M. V. Zhurina

Role of the extracellular polymer matrix in resistance of bacterial biofilms to extreme environmental factors

Biofilms of a number of gram-positive and gram-negative bacteria (both environmental strains from the stratal waters of oil fields and collection strains) were found to exhibit higher resistance to extreme physicochemical factors (unfavorable temperature, pH, and salt concentration) than planktonic cultures. The extracellular polymers forming the structure of the biofilm matrix were shown to contribute significantly to this resistance, since suppression of matrix formation by subbacteriostatic concentrations of azithromycin (for Pseudomonas acephalitica) or mutation in the cvil gene encoding N-hexanoyl homoserine lactone synthetase (for Chromobacterium violaceum CV026) resulted in the resistance of biofilms being decreased almost to the level of planktonic cultures. The role of the biofilm matrix for bacterial survival under extreme conditions is discussed.

Persistence and adaptive mutagenesis in biofilms

The mechanisms of formation of persisting cells in planktonic and structured microbial populations are considered. The relations between persistence and adaptive mutagenesis are discussed. Persisting cells are suggested to be among the major objects of adaptive mutagenesis and act as an instrument of the microevolution processes in microbial biofilms.

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 Rhodococcus) and gram-negative bacteria (Pseudomonas and Chromobacterium) in biofilms exhibited higher resistance to a 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 of azithromycin 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.

Visualization of the extracellular polymeric matrix of Chromobacterium violaceum biofilms by microscopic methods

517 We have previously presented results that indicated direct correlation between the ability to form a fully functional matrix and the sensitivity of microbial bio films to thermal, hyperosmotic, and acidic shock [1]. In particular, we compared the sensitivity to these fac tors in the pigment free (producing no violacein) strain Chromobacterium violaceum WT, which pos sesses a normal quorum sensing system and matrix forming ability, and in the mutant strain C. violaceum CV026, with impaired synthesis of N acyl homoserine lactones (AHL), components of the regulatory quo rum sensing system, and with a decreased capacity for matrix formation. Violacein synthesis by strain CV026 is induced by N hexanoyl homoserine lactone (C6 AHL), as well as by other AHLs with acyl side chains containing from 4 to 8 carbon atoms. This strain is therefore used as a biosensor for AHLs [2]. In the previously reported experiments [1], biofilm matrix production was quantified according to the standard technique [3] with minor modifications: the matrix was stained with 1,9 dimethylmethylene blue (DMMB) specific to the matrix components, DMMB was extracted with 96% ethanol, and optical density of the extract was measured at 540 nm [1]. Earlier, such dyes as Congo red (staining only cellulose and cellu lose like glycans) [4] and Alcian blue (staining acidic polysaccharides) [5] were proposed to visualize the biofilm matrix. However, the results of the cited work [3], as well as our data (see below) show that DMMB has the highest selectivity toward the matrix compo nents and practically does not stain matrix free bacte rial cells.

Resistance of the oil-oxidizing microorganism Dietzia sp. to hyperosmotic shock in reconstituted biofilms

A number of halotolerant and halophilic bacterial strains were isolated from the Romashkinskoe oil field (Tatarstan) stratal waters having a salinity of up to 100 g/l. The isolation of pure cultures involved biofilm reconstitution on M9 medium with paraffins. The associations obtained were dispersed and reinoculated onto solid media that contained either peptone and yeast extract (PY medium) or paraffins. It was shown that such associations included both oil-oxidizing bacteria and accompanying chemoheterotrophic bacteria incapable of oil oxidation. The pure cultures that were isolated were used for creating binary biofilms. In these biofilms, interactions between halophilic and nonhalophilic bacteria under hypo-and hyperosmotic shocks were investigated. We conducted a detailed study of a biofilm obtained from an oil-oxidizing halotolerant species (with an upper growth limit of 10–12% NaCl) identified as Dietzia sp. and an extremely halophilic gram-negative bacterium (growing within the 5–20% NaCl concentration range) of the genus Chromohalobacter that did not oxidize paraffins. If these microorganisms were grown in a mixed suspension (planktonic) culture that was not supplemented with an additional amount of NaCl, no viable cells of the halophilic microorganism were detected after reinoculation. In contrast, only halophilic cells were detected at a NaCl concentration of 15%. Thus, no mutual protective influence of the microorganisms manifested itself in suspension culture, either under hypoor under hyperosmotic shock. Neither could halophile cells be detected after reinoculating a biofilm obtained on a peptone medium without the addition of NaCl. However, biofilms produced at a NaCl concentration of 15% contained approximately equal numbers of cells of the halophilic and halotolerant organisms. Thus, the halophile in biofilms sustaining a hyperosmotic shock exerts a protective influence on the halotolerant microorganism. Preliminary data suggest that this effect is due to release by the halophile of osmoprotective substances (ectoine and glutamate), which are taken up by the halotolerant species. Such substances are diluted by a large medium volume in suspension cultures, whereas, in biofilms, their diffusion into the medium is apparently hampered by their interaction with the intercellular polymer matrix.

Composition and functions of the extracellular polymer matrix of bacterial biofilms

The data on the composition and structure of the components comprising the extracellular polymer matrix of bacterial biofilms, the role of these components, and their functions in the biofilm are reviewed. The main biochemical mechanisms regulating the biosynthesis of biofilm matrix are discussed.

Regulation of biofilm formation by Pseudomonas chlororaphis in an in vitro system

The mutants of Pseudomonas chlororaphis 449 with completely or partially suppressed accumulation of N-acyl homoserine lactones exhibited the absence or a pronounced decrease of their capacity for stimulation of biofilm growth in the presence of azithromycin. Biofilms of the wild type strain preformed in the presence of the stimulatory azithromycin concentrations exhibited more intense staining with a polysaccharide-specific dye 1,9dimethyl methylene blue (DMMB) and were more resistant to heat shock. These findings indicate accumulation of the structural matrix polysaccharides, which play a protective role under conditions of thermal shock. Extremely low azithromycin concentrations (0.001–0.01 μg/mL) inhibit biofilm formation by P. chlororaphis 449 and P. chlororaphis 66 with suppression of the synthesis of DMMBstaining polysaccharides.

Identification of hydrocarbon-oxidizing Dietzia bacteria from petroleum reservoirs based on phenotypic properties and analysis of the 16S rRNA and gyrB genes

The taxonomic position of hydrocarbon-oxidizing bacterial strains 263 and 32d isolated from formation water of the Daqing petroleum reservoir (PRC) was determined by polyphasic taxonomy techniques, including analysis of the 16S rRNA and the gyrB genes. The major chemotaxonomic characteristics of both strains, including the IV type cell wall, composition of cell wall fatty acids, mycolic acids, and menaquinones, agreed with those typical of Dietzia strains. The DNA G+C content of strains 263 and 32d were 67.8 and 67.6 mol %, respectively. Phylogenetic analysis of the 16S rRNA gene of strain 32d revealed 99.7% similarity to the gene of D. maris, making it possible to identify strain 32d as belonging to this species. The 16S rRNA gene sequence of strain 263 exhibited 99.7 and 99.9% similarity to those of D. natronolimnaea and D. cercidiphylli YIM65002T, respectively. Analysis of the gyrB genes of the subterranean isolates and of a number of Dietzia type strains confirmed classification of strain 32d as a D. maris strain and strain 263 as a D. natronolimnaea strain. A conclusion was made concerning higher resolving power of phylogenetic analysis of the gyrB gene compared to the 16S rRNA gene analysis in the case of determination of the species position of Dietzia isolates.

Niclosamide as a promising antibiofilm agent

Search for inexpensive efficient compounds with antibiofilm activity, which could be applied both for treatment of biofilm-associated infections and in other cases requiring biofilm elimination, is presently of importance. For this purpose we chose niclosamide. Niclosamide is a widespread and available anthelmintic drug, which was also recently shown to suppress bacterial growth. High antibiofilm activity of niclosamide against a range of gram-positive bacteria isolated from different ecological niches was shown. According to our results, niclosamide may be a promising component of composite antibiofilm preparations.

Controlling of microbial biofilms formation: Anti- and probiofilm agents

Controlling the formation and reconstruction of microbial biofilms is of ever increasing importance for the ecological, medical, and biotechnological aspects of biofilm studies. The goal of this review was to provide systematization and analysis of the results obtained in recent years on the modes and mechanisms of the stimulatory or inhibitory effect of extreme factors and biocidal agents on biofilm formation. Special attention is paid to controlling the formation of medically (infective diseases, implant biofouling) and technologically or biotechnologically important biofilms (bioremediation, biocorrosion, and biosynthesis of biologically active compounds).