Influence of Acrylamide on Energy Status and Survival of Bacteria of Different Systematic Groups.

We studied the effect of acrylamide on the content of intracellular ATP in the cells of bacteria of the genera Rhodococcus and Alcaligenes, the luminescence of the genetically engineered strain Escherichia coli K12 TG1 (pXen7), and the survival of bacteria of various systematic groups. According to the level of decrease in the concentration of intracellular ATP, it was found that the strain with lower amidase activity (R. erythropolis 6-21) and Gram-negative proteobacteria A. faecalis 2 were the most sensitive to acrylamide after a 20-min exposure, while the strain R. ruber gt 1 was stable, having a high nitrile hydratase activity in combination with a low amidase activity. EC50 of acrylamide for 2 h was 7.1 g/L for E. coli K12 TG1 (pXen7). Acrylamide at a concentration of 10-20 mM added to the culture medium led to a slight decrease in the number of CFUs of Rhodococcus, A. faecalis 2, and E. coli compared to the control. At an acrylamide concentration of 250 mM, from 0.016 to 0.116% of viable bacterial cells remained, and a solution of 500 mM and higher inhibited the growth of the majority of the studied strains. The results confirm that acrylamide is much less toxic to prokaryotes than to eukaryotes.

[Comparative characteristics of uropathogenic scherichia coli strains, allocated in polyclinic and stationary conditions].

The etiological structure of urinary tract infections (UTI) is determined by the leading role of uropathogenic Escherichia coli (UPEC). The aim of the work is to study the biological properties and phylogenetic diversity of E. coli strains isolated from UTI in outpatient and inpatient patients. METHOD: s and materials. 198 clinical UPEC strains were studied, 105 of which were designated as polyclinic and 93 as nosocomial (73 are isolated from urine and 20 are from catheter surface 48 hours after hospitalization). UPEC phylogenetic groups were determined by polymerase chain reaction (quadruplex PCR) according to O. Clermont et al. (2013). RESULTS: Among polyclinic cultures, representatives of all eight recognizable phylogroups were found; strains of UPEC phylogroup B2 (37.1%), E (13.3%) and F (8.6%) were most often found. Nosocomial cultures in almost 90% of cases belonged to the phylogroup B2, to which all the catheter-associated strains were assigned. The E. coli of the phylogroup B2, both in the mono-species and in the polymicrobial associations, was authentically more often isolated in the hospital than in the polyclinic (p<0.00001), whereas the bacteria of the phylogroup E, on the contrary, in the polyclinic (p<0.0001) . The hemolytic activity and biofilm-forming ability of UPEC strains did not differ in the two groups, while in the hospital hemolytic E. coli of the B2 phylogroup was significantly more likely than the polyclinic (p<0.001). In addition, B2 strainsformed biofilms in more than 60% cases. Regardless of the source of isolation, the strains were resistant to ampicillin (62.1%), amoxicillin/clavulanate (27.8%), cefotaxime (37.9%) and ciprofloxacin (36.9%). The production of ESBL was detected in fifty-one (25.8%) cultures, with a statistically significant difference in nosocomial strains: urinary

Stereoselective biotransformation of phenylglycine nitrile by heterogeneous biocatalyst based on immobilized bacterial cells and enzyme preparation.

We studied the effect of a heterogeneous environment on the stereoselectivity of transformation of racemic phenylglycine nitrile. Immobilized biocatalysts were prepared by adhesion of Pseudomonas fluorescens C2 cells on carbon-containing supports and covalent crosslinking of nitrile hydratase and amidase of Rhodococcus rhodochrous 4-1 to activated chitosan as well as by the method of cross-linked aggregates. At a reaction duration of 20 h, the ratio of phenylglycine stereoisomers changes depending on the presence of support in medium. The highest optical purity of the product (enantiomeric excess of L-phenylglycine solution, 98%) is achieved when enzyme aggregates of nitrile hydratase and amidase cross-linked with 0.1% glutaraldehyde are used as a biocatalyst.

[The epidemiological microbiological characteristics of cultures of Candida albicans circulating in hospital for HIV-infected patients.]

The study was organized relating to 70 cultures of Candida albicans isolates from 19 patients (n=45) and from objects of hospital environment (n=25) in July 2013 and October2014 in specialized department for HIV-infected patients. The genetypical characteristic was given related to candida on the basis of polymerase chain reaction of intronic area of gene 25s pRNA and also their biological characteristics taking into account genotype. The rate of prevalence of Candida genotypes A, B, С and В made up to 58.6%, 10%, 18.6% and 12,8% correspondingly. It was established significant predominance of C.albicans genotype A in various commensal (pharynx) and transitory (skin) loci. In most cases, they were a cause of candidiasis, including invasive candidiasis. Besides, epresentatives of this group more frequently (85%) contaminated objects of hospital environment (p=0.0013). The cultures of C.albicans genotype В (Candida dubliniensis) were small in numbers and were isolated only from ecological biotypes of patients without clinic of candidiasis, were not detected in environment, were least active with exo-enzymes (phospholipase and protease) and were sensitive to tested anti-mycotic. In various biotops of single patients as a rule the only one strain persisted. In a number of cases genotypically different cultures were isolated. The residence of patient in hospital enhanced joining of C.albicans of other genotypes and in two cases were established a total change of cultures. Thus, in hospital environment of specialized hospitals can develop and long-time circulate hospital strains of C.albicans with high enzyme activity. The source are HIV-infected patients themselves that increases risk of of development of candidiasis.

[Activated Sludge Bacteria Transforming Cyanopyridines and Amides of Pyridinecarboxylic Acids].

Species diversity of bacteria from the activated sludge of Perm biological waste treatment facilities capable of transformation of cyanopyridines and amides of pyridinecarboxylic acids was investigated. Enrichment cultures in mineral media with 3-cyanopyridine as the sole carbon and nitrogen source were used to obtain 32 clones of gram-negative heterotrophic bacteria exhibiting moderate growth on solid and liquid media with 3- and 4-cyanopyridine. Sequencing of the 16S rRNA gene fragments revealed that the clones with homology of at least 99% belonged to the genera Acinetobacte, Alcaligenes, Delftia, Ochrobactrum, Pseudomonas, Stenotrophomonas, and Xanthobacter. PCR analysis showed that 13 out of 32 isolates contained the sequences (-1070 bp) homologous to the nitrilase genes reported previously in Alcaligenes faecalis JM3 (GenBank, D13419.1). Nine clones were capable of nitrile and amide transformation in minimal salt medium. Acinetobacter sp. 11 h and Alcaligenes sp. osv transformed 3-cyanopyridine to nicotinamide, while most of the clones possessed amidase activity (0.5 to 46.3 mmol/(g h) for acetamide and 0.1 to 5.6 mmol/(g h) for nicotinamide). Nicotinamide utilization by strain A. faecalis 2 was shown to result in excretion of a secondary metabolite, which was identified as dodecyl acrylate at 91% probability.

[Transformation of amides by adherent rhodococcus cells possessing amidase activity].

We report the development of a heterogeneous biocatalyst for the hydrolysis of amides that is based on cell adhesion ofamidase-containing Rhodococci on activated birch carbon (ABC) and crude carbon. We investigated the properties of the obtained biocatalyst in the hydrolysis reaction of acrylamide to acrylic acid and nicotinamide to nicotinic acid, as well as in a model reaction of racemic lactamide hydrolysis to a mixture of D- and L-isomers of lactic acid. We show that a six- and threefold increase in the concentrations of adherent and suspended cells, respectively, results in a reduction of amidase activity by 3 and 30 times, respectively. ABC Cells adherent on ABC maintained more than 50% of enzymatic activity for seven 24-hour cycles of acrylamide hydrolysis, while suspended cells lost more than 60% of activity already in the second cycle. We also noted that cell adhesion on ABC reduced the stereoselectivity of hydrolysis reaction of racemic lactamide.

[Prevalence of cytotoxicity effectors in nosocomial Pseudomonas Aeruginosa strains].

AIM: Analysis of occurrence of the third type secretory system (TTSS) effectors in clinical P. aeruginosa strains. MATERIALS AND METHODS: Intra-hospital (n = 164) and extra-hospital (n = 30) strains of P. aeruginosa were studied. Detection of exoS and exoU genes was carried out by PCR in DNA Engine Dyad Thermal Cycler ("Bio-Rad", USA). Metallo-beta-lactamase (MBL) producers were detected by the presence of blaVIM-2 gene. RESULTS: Screening of intra- and extra-hospital strains for the presence of genes coding ExoS and ExoU showed, that exoS is detected in genome of clinical isolates in 59.8% and exoU--31.1% of cases. At the same time, strains with exoS-/exoU+ genotype predominated in lCU (Φ = 0.466; p = 0.0000). A significant association between the presence of the respective effectors and material of strain isolation was not detected. exoU gene was more frequently detected in genome of MBL producers (Φ = 0.784; p = 0.0004). CONCLUSION: A significant association between exoU and blaVIM-2 could be explained by clonal prevalence of P. aeruginosa ST235 VIM-2, circulation of those is noted on all the territory of Russia. As a rule, ExoU is produced by highly virulent poly-antibiotic resistant hospital isolates that determine unfavorable outcomes of pseudomonas infection.

[Change in the concentration of intracellular ATP during adhesion of Rhodococcus ruber gt1 and pseudomonas fluorescens C2 cells on carbon supports].

The effect of immobilization of nitrile-utilizing bacteria Rhodococcus ruber gt1 and Pseudomonasfluorescens C2 by adhesion on carbon supports on the content of the intracellular ATP immediately after adsorption and after 2 h and 24-48 h after transfer of the adhered cells into a fresh nutrient medium was studied. Adhesion was shown to lead to a decreased concentration ofATP in a cell by one order of magnitude or more in the first hours in a fresh nutrient medium that'can be attributed to energetic consumption upon the initiation of biofilm formation. A gradual rise in the quantity of ATP, which was calculated per 1 mg of adsorbed cells, was reported to take place in daily and two-daily biofilms, which confirms the cells remain viable.

Bioaugmentation of a polychlorobiphenyl contaminated soil with two aerobic bacterial strains.

The consortium of aerobic bacterial strains Rhodococcus ruber P25 and Microbacterium sp. B51 was bioaugmented in natural and industrial soils, contaminated by commercial mixture of polychlorinated biphenyls (PCBs) Sovol. The results showed that the bioaugmentation of bacterial strains led to PCBs degradation in soil. Sovol at the initial concentration of about 100 mg kg(-1) was removed by 72.2% in the bioaugmented system with natural soil within 90 days, while the system with industrial soil removed 96.4% of this compound within the same period. The biodegradation kinetics of PCBs in the bioaugmented soil systems was not dependent on the presence of indigenous microflora. It was found that the growth dynamics of the strains R. ruber P25 and Microbacterium sp. B51 correlated with the specific degradation of Sovol. The strains R. ruber P25 and Microbacterium sp. B51 displayed high degradative activity to all congeners (ortho-, meta- and para-substituent) contained in Sovol. Removal percentage for each congeners amounted to 59-100% in the bioaugmented systems. This study suggests that augmentation of PCB-contaminated soils with strain R. ruber P25 and Microbacterium sp. B51 is promising in PCB bioremediation.

[Destruction of aromatic hydrocarbons by the Rhodococcus wratislaviensis KT112-7 strain isolated from waste products of a salt-mining factory].

The destruction of aromatic hydrocarbons by the Rhodococcus wratislaviensis KT112-7 strain isolated from technogenic mineral waste products of the BKRU1 Uralkalii factory has been investigated (city of Berezniki, Perm krai). The R. wratislaviensis KT112-7 was shown to utilize increased concentrations of ophthalic (o-PA) (8 g/L) and benzoic (BA) (3.4 g/L) acids. The strain grows with o-FA, BA, and biphenyl at a NaCl content of up to 50, 90, and 75 g/L in the culture medium, respectively. Based on an analysis of the metabolic profile and nucleotide sequences of the bphA1, benA, and phtB genes, the KT112-7 strain was established to decompose o-PA via the formation of 3,4-dihydroxyphthalic and 3,4-dihydroxybenzoic acids. The decomposition of biphenyl is carried out via the formation of BA and then at low concentrations of NaCl (up to 50 g/L) via the formation of 4-hydroxybenzoic acid followed by its oxidation; at high concentrations of NaCl (over 60 g/L), via the direct oxidation of benzoic acid with the production of catechol. These data indicate that the Rhodococcus wratislaviensis KT112-7 destructor strain is a promising strain for the development of new biotechnologies directed at the utilization (transformation) of aromatic compounds, including under the conditions of increased mineralization.

[The experience of applying techniques of molecular genetics in identification of clinical strains Pseudomonas aeruginosa].

The article presents comparative analysis of application of common bacteriologic and molecular techniques to identify P. aerugenosa. The genetic detection was applied using polymerase chain reaction with genus-specific and species-specific primers and amplification of conservative sites of gens 16S pRNA with successive identification of bacteria by sequenation. It is established that 95% (151) of strains correspond to species of P. aerugenosa detected in primary bacteriologic laboratories and only 8 strains were not blue pus bacillus. Most of strains were closely congenial species: 2 isolates belonged to Pseudomonas aeruginosa group, 3 isolates to Pseudomonas putida group, 2 strains to Comamonas species and 1 isolate to Stenotrophomonas maltophilia species. The effectiveness of cultural technique of laboratory diagnostic was demonstrated concerning infections conditioned by P. aerugenosa. This conclusion does not eliminate application of molecular genetic trechnologies in complicated arbitral cases of bacteriologic analysis during monitoring of nosocomial infections.

[Transformation of 2- and 4-cyanopyridines by free and immobilized cells of nitrile-hydrolyzing bacteria].

The transformation dynamics of 2- and 4-cyanopyridines by cells suspended and adsorbed on inorganic carriers has been studied in the Rhodococcus ruber gt 1 strain possessing nitrile hydratase activity and the Pseudomonas fluorescens C2 strain containing nitrilase. It was shown that both nitrile hydratase and nitrilase activities of immobilized cells against 2-cyanopyridine were 1.5-4 times lower compared to 4-cyanopyridine and 1.6-2 times lower than the activities of free cells against 2-cyanpopyridine. The possibility of obtaining isonicotinic acid during the combined conversion of 4-cyanopyridine by a mixed suspension of R. ruber gt 1 cells with a high level of nitrile hydratase activity and R. erythropolis 11-2 cells with a pronounced activity of amidase has been shown. Immobilization of Rhodococcus cells on raw coal and Pseudomonas cells on china clay was shown to yield a heterogeneous biocatalyst for the efficient transformation of cyanopyridines into respective amides and carbonic acids.

[Catalytic properties of a nitrile hydratase immobilized on activated chitosan].

The catalytic properties of a nitrile hydratase, isolated from a strain of Rhodococcus ruber gt1 and immobilized by covalent cross-linking with chitosan activated with 0.1% benzoquinone solution, have been investigated. The kinetic parameters ofacrylonitrile hydration catalyzed by immobilized nitrile hydratase and the enzyme in a solution have been determined. It is found that the immobilization does not lead to a decrease in the maximum reaction rate (Vmax), whereas the Michaelis constant (K(M)) is reduced by a factor of 2.4. The possibility of reusing an immobilized enzyme for 50 consecutive cycles of acrylonitrile transformation was shown, and the nitrile hydratase activity in the 50th cycle exceeded that in the first cycle by 3.5 times. It is shown that the effect of temperature on activity depended on the concentration of the enzyme, which confirms the dissociative nature of nitrile hydratase inactivation. It was found that immobilized nitrile hydratases remain active at pH 3.0-4.0, whereas the enzyme is inactivated in a solution under these conditions. The resulting biocatalyst can be effectively used to receive acrylamide from acrylonitrile.

[Effect of Pseudomonas aeruginosa exometabolites on planktonic and biofilm cultures of Escherichia coli].

AIM: Study the effect of P. aeruginosa exometabolites on planktonic and biofilm cultures of bioluminescent E. coli strain. MATERIALS AND METHODS: E. coli K12 TG1 (pF1 lux+ Ap(r)) recombinant bioluminescent strain, P. aeruginosa ATCC 27853 reference strain and 2 nosocomial isolates were used. Pyocyanin and pyoverdin content in supernatant of P. aeruginosa over-night cultures was evaluated according to E. Deziel et al. (2001). Planktonic and biofilm cultures of E. coli were obtained in 96-well plates (LB, statically, 37 degrees C), optical density of plankton, film biomass (OD600, OD580) and bioluminescence in plankton and biofilm were evaluated in microplate reader Infiniti M200 (Tecan, Austria). RESULTS: P. aeruginosa exometabolites increased the duration of lag-phase in E. coli, and short term exposition inhibited luminescence of planktonic cells. These effects are determined by bactericidal action ofpyocyanin and pyoverdin. Supernatants ofover-night cultures of P. aeruginosa inhibit formation of biofilm and disrupt the formed biofilm of E. coli. Effect of pyocyanin and pyoverdin on these processes is not established, other factors may have higher significance. CONCLUSION: Bioluminescence of E. coli K12 TGI that reflects the energetic status of the cell allows to evaluate and prognose the character of coexistence of P. aeruginosa in combined with E. coli planktonic and biofilm culture.

Thalassospira permensis sp. nov., a new terrestrial halotolerant bacterium isolated from a naphthalene-utilizing microbial consortium.

A halotolerant bacterium, strain SMB34T, was isolated from a naphthalene-utilizing bacterial consortium obtained from primitive technogeneous soil (Vrkhnekamsk salt deposit, Perm region, Russia) by enrichment procedure. The strain itself was unable to degrade naphthalene and grew at NaCl concentrations up to 11% (w/v). The 16S rRNA-based phylogenetic analysis showed that the strain belongs to the genus Thalassospira. The DNA-DNA hybridization values between SMB34T and the type strains of phylogenetically closest species (T. xiamenensis, T. profundimaris and T. tepidiphila) did not exceed 50%. The novel strain could be distinguished from the above species by the cell motility, MALDI/TOF mass spectra of whole cells and a range of physiological and biochemical characteristics. SMB34T also considerably differs from the recently described species T. xianhensis, with the most striking differences in the DNA G + C content (53.7 +/- 1.0 vs. 61.2 +/- 1.0 mol.%) and predominant ubiquinones (Q-10 vs. Q-9). The data obtained suggest strain SMB34T (=VKM B-2527T = NBRC 106175T), designated as the type strain, represents a novel species, named Thalassospira permensis sp. nov.

[Destruction of mixture of tri-hexa-chlorinated biphenyls by Rhodococcus genus strains].

Destruction of polychlorinated biphenyls (PCBs) by strain-destructors Rhodococcus sp. B7a and Rhodococcus sp. G12a has been studied. It was shown that these strains destruct 78-95% of PCB mixture containing tri-hexa-chlorinated biphenyls. Rhodococcus destruct all components of the mixture of tri-, tetra-, penta-, and hexa-chlorinated biphenyls without accumulation of toxic chlorinated metabolites. The studied bacteria destruct PCB that are the most stable for oxidation, such as 2,5,2',5'-CB; 3,4,3',4'-CB; and 2,4,5,2',4',5'-CB. The most perspective strains are R. rubber P25, Rhodococcus sp. B7a and Rhodococcus sp. G12a whose metabolic potential can be used for biotechnological refinement of the environment from highly toxic pollutants.

[A study of the catalytic properties of the nitrile hydratase immobilized on aluminum oxides and carbon-containing adsorbents].

The nitrile hydratase isolated from Rhodococcus ruber strain gt1, displaying a high nitrile hydratase activity, was immobilized on unmodified aluminum oxides and carbon-containing adsorbents, including the carbon carrier Sibunit. The activity and operational stability of the immobilized nitrile hydratase were studied in the reaction of acrylonitrile transformation into acrylamide. It was demonstrated that an increase in the carbon content in the carrier led to an increase in the amount of adsorbed enzyme and, concurrently, to a decrease in its activity. The nitrile hydratase immobilized on Sibunit and carbon-containing aluminum alpha-oxide having a "crust" structure displayed the highest operational stability in acrylonitrile hydration. It was shown that the thermostability of adsorbed nitrile hydratase increased by one order of magnitude.

[Degradation of chlorinated biphenyls and products of their bioconversion by Rhodococcus sp. B7a strain].

Strain Rhodococcus sp. B7a isolated from artificially polluted soil destructs mono- and di-substituted ortho- and/or para-chlorinated biphenyls with utilization of chlorinated benzoic acids and shows high degradation activity as regards trichlorinated biphenyls. It is shown that p-hydroxybenzoic and protocatehoic acids are the products of p-chlorobenzoic acid catabolism.

[Salinicola socius gen. nov., sp. nov., a moderately halophilic bacterium from a naphthalene-utilizing microbial association].

A chemoorganotrophic, moderately halophilic bacterium (strain SMB35) has been isolated from a naphthalene-utilizing microbial community obtained from salt mines (Perm region of Russia). Strain SMB35 grows in a wide salinity range, 0.5 to 30% (wt/vol) NaCl. Cells are gram-negative rods motile by means of a single polar flagellum. The predominant fatty acids are 16:1omega7, 16:0, 18:1omega7, and 19 cy. The major lipoquinone is an unsaturated ubiquinone with nine isoprene units (Q-9). The DNA G+C content is 63.0 mol%. The 16S rDNA-based phylogenetic analysis has shown that strain SMB35 formed a separate clade in the cluster of the family Halomonadaceae. The 16S rDNA sequence similarity of the isolate to the members of the family is in the range from 90.6% to 95.1%. The phylogenetic and phenotypic differences from Halomonas elongata (the type species of the genus) and from other members of the family suggest that the isolate represents a novel genus and species, for which the name Salinicola socius gen. nov., sp. nov. is proposed. The type strain is SMB35(T) (=VKM B-2397(T)).

[Immobilization of Rhodococcus ruber strain gt1, possessing nitrile hydratase activity, on carbon sorbents].

Rhodococcus ruber strain gtl, possessing nitrile hydratase activity, was immobilized by adsorption on carbon supports differing in structure and porosity. The adsorption capacity of the supports towards cells, the substrate of the nitrile hydratase reaction (acrylonitrile), and the product (acrylamide) was studied. Also, the effect of immobilization and nitrile hydratase activity of bacteria was investigated, and the operational stability of the immobilized biocatalyst was determined. It was shown that crushed and granulated active coals were more appropriate for immobilization than fibrous carbon adsorbents.