Antibiotic Adjuvant 4-Hexylresorcinol Enhances the Efficiency of Antituberculosis Drugs.

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.

Stress-Tolerant Dormant Bacterial Forms: Biological and Ultrastructural Properties of Moraxella catarrhalis and Kocuria rhizophila.

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.

Effect of 4-Hexylresorcinol on the Efficiency of Antibiotic Treatment of Experimental Sepsis Caused by Antibiotic-Resistant Klebsiella pneumoniae Strain in Mice.

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.

The use of 4-Hexylresorcinol as antibiotic adjuvant.

Ever decreasing efficiency of antibiotic treatment due to growing antibiotic resistance of pathogenic bacteria is a critical issue in clinical practice. The two generally accepted major approaches to this problem are the search for new antibiotics and the development of antibiotic adjuvants to enhance the antimicrobial activity of known compounds. It was therefore the aim of the present study to test whether alkylresorcinols, a class of phenolic lipids, can be used as adjuvants to potentiate the effect of various classes of antibiotics. Alkylresorcinols were combined with 12 clinically used antibiotics. Growth-inhibiting activity against a broad range of pro- and eukaryotic microorganisms was determined. Test organisms did comprise 10 bacterial and 2 fungal collection strains, including E. coli and S. aureus, and clinical isolates of K. pneumoniae. The highest adjuvant activity was observed in the case of 4-hexylresorcinol (4-HR), a natural compound found in plants with antimicrobial activity. 50% of the minimal inhibitory concentration (MIC) of 4-HR caused an up to 50-fold decrease in the MIC of antibiotics of various classes. Application of 4-HR as an adjuvant revealed its efficiency against germination of bacterial dormant forms (spores) and prevented formation of antibiotic-tolerant persister cells. Using an in vivo mouse model of K. pneumoniae-induced sepsis, we could demonstrate that the combination of 4-HR and polymyxin was highly effective. 75% of animals were free of infection after treatment as compared to none of the animals receiving the antibiotic alone. We conclude that alkylresorcinols such as 4-HR can be used as an adjuvant to increase the efficiency of several known antibiotics. We suggest that by this approach the risk for development of genetically determined antibiotic resistance can be minimized due to the multimodal mode of action of 4-HR.

[Model of the Regulation of Activity of Immobilized Enzymes (Amylases) in Soil].

The preservation of activity of extracellular enzymes in soil is presently associated with their immobilization on organic or inorganic carriers. Enzyme immobilization results, however, in a significant decrease in enzymatic activity. In the present work, the mechanism responsible for promotion of the catalytic activity was revealed, as well as the favorable effect of low-molecular alkylhydrozybenzenes of the class of alkylresorcinols, which are common in soil organic matter, on stability of immobilized enzymes (exemplified by amylases) by their post-translational modification. Optimal conditions (enzyme to sorbent ratio, pH optimum, CaCl2 concentration, and sorption time) for amylase sorption on a biological sorbent (yeast cell walls) were determined and decreased activity of the immobilized enzyme compared to its dissolved state was confirmed. Alkylresorcinols (C7AHB) at concentrations of 1.6 to 80 mM were found to cause an increase of amylase activity both in the case of already sorbed enzymes (by 30%) and in the case of a free dissolved enzyme with its subsequent immobilization (by 50­60%). In both cases, the optimal C7AHB concentration was 16 mM. Amylase stability was determined for C7AHB-modified and unmodified enzymes immobilized on the biological sorbent after two cycles of freezing (­20°C) and thawing (4°C). Inverse dependence was revealed between increasing stability of C7AHB-modified enzymes and an increase in their activity, as well as higher stability of immobilized modified amylases than of the dissolved modified enzyme. Investigation of the effect of C7HOB-modification in the preservation of activity in immobilized amylases after four freeze­thaw cycles revealed: (1) better preservation of activity by the modified immobilized enzymes compared to immobilized ones; (2) differences in the dynamics of activity loss within compared pairs, with activity of immobilized amylases decreasing after the second cycle to a lower level (42%) than activity of the modified immobilized enzymes after the fourth cycle (48%). These results demonstrate that in the preservation of activity of extracellular enzymes in soil both stabilization mechanisms are of importance: immobilization on organic carriers and modification of the enzyme conformation by low-molecular compounds with the functions of chemical chaperones.

[Role of Neuromediators in the Functioning of the Human Microbiota: "Business Talks" among Microorganisms and the Microbiota-Host Dialogue].

Current concepts concerning social behavior of the microorganisms inhabiting human gastrointestinal tract, as well as their role in the formation of integrated supracellular structures and in intercellular communication in the host-microbiota system are reviewed. Analysis of the literature data and the results obtained by the authors indicate an important role of neuromediators (biogenic amines, amino acids, peptides, and nitric oxide) in the intra- and interspecies microbial communication, as well as in the microbiota-host dialogue. The role of this dialogue for human health, its effect on human psyche and social behavior, and the possibility of construction of probiotic preparations with a goal-directed neurochemical effect are discussed.

[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.

[Activation of formation of bacterial biofilms by azithromycin and prevention of this effect].

Growth of members of most of the studied genera of gram-positive (Dietzia, Kocuria, and Rhodo- coccus) and gram-negative bacteria (Pseudomonas and Chromobacterium) in biofilms exhibited higher resistance to an translation inhibitor, azithromycin compared to the growth of planktonic cultures of the same strains. Low concentrations of azithromycin were found to stimulate biofilm formation by the studied saprotrophic strains. The rate of synthesis of the polysaccharide matrix component exceeded the rate of cell growth, indicating implementation of the biofilm phenotype under these conditions. It was found that an alkylhydroxybenzene (AHB) compound 4-hexylresorcinol was capable of almost uniform suppression of growth of both planktonic cultures and biofilms of the saprotrophic strains under study. In some cases, combined action ofazithromycin and AHB resulted in an additive inhibitory effect and prevented the stimulation of biofilm growth by subinhibitory azithromycin concentrations. Thus, AHB may be considered a promising antibiofilm agent.

[Effect of Stress on Emergence of Antibiotic-resistant Escherichia coli Cells].

Effect of sublethal doses of physical and chemical stressors (heat shock for 2 h at 45 degrees C and addition of C12-alkylhydroxybenzene, a microbial alarmone) on development of resistance to the subsequent lethal antibiotic attack and the role of the time interval between these treatments were studied on a submerged batch culture of Escherichia coli 12. The interval sufficient for the development of stress response provides for development of temporary adaptive resistance to the antibiotic attack, resulting in increased number of surviving persister cells. The interval below the time required for the stress response potentiates cell death and results in a decreased number of persisters. Heterogeneity of the fractions (10(-4) to 10(-2)% of the intial CFU number) surviving lethal doses of an antibiotic (a mpicillin or ciprofloxacin) was found. Apart from a low number of antibiotic-resistant cells (up to 0.005% of surviving cells), the fractions contained antibiotic-tolerant forms, such as temporarily resistant metabolically adapted cells, long-term persisters, and the cells of slowly growing SCV variants with small colonies (d ≤ 1 mm). Persisters are hypothesized to act as precursors for cystlike dormant cells (CLC), in which the cell differentiation stage is completed and the processes of cell ametabolism (transition to the anabiotic state) are still incomplete.

[Surviving Forms in Antibiotic-Treated Pseudomonas aeruginosa].

Survival of bacterial populations treated with lethal doses of antibiotics is ensured by the presence of very small numbers of persister cells. Unlike antibiotic-resistant cells, antibiotic tolerance of persisters is not inheritable and reversible. The present work provides evidence supporting the hypothesis of transformation (maturation) of persisters of an opportunistic pathogen Pseudomonas aeruginosa revealed by ciprofloxacin (CF) treatment (25-100 µg/mL) into dormant cystlike cells (CLC) and non-culturable cells (NC), as was described previously for a number. of non-spore-forming bacteria. Subpopulations of type 1 and type 2 persisters, which survived antibiotic treatment and developed into dormant forms, were heterogeneous in their capacity to form colonies or microcolonies upon germination, in resistance to heating at 70 degrees C, and in cell morphology Type 1 persisters, which were formed after 1-month incubation in the stationary-phase cultures in the medium with decreased C and N concentrations, developed in several types of surviving cells, including those similar to CLC in cell morphology. In the course of 1-month incubation of type 2 persisters, which were formed in exponentially growing cultures, other types of surviving cells developed: immature CLC and L-forms. Unlike P. aeruginosa CLC formed in the control post-stationary phase cultures without antibiotic treatment, most of 1-month persisters, especially type 2 ones, were characterized by the loss of colony-forming capacity, probably due to transition into an uncultured state with relatively high numbers of live intact cells (Live/Dead test). Another survival strategy of P. aeruginosa populations was ensured by a minor subpopulation of CF-tolerant and CF-resistant cells able to grow in the form of microcolonies or regular colonies of decreased size in the presence of the antibiotic. The described P. aeruginosa dormant forms may be responsible for persistent forms in bacteria carriers and latent infections and, together with antibiotic-resistant cells, are important as components of test systems to assay the of efficiency of potential pharmaceuticals against resistant infections.

[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.

[Microbial dormancy and prevention of healthcare-associated infections].

Healthcare-associated infections (HCAI) remain one of the most challenges of modern health care and assume increasing social and medical significance. The specific features of HCAI are frequent recurrences and inefficiency of antibiotic therapy, a reason for which is antibiotic resistance in microorganisms. The review discusses antibiotic resistance, a form of antibiotic tolerance (AT), and its role in the development of HCAI. It also describes essential differences between AT and antibiotic tolerance at the cellular and molecular genetic levels. Relationships between AT and dormancy of microorganisms, pathogens of HCAI, are discussed. The paper gives the data available in the literature on how AT occurs in HCAI pathogens and discusses the diagnosis of this condition. It also analyzes the literature data on pharmacological attempts to overcome AT and discusses novel approaches to antibiotic therapy for HCAI.

[Dormant state and phenotypic variability of Staphylococcus aureus and Corynebacterium pseudodiphtheriticum].

Ability to produce dormant forms (DF) was demonstrated for non-spore-forming bacteria Staphylococcus aureus (a nonpathogenic strain) and Corynebacterium pseudodiphtheriticum (an organism of the normal oropharyngeal flora). The salient features of the sthaphylococcal and corynebacterial DF were (1) prolonged preservation of viability; (2) resistance to damaging factors (heat treatment); and (3) specific morplology and ultrastructure. The optimal conditions for DF formation were (1) transfer of the stationary-phase cultures into saline solution with CaCl2 (10-300 mM) (for S. aureus); (2) growth in SR1 synthetic medium with fivefold nitrogen limitation (for C. pseudodiphtheriticum); and (3) incubation with (1-5) x 10(-4) M) of C12-AHB, an alkylhydroxybenzene akin to microbial anabiosis autoinducers. Increase of C12-AHB concentration to 7 x 10(-4) -2 x 10(-3) M resulted in "mummification" with irreversible loss of viability without autolytic processes. Germination of the dormant forms was followed by increased phenotypic variability, as seen from (1) diversity of colony types and (2) emergence of antibiotic-resistant clones on selective media. The share of kanamycin-resistant S. aureus variants was most numerous 0.002-0.01% in 4-month DF suspensions in saline with CaCl2. In the C. pseudodiphtheriticum DF produced under the effect of C12-AHB, the share of kanamycin-resistant variants was also found to increase. These data point to association between emergence of antibiotic-resistant variants and their persistence in dormant state mediated by starvation stress and regulated by AHB.

[The effects of alkylhydroxybenzenes on homoserine lactone-induced manifestations of quorum sensing in bacteria].

The effects of four alkylhydroxybenzene (AHB) homologs of different hydrocarbon chain lengths on synthesis of the pigment violacein induced by C6-homoserine lactone (HSL) and biofilm formation by Chromobacterium violaceum NCTC 13274 and on Escherichia coli pAL103 bioluminescence in the presence of C6-oxo-HSL were studied. It was shown that the inhibitory effect of alkylhydroxybenzenes on the growth of C. violaceum increased in the C5-AHB --> C12-AHB series in the absence of this activity in C1-AHB. Sub-inhibitory AHB concentrations reduced violacein production and suppressed biofilm formation. These effects were presented as individual and group regression dependencies between the analyzed parameters. It was shown using the bioluminescent model that the regulatory effects of AHBs are not associated with their direct competition with HSL and that they develop as a result of changes in the sensitivity of bacterial cells to the respective quorum sensing inducer.

[Changes in the phase variant spectra in the populations of lactic acid bacteria under antibiotic treatment].

Effect of the antibiotics kanamycin and ampicillin on growth and phase variation of the populations of four strains of lactic acid bacteria (Lactobacillus sp. M67AT, L. casei MB, Enterococcus faecium M, and E. faecium M3185) was studied. The presence of antibiotics in the medium resulted in a dose-dependent decrease in viable cell numbers and in partial or complete substitution of the dominant S variant with the minor Sm and Sb variants. The variants differed in colony morphology, as well as in some physiological, biochemical, biotechnological, and probiotic characteristics. The Sm type variants of all strains exhibited the highest resistance to antibiotics. High production of exopolysaccharides was found in Sb variants of lactobacilli and in S variants of enterococci. The highest antibacterial activity was found in Sm variants of lactobacilli, especially in Lactobacillus sp. M76AT The latter is biotechnologically the most promising strain, since all variants fermented milk yielding the products with uniformly pronounced functional and organoleptic properties. These patterns are of importance for the understanding of the mechanisms of antibiotic resistance and for selection of the variants with desired properties, as well as for quality control of the lactic acid bacteria starter cultures.

[Effect of alkylresorcinols on thermal denaturation and refolding of bacterial luciferase and synthesis of heat shock proteins revealed in the luminescent molecular and cellular test systems].

Molecular and cellular luminescent biotests were used to reveal the effects of five alkylresorcinol homologues (C7-, C9-, C11-, C12-, and C18-AR) on the thermally induced denaturation and refolding ofbac- terial luciferases, as well as on the synthesis of heat shock proteins. The ARs activities were found to depend on their fine structure and concentration. Direct heat-protective effect of short-chain C7- and C9-AR on the chromatographically pure Photobactrium leiognathii luciferase/oxidoreductase was shown within broad range of concentration (10(-6)-10(-3) M). The long-chain ARs homologues exhibited a similar heat-protective effect at micromolar concentrations only, while their millimolarconcentrations have increased the sensitivity of the model proteins to thermal treatment. The recombinant strain Escherichia coli K12 MG1655 bearing constitutively expressed Vibrio fischieri luxAB genes was used to investigate theARs effect on the intracellular chaperone-independent refolding of bacterial luciferase. The functional activity of heat-inactivated enzyme was restored by micromolar concentrations of short-chain ARs, while long-chain homologues inhibited re- folding in the wide concentration range. The recombinant luminescent E. coli strain bearing the inducible ib- pA'::luxCDABE genetic construction was used to determine the effect of ARs on the synthesis of heat shock proteins (HSP). The preincubation mode of bacterial cells with long-chain alkylresorcinols led to dose-de- pendent stimulation of HSP synthesis (2.7 to 4 times) that confirmed some ARs function as "alarmones". Subsequent thermal treatment resulted in a 5-15-fold decrease of the following HSP induction compared to the control, while the number of viable cells opposite increased 1.5-4-fold. Thus, pretreatment of the bacte- rial cells with long-chain ARs resulted in their preadaptation to subsequent thermally induced stress. Short- chainARs caused less pronounced HSP suppression, although still was accompanied by increased heat resis- tance of the AR-pretreated bacterial cells.