials (poultry, pork, or mixed materials) were investigated. Mature biofilms were revealed both at the sites subject to routine sanitary control (equipment, sewage traps, and walls) and at other locations (ceilings, reverse (lower) side of bridges, vehicle wheels, and tunnel walls at subzero temperatures), indicating high adaptability of microbial communities. This finding indicates the urgency of modification (extension) of the list of cleansing sites and of the relevant protocols. The information on the composition and diversity of microbial biofilms in this ecotope, as well as on the physiological state of component microorganisms (e.g., active, dormant, and lysing cells) was obtained using transmission electron microscopy. The morphotypes facilitating bacterial survival in these biofilms were described (old cells and several types of specialized dormant forms).

— It was shown that immobilization of lactic acid bacteria into organic gels (gelatin and gum) increased the titers of viable cells after 2 months of storage by 2.5–4 times in comparison to the control sample, and immobilization in gels of mixed composition (silanol-humate) led to their increase by 1–2 orders of magnitude. Storage of bacterial preparations in gels is promising for application in food industry and agrobiotechnologies.

-Lactic acid bacteria (LAB) are important components of the human microbiome. While they are capable both of synthesis and response to the signals of the human humoral regulatory system (hormones and neuromediators), the phenomenology and mechanisms of the LAB response to these mediators are insufficiently studied. This work showed estrogen to hinder the growth and development of E. durans, while norepinephrine, estrogen, and the brain natriuretic peptide caused dose-dependent extension of the stationary growth phase. This is the first report on stimulation of E. durans biofilm formation by the atrial natriuretic peptide and estrogen. The frequency of persister formation depended on the type of bacterial growth (planktonic or biofilm one) and was higher in the case of biofilm growth. Epinephrine and norepinephrine exhibited dose-dependent stimulation of persister formation in planktonic LAB cultures, while other tested hormones inhibited it. The effect on persister formation in biofilms was different: natriuretic peptides exhibited dose-dependent stimulation of persister formation, and none of the hormones inhibited it significantly. After several months of incubation, E. durans persister cells matured to anabiotic dormant forms with the typical ultrastructural features. The population of E. durans dormant forms was first shown to contain the form with different dormancy depth, including the viable uncultured ones.

Effects of the human hormones and human microbiota on the growth of symbiotrophic and saprotrophic bacteria are well-known. However, no information is available on the effects of hormones on formation and survival of persister cells. Persistence is a phenotype providing the survival of the population in the presence of biocidal doses of antibiotics. Hence, the aim of this work was to investigate in vitro the effects of the human humoral regulation factors catecholamines (epinephrine (E) and norepinephrine (NE)) and the steroid hormone estradiol on the frequency of persisters’ (P) formation and their survival under abrupt changes of cultivation conditions—starvation with subsequent transfer of P into a fresh rich medium. This is the first study demonstrating E and NE to impact the growth of microorganisms that inhabit human skin, Staphylococcus aureus and S. epidermidis , as well as the frequency of formation of antibiotic-tolerant and lysing agents-resistant persister cells. The effect of E and NE depended on their concentration. New data were obtained on the effect of catecholamines and estradiol on survival of S. epidermidis P under drastic changes of incubation conditions. Under conditions of long-term (12 h) starvation in 0.9% NaCl, E and NE at both physiological (ph) concentrations (4.9 and 3.6 nM) and higher ones (10 ph and 100 ph) did not affect P survival; the P amounts in the control and experimental variants decreased by two orders of magnitude (from 10 8 to 10 6 CFU/mL). E had no effect on the rich medium stress, and caused the so-called substrate-accelerated cell death, while NE at all the tested concentrations induced transition of P to an uncultured state with subsequent resuscitation (return of the colony-forming ability). Estradiol in the physiological concentration (0.22 nM) had no effect on P survival under starvation, while higher doses (10 ph and 100 ph) reduced starvation stress in P, and the amount of P decreased only 2 times. Higher estradiol concentrations (10 ph and 100 ph) alleviated the rich medium stress. Data were obtained concerning the effect of human humoral regulation factors on: (1) frequency of P formation in the cultures of S. aureus and S. epidermidis ; (2) P survival under starvation; and (3) their reversion to growth in fresh medium, i.e., under conditions closer to that in the human organism.

An Erratum to this paper has been published: https://doi.org/10.1134/S0026261722300142

— The intestinal microbiota is known to perform an important role in terms of the host organism’s metabolism and life-sustaining activities. One of the main mechanisms of the interactions between microbiota and the human organism is based upon biogenic amines (BAs). Therefore, BA synthesis by lactic acid bacteria (LABs) is extensively researched currently, and the BA-forming capacity is considered an essential feature of LAB probiotics. The present work is concerned with BA production by 13 strains of three LAB species that were isolated from various habitats. They perform diverse ecophysiological functions and were grown on four different media. The media were characterized by different initial BA concentrations. It was established that the LABs could both consume BAs present in the medium and synthesize them de novo during their growth. The data should be taken into account while developing new probiotics. Based on the results of this work, a modified version of BA biosynthesis pathways by LABs was suggested. This work also deals with the implications of the data obtained in terms of the involvement of microbially produced neuroactive compounds in the functioning of the microbial consortium of the human organism and the operation of human nervous and the immune systems. Prospects for developing “neurotransmitters-producing biofactories” on the basis of the tested symbiotic and probiotic microorganisms are discussed.

The three-dimensional (3D) architecture of the genome determines cell function. The study of DNA condensation in the cell is important for understanding the mechanisms of bacterial survival and for medicine, since ordered DNA condensation provides antibiotic resistance to pathogenic bacteria . In a dilute solution, the length of DNA is several centimeters. The length of an Escherichia coli bacterium is approximately 2.0 μm. Such a dramatic decrease in the volume occupied by DNA is a consequence of its condensation. It was found that DNA is hierarchically organized in the nucleoid with three levels of DNA compaction: the lowest level (small scale ≥1 kb bp) is provided by histone-like NAP proteins. Unlike actively growing bacteria, bacteria under starvation stress use an energy-independent mechanism to maintain the order and protect vital structures (DNA), as in inanimate nature. The study of the DNA structure in the nucleoid of E. coli bacterium was carried out using synchrotron X-ray diffraction and transmission electron microscopy. The experimental results allowed visualization of the structures of the lowest hierarchical level of DNA compaction in the nucleoid of dormant cells. The series of diffraction experiments performed for the first time indicates the presence of a periodic ordered organization of DNA in all studied bacteria. The transmission electron microscopy provided more subtle visual information about the type of DNA condensation in the nucleoid of E. coli bacterium. Intracellular nanocrystalline, liquid-crystalline, and folded nucleosome-like structures of DNA were found. A folded nucleosome-like structure was observed for the first time, it is a result of multiple folding of long DNA molecules around Dps protein and its associates.

— Microorganisms inhabiting natural environments are periodically subject to growth-inhibiting and lethal action of stress factors. Bacterial populations are known to survive and preserve its species identity in the presence of lethal doses of antibiotics due to viable nondividing dormant persister cells. Since the mechanisms of population survival under lethal and especially combined action of chemical and physical stress agents are unknown, this was the goal of the present work. Comparative analysis of survival of the stationary phase cultures of Escherichia coli К-12 MG1655 under lethal impacts of ciprofloxacin (CIP) and a lysing solution, as well as of heat and alkaline shocks, was carried out. Higher resistance to stress impacts was shown for (1) aging stationary phase cells containing a developed biocrystalline nucleoid, compared to the cells just entering the stationary phase; and (2) the populations incubated in a nutrients-free buffer, rather than in a growth medium. CIP treatment of the populations of statically incubated cultures revealed their higher resistance to extreme CIP concentrations (100 µg/mL) compared to the biocidal dose (10 µg/mL) (the Eagle effect). The subpopulations surviving the lethal impacts of heat and pH shock were found to exhibit heterogeneity due to reversion to growth of the cells temporary lacking ability to form colonies on solid media (after 10‒30 days of incubation). Resistance of the cells surviving the heat or pH stresses to the subsequent biocidal treatment with CIP suggests their identification as persister cells. Heterogeneity of the persister subpopulation in their stress resistance may be important for adjustment of the modes for sterilization and antibiotic therapy.

— Due to frequent and large-scale oil spills, increasing the stability of biopreparations with hydrocarbon-oxidizing bacteria (HOB), which are used for bioremediation of oil-contaminated environmental objects, is presently an important task. In the present work, a new approach for maintaining the viable HOB cell titer under unfavorable storage conditions (oxygen availability at 18−24°C) was developed, which implies constructing a strain with increased number of persister cells (High Persistence Strain, HPS). The HPS of a HOB bacterium Acinetobacter seifertii WS1 was obtained by antibiotic selection with ciprofloxacin. The share of persister cells in the population increased after 13 sequential selection cycles from 1.2 to 52%. Several months of storage of the A. seifertii HPS resulted in 2−4 times better survival than in the control. The new strain retained ability to produce high numbers of persister cells after numerous transfers without antibiotic selection. The rate of oil oxidation by the HPS culture after 4-month storage was 2−4 times higher than in the control culture. The developed approach to increasing the viable cell titer of long-term-stored cells has not been applied previously and may be used in ecobiotechnology.

High efficiency of a combined preparation including synergistic polymyxin B and 4-hexylresorcinol was shown for treatment of experimental sepsis caused by an antibiotic-resistant highly virulent hypermucoid Klebsiella pneumoniae strain KPM9Pmr in mice. Complex therapy with polymyxin B (1 mg/kg) and 4-hexylresorcinol (30 mg/kg) led to cure in 80%; in 20% of these mice, no bacterial cells were found. After treatment with polymyxin B alone, only 50% animals survived and all of them contained bacterial cells. Comparative analysis of the results of monotherapy and combined treatment indicates that 4-hexylresorcinol not only increases the efficiency of antibiotic, but also minimizes persistence of the infection agent and therefore, the risk of development of antibiotic resistance.