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Sample records for alcaligenes faecalis s-6

  1. The blue copper protein gene of Alcaligenes faecalis S-6 directs secretion of blue copper protein from Escherichia coli cells.

    PubMed Central

    Yamamoto, K; Uozumi, T; Beppu, T

    1987-01-01

    The gene encoding a blue copper protein (a member of the pseudoazurins) of 123 amino acid residues, containing a single type I Cu2+ ion, was cloned from Alcaligenes faecalis S-6. The nucleotide sequence of the coding region, as well as the 5'- and 3'-flanking regions, was determined. The deduced amino acid sequence after Glu-24 coincided with the reported sequence of the blue protein, and its NH2-terminal sequence of 23 residues resembled a typical signal peptide. The cloned gene was expressed under the control of the tac promoter in Escherichia coli, and the correctly processed blue protein was secreted into the periplasm. The blue protein produced in E. coli possessed the activity to transfer electrons to the copper-containing nitrite reductase of A. faecalis S-6 in vitro. Images PMID:2824441

  2. Directing the mode of nitrite binding to a copper-containing nitrite reductase from Alcaligenes faecalis S-6: characterization of an active site isoleucine.

    PubMed

    Boulanger, Martin J; Murphy, Michael E P

    2003-02-01

    Unlike the heme cd(1)-based nitrite reductase enzymes, the molecular mechanism of copper-containing nitrite reductases remains controversial. A key source of controversy is the productive binding mode of nitrite in the active site. To identify and characterize the molecular determinants associated with nitrite binding, we applied a combinatorial mutagenesis approach to generate a small library of six variants at position 257 in nitrite reductase from Alcaligenes faecalis S-6. The activities of these six variants span nearly two orders of magnitude with one variant, I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. High-resolution (> 1.8 A) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies identify for the first time that the highly conserved Ile257 in the native enzyme is a key molecular determinant in directing a catalytically competent mode of nitrite binding in the active site. The O-coordinate bidentate binding mode of nitrite observed in native and mutant forms with high activity supports a catalytic model distinct from the heme cd(1) NiRs. (The atomic coordinates for I257V[NO(2)(-)], I257L[NO(2)(-)], I257A[NO(2)(-)], I257T[NO(2)(-)], I257M[NO(2)(-)] and I257G[NO(2)(-)] AfNiR have been deposited in the Protein Data Bank [PDB identification codes are listed in Table 2].)

  3. Pancreatic abscess secondary to Alcaligenes faecalis.

    PubMed

    Ashwath, Mahi Lakshmi; Katner, Harold P

    2005-01-01

    We report a patient with pancreatic abscesses and necrosis secondary to Alcaligenes faecalis infection. He initially presented with alcohol-induced acute pancreatitis. Twenty days after the initial presentation, he re-presented with increasing pain and was found to have pancreatic necrosis and abscesses. Treatment was initiated with meropenem. Because of persistent fevers, computed tomography-guided drainage was performed. The fluid grew A faecalis resistant to meropenem and the patient continued to be febrile. He recovered only after adequate surgical intervention and appropriate antibiotic coverage. Although this is the first case of A faecalis reported to cause pancreatic abscess, we believe selection of this organism occurred because surgical drainage was delayed while the patient was on the recommended treatment with meropenem. This case emphasizes the need for early surgical drainage of pancreatic abscesses to avoid the selection of such resistant pathogens.

  4. Size of diffusion pore of Alcaligenes faecalis.

    PubMed Central

    Ishii, J; Nakae, T

    1988-01-01

    The diffusion pore of the outer membrane of Alcaligenes faecalis was shown to be substantially smaller than the Escherichia coli porin pore. In experiments with intact cells, pentoses and hexoses penetrated into the NaCl-expanded periplasm, whereas saccharides of Mr greater than 342 did not. Cells treated with 0.5 M saccharides of Mr greater than 342 weighed 33 to 38% less than cells treated with isotonic solution, suggesting that these saccharides do not permeate through the outer membrane. The diffusion rates of various solutes through the liposome membranes reconstituted from the Mr-43,000 outer membrane protein showed the following characteristics. (i) The relative diffusion rates of pentoses, hexoses, and methylhexoses appeared to be about 1.0, 0.6, and negligibly small, respectively. (ii) The diffusion rate of glucose appeared to be about 1/10th that with the E. coli B porin. (iii) The diffusion rate of gluconic acid was five to seven times higher than that of glucose. (iv) The diffusion rates of beta-lactam antibiotics appeared to be 40 to less than 10% of those with the E. coli B porin. Images PMID:2835003

  5. Biodegradation of phenol at high initial concentration by Alcaligenes faecalis.

    PubMed

    Jiang, Yan; Wen, Jianping; Bai, Jing; Jia, Xiaoqiang; Hu, Zongding

    2007-08-17

    Strain Alcaligenes faecalis was isolated and identified as a member of the genus Alcaligenes by using BIOLOG and 16S rDNA sequence analysis. The phenol biodegradation tests showed that the phenol-degrading potential of A. faecalis related greatly to the different physiological phases of inoculum. The maximum phenol degradation occurred at the late phase of the exponential growth stages, where 1600 mg L(-1) phenol was completely degraded within 76 h. A. faecalis secreted and accumulated a vast quantity of phenol hydroxylase in this physiological phase, which ensured that the cells could quickly utilize phenol as a sole carbon and energy source. In addition, the kinetic behavior of A. faecalis in batch cultures was also investigated over a wide range of initial phenol concentrations (0-1600 mg L(-1)) by using Haldane model. It was clear that the Haldane kinetic model adequately described the dynamic behavior of the phenol biodegradation by the strain of A. faecalis.

  6. Detoxification of lantana hepatotoxin, lantadene A, using Alcaligenes faecalis.

    PubMed

    Singh, A; Sharma, O P; Kurade, N P; Ojha, S

    2001-01-01

    Detoxification of lantadene A (LA), the hepatotoxin from Lantana camara var. aculeata, by the bacterial strain Alcaligenes faecalis has been investigated. Lantadene A induced hepatotoxicity concomitant with increases in plasma bilirubin, blood plasma enzymes and histopathological lesions that typify lantana toxicity. The extract of fermentation broth in which LA was incubated with A. faecalis did not elicit any alterations in blood enzyme prolife or liver histopathology, which were comparable with the control group. It is concluded that A. faecalis detoxified LA and no noxious product was formed on incubation of LA with A. faecalis.

  7. Draft genome sequence of Alcaligenes faecalis subsp. faecalis NCIB 8687 (CCUG 2071).

    PubMed

    Phung, Le T; Trimble, William L; Meyer, Folker; Gilbert, Jack A; Silver, Simon

    2012-09-01

    Alcaligenes faecalis subsp. faecalis NCIB 8687, the betaproteobacterium from which arsenite oxidase had its structure solved and the first "arsenate gene island" identified, provided a draft genome of 3.9 Mb in 186 contigs (with the largest 15 comprising 90% of the total) for this opportunistic pathogen species.

  8. Purification of siderophores of Alcaligenes faecalis on Amberlite XAD.

    PubMed

    Sayyed, R Z; Chincholkar, S B

    2006-05-01

    Studies on siderophore production using Alcaligenes faecalis BCCM ID 2374 revealed hydroxamate and catecholate type of siderophores at 347 microg mL-1. These fractions were purified on Amberlite XAD-4 column, which resulted in the separation of two bands having absorption maxima at 264 and 224 nm. The amount of pure siderophore obtained in powdered form from first and second fraction was 297 and 50 microg mL-1 respectively.

  9. Metabolic energy from arsenite oxidation in Alcaligenes faecalis

    NASA Astrophysics Data System (ADS)

    Anderson, G. L.; Love, M.; Zeider, B. K.

    2003-05-01

    The aerobic soil bacterium, Alcaligenes faecalis, survives in cultures containing greater than 10 g/L of aqueous arsenic. Toleration of arsenite occurs by the enzymatic oxidation of arsenite (As^III), to the less toxic arsenate (As^V). In defined media, the bacterium grows faster in the presence of arsenite than in its absence. This suggests that the bacterium uses the redox potential of arsenite oxidation as metabolic energy. The oxidation occurs via periplasmic arsenite oxidase, azurin, and cytochrome c [11] which presumably pass electron equivalents through an electron transport chain involving cytochrome c oxidase aud oxygen as the terminal electron acceptor. The associated proton translocation would allow synthesis of ATP and provide a useful means of harnessing the redox potential of arsenite oxidation. Arsenite and arsenate assays of the media during bacterial growth indicate that arsenite is depleted during the exponential growth phase and occurs concomitantly with the expression of arsenite oxidase. These results suggest that arsenite is detoxified to arsenate during bacterial growth and are inconsistent with previous reported interpretations of growth data. Alcaligenes faecalis is dependent on organic carbon sources and is therefore not chemolithoautotrophic. The relationship between succinate and arsenite utilisation provides evidence for the use of arsenite as a supplemental energy source. Because Alcaligenes faecalis not only tolerates, but thrives, in very high concentrations of arsenic has important implications in bioremediation of environments contaminated by aqueous arsenic.

  10. Site-directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron-transfer complex with a copper-containing nitrite reductase from Alcaligenes faecalis S-6.

    PubMed

    Kukimoto, M; Nishiyama, M; Tanokura, M; Murphy, M E; Adman, E T; Horinouchi, S

    1996-09-23

    Kinetic analysis of electron transfer between azurin from Pseudomonas aeruginosa and copper-containing nitrite reductase (NIR) from Akaligenes faecalis S-6 was carried out to investigate the specificity of electron transfer between copper-containing proteins. Apparent values of kcat and Km of NIR for azurin were 300-fold smaller and 172-fold larger than those for the physiological redox partner, pseudoazurin from A. faecalis S-6, respectively, suggesting that the electron transfer between azurin and NIR was less specific than that between pseudoazurin and NIR. One of the major differences in 3-D structure between these redox proteins, azurin and pseudoazurin, is the absence and presence of lysine residues near their type 1 copper sites, respectively. Three mutated azurins, D11K, P36K, and D11K/P36K, were constructed to evaluate the importance of lysine residues in the interaction with NIR. The redox potentials of D11K, P36K, and D11K/P36K azurins were higher than that of wild-type azurin by 48, 7, and 55 mV, respectively. As suggested by the increase in the redox potential, kinetic analysis of electron transfer revealed reduced ability of electron transfer in the mutated azurins. On the other hand, although each of the single mutations caused modest effects on the decrease in the Km value, the simultaneous mutations of D11K and P36K caused significant decrease in the Km value when compared to that for wild-type azurin. These results suggest that the introduction of two lysine residues into azurin facilitated docking to NIR.

  11. Unusual causes of peritonitis in a peritoneal dialysis patient: Alcaligenes faecalis and Pantoea agglomerans.

    PubMed

    Kahveci, Arzu; Asicioglu, Ebru; Tigen, Elif; Ari, Elif; Arikan, Hakki; Odabasi, Zekaver; Ozener, Cetin

    2011-04-10

    An 87 -year-old female who was undergoing peritoneal dialysis presented with peritonitis caused by Alcaligenes faecalis and Pantoea agglomerans in consecutive years. With the following report we discuss the importance of these unusual microorganisms in peritoneal dialysis patients.

  12. Draft Genome Sequence of Alcaligenes faecalis Strain IITR89, an Indole-Oxidizing Bacterium.

    PubMed

    Regar, Raj Kumar; Gaur, Vivek Kumar; Mishra, Gayatri; Jadhao, Sudhir; Kamthan, Mohan; Manickam, Natesan

    2016-03-03

    We report the draft genome sequence of Alcaligenes faecalis strain IITR89, a bacterium able to form indigo by utilizing indole as the sole carbon source. The Alcaligenes species is increasingly reported for biodegradation of diverse toxicants and thus complete sequencing may provide insight into biodegradation capabilities and other phenotypes.

  13. [Comparative susceptibility of Ochrobactrum anthropi, Agrobacterium tumefaciens, Alcaligenes faecalis, Alcaligenes denitrificans subsp. denitrificans, Alcaligenes denitrificans subsp. xylosidans and Bordetella bronchiseptica against 35 antibiotics including 17 beta-lactams].

    PubMed

    Bizet, C; Bizet, J

    1995-04-01

    Ochrobactrum anthropi, formerly known as "Achromobacter sp." or CDC group Vd has been isolated from water, hospital environment (antiseptic solutions, dialysis fluids ... ). O. anthropi is a Gram negative, motile, strictly aerobic, oxydase positive and non-fermentative bacteria with a strong urease activity. The susceptibility of 13 strains of O. anthropi was determined by agar diffusion method and compared to those of type strains of Agrobacterium tumefaciens, Alcaligenes faecalis, Alcaligenes denitrificans subsp. denitrificans, Alcaligenes denitrificans subsp. xylosoxydans and Bordetella bronchiseptica. The MICs of 20 antimicrobial agents confirmed the distinct phenotype susceptibility of O. anthropi. All the strains of O. anthropi are sensitive to imipenem, amikacin, gentamicin, netilmicin, nalidixic acid, pefloxacin, ciprofloxacin, tetracyclin, colistin, sulphonamides and rifampicin and resistant to ampicillin, amoxycillin + clavulanic acid, ticarcillin, mezlocillin, cefuroxime, cefamandol, cefoxitin, cefotaxime, cefoperazon, ceftazidime, cefsulodin, aztreonam, streptomycin, kanamycin, pipemidic acid, chloramphenicol, erythromicin, pristinamycin, trimethoprim and fosfomycin. O. anthropi is implicated in nosocomial infections. O. anthropi was the species with the greatest resistance to beta-lactamins.

  14. A Newly Sequenced Alcaligenes faecalis Strain: Implications for Novel Temporal Symbiotic Relationships.

    PubMed

    Hernández-Mendoza, Armando; Lozano-Aguirre Beltrán, Luis Fernando; Martínez-Ocampo, Fernando; Quiroz-Castañeda, Rosa Estela; Dantán-González, Edgar

    2014-12-24

    We report here the draft genome sequence of Alcaligenes faecalis strain MOR02, a bacterium that is able to colonize nematodes in a temporary fashion and kill insects for their own benefit. The availability of the genome should enable us to explain these phenotypes.

  15. The complete genome sequence of Alcaligenes faecalis ZD02, a novel potential bionematocide.

    PubMed

    Ju, Shouyong; Zheng, Jinshui; Lin, Jian; Geng, Ce; Zhu, Lei; Guan, Ziyu; Zheng, Ziqiang; Sun, Ming

    2016-01-20

    Root-knot nematodes (RKNs) can infect almost all crops, and result in huge economic losses in agriculture. There is no effective and environmentally safe means available to control RKNs. Alcaligenes faecalis ZD02 isolated from free living nematode Caenorhabditis elegans cadavers shows toxicity against RKN Meloidogyne incognita, that makes this strain to be a good bionematicide candidate for controlling of RKNs. Here, we firstly report the complete genome of A. faecalis ZD02 and describe its features. Additionally, we found two potential virulence factors in this genome, which play important roles for the nematocidal activity of A. faecalis ZD02.

  16. Chemical Characterization, Crossfeeding and Uptake Studies on Hydroxamate Siderophore of Alcaligenes faecalis.

    PubMed

    Sayyed, R Z; Chincholkar, S B; Meyer, J M; Kale, S P

    2011-06-01

    We report the production of two types of siderophores namely catecholate and hydroxamate in modified succinic acid medium (SM) from Alcaligenes faecalis. Two fractions of siderophores were purified on amberlite XAD, major fraction was hydroxamate type having a λ(max) at 224 nm and minor fraction appeared as catecholate with a λ(max) of 264 nm. The recovery yield obtained from major and minor fractions was 297 and 50 mg ml(-1) respectively. The IEF pattern of XAD-4 purified siderophore suggested the pI value of 6.5. Cross feeding studies revealed that A. faecalis accepts heterologous as well as self (hydroxamate) siderophore in both free and iron complexed forms however; the rate of siderophore uptake was more in case of siderophores complexed to iron. Siderophore iron uptake studies indicated the differences between hydroxamate siderophore of A. faecalis and Alc E, a siderophore of Alcaligenes eutrophus.

  17. Alcaligenes faecalis subsp. parafaecalis subsp. nov., a bacterium accumulating poly-beta-hydroxybutyrate from acetone-butanol bioprocess residues.

    PubMed

    Schroll, G; Busse, H J; Parrer, G; Rölleke, S; Lubitz, W; Denner, E B

    2001-04-01

    The authors have previously isolated a solvent tolerant bacterium, strain G(T), (T = type strain) capable to convert acetone-butanol bioprocess residues into poly-beta-hydroxybutyrate. Strain G(T) was initially identified as Alcaligenes spp by standard bacteriological tests. In this study the taxonomic position of the bacterium was investigated in detail. The 165 rDNA sequence analysis, the G + C content of DNA (56 mol%) and the presence of ubiquinone Q-8 confirmed strain G(T) as a representative of the genus Alcaligenes. In the polyamine pattern of the bacterium putrescine and cadaverine were detected, but only trace amounts of 2-hydroxyputrescine. The extremely low content of 2-hydroxyputrescine is remarkable, since this unique diamine is a common marker for beta-proteobacteria. Phylogenetic analyses of 16S rDNA demonstrated that Alcaligenes sp. G(T) is most closely related to the species Alcaligenes faecalis (99.6% sequence similarity to A. faecalis HR4 and 98.7% sequence similarity to A. faecalis [ATCC 8750T = DSM 30030T]. On the basis of DNA-DNA relatedness (56% similarity), the unique polyamine pattern, the physiological and biochemical differences strain G(T) could be distinguished from the species A. faecalis. Therefore, a new subspecies for the species Alcaligenes faecalis is proposed; Alcaligenes faecalis subsp. parafaecalis subsp. nov.

  18. Immobilization of Alcaligenes faecalis penicillin G acylase on epoxy-type supports.

    PubMed

    Sun, J; Zhou, Y; Yuan, Z; Xu, G

    2009-01-01

    Alcaligenes faecalis penicillin G acylase has several desired features over other penicillin G acylases and its use in industry requires immobilization. In this work, two novel supports ZH-EP (epoxy-type) and ZH-HA (epoxy-amino type) were used to immobilize Alcaligenes faecalis penicillin G acylase (AfPGA) with Eupergit C as reference. The saturation of immobilized protein on ZH-EP (269 mg/g, 116 h) and ZH-HA (296 mg/g, 15 h) was obtained more rapidly than Eupergit C (197 mg/g, 260 h). And the activity of immobilized AfPGA on ZH-EP (520 U/g) and ZH-HA (2200 U/g) was higher than that on Eupergit C (310 U/g). The properties of three immobilized enzymes were compared and no obvious difference was observed, which indicated that ZH-EP and ZH-HA were promised in industry.

  19. N2O and N2 production during heterotrophic nitrification by Alcaligenes faecalis strain NR.

    PubMed

    Zhao, Bin; An, Qiang; He, Yi Liang; Guo, Jin Song

    2012-07-01

    A heterotrophic nitrifier, strain NR, was isolated from a membrane bioreactor. Strain NR was identified as Alcaligenes faecalis by Auto-Microbic system and 16S rRNA gene sequence analysis. A. faecalis strain NR shows a capability of heterotrophic nitrification and N(2)O and N(2) production as well under the aerobic condition. Further tests demonstrated that neither nitrite nor nitrate could be denitrified aerobically by strain NR. However, when hydroxylamine was used as the sole nitrogen source, nitrogenous gases were detected. With an enzyme assay, a 0.063 U activity of hydroxylamine oxidase was observed, while nitrate reductase and nitrite reductase were undetectable. Thus, nitrogenous gas was speculated to be produced via hydroxylamine. Therefore, two different metabolic pathways might exist in A. faecalis NR. One is heterotrophic nitrification by oxidizing ammonium to nitrite and nitrate. The other is oxidizing ammonium to nitrogenous gas directly via hydroxylamine.

  20. Alcaligenes faecalis Cellulitis After a Dog Bite: Case Report and Literature Review.

    PubMed

    Chu, Andrew S; Harkness, Julia

    2016-01-12

    Alcaligenes faecalis is a gram-negative organism that is commonly found in the environment and may also be a part of normal fecal flora in humans. Although various infections with this bacteria have been described in the pediatric population, it has not been previously identified in infections as the pathogen after a dog bite. A case of a 19-month-old boy is presented with a cellulitis secondary to a dog bite, which failed oral antibiotic therapy, and progressed to worsening fever and swelling. The patient ultimately required hospitalization, intravenous antibiotics, and incision and drainage. The wound culture grew A. faecalis, whose identity was confirmed through recombinant DNA sequence analysis. Although it has been identified in cat bite wounds, A. faecalis has not been cited in the literature before in an infection after a dog bite.

  1. Alcaligenes faecalis ZD02, a Novel Nematicidal Bacterium with an Extracellular Serine Protease Virulence Factor.

    PubMed

    Ju, Shouyong; Lin, Jian; Zheng, Jinshui; Wang, Shaoying; Zhou, Hongying; Sun, Ming

    2016-01-29

    Root knot nematodes (RKNs) are the world's most damaging plant-parasitic nematodes (PPNs), and they can infect almost all crops. At present, harmful chemical nematicides are applied to control RKNs. Using microbial nematicides has been proposed as a better management strategy than chemical control. In this study, we describe a novel nematicidal bacterium named Alcaligenes faecalis ZD02. A. faecalis ZD02 was isolated from Caenorhabditis elegans cadavers and has nematostatic and nematicidal activity, as confirmed by C. elegans growth assay and life span assay. In addition, A. faecalis ZD02 fermentation broth showed toxicity against C. elegans and Meloidogyne incognita. To identify the nematicidal virulence factor, the genome of strain ZD02 was sequenced. By comparing all of the predicted proteins of strain ZD02 to reported nematicidal virulence factors, we determined that an extracellular serine protease (Esp) has potential to be a nematicidal virulence factor, which was confirmed by bioassay on C. elegans and M. incognita. Using C. elegans as the target model, we found that both A. faecalis ZD02 and the virulence factor Esp can damage the intestines of C. elegans. The discovery that A. faecalis ZD02 has nematicidal activity provides a novel bacterial resource for the control of RKNs.

  2. Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis.

    PubMed Central

    Verhaert, R M; Riemens, A M; van der Laan, J M; van Duin, J; Quax, W J

    1997-01-01

    Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge. PMID:9292993

  3. Alcaligenes faecalis ZD02, a Novel Nematicidal Bacterium with an Extracellular Serine Protease Virulence Factor

    PubMed Central

    Ju, Shouyong; Lin, Jian; Zheng, Jinshui; Wang, Shaoying; Zhou, Hongying

    2016-01-01

    Root knot nematodes (RKNs) are the world's most damaging plant-parasitic nematodes (PPNs), and they can infect almost all crops. At present, harmful chemical nematicides are applied to control RKNs. Using microbial nematicides has been proposed as a better management strategy than chemical control. In this study, we describe a novel nematicidal bacterium named Alcaligenes faecalis ZD02. A. faecalis ZD02 was isolated from Caenorhabditis elegans cadavers and has nematostatic and nematicidal activity, as confirmed by C. elegans growth assay and life span assay. In addition, A. faecalis ZD02 fermentation broth showed toxicity against C. elegans and Meloidogyne incognita. To identify the nematicidal virulence factor, the genome of strain ZD02 was sequenced. By comparing all of the predicted proteins of strain ZD02 to reported nematicidal virulence factors, we determined that an extracellular serine protease (Esp) has potential to be a nematicidal virulence factor, which was confirmed by bioassay on C. elegans and M. incognita. Using C. elegans as the target model, we found that both A. faecalis ZD02 and the virulence factor Esp can damage the intestines of C. elegans. The discovery that A. faecalis ZD02 has nematicidal activity provides a novel bacterial resource for the control of RKNs. PMID:26826227

  4. Bioreactor cultivation of Escherichia coli for production of recombinant penicillin G amidase from Alcaligenes faecalis.

    PubMed

    Deak, Peter M; Lutz-Wahl, Sabine; Bothe, Harald; Fischer, Lutz

    2003-03-01

    The penicillin G amidase (PGA) from Alcaligenes faecalis, which has interesting properties for use in combinatorial biochemistry, was produced by recombinant expression in Escherichia coli. The corresponding gene was cloned into a multicopy vector under the strict regulatory control of the rhamnose inducible promoter. Cells were grown in a synthetic minimal medium in a bioreactor (5 l working vol.), and production of PGA was induced by repeated addition of the inducer rhamnose, that served also as a carbon source. The fermentation yield was about 4500 units PGA activity per liter of culture medium.

  5. Structure-based stabilization of an enzyme: the case of penicillin acylase from Alcaligenes faecalis.

    PubMed

    Wang, Tianwen; Zhu, Hu; Ma, Xingyuan; Ma, Yushu; Wei, Dongzhi

    2006-01-01

    The modeled structure of penicillin acylase from Alcaligenes faecali (AFPGA) was constructed by comparative modeling with the Modeller program. Candidate positions that could be replaced with cysteine were estimated by scanning the modeled structure of AFPGA with the program MODIP (modeling disulfide bond in protein). The mutant Q3C/P751C had a higher optimum temperature by three degrees than that of the wild type AFPGA. The half life of the double mutant Q3C/P751C at 55 degrees C was increased by 50%. To our knowledge, this was the first structure-based genetic modification of AFPGA.

  6. Nitrous oxide production by Alcaligenes faecalis under transient and dynamic aerobic and anaerobic conditions

    SciTech Connect

    Otte, S.; Grobben, N.G.; Robertson, L.A.; Jetten, M.S.M.; Kuenen, J.G.

    1996-07-01

    Nitrous oxide production contributes to both greenhouse effect and ozone depletion in the stratosphere. A significant part of the global N2O emission can be attributed to microbial processes, especially nitrification and denitrification, used in biological wastewater treatment systems. This study looks at the efficiency of denitrification and the enzymes involved, with the emphasis on N2O production during the transient phase from aerobic to anaerobic conditions and vice versa. The effect of repetitive changing aerobic-anaerobic conditions on N2O was also studied. Alcaligenes faecalis was used as the model denitrofing organism. 35 refs., 3 figs., 1 tab.

  7. Alcaligenes faecalis: an unusual cause of skin and soft tissue infection.

    PubMed

    Tena, Daniel; Fernández, Cristina; Lago, María R

    2015-01-01

    Skin and soft tissue infection (SSTI) due to Alcaligenes faecalis is very rare and has never been studied. The aim of the present study was to investigate the clinical and microbiological characteristics of this infection. We conducted a retrospective review of 5 cases that occurred at our institution over a period of 6 years. All patients had underlying diseases, and infection was secondary to vascular disease or recent surgery in 4 of them. The most common clinical presentations were vascular ulcer infection and surgical site infection. The clinical outcome was uniformly good after treatment, except in 1 patient. In conclusion, A. faecalis should be considered a potential pathogen of SSTI, particularly in patients with vascular diseases or after surgery. The history of contact with water or aqueous solutions should be investigated in all cases. The clinical outcome is usually good, but treatment can be difficult in some cases due to the high level of resistance to commonly used antibiotics.

  8. Effect of nitrogen source on curdlan production by Alcaligenes faecalis ATCC 31749.

    PubMed

    Jiang, Longfa

    2013-01-01

    This study aims to investigate the effect of nitrogen source on curdlan production by Alcaligenes faecalis ATCC 31749. Curdlan production fell when excess nitrogen source was present, while biomass accumulation increased as the level of nitrogen source raised. Curdlan production and biomass accumulation were greater with urea compared with those with other nitrogen sources. The highest production of curdlan and biomass accumulation by A. faecalis ATCC 31749 was 28.16 g L(-1) and 9.58 g L(-1), respectively, with urea, whereas those with NH(4)Cl were 15.17 g L(-1) and 6.25 g L(-1), respectively. The optimum fermentation time for curdlan production was also affected by the nitrogen source in the medium.

  9. Potential application of Alcaligenes faecalis strain No. 4 in mitigating ammonia emissions from dairy wastewater.

    PubMed

    Neerackal, George M; Ndegwa, Pius M; Joo, Hung-Soo; Wang, Xiang; Frear, Craig S; Harrison, Joseph H; Beutel, Marc W

    2016-04-01

    This research examined the potential mitigation of NH3 emissions from dairy manure via an enhanced aerobic bio-treatment with bacterium Alcaligenes faecalis strain No. 4. The studies were conducted in aerated batch reactors using air and pure oxygen. Aeration with air and oxygen removed approximately 40% and 100% total ammoniacal nitrogen (TAN), respectively. Intermittent oxygenation (every 2 or 4 h) reduced oxygen consumption by 95%, while attaining nearly identical TAN removal to continuous aeration. The results revealed that adequate oxygen supply and supplementing dairy wastewater with carbon are essential for this bioprocess. Based on the nitrogen mass balance, only 4% of TAN was released as NH3 gas, while the majority was retained in either the microbial biomass (58%) or converted to nitrogen gas (36%). The mass balance results reveal high potential for environmentally friendly bio-treatment of dairy wastewater using A. faecalis strain No. 4 with respect to NH3 emissions.

  10. Production of R-(-)-mandelic acid from mandelonitrile by Alcaligenes faecalis ATCC 8750.

    PubMed Central

    Yamamoto, K; Oishi, K; Fujimatsu, I; Komatsu, K

    1991-01-01

    R-(-)-Mandelic acid was produced from racemic mandelonitrile by Alcaligenes faecalis ATCC 8750. Ammonium acetate or L-glutamic acid as the carbon source and n-butyronitrile as the inducer in the culture medium were effective for bacterial growth and the induction of R-(-)-mandelic acid-producing activity. The R-(-)-mandelic acid formed from mandelonitrile by resting cells was present in a 100% enantiomeric excess. A. faecalis ATCC 8750 has an R-enantioselective nitrilase for mandelonitrile and an amidase for mandelamide. As R-(-)-mandelic acid was produced from racemic mandelonitrile in a yield of 91%, whereas no S-mandelonitrile was left, the S-mandelonitrile remaining in the reaction is spontaneously racemized because of the chemical equilibrium and is used as the substrate. Consequently, almost all the mandelonitrile is consumed and converted to R-(-)-mandelic acid. R-(-)-Mandelic acid was also produced when benzaldehyde plus HCN was used as the substrate. PMID:1660699

  11. Characterization of Alcaligenes faecalis strain AD15 indicating biocontrol activity against plant pathogens.

    PubMed

    Yokoyama, Shin-ichiro; Adachi, Yoshitomi; Asakura, Shuichi; Kohyama, Erina

    2013-01-01

    Bacterial strain possessing both bacteriostatic and fungistatic activity (biocontrol activity) against pathogens of cyclamen (Cyclamen sp.) was isolated from the soil in Gifu Prefecture, Japan, and characterized with respect to its taxonomic and biocontrol properties. The sequence of its 16S rRNA gene, morphology, biochemistry, and fatty acid composition demonstrated that it is a strain most closely related to Alcaligenes faecalis subsp. faecalis LMG 1229(T). The isolate was named A. faecalis strain AD15. A. faecalis AD15 produced hydroxylamine at maximum yields of 33.3±1.7 mg/L after 16 h cultivation in LB medium and 19.0±0.44 mg/L after 19 h cultivation in synthetic medium. Moreover, minimum inhibitory concentrations of hydroxylamine against the cyclamen pathogens Pantoea agglomerans and Colletotrichum gloeosporioides were 4.20±0.98 and 16.5±0.67 mg/L. These results indicated that the biocontrol activity of strain AD15 might be attributed to hydroxylamine, a metabolite in the culture medium, and it had the potential for biopesticide application.

  12. Inhibition of Serratia marcescens Smj-11 biofilm formation by Alcaligenes faecalis STN17 crude extract

    NASA Astrophysics Data System (ADS)

    Lutfi, Zainal; Usup, Gires; Ahmad, Asmat

    2014-09-01

    Serratia marcescens biofilms are formed when they are bound to surfaces in aqueous environments. S. marcescens utilizes N-acylhomoserine lactone (AHL) as its quorum sensing signal molecule. The accumulation of AHL indicates the bacteria to produce matrices to form biofilms. Prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosin), which causes red pigmentation in the colonies, are also produced when the AHL reaches a certain threshold. The Alcaligenes faecalis STN17 crude extract is believed to inhibit quorum sensing in the S. marcescens Smj-11 and, thus, impedes its biofilm formation ability. A. faecalis STN17 was grown in marine broth, and ethyl acetate extraction was carried out. The crude compound of A. faecalis STN17 was diluted at high concentration (0.2-6.4 mg/mL) and was taken to confirm anti-biofilm activity through the crystal violet method in 96-wells plate. Then, the crude extract underwent purification using simple solvents partitioning test to discern the respective compounds that had the anti-biofilm activity under the crystal violet method. The crystal violet test showed that the crude did have anti-biofilm activity on S. marcescens Smj-11, but did not kill the cells. This finding signifies that the suppression of biofilm formation in S. marcescens by A. faecalis STN17 has a strong correlation. The partitioning test showed that A. faecalis STN17 crude extract has several compounds and only the compound(s) in chloroform showed activities. In conclusion, the crude extract of A. faecalis STN17 has the ability to inhibit S. marcescens Smj-11 biofilm formation.

  13. Inhibition of Serratia marcescens Smj-11 biofilm formation by Alcaligenes faecalis STN17 crude extract

    SciTech Connect

    Lutfi, Zainal; Ahmad, Asmat; Usup, Gires

    2014-09-03

    Serratia marcescens biofilms are formed when they are bound to surfaces in aqueous environments. S. marcescens utilizes N-acylhomoserine lactone (AHL) as its quorum sensing signal molecule. The accumulation of AHL indicates the bacteria to produce matrices to form biofilms. Prodigiosin (2-methyl-3-pentyl-6-methoxyprodigiosin), which causes red pigmentation in the colonies, are also produced when the AHL reaches a certain threshold. The Alcaligenes faecalis STN17 crude extract is believed to inhibit quorum sensing in the S. marcescens Smj-11 and, thus, impedes its biofilm formation ability. A. faecalis STN17 was grown in marine broth, and ethyl acetate extraction was carried out. The crude compound of A. faecalis STN17 was diluted at high concentration (0.2-6.4 mg/mL) and was taken to confirm anti-biofilm activity through the crystal violet method in 96-wells plate. Then, the crude extract underwent purification using simple solvents partitioning test to discern the respective compounds that had the anti-biofilm activity under the crystal violet method. The crystal violet test showed that the crude did have anti-biofilm activity on S. marcescens Smj-11, but did not kill the cells. This finding signifies that the suppression of biofilm formation in S. marcescens by A. faecalis STN17 has a strong correlation. The partitioning test showed that A. faecalis STN17 crude extract has several compounds and only the compound(s) in chloroform showed activities. In conclusion, the crude extract of A. faecalis STN17 has the ability to inhibit S. marcescens Smj-11 biofilm formation.

  14. Fluorescence in situ hybridization for rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis recovered from cystic fibrosis patients.

    PubMed

    Wellinghausen, Nele; Wirths, Beate; Poppert, Sven

    2006-09-01

    Achromobacter xylosoxidans is frequently isolated from the respiratory secretions of cystic fibrosis (CF) patients, but identification with biochemical tests is unreliable. We describe fluorescence in situ hybridization assays for the rapid identification of Achromobacter xylosoxidans and Alcaligenes faecalis. Both assays showed high sensitivities and high specificities with a collection of 155 nonfermenters from CF patients.

  15. The Genome Sequence of Alcaligenes faecalis NBIB-017 Contains Genes with Potentially High Activities against Erwinia carotovora.

    PubMed

    Liu, Xiaoyan; Huang, Daye; Wu, Jinping; Yu, Cui; Zhou, Ronghua; Liu, Cuijun; Zhang, Wei; Yao, Jingwu; Cheng, Meng; Guo, Suxia

    2016-04-07

    Alcaligenes faecalisNBIB-017, a Gram-negative bacterium, was isolated from soil in China. Here, we provide the complete genome sequence of this bacterium, which possesses a high number of genes encoding antibacterial factors, including proteins and small molecular peptides.

  16. The Genome Sequence of Alcaligenes faecalis NBIB-017 Contains Genes with Potentially High Activities against Erwinia carotovora

    PubMed Central

    Liu, Xiaoyan; Huang, Daye; Wu, Jinping; Yu, Cui; Zhou, Ronghua; Liu, Cuijun; Zhang, Wei; Yao, Jingwu; Cheng, Meng

    2016-01-01

    Alcaligenes faecalis NBIB-017, a Gram-negative bacterium, was isolated from soil in China. Here, we provide the complete genome sequence of this bacterium, which possesses a high number of genes encoding antibacterial factors, including proteins and small molecular peptides. PMID:27056227

  17. Alcaligenes faecalis subsp. phenolicus subsp. nov. a phenol-degrading, denitrifying bacterium isolated from a graywater bioprocessor.

    PubMed

    Rehfuss, Marc; Urban, James

    2005-07-01

    A Gram (-) coccobacillary bacterium, J(T), was isolated from a graywater bioprocessor. 16S rRNA and biochemical analysis has revealed strain J(T) closely resembles Alcaligenes faecalis ATCC 8750T and A. faecalis subsp. parafaecalis DSM 13975T, but is a distinct, previously uncharacterized isolate. Strain J(T), along with the type strain of A. faecalis and its previously described subspecies share the ability to aerobically degrade phenol. The degradation rates of phenol for strain J(T) and reference phenol degrading bacteria were determined by photometrically measuring the change in optical density when grown on 0.1% phenol as the sole carbon source, followed by addition of Gibb's reagent to measure depletion of substrate. The phenol degradation rates of strain J(T) was found to exceed that of the phenol hydroxylase group III bacterium Pseudomonas pseudoalcaligenes, with isolate J(T) exhibiting a doubling time of 4.5 h. The presence of the large subunit of the multicomponent phenol hydroxylase gene in strain J(T) was confirmed by PCR. The presence of the nirK nitrite reductase gene as demonstrated by PCR as well as results obtained from nitrite media indicated denitrification at least to N2O. Based on phenotypic, phylogenetic, fatty acid analysis and results from DNA DNA hybridization, we propose assigning a novel subspecies of Alcaligenes faecalis, to be named Alcaligenes faecalis subsp. phenolicus with the type strain J(T) (= DSM 16503) (= NRRL B-41076).

  18. Fragments of pro-peptide activate mature penicillin amidase of Alcaligenes faecalis.

    PubMed

    Kasche, Volker; Galunsky, Boris; Ignatova, Zoya

    2003-12-01

    Penicillin amidase from Alcaligenes faecalis is a recently identified N-terminal nucleophile hydrolase, which possesses the highest specificity constant (kcat/Km) for the hydrolysis of benzylpenicillin compared with penicillin amidases from other sources. Similar to the Escherichia coli penicillin amidase, the A. faecalis penicillin amidase is maturated in vivo from an inactive precursor into the catalytically active enzyme, containing one tightly bound Ca2+ ion, via a complex post-translational autocatalytic processing with a multi-step excision of a small internal pro-peptide. The function of the pro-region is so far unknown. In vitro addition of chemically synthesized fragments of the pro-peptide to purified mature A. faecalis penicillin amidase increased its specific activity up to 2.3-fold. Mutations were used to block various steps in the proteolytic processing of the pro-peptide to obtain stable mutants with covalently attached fragments of the pro-region to their A-chains. These extensions of the A-chain raised the activity up to 2.3-fold and increased the specificity constants for benzylpenicillin hydrolysis mainly by an increase of the turnover number (kcat).

  19. Purification and characterization of Alcaligenes faecalis penicillin G acylase expressed in Bacillus subtilis.

    PubMed

    Zhou, Zheng; Zhou, Li-Ping; Chen, Mei-Juan; Zhang, Yan-Liang; Li, Ren-Bao; Yang, Sheng; Yuan, Zhong-Yi

    2003-05-01

    The Alcaligenes faecalis PGA gene encoding heterodimeric penicillin G acylase (PGA) was cloned and successfully expressed in Escherichia coli and Bacillus subtilis respectively. In contrast to E.coli hosts where the enzymes were retained in the periplasm, B. subtilis cell secreted the recombinant enzyme into the medium. Contrary to limited expression yield of E. coli (pETAPGA), PGA extracellularly expressed by B. subtilis (pBAPGA) and B. subtilis (pMAPGA) reached the highest yield of 653 u/L. This yield increased 109-fold higher than the native expression of A. faecalis CICC AS1.767. The enzyme was fractionated with (NH(4))(2)SO(4) and purified by DEAE-Sepharose CL-6B with a yield of 81%. The purified enzyme had a specific activity of 1.469 u/mg. Furthermore, some enzyme characteristics, such as the pH and temperature optimum, the stability against organic solvent and the ratio of cepholexin synthesis to hydrolysis were determined. By overexpressing A. faecalis PGA in B. subtilis and purifying secreted enzyme from culture medium one could readily obtain a large amount of an alternative source of PGA.

  20. Exogenous cofactors for the improvement of bioremoval and biotransformation of sulfamethoxazole by Alcaligenes faecalis.

    PubMed

    Zhang, Yi-Bi; Zhou, Jiao; Xu, Qiu-Man; Cheng, Jing-Sheng; Luo, Yu-Lu; Yuan, Ying-Jin

    2016-09-15

    Sulfamethoxazole (SMX), an extensively prescribed or administered antibiotic pharmaceutical product, is usually detected in aquatic environments, because of its incomplete metabolism and elimination. This study investigated the effects of exogenous cofactors on the bioremoval and biotransformation of SMX by Alcaligenes faecalis. High concentration (100mg·L(-1)) of exogenous vitamin C (VC), vitamin B6 (VB6) and oxidized glutathione (GSSG) enhanced SMX bioremoval, while the additions of vitamin B2 (VB2) and vitamin B12 (VB12) did not significantly alter the SMX removal efficiency. Globally, cellular growth of A. faecalis and SMX removal both initially increased and then gradually decreased, indicating that SMX bioremoval is likely dependent on the primary biomass activity of A. faecalis. The decreases in the SMX removal efficiency indicated that some metabolites of SMX might be transformed into parent compound at the last stage of incubation. Two transformation products of SMX, N-hydroxy sulfamethoxazole (HO-SMX) and N4-acetyl sulfamethoxazole (Ac-SMX), were identified by a high-performance liquid chromatograph coupled with mass spectrometer. High concentrations of VC, nicotinamide adenine dinucleotide hydrogen (NADH, 7.1mg·L(-1)), and nicotinamide adenine dinucleotide (NAD(+), 6.6mg·L(-1)), and low concentrations of reduced glutathione (GSH, 0.1 and 10mg·L(-1)) and VB2 (1mg·L(-1)) remarkably increased the formation of HO-SMX, while VB12 showed opposite effects on HO-SMX formation. In addition, low concentrations of GSH and NADH enhanced Ac-SMX formation by the addition of A. faecalis, whereas cofactors (VC, VB2, VB12, NAD(+), and GSSG) had no obvious impact on the formation of Ac-SMX compared with the controls. The levels of Ac-SMX were stable when biomass of A. faecalis gradually decreased, indicating the direct effect of biomass on the formation of Ac-SMX by A. faecalis. In sum, these results help us understand the roles played by exogenous cofactors in

  1. Characteristics of ammonium removal by heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis No. 4.

    PubMed

    Joo, Hung-Soo; Hirai, Mitsuyo; Shoda, Makoto

    2005-08-01

    Alcaligenes faecalis no. 4 has heterotrophic nitrification and aerobic denitrification abilities. By taking the nitrogen balance under different culture conditions, 40-50% of removed NH4+-N was denitrified and about one-half of removed NH4+-N was converted to intracellular nitrogen. The maximum ammonium removal rate of no. 4 (28.9 mg-N/l/h) and its denitrification rate at high-strength NH4+-N of about 1200 ppm in aerated batch experiments at a C/N ratio of 10 were 5-40 times higher than those of other bacteria with the same ability. Only a few percent of the removed ammonium was converted to nitrite, and the main denitrification process was speculated to be via hydroxylamine which was produced by ammonium oxidation.

  2. Production of bioemulsifier by Bacillus subtilis, Alcaligenes faecalis and Enterobacter species in liquid culture.

    PubMed

    Toledo, F L; Gonzalez-Lopez, J; Calvo, C

    2008-11-01

    Three bacterial strains isolated from waste crude oil were selected due to their capacity of growing in the presence of hydrocarbons and production of bioemulsifier. The genetic identification (PCR of the 16S rDNA gene using fD1 and rD1 primers) of these strains showed their affiliation to Bacillus subtilis, Alcaligenes faecalis and Enterobacter sp. These strains were able to emulsify n-octane, toluene, xylene, mineral oils and crude oil, look promising for bioremediation application. Finally, chemical composition, emulsifying activity and surfactant activity of the biopolymers produced by the selected strains were studies under different culture conditions. Our results showed that chemical and functional properties of the bioemulsifiers were affected by the carbon source added to the growth media.

  3. Tributyltin chloride (TBTCl)-enhanced exopolysaccharide and siderophore production in an estuarine Alcaligenes faecalis strain.

    PubMed

    Khanolkar, Dnyanada; Dubey, S K; Naik, Milind Mohan

    2015-05-01

    Tributyltin chloride (TBTCl) has been used extensively as an antifouling agent in ship paints, which results in the contamination of aquatic sites. These contaminated sites serve as enrichment areas for TBTCl-resistant bacterial strains. One TBTCl-resistant bacterial strain was isolated from the sediments of Zuari estuary, Goa, India, which is a major hub of various ship-building activities. Based on biochemical characteristics and 16S rDNA sequence analysis, this bacterial strain was identified as Alcaligenes faecalis and designated as strain SD5. It could degrade ≥3 mM TBTCl by using it as a sole carbon source and transform it into the less toxic dibutyltin chloride, which was confirmed by nuclear magnetic resonance and mass spectroscopy. Interestingly, this bacterial strain also showed enhanced exopolysaccharide and siderophore production when cells were exposed to toxic levels of TBTCl, suggesting their involvement in conferring resistance to this antifouling biocide as well as degradative capability respectively.

  4. A novel synthesis of iminodiacetic acid: biocatalysis by whole Alcaligenes faecalis ZJB-09133 cells from iminodiacetonitrile.

    PubMed

    Liu, Zhi-Qiang; Li, Fei-Fei; Cheng, Feng; Zhang, Tao; You, Zhong-Yu; Xu, Jian-Miao; Xue, Ya-Ping; Zheng, Yu-Guo; Shen, Yin-Chu

    2011-01-01

    Iminodiacetic acid (IDA) has been widely used as an important intermediate in the fine chemical industry. In this study, a novel synthesis route of IDA from iminodiacetonitrile by whole microorganisms was investigated. A strain with the capability of producing nitrilase, ZJB-09133, was isolated and identified, and later named Alcaligenes faecalis ZJB-09133. In addition, the detailed biocatalysis of iminodiacetonitrile to produce IDA using ZJB-09133 was investigated. The results showed that the conversion reached 65.3% in Na(2)HPO(4)-NaH(2)PO(4) buffer of pH 8.0 under the following conditions: cells in the amount of 0.075-g DCW/L, 1.5% substrate, conversion time of 8 h, and a reaction temperature of 35°C. To the best of our knowledge, this is the first time that the production of IDA using a biocatalysis method has been reported.

  5. Hypochlorite digestion method for efficient recovery of PHB from Alcaligenes faecalis.

    PubMed

    Sayyed, R Z; Gangurde, N S; Chincholkar, S B

    2009-09-01

    We reported the optimum amount of PHB accumulated by Alcaligenes faecalis during its 24 h growth under nitrogen deficient conditions. After 24 h incubation decrease in the amount of PHB was recorded. Hypochlorite digestion of biomass of organism followed by extraction with a solvent system consisting of 1:1 mixture of ethanol and acetone resulted in efficient recovery of PHB vis-à-vis earlier methods. This solvent system gave a high recovery yield, i.e. 5.6 gL(-1) vis-à-vis earlier reported yield, 1.34 gL(-1) (by same method), 0.63 gL(-1) (by chloroform extraction method) and 1.1 gL(-1) (by dispersion method).

  6. Piggery wastewater treatment using Alcaligenes faecalis strain No. 4 with heterotrophic nitrification and aerobic denitrification.

    PubMed

    Joo, Hung-Soo; Hirai, Mitsuyo; Shoda, Makoto

    2006-09-01

    Alcaligenes faecalis strain No. 4, which has heterotrophic nitrification and aerobic denitrification abilities, was used to treat actual piggery wastewater containing high-strength ammonium under aerobic conditions. In a continuous experiment using a solids-free wastewater (SFW) mixed with feces, almost all of the 2000 NH4+ -N mg/L and 12,000 COD mg/L in the wastewater was removed and the ammonium removal rate was approximately 30 mg-N/L/h, which was 5-10 times higher than the rates achieved by other bacteria with the same abilities. The denitrification ratio was more than 65% of removed NH4+ -N, indicating that strain No. 4 exhibited its heterotrophic nitrification and aerobic denitrification abilities in the piggery wastewater.

  7. Nitrification and denitrification in high-strength ammonium by Alcaligenes faecalis.

    PubMed

    Joo, Hung-Soo; Hirai, Mitsuyo; Shoda, Makoto

    2005-06-01

    Alcaligenes faecalis sp. No. 4, that has the ability of heterotrophic nitrification and aerobic denitrification in high-strength ammonium at about 1200 mg-N/l, converted about one-half of removed NH4+-N to intracellular nitrogen and nitrified only 3% of the removed NH4+. From the nitrogen balance, 40-50% of removed NH4+-N was estimated to be denitrified. Production of N2 was confirmed by GC-MS and 90% of denitrified products was N2. The maximum ammonium removal rate, 29 mg-N/l h and its denitrification rate in aerated batch experiments, were 5-40 times higher than those of other bacteria with the same ability.

  8. Metal induced changes in trivalent chromium resistant Alcaligenes faecalis VITSIM2.

    PubMed

    Matilda, Shiny C; Shanthi, Chittibabu

    2017-02-20

    The changes induced in bacterial strains under stress conditions provide an insight into metal resistance strategies. Trivalent chromium resistant bacterium were isolated and identified by 16S rRNA gene sequencing and designated as Alcaligenes faecalis VITSIM2. The growth pattern was monitored. The organism also showed resistance to copper, cadmium, and certain antibiotics. The differentially expressed proteins in SDS PAGE were identified by mass spectrometry as flagellin and 50S ribosomal L36 protein. The morphological changes were identified by scanning electron microscopy. The changes in the cell wall content were estimated by peptidoglycan analysis and transformation of phosphates was detected by (31) P NMR. Flow cytometry was employed to measure the membrane integrity, esterase activity and intracellular pH. In conclusion spectrum of proteomic, physiological, and morphological alterations was observed that aid the organism to overcome chromium stress.

  9. The rice inoculant strain Alcaligenes faecalis A15 is a nitrogen-fixing Pseudomonas stutzeri.

    PubMed

    Vermeiren, H; Willems, A; Schoofs, G; de Mot, R; Keijers, V; Hai, W; Vanderleyden, J

    1999-05-01

    The taxonomic position of the nitrogen-fixing rice isolate A15, previously classified as Alcaligenes faecalis, was reinvestigated. On the basis of its small subunit ribosomal RNA (16S rRNA) sequence this strain identifies as Pseudomonas stutzeri. Phenotyping and fatty acid profiling confirm this result. DNA:DNA hybridisations, using the optical renaturation rate method, between strain A15 and Pseudomonas stutzeri LMG 11199T revealed a mean DNA-binding of 77%. The identification was further corroborated by comparative sequence analysis of the oprF gene, which encodes the major outer membrane protein of rRNA homology group I pseudomonads. Furthermore we determined the nifH sequence of this strain and of two putative diazotrophic Pseudomonas spp. and made a comparative analysis with sequences of other diazotrophs. These Pseudomonas NifH sequences cluster with NifH sequences isolated from the rice rhizosphere by PCR and of proteobacteria from the beta and gamma subclasses.

  10. Colonization of Alcaligenes faecalis strain JBW4 in natural soils and its detoxification of endosulfan.

    PubMed

    Kong, Lingfen; Zhu, Shaoyuan; Zhu, Lusheng; Xie, Hui; Wei, Kai; Yan, Tongxiang; Wang, Jun; Wang, Jinhua; Wang, Fenghua; Sun, Fengxia

    2014-02-01

    Alcaligenes faecalis strain JBW4, a strain of bacteria that is capable of degrading endosulfan, was inoculated into sterilized and natural soils spiked with endosulfan. JBW4 degraded 75.8 and 87.0 % of α-endosulfan and 58.5 and 69.5 % of β-endosulfan in sterilized and natural soils, respectively, after 77 days. Endosulfan ether and endosulfan lactone were the major metabolites that were detected by gas chromatography-mass spectrometry. This result suggested that A. faecalis strain JBW4 degrades endosulfan using a non-oxidative pathway in soils. The ability of strain JBW4 to colonize endosulfan-contaminated soils was confirmed by polymerase chain reaction-denaturing gradient gel electrophoresis. This result suggested that strain JBW4 competed with the original inhabitants in the soil to establish a balance and successfully colonize the soils. In addition, the detoxification of endosulfan by strain JBW4 was evaluated using single-cell gel electrophoresis and by determining the soil microbial biomass carbon and enzymatic activities. The results showed that the genotoxicity and ecotoxicity of endosulfan in soil were reduced after degradation. The natural degradation of endosulfan in soil is inadequate; therefore, JBW4 shows potential for the bioremediation of industrial soils that are contaminated with endosulfan residues.

  11. Biodegradation of Picolinic Acid by a Newly Isolated Bacterium Alcaligenes faecalis Strain JQ135.

    PubMed

    Qiu, Jiguo; Zhang, Junjie; Zhang, Yanting; Wang, Yuhong; Tong, Lu; Hong, Qing; He, Jian

    2017-04-01

    We isolated a bacterial strain JQ135 from municipal wastewater, which was capable of efficiently degrading picolinic acid (PA). Based on the physico-biochemical characteristics and 16S rDNA analysis, strain JQ135 was identified as Alcaligenes faecalis. In addition, strain JQ135 produced an orange pigment when cultured in the Luria-Bertani medium, which is different from the previously reported strains of A. faecalis. During the degradation of PA by the resting strain JQ135 cells, only one intermediate, 6-hydroxypicolinic acid (6HPA), was detected by ultraviolet spectrophotometry, high-pressure liquid chromatography, and liquid chromatography-mass spectrometry. A random transposon mutagenesis library of strain JQ135 was constructed. One mutant, Mut-G31, could convert PA into 6HPA without further degradation. The disrupted gene (orf2) was amplified from Mut-G31, and its product showed 32% identity to the 3-deoxy-D-manno-octulosonic acid kinase (KdkA) from Haemophilus influenzae. Results from complementation analysis confirmed that GTG was the initiation codon of the kdkA-like orf2, and that it was essential for PA biodegradation by strain JQ135. This study provides the first genetic evidence for the bacterial degradation of PA.

  12. Kinetic characteristics and modelling of growth and substrate removal by Alcaligenes faecalis strain NR.

    PubMed

    Chen, Jie; Zhao, Bin; An, Qiang; Wang, Xia; Zhang, Yi Xin

    2016-04-01

    Alcaligenes faecalis strain NR has the capability of simultaneous ammonium and organic carbon removal under sole aerobic conditions. The growth and substrate removal characteristics of A. faecalis strain NR were studied and appropriate kinetic models were developed. The maximum substrate removal rate of NH4 (+)-N and TOC were determined as 2.27 mg NH4 (+)-N/L/h and 30.00 mg TOC/L/h, respectively with initial NH4 (+)-N = 80 mg/L and TOC = 800 mg/L. Single-substrate models and double-substrate models based on Monod, Contois, Moser and Teissier were employed to describe the bioprocess kinetic coefficients. As a result, two double-substrate models, Teissier-Contois and Contois-Contois, were considered to be appropriate to model growth kinetics with both NH4 (+)-N and TOC as limiting substrates. The kinetic constants of maximum growth rate (μ max) and half-saturation constant (K S and B S) were obtained by solving multiple equations with regression. This work can be used to further understand and predict the performance of heterotrophic nitrifiers, and thus provides specific guidance of these functional strains in practical wastewater treatment process.

  13. Enhanced Alcaligenes faecalis Denitrification Rate with Electrodes as the Electron Donor.

    PubMed

    Wang, Xin; Yu, Ping; Zeng, Cuiping; Ding, Hongrui; Li, Yan; Wang, Changqiu; Lu, Anhuai

    2015-08-15

    The utilization by Alcaligenes faecalis of electrodes as the electron donor for denitrification was investigated in this study. The denitrification rate of A. faecalis with a poised potential was greatly enhanced compared with that of the controls without poised potentials. For nitrate reduction, although A. faecalis could not reduce nitrate, at three poised potentials of +0.06, -0.06, and -0.15 V (versus normal hydrogen electrode [NHE]), the nitrate was partially reduced with -0.15- and -0.06-V potentials at rates of 17.3 and 28.5 mg/liter/day, respectively. The percentages of reduction for -0.15 and -0.06 V were 52.4 and 30.4%, respectively. Meanwhile, for nitrite reduction, the poised potentials greatly enhanced the nitrite reduction. The nitrite reduction rates for three poised potentials (-0.06, -0.15, and -0.30 V) were 1.98, 4.37, and 3.91 mg/liter/h, respectively. When the potentials were cut off, the nitrite reduction rate was maintained for 1.5 h (from 2.3 to 2.25 mg/liter/h) and then greatly decreased, and the reduction rate (0.38 mg/liter/h) was about 1/6 compared with the rate (2.3 mg/liter/h) when potential was on. Then the potentials resumed, but the reduction rate did not resume and was only 2 times higher than the rate when the potential was off.

  14. Enhanced Alcaligenes faecalis Denitrification Rate with Electrodes as the Electron Donor

    PubMed Central

    Wang, Xin; Yu, Ping; Zeng, Cuiping; Ding, Hongrui; Wang, Changqiu

    2015-01-01

    The utilization by Alcaligenes faecalis of electrodes as the electron donor for denitrification was investigated in this study. The denitrification rate of A. faecalis with a poised potential was greatly enhanced compared with that of the controls without poised potentials. For nitrate reduction, although A. faecalis could not reduce nitrate, at three poised potentials of +0.06, −0.06, and −0.15 V (versus normal hydrogen electrode [NHE]), the nitrate was partially reduced with −0.15- and −0.06-V potentials at rates of 17.3 and 28.5 mg/liter/day, respectively. The percentages of reduction for −0.15 and −0.06 V were 52.4 and 30.4%, respectively. Meanwhile, for nitrite reduction, the poised potentials greatly enhanced the nitrite reduction. The nitrite reduction rates for three poised potentials (−0.06, −0.15, and −0.30 V) were 1.98, 4.37, and 3.91 mg/liter/h, respectively. When the potentials were cut off, the nitrite reduction rate was maintained for 1.5 h (from 2.3 to 2.25 mg/liter/h) and then greatly decreased, and the reduction rate (0.38 mg/liter/h) was about 1/6 compared with the rate (2.3 mg/liter/h) when potential was on. Then the potentials resumed, but the reduction rate did not resume and was only 2 times higher than the rate when the potential was off. PMID:26048940

  15. [Influence of pH control on the production of curdlan by Alcaligenes faecalis strain].

    PubMed

    Wang, Lei; Zhan, Xiao-Bei; Zhu, Yi-Hui; Li, Zhen-Yu; Yang, Ye

    2002-09-01

    A two-stage pH control method was employed in batch fermentation of curdlan by Alcaligenes faecalis WX-C12 where cell-growth stage was constantly controlled at pH 7.0 and stationary stage was controlled at a constant pH as well. The influence of pH control on the curdlan production was investigated. The optimal pH control of batch process for curdlan production was obtained when cell-growth stage was controlled at pH 7.0 and stationary stage was constantly controlled at pH 5.6. Production and productivity of curdlan, QP and YP/S reached 28.19 g/L, 291 mg/(L.h), 132.27 mg/(L.h.g) and 0.659, an improvement of 20.4%, 38.1%, 38.1% and 29.5% compared to a pH uncontrolled operation respectively.

  16. Biodegradation of nicosulfuron by a novel Alcaligenes faecalis strain ZWS11.

    PubMed

    Zhao, Weisong; Wang, Chen; Xu, Li; Zhao, Chunqing; Liang, Hongwu; Qiu, Lihong

    2015-09-01

    A bacterial strain ZWS11 was isolated from sulfonylurea herbicide-contaminated farmland soil and identified as a potential nicosulfuron-degrading bacterium. Based on morphological and physicochemical characterization of the bacterium and phylogenetic analysis of the 16S rRNA sequence, strain ZWS11 was identified as Alcaligenes faecalis. The effects of the initial concentration of nicosulfuron, inoculation volume, and medium pH on degradation of nicosulfuron were investigated. Strain ZWS11 could degrade 80.56% of the initial nicosulfuron supplemented at 500.0mg/L under the conditions of pH7.0, 180r/min and 30°C after incubation for 6days. Strain ZWS11 was also capable of degrading rimsulfuron, tribenuron-methyl and thifensulfuron-methyl. Four metabolites from biodegradation of nicosulfuron were identified, which were 2-aminosulfonyl-N, N-dimethylnicotinamide (M1), 4, 6-dihydroxypyrimidine (M2), 2-amino-4, 6-dimethoxypyrimidine (M3) and 2-(1-(4,6-dimethoxy-pyrimidin-2-yl)-ureido)-N,N-dimethyl-nicotinamide (M4). Among the metabolites detected, M2 was reported for the first time. Possible biodegradation pathways of nicosulfuron by strain ZWS11 were proposed. The degradation proceeded mainly via cleavage of the sulfonylurea bridge, O-dealkylation, and contraction of the sulfonylurea bridge by elimination of a sulfur dioxide group. The results provide valuable information for degradation of nicosulfuron in contaminated environments.

  17. The crystal structure of cobalt-substituted pseudoazurin from Alcaligenes faecalis.

    PubMed

    Gessmann, Renate; Kyvelidou, Christiana; Papadovasilaki, Maria; Petratos, Kyriacos

    2011-03-01

    The Cu(II) center at the active site of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by Co(II) via denaturing of the protein, chelation and removal of copper by EDTA and refolding of the apo-protein, followed by addition of an aqueous solution of CoCl(2). Sitting drop vapour diffusion experiments produced green hexagonal crystals, which belong to space group P6(5), with unit cell dimensions a = b = 50.03, c = 98.80 Å. Diffraction data, collected at 291 K on a copper rotating anode X-ray source, were phased by the anomalous signal of the cobalt atom. The structure was built automatically, fitted manually and subsequently refined to 1.86 Å resolution. The Co-substituted protein exhibits similar overall geometry to the native structure with copper. Cobalt binds more strongly to the axial Met86-Sδ and retains the tetrahedral arrangement with the four ligand atoms, His40-Nδ(1), Cys78-Sγ, His81-Nδ(1), and 86Met-Sδ, although the structure is less distorted than the native copper protein. The structure reported herein, is the first crystallographic structure of a Co(II)-substituted pseudoazurin.

  18. Mutant AFM 2 of Alcaligenes faecalis for phenol biodegradation using He-Ne laser irradiation.

    PubMed

    Jiang, Yan; Wen, Jianping; Caiyin, Qinggele; Lin, Liangcai; Hu, Zongding

    2006-11-01

    He-Ne laser technology was utilized in this study to investigate the response of Alcaligenes faecalis to laser stimulation. The irradiation experiments were conducted by the adjustment of the output power from 5 to 25 mW and the exposure time from 5 to 25 min. The results showed that the survival rate changed regularly with the variety of irradiation dose, and high positive mutation frequency was determined by both the energy density and the output power. The mutant strain AFM 2 was obtained. Phenol biodegradation assay demonstrated that AFM 2 possessed a more prominent phenol-degrading potential than its parent strain, which presumably attributed to the improvements of phenol hydroxylase and catechol 1,2-dioxygenase activities. The phenol of 2000 mgl(-1) was completely degraded by AFM 2 within 85.5h at 30 degrees C. In addition, the cell growth and phenol degradation kinetics of the mutant strain AFM 2 and its parent strain in batch cultures were also investigated at the wide initial phenol concentration ranging from 0 to 2000 mgl(-1) by Haldane model. The results of these experiments further demonstrated that the mutant strain AFM 2 possessed a higher capacity to resist phenol.

  19. Crystallization and X-ray structure analysis of a thermostable penicillin G acylase from Alcaligenes faecalis.

    PubMed

    Varshney, Nishant Kumar; Kumar, R Suresh; Ignatova, Zoya; Prabhune, Asmita; Pundle, Archana; Dodson, Eleanor; Suresh, C G

    2012-03-01

    The enzyme penicillin G acylase (EC 3.5.1.11) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C222(1), with unit-cell parameters a = 72.9, b = 86.0, c = 260.2 , and P4(1)2(1)2, with unit-cell parameters a = b = 85.6, c = 298.8 . Data were collected at 293 and the structure was determined using the molecular-replacement method. Like other penicillin acylases, AfPGA belongs to the N-terminal nucleophilic hydrolase superfamily, has undergone post-translational processing and has a serine as the N-terminal residue of the β-chain. A disulfide bridge has been identified in the structure that was not found in the other two known penicillin G cylase structures. The presence of the disulfide bridge is perceived to be one factor that confers higher stability to this enzyme.

  20. Alcaligenes faecalis kw-a biofilm for denitrification of nitrate-rich effluent.

    PubMed

    Jadhav, T S; Faldu, N J; Patel, P; Narolkar, S N; Nerurkar, A S

    2005-06-01

    Alcaligenes faecalis kw-A selected for possessing good denitrification efficiency was used for biofilm development. The biofilm could be developed on a glass surface within 12 hr when 5%, Ix 10(8) cells/ml was used as inoculum. The microcolonies were seen in 6 hr and glycocalyx in 9 hr stage. At 24 hr the biofilm was developed fully and hence was visualised as dense mass. The biofilm protein content showed 48.5% increase in shake flask than in static condition. The exopoplymer is produced in larger amounts in biofilm as compared to the suspended cells. Also, its amount was more by 43% in the biofilm produced in shake flask condition than in static condition. The biofilm could remove 95% nitrate from nitrate-rich effluent in a bench-scale process in 36 hr. The attached growth technique demonstrated here can be utilised to study the effect of favourable as well as adverse conditions on the denitrification efficiency of a culture. The ultimate application of a denitrifying biofilm would be in attached growth or biofilm reactor.

  1. Enzymatic properties of immobilized Alcaligenes faecalis cells with cell-associated beta-glucosidase activity

    SciTech Connect

    Wheatly, M.A.; Phillips, C.R.

    1984-06-01

    Enzymatic properties of Alcaligenes faecalis cells immobilized in polyacrylamide were characterized and compared with those reported for the extracted enzyme, and with those measured for free cells. Many of the properties reflected those of the extracted enzyme rather than those measured in the free whole cells prior to immobilization, suggesting cell disruption during immobilization. These properties included the pH activity profile, a slightly broader pH stability profile, and the activation energy. Electron micrographs showed evidence of cell debris among the polymer matrix. The immobilized cells were not viable, and did not consume glucose. Thermal stability was less after immobilization with a half-line of 16 h at 45 degrees C, and 3.5 h at 50 degrees C. The immobilized preparation was more stable when stored lyophilized rather than in buffer, losing 23 and 52% activity, respectively, after six months. The enzyme was irreversibly inhibited by both acetate and citrate buffers. If the immobilized enzyme is to be used in conjunction with cellulases from Trichoderma reesei for cellulase saccharification, the optimal conditions would be pH 5.5 and 45 degrees C in a buffer containing no carboxylic acid groups.

  2. Aspartate beta-decarboxylase from Alcaligenes faecalis: carbon-13 kinetic isotope effect and deuterium exchange experiments

    SciTech Connect

    Rosenberg, R.M.; O'Leary, M.H.

    1985-03-26

    The authors have measured the /sup 13/C kinetic isotope effect at pH 4.0, 5.0, 6.0, and 6.5 and in D/sub 2/O at pH 5.0 and the rate of D-H exchange of the alpha and beta protons of aspartic acid in D/sub 2/O at pH 5.0 for the reaction catalyzed by the enzyme aspartate beta-decarboxylase from Alcaligenes faecalis. The /sup 13/C kinetic isotope effect, with a value of 1.0099 +/- 0.0002 at pH 5.0, is less than the intrinsic isotope effect for the decarboxylation step, indicating that the decarboxylation step is not entirely rate limiting. The authors have been able to estimate probable values of the relative free energies of the transition states of the enzymatic reaction up to and including the decarboxylation step from the /sup 13/C kinetic isotope effect and the rate of D-H exchange of alpha-H. The pH dependence of the kinetic isotope effect reflects the pKa of the pyridine nitrogen of the coenzyme pyridoxal 5'-phosphate but not that of the imine nitrogen. A mechanism is proposed for the exchange of aspartate beta-H that is consistent with the stereochemistry suggested earlier.

  3. Release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126: a comparative study.

    PubMed

    Singh, Renu; Banerjee, Anirban; Kaul, Praveen; Barse, Brajesh; Banerjee, U C

    2005-10-01

    Nitrilases constitute an important class of hydrolases, however, cheap and ready availability of enzyme sources limit their practical synthetic applications. The present investigation was directed to compare the applicability of various physical cell disintegration methods namely, solid shear, liquid shear and sonication, for the release of an enantioselective nitrilase from Alcaligenes faecalis MTCC 126. Different parameters associated with each method were optimized in order to ensure maximal release of active nitrilase. The methods were also compared under optimal conditions for their efficiency of nitrilase release and extent of cell disruption, and enzyme release were visualized under a differential interference contrast microscope (DIC) and SDS-PAGE, respectively. Maximum release of the enzyme protein from the cells was observed in case of liquid shear method employing high-pressure homogenization, however, the specific activity of nitrilase was highest in cell-free extract (CFE) generated by sonication. Both the solid shear and liquid shear proved to be equally effective for maximum release of intracellular enzymes, however, from the specific activity point of view, sonication was found to be a better one compared to other two methodologies. The generated cell-free extract can be further employed for the production of enantiopure chiral carboxylic acids, which are important chiral building blocks.

  4. Efficient enzymatic synthesis of ampicillin by mutant Alcaligenes faecalis penicillin G acylase.

    PubMed

    Deng, Senwen; Su, Erzheng; Ma, Xiaoqiang; Yang, Shengli; Wei, Dongzhi

    2015-04-10

    Semi-synthetic β-lactam antibiotics (SSBAs) are one of the most important antibiotic families in the world market. Their enzymatic synthesis can be catalyzed by penicillin G acylases (PGAs). In this study, to improve enzymatic synthesis of ampicillin, site-saturating mutagenesis was performed on three conserved amino acid residues: βF24, αR146, and αF147 of thermo-stable penicillin G acylase from Alcaligenes faecalis (Af PGA). Four mutants βF24G, βF24A, βF24S, and βF24P were recovered by screening the mutant bank. Kinetic analysis of them showed up to 800-fold increased kcat/Km value for activated acyl donor D-phenylglycine methyl ester (D-PGME). When βF24G was used for ampicillin synthesis under kinetic control at industrially relevant conditions, 95% of nucleophile 6-aminopenicillanic acid (6-APA) was converted to ampicillin in aqueous medium at room temperature while 12% process time is needed to reach maximum product accumulation at 25% enzyme concentration compared with the wild-type Af PGA. Consequently, process productivity of enzymatic synthesis of ampicillin catalyzed by Af PGA was improved by more than 130 times, which indicated an enzyme viable for efficient SSBAs synthesis.

  5. Crystallization and X-ray structure analysis of a thermostable penicillin G acylase from Alcaligenes faecalis

    PubMed Central

    Varshney, Nishant Kumar; Suresh Kumar, R.; Ignatova, Zoya; Prabhune, Asmita; Pundle, Archana; Dodson, Eleanor; Suresh, C. G.

    2012-01-01

    The enzyme penicillin G acylase (EC 3.5.1.11) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C2221, with unit-cell parameters a = 72.9, b = 86.0, c = 260.2 Å, and P41212, with unit-cell parameters a = b = 85.6, c = 298.8 Å. Data were collected at 293 K and the structure was determined using the molecular-replacement method. Like other penicillin acylases, AfPGA belongs to the N-terminal nucleophilic hydrolase superfamily, has undergone post-translational processing and has a serine as the N-­terminal residue of the β-chain. A disulfide bridge has been identified in the structure that was not found in the other two known penicillin G acylase structures. The presence of the disulfide bridge is perceived to be one factor that confers higher stability to this enzyme. PMID:22442220

  6. Optimization of nitrilase production from Alcaligenes faecalis MTCC 10757 (IICT-A3): effect of inducers on substrate specificity.

    PubMed

    Nageshwar, Y V D; Sheelu, Gurrala; Shambhu, Rekha Rao; Muluka, Hemalatha; Mehdi, Nooreen; Malik, M Shaheer; Kamal, Ahmed

    2011-06-01

    Microbial nitrilases are biocatalysts of interest and the enzyme produced using various inducers exhibits altered substrate specificity, which is of great interest in bioprocess development. The aim of the present study is to investigate the nitrilase-producing Alcaligenes faecalis MTCC 10757 (IICT-A3) for its ability to transform various nitriles in the presence of different inducers after optimization of various parameters for maximum enzyme production and activity. The production of A. faecalis MTCC 10757 (IICT-A3) nitrilase was optimum with glucose (1.0%), acrylonitrile (0.1%) at pH 7.0. The nitrilase activity of A. faecalis MTCC 10757 (IICT-A3) was optimum at 35 °C, pH 8.0 and the enzyme was stable up to 6 h at 50 °C. The nitrilase enzyme produced using different inducers was investigated for substrate specificity. The enzyme hydrolyzed aliphatic, heterocyclic and aromatic nitriles with different substitutions. Acrylonitrile was the most preferred substrate (~40 U) as well as inducer. Benzonitrile was hydrolyzed with almost twofold higher relative activity than acrylonitrile when it was used as an inducer. The versatile nitrilase-producing A. faecalis MTCC 10757 (IICT-A3) exhibits efficient conversion of both aliphatic and aromatic nitriles. The aromatic nitriles, which show not much or no affinity towards nitrilase from A. faecalis, are hydrolyzed effectively with this nitrilase-producing organism. Studies are in progress to exploit this organism for synthesis of industrially important compounds.

  7. [Constitutive expression and purification of Alcaligenes faecalis penicillin G acylase in Escherichia coli].

    PubMed

    Yang, Zhi-Jian; Cai, Jin; Sun, Jian; Yuan, Zhong-Yi

    2004-09-01

    Considering Alcaligenes faecalis pencillin G acylase(AfPGA), which possesses the attractive characteristics for beta-lactam antibiotics conversions, the gene of PGA was cloned into an expressing vector pKKFPGA. The recombinant plasmid contained multicopy replicon(COLE 1), trc promoter, AfPGA gene, rrnB transcript terminator and ampicillin marker transformed Escherichia coli DH5alpha. As both the recombinant plasmid and the host DH5alpha had no laclq gene, the trc promoter was always active and the AfPGA could be constitutively expressed without IPTG induction in the host DH5alpha. In the shaking flask, the recombinant cell was inoculated into the fermentation medium (tryptone 10g/L, yeast extract 5g/L, MgSO4 x 7 H2O 1g, KH2 PO4 2g/L, K2HPO4 x 3H2O 5g/L, Na2HPO4 x 12H2O 7g/L, (NH4)2SO4 1.2g/L, NH4Cl 0.2 g/L, NaCl 0.1g/L, dextrin 30g/L) and cultured at 28 degrees C for 20h. The production of AfPGA reached 2,590u/L(NIPAB method), with a cell-density-specific activity of more than 300(u/L)/A600, this yield increased 432 fold higher than the native expression of Alcaligenes faecalis . Without ammonium sulphate fractionation and dialysis, the supernatant of crude extract was directly loaded on DEAE-Sepharose CL 6B column equilibrated by phosphate buffer (50mmol/L, pH7.8), and the enzyme fraction was not absorbed on the column but impurities were absorbed. Subsequently the effluent was added ammonium sulphate to 1mol/L and loaded on Butyl-Sepharose CL 4B column equilibrated by 50mmol/L phosphate buffer pH7.8-1mol/L ammonium sulphate. The enzyme was eluted as concentration of ammonium sulphate in phosphate buffer decreased to 0, PGA was eluted. After these two column chromatography, the enzyme was enriched 20 times with a 91% activity recovery. The purified enzyme had a specific activity of 68.6u/mg protein. However, the overproduction of PGA was often limited by translocation and/or periplasmic processing steps, subsequently resulted in intracellular accumulation of

  8. Classification of Alcaligenes faecalis-like isolates from the environment and human clinical samples as Ralstonia gilardii sp. nov.

    PubMed

    Coenye, T; Falsen, E; Vancanneyt, M; Hoste, B; Govan, J R; Kersters, K; Vandamme, P

    1999-04-01

    A polyphasic taxonomic study that included DNA-DNA hybridizations, DNA base ratio determinations, 16S rDNA sequence analysis, whole-cell protein and fatty acid analyses, AFLP (amplified fragment length polymorphism) fingerprinting and an extensive biochemical characterization was performed on 10 strains provisionally identified as Alcaligenes faecalis-like bacteria. The six environmental and four human isolates belonged to the genus Ralstonia and were assigned to a new species for which the name Ralstonia gilardii sp. nov. is proposed. The type strain is LMG 5886T.

  9. Immunostimulatory activities of a decapeptide derived from Alcaligenes faecalis FY-3 to crucian carp.

    PubMed

    Wang, G-X; Li, F-Y; Cui, J; Wang, Y; Liu, Y-T; Han, J; Lei, Y

    2011-07-01

    A strain was isolated from a soil sample collected from Weihe river in Shaanxi province (108°03'E 34°14'N), which was identified as Alcaligenes faecalis by 16S rRNA analysis. A compound M showing potent immune activity was isolated from secondary metabolites of the strain through bioassay-guided isolation techniques. The structure of the compound M was elucidated using FT-IR, EI-MS, 1H NMR and 13C NMR spectra and identified as cyclo-(L-Pro-Gly)5 which was first time reported as a natural product. We evaluated the immune effects of the cyclo-(L-Pro-Gly)5 on the basis of serum lysozyme activity, bacterial agglutination titre assay, superoxide anion production and phagocytic activity assay, and they were found to be significantly increased by cyclo-(L-Pro-Gly)5. The effects of cyclo-(L-Pro-Gly)5 on immune-related gene expression were further investigated. The outcomes of real-time quantitative polymerase chain reaction (RQ-PCR) proved that the transcribing level of interleukin 6β (IL-6β) and inducible nitric oxide synthase 1β (iNOS-1β) mRNA in the blood leucocytes have been augmented by cyclo-(L-Pro-Gly)5. The challenge experiment showed that crucian carp injected the cyclo-(L-Pro-Gly)5 had significantly (P < 0.05) lower cumulative mortality (13.0%) compared with the control (45.4%) after infection with live Aeromonas hydrophila. These results suggested that cyclo-(L-Pro-Gly)5 is a possible immunostimulant and may strengthen the immune response and protect the heath status of crucian carp against A. hydrophila.

  10. Characterization of a novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis.

    PubMed

    Shahi, Puja; Kumar, Ish; Sharma, Ritu; Sanger, Shefali; Jolly, Ravinder S

    2006-06-01

    A novel long-chain acyl-CoA thioesterase from Alcaligenes faecalis has been isolated and characterized. The protein was extracted from the cells with 1 m NaCl, which required 1.5-fold, single-step purification to yield near-homogeneous preparations. In solution, the protein exists as homomeric aggregates, of mean diameter 21.6 nm, consisting of 22-kDa subunits. MS/MS data for peptides obtained by trypsin digestion of the thiosterase did not match any peptide from Escherichia coli thioesterases or any other thioesterases in the database. The thioesterase was associated exclusively with the surface of cells as revealed by ultrastructural studies using electron microscopy and immunogold labeling. It hydrolyzed saturated and unsaturated fatty acyl-CoAs of C12 to C18 chain length with Vmax and Km of 3.58-9.73 micromol x min(-1) x (mg protein)(-1) and 2.66-4.11 microm, respectively. A catalytically important histidine residue is implicated in the active site of the enzyme. The thioesterase was active and stable over a wide range of temperature and pH. Maximum activity was observed at 65 degrees C and pH 10.5, and varied between 60% and 80% at temperatures of 25-70 degrees C and pH 6.5-10. The thioesterase also hydrolyzed p-nitrophenyl esters of C2 to C12 chain length, but substrate competition experiments demonstrated that the long-chain acyl-CoAs are better substrates for thioesterase than p-nitrophenyl esters. When assayed at 37 and 20 degrees C, the affinity and catalytic efficiency of the thioesterase for palmitoleoyl-CoA and cis-vaccenoyl-CoA were reduced approximately twofold at the lower temperature, but remained largely unaltered for palmitoyl-CoA.

  11. Structural-based mutational analysis of D-aminoacylase from Alcaligenes faecalis DA1.

    PubMed

    Hsu, Cheng-Sheng; Lai, Wen-Lin; Chang, Wei-Wei; Liaw, Shwu-Huey; Tsai, Ying-Chieh

    2002-11-01

    D-Aminoacylase is an attractive candidate for commercial production of D-amino acids through its catalysis in the zinc-assistant hydrolysis of N-acyl-D-amino acids. We report here the cloning, expression, and structural-based mutation of the D-aminoacylase from Alcaligenes faecalis DA1. A 1,007-bp PCR product amplified with degenerate primers, was used to isolate a 4-kb genomic fragment, encoding a 484-residue D-aminoacylase. The enzyme amino-terminal segment shared significant homology within a variety of enzymes including urease. The structural fold was predicted by 3D-PSSM to be similar to urease and dihydroorotase, which have grouped into a novel alpha/beta-barrel amidohydrolase superfamily with a virtually indistinguishable binuclear metal centers containing six ligands, four histidines, one aspartate, and one carboxylated lysine. Three histidines, His-67, His-69, and His-250, putative metal ligands in D-aminoacylase, have been mutated previously, the remaining histidine (His-220) and aspartate (Asp-366) Asp-65, and four cysteines were then characterized. Substitution of Asp-65, Cys-96, His-220, and Asp-366 with alanine abolished the enzyme activity. The H220A mutant bound approximately half the normal complement of zinc ion as did H250N. However, the C96A mutant showed little zinc-binding ability, revealing that Cys-96 may replace the carboxylated lysine to serve as a bridging ligand. According to the urease structure, the conserved amino-terminal segment including Asp-65 may be responsible for structural stabilization.

  12. Antimicrobial activity and determination of bioactive components from marine Alcaligenes faecalis extract against a sulfate-reducing bacteria

    NASA Astrophysics Data System (ADS)

    AbdSharad, Ali; Usup, Gires; Sahrani, Fathul Karim; Ahmad, Asmat

    2016-11-01

    Biogenic souring and microbial-influenced corrosion is a common scenario in petroleum reservoir. The serious threat normally comes from sulfate-reducing bacteria (SRB). Alcaligenes faecalis was tested in this study for the ability to inhibit the growth of SRB. Ethyl acetate extraction of A. faecalis grown in marine broth was carried out to produce crude ethyl acetate of A. faecalis (CEAF). CEAF was diluted at concentrations 0.2-12.8 mg/mL and was tested for anti-microbial activity by microdilution susceptibility tests in 96-wells plate. CEAF was then analyzed by Gas Chromatography Mass Spectrometry (GC-MS). The microdilution susceptibility tests showed that the crude have anti- microbial activities on SRB. CEAF showed immediate killing effect against SRB in liquid medium which suggest the presence of active chemical compounds with antimicrobial activity. The GC-MS analysis showed the presence of 20 different chemical compounds in CEAF, The major components in CEAF can be related to antimicrobial, antifungal, antioxidant, pesticide, metabolism, toxicity, anticancer and corrosion inhibition activities. In conclusion, crude ethyl acetate extract of A. faecalis has the ability to inhibit SRB growth.

  13. [Enhanced production of curdlan by Alcaligenes faecalis by selective feeding with ammonia water during the cell growth phase of fermentation].

    PubMed

    Wu, Jianrong; Zhan, Xiaobei; Liu, Hui; Zheng, Zhiyong

    2008-06-01

    Curdlan is a water insoluble exopolysaccharide produced by Alcaligenes faecalis under nitrogen-limiting conditions. After excretion, the polysaccharide is attached the cell wall. Thus enhancement of biomass production during the cell growth phase is important to curdlan production. A strategy of increasing nitrogen source to improve biomass production was adopted for curdlan production by Alcaligenes faecalis (ATCC 31749). In the batch fermentation of curdlan, a relatively higher NH4Cl level of 3.6 g/L with continuous glucose feeding increased the cell density leading to improvement of curdlan production. However, excessive NH4Cl would inhibit curdlan production and biomass production was not improved significantly. In addition, feeding of ammonia water at the initial phase replaced NaOH solution to control pH at 7.0. Subsequently, feeding of NaOH solution was resumed to control pH at 5.6 for curdlan production after ammonia was consumed. As a result, biomass production and curdlan yield were both enhanced remarkably. Feeding of ammonia water during the first 24 h led to biomass production of 18.8 g/L. However, higher cell density did not lead to increase in curdlan production. The maximum curdlan production (72 g/L) was obtained by feeding ammonia water for the first 14 h, during which the cell density was about 11.9 g/L.

  14. Effect of Alcaligenes faecalis on nitrous oxide emission and nitrogen removal in three phase fluidized bed process.

    PubMed

    Kim, Jeong-Sook; Kim, Shi-Jun; Lee, Byung-Hun

    2004-01-01

    Nitrous oxide (N2O), one of the greenhouse effect gases, has not been known that how much N2O is produced from municipal wastewater treatment and what its management should be. In this study, for controlling nitrous oxide emission and removing nitrogen from municipal wastewater, we experimented the three phase fluidized bed process equipped with draft tube along with immobilized Alcaligenes faecalis, a typical heterotrophic nitrifer and a predominant genus. Also we evaluated the optimum treatment condition of the three phase fluidized bed process for emitting nitrous oxide. The results of this study showed that the three phase fluidized bed process was more effective than the activated sludge process for controlling nitrous oxide emission and removing nitrogen. Increasing amount of A. faecalis in reactor should be encouraged for controlling nitrous oxide emission and removing nitrogen. In addition, the activated sludge process using immobilized A. faecalis as a carrier had more nitrogen removal efficiency than conventional activated sludge process. The accumulation of NO2-N, NO3-N resulted in high N2O emission. Therefore, we suggested that it is necessary to reduce NO2-N and NO3-N for both reducing N2O emission and improving nitrogen removal.

  15. Improvement in ammonium removal efficiency in wastewater treatment by mixed culture of Alcaligenes faecalis no. 4 and L1.

    PubMed

    Joo, Hung-Soo; Hirai, Mitsuyo; Shoda, Makoto

    2007-01-01

    To improve ammonium removal efficiency in wastewater treatment, a mixed culture of Alcaligenes faecalis no. 4 and its mutant L1, both of which have heterotrophic nitrification and aerobic denitrification abilities, was performed. In a batch culture, no. 4 has a higher denitrification ability than L1, but its ammonium removal rate was lower. In a mixed continuous culture in the ammonium loading range of 750 to 3500 mg-N/l/d, the average ammonium removal rate and the average denitrification ratio were 61 mg-N/l/h and 31%, respectively. In the mixed culture, the ammonium removal rate was twofold higher than that in a single culture of no. 4, the rate was similar to that in a single culture of L1, and the denitrification ratio was very high compared with that in the single culture of L1.

  16. Cloning, sequencing and mutagenesis of the genes for aromatic amine dehydrogenase from Alcaligenes faecalis and evolution of amine dehydrogenases.

    PubMed

    Chistoserdov, A Y

    2001-08-01

    The nucleotide sequence of the aromatic amine utilization (aau) gene region from Alcaligenes faecalis contained nine genes (orf-1, aauBEDA, orf-2, orf-3, orf-4 and hemE) transcribed in the same direction. The aauB and aauA genes encode the periplasmic aromatic amine dehydrogenase (AADH) large and small subunit polypeptides, respectively, and were homologous to mauB and mauA, the genes for the large and small subunits of methylamine dehydrogenase (MADH). aauE and aauD are homologous to mauE and mauD and apparently carry out the same function of transport and folding of the small subunit polypeptide in the periplasm. No analogues of the mauF, mauG, mauL, mauM and mauN genes responsible for biosynthesis of tryptophan tryptophylquinone (the prosthetic group of amine dehydrogenases) were found in the aau cluster. orf-2 was predicted to encode a small periplasmic monohaem c-type cytochrome. No biological function can be assigned to polypeptides encoded by orf-1, orf-3 and orf-4 and mutations in these genes appeared to be lethal. Mutants generated by insertions into mauD were not able to use phenylethylamine, tyramine and tryptamine as a source of carbon and phenylethylamine, 3'-hydroxytyramine (dopamine) and tyramine as a source of nitrogen, indicating that AADH is the only enzyme involved in utilization of primary amines in A. faecalis. AADH genes are present in Alcaligenes xylosoxydans subsp. xylosoxydans, but not in other beta- and gamma-proteobacteria. Phylogenetic analysis of amine dehydrogenases (MADH and AADH) indicated that AADH and MADH evolutionarily diverged before separation of proteobacteria into existing subclasses.

  17. Genetic Diversity and Horizontal Transfer of Genes Involved in Oxidation of Reduced Phosphorus Compounds by Alcaligenes faecalis WM2072

    PubMed Central

    Wilson, Marlena M.; Metcalf, William W.

    2005-01-01

    Enrichment was performed to isolate organisms that could utilize reduced phosphorus compounds as their sole phosphorus sources. One isolate that grew well with either hypophosphite or phosphite was identified by 16S rRNA gene analysis as a strain of Alcaligenes faecalis. The genes required for oxidation of hypophosphite and phosphite by this organism were identified by using transposon mutagenesis and include homologs of the ptxD and htxA genes of Pseudomonas stutzeri WM88, which encode an NAD-dependent phosphite dehydrogenase (PtxD) and 2-oxoglutarate-dependent hypophosphite dioxygenase (HtxA). This organism also has the htxB, htxC, and htxD genes that comprise an ABC-type transporter, presumably for hypophosphite and phosphite transport. The role of these genes in reduced phosphorus metabolism was confirmed by analyzing the growth of mutants in which these genes were deleted. Sequencing data showed that htxA, htxB, htxC, and htxD are virtually identical to their homologs in P. stutzeri at the DNA level, indicating that horizontal gene transfer occurred. However, A. faecalis ptxD is very different from its P. stutzeri homolog and represents a new ptxD lineage. Therefore, this gene has ancient evolutionary roots in bacteria. These data suggest that there is strong evolutionary selection for the ability of microorganisms to oxidize hypophosphite and phosphite. PMID:15640200

  18. Purification and characterization of chitinase from Alcaligenes faecalis AU02 by utilizing marine wastes and its antioxidant activity.

    PubMed

    Annamalai, Neelamegam; Veeramuthu Rajeswari, Mayavan; Vijayalakshmi, Shanmugam; Balasubramanian, Thangavel

    2011-12-01

    Marine waste is an abundant renewable source for the recovery of several value added metabolites with potential industrial applications. This study describes the production of chitinase on marine waste, with the subsequent use of the same marine waste for the extraction of antioxidants. A chitinase-producing bacterium isolated from seafood effluent was identified as Alcaligenes faecalis AU02. Optimal chitinase production was obtained in culture conditions of 37°C for 72 h in 100 ml medium containing 1% shrimp and crab shell powder (1:1) (w/v), 0.1% K(2)HPO(4), and 0.05% MgSO(4)·7H(2)O. The molecular weight of chitinase was determined by SDS-PAGE to be 36 kDa. The optimum pH, temperature, pH stability, and thermal stability of chitinase were about 8, 37°C, 5-12, and 40-80°C, respectively. The antioxidant activity of A. faecalis AU02 culture supernatant was determined through scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) as 84%, and the antioxidant compound was characterized by TLC and its FT-IR spectrum. The present study proposed that marine wastes can be utilized to generate a high-value-added product and that pharmacological studies can extend its use to the field of medicine.

  19. Heterotrophic nitrification and aerobic denitrification of high-strength ammonium in anaerobically digested sludge by Alcaligenes faecalis strain No. 4.

    PubMed

    Shoda, Makoto; Ishikawa, Yoichi

    2014-06-01

    Alcaligenes faecalis strain No. 4 which is capable of heterogeneous nitrification and aerobic denitrification, was used to remove high-strength ammonium (approximately 1 g NH4(+)-N/l) from digested sludge, the product of an anaerobic digestion reactor, in which methane was produced from excess municipal sewage sludge. Repeated batch operations were conducted at 20°C and 30°C for 550 h, using a jar fermentor. The removal ratios of high-strength ammonium reached 90-100% within 24 h, and the average ammonium removal rate was 2.9 kg-N/m(3)/day, more than 200 times higher than that in conventional nitrification-denitrification processes. During these operations, the cell density was maintained at 10(8)-10(9) cells of A. faecalis strain No. 4/ml. At 3% NaCl in the digested sludge, strain No. 4 exhibited an ammonium removal rate of 3 kg-N/m(3)/day.

  20. Fed batch bioconversion of 2-propanol by a solvent tolerant strain of Alcaligenes faecalis entrapped in Ca-alginate gel.

    PubMed

    Mohammad, Balsam T; Bustard, Mark T

    2008-07-01

    A gram-negative, rod-shaped, aerobe, capable of converting 2-propanol (isopropanol, IPA) to acetone was isolated from an oil/sump, and identified by 16 S rDNA analysis as Alcaligenes faecalis. Investigations showed this strain to be extremely solvent-tolerant and it was subsequently named ST1. In this study, A. faecalis ST1 cells were immobilized by entrapment in Ca-alginate beads (3 mm in diameter), and used in the bioconversion of high concentration IPA. The biodegradation rates and the corresponding microbial growth inside the beads were measured at four different IPA concentration ranges from 2 to 15 g l(-1). The maximum cell concentration obtained was 9.59 g dry cell weight (DCW) l(-1) medium which equated to 66 g DCW l(-1) gel, at an initial IPA concentration of 15 g l(-1) after 216 h of incubation. A maximum biodegradation rate of 0.067 g IPA g cells(-1) h(-1) was achieved for 5 g l(-1) IPA where an increase in IPA concentration to 38 g l(-1) caused reduction in bead integrity. A modified growth medium was developed which allowed repeated use of the beads for more than 42 days without any loss of integrity and continued bioconversion activity.

  1. Identification of a new Alcaligenes faecalis strain MOR02 and assessment of its toxicity and pathogenicity to insects.

    PubMed

    Quiroz-Castañeda, Rosa Estela; Mendoza-Mejía, Ared; Obregón-Barboza, Verónica; Martínez-Ocampo, Fernando; Hernández-Mendoza, Armando; Martínez-Garduño, Felipe; Guillén-Solís, Gabriel; Sánchez-Rodríguez, Federico; Peña-Chora, Guadalupe; Ortíz-Hernández, Laura; Gaytán-Colín, Paul; Dantán-González, Edgar

    2015-01-01

    We report the isolation of a bacterium from Galleria mellonella larva and its identification using genome sequencing and phylogenomic analysis. This bacterium was named Alcaligenes faecalis strain MOR02. Microscopic analyses revealed that the bacteria are located in the esophagus and intestine of the nematodes Steinernema feltiae, S. carpocapsae, and H. bacteriophora. Using G. mellonella larvae as a model, when the larvae were injected with 24,000 CFU in their hemocoel, more than 96% mortality was achieved after 24 h. Additionally, toxicity assays determined that 1 μg of supernatant extract from A. faecalis MOR02 killed more than 70% G. mellonella larvae 96 h after injection. A correlation of experimental data with sequence genome analyses was also performed. We discovered genes that encode proteins and enzymes that are related to pathogenicity, toxicity, and host/environment interactions that may be responsible for the observed phenotypic characteristics. Our data demonstrates that the bacteria are able to use different strategies to colonize nematodes and kill insects to their own benefit. However, there remains an extensive group of unidentified microorganisms that could be participating in the infection process. Additionally, a nematode-bacterium association could be established probably as a strategy of dispersion and colonization.

  2. [Evaluation of occurrence of Alcaligenes faecalis in clinical samples of patients of the university hospital in Bydgoszcz].

    PubMed

    Jachna-Sawicka, Katarzyna; Gospodarek, Eugenia

    2009-01-01

    Alcaligenes faecalis is an aerobic Gram-negative, non-fermentative rod. It's saprophyte of water and soil. It may be recovered from wet places of hospital environment. It is considered as an opportunistic pathogen. The aim of this review was evaluation of occurrence in clinical samples and susceptibility to antibiotics of 72 A. faecalis strains isolated in years 2003-2008. Over 30% of strains were isolated from patients in surgical ward, 19.6% from patients in outpatient clinic and almost 14% from patients in Department of Dermatology. 70.8% of strains were isolated from purulent material samples, whereas from urine--16.7% of strains. Nearly 88% out of examined strains were grown in mixed culture together with one (26.4%), two (32.0%), three (23.6%) or four (5.6%) microorganisms. All out of strains were sensitive to piperacyline, piperacyline/tazobactam and carbapenems. Sensitivity to aztreonam was observed at 22.2% of strains and to co-trimoxazole at 57.1% of strains.

  3. Genetic diversity and horizontal transfer of genes involved in oxidation of reduced phosphorus compounds by Alcaligenes faecalis WM2072.

    PubMed

    Wilson, Marlena M; Metcalf, William W

    2005-01-01

    Enrichment was performed to isolate organisms that could utilize reduced phosphorus compounds as their sole phosphorus sources. One isolate that grew well with either hypophosphite or phosphite was identified by 16S rRNA gene analysis as a strain of Alcaligenes faecalis. The genes required for oxidation of hypophosphite and phosphite by this organism were identified by using transposon mutagenesis and include homologs of the ptxD and htxA genes of Pseudomonas stutzeri WM88, which encode an NAD-dependent phosphite dehydrogenase (PtxD) and 2-oxoglutarate-dependent hypophosphite dioxygenase (HtxA). This organism also has the htxB, htxC, and htxD genes that comprise an ABC-type transporter, presumably for hypophosphite and phosphite transport. The role of these genes in reduced phosphorus metabolism was confirmed by analyzing the growth of mutants in which these genes were deleted. Sequencing data showed that htxA, htxB, htxC, and htxD are virtually identical to their homologs in P. stutzeri at the DNA level, indicating that horizontal gene transfer occurred. However, A. faecalis ptxD is very different from its P. stutzeri homolog and represents a new ptxD lineage. Therefore, this gene has ancient evolutionary roots in bacteria. These data suggest that there is strong evolutionary selection for the ability of microorganisms to oxidize hypophosphite and phosphite.

  4. Biocatalytic synthesis of (R)-(-)-mandelic acid from racemic mandelonitrile by cetyltrimethylammonium bromide-permeabilized cells of Alcaligenes faecalis ECU0401.

    PubMed

    He, Yu-Cai; Zhang, Zhi-Jun; Xu, Jian-He; Liu, You-Yan

    2010-07-01

    The nitrilase from Alcaligenes faecalis ECU0401 belongs to the category of arylacetonitrilase, which could hydrolyze 2-chloromandelonitrile, 3,4-dimethoxyphenylacetonitrile, mandelonitrile, and phenylacetonitrile into the corresponding arylacetic acids. To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Alcaligenes faecalis ECU0401 in relation to nitrilase activity was optimized by using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. The nitrilase activity from Alcaligenes faecalis ECU0401 increased 4.5-fold when the cells were permeabilized with 0.3% (w/v) CTAB for 20 min at 25 degrees C and pH 6.5. Consequently, almost all the mandelonitrile was consumed and converted to (R)-(-)-mandelic acid with greater than 99.9% enantiomeric excess (e.e.) by the CTAB-permeabilized cells. The permeability barrier has been significantly reduced in the hydrolysis of mandelonitrile by using CTAB-permeabilized cells and a dynamic resolution was successfully achieved, giving a 100% theoretical yield of (R)-(-)-mandelic acid. Efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 3.82 g (R)-(-)-mandelic acid g(-1) dry cell weight (dcw) cell after 20 cycles of repeated use.

  5. Biochemical and histochemical analyses revealing endophytic Alcaligenes faecalis mediated suppression of oxidative stress in Abelmoschus esculentus challenged with Sclerotium rolfsii.

    PubMed

    Ray, Shatrupa; Singh, Vivek; Singh, Surendra; Sarma, Birinchi Kumar; Singh, Harikesh Bahadur

    2016-12-01

    Sclerotium rolfsii is a highly aggressive pathogen that causes huge economic losses, especially in temperate climates. Alcaligenes faecalis, particularly in endophytic form, has rarely been used to control this fungus. In this study, endophytic Alcaligenes sp. strain BHU 12, BHU 16 (isolated from Abelmoschus esculentus leaf) and BHU M7 (isolated from Andrographis paniculata leaf) were reported to trigger a wide range of host defenses in Okra plant against the collar-rot pathogen S. rolfsii. Endophytic colonization of the strains in ten days old plants was assessed through re-isolation of the rif-tagged strains on rifampicin augmented nutrient agar media. The ability of the endophytic strains to induce systemic defense responses in above-ground organs was assessed by collecting leaf tissues of the Okra plants grown under non-gnotobiotic conditions at different time intervals post seedling bacterization with the endophytic biocontrol agents. The pathogen challenged unprimed plants exhibited flaccidity of the stem and leaves at 48 h post infection (hpi) in contrast to the bioprimed and challenged plants. Biochemical and histochemical analyses explained the above phenomenon as activation of phyto-peroxidases leading to an increased metabolism of the reactive oxygen species (ROS), accompanied by activation of the phenylpropanoid network and a subsequent enhancement in plant phenolics. Interestingly, though the maximum increase in the defense pathways was observed in treatments with native endophytes of Okra plant, yet the enhancement in antioxidant pathway due to A. paniculata borne endophytes was also quite significant. Thus, this work clearly demonstrates how Okra plants respond to the "non-hostile" colonization of bacterial endophytes and how induced defense response can contribute to the biocontrol activity of the endophytic strains.

  6. Determination of the active sites serine of the poly (3-hydroxybutyrate) depolymerases of Pseudomonas lemoignei (PhaZ5) and of Alcaligenes faecalis.

    PubMed

    Shinohe, T; Nojiri, M; Saito, T; Stanislawski, T; Jendrossek, D

    1996-07-15

    Mutational analysis of the poly(3-hydroxybutyrate) (PHB) depolymerase A of Pseudomonas lemoignei and of the poly(3-hydroxybutyrate) depolymerase of Alcaligenes faecalis revealed that S138 (P. lemoignei) and S139 (A. faecalis) are essential for activity. Both serines are part of a strictly conserved pentapeptide sequence which is present in all poly(3-hydroxybutyrate) depolymerases analyzed so far (G-L-S-S(A)-G) and which resembles the lipase box of lipases and other serine hydrolases (G-X-S-X-G). Mutation of another conserved serine, namely S195 (P. lemoignei) and S196 (A. faecalis), resulted in mutant proteins with almost full activity and proved that S195 and S196 are not essential for activity. The results indicate the structural and functional relationship of poly(3-hydroxybutyrate) depolymerases to the family of serine hydrolases.

  7. A comparison of NO and N2O production by the autotrophic nitrifier Nitrosomonas europaea and the heterotrophic nitrifier Alcaligenes faecalis.

    PubMed Central

    Anderson, I C; Poth, M; Homstead, J; Burdige, D

    1993-01-01

    Soil microorganisms are important sources of the nitrogen trace gases NO and N2O for the atmosphere. Present evidence suggests that autotrophic nitrifiers such as Nitrosomonas europaea are the primary producers of NO and N2O in aerobic soils, whereas denitrifiers such as Pseudomonas spp. or Alcaligenes spp. are responsible for most of the NO and N2O emissions from anaerobic soils. It has been shown that Alcaligenes faecalis, a bacterium common in both soil and water, is capable of concomitant heterotrophic nitrification and denitrification. This study was undertaken to determine whether heterotrophic nitrification might be as important a source of NO and N2O as autotrophic nitrification. We compared the responses of N. europaea and A. faecalis to changes in partial O2 pressure (pO2) and to the presence of typical nitrification inhibitors. Maximal production of NO and N2O occurred at low pO2 values in cultures of both N. europaea (pO2, 0.3 kPa) and A. faecalis (pO2, 2 to 4 kPa). With N. europaea most of the NH4+ oxidized was converted to NO2-, with NO and N2O accounting for 2.6 and 1% of the end product, respectively. With A. faecalis maximal production of NO occurred at a pO2 of 2 kPa, and maximal production of N2O occurred at a pO2 of 4 kPa. At these low pO2 values there was net nitrite consumption. Aerobically, A. faecalis produced approximately the same amount of NO but 10-fold more N2O per cell than N. europaea did. Typical nitrification inhibitors were far less effective for reducing emissions of NO and N2O by A. faecalis than for reducing emissions of NO and N2O by N. europaea.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8285659

  8. Enantioselective acylation of β-phenylalanine acid and its derivatives catalyzed by penicillin G acylase from Alcaligenes faecalis.

    PubMed

    Li, Dengchao; Ji, Lilian; Wang, Xinfeng; Wei, Dongzhi

    2013-01-01

    This study developed a simple, efficient method for producing racemic β-phenylalanine acid (BPA) and its derivatives via the enantioselective acylation catalyzed by the penicillin G acylase from Alcaligenes faecalis (Af-PGA). When the reaction was run at 25°C and pH 10 in an aqueous medium containing phenylacetamide and BPA in a molar ratio of 2:1, 8 U/mL enzyme and 0.1 M BPA, the maximum BPA conversion efficiency at 40 min only reached 36.1%, which, however, increased to 42.9% as the pH value and the molar ratio of phenylacetamide to BPA were elevated to 11 and 3:1, respectively. Under the relatively optimum reaction conditions, the maximum conversion efficiencies of BPA derivatives all reached about 50% in a relatively short reaction time (45-90 min). The enantiomeric excess value of product (ee(p)) and enantiomeric excess value of substrate (ee(s)) were all above 98% and 95%, respectively. These results suggest that the method established in this study is practical, effective, and environmentally benign and may be applied to industrial production of enantiomerically pure BPA and its derivatives.

  9. The sulfonated osmolyte N-methyltaurine is dissimilated by Alcaligenes faecalis and by Paracoccus versutus with release of methylamine.

    PubMed

    Weinitschke, Sonja; Denger, Karin; Smits, Theo H M; Hollemeyer, Klaus; Cook, Alasdair M

    2006-04-01

    Selective enrichments yielded bacterial cultures able to utilize the osmolyte N-methyltaurine as sole source of carbon and energy or as sole source of fixed nitrogen for aerobic growth. Strain MT1, which degraded N-methyltaurine as a sole source of carbon concomitantly with growth, was identified as a strain of Alcaligenes faecalis. Stoichiometric amounts of methylamine, whose identity was confirmed by matrix-assisted, laser-desorption ionization time-of-flight mass spectrometry, and of sulfate were released during growth. Inducible N-methyltaurine dehydrogenase, sulfoacetaldehyde acetyltransferase (Xsc) and a sulfite dehydrogenase could be detected. Taurine dehydrogenase was also present and it was hypothesized that taurine dehydrogenase has a substrate range that includes N-methyltaurine. Partial sequences of a tauY-like gene (encoding the putative large component of taurine dehydrogenase) and an xsc gene were obtained by PCR with degenerate primers. Strain N-MT utilized N-methyltaurine as a sole source of fixed nitrogen for growth and could also utilize the compound as sole source of carbon. This bacterium was identified as a strain of Paracoccus versutus. This organism also expressed inducible (N-methyl)taurine dehydrogenase, Xsc and a sulfite dehydrogenase. The presence of a gene cluster with high identity to a larger cluster from Paracoccus pantotrophus NKNCYSA, which is now known to dissimilate N-methyltaurine via Xsc, allowed most of the overall pathway, including transport and excretion, to be defined. N-Methyltaurine is thus another compound whose catabolism is channelled directly through sulfoacetaldehyde.

  10. New molecular packing in a crystal of pseudoazurin from Alcaligenes faecalis: a double-helical arrangement of blue copper.

    PubMed

    Fukuda, Yohta; Mizohata, Eiichi; Inoue, Tsuyoshi

    2017-03-01

    Pseudoazurin from the denitrifying bacterium Alcaligenes faecalis (AfPAz) is a blue copper protein and functions as an electron donor to copper-containing nitrite reductase (CuNIR). Conventionally, AfPAz has been crystallized using highly concentrated ammonium sulfate as a precipitant. Here, a needle-like crystal of AfPAz grown in a solution containing a macromolecular precipitant, polyethylene glycol 8000 (PEG 8000), is reported. The crystal belonged to space group P61, with unit-cell parameters a = b = 68.7, c = 94.2 Å. The structure has been determined and refined at 2.6 Å resolution. The asymmetric unit contained two AfPAz molecules contacting each other on negatively charged surfaces. The molecular packing of the crystal showed a right-handed double-helical arrangement of AfPAz molecules and hence of blue copper sites. This structure provides insight into the excluded-volume effect of PEG and the manner of assembly of AfPAz.

  11. Efficient cascade synthesis of ampicillin from penicillin G potassium salt using wild and mutant penicillin G acylase from Alcaligenes faecalis.

    PubMed

    Deng, Senwen; Ma, Xiaoqiang; Su, Erzheng; Wei, Dongzhi

    2016-02-10

    To avoid isolation and purification of the intermediate 6-aminopenicillanic acid (6-APA), a two-enzyme two-step cascade synthesis of ampicillin from penicillin G was established. In purely aqueous medium, penicillin G hydrolysis and ampicillin synthesis were catalyzed by immobilized wild-type and mutagenized penicillin G acylases from Alcaligenes faecalis (Af PGA), respectively (Fig. 1). The βF24 G mutant Af PGA (the 24th Phenylalanine of the β-subunit was replaced by Glycine) was employed for its superior performance in enzymatic synthesis of ampicillin. By optimizing the reaction conditions, including enzyme loading, temperature, initial pH and D-PGME/6-APA ratio, the conversion of the second step of ampicillin synthesis reached approximately 90% in 240 min and less than 1.7 mole D-PGME were required to produce 1 mole ampicillin. Overall, in a 285 min continuous two-step procedure, an ampicillin yield of 87% was achieved, demonstrating the possibility of improving the cascade synthesis of ampicillin by mutagenized PGA, providing an economically efficient and environmentally benign procedure for semi-synthetic penicillins antibiotics synthesis.

  12. Arsenite oxidation by immobilized cells of Alcaligenes faecalis strain O1201 in a fluidized-bed reactor.

    PubMed

    Wang, Yi-Tin; Suttigarn, Arthon; Dastidar, Aniruddha

    2009-02-01

    Arsenic(III) oxidation was evaluated in a continuous-flow fluidized-bed reactor (FBR) with Alcaligenes faecalis strain 01201 immobilized in gel beads. The FBR was operated under 300 mg/L citrate and a range of influent As(III) concentrations (75 to 3000 mg/L) at short hydraulic retention times (1.06 to 3.17 hours). The pH and temperature in the FBR were maintained at optimal growth conditions for strain O1201 (pH 7 and 30 degrees C) throughout the study. A total of 10 quasi-steady-state operating conditions were obtained after 54 days of operation under an As(III) concentration of 441 mg/L (10 000 mg/L/d loading rate), with As (III) removal efficiency ranging from 76% to near complete. Material balance analysis over the FBR revealed that the difference between the cumulative influent As (III) and the sum of cumulative effluent As(III) and As(V) was insignificant. The major mechanism of As(III) removal from the FBR is biological oxidation to As(V).

  13. Growth and siderophores production in Alcaligenes faecalis is regulated by metal ions.

    PubMed

    Sayyed, R Z; Chincholkar, S B

    2010-06-01

    During stationary phase of growth under low stress of iron in succinic acid medium, Alcaligenes feacalis BCCM ID 2374 produced microbial iron chelators. Increase in iron concentration supported bacterial growth but suppressed siderophores production, 1 μM and 2 μM of iron was optimum for maximum siderophore yield, i.e. 354 and 360 μg/ml in untreated and deferrated medium, respectively. Threshold level of iron, which suppressed siderophores production in A. feacalis BCCM ID 2374, was 20 μM. Ten micromoles and above concentration of CuCl(2) and CoCl(2), and 20 μM of MgCl(2), MgSO(4), ZnCl(2) and ZnSO(4) severely affected siderophores production.

  14. Biodegradation of organochlorine pesticide endosulfan by bacterial strain Alcaligenes faecalis JBW4.

    PubMed

    Kong, Lingfen; Zhu, Shaoyuan; Zhu, Lusheng; Xie, Hui; Su, Kunchang; Yan, Tongxiang; Wang, Jun; Wang, Jinhua; Wang, Fenghua; Sun, Fengxia

    2013-11-01

    The recently discovered endosulfan-degrading bacterial strain Alcaligenesfaecalis JBW4 was isolated from activated sludge. This strain is able to use endosulfan as a carbon and energy source. The optimal conditions for the growth of strain JBW4 and for biodegradation by this strain were identified, and the metabolic products of endosulfan degradation were studied in detail. The maximum level of endosulfan biodegradation by strain JBW4 was obtained using broth at an initial pH of 7.0, an incubation temperature of 40 degreeC and an endosulfan concentration of 100 mg/L. The concentration of endosulfan was determined by gas chromatography. Strain JBW4 was able to degrade 87.5% of alpha-endosulfan and 83.9% of beta-endosulfan within 5 days. These degradation rates are much higher than the previously reported bacterial strains. Endosulfan diol and endosulfan lactone were the major metabolites detected by gas chromatography-mass spectrometry; endosulfan sulfate, which is a persistent and toxic metabolite, was not detected. These results suggested that A. faecalis JBW4 degrades endosulfan via a non-oxidative pathway. The biodegradation of endosulfan by A. faecalis is reported for the first time. Additionally, the present study indicates that strain JBW4 may have potential for the biodegradation of endosulfan residues.

  15. Gene cloning, expression, and characterization of a nitrilase from Alcaligenes faecalis ZJUTB10.

    PubMed

    Liu, Zhi-Qiang; Dong, Li-Zhu; Cheng, Feng; Xue, Ya-Ping; Wang, Yuan-Shan; Ding, Jie-Nv; Zheng, Yu-Guo; Shen, Yin-Chu

    2011-11-09

    Nitrilases are important industrial enzymes that convert nitriles directly into the corresponding carboxylic acids. In the current work, the fragment with a length of 1068 bp that encodes the A. faecalis ZJUTB10 nitrilase was obtained. Moreover, a catalytic triad was proposed and verified by site-directed mutagenesis, and the detailed mechanism of this nitrilase was clarified. The substrate specificity study demonstrated that the A. faecalis ZJUTB10 nitrilase belongs to the family of arylacetonitrilases. The optimum pH and temperature for the purified nitrilase was 7-8 and 40 °C, respectively. Mg(2+) stimulated hydrolytic activity, whereas Cu(2+), Co(2+), Ni(2+), Ag(+), and Hg(2+) showed a strong inhibitory effect. The K(m) and v(max) for mandelonitrile were 4.74 mM and 15.85 μmol min(-1) mg(-1) protein, respectively. After 30 min reaction using the nitrilase, mandelonitrile at the concentration of 20 mM was completely hydrolyzed and the enantiomeric excess against (R)-(-)-mandelic acid was >99%. Characteristics investigation indicates that this nitrilase is promising in catalysis applications.

  16. Kerstersia gyiorum gen. nov., sp. nov., a novel Alcaligenes faecalis-like organism isolated from human clinical samples, and reclassification of Alcaligenes denitrificans Rüger and Tan 1983 as Achromobacter denitrificans comb. nov.

    PubMed

    Coenye, Tom; Vancanneyt, Marc; Cnockaert, Margo C; Falsen, Enevold; Swings, Jean; Vandamme, Peter

    2003-11-01

    A polyphasic taxonomic study was performed on nine isolates recovered from various human clinical samples. Phenotypically, these isolates resembled Alcaligenes faecalis. Whole-cell protein analysis distinguished two different species, and this was confirmed by DNA-DNA hybridizations. Cellular fatty acid analysis and 16S rDNA sequence analysis indicated that these isolates were related to the genera Alcaligenes, Bordetella, Achromobacter and Pigmentiphaga and belonged to the family Alcaligenaceae. On the basis of the results of this study, the organisms were classified in a novel genus, Kerstersia gen. nov. This genus comprises one species, Kerstersia gyiorum sp. nov. (type strain LMG 5906(T)=API 184-2-84(T)=CCUG 47000(T)), and several unnamed isolates. The DNA G+C content of members of the genus Kerstersia is between 61.5 and 62.9 mol%. On the basis of previously published DNA-DNA hybridization results and data from chemotaxonomic studies, it is proposed that Alcaligenes denitrificans Rüger and Tan 1983 be reclassified as Achromobacter denitrificans comb. nov.

  17. Heterotrophic nitrification by Alcaligenes faecalis: NO sub 2 sup minus , NO sub 3 sup minus , N sub 2 O, and NO production in exponentially growing cultures

    SciTech Connect

    Papen, H.; von Berg, R.; Hinkel, I.; Thoene, B.; Rennenberg, H. )

    1989-08-01

    Heterotrophic nitrification by Alcaligenes faecalis DSM 30030 was not restricted to media containing organic forms of nitrogen. In both peptone-meat extract and defined media with ammonium and citrate as the sole nitrogen and carbon sources, respectively, NO{sub 2}{sup {minus}}, NO{sub 3}{sup {minus}}, NO, and N{sub 2}O were produced under aerobic growth conditions. Heterotrophic nitrification was not attributable to old or dying cell populations. Production of NO{sub 2}{sup {minus}}, NO{sub 3}{sup {minus}}, NO, and N{sub 2}O was detectable shortly after cultures started growth and proceeded exponentially during the logarithmic growth phase. NO{sub 2}{sup {minus}} and NO{sub 3}{sup {minus}} production rates were higher for cultures inoculated in media with pH values below 7 than for those in media at alkaline pH. Neither assimilatory nor dissimilatory nitrate or nitrite reductase activities were detectable in aerobic cultures.

  18. Metabolism of acrylate to {beta}-hydroxypropionate and its role in dimethylsulfoniopropionate lyase induction by a salt marsh sediment bacterium, Alcaligenes faecalis M3A

    SciTech Connect

    Ansede, J.H.; Pellechia, P.J.; Yoch, D.C.

    1999-11-01

    Dimethylsulfoniopropionate (DMSP) is degraded to dimethylsulfide (DMS) and acrylate by the enzyme DMSP lyase. DMS or acrylate can serve as a carbon source for both free-living and endophytic bacteria in the marine environment. In this study, the authors report on the mechanism of DMSP-acrylate metabolism by Alcaligenes faecalis M3A. Suspensions of citrate-grown cells expressed a low level of DMSP lyase activity that could be induced to much higher levels in the presence of DMSP, acrylate, and its metabolic product, {beta}-hydroxypropionate. DMSP was degraded outside the cell, resulting in an extracellular accumulation of acrylate, which in suspensions of citrate-grown cells was then metabolized at a low endogenous rate. The inducible nature of acrylate metabolism was evidenced by both an increase in the rate of its degradation over time and the ability of acrylate-grown cells to metabolize this molecule at about an eight times higher rate than citrate-grown cells. Therefore, acrylate induces both its production (from DMSP) and its degradation by an acrylase enzyme. {sup 1}H and {sup 13}C nuclear magnetic resonance analyses were used to identify the products resulting from [1-{sup 13}C]acrylate metabolism. The results indicated that A.faecalis first metabolized acrylate to {beta}-hydroxypropionate outside the cell, which was followed by its intracellular accumulation and subsequent induction of DMSP lyase activity. In summary, the mechanism of DMSP degradation to acrylate and the subsequent degradation of acrylate to {beta}-hydroxypropionate in the aerobic {beta}-Proteobacterium A.faecalis has been described.

  19. Induction of immune-related gene expression in Ctenopharyngodon idella kidney cells by secondary metabolites from immunostimulatory Alcaligenes faecalis FY-3.

    PubMed

    Wu, Z-F; Liu, G-L; Zhou, Z; Wang, G-X; Xia, L; Liu, J-L

    2012-08-01

    This study was undertaken to isolate active secondary metabolites from immunostimulatory Alcaligenes faecalis FY-3 and evaluate their activities using grass carp Ctenopharyngodon idella kidney (CIK) cells. By applying chromatography techniques and successive recrystallization, three purified metabolites were obtained and identified by spectral data (mass spectrometry and nuclear magnetic resonance) as: (1) phenylacetic acid, (2) p-hydroxyphenylacetylamide and (3) cyclo-(Gly-(L)-Pro). CIK cells were stimulated by different concentrations (1, 10 and 100 μg/ml) of the isolated compounds, and expression of MyD88, IL-1β, TNF-α, type I-IFN and IL-8 genes at different time points (2, 8 and 24 h) post-stimulation was quantified by real-time PCR. The known immunostimulatory agent lipopolysaccharide (LPS) was used as a positive control. To analyse whether these compounds are toxic to the cells, the methyl tetrazolium assay was employed to measure changes in cell viability. The obtained results revealed that transcribing level of MyD88, an important adaptor molecule in toll-like receptor signalling pathway, was augmented remarkably by all the three isolated compounds and LPS as early as 2-h exposure. These compounds also induced gene expression of cytokines such as IL-1β, TNF-α and type I-IFN. Under the experimental conditions, none of the test compounds is toxic to the CIK cells. These findings demonstrate that the immunostimulatory properties of the three metabolites [phenylacetic acid, p-hydroxyphenylacetylamide and cyclo-(Gly-(L)-Pro)] from A. faecalis FY-3 in CIK cells and highlight the potential of using these metabolites as immunostimulants in fish aquaculture.

  20. Improvement of Alcaligenes faecalis nitrilase by gene site saturation mutagenesis and its application in stereospecific biosynthesis of (R)-(-)-mandelic acid.

    PubMed

    Liu, Zhi-Qiang; Zhang, Xin-Hong; Xue, Ya-Ping; Xu, Ming; Zheng, Yu-Guo

    2014-05-21

    Nitrilases have recently received considerable attention as the biocatalysts for stereospecific production of carboxylic acids. To improve the activity, the nitrilase from Alcaligenes faecalis was selected for further modification by the gene site saturation mutagenesis method (GSSM), based on homology modeling and previous reports about mutations. After mutagenesis, the positive mutants were selected using a convenient two-step high-throughput screening method based on product formation and pH indicator combined with the HPLC method. After three rounds of GSSM, Mut3 (Gln196Ser/Ala284Ile) with the highest activity and ability of tolerance to the substrate was selected. As compared to the wild-type A. faecalis nitrilase, Mut3 showed 154% higher specific activity. Mut3 could retain 91.6% of its residual activity after incubation at pH 6.5 for 6 h. In a fed-batch reaction with 800 mM mandelonitrile as the substrate, the cumulative production of (R)-(-)-mandelic acid after 7.5 h of conversion reached 693 mM with an enantiomeric excess of 99%, and the space-time productivity of Mut3 was 21.50-fold higher than that of wild-type nitrilase. The Km, Vmax, and k(cat) of wild-type and Mut3 for mandelonitrile were 20.64 mM, 33.74 μmol mg(-1) min(-1), 24.45 s(-1), and 9.24 mM, 47.68 μmol mg(-1) min(-1), and 34.55 s(-1), respectively. A homology modeling and molecular docking study showed that the diameter of the catalytic tunnel of Mut3 became longer and that the tunnel volume was smaller. These structural changes are proposed to improve the hydrolytic activity and pH stability of Mut3. Mut3 has the potential for industrial applications in the upscale production of (R)-(-)-mandelic acid.

  1. Long-term analysis of diesel fuel consumption in a co-culture of Acinetobacter venetianus, Pseudomonas putida and Alcaligenes faecalis.

    PubMed

    Pepi, Milva; Minacci, Andrea; Di Cello, Francescopaolo; Baldi, Franco; Fani, Renato

    2003-01-01

    The dynamics of a microbial population isolated from superficial waters of Venice Lagoon and the ability to utilise diesel fuel (n-alkanes mixture C12-C28) as the sole carbon and energy source were studied in a long-term reconstruction experiment. The reconstructed microbial population consisted of three bacterial strains belonging to the species Acinetobacter venetianus, Pseudomonas putida, and Alcaligenes faecalis, which were able to oxidise n-alkanes to alkanoates, n-alkanols to alkanoates, or only n-alkanoates, respectively. Three different approaches: plate counting, cell counting by epifluorescence microscopy with DAPI staining, and by fluorescence in situ hybridisation (FISH) by using a probe conjugate with fluoresceine isothiocyanate specifically targeted towards the 16S rRNA of bacteria belonging to the genus Acinetobacter were used to monitor the growth of the bacterial population. The growth of A. venetianus was stimulated by the presence of other strains, suggesting a beneficial interaction. After the first week of growth A. venetianus cells formed aggregates, as confirmed by confocal microscopy (CLSM), which allowed them to be distinguished from free cells. A relationship between cell number and measured areas (microm2) per aggregate was found. Each cell presented an average surface of 1.21 microm2. Each aggregate was formed by a cellular monolayer biofilm consisting of up to several thousands of cells. The A. venetianus aggregates increased in number and size over time, but after two weeks fragmentation events, which had a beneficial effect on the growth of P. putida and A. faecalis, occurred.

  2. Improving the bioremoval of sulfamethoxazole and alleviating cytotoxicity of its biotransformation by laccase producing system under coculture of Pycnoporus sanguineus and Alcaligenes faecalis.

    PubMed

    Li, Xin; Xu, Qiu-Man; Cheng, Jing-Sheng; Yuan, Ying-Jin

    2016-11-01

    The occurrence of sulfamethoxazole (SMX) in aquatic environment is a health concern. The presence of SMX significantly inhibited the laccase activity of Pycnoporus sanguineus with a lower removal efficiency of SMX. Although a laccase system with 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) eliminated 100% SMX within 6h, ABTS might cause an environmental issue. An alternative to SMX elimination is the coculture of Alcaligenes faecalis and P. sanguineus. The SMX removal efficiency at 48h under the coculture with vitamins was higher than that under their pure culture alone, indicating that a coculture was more efficient in eliminating SMX than a pure culture. Only 1% SMX was detected in mycelia, indicating that SMX elimination is achieved primarily through biotransformation rather than adsorption. Laccase production by the coculture effectively inhibited the accumulations of N4-acetyl-SMX and N-hydroxy-SMX and alleviated the cytotoxicity of SMX transformation products. The mixture of SMX and sulfadiazine inhibited their removal efficiency.

  3. Pyruvic oxime dioxygenase from heterotrophic nitrifier Alcaligenes faecalis is a nonheme Fe((II))-dependent enzyme homologous to class II aldolase.

    PubMed

    Tsujino, Shuhei; Uematsu, Chisato; Dohra, Hideo; Fujiwara, Taketomo

    2017-01-06

    Pyruvic oxime dioxygenase (POD), a key enzyme in heterotrophic nitrification, was purified from Alcaligenes faecalis, and the molecular and catalytic characteristics were reexamined. POD was purified as the homotetramer of the subunit whose molecular weight was 30,000. The deduced amino acid sequence of POD was homologous with a class II aldolase that has been regarded as the Zn((II))-dependent enzyme catalyzing aldol reactions. The recombinant protein showed weak POD activity, and was activated by reconstitution with Fe((II)). Affinity and catalytic constants were estimated at 470 μM and 4.69 sec(-1), respectively. The POD was inactivated by EDTA to remove bound divalent metal cations. A reconstitution experiment demonstrated that Fe((II)), not Zn((II)), is essential for POD activity and that Mn((II)) could partially fulfill the function of Fe((II)). A mutant POD with replacement of His(183), corresponding to one of three Zn((II))-binding ligands in the class II aldolase, by Asn was purified as a homotetrameric protein but showed no catalytic activities. Those results suggest that the POD is homologous to class II aldolase having non-heme Fe((II)) as a catalytic center instead of Zn((II)). A possible mechanism of the POD reaction is discussed on the basis of that of a known Fe((II))-dependent dioxygenase.

  4. The structures of Alcaligenes faecalis D-3-hydroxybutyrate dehydrogenase before and after NAD+ and acetate binding suggest a dynamical reaction mechanism as a member of the SDR family.

    PubMed

    Hoque, Md Mominul; Shimizu, Satoru; Hossain, Md Tofazzal; Yamamoto, Tamotsu; Imamura, Shigeyuki; Suzuki, Kaoru; Tsunoda, Masaru; Amano, Hitoshi; Sekiguchi, Takeshi; Takénaka, Akio

    2008-05-01

    D-3-Hydroxybutyrate dehydrogenase, which catalyzes the reversible reaction between D-3-hydroxybutyrate and acetoacetate, has been classified into the short-chain dehydrogenase/reductase family and is a useful marker in the assay of diabetes mellitus and/or ketoacidosis. The enzyme from Alcaligenes faecalis was crystallized in the apo form and in the holo form with acetate as a substrate analogue. The crystal structures of both forms were determined at 2.2 angstroms resolution. The enzyme is a tetramer composed of four subunits assembled with noncrystallographic 222 point symmetry. Each subunit has two domains. The principal domain adopts the Rossmann fold essential for nucleotide binding, which is a common feature of the SDR family. NAD+ is bound in a large cleft in the domain. The pyrophosphate group of NAD+ is covered by the small additional domain, which is supported by two extended arms allowing domain movement. In the catalytic site, a water molecule is trapped by the catalytic Tyr155 and Ser142 residues in the vicinity of the bound NAD+ and acetate. The substrate analogue acetate is bound above the nicotinamide plane. A substrate (D-3-hydroxybutylate) bound model can reasonably be constructed by adding two C atoms into the void space between the water O atom and the methyl group of the acetate, suggesting a substrate-bound state before enzymatic reaction occurs. Based on these structural features, a reaction mechanism has been proposed.

  5. Pyruvic oxime dioxygenase from heterotrophic nitrifier Alcaligenes faecalis is a nonheme Fe(II)-dependent enzyme homologous to class II aldolase

    PubMed Central

    Tsujino, Shuhei; Uematsu, Chisato; Dohra, Hideo; Fujiwara, Taketomo

    2017-01-01

    Pyruvic oxime dioxygenase (POD), a key enzyme in heterotrophic nitrification, was purified from Alcaligenes faecalis, and the molecular and catalytic characteristics were reexamined. POD was purified as the homotetramer of the subunit whose molecular weight was 30,000. The deduced amino acid sequence of POD was homologous with a class II aldolase that has been regarded as the Zn(II)-dependent enzyme catalyzing aldol reactions. The recombinant protein showed weak POD activity, and was activated by reconstitution with Fe(II). Affinity and catalytic constants were estimated at 470 μM and 4.69 sec−1, respectively. The POD was inactivated by EDTA to remove bound divalent metal cations. A reconstitution experiment demonstrated that Fe(II), not Zn(II), is essential for POD activity and that Mn(II) could partially fulfill the function of Fe(II). A mutant POD with replacement of His183, corresponding to one of three Zn(II)-binding ligands in the class II aldolase, by Asn was purified as a homotetrameric protein but showed no catalytic activities. Those results suggest that the POD is homologous to class II aldolase having non-heme Fe(II) as a catalytic center instead of Zn(II). A possible mechanism of the POD reaction is discussed on the basis of that of a known Fe(II)-dependent dioxygenase. PMID:28059164

  6. Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis. Cofactor assembly and catalytic properties of recombinant enzyme expressed in Paracoccus denitrificans.

    PubMed

    Hothi, Parvinder; Khadra, Khalid Abu; Combe, Jonathan P; Leys, David; Scrutton, Nigel S

    2005-11-01

    The heterologous expression of tryptophan trytophylquinone (TTQ)-dependent aromatic amine dehydrogenase (AADH) has been achieved in Paracoccus denitrificans. The aauBEDA genes and orf-2 from the aromatic amine utilization (aau) gene cluster of Alcaligenes faecalis were placed under the regulatory control of the mauF promoter from P. denitrificans and introduced into P. denitrificans using a broad-host-range vector. The physical, spectroscopic and kinetic properties of the recombinant AADH were indistinguishable from those of the native enzyme isolated from A. faecalis. TTQ biogenesis in recombinant AADH is functional despite the lack of analogues in the cloned aau gene cluster for mauF, mauG, mauL, mauM and mauN that are found in the methylamine utilization (mau) gene cluster of a number of methylotrophic organisms. Steady-state reaction profiles for recombinant AADH as a function of substrate concentration differed between 'fast' (tryptamine) and 'slow' (benzylamine) substrates, owing to a lack of inhibition by benzylamine at high substrate concentrations. A deflated and temperature-dependent kinetic isotope effect indicated that C-H/C-D bond breakage is only partially rate-limiting in steady-state reactions with benzylamine. Stopped-flow studies of the reductive half-reaction of recombinant AADH with benzylamine demonstrated that the KIE is elevated over the value observed in steady-state turnover and is independent of temperature, consistent with (a) previously reported studies with native AADH and (b) breakage of the substrate C-H bond by quantum mechanical tunnelling. The limiting rate constant (k(lim)) for TTQ reduction is controlled by a single ionization with pK(a) value of 6.0, with maximum activity realized in the alkaline region. Two kinetically influential ionizations were identified in plots of k(lim)/K(d) of pK(a) values 7.1 and 9.3, again with the maximum value realized in the alkaline region. The potential origin of these kinetically influential

  7. Relative rates of nitric oxide and nitrous oxide production by nitrifiers, denitrifiers, and nitrate respirers. [Pseudomonas fluorescens; Serratia marcescens; Alcaligenes faecalis

    SciTech Connect

    Anderson, I.C.; Levine, J.S.

    1986-05-01

    The authors investigated the effect of the partial pressure of oxygen (pO/sub 2/) on the production of NO and N/sub 2/O by a wide variety of common soil nitrifying, denitrifying, and nitrate-respiring bacteria under laboratory conditions. The production of NO per cell was highest by autotrophic nitrifiers and was independent of pO/sub 2/ in the range tested (0.5 to 10%), whereas N/sub 2/O production was inversely proportional to pO/sub 2/. Nitrous oxide production was highest in the denitrifier Pseudomonas fluorescens, but only under anaerobic conditions. The molar ratio of NO/N/sub 2/O produced was usually greater than unity for nitrifiers and much less than unity for denitrifiers. Chemodenitrification was the major source of both the NO and N/sub 2/O produced by the nitrate respirer Serratia marcescens. Chemodenitrification was also a possible source of NO and N/sub 2/O produced by the nitrate respirer Serratia marcescens. Chemodenitrification was also a possible source of No and N/sub 2/O in nitrifier cultures but only when high concentrations of nitrite had accumulated or were added to the medium. Although most of the denitrifiers produced NO and N/sub 2/O only under anaerobic conditions, chemostat cultures of Alcaligenes faecalis continued to emit these gases even when the cultures were sprayed with air. Based upon these results, we predict that aerobic soils are primary sources of NO and that N/sub 2/O is produced only when there is sufficient soil moisture to provide the anaerobic microsites necessary for denitrification by either denitrifiers or nitrifiers.

  8. Molecular weight-dependent degradation of D-lactate-containing polyesters by polyhydroxyalkanoate depolymerases from Variovorax sp. C34 and Alcaligenes faecalis T1.

    PubMed

    Sun, Jian; Matsumoto, Ken'ichiro; Tabata, Yuta; Kadoya, Ryosuke; Ooi, Toshihiko; Abe, Hideki; Taguchi, Seiichi

    2015-11-01

    Polyhydroxyalkanoate depolymerase derived from Variovorax sp. C34 (PhaZVs) was identified as the first enzyme that is capable of degrading isotactic P[67 mol% (R)-lactate(LA)-co-(R)-3-hydroxybutyrate(3HB)] [P(D-LA-co-D-3HB)]. This study aimed at analyzing the monomer sequence specificity of PhaZVs for hydrolyzing P(LA-co-3HB) in comparison with a P(3HB) depolymerase from Alcaligenes faecalis T1 (PhaZAf) that did not degrade the same copolymer. Degradation of P(LA-co-3HB) by action of PhaZVs generated dimers, 3HB-3HB, 3HB-LA, LA-3HB, and LA-LA, and the monomers, suggesting that PhaZVs cleaved the linkages between LA and 3HB units and between LA units. To provide a direct evidence for the hydrolysis of these sequences, the synthetic methyl trimers, 3HB-3HB-3HB, LA-LA-3HB, LA-3HB-LA, and 3HB-LA-LA, were treated with the PhaZs. Unexpectedly, not only PhaZVs but also PhaZAf hydrolyzed all of these substrates, namely PhaZAf also cleaved LA-LA linkage. Considering the fact that both PhaZs did not degrade P[(R)-LA] (PDLA) homopolymer, the cleavage capability of LA-LA linkage by PhaZs was supposed to depend on the length of the LA-clustering region in the polymer chain. To test this hypothesis, PDLA oligomers (6 to 40 mer) were subjected to the PhaZ assay, revealing that there was an inverse relationship between molecular weight of the substrates and their hydrolysis efficiency. Moreover, PhaZVs exhibited the degrading activity toward significantly longer PDLA oligomers compared to PhaZAf. Therefore, the cleaving capability of PhaZs used here toward the D-LA-based polymers containing the LA-clustering region was strongly associated with the substrate length, rather than the monomer sequence specificity of the enzyme.

  9. Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid.

    PubMed

    Mishra, Pradeep; Kaur, Suneet; Sharma, Amar Nath; Jolly, Ravinder S

    2016-01-01

    Both the enantiomers of 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid are valuable chiral synthons for enantiospecific synthesis of therapeutic agents such as (S)-doxazosin mesylate, WB 4101, MKC 242, 2,3-dihydro-2-hydroxymethyl-1,4-benzodioxin, and N-[2,4-oxo-1,3-thiazolidin-3-yl]-2,3-dihydro-1,4-benzodioxin-2-carboxamide. Pharmaceutical applications require these enantiomers in optically pure form. However, currently available methods suffer from one drawback or other, such as low efficiency, uncommon and not so easily accessible chiral resolving agent and less than optimal enantiomeric purity. Our interest in finding a biocatalyst for efficient production of enantiomerically pure 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid lead us to discover an amidase activity from Alcaligenes faecalis subsp. parafaecalis, which was able to kinetically resolve 2,3-dihydro-1,4-benzodioxin-2-carboxyamide with E value of >200. Thus, at about 50% conversion, (R)-2,3-dihydro-1,4-benzodioxin-2-carboxylic acid was produced in >99% e.e. The remaining amide had (S)-configuration and 99% e.e. The amide and acid were easily separated by aqueous (alkaline)-organic two phase extraction method. The same amidase was able to catalyse, albeit at much lower rate the hydrolysis of (S)-amide to (S)-acid without loss of e.e. The amidase activity was identified as indole-3-acetamide hydrolase (IaaH). IaaH is known to catalyse conversion of indole-3-acetamide (IAM) to indole-3-acetic acid (IAA), which is phytohormone of auxin class and is widespread among plants and bacteria that inhabit plant rhizosphere. IaaH exhibited high activity for 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which was about 65% compared to its natural substrate, indole-3-acetamide. The natural substrate for IaaH indole-3-acetamide shared, at least in part a similar bicyclic structure with 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which may account for high activity of enzyme towards this un-natural substrate. To the best of

  10. Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid

    PubMed Central

    Mishra, Pradeep; Kaur, Suneet; Sharma, Amar Nath; Jolly, Ravinder S.

    2016-01-01

    Both the enantiomers of 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid are valuable chiral synthons for enantiospecific synthesis of therapeutic agents such as (S)-doxazosin mesylate, WB 4101, MKC 242, 2,3-dihydro-2-hydroxymethyl-1,4-benzodioxin, and N-[2,4-oxo-1,3-thiazolidin-3-yl]-2,3-dihydro-1,4-benzodioxin-2-carboxamide. Pharmaceutical applications require these enantiomers in optically pure form. However, currently available methods suffer from one drawback or other, such as low efficiency, uncommon and not so easily accessible chiral resolving agent and less than optimal enantiomeric purity. Our interest in finding a biocatalyst for efficient production of enantiomerically pure 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid lead us to discover an amidase activity from Alcaligenes faecalis subsp. parafaecalis, which was able to kinetically resolve 2,3-dihydro-1,4-benzodioxin-2-carboxyamide with E value of >200. Thus, at about 50% conversion, (R)-2,3-dihydro-1,4-benzodioxin-2-carboxylic acid was produced in >99% e.e. The remaining amide had (S)-configuration and 99% e.e. The amide and acid were easily separated by aqueous (alkaline)-organic two phase extraction method. The same amidase was able to catalyse, albeit at much lower rate the hydrolysis of (S)-amide to (S)-acid without loss of e.e. The amidase activity was identified as indole-3-acetamide hydrolase (IaaH). IaaH is known to catalyse conversion of indole-3-acetamide (IAM) to indole-3-acetic acid (IAA), which is phytohormone of auxin class and is widespread among plants and bacteria that inhabit plant rhizosphere. IaaH exhibited high activity for 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which was about 65% compared to its natural substrate, indole-3-acetamide. The natural substrate for IaaH indole-3-acetamide shared, at least in part a similar bicyclic structure with 2,3-dihydro-1,4-benzodioxin-2-carboxamide, which may account for high activity of enzyme towards this un-natural substrate. To the best of

  11. Alcaligenes endophyticus sp. nov., isolated from roots of Ammodendron bifolium.

    PubMed

    Lu, Chun-Yan; Li, Yu-Qian; Tian, Ye; Han, Ming-Xian; Narsing Rao, Manik Prabhu; Li, Yan-Ru; Zhu, Zhi-Nan; Wei, Da-Qiao; An, Deng-Di; Li, Wen-Jun

    2016-12-12

    A Gram-staining negative, rod-shaped, motile bacterium, designated AER10T, was isolated from roots of Ammodendron bifolium collected from Takeermohuer desert in Xinjiang Uygur Autonomous Region, north western China. Growth was found to occur from 10°C to 45°C, at pH 5.0-9.0 and could tolerate NaCl up to 10 % (w/v). 16S rRNA gene sequence result indicated that the strain AER10T belongs to the genus Alcaligenes and was closely related to Alcaligenes aquatilis (98.4 %), Alcaligenes faecalis subsp. parafaecalis (98.4 %), Alcaligenes faecalis subsp. faecalis (98.1 %) and Alcaligenes faecalis subsp. phenolicus (97.9 %). However, the DNA-DNA hybridization values between the strain AER10T and the above strains were less than the threshold value (below 70 %) for the delineation of genomic species. The DNA G+C content was 53.3 mol %. Ubiquinone-8 (Q-8) was the only quinone system present. The major fatty acid were summed feature 8 (C18:1ω7c, 25 %), C16:0 (24.2 %), summed feature 3 (C16:1ω7c /C16:1ω6c, 19.3 %) and cyclo-C17:0 (10.5 %). The polar lipid profile of the strain AER10T consist diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), two unidentified aminolipid (AL) and five unknown polar lipids (UL). On the basis of the evidence presented in this study, strain AER10T is a representative of a novel species in the genus Alcaligenes, for which the name Alcaligenes endophyticus sp. nov. is proposed. The type strain is AER10T (=DSM 100498T=KCTC 42688T).

  12. Castellaniella gen. nov., to accommodate the phylogenetic lineage of Alcaligenes defragrans, and proposal of Castellaniella defragrans gen. nov., comb. nov. and Castellaniella denitrificans sp. nov.

    PubMed

    Kämpfer, P; Denger, K; Cook, A M; Lee, S-T; Jäckel, U; Denner, E B M; Busse, H-J

    2006-04-01

    Comparative 16S rRNA gene sequence analysis indicates that two distinct sublineages exist within the genus Alcaligenes: the Alcaligenes faecalis lineage, comprising Alcaligenes aquatilis and A. faecalis (with the three subspecies A. faecalis subsp. faecalis, A. faecalis subsp. parafaecalis and A. faecalis subsp. phenolicus), and the Alcaligenes defragrans lineage, comprising A. defragrans. This phylogenetic discrimination is supported by phenotypic and chemotaxonomic differences. It is proposed that the A. defragrans lineage constitutes a distinct genus, for which the name Castellaniella gen. nov. is proposed. The type strain for Castellaniella defragrans gen. nov., comb. nov. is 54PinT (=CCUG 39790T = CIP 105602T = DSM 12141T). Finally, on the basis of data from the literature and new DNA-DNA hybridization and phenotypic data, the novel species Castellaniella denitrificans sp. nov. (type strain NKNTAUT = DSM 11046T = CCUG 39541T) is proposed for two strains previously identified as strains of A. defragrans.

  13. Specific and sensitive detection of Alcaligenes species from an agricultural environment.

    PubMed

    Nakano, Miyo; Niwa, Masumi; Nishimura, Norihiro

    2013-03-01

    A quantitative real-time PCR assay to specifically detect and quantify the genus Alcaligenes in samples from the agricultural environment, such as vegetables and farming soils, was developed. The minimum detection sensitivity was 106 fg of pure culture DNA, corresponding to DNA extracted from two cells of Alcaligenes faecalis. To evaluate the detection limit of A. faecalis, serially diluted genomic DNA from this organism was mixed with DNA extracted from soil and vegetables and then a standard curve was constructed. It was found that Alcaligenes species are present in the plant phytosphere at levels 10(2)-10(4) times lower than those in soil. The approach presented here will be useful for tracking or quantifying species of the genus Alcaligenes in the agricultural environment.

  14. The characterisation of Bordetella/Alcaligenes-like organisms and their effects on turkey poults and chicks.

    PubMed

    Varley, J

    1986-01-01

    Eight isolates of the Bordetella or Alcaligenes-like organisms associated with turkey rhino-tracheitis were examined. Five of these isolates had been recovered from the United Kingdom and three were foreign isolates. Four of the UK isolates came from flocks with mild respiratory disease. The fifth isolate came from birds with no respiratory signs and this appears to be the first report of the recovery of Bordetella/Alcaligenes from apparently normal turkeys. The field isolates and type strains Alcaligenes faecalis NCTC 415 and Bordetella bronchiseptica NCTC 452 were characterised by biochemical tests, but these did not include any electrophoresis or nucleic acid studies. Cluster analysis using the group average method and the similarly coefficient of Sokal and Sneath indicated that all the strains were distinct from Alcaligenes faecalis but were quite closely related to Bordetella bronchiseptica. Each field isolate was used to infect separate groups of day-old turkey poults and chicks, and each group contained birds which were experimentally infected and others which were in-contact. Observations were made over a 32-day period. In turkey poults, some of the isolates induced severe respiratory disease and mortality, and others very little or none. The UK isolates were less pathogenic than the foreign isolates. It was not possible to correlate the pathogenicity of the isolates for turkey poults with their biochemical characteristics. Chicks infected with two of the eight isolates showed slight respiratory signs, but there was no significant mortality.

  15. Alcaligenes infection in cystic fibrosis.

    PubMed

    Tan, Kenneth; Conway, Steven P; Brownlee, Keith G; Etherington, Christine; Peckham, Daniel G

    2002-08-01

    The aim of this study was to investigate the effect of chronic Alcaligenes species infection of the respiratory tract on the clinical status of patients with cystic fibrosis. We conducted a retrospective case-controlled study. The microbiological records of all patients attending the Leeds Regional Pediatric and Adult Cystic Fibrosis Units from 1992-1999 were examined. Chronic Alcaligenes infection was defined as a positive sputum culture on at least three occasions over a 6-month period. These patients were compared with controls matched for age, gender, respiratory function, and Pseudomonas aeruginosa infection status. Respiratory function tests, anthropometric data, Shwachman-Kulczycki score, Northern chest x-ray score, intravenous and nebulized antibiotic treatment, and corticosteroid treatment were compared from 2 years before to 2 years after Alcaligenes infection. From a clinic population of 557, 13 (2.3%) fulfilled the criteria for chronic infection. The median age at acquisition of infection was 17.2 years (range, 6.5-33.6). There was no significant difference in the changes of percentage predicted values for FEV(1), FVC, FEF(25-75), or Shwachman-Kulczycki and Northern chest x-ray scores, or in weight, height, and body mass index z-scores between Alcaligenes-infected cases and controls. There was also no significant difference in the use of antibiotics (intravenous and nebulized) or corticosteroids (inhaled and oral). We conclude that in our clinic, chronic infection with Alcaligenes species was uncommon. Chronically infected patients showed no excess deterioration in clinical or pulmonary function status from 2 years before to 2 years after primary acquisition.

  16. Development of a PCR-based method for monitoring the status of Alcaligenes species in the agricultural environment.

    PubMed

    Nakano, Miyo; Niwa, Masumi; Nishimura, Norihiro

    2014-01-01

    To analyze the status of the genus Alcaligenes in the agricultural environment, we developed a PCR method for detection of these species from vegetables and farming soil. The selected PCR primers amplified a 107-bp fragment of the 16S rRNA gene in a specific PCR assay with a detection limit of 1.06 pg of pure culture DNA, corresponding to DNA extracted from approximately 23 cells of Alcaligenes faecalis. Meanwhile, PCR primers generated a detectable amount of the amplicon from 2.2×10(2) CFU/ml cell suspensions from the soil. Analysis of vegetable phylloepiphytic and farming soil microbes showed that bacterial species belonging to the genus Alcaligenes were present in the range from 0.9×10(0) CFU per gram (or cm(2)) (Japanese radish: Raphanus sativus var. longipinnatus) to more than 1.1×10(4) CFU/g (broccoli flowers: Brassica oleracea var. italic), while 2.4×10(2) to 4.4×10(3) CFU/g were detected from all soil samples. These results indicated that Alcaligenes species are present in the phytosphere at levels 10-1000 times lower than those in soil. Our approach may be useful for tracking or quantifying species of the genus Alcaligenes in the agricultural environment.

  17. Septic arthritis caused by a gram-negative bacterium representing a new species related to the Bordetella-Alcaligenes complex.

    PubMed

    Kronvall, G; Hanson, H S; von Stedingk, L V; Törnqvist, E; Falsen, E

    2000-03-01

    A knee-joint exudate culture yielded on two occasions a gram-negative bacterium. Regular methods for speciation did not provide an identification. The infection was successfully treated with ciprofloxacin. The unknown isolate, CCUG 36768, was subjected to further investigation, including 16S rDNA sequencing, protein profiling, cellular fatty acid analysis, and various biochemical tests, in order to produce a species identification. The 1469 bp-long 16S rDNA sequence did not reveal identity with any known species sequence. CCUG 36768 clustered in a group of species, including Alcaligenes defragrans, Denitrobacter permanens, Taylorella equigenitalis, Alcaligenes faecalis, and four strains of Alcaligenes species without a specific species name. Bordetella species also showed a high degree of similarity with CCUG 36768. Protein profiling, cellular fatty acid analysis and computer-assisted analysis of biochemical profiles indicated similarity with Bordetella-Alcaligenes species, often close to B. holmesii and B. avium. API 20 NE indicated the profile of Moraxella species of poor identity. It is concluded that CCUG 36768 represents a new bacterial species of pathogenic potential in humans. It is related to the Bordetella-Alcaligenes group. Powerful new methods for speciation are available and it is recommended that unknown isolates from normally sterile sites be submitted for further analysis. Several isolates are required for the definition of new species.

  18. Alcaligenes defragrans sp. nov., description of four strains isolated on alkenoic monoterpenes ((+)-menthene, alpha-pinene, 2-carene, and alpha-phellandrene) and nitrate.

    PubMed

    Foss, S; Heyen, U; Harder, J

    1998-06-01

    Four pseudomonad strains 51Men, 54Pin, 62Car and 65Phen were recently isolated on the monoterpenes (+)-menthene, alpha-pinene, 2-carene and alpha-phellandrene as sole carbon source and nitrate as electron acceptor. These bacteria were characterised. The motile, mesophilic, Gram-negative rods had a strictly respiratory metabolism. Monoterpenes as carbon sources were completely mineralised to carbon dioxide. The physiology of all strains was very similar, but displayed an individual utilisation preference for the isolation substrate. The fatty acid composition of whole cells showed a high degree of similarity to that of Alcaligenes faecalis. Comparative 16S rDNA data analysis placed the isolates into the beta-subclass of Proteobacteria in a common offshoot together with Alcaligenes and Bordetella species. On the basis of these characteristics, the strains are described as a new species belonging to the genus Alcaligenes, A. defragrans sp. nov., with strain 54Pin (DSM 12141T) as type strain.

  19. Alcaligenes aquatilis sp. nov., a novel bacterium from sediments of the Weser Estuary, Germany, and a salt marsh on Shem Creek in Charleston Harbor, USA.

    PubMed

    Van Trappen, Stefanie; Tan, Tjhing-Lok; Samyn, Emly; Vandamme, Peter

    2005-11-01

    Four nitrite-dissimilating strains, isolated from Weser Estuary sediments, were investigated using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these strains belong to the 'Betaproteobacteria' and are related to the genus Alcaligenes. The highest level of sequence similarity (100 %) was found with strain M3A (=ATCC 700596), a dimethyl sulfide-producing marine isolate that was included in this study. DNA-DNA hybridizations between the five strains and related Alcaligenes faecalis strains confirmed that the former belong to a single and novel species within the genus Alcaligenes. The isolates are Gram-negative, motile, rod-shaped cells with a DNA G+C content of about 56 mol%. The whole-cell fatty acid profiles of the isolates were very similar and included C(16 : 0), C(17 : 0) cyclo, C(18 : 1)omega7c, summed feature 2 (comprising any combination of C(12 : 0) aldehyde, an unknown fatty acid of equivalent chain length 10.928, C(16 : 1) iso I and C(14 : 0) 3-OH) and summed feature 3 (C(15 : 0) iso 2-OH and/or C(16 : 1)omega7c) as the major fatty acid components. On the basis of their phylogenetic, genomic and phenotypic properties, the five novel strains can be assigned to the genus Alcaligenes as a novel species, for which the name Alcaligenes aquatilis sp. nov. is proposed. The type strain is LMG 22996T (=CCUG 50924T).

  20. Crystal structure of an electron transfer complex between aromatic amine dehydrogenase and azurin from Alcaligenes faecalis.

    PubMed

    Sukumar, Narayanasami; Chen, Zhi-wei; Ferrari, Davide; Merli, Angelo; Rossi, Gian Luigi; Bellamy, Henry D; Chistoserdov, Andrei; Davidson, Victor L; Mathews, F Scott

    2006-11-14

    The crystal structure of an electron transfer complex of aromatic amine dehydrogenase (AADH) and azurin is presented. Electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of AADH to the type I copper of the cupredoxin azurin. This structure is compared with the complex of the TTQ-containing methylamine dehydrogenase (MADH) and the cupredoxin amicyanin. Despite significant similarities between the two quinoproteins and the two cupredoxins, each is specific for its respective partner and the ionic strength dependence and magnitude of the binding constant for each complex are quite different. The AADH-azurin interface is largely hydrophobic, covering approximately 500 A(2) of surface on each molecule, with one direct hydrogen bond linking them. The closest distance from TTQ to copper is 12.6 A compared with a distance of 9.3 A in the MADH-amicyanin complex. When the MADH-amicyanin complex is aligned with the AADH-azurin complex, the amicyanin lies on top of the azurin but is oriented quite differently. Although the copper atoms differ in position by approximately 4.7 A, the amicyanin bound to MADH appears to be rotated approximately 90 degrees from its aligned position with azurin. Comparison of the structures of the two complexes identifies features of the interface that dictate the specificity of the protein-protein interaction and determine the rate of interprotein electron transfer.

  1. [Influence of nitrogen source NH4 Cl concentration on curdlan production in Alcaligenes faecalis].

    PubMed

    Sun, Yong-Sheng; Wang, Lei; Zhan, Xiao-Bei; Zheng, Zhi-Yong; Chen, Yuan-Zhi

    2005-03-01

    The effect of initial ammonium chloride level on production of curdlan in Alcaligenesfaecalis was investigated. It was found that ammonium chloride was the limiting substrate for cell growth during the batch fermentation process. However, the cell growth and curdlan production could not be enhanced by solely increasing the initial ammonium chloride level. The pH drop in the broth due to the consumption of ammonium chloride also effected the cell growth and curdlan production. By simultaneously increasing the initial ammonium chloride concentration and implementing an optimal pH control strategy, which is to control pH at 7.0 in the growth phase, and then shift to 5.6 in the production phase, the biomass and curdlan production in batch fermentation were increased markedly. If the initial ammonium chloride concentration was increased from 1.1 g/L to 3.6 g/L, biomass concentration of 7.2 g/L was obtained, and the final curdlan concentration reached 30.5 g/L, which was 51.7% higher than that of the former case. As the cell growth was improved due to the increase of the initial ammonium chloride concentration, the agitation speed and aeration rates must be enhanced to suit the higher oxygen uptake requirement. However, as curdlan molecules is subject to the structural breakage due to the high shear stress at higher agitation speed, an overall optimal condition for both productivity and quality of curdlan should be considered comprehensively.

  2. Effect of Tween 80 on the production of curdlan by Alcaligenes faecalis ATCC 31749.

    PubMed

    Xia, Zhenqiang

    2013-10-15

    This study aims to investigate the effects of Tween 80 on curdlan production, cell growth, and glucosyltransferase activity. The addition of Tween 80 to the culture medium increased curdlan production. However, curdlan production did not increase further when excessive Tween 80 (>0.3% Tween 80) was added to the culture medium. The addition of Tween 80 to the culture medium did not affect cell growth. The glucosyltransferase activity involved in the curdlan synthesis increased with the increase of Tween 80 concentration. The glucosyltransferase activity did not increase further when excessive Tween 80 (>0.3% Tween 80) was added to the culture medium. Maximum curdlan was observed at day 5 and then levelled off. The biomass continued to increase until the end of the experimental period (6d). Maximum glucosyltransferase activity was also observed at day 5 and decreased thereafter. The results indicate that the enhanced curdlan production by Tween 80 is highly correlated with glucosyltransferase activity.

  3. High-Level Nickel Resistance in Alcaligenes xylosoxydans 31A and Alcaligenes eutrophus KTO2

    PubMed Central

    Schmidt, Thomas; Stoppel, Ralf-D.; Schlegel, Hans G.

    1991-01-01

    Two new nickel-resistant strains of Alcaligenes species were selected from a large number (about 400) of strains isolated from ecosystems polluted by heavy metals and were studied on the physiological and molecular level. Alcaligenes xylosoxydans 31A is a heterotrophic bacterium, and Alcaligenes eutrophus KTO2 is an autotrophic aerobic hydrogen-oxidizing bacterium. Both strains carry—among other plasmids—a megaplasmid determining resistance to 20 to 50 mM NiCl2 and 20 mM CoCl2 (when growing in defined Tris-buffered media). Megaplasmids pTOM8, pTOM9 from strain 31A, and pGOE2 from strain KTO2 confer nickel resistance to the same degree to transconjugants of all strains of A. eutrophus tested but were not transferred to Escherichia coli. However, DNA fragments carrying the nickel resistance genes, cloned into broad-hostrange vector pVDZ'2, confer resistance to A. eutrophus derivatives as well as E. coli. The DNA fragments of both bacteria, TBA8, TBA9, and GBA (14.5-kb BamHI fragments), appear to be identical. They share equal size, restriction maps, and strong DNA homology but are largely different from fragment HKI of nickel-cobalt resistance plasmid pMOL28 of A. eutrophus CH34. Images PMID:16348590

  4. Genetic Diversity among Enterococcus faecalis

    PubMed Central

    McBride, Shonna M.; Fischetti, Vincent A.; LeBlanc, Donald J.; Moellering, Robert C.; Gilmore, Michael S.

    2007-01-01

    Enterococcus faecalis, a ubiquitous member of mammalian gastrointestinal flora, is a leading cause of nosocomial infections and a growing public health concern. The enterococci responsible for these infections are often resistant to multiple antibiotics and have become notorious for their ability to acquire and disseminate antibiotic resistances. In the current study, we examined genetic relationships among 106 strains of E. faecalis isolated over the past 100 years, including strains identified for their diversity and used historically for serotyping, strains that have been adapted for laboratory use, and isolates from previously described E. faecalis infection outbreaks. This collection also includes isolates first characterized as having novel plasmids, virulence traits, antibiotic resistances, and pathogenicity island (PAI) components. We evaluated variation in factors contributing to pathogenicity, including toxin production, antibiotic resistance, polymorphism in the capsule (cps) operon, pathogenicity island (PAI) gene content, and other accessory factors. This information was correlated with multi-locus sequence typing (MLST) data, which was used to define genetic lineages. Our findings show that virulence and antibiotic resistance traits can be found within many diverse lineages of E. faecalis. However, lineages have emerged that have caused infection outbreaks globally, in which several new antibiotic resistances have entered the species, and in which virulence traits have converged. Comparing genomic hybridization profiles, using a microarray, of strains identified by MLST as spanning the diversity of the species, allowed us to identify the core E. faecalis genome as consisting of an estimated 2057 unique genes. PMID:17611618

  5. Fluoranthene degradation in Pseudomonas alcaligenes PA-10.

    PubMed

    Gordon, L; Dobson, A D

    2001-01-01

    Pseudomonas alcaligenes strain PA-10 degrades the four-ring polycyclic aromatic hydrocarbon fluoranthene, co-metabolically. HPLC analysis of the growth medium identified four intermediates, 9-fluorenone-1-carboxylic acid; 9-hydroxy-1-fluorene carboxylic acid; 9-fluorenone and 9-fluorenol, formed during fluoranthene degradation. Pre-exposure of PA-10 to 9-fluorenone-1-carboxylic acid and 9-hydroxy-1-fluorene-carboxylic acid resulted in increases in fluoranthene removal, while pre-exposure to 9-fluorenone and 9-fluorenol resulted in a decrease in fluoranthene degradation. The rate of indole transformation was similarly affected by pre-exposure to these metabolic intermediates, indicating a link between fluoranthene degradation and indigo formation in this strain.

  6. Dense autotrophic cultures of Alcaligenes eutrophus.

    PubMed Central

    Repaske, R; Mayer, R

    1976-01-01

    Alcaligenes eutrophus was grown autotrophically in 23-liter batch cultures in a controlled H2-O2-CO2 atmosphere. It was demonstrated that the need for periodic supplements of individual nutrients could be anticipated before cell growth depleted these nutrients to the point of becoming growth rate limiting. As a result, exponential growth was extended to optical densities of 44, with doubling times maintained at 2 h. Cultures having an initial optical density of 0.040 to 0.70 reached the final optical density of 60 in about 25 h. The final viable count was 1.2 X 10(11) cells per ml, and the dry weight was 25 g/liter. PMID:10840

  7. Alcaligenes Eutrophus as a Source of Hydrogenase: An Evaluation of Techniques for Its Large Scale Production

    DTIC Science & Technology

    1990-08-01

    block number) -A Alcaligenes eutroplus H16 (AICC 17699), an aerobic H, oxidizing bacteria, has been selected as the most sutable enzyme source for a NAD...laboratory animals (Wistar rats). 2. SELECTION OF Alcaligenes eutrophus AS THE HYDROGENASE SOURCE. 2.1. Hydrogen-oxidizing bacteria: Historical...among strains, and therefore 2 the indMdual species have been placed later under preexisting genera of heterotrophic bacteria - such as Alcaligenes

  8. Targeting Enterococcus faecalis Biofilms with Phage Therapy

    PubMed Central

    Khalifa, Leron; Brosh, Yair; Gelman, Daniel; Coppenhagen-Glazer, Shunit; Beyth, Shaul; Poradosu-Cohen, Ronit; Que, Yok-Ai; Beyth, Nurit

    2015-01-01

    Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment. PMID:25662974

  9. Targeting Enterococcus faecalis biofilms with phage therapy.

    PubMed

    Khalifa, Leron; Brosh, Yair; Gelman, Daniel; Coppenhagen-Glazer, Shunit; Beyth, Shaul; Poradosu-Cohen, Ronit; Que, Yok-Ai; Beyth, Nurit; Hazan, Ronen

    2015-04-01

    Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment.

  10. Characterization of S6K2, a novel kinase homologous to S6K1.

    PubMed

    Lee-Fruman, K K; Kuo, C J; Lippincott, J; Terada, N; Blenis, J

    1999-09-09

    Rapamycin is an immunosuppressant which antagonizes cellular proliferation by inhibiting the function of mTOR. The mTOR:FKBP12: rapamycin complex blocks G1/S transition by inhibiting downstream targets essential for cell cycle progression. One such target is p70S6k1 (S6K1), a serine/threonine kinase which is inactivated by the mTOR : FKBP12 : rapamycin complex, and which has been linked to translational control by virtue of its ability to phosphorylate the ribosomal protein S6. In the current work, we describe cloning and characterization of a novel S6K1 homolog, p54 S6 kinase 2 (p54S6k2/S6K2). Similar to S6K1, S6K2 is activated by mitogens and by constitutively active PI3K, and is inhibited by rapamycin as well as wortmannin. Differences between activation of S6K1 and S6K2 by PDK1 were observed, suggesting potential differences in the regulation of these homologs. Strikingly, S6K2 activity and S6 phosphorylation were both intact in S6K1-/-ES cell, indicating a possible role for S6K2 in in vivo S6 phosphorylation. Interestingly, we found two isoforms of S6K2 which are localized to distinct cellular compartments; the smaller form resides in the detergent-soluble fraction, whereas the larger form is found in the particulate fraction. Our findings demonstrate the existence of a family of rapamycin-sensitive protein kinases potentially involved in S6 phosphorylation, translational control, and transduction of mTOR signals.

  11. Isolation and identification of dexamethasone sodium phosphate degrading Pseudomonas alcaligenes.

    PubMed

    Yi, Wang; Zhibang, Yang; Lili, Zhu; Zhongquan, Shi; Lianju, Ma; Ziwei, Tang; Renju, Jiang

    2015-02-01

    Glucocorticosteroids such as dexamethasone have polluted hospital wastewater, urban sewage, and river water in varying degrees. However, dexamethasone degradation by bioremediation technology is less understood. This study aims to isolate bacteria that could degrade dexamethasone and to identify their degradation characteristics. Hospital wastewater contaminated by dexamethasone was collected. After culturing in inorganic salt medium and in carbon deficient medium containing dexamethasone sodium phosphate, a bacterial strain with dexamethasone sodium phosphate as the sole carbon and energy source was enriched and isolated from the contaminated wastewater. The strain was identified as Pseudomonas alcaligenes by morphology, Gram staining, biochemical test, and 16S rDNA sequencing. Isolated bacteria were domesticated. Then its degradation characteristic was determined by high-performance liquid chromatography method. The degradation rate of P. alcaligenes on dexamethasone sodium phosphate was 50.86%. Of the degraded dexamethasone sodium phosphate, 75.23% of dexamethasone sodium phosphate was degraded to dexamethasone and 23.63% was degraded to other metabolites. In conclusion, the isolated P. alcaligenes in this study would provide experimental evidence for further research on the bioremediation technology to treat dexamethasone sodium phosphate and dexamethasone polluted water and further for the elimination of side effects of dexamethasone.

  12. Genome Sequence of a Strain of the Human Pathogenic Bacterium Pseudomonas alcaligenes That Caused Bloodstream Infection.

    PubMed

    Suzuki, Masato; Suzuki, Satowa; Matsui, Mari; Hiraki, Yoichi; Kawano, Fumio; Shibayama, Keigo

    2013-10-31

    Pseudomonas alcaligenes, a Gram-negative aerobic bacterium, is a rare opportunistic human pathogen. Here, we report the whole-genome sequence of P. alcaligenes strain MRY13-0052, which was isolated from a bloodstream infection in a medical institution in Japan and is resistant to antimicrobial agents, including broad-spectrum cephalosporins and monobactams.

  13. Biodegradation and detoxification of melanoidin from distillery effluent using an aerobic bacterial strain SAG5 of Alcaligenes faecalis.

    PubMed

    Santal, Anita Rani; Singh, N P; Saharan, Baljeet Singh

    2011-10-15

    Distillery effluent retains very dark brown color even after anaerobic treatment due to presence of various water soluble, recalcitrant and coloring compounds mainly melanoidins. In laboratory conditions, melanoidin decolorizing bacteria was isolated and optimized the cultural conditions at various incubation temperatures, pH, carbon sources, nitrogen sources and combined effect of both carbon and nitrogen sources. The optimum decolorization (72.6 ± 0.56%) of melanoidins was achieved at pH 7.5 and temperature 37 °C on 5th day of cultivation. The toxicity evaluation with mung bean (Vigna radiata) revealed that the raw distillery effluent was environmentally highly toxic as compared to biologically treated distillery effluent, which indicated that the effluent after bacterial treatment is environmentally safe. This proves to be novel biological treatment technique for biodegradation and detoxification of melanoidin from distillery effluent using the bacterial strain SAG(5).

  14. Transcriptional response of Enterococcus faecalis to sunlight.

    PubMed

    Sassoubre, Lauren M; Ramsey, Matthew M; Gilmore, Michael S; Boehm, Alexandria B

    2014-01-05

    Microarrays were used to investigate the transcriptional response of Enterococcus faecalis to photostress. E. faecalis are Gram-positive bacteria used as indicators of water quality and have been shown to vary diurnally in response to sunlight. E. faecalis in filtered seawater microcosms were exposed to artificial sunlight for 12h and then placed in the dark for 12h. Transcript abundance was measured at 0, 2, 6, 12, and 24h in the sunlit microcosm and a dark control using microarrays. Culturable E. faecalis concentrations decreased 6-7 orders of magnitude within the first 6h of light exposure. After 12h in the dark, no evidence of dark-repair was observed. Expression data collected after 12h of sunlight exposure revealed a difference in transcript abundance in the light relative to dark microcosms for 35 unique ORFs, 33 ORFs showed increased transcript abundance and 2 ORFs showed reduced transcript abundance. A majority (51%) of the ORFs with increased transcript abundance in the sunlit relative to dark microcosms encoded hypothetical proteins; others were associated with protein synthesis, oxidative stress and DNA repair. Results suggest that E. faecalis exposed to sunlight actively transcribe RNA in response to photostress.

  15. Characterization of monolaurin resistance in Enterococcus faecalis.

    PubMed

    Dufour, Muriel; Manson, Janet M; Bremer, Philip J; Dufour, Jean-Pierre; Cook, Gregory M; Simmonds, Robin S

    2007-09-01

    There is increasing concern regarding the presence of vancomycin-resistant enterococci in domestically farmed animals, which may act as reservoirs and vehicles of transmission for drug-resistant enterococci to humans, resulting in serious infections. In order to assess the potential for the use of monolaurin as a food preservative, it is important to understand both its target and potential mechanisms of resistance. A Tn917 mutant library of Enterococcus faecalis AR01/DGVS was screened for resistance (MIC, >100 microg/ml) to monolaurin. Three mutants were identified as resistant to monolaurin and were designated DGRM2, DGRM5, and DGRM12. The gene interrupted in all three mutants was identified as traB, which encodes an E. faecalis pheromone shutdown protein and whose complementation in trans restored monolaurin sensitivity in all three mutants. DGRM2 was selected for further characterization. E. faecalis DGRM2 showed increased resistance to gentamicin and chloramphenicol (inhibitors of protein synthesis), while no difference in the MIC was observed with the cell wall-active antibiotics penicillin and vancomycin. E. faecalis AR01/DGVS and DGRM2 were shown to have similar rates (30% cell lysis after 4 h) of cell autolytic activity when activated by monolaurin. Differences in cell surface hydrophobicity were observed between the wild type and the mutant, with the cell surface of the parent strain being significantly more hydrophobic. Analysis of the cell wall structure of DGRM2 by transmission electron microscopy revealed an increase in the apparent cell wall thickness and contraction of its cytoplasm. Taken together, these results suggest that the increased resistance of DGRM2 was due to a change in cell surface hydrophobicity, consequently limiting the diffusion of monolaurin to a potential target in the cytoplasmic membrane and/or cytoplasm of E. faecalis.

  16. Alcaligenes xylosoxidans endophthalmitis following phacoemulsification and intraocular lens implantation.

    PubMed

    Robert, Pierre-Yves; Chainier, Delphine; Garnier, Fabien; Ploy, Marie-Cécile; Parneix, Pierre; Adenis, Jean-Paul; Martin, Christian

    2008-01-01

    Five consecutive cases of endophthalmitis that developed after cataract extraction by a single surgeon using the same operating room during one morning session are described. Following preoperative topical administration of ciprofloxacin, surgery consisted of phacoemulsification with peristaltic pump and fluid venting, polymethylmethacrylate intraocular lens implantation, and corneal suture. No complications occurred during surgery. All five patients developed endophthalmitis caused by infection with Alcaligenes xylosoxidans in less than 24 hours. Pulsed-field gel electrophoresis was used to prove similarity between strains. Bacterial inquiry on contamination of the operating room environment revealed massive colonization of phacoemulsifier irrigation channels by Pseudomonas fluorescens bacteria from an unestablished source. Four of the five patients ultimately recovered visual acuity better than 20/60.

  17. Molecular and genetic characterization of an Alcaligenes eutrophus insertion element.

    PubMed Central

    Kung, S S; Chen, J; Chow, W Y

    1992-01-01

    An insertion element, ISAE1, was discovered during the molecular analysis of mutants defective in the autotrophic growth (Aut-) of Alcaligenes eutrophus H1-4, a mitomycin C-generated derivative of strain H1. ISAE1 is 1,313 bp long, has 12-bp nearly perfect inverted terminal repeats, and contains an open reading frame that has a coding capacity of 408 amino acids. Direct repeats of 8 bp were generated by insertion of ISAE1 into chromosomes or plasmids. Most insertion were found in the AT-rich target sites. The distribution of ISAE1 is limited to A. eutrophus H1 (ATCC 17698) and H16 (ATCC 17699). Variants with newly transposed copies of ISAE1 could be isolated at an elevated frequency by changing the growth conditions. Images PMID:1334068

  18. [Investigation of Enterococcus faecalis antimicrobial resistance].

    PubMed

    Casal, M M; Cause, M; Solís, F; Rodríz, F; Casal, M

    2009-09-01

    We performed an antibiotic resistance study on Enterococcus faecalis isolated from intrahospitalary and extrahospitalary samples between january 2004 and january 2008. Three different samples were studied; urine, blood and wound swabs, considering a strain per patient. We included in the study a global amount of 3,641 Enterococcus faecalis isolations from clinical samples received at Hospital Universitario Reina Sofía microbiology service in Córdoba (Spain). We employed semiautomatic system WIDER I (Soria Melguizo) for identification and sensibility testing. We considered sensibility and resistance criteria recommended by MENSURA group. We found a sensitivity rate of 98.04% to betalactamics.The highest resistance rates were obtained with aminoglycosides, between 33.82% and 48.01%. Linezolid and Vancomycin sensitivity was 100%. It seems that vancomycin resistance is not a worrying issue today, but it should be controlled.

  19. Development of a lipase fermentation process that uses a recombinant Pseudomonas alcaligenes strain.

    PubMed

    Gerritse, G; Hommes, R W; Quax, W J

    1998-07-01

    Pseudomonas alcaligenes M-1 secretes an alkaline lipase, which has excellent characteristics for the removal of fatty stains under modern washing conditions. A fed-batch fermentation process based on the secretion of the alkaline lipase from P. alcaligenes was developed. Due to the inability of P. alcaligenes to grow on glucose, citric acid and soybean oil were applied as substrates in the batch phase and feed phase, respectively. The gene encoding the high-alkaline lipase from P. alcaligenes was isolated and characterized. Amplification of lipase gene copies in P. alcaligenes with the aid of low- and high-copy-number plasmids resulted in an increase of lipase expression that was apparently colinear with the gene copy number. It was found that overexpression of the lipase helper gene, lipB, produced a stimulating effect in strains with high copy numbers (> 20) of the lipase structural gene, lipA. In strains with lipA on a low-copy-number vector, the lipB gene did not show any effect, suggesting that LipB is required in a low ratio to LipA only. During scaling up of the fermentation process to 100 m3, severe losses in lipase productivity were observed. Simulations have identified an increased level of dissolved carbon dioxide as the most probable cause for the scale-up losses. A large-scale fermentation protocol with a reduced dissolved carbon dioxide concentration resulted in a substantial elimination of the scale-up loss.

  20. Genome Sequence of Enterococcus faecalis Strain CG_E.

    PubMed

    Gabris, Christina; Poehlein, Anja; Bengelsdorf, Frank R; Daniel, Rolf; Dürre, Peter

    2017-01-12

    Enterococcus faecalis CG_E is a Gram-positive, lactic acid-producing coccus. The draft genome of E. faecalis strain CG_E comprises 2,969,881 bp and exhibits a G+C content of 37.34%. The genome encodes 2,848 predicted protein-encoding and 97 RNA genes.

  1. Enterococcus faecalis promotes osteoclastogenesis and semaphorin 4D expression.

    PubMed

    Wang, Shuai; Deng, Zuhui; Seneviratne, Chaminda J; Cheung, Gary S P; Jin, Lijian; Zhao, Baohong; Zhang, Chengfei

    2015-10-01

    Enterococcus faecalis is considered a major bacterial pathogen implicated in endodontic infections and contributes considerably to periapical periodontitis. This study aimed to investigate the potential mechanisms by which E. faecalis accounts for the bone destruction in periapical periodontitis in vitro. Osteoclast precursor RAW264.7 cells were treated with E. faecalis ATCC 29212 and a wild strain of E. faecalis derived clinically from an infected root canal. The results showed that, to some extent, E. faecalis induced the RAW264.7 cells to form tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclast-like cells. This pathogen markedly stimulated RAW264.7 cells to express semaphorin 4D (Sema4D), which inhibits bone formation. Once RAW264.7 cells were primed by low-dose receptor activator of nuclear factor-kappa B ligand (RANKL), E. faecalis could significantly increase the production of TRAP-positive multinucleated cells and up-regulate the expression of osteoclast-specific markers, including NFATc1, TRAP and cathepsin K. Both p38 and ERK1/2 MAPK signaling pathways were activated by E. faecalis in RANKL-primed RAW264.7 cells, and meanwhile the expression of Sema4D was highly increased. In conclusion, E. faecalis may greatly contribute to the bone resorption in periapical periodontitis by promoting RANKL-dependent osteoclastogenesis and expression of Sema4D through activation of p38 and ERK1/2 MAPK signaling pathways.

  2. Genome Sequence of Enterococcus faecalis Strain CG_E

    PubMed Central

    Gabris, Christina; Daniel, Rolf

    2017-01-01

    ABSTRACT Enterococcus faecalis CG_E is a Gram-positive, lactic acid-producing coccus. The draft genome of E. faecalis strain CG_E comprises 2,969,881 bp and exhibits a G+C content of 37.34%. The genome encodes 2,848 predicted protein-encoding and 97 RNA genes. PMID:28082508

  3. Pheromone-inducible conjugation in Enterococcus faecalis

    PubMed Central

    Kozlowicz, Briana K.; Dworkin, Martin; Dunny, Gary M.

    2009-01-01

    Pheromone-inducible transfer of the plasmid pCF10 in Enterococcus faecalis is regulated using a complicated network of proteins and RNAs. The plasmid itself has been assembled from parts garnered from a variety of sources, and many aspects of the system resemble a biological kluge. Recently several new functions of various pCF10 gene products that participate in regulation of plasmid transfer have been identified. The results indicate that selective pressures controlling the evolution of the plasmid have produced a highly complex regulatory network with multiple biological functions that may serve well as a model for the evolution of biological complexity. PMID:16503196

  4. Comparison of aspartate transcarbamoylase regulation in Pseudomonas alcaligenes and Pseudomonas mendocina.

    PubMed

    Santiago, Manuel F; West, Thomas P

    2003-01-01

    The regulation of aspartate transcarbamoylase activity in cell extracts of Pseudomonas alcaligenes ATCC 14909 and Pseudomonas mendocina ATCC 25411 was compared. Under saturating substrate concentrations, pyrophosphate, CTP, UDP and ADP were highly inhibitory of the P. alcaligenes transcarbamoylase activity while pyrophosphate, UDP, ADP, ATP and GTP were the most effective inhibitors of the P. mendocina transcarbamoylase. By examining transcarbamoylase inhibition by ribonucleotide triphosphates, it was possible to differentiate these species assigned to different DNA homology groups and such an analysis might prove useful in the reclassification of Pseudomonas species.

  5. Metabolism of Cyclohexane Carboxylic Acid by Alcaligenes Strain W1

    PubMed Central

    Taylor, David G.; Trudgill, Peter W.

    1978-01-01

    Thirty-three microorganisms capable of growth with cyclohexane carboxylate as the sole source of carbon were isolated from mud, water, and soil samples from the Aberystwyth area. Preliminary screening and whole-cell oxidation studies suggested that, with one exception, all of the strains metabolized the growth substrate by beta-oxidation of the coenzyme A ester. This single distinctive strain, able to oxidize rapidly trans-4-hydroxycyclohexane carboxylate, 4-ketocyclohexane carboxylate, p-hydroxybenzoate, and protocatechuate when grown with cyclohexane carboxylate, was classified as a strain of Alcaligenes and given the number W1. Enzymes capable of converting cyclohexane carboxylate to p-hydroxybenzoate were induced by growth with the alicyclic acid and included the first unambiguous specimen of a cyclohexane carboxylate hydroxylase. Because it is a very fragile protein, attempts to stabilize the cyclohexane carboxylate hydroxylase so that a purification procedure could be developed have consistently failed. In limited studies with crude cell extracts, we found that hydroxylation occurred at the 4 position, probably yielding the trans isomer of 4-hydroxycyclohexane carboxylate. Simultaneous measurement of oxygen consumption and reduced nicotinamide adenine dinucleotide oxidation, coupled with an assessment of reactant stoichiometry, showed the enzyme to be a mixed-function oxygenase. Mass spectral analysis enabled the conversion of cyclohexane carboxylate to p-hydroxybenzoate by cell extracts to be established unequivocally, and all of our data were consistent with the pathway: cyclohexane carboxylate → trans-4-hydroxycyclohexane carboxylate → 4-ketocyclohexane carboxylate → p-hydroxybenzoate. The further metabolism of p-hydroxybenzoate proceeded by meta fission and by the oxidative branch of the 2-hydroxy-4-carboxymuconic semialde-hyde-cleaving pathway. PMID:207665

  6. Degradation of dexamethasone by acclimated strain of Pseudomonas Alcaligenes.

    PubMed

    Zhu, Lili; Yang, Zhibang; Yang, Qian; Tu, Zeng; Ma, Lianju; Shi, Zhongquan; Li, Xiaoyu

    2015-01-01

    This study is to investigate the use of microbial remediation technology for degradation of dexamethasone in polluted water. A strain of Pseudomonas Alcaligenes with the ability of dexamethasone degradation was isolated from hospital polluted water. This strain was further acclimated into a bacterial strain that could highly degrade dexamethasone. Domesticated bacterial proteins were separated by osmotic shock method and were analyzed using SDS-PAGE. Enzyme activity of dexamethasone degradation was detected by high performance liquid chromatography. Protein bands with different molecular weight were found in all regions of the bacteria and a band with molecular weight of about 100 kDa was most obvious. In intracellular and periplasmic liquid, there was a band with molecular weight of about 41 kDa. Enzyme activity mainly existed in intracellular liquid. The 41 kDa protease was purified using ammonium sulfate precipitation, DEAE-52 ion exchange column and Sephadex G-100 column. Dexamethasone and dexamethasone sodium phosphate degrading rates of the purified enzyme were 36% and 95%, respectively. The 100 kDa protein had a 19% coverage rate to TonB receptor dependent protein, with 11 peptides matching. The 41 kDa protein had a 56% coverage rate to isovaleryl coenzyme A dehydrogenase, with 5 peptides matching. The 41 kDa protein had good degradation between the temperature of 25-40°C and PH value of 6.5-8.5. The enzyme kinetics equation was Ct = C0 e(-0.1769t), in accordance with the first-order kinetic equation. This study laid the foundation for further preparation of bioremediation agents for clearance of dexamethasone pollution in water.

  7. Probiotic potential of Enterococcus faecalis strains isolated from meconium

    PubMed Central

    Al Atya, Ahmed K.; Drider-Hadiouche, Karima; Ravallec, Rozenn; Silvain, Amadine; Vachee, Anne; Drider, Djamel

    2015-01-01

    107 bacterial isolates with Gram positive staining and negative catalase activity, presumably assumed as lactic acid bacteria, were isolated from samples of meconium of 6 donors at Roubaix hospital, in the north of France. All these bacterial isolates were identified by MALDI-TOF mass spectrometry as Enterococcus faecalis. However, only six isolates among which E. faecalis 14, E. faecalis 28, E. faecalis 90, E. faecalis 97, and E. faecalis 101 (obtained from donor 3), and E. faecalis 93 (obtained from donor 5) were active against some Gram-negative bacteria and Gram-positive bacteria , through production of lactic acid, and bacteriocin like inhibitory substances. The identification of these isolates was confirmed by 16rDNA sequencing and their genetic relatedness was established by REP-PCR and pulsed field gel electrophoresis methods. Importantly, the aforementioned antagonistic isolates were sensitive to various classes of antibiotics tested, exhibited high scores of coaggregation and hydrophobicity, and were not hemolytic. Taken together, these properties render these strains as potential candidates for probiotic applications. PMID:25883590

  8. Mitogen-independent phosphorylation of S6K1 and decreased ribosomal S6 phosphorylation in senescent human fibroblasts.

    PubMed

    Zhang, H; Hoff, H; Marinucci, T; Cristofalo, V J; Sell, C

    2000-08-25

    The p70 ribosomal S6 kinase (S6K1) is rapidly activated following growth factor stimulation of quiescent fibroblasts and inhibition of this enzyme results in a G(1) arrest. Phosphorylation of the ribosomal S6 protein by S6K1 regulates the translation of both ribosomal proteins and initiation factors, leading to an increase in protein synthesis. We have examined the activation of S6K1 in human fibroblasts following mitogen stimulation. In early passage fibroblasts S6K1 is activated following serum stimulation as evidenced by increased kinase activity and site-specific phosphorylation. In contrast, site-specific phosphorylation of S6K1 at Thr421/Ser424 is diminished in senescent fibroblast cultures. A second phosphorylation site within S6K1 (Ser411) is phosphorylated even in the absence of serum stimulation and the enzyme shows increased phosphorylation as judged by decreased electrophoretic mobility. Inhibitor studies indicate that this phosphorylation is dependent upon the mammalian target of rapamycin, PI 3-kinase, and the MAPK pathway. In order to understand the consequences of the altered phosphorylation of the S6K1, we examined the phosphorylation state of the ribosomal S6 protein. In early passage fibroblasts the ribosomal S6 protein is phosphorylated upon serum stimulation while the phosphorylation of the ribosomal S6 protein is drastically reduced in senescent fibroblasts. These results suggest that the intracellular regulators of S6K1 are altered during replicative senescence leading to a deregulation of the enzyme and a loss of ribosomal S6 phosphorylation.

  9. Treatment of dairy wastewater using a selected bacterial isolate, Alcaligenes sp. MMRR7.

    PubMed

    Rajeshkumar, K; Jayachandran, K

    2004-01-01

    Physicochemical and biologic analysis of dairy wastewater showed that the effluent had a high organic load (chemical oxygen demand [COD]: 5095 mg/L), an acidic pH (6.4), and a high probability of coliforms (most probable number [MPN] >1100). The various bacterial strains isolated and purified were identified as Sporolactobacillus sp., Citrobacter sp., Pseudomonas sp., Alcaligenes sp., Bacillus sp., Staphylococcus sp., and Proteus sp., as per the Bergey's manual of systematic bacteriology. Among the five selected bacterial strains, the strain designated as MMRR7 and identified as Alcaligenes sp. was found to give a maximum reduction in COD (62%) in 5 d of incubation. Chemical coagulation using alum at a concentration of 0.5 g/100 mL was found to be effective in the primary treatment of the effluent. Studies on free-cell treatment of the coagulated effluent with the selected bacterial strain Alcaligenes sp. MMRR7 gave a maximum COD reduction of 91% in 120 h. This study clearly indicates the possibility of using Alcaligenes sp. MMRR7 for the effective treatment of dairy wastewater.

  10. Cholecystokinin (CCK) stimulates S6 phosphorylation and induced activation of S6 protein kinase in rat pancreatic acini

    SciTech Connect

    Sung, C.; Okabayashi, Y.; Williams, J.

    1987-05-01

    CCK and insulin stimulate pancreatic protein synthesis at a post transcriptional step. To better understand this regulation the authors evaluated the phosphorylation state of ribosomal protein S6 and the presence of a specific S6 protein kinase in pancreatic acini from diabetic rats. Both CCK and insulin increased S6 phosphorylation by up to 400% in intact TSP-labelled acini. The phorbol ester 12-0-tetradecanoylphorbol 13-acetate also stimulated both protein synthesis and S6 phosphorlyation suggesting a role for protein kinase C in mediating the effect of CCK. By contrast, the CaS ionophore ionomycin had no effect on either parameter. Recently, insulin has been shown to activate a unique S6 kinase in various cells. To test for its presence, cytosolic extracts were prepared from acini stimulated with CCK and insulin by homogenization in US -glycerophosphate buffer and assayed for the kinase using el-TSP ATP and rat pancreatic ribosomes followed by SDS-polyacrylamide gel electrophoresis. CCK and insulin both increased S6 kinase activity which required neither CaS or phospholipid. The dose response for CCk was similar to S6 phosphorlyation in the intact acini. TPA did not stimulate the S6 kinase. Thus, CCK may induce S6 phosphorylation both via C kinase and by activation of a unique S6 kinase.

  11. Nuclear and nucleolar targeting of human ribosomal protein S6.

    PubMed Central

    Schmidt, C; Lipsius, E; Kruppa, J

    1995-01-01

    Chimeric proteins were constructed to define the nuclear localization signals (NLSs) of human ribosomal protein S6. The complete cDNA sequence, different cDNA fragments and oligonucleotides of the human ribosomal proteins S6, respectively, were joined to the 5' end of the entire LacZ gene of Escherichia coli by using recombinant techniques. The hybrid genes were transfected into L cells, transiently expressed, and the intracellular location of the fusion proteins was determined by their beta-galactosidase activity. Three NLSs were identified in the C-terminal half of the S6 protein. Deletion mutagenesis demonstrated that a single NLS is sufficient for targeting the corresponding S6-beta-galactosidase chimera into the nucleus. Removal of all three putative NLSs completely blocked the nuclear import of the resulting S6-beta-galactosidase fusion protein, which instead became evenly distributed in the cytoplasm. Chimeras containing deletion mutants of S6 with at least one single NLS or unmodified S6 accumulated in the nucleolus. Analysis of several constructs reveals the existence of a specific domain that is essential but not sufficient for nucleolar accumulation of S6. Images PMID:8590812

  12. Saporin-S6: A Useful Tool in Cancer Therapy

    PubMed Central

    Polito, Letizia; Bortolotti, Massimo; Mercatelli, Daniele; Battelli, Maria Giulia; Bolognesi, Andrea

    2013-01-01

    Thirty years ago, the type 1 ribosome-inactivating protein (RIP) saporin-S6 (also known as saporin) was isolated from Saponaria officinalis L. seeds. Since then, the properties and mechanisms of action of saporin-S6 have been well characterized, and it has been widely employed in the construction of conjugates and immunotoxins for different purposes. These immunotoxins have shown many interesting results when used in cancer therapy, particularly in hematological tumors. The high enzymatic activity, stability and resistance to conjugation procedures and blood proteases make saporin-S6 a very useful tool in cancer therapy. High efficacy has been reported in clinical trials with saporin-S6-containing immunotoxins, at dosages that induced only mild and transient side effects, which were mainly fever, myalgias, hepatotoxicity, thrombocytopenia and vascular leak syndrome. Moreover, saporin-S6 triggers multiple cell death pathways, rendering impossible the selection of RIP-resistant mutants. In this review, some aspects of saporin-S6, such as the chemico-physical characteristics, the structural properties, its endocytosis, its intracellular routing and the pathogenetic mechanisms of the cell damage, are reported. In addition, the recent progress and developments of saporin-S6-containing immunotoxins in cancer immunotherapy are summarized, including in vitro and in vivo pre-clinical studies and clinical trials. PMID:24105401

  13. Phosphorylation of ribosomal protein S6 mediates compensatory renal hypertrophy.

    PubMed

    Xu, Jinxian; Chen, Jianchun; Dong, Zheng; Meyuhas, Oded; Chen, Jian-Kang

    2015-03-01

    The molecular mechanism underlying renal hypertrophy and progressive nephron damage remains poorly understood. Here we generated congenic ribosomal protein S6 (rpS6) knock-in mice expressing nonphosphorylatable rpS6 and found that uninephrectomy-induced renal hypertrophy was significantly blunted in these knock-in mice. Uninephrectomy-induced increases in cyclin D1 and decreases in cyclin E in the remaining kidney were attenuated in the knock-in mice compared with their wild-type littermates. Uninephrectomy induced rpS6 phosphorylation in the wild-type mice; however, no rpS6 phosphorylation was detected in uninephrectomized or sham-operated knock-in mice. Nonetheless, uninephrectomy stimulated comparable 4E-BP1 phosphorylation in both knock-in and wild-type mice, indicating that mTORC1 was still activated in the knock-in mice. Moreover, the mTORC1 inhibitor rapamycin prevented both rpS6 and 4E-BP1 phosphorylation, significantly blunted uninephrectomy-induced renal hypertrophy in wild-type mice, but did not prevent residual renal hypertrophy despite inhibiting 4E-BP1 phosphorylation in uninephrectomized knock-in mice. Thus, both genetic and pharmacological approaches unequivocally demonstrate that phosphorylated rpS6 is a downstream effector of the mTORC1-S6K1 signaling pathway mediating renal hypertrophy. Hence, rpS6 phosphorylation facilitates the increase in cyclin D1 and decrease in cyclin E1 that underlie the hypertrophic nature of uninephrectomy-induced kidney growth.

  14. Esp-independent biofilm formation by Enterococcus faecalis.

    PubMed

    Kristich, Christopher J; Li, Yung-Hua; Cvitkovitch, Dennis G; Dunny, Gary M

    2004-01-01

    Enterococcus faecalis is a gram-positive opportunistic pathogen known to form biofilms in vitro. In addition, this organism is often isolated from biofilms on the surfaces of various indwelling medical devices. However, the molecular mechanisms regulating biofilm formation in these clinical isolates are largely unknown. Recent work has suggested that a specific cell surface protein (Esp) of E. faecalis is critical for biofilm formation by this organism. However, in the same study, esp-deficient strains of E. faecalis were found to be capable of biofilm formation. To test the hypothesis that Esp is dispensable for biofilm formation by E. faecalis, we used microtiter plate assays and a chemostat-based biofilm fermentor assay to examine biofilm formation by genetically well-defined, non-Esp-expressing strains. Our results demonstrate that in vitro biofilm formation occurs, not only in the absence of esp, but also in the absence of the entire pathogenicity island that harbors the esp coding sequence. Using scanning electron microscopy to evaluate biofilms of E. faecalis OG1RF grown in the fermentor system, biofilm development was observed to progress through multiple stages, including attachment of individual cells to the substratum, microcolony formation, and maturation into complex multilayered structures apparently containing water channels. Microtiter plate biofilm analyses indicated that biofilm formation or maintenance was modulated by environmental conditions. Furthermore, our results demonstrate that expression of a secreted metalloprotease, GelE, enhances biofilm formation by E. faecalis. In summary, E. faecalis forms complex biofilms by a process that is sensitive to environmental conditions and does not require the Esp surface protein.

  15. Thermal Injury and Recovery of Streptococcus faecalis

    PubMed Central

    Clark, Carol W.; Witter, Lloyd D.; Ordal, Z. John

    1968-01-01

    Exposure of Streptococcus faecalis R57 to sublethal heating produced a temporary change in the salt tolerance and growth of the organism. After sublethal heat treatment at 60 C for 15 min, greater than 99.0% of the viable population was unable to reproduce on media containing 6% NaCl. In addition, the heated cells displayed a sensitivity to incubation temperature, pH, and 0.01% methylene blue. When the injured cells were placed in a synthetic medium, recovery occurred at a much slower rate than in a complex medium. However, both media supported comparable growth of the uninjured organism. Various media used for the enrichment of streptococci also provided a suitable environment for the recovery of the injured cells. Generally, as more selective agents were present in the medium, the rates of recovery decreased. Metabolic inhibitor studies with chloramphenicol, penicillin, and actinomycin D substantiated the fact that the process involved was recovery and not growth, and that this recovery was linked to ribonucleic acid synthesis. PMID:4973066

  16. Fumarate Reductase Activity of Streptococcus faecalis

    PubMed Central

    Aue, B. J.; Diebel, R. H.

    1967-01-01

    Some characteristics of a fumarate reductase from Streptococcus faecalis are described. The enzyme had a pH optimum of 7.4; optimal activity was observed when the ionic strength of the phosphate buffer was adjusted to 0.088. The Km value of the enzyme for reduced flavin mononucleotide was 2 × 10−4 m as determined with a 26-fold preparation. In addition to fumarate, the enzyme reduced maleate and mesaconate. No succinate dehydrogenase activity was detected, but succinate did act as an inhibitor of the fumarate reductase activity. Other inhibitors were malonate, citraconate, and trans-, trans-muconate. Metal-chelating agents did not inhibit the enzyme. A limited inhibition by sulfhydryl-binding agents was observed, and the preparations were sensitive to air oxidation and storage. Glycine, alanine, histidine, and possibly lysine stimulated fumarate reductase activity in the cell-free extracts. However, growth in media supplemented with glycine did not enhance fumarate reductase activity. The enzymatic activity appears to be constitutive. PMID:4960892

  17. Sodium-stimulated ATPase in Streptococcus faecalis.

    PubMed Central

    Kinoshita, N; Unemoto, T; Kobayashi, H

    1984-01-01

    We measured Na+-stimulated ATPase activity in a mutant of Streptococcus faecalis defective in the generation of proton motive force. The activity in membrane vesicles was 62.1 +/- 5.9 nmol of phosphate produced per min per mg of protein when cells were grown on medium containing 0.12 M Na+. Activity decreased as the concentration of Na+ in the growth medium decreased. The decrease in enzyme activity corresponded to the decrease in transport activity for Na+ in both whole cells and membrane vesicles. The effects of pH on both activities were identical. Thus, it is suggested that Na+ movement is mediated by this enzyme. Sodium extrusion and ATPase activity in the wild-type strain were markedly lower than those observed in the mutant strain. Elevated activities of both Na+ extrusion and Na+-stimulated ATPase could be detected in the wild-type strain when cells were grown in the absence of proton motive force. Thus, we propose that the level of ATPase is increased by dissipation of the proton motive force. PMID:6144668

  18. Meningitis caused by Alcaligenes xylosoxidans in a patient with HIV/AIDS.

    PubMed

    Espinoza-Gómez, F; Newton-Sánchez, O A; Melnikov, V; Virgen-González, O; Unrau, J

    2007-12-01

    The purpose of the present work was to inform about the first case of meningitis associated to the bacteria Alcaligenes xylosoxidans in a patient with HIV/AIDS. The patient was a 46-year-old male, with the antecedent of have been diagnosed for HIV/ AIDS, who attended in the Hospital Universitario de Colima, Mexico, with fever, shock and meningismus. The study of the cerebrospinal fluid showed pleocytosis, elevated protein levels and hypoglycorrhachia. The culture yielded the presence of Alcaligenes xylosoxidans with sensitivity to ciprofloxacin. After 14 days of treatment with this antibiotic, the patient showed neurologic improvement and was able to continue with his outpatient antiretroviral treatment. The present case shows the importance of the inclusion of this bacterium in the differential diagnosis of the neurological infections in HIV/AIDS patients and emphasizes the importance of considering the bacterial meningitis in this population.

  19. Effect of surfactants on fluoranthene degradation by Pseudomonas alcaligenes PA-10.

    PubMed

    Hickey, Anne Marie; Gordon, Linda; Dobson, Alan D W; Kelly, Catherine T; Doyle, Evelyn M

    2007-03-01

    Two surfactants, Tween 80 and JBR, were investigated for their effect on fluoranthene degradation by a Pseudomonad. Both surfactants enhanced fluoranthene degradation by Pseudomonas alcaligenes PA-10 in shake flask culture. This bacterium was capable of utilising the synthetic surfactant and the biosurfactant as growth substrates and the critical micelle concentration of neither compound inhibited bacterial growth. The biosurfactant JBR significantly increased polycyclic aromatic hydrocarbon (PAH) desorption from soil. Inoculation of fluoranthene-contaminated soil microcosms with P. alcaligenes PA-10 resulted in the removal of significant amounts (45 +/- 5%) of the PAH after 28 days compared to an uninoculated control. Addition of the biosurfactant increased the initial rate of fluoranthene degradation in the inoculated microcosm. The presence of a lower molecular weight PAH, phenanthrene, had a similar effect on the rate of fluoranthene removal.

  20. [Case report: respiratory infection due to Alcaligenes xylosoxidans in a patient with Mounier-Kuhn syndrome].

    PubMed

    Arroyo-Cózar, Marta; Ruiz-García, Montserrat; Merlos, Eva M; Vielba, David; Macías, Enrique

    2012-10-01

    Mounier-Kuhn syndrome is a rare entity characterized by abnormal dilatation of the trachea and main bronchi (tracheobronchomegaly). Alcaligenes xylosoxidans is a non fermenting gram-negative pathogen common in extra-and intra-hospital environment, which may be related to immunosuppression states. We describe the case of a 75 years old male, ex-smoker with moderate functional obstruction, chronic respiratory failure and chronic colonization by Pseudomonas aeuriginosa. He had an infectious exacerbation of his disease, reason that previously required several hospital admissions. The patient was treated with antibiotics and his evolution was favourable with negativization in cultures of the pathogen. This is the first description of the isolation of Alcaligenes xylosoxidans as a cause of respiratory infection in a patient with Mounier-Kuhn syndrome.

  1. Effect of carbon source on pyrimidine biosynthesis in Pseudomonas alcaligenes ATCC 14909.

    PubMed

    Santiago, Manuel F; West, Thomas P

    2003-01-01

    The effect of carbon source on the regulation of the de novo pyrimidine biosynthetic enzymes in Pseudomonas alcaligenes ATCC 14909 was investigated. The de novo pyrimidine biosynthetic enzymes were measured in extracts of P. alcaligenes ATCC 14909 cells and of cells from an auxotroph deficient for orotate phosphoribosyltransferase activity. Pyrimidine biosynthetic enzyme activities in ATCC 14909 were influenced by pyrimidine supplementation to the culture medium but not by the carbon source present. Pyrimidine limitation of the auxotroph elevated the de novo enzyme activities indicating that this pathway may be controlled at the transcriptional level by a pyrimidine-related compound. Its regulation seemed to be subject to less transcriptional control by a pyrimidine-related compound than what was observed in the closely related species Pseudomonas pseudoalcaligenes.

  2. Purification, crystallization and preliminary X-ray studies of two isoforms of Rubisco from Alcaligenes eutrophus.

    PubMed

    Hansen, S; Hough, E; Andersen, K

    1999-01-01

    Two different isoforms of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Alcaligenes eutrophus have been purified and crystallized. Both isoforms crystallize in space group P43212. Crystals of isoform I (unit-cell dimensions a = 112.0 and c = 402.7 A) diffract to 2.7 A, whereas isoform II (unit-cell dimensions a = 111.8 and c = 400.0 A) presently diffract to 3.2 A, using synchrotron radiation in both cases.

  3. Lipase expression in Pseudomonas alcaligenes is under the control of a two-component regulatory system.

    PubMed

    Krzeslak, Joanna; Gerritse, Gijs; van Merkerk, Ronald; Cool, Robbert H; Quax, Wim J

    2008-03-01

    Preliminary observations in a large-scale fermentation process suggested that the lipase expression of Pseudomonas alcaligenes can be switched on by the addition of certain medium components, such as soybean oil. In an attempt to elucidate the mechanism of induction of lipase expression, we have set up a search method for genes controlling lipase expression by use of a cosmid library containing fragments of P. alcaligenes genomic DNA. A screen for lipase hyperproduction resulted in the selection of multiple transformants, of which the best-producing strains comprised cosmids that shared an overlapping genomic fragment. Within this fragment, two previously unidentified genes were found and named lipQ and lipR. Their encoded proteins belong to the NtrBC family of regulators that regulate gene expression via binding to a specific upstream activator sequence (UAS). Such an NtrC-like UAS was identified in a previous study in the P. alcaligenes lipase promoter, strongly suggesting that LipR acts as a positive regulator of lipase expression. The regulating role could be confirmed by down-regulated lipase expression in a strain with an inactivated lipR gene and a threefold increase in lipase yield in a large-scale fermentation when expressing the lipQR operon from the multicopy plasmid pLAFR3. Finally, cell extracts of a LipR-overexpressing strain caused a retardation of the lipase promoter fragment in a band shift assay. Our results indicate that lipase expression in Pseudomonas alcaligenes is under the control of the LipQR two-component system.

  4. Introduction of Pseudomonas aeruginosa mutator phage D3112 into Alcaligenes eutrophus strain CH34.

    PubMed

    Krylov, V; Merlin, C; Toussaint, A

    1995-01-01

    We have investigated the possibility of growing mutator phages from Pseudomonas aeruginosa on various isolates of Alcaligenes eutrophus. Although none out of 10 A. eutrophus strains were susceptible to infection with any of the phages tested, phage D3112 could be readily transferred in our model strain CH34 by means of an RP4::D3112 plasmid. CH34/RP4::D3112 lysogens were stable and produced phages. However, neither mitomycin C nor UV treatment increased the phage yield.

  5. The effect of berberine hydrochloride on Enterococcus faecalis biofilm formation and dispersion in vitro.

    PubMed

    Chen, Lihua; Bu, Qianqian; Xu, Huan; Liu, Yuan; She, Pengfei; Tan, Ruichen; Wu, Yong

    2016-01-01

    Enterococcus faecalis (E. faecalis) is one of the major causes of biofilm infections. Berberine hydrochloride (BBH) has diverse pharmacological effects; however, the effects and mechanisms of BBH on E. faecalis biofilm formation and dispersion have not been reported. In this study, 99 clinical isolates from the urine samples of patients with urinary tract infections (UTIs) were collected and identified. Ten strains of E. faecalis with biofilm formation ability were studied. BBH inhibited E. faecalis biofilm formation and promoted the biofilm dispersion of E. faecalis. In addition, sortase A and esp expression levels were elevated during early E. faecalis biofilm development, whereas BBH significantly reduced their expression levels. The results of this study indicated that BBH effectively prevents biofilm formation and promotes biofilm dispersion in E. faecalis, most likely by inhibiting the expressions of sortase A and esp.

  6. Identification of Strains of Alcaligenes and Agrobacterium by a Polyphasic Approach

    PubMed Central

    Clermont, Dominique; Harmant, Christine; Bizet, Chantal

    2001-01-01

    The number of stable discriminant biochemical characters is limited in the genera Alcaligenes and Agrobacterium, whose species are consequently difficult to distinguish from one another by conventional tests. Moreover, genomic studies have recently drastically modified the nomenclature of these genera; for example, Alcaligenes xylosoxidans was transferred to the genus Achromobacter in 1998. Twenty-five strains of Achromobacter xylosoxidans, three strains of an Agrobacterium sp., five strains of an Alcaligenes sp., and four unnamed strains belonging to the Centers for Disease Control and Prevention group IVc-2 were examined. These strains were characterized by conventional tests, including biochemical tests. The assimilation of 99 carbohydrates, organic acids, and amino acids was studied by using Biotype-100 strips, and rRNA gene restriction patterns were obtained with the automated Riboprinter microbial characterization system after cleavage of total DNA with EcoRI or PstI restriction endonuclease. This polyphasic approach allowed the two subspecies of A. xylosoxidans to be clearly separated. Relationships between five strains and the Ralstonia paucula type strain were demonstrated. Likewise, three strains were found to be related to the Ochrobactrum anthropi type strain. We showed that substrate assimilation tests and automated ribotyping provide a simple, rapid, and reliable means of identifying A. xylosoxidans subspecies and that these two methods can be used as alternative methods to characterize unidentified strains rapidly when discriminant biochemical characters are missing. PMID:11526136

  7. Arsenic methylation and volatilization by arsenite S-adenosylmethionine methyltransferase in Pseudomonas alcaligenes NBRC14159.

    PubMed

    Zhang, Jun; Cao, Tingting; Tang, Zhu; Shen, Qirong; Rosen, Barry P; Zhao, Fang-Jie

    2015-04-01

    Inorganic arsenic (As) is highly toxic and ubiquitous in the environment. Inorganic As can be transformed by microbial methylation, which constitutes an important part of the As biogeochemical cycle. In this study, we investigated As biotransformation by Pseudomonas alcaligenes NBRC14159. P. alcaligenes was able to methylate arsenite [As(III)] rapidly to dimethylarsenate and small amounts of trimethylarsenic oxide. An arsenite S-adenosylmethionine methyltransferase, PaArsM, was identified and functionally characterized. PaArsM shares low similarities with other reported ArsM enzymes (<55%). When P. alcaligenes arsM gene (PaarsM) was disrupted, the mutant lost As methylation ability and became more sensitive to As(III). PaarsM was expressed in the absence of As(III) and the expression was further enhanced by As(III) exposure. Heterologous expression of PaarsM in an As-hypersensitive strain of Escherichia coli conferred As(III) resistance. Purified PaArsM protein methylated As(III) to dimethylarsenate as the main product in the medium and also produced dimethylarsine and trimethylarsine gases. We propose that PaArsM plays a role in As methylation and detoxification of As(III) and could be exploited in bioremediation of As-contaminated environments.

  8. Identification and antimicrobial susceptibility of Alcaligenes xylosoxidans isolated from patients with cystic fibrosis.

    PubMed

    Saiman, L; Chen, Y; Tabibi, S; San Gabriel, P; Zhou, J; Liu, Z; Lai, L; Whittier, S

    2001-11-01

    In the past decade, potential pathogens, including Alcaligenes species, have been increasingly recovered from cystic fibrosis (CF) patients. Accurate identification of multiply antibiotic-resistant gram-negative bacilli is critical to understanding the epidemiology and clinical implications of emerging pathogens in CF. We examined the frequency of correct identification of Alcaligenes spp. by microbiology laboratories affiliated with American CF patient care centers. Selective media, an exotoxin A probe for Pseudomonas aeruginosa, and a commercial identification assay, API 20 NE, were used for identification. The activity of antimicrobial agents against these clinical isolates was determined. A total of 106 strains from 78 patients from 49 CF centers in 22 states were studied. Most (89%) were correctly identified by the referring laboratories as Alcaligenes xylosoxidans. However, 12 (11%) strains were misidentified; these were found to be P. aeruginosa (n = 10), Stenotrophomonas maltophilia (n = 1), and Burkholderia cepacia (n = 1). Minocycline, imipenem, meropenem, piperacillin, and piperacillin-tazobactam were the most active since 51, 59, 51, 50, and 55% of strains, respectively, were inhibited. High concentrations of colistin (100 and 200 microg/ml) inhibited 92% of strains. Chloramphenicol paired with minocycline and ciprofloxacin paired with either imipenem or meropenem were the most active combinations and inhibited 40 and 32%, respectively, of strains. Selective media and biochemical identification proved to be useful strategies for distinguishing A. xylosoxidans from other CF pathogens. Standards for processing CF specimens should be developed, and the optimal method for antimicrobial susceptibility testing of A. xylosoxidans should be determined.

  9. Isolation and characterization of group II introns from Pseudomonas alcaligenes and Pseudomonas putida.

    PubMed

    Yeo, C C; Yiin, S; Tan, B H; Poh, C L

    2001-05-01

    Group II introns isolated from Pseudomonas alcaligenes NCIB 9867, Pseudomonas putida NCIB 9869, and P. putida KT2440 were closely related with nucleotide sequence identities of between 87 and 96%. The genome of P. alcaligenes also harbored a truncated group II intron of 682 bp that lacks the gene for the intron-encoded protein (IEP). Unlike most bacterial group II introns, the Pseudomonas introns were found to lack the Zn domains in their IEPs, did not appear to interrupt any genes, and were located downstream of open reading frames which were adjacent to hairpin loop structures that resemble rho-independent terminators. These structures also contain the intron binding sites 1 and 2 (IBS1 and IBS2 sequences) that were required for intron target site recognition in transposition. One of the group II introns found in P. alcaligenes, Xln3, was shown to have transposed from the chromosome to the endogenous pRA2 plasmid at a site adjacent to IBS1- and IBS2-like sequences.

  10. In vitro susceptibility studies of vancomycin-resistant Enterococcus faecalis.

    PubMed Central

    Sahm, D F; Kissinger, J; Gilmore, M S; Murray, P R; Mulder, R; Solliday, J; Clarke, B

    1989-01-01

    Vancomycin resistance exhibited by Enterococcus faecalis isolates V583, V586, and V587 is described. The vancomycin MICs ranged from 32 to 64 micrograms/ml. Although resistant to vancomycin, the isolates were susceptible to teicoplanin (MIC, less than or equal to 0.5 micrograms/ml). Such a glycopeptide susceptibility profile has not been previously described for E. faecalis. Time kill studies showed that vancomycin resistance adversely affected the synergistic activity that vancomycin and aminoglycoside combinations usually demonstrate against enterococci. However, the ability to detect vancomycin resistance varied with the susceptibility testing method used. Whereas broth microdilution, broth macrodilution, and agar dilution methods detected resistance, disk-agar diffusion and the AutoMicrobic system Gram-Positive GPS-A susceptibility card (Vitek Systems Inc., Hazelwood, Mo.) did not. To detect vancomycin resistance reliably and establish the incidence of such E. faecalis isolates, adjustments in some susceptibility testing methods may be necessary. PMID:2554802

  11. Enterococcus faecalis Constitutes an Unusual Bacterial Model in Lysozyme Resistance▿

    PubMed Central

    Hébert, Laurent; Courtin, Pascal; Torelli, Riccardo; Sanguinetti, Maurizio; Chapot-Chartier, Marie-Pierre; Auffray, Yanick; Benachour, Abdellah

    2007-01-01

    Lysozyme is an important and widespread compound of the host constitutive defense system, and it is assumed that Enterococcus faecalis is one of the few bacteria that are almost completely lysozyme resistant. On the basis of the sequence analysis of the whole genome of E. faecalis V583 strain, we identified two genes that are potentially involved in lysozyme resistance, EF_0783 and EF_1843. Protein products of these two genes share significant homology with Staphylococcus aureus peptidoglycan O-acetyltransferase (OatA) and Streptococcus pneumoniae N-acetylglucosamine deacetylase (PgdA), respectively. In order to determine whether EF_0783 and EF_1843 are involved in lysozyme resistance, we constructed their corresponding mutants and a double mutant. The ΔEF_0783 mutant and ΔEF_0783 ΔEF_1843 double mutant were shown to be more sensitive to lysozyme than the parental E. faecalis JH2-2 strain and ΔEF_1843 mutant were. However, compared to other bacteria, such as Listeria monocytogenes or S. pneumoniae, the tolerance of ΔEF_0783 and ΔEF_0783 ΔEF_1843 mutants towards lysozyme remains very high. Peptidoglycan structure analysis showed that EF_0783 modifies the peptidoglycan by O acetylation of N-acetyl muramic acid, while the EF_1843 deletion has no obvious effect on peptidoglycan structure under the same conditions. Moreover, the EF_0783 and EF_1843 deletions seem to significantly affect the ability of E. faecalis to survive within murine macrophages. In all, while EF_0783 is currently involved in the lysozyme resistance of E. faecalis, peptidoglycan O acetylation and de-N-acetylation are not the main mechanisms conferring high levels of lysozyme resistance to E. faecalis. PMID:17785473

  12. Faraday effect in Sn2P2S6 crystals.

    PubMed

    Krupych, Oleh; Adamenko, Dmytro; Mys, Oksana; Grabar, Aleksandr; Vlokh, Rostyslav

    2008-11-10

    We have revealed a large Faraday rotation in tin thiohypodiphosphate (Sn(2)P(2)S(6)) crystals, which makes this material promising for magneto-optics. The effective Faraday tensor component and the Verdet constant for the direction of the optic axis have been determined by measuring the pure Faraday rotation in Sn(2)P(2)S(6) crystals with both the single-ray and small-angular polarimetric methods at the normal conditions and a wavelength of 632.8 nm. The effective Verdet constant is found to be equal to 115 rad/T x m.

  13. Ligand-Signaled Upregulation of Enterococcus faecalis ace Transcription, a Mechanism for Modulating Host-E. faecalis Interaction

    PubMed Central

    Nallapareddy, Sreedhar R.; Murray, Barbara E.

    2006-01-01

    Enterococcus faecalis, the third most frequent cause of bacterial endocarditis, appears to be equipped with diverse surface-associated proteins showing structural-fold similarity to the immunoglobulin-fold family of staphylococcal adhesins. Among the putative E. faecalis surface proteins, the previously characterized adhesin Ace, which shows specific binding to collagen and laminin, was detectable in surface protein preparations only after growth at 46°C, mirroring the finding that adherence was observed in 46°C, but not 37°C, grown E. faecalis cultures. To elucidate the influence of different growth and host parameters on ace expression, we investigated ace expression using E. faecalis OG1RF grown in routine laboratory media (brain heart infusion) and found that ace mRNA levels were low in all growth phases. However, quantitative reverse transcription-PCR showed 18-fold-higher ace mRNA amounts in cells grown in the presence of collagen type IV compared to the controls. Similarly, a marked increase was observed when cells were either grown in the presence of collagen type I or serum but not in the presence of fibrinogen or bovine serum albumin. The production of Ace after growth in the presence of collagen type IV was demonstrated by immunofluorescence microscopy, mirroring the increased ace mRNA levels. Furthermore, increased Ace expression correlated with increased collagen and laminin adhesion. Collagen-induced Ace expression was also seen in three of three other E. faecalis strains of diverse origins tested, and thus it appears to be a common phenomenon. The observation of host matrix signal-induced adherence of E. faecalis may have important implications on our understanding of this opportunistic pathogen. PMID:16926389

  14. Outbreak of mastitis in sheep caused by multi-drug resistant Enterococcus faecalis in Sardinia, Italy.

    PubMed

    Sanciu, G; Marogna, G; Paglietti, B; Cappuccinelli, P; Leori, G; Rappelli, P

    2013-03-01

    An outbreak of infective mastitis due to Enterococcus faecalis occurred in an intensive sheep farm in north Sardinia (Italy). E. faecalis, which is only rarely isolated from sheep milk, was unexpectedly found in 22·3% of positive samples at microbiological examination. Forty-five out of the 48 E. faecalis isolates showed the same multi-drug resistance pattern (cloxacillin, streptomycin, kanamycin, clindamycin, oxytetracycline). E. faecalis isolates were analysed by pulsed-field gel electrophoresis, and all 45 multi-drug resistant strains showed an indistinguishable macrorestiction profile, indicating their clonal origin. To our knowledge, this is the first report of an outbreak of mastitis in sheep caused by E. faecalis.

  15. Phage therapy against Enterococcus faecalis in dental root canals

    PubMed Central

    Khalifa, Leron; Shlezinger, Mor; Beyth, Shaul; Houri-Haddad, Yael; Coppenhagen-Glazer, Shunit; Beyth, Nurit; Hazan, Ronen

    2016-01-01

    Antibiotic resistance is an ever-growing problem faced by all major sectors of health care, including dentistry. Recurrent infections related to multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, and vancomycin-resistant enterococci (VRE) in hospitals are untreatable and question the effectiveness of notable drugs. Two major reasons for these recurrent infections are acquired antibiotic resistance genes and biofilm formation. None of the traditionally known effective techniques have been able to efficiently resolve these issues. Hence, development of a highly effective antibacterial practice has become inevitable. One example of a hard-to-eradicate pathogen in dentistry is Enterococcus faecalis, which is one of the most common threats observed in recurrent root canal treatment failures, of which the most problematic to treat are its biofilm-forming VRE strains. An effective response against such infections could be the use of bacteriophages (phages). Phage therapy was found to be highly effective against biofilm and multidrug-resistant bacteria and has other advantages like ease of isolation and possibilities for genetic manipulations. The potential of phage therapy in dentistry, in particular against E. faecalis biofilms in root canals, is almost unexplored. Here we review the efforts to develop phage therapy against biofilms. We also focus on the phages isolated against E. faecalis and discuss the possibility of using phages against E. faecalis biofilm in root canals. PMID:27640530

  16. An antimicrobial peptidoglycan hydrolase for treating Enterococcus faecalis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enterococcus faecalis is an intestinal bacteria species that can become an opportunistic pathogen in humans and farm animals with antibiotic resistant strains becoming increasingly common. In farm animals, strong antimicrobials, such as Vancomycin, should not be used due to the risk of propagation ...

  17. Phage therapy against Enterococcus faecalis in dental root canals.

    PubMed

    Khalifa, Leron; Shlezinger, Mor; Beyth, Shaul; Houri-Haddad, Yael; Coppenhagen-Glazer, Shunit; Beyth, Nurit; Hazan, Ronen

    2016-01-01

    Antibiotic resistance is an ever-growing problem faced by all major sectors of health care, including dentistry. Recurrent infections related to multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus, carbapenem-resistant Enterobacteriaceae, and vancomycin-resistant enterococci (VRE) in hospitals are untreatable and question the effectiveness of notable drugs. Two major reasons for these recurrent infections are acquired antibiotic resistance genes and biofilm formation. None of the traditionally known effective techniques have been able to efficiently resolve these issues. Hence, development of a highly effective antibacterial practice has become inevitable. One example of a hard-to-eradicate pathogen in dentistry is Enterococcus faecalis, which is one of the most common threats observed in recurrent root canal treatment failures, of which the most problematic to treat are its biofilm-forming VRE strains. An effective response against such infections could be the use of bacteriophages (phages). Phage therapy was found to be highly effective against biofilm and multidrug-resistant bacteria and has other advantages like ease of isolation and possibilities for genetic manipulations. The potential of phage therapy in dentistry, in particular against E. faecalis biofilms in root canals, is almost unexplored. Here we review the efforts to develop phage therapy against biofilms. We also focus on the phages isolated against E. faecalis and discuss the possibility of using phages against E. faecalis biofilm in root canals.

  18. Susceptibilities of Enterococcus faecalis biofilms to some antimicrobial medications.

    PubMed

    Lima, K C; Fava, L R; Siqueira, J F

    2001-10-01

    Enterococcus faecalis has been suggested to be an important etiological agent in endodontic failures. The purpose of this study was to evaluate the effectiveness of chlorhexidine- or antibiotics-based medications in eliminating E. faecalis biofilms. One-day and three-day biofilms of E. faecalis were induced on cellulose nitrate membrane filters. Each biofilm-containing membrane was thoroughly covered with 1 ml of the test medications and incubated for 1 day at 37 degrees C. Treated biofilms were then aseptically transferred to vials containing a neutralizing agent in saline solution and vortexed. Suspensions were 10-fold diluted, seeded onto Mitis salivarius agar plates, and the colony-forming units counted after 48 h of incubation. There were significant differences between the formulations tested. The association of clindamycin with metronidazole significantly reduced the number of cells in 1-day biofilms. However of all medications tested, only 2% chlorhexidine-containing medications were able to thoroughly eliminate most of both 1-day and 3-day E. faecalis biofilms.

  19. Enterococcus faecalis prophage dynamics and contributions to pathogenic traits.

    PubMed

    Matos, Renata C; Lapaque, Nicolas; Rigottier-Gois, Lionel; Debarbieux, Laurent; Meylheuc, Thierry; Gonzalez-Zorn, Bruno; Repoila, Francis; Lopes, Maria de Fatima; Serror, Pascale

    2013-06-01

    Polylysogeny is frequently considered to be the result of an adaptive evolutionary process in which prophages confer fitness and/or virulence factors, thus making them important for evolution of both bacterial populations and infectious diseases. The Enterococcus faecalis V583 isolate belongs to the high-risk clonal complex 2 that is particularly well adapted to the hospital environment. Its genome carries 7 prophage-like elements (V583-pp1 to -pp7), one of which is ubiquitous in the species. In this study, we investigated the activity of the V583 prophages and their contribution to E. faecalis biological traits. We systematically analyzed the ability of each prophage to excise from the bacterial chromosome, to replicate and to package its DNA. We also created a set of E. faecalis isogenic strains that lack from one to all six non-ubiquitous prophages by mimicking natural excision. Our work reveals that prophages of E. faecalis V583 excise from the bacterial chromosome in the presence of a fluoroquinolone, and are able to produce active phage progeny. Intricate interactions between V583 prophages were also unveiled: i) pp7, coined EfCIV583 for E. faecalis chromosomal island of V583, hijacks capsids from helper phage 1, leading to the formation of distinct virions, and ii) pp1, pp3 and pp5 inhibit excision of pp4 and pp6. The hijacking exerted by EfCIV583 on helper phage 1 capsids is the first example of molecular piracy in Gram positive bacteria other than staphylococci. Furthermore, prophages encoding platelet-binding-like proteins were found to be involved in adhesion to human platelets, considered as a first step towards the development of infective endocarditis. Our findings reveal not only a role of E. faecalis V583 prophages in pathogenicity, but also provide an explanation for the correlation between antibiotic usage and E. faecalis success as a nosocomial pathogen, as fluoriquinolone may provoke release of prophages and promote gene dissemination among

  20. Supersymmetric AdS_6 solutions of type IIB supergravity

    NASA Astrophysics Data System (ADS)

    Kim, Hyojoong; Kim, Nakwoo; Suh, Minwoo

    2015-10-01

    We study the general requirement for supersymmetric AdS_6 solutions in type IIB supergravity. We employ the Killing spinor technique and study the differential and algebraic relations among various Killing spinor bilinears to find the canonical form of the solutions. Our result agrees precisely with the work of Apruzzi et al. (JHEP 1411:099, 2014), which used the pure spinor technique. Hoping to identify the geometry of the problem, we also computed four-dimensional theory through the dimensional reduction of type IIB supergravity on AdS_6. This effective action is essentially a non-linear sigma model with five scalar fields parametrizing {SL}(3,{R})/{SO}(2,1), modified by a scalar potential and coupled to Einstein gravity in Euclidean signature. We argue that the scalar potential can be explained by a subgroup CSO(1,1,1) subset {SL}(3,{R}) in a way analogous to gauged supergravity.

  1. Growth kinetics of Pseudomonas alcaligenes C-0 relative to inoculation and 3-chlorobenzoate metabolism in soil.

    PubMed Central

    Focht, D D; Shelton, D

    1987-01-01

    Pseudomonas alcaligenes C-0 was isolated from activated sewage sludge by enrichment with 3-chlorobenzoate (3CB) as the sole carbon source. The carbon balance from [14C]3CB in pure culture could be accounted for in substrate, biomass, and CO2 from all sampling periods and inoculum densities (0.012, 0.092, 0.20, and 0.92 micrograms of dry cells X ml-1), and inorganic chloride was produced stoichiometrically. Monod parameters as determined in culture were compared with the kinetics of 3CB metabolism in soil with decreasing inoculum densities (1.9 X 10(-1), 1.9 X 10(-3), and 1.9 X 10(-5) micrograms of cells X g-1). 3CB was refractile to attack in soil by indigenous microflora, but it was completely metabolized upon inoculation with P. alcaligenes C-0. The saturation constant KS was much higher in soil than in culture, but the yield coefficient Y and the growth rate constant were the same in both systems: mu max = 0.32 h-1; Y = 34 micrograms cells X mumol-1; KS = 0.18 mM in culture and 6.0 mM in soil solution (1.1 mumol X g-1 of soil). The parameter estimates obtained from the highest inoculum density could be used for the lower inoculum densities with reasonable agreement between predicted and observed 3CB concentrations in soil, although the residual sum of squares was progressively higher. Since the growth rate of P. alcaligenes C-0 in soil was comparable to its growth rate in culture, inoculation should be a viable strategy for biodegradation of 3CB in soil if indigenous microflora are unable to exploit this metabolic niche. PMID:3662518

  2. Proteome analysis of gentisate-induced response in Pseudomonas alcaligenes NCIB 9867.

    PubMed

    Zhao, Bing; Yeo, Chew Chieng; Lee, Chee Chow; Geng, Anli; Chew, Fook Tim; Poh, Chit Laa

    2004-07-01

    Pseudomonas alcaligenes NCIB 9867 (P25X wild-type) is capable of degrading aromatic hydrocarbons via the gentisate pathway. Biochemical characterization of P25X mutants indicated that it has isofunctional enzymes for the mono- and dioxygenase-catalyzed reactions. One set of the enzymes is constitutive whereas the other is strictly inducible. To date, only the gene encoding the constitutively-expressed gentisate dioxygenase had been cloned and characterized. A mutant strain of P25X, designated G56, which had the constitutive copy of the gentisate 1,2-dioxygenase gene interrupted by a streptomycin/spectinomycin resistance gene cassette, was found to express gentisate dioxygenase, but only when the cells were induced by gentisate. The proteome profiles of P. alcaligenes P25X and mutant G56 cells grown in the presence and absence of gentisate were compared after two-dimensional polyacrylamide gel electrophoresis. Eight distinctive protein spots (designated M1-M8) which were observed only in induced cells of strain G56 but absent in noninduced cells were further analyzed by matrix-assisted laser desorption/ionization-time of flight, quadrupole-TOF and N-terminal sequencing. Of the 15 proteins (including seven up-regulated) examined, 13 showed sequence similarities to proteins with assigned functions in other microorganisms. The identification of protein M5 which showed high homology to a gentisate dioxygenase from Ralstonia sp. U2 indicated the putative function of this protein being consistent with the inducible gentisate 1,2-dioxygenase in P. alcaligenes. In addition, the induction of stress proteins and other adaptation phenomena were also observed.

  3. Characterization of the endogenous plasmid from Pseudomonas alcaligenes NCIB 9867: DNA sequence and mechanism of transfer.

    PubMed

    Kwong, S M; Yeo, C C; Suwanto, A; Poh, C L

    2000-01-01

    The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The Pac25I restriction-modification system and two mobile elements, Tn5563 and IS1633, were physically localized. An additional eight open reading frames with unknown functions were also detected. pRA2 was genetically tagged with the OmegaStr(r)/Spc(r) gene cassette by homologous recombination. Intrastrain transfer of pRA2-encoded genetic markers between isogenic mutants of P. alcaligenes NCIB 9867 were observed at high frequencies (2.4 x 10(-4) per donor). This transfer was determined to be mediated by a natural transformation process that required cell-cell contact and was completely sensitive to DNase I (1 mg/ml). Efficient transformation was also observed when pRA2 DNA was applied directly onto the cells, while transformation with foreign plasmid DNAs was not observed. pRA2 could be conjugally transferred into Pseudomonas putida RA713 and KT2440 recipients only when plasmid RK2/RP4 transfer functions were provided in trans. Plasmid stability analysis demonstrated that pRA2 could be stably maintained in its original host, P. alcaligenes NCIB 9867, as well as in P. putida RA713 after 100 generations of nonselective growth. Disruption of the pRA2 pac25I restriction endonuclease gene did not alter plasmid stability, while the pRA2 minireplicon exhibited only partial stability. This indicates that other pRA2-encoded determinants could have significant roles in influencing plasmid stability.

  4. Construction and characterization of heavy metal-resistant haloaromatic-degrading Alcaligenes eutrophus strains

    SciTech Connect

    Springael, D.; Diels, L.; Hooyberghs, L.; Kreps, S.; Mergeay, M. )

    1993-01-01

    The use of specialized bacterial strains that are able to degrade organic pollutants, such as polycholorinated biphenyls (PCBs) and pesticides, is becoming increasingly important for decontaminating polluted soils, sludges, and groundwaters. However, bioremediation of sites polluted with organic compounds might be impaired by the presence of heavy metals. The researchers used Alcaligenes eutrophus strains to test responses to heavy metals by evaluating their capacity to degrade organic xenobiotic compounds. The constructed strains were able to grow in the presence of nickel and zinc and exhibited unimpaired biodegradation activities. Both properties are important in considering microorganisms for decontamination of areas with organic xenobiotic and heavy metal pollution. 36 refs., 1 fig., 6 tabs.

  5. Modification of cell surface properties of Pseudomonas alcaligenes S22 during hydrocarbon biodegradation.

    PubMed

    Kaczorek, Ewa; Moszyńska, Sylwia; Olszanowski, Andrzej

    2011-04-01

    Biodegradation of water insoluble hydrocarbons can be significantly increased by the addition of natural surfactants one. Very promising option is the use of saponins. The obtained results indicated that in this system, after 21 days, 92% biodegradation of diesel oil could be achieved using Pseudomonas alcaligenes. No positive effect on the biodegradation process was observed using synthetic surfactant Triton X-100. The kind of carbon source influences the cell surface properties of microorganisms. Modification of the surface cell could be observed by control of the sedimentation profile. This analytical method is a new approach in microbiological analysis.

  6. The enterococcal surface protein, Esp, is involved in Enterococcus faecalis biofilm formation.

    PubMed

    Toledo-Arana, A; Valle, J; Solano, C; Arrizubieta, M J; Cucarella, C; Lamata, M; Amorena, B; Leiva, J; Penadés, J R; Lasa, I

    2001-10-01

    The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

  7. The Enterococcal Surface Protein, Esp, Is Involved in Enterococcus faecalis Biofilm Formation

    PubMed Central

    Toledo-Arana, Alejandro; Valle, Jaione; Solano, Cristina; Arrizubieta, María Jesús; Cucarella, Carme; Lamata, Marta; Amorena, Beatriz; Leiva, José; Penadés, José Rafael; Lasa, Iñigo

    2001-01-01

    The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces. PMID:11571153

  8. Cloning and expression of plasmid genes encoding resistances to chromate and cobalt in Alcaligenes eutrophus.

    PubMed Central

    Nies, A; Nies, D H; Silver, S

    1989-01-01

    Resistances to chromate and cobalt were cloned on a 30-kilobase-pair (kb) DNA region from the large Alcaligenes eutrophus plasmid pMOL28 into the broad-host-range mobilizable cosmid vector pVK102. A restriction nuclease map of the 30-kb region was generated. The resistances expressed from the hybrid plasmids after transfer back into A. eutrophus were inducible and conferred the same degree of resistance as the parent plasmid pMOL28. Resistances were expressed in metal-sensitive Alcaligenes strains and related bacteria but not in Escherichia coli. Resistance to chromate was further localized on a 2.6-kb EcoRI fragment, and resistance to cobalt was localized on an adjoining 8.5-kb PstI-EcoRI fragment. When the 2.6-kb EcoRI fragment was expressed in E. coli under the control of a bacteriophage T7 promoter, three polypeptides with molecular masses of 31,500, 21,000, and 14,500 daltons were visible on autoradiograms. The 31,500- and 21,000-dalton polypeptides were membrane bound; the 14,500-dalton polypeptide was soluble. Images PMID:2549011

  9. IS1491 from Pseudomonas alcaligenes NCIB 9867: characterization and distribution among Pseudomonas species.

    PubMed

    Yeo, C C; Wong, D T; Poh, C L

    1998-01-01

    A new insertion sequence, IS1491, has been cloned and sequenced. The 2489-bp IS1491 was isolated from a Pseudomonas alcaligenes NCIB 9867 (strain P25X) 4.8-kb PstI chromosomal fragment. IS1491 is flanked by an imperfect inverted repeat of 23 bp and carries two overlapping open reading frames, ORF1 and ORF2. Both ORF1 and ORF2 displayed homology to the IstA-like and IstB-like transposases encoded by the IS21 family of insertion sequences, which include two IS elements previously isolated from P. alcaligenes P25X, IS1474, and IS1475 (Yeo, C. C., and Poh, C. L. (1997). FEMS Microbiol. Lett. 149, 257-263). Transposition assays showed that IS1491 transposed at a frequency of approximately 1.4 x 10(-6). Transposition of IS1491 into the target pRK415 replicon was observed but when ORF2 was disrupted, a fusion between the donor and target replicons was detected. IS1491-like sequences were detected in total DNA of Pseudomonas putida NCIB 9869 (strain P35X), Pseudomonas aeruginosa, Pseudomonas stutzeri, Pseudomonas syringae, Pseudomonas mendocina, Comomonas acidovorans, and Comomonas testosteroni by hybridization with IS1491 DNA.

  10. [Extraction, Purification and Identification of a Dexamethasone-degrading Enzymes Generated by Pseudomonas Alcaligenes].

    PubMed

    Zhu, Lili; Yang, Zhibang; Yang, Qian; Shi, Zhongquan; Deng, Xichuan

    2015-10-01

    In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock method. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63% to 52.84%. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrading enzymes were achieved to 1.02 U x mg(-1). Its 5-peptide amino acid sequences were consistent with some sequences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.

  11. Plasmid-determined inducible efflux is responsible for resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus.

    PubMed Central

    Nies, D H; Silver, S

    1989-01-01

    In Alcaligenes eutrophus CH34, resistance to chromate is plasmid determined, inducible, and based on decreased net accumulation of the metal anion. Plasmid-encoded resistances to zinc, cadmium, cobalt, and nickel are resulting from inducible, energy-dependent cation efflux systems. PMID:2914875

  12. The phenotype enhancement method identifies the Xcp outer membrane secretion machinery from Pseudomonas alcaligenes as a bottleneck for lipase production.

    PubMed

    Gerritse, G; Ure, R; Bizoullier, F; Quax, W J

    1998-09-17

    Pseudomonas alcaligenes M-1 has been selected from an intensive screening for micro-organisms that can naturally produce a lipase active in detergent formulations. The lipase expression has been increased to allow high level secretion from Pseudomonas alcaligenes, via the introduction of multi-copy plasmids. In order to improve the lipase yield further, the phenotype enhancement method has been developed. This idea comprises the reintroduction of a cosmid library with random chromosomal fragments in a P. alcaligenes strain with already high lipase productivity. One of the strains which showed an enhanced lipase production appeared to contain a cosmid encoding the outer membrane secretion genes. These xcp-genes are clustered in two divergently transcribed operons similar to the situation in Pseudomonas aeruginosa. Remarkably and dissimilar to P. aeruginosa, in between the two xcp gene clusters, two reading frames of unknown function--OrfV and OrfX--are present. For OrfX no equivalent can be found in the known protein data bases. On the other hand, OrfV shows homology to the regulatory proteins MalT and AcoK. Some evidence is provided that suggests that OrfV acts as a regulator of the xcp operons. A model is proposed for the regulation of the secretion system from P. alcaligenes.

  13. Exchange of Xcp (Gsp) secretion machineries between Pseudomonas aeruginosa and Pseudomonas alcaligenes: species specificity unrelated to substrate recognition.

    PubMed

    de Groot, A; Koster, M; Gérard-Vincent, M; Gerritse, G; Lazdunski, A; Tommassen, J; Filloux, A

    2001-02-01

    Pseudomonas aeruginosa and Pseudomonas alcaligenes are gram-negative bacteria that secrete proteins using the type II or general secretory pathway, which requires at least 12 xcp gene products (XcpA and XcpP to -Z). Despite strong conservation of this secretion pathway, gram-negative bacteria usually cannot secrete exoproteins from other species. Based on results obtained with Erwinia, it has been proposed that the XcpP and/or XcpQ homologs determine this secretion specificity (M. Linderberg, G. P. Salmond, and A. Collmer, Mol. Microbiol. 20:175-190, 1996). In the present study, we report that XcpP and XcpQ of P. alcaligenes could not substitute for their respective P. aeruginosa counterparts. However, these complementation failures could not be correlated to species-specific recognition of exoproteins, since these bacteria could secrete exoproteins of each other. Moreover, when P. alcaligenes xcpP and xcpQ were expressed simultaneously in a P. aeruginosa xcpPQ deletion mutant, complementation was observed, albeit only on agar plates and not in liquid cultures. After growth in liquid culture the heat-stable P. alcaligenes XcpQ multimers were not detected, whereas monomers were clearly visible. Together, our results indicate that the assembly of a functional Xcp machinery requires species-specific interactions between XcpP and XcpQ and between XcpP or XcpQ and another, as yet uncharacterized component(s).

  14. SEQUENCE SIMILARITIES IN THE GENES ENCODING POLY- CHLORINATED BIPHENYL DEGRADATION BY PSEUDOMONAS STRAIN LB400 AND ALCALIGENES EUTROPHUS H850

    EPA Science Inventory

    DNA-DNA hybridization was used to compare the Pseudomonas strain LB400 genes for polychlorinated biphenyl (PCB) degradation with those from seven other PCB-degrading strains. Significant hybridization was detected to the genome of Alcaligenes eutrophus H850, a strain similar to L...

  15. Photocatalytic inactivation of E. faecalis in secondary wastewater plant effluents.

    PubMed

    Backhaus, Karin; Marugán, Javier; van Grieken, Rafael; Sordo, Carlos

    2010-01-01

    Photocatalytic inactivation of Enterococcus faecalis using TiO(2) suspensions was investigated and compared to the inactivation of the most commonly used faecal indicator strain Escherichia coli. In contrast to the inactivation in pure deionized water, disinfection of effluents from the biological process of an urban wastewater plant showed a longer initial lag phase and higher survival fractions after several hours of irradiation. Moreover, the fluctuation of the composition of the effluents strongly affects the overall inactivation rate, not directly related to changes in the values of organic matter content. Additionally, it was found that E. faecalis seems to be more resistant than E.coli towards the photocatalytic treatment. These results could be related to the differences in the cell wall structure of both microorganisms. The main conclusion of this work is that attention must be paid when transferring results obtained for model organism to real bacteria consortia and from laboratory experiments with deionized water to effluents from sewage plants.

  16. Candida albicans and Enterococcus faecalis in the gut

    PubMed Central

    Garsin, Danielle A; Lorenz, Michael C

    2013-01-01

    The fungus Candida albicans and the gram-positive bacterium Enterococcus faecalis are both normal residents of the human gut microbiome and cause opportunistic disseminated infections in immunocompromised individuals. Using a nematode infection model, we recently showed that co-infection resulted in less pathology and less mortality than infection with either species alone and this was partly explained by an interkingdom signaling event in which a bacterial-derived product inhibits hyphal morphogenesis of C. albicans. In this addendum we discuss these findings in the contest of other described bacterial-fungal interactions and recent data suggesting a potentially synergistic relationship between these two species in the mouse gut as well. We suggest that E. faecalis and C. albicans promote a mutually beneficial association with the host, in effect choosing a commensal lifestyle over a pathogenic one. PMID:23941906

  17. Synaptic Activation of Ribosomal Protein S6 Phosphorylation Occurs Locally in Activated Dendritic Domains

    ERIC Educational Resources Information Center

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-01-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6)…

  18. Mechanisms of clinical resistance to fluoroquinolones in Enterococcus faecalis.

    PubMed Central

    Nakanishi, N; Yoshida, S; Wakebe, H; Inoue, M; Mitsuhashi, S

    1991-01-01

    About 10% of 100 clinical isolates of Enterococcus faecalis were resistant to greater than or equal to 25 micrograms of norfloxacin, ofloxacin, ciprofloxacin, and temafloxacin per ml. In this study, the DNA gyrase of E. faecalis was purified from a fluoroquinolone-susceptible strain (ATCC 19433) and two resistant isolates, MS16968 and MS16996. Strains MS16968 and MS16996 were 64- to 128-fold and 16- to 32-fold less susceptible, respectively, to fluoroquinolones than was ATCC 19433; MICs of nonquinolone antibacterial agents for these strains were almost equal. The DNA gyrase from ATCC 19433 had two subunits, designated A and B, with properties similar to those of DNA gyrase from other gram-positive bacteria such as Bacillus subtilis and Micrococcus luteus. Inhibition of the supercoiling activity of the enzyme from ATCC 19433 by the fluoroquinolones correlated with their antibacterial activities. In contrast, preparations of DNA gyrase from MS16968 and MS16996 were at least 30-fold less sensitive to inhibition of supercoiling by the fluoroquinolones than the gyrase from ATCC 19433 was. Experiments that combined heterologous gyrase subunits showed that the A subunit from either of the resistant isolates conferred resistance to fluoroquinolones. These findings indicate that an alteration in the gyrase A subunit is the major contributor to fluoroquinolone resistance in E. faecalis clinical isolates. A difference in drug uptake may also contribute to the level of fluoroquinolone resistance in these isolates. Images PMID:1656852

  19. Enterococcal surface protein, Esp, enhances biofilm formation by Enterococcus faecalis.

    PubMed

    Tendolkar, Preeti M; Baghdayan, Arto S; Gilmore, Michael S; Shankar, Nathan

    2004-10-01

    Enterococci play a dual role in human ecology. They serve as commensal organisms of the gastrointestinal tract and are also leading causes of multiple antibiotic-resistant hospital-acquired infection. Many nosocomial infections result from the ability of microorganisms to form biofilms. The molecular mechanisms involved in enterococcal biofilm formation are only now beginning to be understood. Enterococcal surface protein, Esp, has been reported to contribute to biofilm formation by Enterococcus faecalis. Recent studies have shown that enterococci form biofilms independently of Esp expression. To precisely determine what role Esp plays in E. faecalis biofilm formation, Esp was expressed on the cell surface of genetically well-defined, natively Esp-deficient strains, and isogenic Esp-positive and Esp-deficient strains were compared for their biofilm-forming ability. The results show that Esp expression leads to a significant increase in biofilm formation, irrespective of the strain tested. The contribution of Esp to biofilm formation was found to be most pronounced in the presence of 0.5% (wt/vol) or greater glucose. These results unambiguously define Esp as a key contributor to the ability of E. faecalis to form biofilms.

  20. FORMATION OF PROTOPLASTS FROM STREPTOCOCCUS FAECALIS BY LYSOZYME1

    PubMed Central

    Bibb, William R.; Straughn, W. R.

    1962-01-01

    Bibb, William R. (University of North Carolina, Chapel Hill) and W. R. Straughn. Formation of protoplasts from Streptococcus faecalis by lysozyme. J. Bacteriol. 84:1094–1098. 1962.—Incubation of whole cells of Streptococcus faecalis F24 in the presence of the crystalline egg-white lysozyme and appropriate sucrose concentration resulted in the formation of discrete spherical structures. On dilution, these osmotically fragile structures lysed immediately. Methyl pentose determinations on isolated cell walls and protoplast membranes verified the presence of rhamnose in the cell walls and its essentially complete absence in protoplast membranes. Cell walls were rendered soluble by lysozyme. After lysozyme treatment of cell walls, 96% of the rhamnose present was not sedimented by centrifugation at 12,500 × g for 30 min. No cell-wall structures were recognized by phasecontrast or electron microscopy. After direct lysis of whole cells of S. faecalis F24 by lysozyme, protoplast membranes were isolated. It is concluded that, in the strain of group D streptococcus studied, lysozyme effectively removes the cell wall. Images PMID:13968087

  1. Antimicrobial effect of alexidine and chlorhexidine against Enterococcus faecalis infection

    PubMed Central

    Kim, Hyun-Shik; Woo Chang, Seok; Baek, Seung-Ho; Han, Seung Hyun; Lee, Yoon; Zhu, Qiang; Kum, Kee-Yeon

    2013-01-01

    A previous study demonstrated that alexidine has greater affinity for the major virulence factors of bacteria than chlorhexidine. The aim of this study was to compare the antimicrobial activity of 1% alexidine with that of 2% chlorhexidine using Enterococcus faecalis-infected dentin blocks. Sixty bovine dentin blocks were prepared and randomly divided into six groups of 10 each. E. faecalis was inoculated on 60 dentin blocks using the Luppens apparatus for 24 h and then the dentin blocks were soaked in 2% chlorhexidine or 1% alexidine solutions for 5 and 10 min, respectively. Sterile saline was used as a control. The antimicrobial efficacy was assessed by counting the number of bacteria adhering to the dentin surface and observing the degradation of bacterial shape or membrane rupture under a scanning electron microscope. Significantly fewer bacteria were observed in the 2% chlorhexidine- or 1% alexidine-soaked groups than in the control group (P<0.05). However, there was no significant difference in the number of bacteria adhering to the dentinal surface between the two experimental groups or between the two soaking time groups (P>0.05). Ruptured or antiseptic-attached bacteria were more frequently observed in the 10-min-soaked chlorhexidine and alexidine groups than in the 5-min-soaked chlorhexidine and alexidine groups. In conclusion, 10-min soaking with 1% alexidine or 2% chlorhexidine can be effective against E. faecalis infection. PMID:23492900

  2. The opportunistic pathogen Enterococcus faecalis resists phagosome acidification and autophagy to promote intracellular survival in macrophages.

    PubMed

    Zou, Jun; Shankar, Nathan

    2016-06-01

    While many strains of Enterococcus faecalis have been reported to be capable of surviving within macrophages for extended periods, the exact mechanisms involved are largely unknown. In this study, we found that after phagocytosis by macrophages, enterococci-containing vacuoles resist acidification, and E. faecalis is resistant to low pH. Ultrastructural examination of the enterococci-containing vacuole by transmission electron microscopy revealed a single membrane envelope, with no evidence of the classical double-membraned autophagosomes. Western blot analysis further confirmed that E. faecalis could trigger inhibition of the production of LC3-II during infection. By employing cells transfected with RFP-LC3 plasmid and infected with GFP-labelled E. faecalis, we also observed that E. faecalis was not delivered into autophagosomes during macrophage infection. While these observations indicated no role for autophagy in elimination of intracellular E. faecalis, enhanced production of reactive oxygen species and nitric oxide were keys to this process. Stimulation of autophagy suppressed the intracellular survival of E. faecalis in macrophages in vitro and decreased the burden of E. faecalis in vivo. In summary, the results from this study offer new insights into the interaction of E. faecalis with host cells and may provide a new approach to treatment of enterococcal infections.

  3. Removal of S6K1 and S6K2 Leads to Divergent Alterations in Learning, Memory, and Synaptic Plasticity

    ERIC Educational Resources Information Center

    Antion, Marcia D.; Merhav, Maayan; Hoeffer, Charles A.; Reis, Gerald; Kozma, Sara C.; Thomas, George; Schuman Erin M.; Rosenblum, Kobi; Klann, Eric

    2008-01-01

    Protein synthesis is required for the expression of enduring memories and long-lasting synaptic plasticity. During cellular proliferation and growth, S6 kinases (S6Ks) are activated and coordinate the synthesis of de novo proteins. We hypothesized that protein synthesis mediated by S6Ks is critical for the manifestation of learning, memory, and…

  4. Anaerobic taurine oxidation: a novel reaction by a nitrate-reducing Alcaligenes sp.

    PubMed

    Denger, K; Laue, H; Cook, A M

    1997-06-01

    Enrichment cultures were prepared under strictly anoxic conditions in medium representing fresh water and containing an organosulfonate as electron donor and carbon source, and nitrate as electron acceptor. The inoculum was from the anaerobic digestor of two communal sewage works. The natural organosulfonates 2-aminoethanesulfonate (taurine), DL-2-amino-3-sulfopropionate (cysteate) and 2-hydroxyethanesulfonate (isethionate) all gave positive enrichments, whereas unsubstituted alkanesulfonates, such as methanesulfonate and arenesulfonates, gave no enrichment. Two representative enrichments were used to obtain pure cultures, and strains NKNTAU (utilizing taurine) and NKNIS (utilizing isethionate) were isolated. Strain NKNTAU was examined in detail. Out of 18 tested organosulfonates, it utilized only one, taurine, and was identified as a novel Alcaligenes sp., a facultatively anaerobic bacterium. Carbon from taurine was converted to cell material and carbon dioxide. The amino group was released as ammonium ion and the sulfonate moiety was recovered as sulfate. Nitrate was reduced to nitrogen gas.

  5. Alcaligenes eutrophus JMP134 "2,4-dichlorophenoxyacetate monooxygenase" is an alpha-ketoglutarate-dependent dioxygenase.

    PubMed Central

    Fukumori, F; Hausinger, R P

    1993-01-01

    The Alcaligenes eutrophus JMP134 tfdA gene, encoding the enzyme responsible for the first step in 2,4-dichlorophenoxyacetic acid (2,4-D) biodegradation, was overexpressed in Escherichia coli, and several enzymatic properties of the partially purified gene product were examined. Although the tfdA-encoded enzyme is typically referred to as 2,4-D monooxygenase, we were unable to observe any reductant-dependent activity. Rather, we demonstrate that this enzyme is a ferrous ion-dependent dioxygenase that uses alpha-ketoglutarate as a cosubstrate. The alpha-ketoglutarate is converted to succinate concomitant with 2,4-D conversion to 2,4-dichlorophenol. By using [1-14C]alpha-ketoglutarate, we established that carbon dioxide is the second product derived from alpha-ketoglutarate. Finally, we verified the proposal that glyoxylate is the second product derived from 2,4-D. PMID:8458850

  6. The purification and chemical composition of the lipopolysaccharide of Pseudomonas alcaligenes

    PubMed Central

    Key, B. A.; Gray, G. W.; Wilkinson, S. G.

    1970-01-01

    1. A method for obtaining lipopolysaccharide free from glycosaminopeptide from isolated cell walls of Pseudomonas alcaligenes is discussed. 2. About 70–75% of the lipopolysaccharide and 86–90% of the isolated lipid A have been accounted for in terms of identifiable components. 3. Hydrolysates of lipid A contain mainly inorganic phosphate, glucosamine, O-phosphorylglucosamine and fatty acids (dodecanoic acid, dodec-2-enoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid), of which the last is the main N-acylating acid of the glucosamine backbone. 4. Material corresponding to the polysaccharide moiety of the lipopolysaccharide is extensively degraded. 5. Solubilization of the lipopolysaccharide by using sodium deoxycholate appreciably affects the chemical composition of the material. PMID:5499968

  7. A cytochrome cd1-type nitrite reductase mediates the first step of denitrification in Alcaligenes eutrophus.

    PubMed

    Sann, R; Kostka, S; Friedrich, B

    1994-01-01

    Respiratory nitrite reductase (NIR) has been purified from the soluble extract of denitrifying cells of Alcaligenes eutrophus strain H16 to apparent electrophoretic homogeneity. The enzyme was induced under anoxic conditions in the presence of nitrite. Purified NIR showed typical features of a cytochrome cd1-type nitrite reductase. It appeared to be a dimer of kDa subunits, its activity was only weakly inhibited by the copper chelator diethyldithiocarbamate, and spectral analysis revealed absorption maxima which were characteristic for the presence of heme c and heme d1. The isoelectric point of 8.6 was considerably higher than the pI determined for cd1 nitrite reductases from pseudomonads. Eighteen amino acids at the N-terminus of the A. eutrophus NIR, obtained by protein sequencing, showed no significant homology to the N-terminal region of nitrite reductases from Pseudomonas stutzeri and Pseudomonas aeruginosa.

  8. Soil bacteria Pseudomonas putida and Alcaligenes xylosoxidans subsp. denitrificans inactivate triclosan in liquid and solid substrates.

    PubMed

    Meade, M J; Waddell, R L; Callahan, T M

    2001-10-16

    Triclosan is a broad-spectrum antimicrobial agent that has been incorporated into many household and medical products. Bacteria with high levels of triclosan resistance were isolated from compost, water, and soil samples. Two of these bacteria, Pseudomonas putida TriRY and Alcaligenes xylosoxidans subsp. denitrificans TR1, were able to use triclosan as a sole carbon source and clear particulate triclosan from agar. A decrease in triclosan concentration was measured by HPLC within 6 h of inoculation with strain TriRY and 24 h with strain TR1. Bioassays demonstrated that triclosan was inactivated in liquid cultures and/or embedded in plastic by the growth of strain TriRY and strain TR1, permitting the growth of triclosan-sensitive bacteria.

  9. Strain of alcaligenes latus bacteria used for the decomposition of polychlorinated biphenyls

    DOEpatents

    Dyadischev, Nikolai Romanovich; Zharikov, Gennady Alekseevich; Kapranov, Vladimir Vladimirovich

    2001-09-11

    Alcaligenes latus bacterial strain TXD-13 VKPM B 75-05 is capable of degrading polychlorinated biphenyls (PCBs). The strain may be employed to detoxicate environment media and PCB-containing industrial waste. To produce biomass, the strain is incubated on media which contain carbon sources, nitrogen sources and mineral salts. The strain is cultivated by a subsurface method up to a titer from 6.0.multidot.10.sup.8 to 2.0.times.10.sup.9 cells per cu cm. The produced biomass is used for degrading PCBs in concentrations from 10.sup.7 to 10.sup.8 cells per cu cm. The strain ensures from 35 to 50% reduction in PCB content in soil and water.

  10. Molecular characterization of extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase genes from Pseudomonas alcaligenes strains.

    PubMed

    Kim, Do Young; Kim, Hyun Chul; Kim, Sun Young; Rhee, Young Ha

    2005-06-01

    A bacterial strain M4-7 capable of degrading various polyesters, such as poly(epsilon-caprolactone), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxyoctanoate), and poly(3-hydroxy-5-phenylvalerate), was isolated from a marine environment and identified as Pseudomonas alcaligenes. The relative molecular mass of a purified extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase (PhaZ(PalM4-7)) from P. alcaligenes M4-7 was 28.0 kDa, as determined by SDS-PAGE. The PhaZ(PalM4-7) was most active in 50 mM glycine-NaOH buffer (pH 9.0) at 35 degrees C. It was insensitive to dithiothreitol, sodium azide, and iodoacetamide, but susceptible to p-hydroxymercuribenzoic acid, N-bromosuccinimide, acetic anhydride, EDTA, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, Tween 80, and Triton X-100. In this study, the genes encoding MCL-PHA depolymerase were cloned, sequenced, and characterized from a soil bacterium, P. alcaligenes LB19 (Kim et al., 2002, Biomacromolecules 3, 291-296) as well as P. alcaligenes M4-7. The structural gene (phaZ(PalLB19)) of MCL-PHA depolymerase of P. alcaligenes LB19 consisted of an 837 bp open reading frame (ORF) encoding a protein of 278 amino acids with a deduced M((r)) of 30,188 Da. However, the MCL-PHA depolymerase gene (phaZ(PalM4-7)) of P. alcaligenes M4-7 was composed of an 834 bp ORF encoding a protein of 277 amino acids with a deduced Mr of 30,323 Da. Amino acid sequence analyses showed that, in the two different polypeptides, a substrate-binding domain and a catalytic domain are located in the N-terminus and in the C-terminus, respectively. The PhaZ(PalLB19) and the PhaZ(PalM4-7) commonly share the lipase box, GISSG, in their catalytic domains, and utilize 111Asn and 110Ser residues, respectively, as oxyanions that play an important role in transition-state stabilization of hydrolytic reactions.

  11. Characterization of PF-4708671, a novel and highly specific inhibitor of p70 ribosomal S6 kinase (S6K1).

    PubMed

    Pearce, Laura R; Alton, Gordon R; Richter, Daniel T; Kath, John C; Lingardo, Laura; Chapman, Justin; Hwang, Catherine; Alessi, Dario R

    2010-10-15

    S6K1 (p70 ribosomal S6 kinase 1) is activated by insulin and growth factors via the PI3K (phosphoinositide 3-kinase) and mTOR (mammalian target of rapamycin) signalling pathways. S6K1 regulates numerous processes, such as protein synthesis, growth, proliferation and longevity, and its inhibition has been proposed as a strategy for the treatment of cancer and insulin resistance. In the present paper we describe a novel cell-permeable inhibitor of S6K1, PF-4708671, which specifically inhibits the S6K1 isoform with a Ki of 20 nM and IC50 of 160 nM. PF-4708671 prevents the S6K1-mediated phosphorylation of S6 protein in response to IGF-1 (insulin-like growth factor 1), while having no effect upon the PMA-induced phosphorylation of substrates of the highly related RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated kinase) kinases. PF-4708671 was also found to induce phosphorylation of the T-loop and hydrophobic motif of S6K1, an effect that is dependent upon mTORC1 (mTOR complex 1). PF-4708671 is the first S6K1-specific inhibitor to be reported and will be a useful tool for delineating S6K1-specific roles downstream of mTOR.

  12. Production optimization of cyanophycinase ChpEal from Pseudomonas alcaligenes DIP1

    PubMed Central

    2011-01-01

    Pseudomonas alcaligenes DIP1 produces an extracellular cyanophycinase (CphEal). The corresponding gene (cphEal) was identified from subclones of a genomic DNA gene library by heterologously expressing the functionally active enzyme in Escherichia coli. The nucleotide sequence of the gene (1260 base pairs) was determined indicating a theoretical mass of 43.6 kDa (mature CphEal) plus a leader peptide of 2,6 kDa which corresponds well to the apparent molecular mass of 45 kDa as revealed by SDS-PAGE. The enzyme exhibited a high sequence identity of 91% with the extracellular cyanophycinase from P. anguilliseptica strain BI and carried an N-terminal Sec secretion signal peptide. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the serine motif GXSXG plus a histidine and a glutamate residue, suggesting a catalytic mechanism similar to serine-type proteases. The cyanophycinase (CphEal) was heterologously produced in two different E. coli strains (Top10 and BL21(DE3)) from two plasmid vectors (pBBR1MCS-4 and pET-23a(+)). The signal peptide of CphEal was cleaved in E. coli, suggesting active export of the protein at least to the periplasm. Substantial enzyme activity was also present in the culture supernatants. The extracellular cyanophycinase activities in E. coli were higher than activities in the wild type P. alcaligenes DIP1 in complex LB medium. Highest extracellular enzyme production was achieved with E. coli BL21(DE3) expressing CphEal from pBBR1MCS-4. Using M9 minimal medium was less effective, but the relatively low cost of mineral salt media makes these results important for the industrial-scale production of dipeptides from cyanophycin. PMID:22060187

  13. Purification and characterization of a novel caffeine oxidase from Alcaligenes species.

    PubMed

    Mohapatra, B R; Harris, N; Nordin, R; Mazumder, A

    2006-09-18

    Alcaligenes species CF8 isolated from surface water of a lake produced a novel serine type metallo-caffeine oxidase. The optimal medium for caffeine oxidase production by this strain was (w/v) NaNO(3), 0.4%; KH(2)PO(4), 0.15%; Na(2)HPO(4), 0.05%; FeCl(3).6H(2)O, 0.0005%; CaCl(2).2H(2)O, 0.001%; MgSO(4).7H(2)O, 0.02%; glucose, 0.2%; caffeine, 0.05%, pH 7.5. The enzyme was purified to 63-fold by using ammonium sulfate precipitation, dialysis, ion exchange (diethylaminoethyl-cellulose) and gel filtration (Sephadex G-100) chromatographic techniques. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified caffeine oxidase was monomeric with a molecular mass of 65 kDa. The purified caffeine oxidase with a half-life of 20 min at 50 degrees C had maximal activity at pH 7.5 and 35 degrees C. The purified caffeine oxidase had strict substrate specificity towards caffeine (K(m) 8.94 microM and V(max) 47.62 U mg protein(-1)) and was not able to oxidize xanthine and hypoxanthine. The enzyme activity was not inhibited by para-chloromercuribenzoic acid, iodoacetamide, n-methylmaleimide, salicylic acid and sodium arsenite indicating the enzyme did not belong to xanthine oxidase family. The enzyme was not affected by Ca(+2), Mg(+2) and Na(+), but was completely inhibited by Co(+2), Cu(+2) and Mn(+2) at 1mM level. The novel caffeine oxidase isolated here from Alcaligenes species CF8 may be useful in biotechnological processes including waste treatment and biosensor development.

  14. Production optimization of cyanophycinase ChpEal from Pseudomonas alcaligenes DIP1.

    PubMed

    Sallam, Ahmed; Kalkandzhiev, Dimitar; Steinbüchel, Alexander

    2011-11-07

    Pseudomonas alcaligenes DIP1 produces an extracellular cyanophycinase (CphEal). The corresponding gene (cphEal) was identified from subclones of a genomic DNA gene library by heterologously expressing the functionally active enzyme in Escherichia coli. The nucleotide sequence of the gene (1260 base pairs) was determined indicating a theoretical mass of 43.6 kDa (mature CphEal) plus a leader peptide of 2,6 kDa which corresponds well to the apparent molecular mass of 45 kDa as revealed by SDS-PAGE. The enzyme exhibited a high sequence identity of 91% with the extracellular cyanophycinase from P. anguilliseptica strain BI and carried an N-terminal Sec secretion signal peptide. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the serine motif GXSXG plus a histidine and a glutamate residue, suggesting a catalytic mechanism similar to serine-type proteases. The cyanophycinase (CphEal) was heterologously produced in two different E. coli strains (Top10 and BL21(DE3)) from two plasmid vectors (pBBR1MCS-4 and pET-23a(+)). The signal peptide of CphEal was cleaved in E. coli, suggesting active export of the protein at least to the periplasm. Substantial enzyme activity was also present in the culture supernatants. The extracellular cyanophycinase activities in E. coli were higher than activities in the wild type P. alcaligenes DIP1 in complex LB medium. Highest extracellular enzyme production was achieved with E. coli BL21(DE3) expressing CphEal from pBBR1MCS-4. Using M9 minimal medium was less effective, but the relatively low cost of mineral salt media makes these results important for the industrial-scale production of dipeptides from cyanophycin.

  15. Identification of the S6 kinase activity stimulated in quiescent brine shrimp embryos upon entry to preemergence development as p70 ribosomal protein S6 kinase: Isolation of Artemia franciscana p70S6k cDNA

    PubMed Central

    Santiago, J.; Sturgill, T.W.

    2010-01-01

    We previously demonstrated that a protein kinase responsible for phosphorylating 40S ribosomal subunits is activated in quiescent Artemia franciscana embryos within 15 min of restoration of normal tonicity and incubation at 30°C. Here, we identify the activated S6 kinase as A. franciscana p70 ribosomal S6 kinase (p70S6k) subsequent to the isolation of an Artemia p70S6k cDNA. The protein conceptually translated from cDNA has 70% similarity and 64% identity to both Drosophila melanogaster and human p70S6k. Southern blot analysis is consistent with presence of a single p70S6k gene. Two transcripts of 5.4 and 2.7 kb were found. Abundance of both mRNAs increased dramatically around 4 h of preemergence development, and exhibited different steady-state level variation thereafter. Stimulated S6 kinase activity, partially purified by Superose 6 chromatography, correlated best with the slowest migrating, ~65 kDa, form detected by Western analysis using a specific polyclonal antibody made to a peptide from the predicted p70S6k NH2-terminus. Furthermore, the A. franciscana p70S6k was immunoprecipitated with the same antibody, showing in parallel an S6 kinase activity similar to peak profiles. We conclude that the stimulated S6 kinase activity is that of an ortholog of human p70S6k that may be involved in the regulation of protein synthesis during preemergence development in A. franciscana species. PMID:11310561

  16. Prevalence of Enterococcus faecalis in saliva and filled root canals of teeth associated with apical periodontitis

    PubMed Central

    Wang, Qian-Qian; Zhang, Cheng-Fei; Chu, Chun-Hung; Zhu, Xiao-Fei

    2012-01-01

    To investigate the prevalence of Enterococcus faecalis in saliva and filled root canals of patients requiring endodontic retreatment for apical periodontitis. Patients with apical periodontitis who were referred for endodontic retreatment were examined. The type and quality of the restoration, symptoms, quality of obturation were recorded. During retreatment, an oral rinse sample and root canal sample were cultured using brain-heart infusion agar and bile esculinazide agar to select for E. faecalis. The 16S rRNA technique was used to identify E. faecalis. A total of 32 women and 22 men (mean age: 38 years; s.d.: 11 years) and 58 teeth were studied. The prevalence of E. faecalis was 19% in the saliva and 38% in the root canals. The odds that root canals harbored E. faecalis were increased if the saliva habored this bacterium (odds ratio=9.7; 95% confidence interval=1.8–51.6; P<0.05). Teeth with unsatisfactory root obturation had more cultivable bacterial species in root canals than teeth with satisfactory root obturation (P<0.05). E. faecalis is more common in root canals of teeth with apical periodontitis than in saliva. The prevalence of E. faecalis in root canals is associated with the presence of E. faecalis in saliva. PMID:22422085

  17. Transmission and genetic diversity of Enterococcus faecalis during hatch of broiler chicks.

    PubMed

    Olsen, Rikke Heidemann; Christensen, Henrik; Bisgaard, Magne

    2012-11-09

    The normal gastrointestinal flora of poultry includes Enterococcus faecalis. E. faecalis is also associated with first week mortality of chickens, but it is not clear whether this is due to vertical or horizontal transmission. Aims of the present study were to investigate transmission and genetic diversity of E. faecalis during hatching of broiler chicks. When hatching started, 15% of the chicks were colonized with E. faecalis. This colonization was interpreted as vertical transmission and was higher than previously reported. Transmission of E. faecalis from parents older than 42 weeks was five times greater than transmission of E. faecalis from younger parents. Seventy percent of broiler chicks were colonized with E. faecalis within 24 h after hatch started, which was interpreted as horizontal transmission. Twenty-one sequence types (STs) were demonstrated among 322 isolates of E. faecalis obtained from newly hatched chicks representing 11 different broiler parent flocks. Furthermore, three STs (ST59, ST82, ST174) made up 50.6% of the isolates, indicating that these STs have adapted successfully to the avian niche. All STs, except those novel to this study, have previously been associated with lesions in poultry, underlining the importance of controlling these particular STs.

  18. Prevalence of Enterococcus faecalis in saliva and filled root canals of teeth associated with apical periodontitis.

    PubMed

    Wang, Qian-Qian; Zhang, Cheng-Fei; Chu, Chun-Hung; Zhu, Xiao-Fei

    2012-03-01

    To investigate the prevalence of Enterococcus faecalis in saliva and filled root canals of patients requiring endodontic retreatment for apical periodontitis. Patients with apical periodontitis who were referred for endodontic retreatment were examined. The type and quality of the restoration, symptoms, quality of obturation were recorded. During retreatment, an oral rinse sample and root canal sample were cultured using brain-heart infusion agar and bile esculinazide agar to select for E. faecalis. The 16S rRNA technique was used to identify E. faecalis. A total of 32 women and 22 men (mean age: 38 years; s.d.: 11 years) and 58 teeth were studied. The prevalence of E. faecalis was 19% in the saliva and 38% in the root canals. The odds that root canals harbored E. faecalis were increased if the saliva habored this bacterium (odds ratio=9.7; 95% confidence interval=1.8-51.6; P<0.05). Teeth with unsatisfactory root obturation had more cultivable bacterial species in root canals than teeth with satisfactory root obturation (P<0.05). E. faecalis is more common in root canals of teeth with apical periodontitis than in saliva. The prevalence of E. faecalis in root canals is associated with the presence of E. faecalis in saliva.

  19. Efficacy of Atmospheric Pressure Plasma as an Antibacterial Agent Against Enterococcus Faecalis in Vitro

    NASA Astrophysics Data System (ADS)

    Cao, Yingguang; Yang, Ping; Lu, Xinpei; Xiong, Zilan; Ye, Tao; Xiong, Qing; Sun, Ziyong

    2011-02-01

    Enterococcus faecalis (E. faecalis) is a microorganism that can survive extreme challenges in obturated root canals. The aim of this study was to evaluate the efficacy of a non-thermal atmospheric pressure plasma plume against E. faecalis in vitro. A non-thermal atmospheric pressure plasma jet device which could generate a cold plasma plume carrying a peak current of 300 mA was used. The antibacterial efficacy of this device against E. faecalis and its biofilm under different conditions was detected. The antibacterial efficacy of the plasma against E. faecalis and Staphylococcus aureus (S. aureus) was also evaluated. After plasma treatment, the average diameter of inhibition zone on S. aureus and E. faecalis was 2.62±0.26 cm and 1.06±0.30 cm, respectively (P < 0.05). The diameter was increased with prolongation of the treatment duration. The diameters of inhibition zone of the sealed Petri dishes were larger than those of the uncovered Petri dishes. There was significant difference in colony-forming units between plasma group and control group on E. faecalis biofilm (P < 0.01). The transmission electron microscopy revealed that the ultrastructural changes cytoderm of E. faecalis were observed after treatment for 2 min. It is concluded that the non-thermal atmospheric pressure plasma could serve as an effective adjunct to standard endodontic microbial treatment.

  20. REAL-TIME PCR METHOD TO DETECT ENTEROCOCCUS FAECALIS IN WATER

    EPA Science Inventory

    A 16S rDNA real-time PCR method was developed to detect Enterococcus faecalis in water samples. The dynamic range for cell detection spanned five logs and the detection limit was determined to be 6 cfu/reaction. The assay was capable of detecting E. faecalis cells added to biof...

  1. Biological changes of Enterococcus faecalis in the viable but nonculturable state.

    PubMed

    E, J; Jiang, Y T; Yan, P F; Liang, J P

    2015-11-23

    Enterococcus faecalis may enter a viable but nonculturable (VBNC) state under adverse conditions. E. faecalis, the major bacterial species present in failed root canal treatments, is thought to survive after endodontic treatment by entering a VBNC state. In this study, we characterized the VBNC state of E. faecalis. We designed 3 different protocols to successfully induce the VBNC state. Approximately one-third of bacteria entered a VBNC state after 15-30 days, and all remained viable for at least 2 months. The morphology, glycometabolism, and adhesion capabilities of VBNC cells differed from those of E. faecalis during the exponential growth phase. Specifically, VBNC E. faecalis cells could not decompose lactose, D-mannitol, or D-sorbitol, although they were able to metabolize sucrose. Transmission electron microscopy showed that the morphology of the VBNC E. faecalis cells changed significantly; the cytoplasmic matrix was unevenly condensed and the overall morphology of the cells became irregular, but the cell membranes remained intact. Although the adhesion ability of the bacteria decreased, VBNC E. faecalis could still adhere to collagen fiber type I and tooth dentine. The persistence of this adhesion ability may be important in the virulence of VBNC E. faecalis.

  2. In vitro inactivation of Enterococcus faecalis with a led device.

    PubMed

    D'Ercole, S; Spoto, G; Trentini, P; Tripodi, D; Petrini, M

    2016-07-01

    Non-coherent light-emitting diodes (LEDs) are effective in a large variety of clinical indications; however, the bactericidal activity of LEDs is unclear, although the effectiveness of such lights is well known. Currently, no studies have examined the effects of NIR-LED on bacteria. The aims of this study were to verify the antibacterial activity of 880-nm LED irradiation on a bacterial suspension of Enterococcus faecalis and to compare it with the actions of sodium hypochlorite (NaOCl) and the concurrent use of both treatments. Before we proceeded with the main experiment, we first performed preliminary tests to evaluate the influence of such parameters as the distance of irradiation, the energy density, the irradiation time and the presence of photosensitizers on the antimicrobial effects of LEDs. After treatment, the colony forming units per milliliter (CFU/mL) was recorded and the data were submitted to ANOVA and Bonferroni post hoc tests at a level of significance of 5%. The results showed that LED irradiation, at the parameters used, is able to significantly decrease E. faecalis viability in vitro. The total inhibition of E. faecalis was obtained throughout concurrent treatment of LED and NaOCl (1%) for 5min. The same antimicrobial activity was confirmed in all of the experiments (p<0.05), but no statistically significant differences were found by varying such parameters as the distance of irradiation (from 0.5mm to 10mm), energy density (from 2.37 to 8.15mJ/s), irradiation time (from 5min to 20min) or by adding toluidine blue O (TBO).

  3. Probiotic properties and adsorption of Enterococcus faecalis PSCT3-7 to vermiculite

    PubMed Central

    Kim, Jin-Yoon; Awji, Elias Gebru; Park, Na-Hye; Park, Ji-Yong; Kim, Jong-Choon; Lee, Sam-Pin

    2017-01-01

    The probiotic properties of Enterococcus (E.) faecalis PSCT3-7, a new strain isolated from the intestines of pigs fed dietary fiber containing 50% sawdust, were investigated. E. faecalis PSCT3-7 tolerated a pH range of 3 to 8 and 0.3% bile salts, and it inhibited the growth of Salmonella Typhimurium in a concentration-dependent manner. In addition, E. faecalis showed resistance to several antibacterial agents. Vermiculite, a nutrient and microbial carrier, increased the bile tolerance of the strain. Scanning electron microscope images revealed good adsorption of E. faecalis PSCT3-7 onto vermiculite. E. faecalis PSCT3-7 represents a potential probiotic candidate to administer with vermiculite to swine. PMID:27456777

  4. Probiotic properties and adsorption of Enterococcus faecalis PSCT3-7 to vermiculite.

    PubMed

    Kim, Jin-Yoon; Awji, Elias Gebru; Park, Na-Hye; Park, Ji-Yong; Kim, Jong-Choon; Lee, Sam-Pin; Suh, Joo-Won; Park, Seung-Chun

    2017-03-30

    The probiotic properties of Enterococcus (E.) faecalis PSCT3-7, a new strain isolated from the intestines of pigs fed dietary fiber containing 50% sawdust, were investigated. E. faecalis PSCT3-7 tolerated a pH range of 3 to 8 and 0.3% bile salts, and it inhibited the growth of Salmonella Typhimurium in a concentration-dependent manner. In addition, E. faecalis showed resistance to several antibacterial agents. Vermiculite, a nutrient and microbial carrier, increased the bile tolerance of the strain. Scanning electron microscope images revealed good adsorption of E. faecalis PSCT3-7 onto vermiculite. E. faecalis PSCT3-7 represents a potential probiotic candidate to administer with vermiculite to swine.

  5. Delivery of a genetically marked Alcaligenes sp. to the glassy-winged sharpshooter for use in a paratransgenic control strategy.

    PubMed

    Bextine, Blake; Lauzon, Carol; Potter, Sarah; Lampe, David; Miller, Thomas A

    2004-05-01

    An artificial feeding system was designed for the glassy-winged sharpshooter (GWSS), Homalodisca coagulata Say (Hemiptera: Cicadellidae). The system, unlike previous systems, provided enough nutrients to GWSS to survive for 48 h. A system like this is a prerequisite to examining the potential use of paratransgenesis to interrupt transmission of Xylella fastidiosa, the bacterial pathogen causing Pierce's disease of grape, by insect vectors. We developed a system for short-term feeding of GWSS that allows for the introduction of bacteria in liquid medium, and we have demonstrated the ability of Alcaligenes xylosoxidans denitrificans, expressing a red fluorescent protein (dsRed), to colonize the cibarial region of the GWSS foregut for up to 5 weeks post-exposure. Alcaligenes xylosoxidans denitrificans thus occupies the same region in the foregut as the pathogen, Xylella fastidiosa.

  6. HbzF catalyzes direct hydrolysis of maleylpyruvate in the gentisate pathway of Pseudomonas alcaligenes NCIMB 9867.

    PubMed

    Liu, Kun; Liu, Ting-Ting; Zhou, Ning-Yi

    2013-02-01

    HbzF from Pseudomonas alcaligenes NCIMB 9867 was purified to homogeneity as a His-tagged protein and likely a dimer by SDS-PAGE and gel filtration. This protein was demonstrated to be a novel maleylpyruvate hydrolase, catalyzing direct hydrolysis of maleylpyruvate to maleate and pyruvate, and belongs to the fumarylacetoacetate hydrolase superfamily. This study reveals the genetic determinate for the direct maleylpyruvate hydrolysis in the gentisate pathway, complementary to the well-studied maleylpyruvate isomerization route.

  7. Susceptibilties of two Enterococcus faecalis phenotypes to root canal medications.

    PubMed

    Abdullah, Mariam; Ng, Yuan-Ling; Gulabivala, Kishor; Moles, David R; Spratt, David A

    2005-01-01

    This study aimed to investigate and compare the efficacy of selected root canal irrigants and a medicament on a clinical isolate of Enterococcus faecalis grown as biofilm or planktonic suspension phenotype. A cell-dense pellet "presentation" prepared from planktonic phenotype was also tested. Each bacterial presentation was exposed to calcium hydroxide (pH 12.3), 0.2% chlorhexidine gluconate, 17% ethylene-diamine-tetra-acetic acid, 10% povidone iodine, or 3.0% sodium hypochlorite (NaOCl) for a range of time periods (1, 2, 4, 8, 15, 30, and 60 min). Phosphate buffered saline was used as a control agent. The difference in gradients of bacterial killing among the biofilm, planktonic suspension or pellet presentation was significant (p < 0.05) and dependent upon the test agent except in the case of NaOCl and calcium hydroxide where no difference could be detected. NaOCl was the most effective agent and achieved 100% kills for all presentations of E. faecalis after a 2 min contact time.

  8. Pulmonary hypertension syndrome in broilers caused by Enterococcus faecalis.

    PubMed

    Tankson, J D; Thaxton, J P; Vizzier-Thaxton, Y

    2001-10-01

    A field strain of Enterococcus faecalis was administered to broiler chicks at doses of 0, 3 x 10(6), 1.5 x 10(7), and 2 x 10(7) bacteria/bird either intra-abdominally or intravenously. In trials 1 to 3, birds were reared communally in a broiler house on pine shaving litter. In trial 4, challenged and control birds were maintained in separate isolation rooms in metal cages with raised wire floors. Challenged birds exhibited a characteristic cavity or depression in the external wall of the right ventricle. A subjective scoring system was devised to quantify challenge effects by assigning each heart a score of 1 to 4. The average number of birds, over all trials and over all dose levels, exhibiting the ventricular cavity was 93%. This value in controls was 5%. The average heart score for challenged birds was 3.1, and that for controls was 0.20. Heart scores of challenged and control chicks were not different in birds reared communally or in separate isolation rooms. Additionally, both routes of administration were equally effective. Results suggest that challenge with E. faecalis caused pulmonary hypertension.

  9. Eradication of Enterococcus faecalis Biofilms on Human Dentin

    PubMed Central

    Rosen, Eyal; Tsesis, Igor; Elbahary, Shlomo; Storzi, Nimrod; Kolodkin-Gal, Ilana

    2016-01-01

    Objectives: This work assesses different methods to interfere with Enterococcus faecalis biofilms formed on human dentin slabs. Methods: First, methods are presented that select for small molecule inhibitors of biofilm targets using multi-well polystyrene biofilm plates. Next, we establish methodologies to study and interfere with biofilm formation on a medically relevant model, whereby biofilms are grown on human root dentin slabs. Results: Non-conventional D-amino acid (D-Leucine) can efficiently disperse biofilms formed on dentin slabs without disturbing planktonic growth. Cation chelators interfere with biofilm formation on dentin slabs and polystyrene surfaces, and modestly impact planktonic growth. Strikingly, sodium hypochlorite, the treatment conventionally used to decontaminate infected root canal systems, was extremely toxic to planktonic bacteria, but did not eradicate biofilm cells. Instead, it induced a viable but non-culturable state in biofilm cells when grown on dentin slabs. Conclusion: Sodium hypochlorite may contribute to bacterial persistence. A combination of the methods described here can greatly contribute to the development of biofilm inhibitors and therapies to treat Enterococcus faecalis infections formed in the root canal system. PMID:28082955

  10. MurAA Is Required for Intrinsic Cephalosporin Resistance of Enterococcus faecalis

    PubMed Central

    Vesić, Dušanka

    2012-01-01

    Enterococcus faecalis is a low-GC Gram-positive bacterium that is intrinsically resistant to cephalosporins, antibiotics that target cell wall biosynthesis. To probe the mechanistic basis for intrinsic resistance, a library of transposon mutants was screened to identify E. faecalis strains that are highly susceptible to ceftriaxone, revealing a transposon mutant with a disruption in murAA. murAA is predicted to encode a UDP-N-acetylglucosamine 1-carboxyvinyl transferase that catalyzes the first committed step in peptidoglycan synthesis: phosphoenolpyruvate (PEP)-dependent conversion of UDP-N-acetylglucosamine to UDP-N-acetylglucosamine-enolpyruvate. In-frame deletion of murAA, but not its homolog in the E. faecalis genome (murAB), led to increased susceptibility of E. faecalis to cephalosporins. Furthermore, expression of murAA enhanced cephalosporin resistance in an E. faecalis mutant lacking IreK (formerly PrkC), a key kinase required for cephalosporin resistance. Further genetic analysis revealed that MurAA catalytic activity is necessary but not sufficient for this role. Collectively, our data indicate that MurAA and MurAB have distinct roles in E. faecalis physiology and suggest that MurAA possesses a unique property or activity that enables it to enhance intrinsic resistance of E. faecalis to cephalosporins. PMID:22290954

  11. Increased Enterococcus faecalis infection is associated with clinically active Crohn disease.

    PubMed

    Zhou, Youlian; Chen, Huiting; He, Hanchang; Du, Yanlei; Hu, Jiaqi; Li, Yingfei; Li, Yuyuan; Zhou, Yongjian; Wang, Hong; Chen, Ye; Nie, Yuqiang

    2016-09-01

    This study was performed to investigate the relationship between the abundance of pathogenic gut microbes in Chinese patients with inflammatory bowel disease (IBD) and disease severity.We collected clinical data and fecal samples from 47 therapy-naive Chinese patients with ulcerative colitis (UC), 67 patients with Crohn disease (CD), and 48 healthy volunteers. Bacteria levels of Fusobacterium species (spp), enterotoxigenic Bacteroides fragilis (B fragilis), enteropathogenic Escherichia coli (E coli), and Enterococcus faecalis (E faecalis) were assessed by quantitative real-time PCR (qRT-PCR). Spearman correlation coefficients were calculated to test associations between bacterial content and clinical parameters.Compared to healthy controls, the levels of both Fusobacterium spp and E faecalis were significantly increased in the feces of patients with IBD (P < 0.01). B fragilis levels were higher (P < 0.05) and E faecalis levels lower (P < 0.05) in patients with CD compared to those with UC. Increased E faecalis colonization in CD associated positively with disease activity (P = 0.015), Crohn disease activity index (CDAI; R = 0.3118, P = 0.0108), and fecal calprotectin (P = 0.016).E faecalis and Fusobacterium spp are significantly enriched in patients with IBD, and increased E faecalis infection is associated with clinically active CD.

  12. Increased Enterococcus faecalis infection is associated with clinically active Crohn disease

    PubMed Central

    Zhou, Youlian; Chen, Huiting; He, Hanchang; Du, Yanlei; Hu, Jiaqi; Li, Yingfei; Li, Yuyuan; Zhou, Yongjian; Wang, Hong; Chen, Ye; Nie, Yuqiang

    2016-01-01

    Abstract This study was performed to investigate the relationship between the abundance of pathogenic gut microbes in Chinese patients with inflammatory bowel disease (IBD) and disease severity. We collected clinical data and fecal samples from 47 therapy-naive Chinese patients with ulcerative colitis (UC), 67 patients with Crohn disease (CD), and 48 healthy volunteers. Bacteria levels of Fusobacterium species (spp), enterotoxigenic Bacteroides fragilis (B fragilis), enteropathogenic Escherichia coli (E coli), and Enterococcus faecalis (E faecalis) were assessed by quantitative real-time PCR (qRT-PCR). Spearman correlation coefficients were calculated to test associations between bacterial content and clinical parameters. Compared to healthy controls, the levels of both Fusobacterium spp and E faecalis were significantly increased in the feces of patients with IBD (P < 0.01). B fragilis levels were higher (P < 0.05) and E faecalis levels lower (P < 0.05) in patients with CD compared to those with UC. Increased E faecalis colonization in CD associated positively with disease activity (P = 0.015), Crohn disease activity index (CDAI; R = 0.3118, P = 0.0108), and fecal calprotectin (P = 0.016). E faecalis and Fusobacterium spp are significantly enriched in patients with IBD, and increased E faecalis infection is associated with clinically active CD. PMID:27684872

  13. Different extracts of Zingiber officinale decrease Enterococcus faecalis infection in Galleria mellonella.

    PubMed

    Maekawa, Lilian Eiko; Rossoni, Rodnei Dennis; Barbosa, Júnia Oliveira; Jorge, Antonio Olavo Cardoso; Junqueira, Juliana Campos; Valera, Marcia Carneiro

    2015-01-01

    Dried, fresh and glycolic extracts of Zingiber officinale were obtained to evaluate the action against G. mellonella survival assay against Enterococcus faecalis infection. Eighty larvae were divided into: 1) E. faecalis suspension (control); 2) E. faecalis + fresh extract of Z. officinale (FEO); 3) E. faecalis + dried extract of Z. officinale (DEO); 4) E. faecalis + glycolic extract of Z. officinale (GEO); 5) Phosphate buffered saline (PBS). For control group, a 5 μL inoculum of standardized suspension (107 cells/mL) of E. faecalis (ATCC 29212) was injected into the last left proleg of each larva. For the treatment groups, after E. faecalis inoculation, the extracts were also injected, but into the last right proleg. The larvae were stored at 37 °C and the number of dead larvae was recorded daily for 168 h (7 days) to analyze the survival curve. The larvae were considered dead when they did not show any movement after touching. E. faecalis infection led to the death of 85% of the larvae after 168 h. Notwithstanding, in treatment groups with association of extracts, there was an increase in the survival rates of 50% (GEO), 61% (FEO) and 66% (DEO) of the larvae. In all treatment groups, the larvae exhibited a survival increase with statistically significant difference in relation to control group (p=0.0029). There were no statistically significant differences among treatment groups with different extracts (p=0.3859). It may be concluded that the tested extracts showed antimicrobial activity against E. faecalis infection by increasing the survival of Galleria mellonella larvae.

  14. Structure of the 2, 4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP

    SciTech Connect

    Keegan, R.; Lebedev, A.; Erskine, P.; Guo, J.; Wood, S. P.; Hopper, D. J.; Rigby, S. E. J.; Cooper, J. B.

    2014-09-01

    The first X-ray structure of a 2, 4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP at a resolution of 2.2 Å is reported. This structure establishes that the enzyme adopts the cupin-fold, forming compact dimers with a pronounced hydrophobic interface between the monomers. Each monomer possesses a catalytic ferrous iron that is coordinated by three histidines (76, 78 and 114) and an additional ligand which has been putatively assigned as a carbonate, although formate and acetate are possibilities. The enzyme 2, 4′-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2, 4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C—C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits, each containing nonhaem iron, and its sequence suggests that it belongs to the cupin family of dioxygenases. In this paper, the first X-ray structure of a DAD enzyme from the Gram-negative bacterium Alcaligenes sp. 4HAP is reported, at a resolution of 2.2 Å. The structure establishes that the enzyme adopts a cupin fold, forming dimers with a pronounced hydrophobic interface between the monomers. The catalytic iron is coordinated by three histidine residues (76, 78 and 114) within a buried active-site cavity. The iron also appears to be tightly coordinated by an additional ligand which was putatively assigned as a carbonate dianion since this fits the electron density optimally, although it might also be the product formate. The modelled carbonate is located in a position which is highly likely to be occupied by the α-hydroxyketone group of the bound substrate during catalysis. Modelling of a substrate molecule in this position indicates that it will interact with many conserved amino acids in

  15. Enterococcus faecalis as multidrug resistance strains in clinical isolates in Imam Reza Hospital in Kermanshah, Iran.

    PubMed

    Mohammadi, F; Ghafourian, S; Mohebi, R; Taherikalani, M; Pakzad, I; Valadbeigi, H; Hatami, V; Sadeghifard, N

    2015-01-01

    The current study aimed to investigate the prevalence of vancomycin-resistant Enterococcus in E. faecalis and E. faecium and antimicrobial susceptibility patterns, then dominant genes responsible for vancomycin resistance were determined. For this propose, 180 clinical isolates of Enterococcus were subjected for identification and antibiotic susceptibility assay. Then, the gene responsible vancomycin resistant strains were determined. The results demonstrated the E. faecalis as a dominant Enterococcus. Resistance to erythromycin was dominant and multidrug resistance strains observed in E. faecalis. vanA was responsible for vancomycin resistance. In conclusion, a high rate of resistance to antibiotics in Enterococcus is clearly problematic, and a novel strategy is needed to decrease resistance in Enterococcus.

  16. Riboflavin-shuttled extracellular electron transfer from Enterococcus faecalis to electrodes in microbial fuel cells.

    PubMed

    Zhang, Enren; Cai, Yamin; Luo, Yue; Piao, Zhe

    2014-11-01

    Great attention has been focused on Gram-negative bacteria in the application of microbial fuel cells. In this study, the Gram-positive bacterium Enterococcus faecalis was employed in microbial fuel cells. Bacterial biofilms formed by E. faecalis ZER6 were investigated with respect to electricity production through the riboflavin-shuttled extracellular electron transfer. Trace riboflavin was shown to be essential for transferring electrons derived from the oxidation of glucose outside the peptidoglycan layer in the cell wall of E. faecalis biofilms formed on the surface of electrodes, in the absence of other potential electron mediators (e.g., yeast extract).

  17. Mechanisms for Photoinactivation of Enterococcus faecalis in Seawater

    PubMed Central

    Sassoubre, Lauren M.; Nelson, Kara L.

    2012-01-01

    Field studies in fresh and marine waters consistently show diel fluctuations in concentrations of enterococci, indicators of water quality. We investigated sunlight inactivation of Enterococcus faecalis to gain insight into photoinactivation mechanisms and cellular responses to photostress. E. faecalis bacteria were exposed to natural sunlight in clear, filtered seawater under both oxic and anoxic conditions to test the relative importance of oxygen-mediated and non-oxygen-mediated photoinactivation mechanisms. Multiple methods were used to assess changes in bacterial concentration, including cultivation, quantitative PCR (qPCR), propidium monoazide (PMA)-qPCR, LIVE/DEAD staining using propidium iodide (PI), and cellular activity, including ATP concentrations and expression of the superoxide dismutase-encoding gene, sodA. Photoinactivation, based on numbers of cultivable cells, was faster in oxic than in anoxic microcosms exposed to sunlight, suggesting that oxygen-mediated photoinactivation dominated. There was little change in qPCR signal over the course of the experiment, demonstrating that the nucleic acid targets were not damaged to a significant extent. The PMA-qPCR signal was also fairly stable, consistent with the observation that the fraction of PI-permeable cells was constant. Thus, damage to the membrane was minimal. Microbial ATP concentrations decreased in all microcosms, particularly the sunlit oxic microcosms. The increase in relative expression of the sodA gene in the sunlit oxic microcosms suggests that cells were actively responding to oxidative stress. Dark repair was not observed. This research furthers our understanding of photoinactivation mechanisms and the conditions under which diel fluctuations in enterococci can be expected in natural and engineered systems. PMID:22941072

  18. Thermodynamics of ligand binding to histone deacetylase like amidohydrolase from Bordetella/Alcaligenes.

    PubMed

    Meyners, Christian; Baud, Matthias G J; Fuchter, Matthew J; Meyer-Almes, Franz-Josef

    2014-03-01

    Thermodynamic studies on ligand-protein binding have become increasingly important in the process of drug design. In combination with structural data and molecular dynamics simulations, thermodynamic studies provide relevant information about the mode of interaction between compounds and their target proteins and therefore build a sound basis for further drug optimization. Using the example of histone deacetylases (HDACs), particularly the histone deacetylase like amidohydrolase (HDAH) from Bordetella/Alcaligenes, a novel sensitive competitive fluorescence resonance energy transfer-based binding assay was developed and the thermodynamics of interaction of both fluorescent ligands and inhibitors to histone deacetylase like amidohydrolase were investigated. The assay consumes only small amounts of valuable target proteins and is suitable for fast kinetic and mechanistic studies as well as high throughput screening applications. Binding affinity increased with increasing length of aliphatic spacers (n = 4-7) between the hydroxamate moiety and the dansyl head group of ligand probes. Van't Hoff plots revealed an optimum in enthalpy contribution to the free energy of binding for the dansyl-ligand with hexyl spacer. The selectivity in the series of dansyl-ligands against human class I HDAC1 but not class II HDACs 4 and 6 increased with the ratio of ΔH(0)/ΔG(0). The data clearly emphasize the importance of thermodynamic signatures as useful general guidance for the optimization of ligands or rational drug design.

  19. Improved welan gum production by Alcaligenes sp. ATCC31555 from pretreated cane molasses.

    PubMed

    Ai, Hongxia; Liu, Min; Yu, Pingru; Zhang, Shaozhi; Suo, Yukai; Luo, Ping; Li, Shuang; Wang, Jufang

    2015-09-20

    Welan gum production by Alcaligenes sp. ATCC31555 from cane molasses was studied in batch fermentation to reduce production costs and enhance gum production. The pretreatment of cane molasses, agitation speed and the addition of supplements were investigated to optimize the process. Sulfuric acid hydrolysis was found to be the optimal pretreatment, resulting in a maximum gum concentration of 33.5 g/L, which is 50.0% higher than those obtained from the molasses' mother liquor. Agitation at 600 rpm at 30°C and addition of 10% n-dodecane following fermentation for 36 h increased the maximum gum production up to 41.0 ± 1.41 g/L, which is 49.1% higher than the greatest welan gum concentration in the literature so far. The welan gum product showed an acceptable molecular weight, similar rheological properties and better thermal stability to that obtained from glucose. These results indicate that cane molasses may be a suitable and inexpensive substrate for cost-effective industrial-scale welan gum production.

  20. Wheat Bran Enhances the Cytotoxicity of Immobilized Alcaligenes aquatilis F8 against Microcystis aeruginosa

    PubMed Central

    Sun, Pengfei; Lin, Hui; Wang, Guan; Zhang, Ximing; Zhang, Qichun; Zhao, Yuhua

    2015-01-01

    Algicidal bacteria offer a promising option for killing cyanobacteria. Therefore, a new Alcaligenes aquatilis strain F8 was isolated to control Microcystis aeruginosa in this study. The algicidal activity of strain F8 was dependent on the cell density of M. aeruginosa, and the maximal algicidal rate of the free bacterium reached 88.45% within 72 h. With a view to its application to the control of M. aeruginosa in the natural environment, strain F8 was immobilized in sodium alginate beads, but immobilization of the strain decreased its algicidal rate compared to that of the free bacterium. However, addition of wheat bran to the sodium alginate matrix used to immobilize strain F8 not only eliminated the adverse effects of immobilization on the bacteria but also resulted in an 8.83% higher algicidal rate of the immobilized than free bacteria. Exclusion and recovery methods were used to identify key ingredients of wheat bran and gain insight into the mechanism underlying the observed enhancement of algicidal activity. This analysis indicated that certain factors in wheat bran, including vitamins B1, B2, B9, and E were responsible for promoting bacterial growth and thereby improving the algicidal rate of immobilized strain F8. Our findings indicate that wheat bran is able to improve the algicidal efficiency of A. aquatilis strain F8 for killing M. aeruginosa and is a good source of not only carbon and nitrogen but also vitamins for bacteria. PMID:26295573

  1. Crystal structure of the flavohemoglobin from Alcaligenes eutrophus at 1.75 A resolution.

    PubMed Central

    Ermler, U; Siddiqui, R A; Cramm, R; Friedrich, B

    1995-01-01

    The molecular structure of the flavohemoglobin from Alcaligenes eutrophus has been determined to a resolution of 1.75 A and refined to an R-factor of 19.6%. The protein comprises two fused modules: a heme binding module, which belongs to the globin family, and an FAD binding oxidoreductase module, which adopts a fold like ferredoxin reductase. The most striking deviation of the bacterial globin structure from those of other species is the movement of helix E in a way to provide more space in the vicinity of the distal heme binding site. A comparison with other members of the ferredoxin reductase family shows similar tertiary structures for the individual FAD and NAD binding domains but largely different interdomain orientations. The heme and FAD molecules approach each other to a minimal distance of 6.3 A and adopt an interplanar angle of 80 degrees. The electron transfer from FAD to heme occurs in a predominantly polar environment and may occur directly or be mediated by a water molecule. Images PMID:8557026

  2. Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals

    SciTech Connect

    Mergeay, M.; Nies, D.; Schlegel, H.G.; Gerits, J.; Charles, P.; Van Gijsegem, F.

    1985-04-01

    Alcaligenes eutrophus strain CH34, which was isolated as a bacterium resistant to cobalt, zinc, and cadmium ions, shares with A. eutrophus strain H16 the ability to grow lithoautotrophically on molecular hydrocarbon, to form a cytoplasmic NAD-reducing and a membrane-bound hydrogenase, and most metabolic attributes; however, it does not grow on fructose. Strain CH34 contains two plasmids, pMOL28 (163 kilobases) specifying nickel, mercury, and cobalt resistance and pMOL30 (238 kilobases) specifying zinc, cadmium, mercury, and cobalt resistance. The plasmids are self-transmissible in homologous matings, but at low frequencies. The transfer frequency was strongly increased with IncP1 plasmids RP4 and pUZ8 as helper plasmids. The phenotypes of the wild type, cured strains, and transconjugants are characterized by the following MICs (Micromolar) in strains with the indicated phenotypes: Nic/sup +/, 2.5; Nic/sup -/, 0.6; Cob/sup +/A, 5.0; Cob/sup +/B, 20.0; Cob/sup -/, < 0.07; Zin/sup +/, 12.0; Zin/sup -/, 0.6; Cad/sup +/, 2.5; and Cad/sup -/, 0.6. Plasmid-free cells of strain CH34 are still able to grow lithoautotrophically and to form both hydrogenases, indicating that the hydrogenase genes are located on the chromosome, in contrast to the Hox structural genes of strain H16, which are located on the megaplasmid pHG1 (450 kilobases).

  3. [Adenosine-dependent death of Hydrogenomonas eutropha (Alcaligenes eutrophus) H 16 (author's transl)].

    PubMed

    Kaltwasser, H; Glaeser, H

    1976-01-01

    Heterotrophic growth with fructose and autotrophic growth with hydrogen and carbon dioxide was entirely inhibited by adenosine at a concentration of 0.6 mg/ml in Hydrogenomonas eutropha (Alcaligenes eutrophus) H 16. Growth inhibition was not accompanied by a detectable uptake of the nucleoside. Adenosine caused a rapid inhibition of protein synthesis, followed by a decrease in nucleic acid formation. Enzyme synthesis was also impared, whilst cell respiration remained uneffected. Adenosin also caused a drastic but temporary decrease in viable cell counts. Cells incubated in presence of adenosine and fructose for several days, however, were no longer susceptable to this nucleoside. Adenosine-dependent growth inhibition turned out to be contingent upon the nature of the organic substrate. Cells growing with succinate, glutamate or peptone were less effected than cells, growing autotrophically or with fructose. No inhibition was observed in fructose-growing cells, if amino acids were also present in the medium. Several other nucleosides tested, did not show such growth inhibition, except desoxyadenosine, which, however, did not effect viable cell counts.

  4. Nitrogen Redox Metabolism of a Heterotrophic, Nitrifying-Denitrifying Alcaligenes sp. from Soil

    PubMed Central

    Castignetti, Domenic; Hollocher, Thomas C.

    1982-01-01

    Metabolic characteristics of a heterotrophic, nitrifier-denitrifier Alcaligenes sp. isolated from soil were further characterized. Pyruvic oxime and hydroxylamine were oxidized to nitrite aerobically by nitrification-adapted cells with specific activities (Vmax) of 0.066 and 0.003 μmol of N × min−1 × mg of protein−1, respectively, at 22°C. Km values were 15 and 42 μM for pyruvic oxime and hydroxylamine, respectively. The greater pyruvic oxime oxidation activity relative to hydroxylamine oxidation activity indicates that pyruvic oxime was a specific substrate and was not oxidized appreciably via its hydrolysis product, hydroxylamine. When grown as a denitrifier on nitrate, the bacterium could not aerobically oxidize pyruvic oxime or hydroxylamine to nitrite. However, hydroxylamine was converted to nearly equimolar amounts of ammonium ion and nitrous oxide, and the nature of this reaction is discussed. Cells grown as heterotrophic nitrifiers on pyruvic oxime contained two enzymes of denitrification, nitrate reductase and nitric oxide reductase. The nitrate reductase was the dissimilatory type, as evidenced by its extreme sensitivity to inhibition by azide and by its ability to be reversibly inhibited by oxygen. Cells grown aerobically on organic carbon sources other than pyruvic oxime contained none of the denitrifying enzymes surveyed but were able to oxidize pyruvic oxime to nitrite and reduce hydroxylamine to ammonium ion. PMID:16346117

  5. Characterization of the promoter and upstream activating sequence from the Pseudomonas alcaligenes lipase gene.

    PubMed

    Cox, M; Gerritse, G; Dankmeyer, L; Quax, W J

    2001-03-09

    Pseudomonas alcaligenes secretes a lipase with a high pH optimum, which has interesting properties for application in detergents. The expression of the lipase is strongly dependent on the presence of lipids in the growth medium such as soybean oil. The promoter of the gene was characterized and found to have resemblance to sigma54 controlled promoters, which are known to be tightly regulated. The transcription start was mapped precisely downstream of a sequence with close similarity to the -12/-24 consensus sequence of sigma54 controlled promoters. Interestingly, a hyperproducer mutant strain was isolated and found to have a C to T mutation in the -12/-24 promoter consensus region. In addition an Upstream Activating Sequence (UAS) with homology to sigma54 UAS consensus sequences was identified. It was demonstrated that an increase of the distance from the UAS to the transcription start or the deletion of the UAS results in significantly lower expression levels of lipase. A systematic mutational analysis of the UAS sequence has resulted in a variant with an increased lipase expression.

  6. Characterization of hbzE-encoded gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867.

    PubMed

    Yeo, Chew Chieng; Tan, Chew Ling; Gao, Xiaoli; Zhao, Bing; Poh, Chit Laa

    2007-09-01

    Pseudomonas alcaligenes NCIMB 9867 (strain P25X) is known to synthesize two isofunctional gentisate 1,2-dioxygenases (GDO; EC 1.13.11.4) as well as other enzymes involved in the degradation of xylenols and cresols via the gentisate pathway. The hbzE gene encoding what is possibly the strictly inducible gentisate 1,2-dioxygenase II (GDO-II) was cloned, overexpressed and purified as a hexahistidine fusion protein from Escherichia coli. Active recombinant GDO-II had an estimated molecular mass of 150kDa and is likely a tetrameric protein with a subunit mass of approximately 40kDa, similar to the previously characterized gentisate 1,2-dioxygenase I (GDO-I) encoded by xlnE. However, GDO-II was unable to utilize gentisate that is substituted at the carbon-4 position, unlike GDO-I which had broader substrate specificity. GDO-II also possessed different kinetic characteristics when compared to GDO-I. The hbzE-encoded GDO-II shared higher sequence identities (53%) with GDOs from Ralstonia sp. U2 and Polaromonas naphthalenivorans CJ2, compared with only 35% identity with the xlnE-encoded GDO-I. The hbzE gene was found to be part of a cluster of nine genes including the putative regulatory gene designated hbzR, which encodes an LysR-type regulator and is divergently transcribed from the other genes of the hbzHIJKLFED cluster.

  7. A gene complex coding for the membrane-bound hydrogenase of Alcaligenes eutrophus H16.

    PubMed Central

    Kortlüke, C; Horstmann, K; Schwartz, E; Rohde, M; Binsack, R; Friedrich, B

    1992-01-01

    One of the key enzymes in the chemolithoautotrophic metabolism of Alcaligenes eutrophus H16 is a dimeric, membrane-associated hydrogenase. The genetic determinants of this enzyme are located on the endogenous megaplasmid pHG1 (G. Eberz, C. Hogrefe, C. Kortlüke, A. Kamienski, and B. Friedrich, J. Bacteriol. 168:636-641, 1986). Complementation studies showed that the information required for the formation of active membrane-bound hydrogenase occupies more than 7.5 kb of megaplasmid DNA. We cloned and sequenced this region and identified the genes encoding the two hydrogenase subunits (hoxK and hoxG). The nucleotide sequence contains nine additional closely spaced open reading frames. Immunoelectron microscopy showed that the gene product of one of these open reading frames (hoxM) is involved in the process leading to the attachment of hydrogenase to the membrane. Other open reading frames may encode additional processing functions and components of a hydrogenase-linked electron transport chain. Analysis of Tn5-B21-mediated transcriptional fusions provided evidence that the structural genes and accessory functions belong to at least three coordinately regulated transcriptional units. Images PMID:1383192

  8. The Alcaligenes eutrophus H16 hoxX gene participates in hydrogenase regulation.

    PubMed Central

    Lenz, O.; Schwartz, E.; Dernedde, J.; Eitinger, M.; Friedrich, B.

    1994-01-01

    Nucleotide sequence analysis revealed a 1,791-bp open reading frame in the hox gene cluster of the gram-negative chemolithotroph Alcaligenes eutrophus H16. In order to investigate the biological role of this open reading frame, we generated an in-frame deletion allele via a gene replacement strategy. The resulting mutant grew significantly more slowly than the wild type under lithoautotrophic conditions (6.1 versus 4.2 h doubling time). A reduction in the level of the soluble NAD-reducing hydrogenase (60% of the wild-type activity) was shown to be the cause of the slow lithoautotrophic growth. We used plasmid-borne gene fusions to monitor the expression of the operons encoding the soluble and membrane-bound hydrogenases. The expression of both operons was lower in the mutant than in the wild-type strain. These results suggest that the newly identified gene, designated hoxX, encodes a regulatory component which, in conjunction with the transcriptional activator HoxA, controls hydrogenase synthesis. Images PMID:8021224

  9. S6K1-mediated disassembly of mitochondrial URI/PP1gamma complexes activates a negative feedback program that counters S6K1 survival signaling.

    PubMed

    Djouder, Nabil; Metzler, Stefan Christian; Schmidt, Alexander; Wirbelauer, Christiane; Gstaiger, Matthias; Aebersold, Ruedi; Hess, Daniel; Krek, Wilhelm

    2007-10-12

    S6 kinase 1 (S6K1) acts to integrate nutrient and growth factor signals to promote cell growth but also cell survival as a mitochondria-tethered protein kinase that phosphorylates and inactivates the proapoptotic molecule BAD. Here we report that the prefoldin chaperone URI represents a mitochondrial substrate of S6K1. In growth factor-deprived or rapamycin-treated cells, URI forms stable complexes with protein phosphatase (PP)1gamma at mitochondria, thereby inhibiting the activity of the bound enzyme. Growth factor stimulation induces disassembly of URI/PP1gamma complexes through S6K1-mediated phosphorylation of URI at serine 371. This activates a PP1gamma-dependent negative feedback program that decreases S6K1 activity and BAD phosphorylation, thereby altering the threshold for apoptosis. These findings establish URI and PP1gamma as integral components of an S6K1-regulated mitochondrial pathway dedicated, in part, to oppose sustained S6K1 survival signaling and to ensure that the mitochondrial threshold for apoptosis is set in accord with nutrient and growth factor availability.

  10. Activation of ribosomal protein S6 phosphorylation during meiotic maturation of Xenopus laevis oocytes: in vitro ordered appearance of S6 phosphopeptides.

    PubMed Central

    Martin-Pérez, J; Rudkin, B B; Siegmann, M; Thomas, G

    1986-01-01

    During meiotic maturation of Xenopus laevis stage 6 oocytes into unfertilized eggs, 40S ribosomal protein S6 undergoes multiple phosphorylation. Extracts prepared from unfertilized eggs are up to 10-fold more efficient in phosphorylating S6 than those prepared from immature oocytes. When analyzed by DEAE chromatography the S6 kinase activity elutes as a single peak. If extracts from unfertilized eggs are prepared in the absence of beta-glycerol phosphate, a putative phosphatase inhibitor, there is a severe reduction in recovered S6 kinase activity. Under optimal conditions, incubation of unfertilized egg extracts with 40S ribosomes in the presence of ATP leads to the average incorporation of 3.5 mol of phosphate/mol of S6. Prior incubation of these extracts with the cAMP-dependent protein kinase inhibitor does not inhibit S6 phosphorylation indicating that another kinase is responsible. Analysis of the in vitro phosphorylated peptides demonstrates that they migrate to the equivalent position of those observed previously in vivo and in vitro. More strikingly, if each of the increasingly phosphorylated derivatives of S6 is analyzed independently, it is found that the phosphopeptides appear in a specific order. Images Fig.1. Fig.4. Fig.5. Fig.6. PMID:3709523

  11. P70S6K 1 regulation of angiogenesis through VEGF and HIF-1{alpha} expression

    SciTech Connect

    Bian, Chuan-Xiu; Shi, Zhumei; Meng, Qiao; Jiang, Yue; Liu, Ling-Zhi; Jiang, Bing-Hua

    2010-07-30

    Research highlights: {yields} P70S6K1 regulates VEGF expression; {yields} P70S6K1 induces transcriptional activation through HIF-1{alpha} binding site; {yields} P70S6K1 regulates HIF-1{alpha}, but not HIF-1{beta} protein expression; {yields} P70S6K1 mediates tumor growth and angiogenesis through HIF-1{alpha} and VEGF expression. -- Abstract: The 70 kDa ribosomal S6 kinase 1 (p70S6K1), a downstream target of phosphoinositide 3-kinase (PI3K) and ERK mitogen-activated protein kinase (MAPK), is an important regulator of cell cycle progression, and cell proliferation. Recent studies indicated an important role of p70S6K1 in PTEN-negative and AKT-overexpressing tumors. However, the mechanism of p70S6K1 in tumor angiogenesis remains to be elucidated. In this study, we specifically inhibited p70S6K1 activity in ovarian cancer cells using vector-based small interfering RNA (siRNA) against p70S6K1. We found that knockdown of p70S6K1 significantly decreased VEGF protein expression and VEGF transcriptional activation through the HIF-1{alpha} binding site at its enhancer region. The expression of p70S6K1 siRNA specifically inhibited HIF-1{alpha}, but not HIF-1{beta} protein expression. We also found that p70S6K1 down-regulation inhibited ovarian tumor growth and angiogenesis, and decreased cell proliferation and levels of VEGF and HIF-1{alpha} expression in tumor tissues. Our results suggest that p70S6K1 is required for tumor growth and angiogenesis through HIF-1{alpha} and VEGF expression, providing a molecular mechanism of human ovarian cancer mediated by p70S6K1 signaling.

  12. The surface rhamnopolysaccharide epa of Enterococcus faecalis is a key determinant of intestinal colonization.

    PubMed

    Rigottier-Gois, Lionel; Madec, Clément; Navickas, Albertas; Matos, Renata C; Akary-Lepage, Elodie; Mistou, Michel-Yves; Serror, Pascale

    2015-01-01

    Enterococcus faecalis is a commensal bacterium of the human intestine and a major opportunistic pathogen in immunocompromised and elderly patients. The pathogenesis of E. faecalis infection relies in part on its capacity to colonize the gut. Following disruption of intestinal homeostasis, E. faecalis can overgrow, cross the intestinal barrier, and enter the lymph and bloodstream. To identify and characterize E. faecalis genes that are key to intestinal colonization, our strategy consisted in screening mutants for the following phenotypes related to intestinal lifestyle: antibiotic resistance, overgrowth, and competition against microbiota. From the identified colonization genes, epaX encodes a glycosyltransferase located in a variable region of the enterococcal polysaccharide antigen (epa) locus. We demonstrated that EpaX acts on sugar composition, promoting resistance to bile salts and cell wall integrity. Given that EpaX is enriched in hospital-adapted isolates, this study points to the importance of the epa variability as a key determinant for enterococcal intestinal colonization.

  13. Antimicrobial resistance and virulence of Enterococcus faecalis isolated from retail food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although enterococci are considered opportunistic nosocomial pathogens, their contribution to food-borne illnesses via dissemination through retail food remains undefined. In this study, prevalence and association of antimicrobial resistance and virulence factors of 80 Enterococcus faecalis isolate...

  14. Enterococcus Faecalis Biofilm. Formation and Development in Vitro Observed by Scanning Electron Microscopy.

    PubMed

    Bulacio, María de Los Á; Galván, Lucas R; Gaudioso, Cristina; Cangemi, Rosa; Erimbaue, Marta I

    2015-12-01

    Biofilm produced by Enterococcus faecalis isolated from root canals was detected by growing it on microplates and using 10% crystal violet stain, elution with alcohol and three procedures: no fixation, heat fixation and 10% formaldehyde fixation. The biofilm was evaluated using a Versamax Microplate Reader (USA). Twenty sterile root portions were incubated in TS broth with E. faecalis (108) for 48 hours, 4, 7, 14 and 30 days, after which they were processed and observed by scanning electron microscopy (SEM). Significantly more biofilm was found on the microplates for formaldehyde fixation than for heat fixation or no fixation (ANOVA p<0.0001). SEM showed E. faecalis growth at all times and biofilm development as from 14 days' incubation. Fixation with 10% formaldehyde was the most appropriate technique for detecting E. faecalis biofilm development on microplates. SEM confirmed biofilm formation after 14 days incubation.

  15. Draft Genome Sequence of an Enterococcus faecalis ATCC 19433 Siphovirus Isolated from Raw Domestic Sewage

    PubMed Central

    Ly, Melissa; Pride, David T.; Toranzos, Gary A.

    2017-01-01

    ABSTRACT We previously isolated and characterized an Enterococcus faecalis ATCC 19433 siphovirus from raw domestic sewage as a viral indicator of human fecal pollution. Here, we report the draft genome sequence of this bacteriophage. PMID:28104647

  16. Survival and activity of Streptococcus faecalis and escherichia coli in tropical freshwater

    SciTech Connect

    Muniz, I; Toranzos, G.A. ); Jimenez, L.; Hazen, T.C.

    1989-01-01

    The survival of Streptococcus faecalis and Escherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Densities were determined by acridine orange direct count and Coulter Counter. Population activity was determined by microautoradiography, cell respiration, and by nucleic acid composition. Densities of S. faecalis and E. coli decreased less than 1 log unit after 105 hours as measured by direct count methods. Activity as measured by respiration, acridine orange activity, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 12 hours, E. coli was more active than S. faecalis as measured by nucleic acid composition. In this tropical rain forest watershed, E. coli and S. faecalis survived and remained active for more than 5 days; consequently, both would seem to be unsuitable as indicators of recent fecal contamination in tropical waters.

  17. Draft Genome Sequence of an Enterococcus faecalis ATCC 19433 Siphovirus Isolated from Raw Domestic Sewage.

    PubMed

    Santiago-Rodriguez, Tasha M; Ly, Melissa; Pride, David T; Toranzos, Gary A

    2017-01-19

    We previously isolated and characterized an Enterococcus faecalis ATCC 19433 siphovirus from raw domestic sewage as a viral indicator of human fecal pollution. Here, we report the draft genome sequence of this bacteriophage.

  18. Drosophila S6 Kinase Like Inhibits Neuromuscular Junction Growth by Downregulating the BMP Receptor Thickveins

    PubMed Central

    Zhao, Guoli; Wu, Yingga; Du, Li; Li, Wenhua; Xiong, Ying; Yao, Aiyu; Wang, Qifu; Zhang, Yong Q.

    2015-01-01

    Synaptic connections must be precisely controlled to ensure proper neural circuit formation. In Drosophila melanogaster, bone morphogenetic protein (BMP) promotes growth of the neuromuscular junction (NMJ) by binding and activating the BMP ligand receptors wishful thinking (Wit) and thickveins (Tkv) expressed in motor neurons. We report here that an evolutionally conserved, previously uncharacterized member of the S6 kinase (S6K) family S6K like (S6KL) acts as a negative regulator of BMP signaling. S6KL null mutants were viable and fertile but exhibited more satellite boutons, fewer and larger synaptic vesicles, larger spontaneous miniature excitatory junctional potential (mEJP) amplitudes, and reduced synaptic endocytosis at the NMJ terminals. Reducing the gene dose by half of tkv in S6KL mutant background reversed the NMJ overgrowth phenotype. The NMJ phenotypes of S6KL mutants were accompanied by an elevated level of Tkv protein and phosphorylated Mad, an effector of the BMP signaling pathway, in the nervous system. In addition, Tkv physically interacted with S6KL in cultured S2 cells. Furthermore, knockdown of S6KL enhanced Tkv expression, while S6KL overexpression downregulated Tkv in cultured S2 cells. This latter effect was blocked by the proteasome inhibitor MG132. Our results together demonstrate for the first time that S6KL regulates synaptic development and function by facilitating proteasomal degradation of the BMP receptor Tkv. PMID:25748449

  19. PHLPP-mediated dephosphorylation of S6K1 inhibits protein translation and cell growth.

    PubMed

    Liu, Jianyu; Stevens, Payton D; Li, Xin; Schmidt, Micheal D; Gao, Tianyan

    2011-12-01

    PHLPP is a family of Ser/Thr protein phosphatases that contains PHLPP1 and PHLPP2 isoforms. We have shown previously that PHLPP functions as a tumor suppressor by negatively regulating Akt signaling in cancer cells. Here we report the identification of ribosomal protein S6 kinase 1 (S6K1) as a novel substrate of PHLPP. Overexpression of both PHLPP isoforms resulted in a decrease in S6K1 phosphorylation in cells, and this PHLPP-mediated dephosphorylation of S6K1 was independent of its ability to dephosphorylate Akt. Conversely, S6K1 phosphorylation was increased in cells depleted of PHLPP expression. Furthermore, we showed that the insulin receptor substrate 1 (IRS-1) expression and insulin-induced Akt phosphorylation were significantly decreased as the result of activation of the S6K-dependent negative feedback loop in PHLPP knockdown cells. Functionally, the phosphorylation of ribosomal protein S6 (rpS6) and the amount of phosphorylated rpS6 bound to the translation initiation complex were increased in PHLPP-knockdown cells. This correlated with increased cell size, protein content, and rate of cap-dependent translation. Taken together, our results demonstrate that loss of PHLPP expression activates the S6K-dependent negative feedback loop and that PHLPP is a novel player involved in regulating protein translation initiation and cell size via direct dephosphorylation of S6K1.

  20. Pharmacologic targeting of S6K1 in PTEN-deficient neoplasia

    PubMed Central

    Liu, Hongqi; Feng, Xizhi; Ennis, Kelli N.; Behrmann, Catherine A.; Sarma, Pranjal; Jiang, Tony T.; Kofuji, Satoshi; Niu, Liang; Stratton, Yiwen; Thomas, Hala Elnakat; Yoon, Sang-Oh; Sasaki, Atsuo T.; Plas, David R.

    2017-01-01

    SUMMARY Genetic S6K1 inactivation can induce apoptosis in PTEN-deficient cells. We analyzed the therapeutic potential of S6K1 inhibitors in PTEN-deficient T cell leukemia and glioblastoma. Results revealed that the S6K1 inhibitor LY-2779964 was relatively ineffective as a single agent, while S6K1-targeting AD80 induced cytotoxicity selectively in PTEN-deficient cells. In vivo, AD80 rescued 50% of mice transplanted with PTEN-deficient leukemia cells. Cells surviving LY-2779964 treatment exhibited inhibitor-induced S6K1 phosphorylation due to increased mTOR-S6K1 co-association, which primed rapid recovery of S6K1 signaling. In contrast, AD80 avoided S6K1 phosphorylation and mTOR co-association, resulting in durable suppression of S6K1-induced signaling and protein synthesis. Kinome analysis revealed that AD80 coordinately inhibits S6K1 together with the TAM family tyrosine kinase AXL. TAM suppression by BMS-777607 or genetic knockdown potentiated cytotoxic responses to LY-2779964 in PTEN-deficient glioblastoma cells. These results reveal that combination targeting of S6K1 and TAMs is a potential strategy for treatment of PTEN-deficient malignancy. PMID:28249155

  1. Bio-preservation of ground beef meat by Enterococcus faecalis CECT7121

    PubMed Central

    Sparo, M.D.; Confalonieri, A.; Urbizu, L.; Ceci, M.; Bruni, S.F. Sánchez

    2013-01-01

    Meat and particularly ground beef is frequently associated with Food Poisoning episodes and breeches in Food Safety. The main goal of this research was to evaluate the bactericide effect of the probiotic Enterococcus faecalis CECT7121, against different pathogens as: Escherichia coli O157:H7, Staphylococcus aureus, Clostridium perfringens and Listeria monocytogenes, inoculated in ground beef meat. Three studies were performed to evaluate the inhibition of E. faecalis CECT7121 on ground beef meat samples inoculated with pathogens: Study I: Samples (100 g meat) were inoculated with pathogens (103 CFU/g)) and E. faecalis CECT7121 (104 CFU/g) simultaneously. Study II: Samples were inoculated with E. faecalis CECT7121 24 h before the pathogens. Study III: E. faecalis CECT7121were inoculated 24 h after pathogens. The viable counts were performed at 0, 24, 48 and 72 h post-inoculation. The simultaneous inoculation of E. faecalis CECT7121 with E. coli O157:H7 strains resulted in the absence of viable counts of bacteria at 72 h post-treatment. However, when the probiotic was added 24 h before and 24 h after the pathogen E. coli O157:H7, viable cells were not detected at 24 h and 48 h post-treatment, respectively. Consistently, neither S. aureus nor Cl. perfringens viable bacteria were detected at 48 h in whole assays when inoculated with E. faecalis CECT7121. The same trend than described before was obtained after applying the 3 models assayed for L. monocytogenes. The current assays demonstrated the bactericide activity of E. faecalis CECT7121 strain on bacterial pathogens in ground beef meat. PMID:24159282

  2. Purification and characterization of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4.

    PubMed Central

    Joosten, H M; Nunez, M; Devreese, B; Van Beeumen, J; Marugg, J D

    1996-01-01

    A simple two-step procedure was developed to obtain pure enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4. Chemical and genetic characterization revealed that the primary structure of enterocin 4 is identical to that of peptide antibiotic AS-48 from Enterococcus faecalis S-48. In contrast to the reported inhibitory spectrum of AS-48, enterocin 4 displayed no activity against gram-negative bacteria. PMID:8900014

  3. Microbiologic Evaluation of Matricaria and Chlorhexidine against E. faecalis and C. albicans

    PubMed Central

    Rahman, Hena; Chandra, Anil

    2015-01-01

    Objective: To evaluate the antimicrobial activity of different concentrations of Matricaria chamomilla and Chlorhexidine gel against Candida albicans and Enterococcus faecalis. Materials and Methods: The agar diffusion test was used to evaluate the antimicrobial activity of 15%, 25% Matricaria chamomilla in aq. base and 2% chlorhexidine gel against C. albicans (ATCC 24433) and E. faecalis (ATCC 24212) strains. Vancomycin was used as the positive control for E. faecalis and fluconazole for C. albicans . The agar plates were incubated at 37°C for 48 h after which the zone of inhibition were measured separately for each material. Data thus obtained were statistically analyzed using the Wilcoxon rank–order test. Results: 2% chlorhexidine showed maximum inhibitory zone for C. albicans (33.26 mm) and E. faecalis (24.54 mm). 25% Matricaria showed zones of 24.16 mm and 20.62 mm for C. albicans and E. faecalis, respectively. 15% Matricaria did not show any antimicrobial activity (0 mm). Conclusion: The results of the current in vitro study suggest that 25% Matricaria can be used as an antimicrobial agent, but it is less effective than 2% chlorhexidine gluconate gel against C. albicans and E. faecalis. Matricaria at a lesser concentration of 15% aq. base is ineffective against both the microorganisms. PMID:26097333

  4. Differences in the chemical composition of Enterococcus faecalis biofilm under conditions of starvation and alkalinity.

    PubMed

    Chen, Weixu; Liang, Jingping; He, Zhiyan; Jiang, Wei

    2017-01-02

    ABSTACT This study aimed to investigate the dynamic changes that occur in the chemical composition of an Enterococcus faecalis (E. faecalis) biofilm under conditions of starvation and in an alkaline environment and to explore the function of chemical composition changes in the resistance of the E. faecalis biofilm to an extreme environment. This study established an in vitro E. faecalis biofilm model under starvation and in an alkaline environment. During the formation of the biofilm, the pH value and nutritional condition of the culture medium were changed, and the changes in chemical composition were observed using biochemical measures. The results showed that, when the pH value of the culture medium was 11, the percentage of water-insoluble polysaccharides in the biofilm was significantly lower than under other conditions. In addition, the percentage of water-soluble polysaccharides in culture medium with pH values of 9 and 11 gradually decreased. The level of the water-soluble polysaccharides in each milligram of dry weight of biofilm at pH 11 increased compared to that under other conditions. The results from this study indicate that the chemical composition of E. faecalis biofilm changed in extreme environments. These changes served as a defensive mechanism for E. faecalis against environmental pressures.

  5. Maintenance and induction of naphthalene degradation activity in Pseudomonas putida and an Alcaligenes sp. under different culture conditions.

    PubMed

    Guerin, W F; Boyd, S A

    1995-11-01

    The expression of xenobiotic-degradative genes in indigenous bacteria or in bacteria introduced into an ecosystem is essential for the successful bioremediation of contaminated environments. The maintenance of naphthalene utilization activity is studied in Pseudomonas putida (ATCC 17484) and an Alcaligenes sp. (strain NP-Alk) under different batch culture conditions. Levels of activity decreased exponentially in stationary phase with half-lives of 43 and 13 h for strains ATCC 17484 and NP-Alk, respectively. Activity half-lives were 2.7 and 5.3 times longer, respectively, in starved cultures than in stationary-phase cultures following growth on naphthalene. The treatment of starved cultures with chloramphenicol caused a loss of activity more rapid than that measured in untreated starved cultures, suggesting a continued enzyme synthesis in starved cultures in the absence of a substrate. Following growth in nutrient medium, activity decreased to undetectable levels in the Alcaligenes sp. but remained at measurable levels in the pseudomonad even after 9 months. The induction of naphthalene degradation activities in these cultures, when followed by radiorespirometry with 14C-labeled naphthalene as the substrate, was consistent with activity maintenance data. In the pseudomonad, naphthalene degradation activity was present constitutively at low levels under all growth conditions and was rapidly (in approximately 15 min) induced to high levels upon exposure to naphthalene. Adaptation in the uninduced Alcaligenes sp. occurred after many hours of exposure to naphthalene. In vivo labeling with 35S, to monitor the extent of de novo enzyme synthesis by naphthalene-challenged cells, provided an independent confirmation of the results.

  6. Proteome investigation of the global regulatory role of sigma 54 in response to gentisate induction in Pseudomonas alcaligenes NCIMB 9867.

    PubMed

    Zhao, Bing; Yeo, Chew Chieng; Poh, Chit Laa

    2005-05-01

    Pseudomonas alcaligenes NCIMB 9867 (strain P25X) utilizes the gentisate pathway for the degradation of aromatic hydrocarbons. The gene encoding the alternative sigma (sigma) factor sigma(54), rpoN, was cloned from strain P25X and a rpoN knock-out strain, designated G54, was constructed by insertional inactivation with a kanamycin resistance gene cassette. The role of sigma(54) in the physiological response of P. alcaligenes P25X to gentisate induction was assessed by comparing the global protein expression profiles of the wild-type P25X with the rpoN mutant strain G54. Analysis of two-dimensional polyacrylamide gel electrophoresis gels showed that 39 out of 355 prominent protein spots exhibited differential expression as a result of the insertional inactivation of rpoN. Identification of the protein spots by matrix-assisted laser desorption/ionization-time of flight/time of flight revealed a wide diversity of proteins that are affected by the sigma(54) mutation, the largest group being proteins that are involved in carbon metabolism. The strictly inducible gentisate 1,2-dioxygenase, one of two isofunctional copies of the key enzyme in the gentisate pathway, and enzymes of the TCA cycle, pyruvate metabolism and gluconeogenesis were part of this group. Other proteins that are part of the sigma(54) regulon include enzymes implicated in nitrogen metabolism, transport proteins, stress-response proteins and proteins involved in cell motility. The results of this study showed that sigma(54) plays a global regulatory role in the expression of a wide variety of genes in P. alcaligenes, including the wild-type response to the presence of the aromatic inducer, gentisate.

  7. Maintenance and induction of naphthalene degradation activity in Pseudomonas putida and an Alcaligenes sp. under different culture conditions

    SciTech Connect

    Guerin, W.F.; Boyd, S.A.

    1995-11-01

    The expression of xenobiotic-degradative genes in indigenous bacteria or in bacteria introduced into an ecosystem is essential for the successful bioremediation of contaminated environments. The maintenance of naphthalene utilization activity is studied in Pseudomonas putida (ATCC 17484) and an Alcaligenes sp. (strain NP-Alk) under different batch culture conditions. Levels of activity decreased exponentially in stationary phase with half-lives of 43 and 13 h for strains ATCC 17484 nad NP-Alk, respectively. Activity half-lives were 2.7 and 5.3 times longer, respectively, in starved cultures than in stationary-phase cultures following growth on naphthalene. The treatment of starved cultures with chloramphenicol caused a loss of activity more rapid than that measured in untreated starved cultures, suggesting a continued enzyme synthesis in starved cultures in the absence of a substrate. Following growth in nutrient medium, activity decreased to undetectable levels in the Alcaligenes sp. but remained at measureable levels int he pseudomonad even after 9 months. The induction of naphthalene degradation activities in these cultures, when followed by radiorespirometry with {sup 14}C-labeled naphthalene as the substrate, was consistent with activity maintenance data. In the pseudomonad, naphthalene degradation activity was present constitutively at low levels under all growth conditions and was rapidly (in approximately 15 min) induced to high levels upon exposure to naphthalene. Adaptation in the uninduced Alcaligenes sp. occurred after many hours of exposure to naphthalene. In vivo labeling with {sup 35}S, to monitor the extent of de novo enzyme synthesis by naphthalene-challenged cells, provided an independent confirmation of the results. 43 refs., 9 figs., 1 tab.

  8. Increased phosphorylation of ribosomal protein S6 during meiotic maturation of Xenopus oocytes.

    PubMed Central

    Nielsen, P J; Thomas, G; Maller, J L

    1982-01-01

    A single ribosomal protein (Mr, 32,000) becomes phosphorylated during progesterone-induced in vitro maturation of Xenopus laevis oocytes. The protein is identified as 40S ribosomal protein S6. Phosphorylation of S6 is monitored by incorporation of 32Pi and by two-dimensional polyacrylamide gel electrophoresis. S6 is minimally phosphorylated in unstimulated oocytes. After progesterone treatment, phosphorylation of S6 precedes germinal vesicle breakdown (GVBD) and is maximal at the time when 50% of the oocytes have undergone GVBD. S6, when maximally phosphorylated, exists in derivatives that correspond to the most highly phosphorylated forms observed in other systems, and the increase in S6 phosphorylation occurs at approximately the same time as the increase in the overall protein synthesis rate reported to occur during oocyte maturation. S6 is also maximally phosphorylated in unfertilized eggs following maturation in vivo. Injection of a partially purified preparation of maturation-promoting factor into immature oocytes induces immediate phosphorylation of S6 and rapidly increases the rate of protein synthesis. Moreover, incubation of ribosomes with this factor and radiolabeled ATP results in labeling of S6. These findings suggest that S6 phosphorylation may be important in the control of protein synthesis during maturation and may also play a role in the mechanism of action of maturation-promoting factor. Images PMID:7045876

  9. Plasmid pCBI carries genes for anaerobic benzoate catabolism in Alcaligenes xylosoxidans subsp. denitrificans PN-1.

    PubMed Central

    Blake, C K; Hegeman, G D

    1987-01-01

    Pseudomonas sp. strain PN-1 is reclassified as Alcaligenes xylosoxidans subsp. denitrificans PN-1. Strain PN-1 is a gram-negative, rod-shaped organism, is motile by means of lateral flagella, is oxidase positive, and does not ferment sugars. Plasmid pCBI, carrying genes for the anaerobic degradation of benzoate in strain PN-1, is 17.4 kilobase pairs in length and is transmissible to a number of denitrifying Pseudomonas aeruginosa and Pseudomonas stutzeri strains. A restriction endonuclease map was constructed. PMID:2822651

  10. Sequence similarities in the genes encoding polychlorinated biphenyl degradation by pseudomonas strain LB400 and alcaligenes eutrophus H850

    SciTech Connect

    Yates, J.R.; Mondello, F.J.

    1989-01-01

    DNA-DNA hybridization was used to compare the Pseudomonas strain LB400 genes for polychlorinated biphenyl (PCB) degradation with those from seven other PCB-degrading strains. Significant hybridization was detected to the genome of Alcaligenes eutrophus H850, a strain similar to LB400 in PCB-degrading capability. These two organisms showed a strong conservation of restriction sites in the region of DNA encoding PCB metabolism. No other sequence similarities were detected in the two genomes. DNA from the other PCB-degrading strains showed no hybridization to the probe, which demonstrated the existence of at least two distinct classes of genes encoding PCB degradation.

  11. Ampicillin in Combination with Ceftaroline, Cefepime, or Ceftriaxone Demonstrates Equivalent Activities in a High-Inoculum Enterococcus faecalis Infection Model.

    PubMed

    Luther, Megan K; Rice, Louis B; LaPlante, Kerry L

    2016-05-01

    Ampicillin-ceftriaxone combination therapy has become a predominant treatment for serious Enterococcus faecalis infections, such as endocarditis. Unfortunately, ceftriaxone use is associated with future vancomycin-resistant enterococcus colonization. We evaluated E. faecalis in an in vitro pharmacodynamic model against simulated human concentration-time profiles of ampicillin plus ceftaroline, cefepime, ceftriaxone, or gentamicin. Ampicillin-cefepime and ampicillin-ceftaroline demonstrated activities similar to those of ampicillin-ceftriaxone against E. faecalis.

  12. Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus.

    PubMed Central

    Eberz, G; Friedrich, B

    1991-01-01

    Random Tn5 mutagenesis of the regulatory region of megaplasmid pHG1 of Alcaligenes eutrophus led to the identification of three distinct loci designated hoxA, hoxD, and hoxE. Sequencing of the hoxA locus revealed an open reading frame which could code for a polypeptide of 482 amino acids with a molecular mass of 53.5 kDa. A protein of comparable apparent molecular mass was detected in heterologous expression studies with a plasmid-borne copy of the hoxA gene. Amino acid alignments revealed striking homologies between HoxA and the transcriptional activators NifA and NtrC of Klebsiella pneumoniae and HydG of Escherichia coli. HoxA- mutants of A. eutrophus lacked both NAD-reducing soluble hydrogenase and membrane-bound hydrogenase. In HoxA- mutants, the synthesis of beta-galactosidase from a hoxS'-'lacZ operon fusion was drastically reduced, indicating that HoxA is essential for the transcription of hydrogenase genes. Mutants defective in hoxD and hoxE also lacked the catalytic activities of the two hydrogenases; however, in contrast to HoxA- mutants, they contained immunologically detectable NAD-reducing soluble hydrogenase and membrane-bound hydrogenase proteins, although at a reduced level. The low hydrogenase content in the HoxD- and HoxE- mutants correlated with a decrease in beta-galactosidase synthesized under the direction of a hoxS'-'lacZ operon fusion. Thus, hoxD and hoxE apparently intervene both in the regulation of hydrogenase synthesis and in subsequent steps leading to the formation of catalytically active enzymes. Images PMID:2001989

  13. Structure of the 2,4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP

    PubMed Central

    Keegan, R.; Lebedev, A.; Erskine, P.; Guo, J.; Wood, S. P.; Hopper, D. J.; Rigby, S. E. J.; Cooper, J. B.

    2014-01-01

    The enzyme 2,4′-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C—C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits, each containing nonhaem iron, and its sequence suggests that it belongs to the cupin family of dioxygenases. In this paper, the first X-ray structure of a DAD enzyme from the Gram-negative bacterium Alcaligenes sp. 4HAP is reported, at a resolution of 2.2 Å. The structure establishes that the enzyme adopts a cupin fold, forming dimers with a pronounced hydrophobic interface between the monomers. The catalytic iron is coordinated by three histidine residues (76, 78 and 114) within a buried active-site cavity. The iron also appears to be tightly coordinated by an additional ligand which was putatively assigned as a carbonate dianion since this fits the electron density optimally, although it might also be the product formate. The modelled carbonate is located in a position which is highly likely to be occupied by the α-hydroxyketone group of the bound substrate during catalysis. Modelling of a substrate molecule in this position indicates that it will interact with many conserved amino acids in the predominantly hydrophobic active-site pocket where it undergoes peroxide radical-mediated heterolysis. PMID:25195757

  14. The role of S6K1 in ER-positive breast cancer.

    PubMed

    Holz, Marina K

    2012-09-01

    The 40S ribosomal S6 kinase 1 (S6K1) is a conserved serine/threonine protein kinase that belongs to the AGC family of protein kinases, which also includes Akt and many others. S6K1 is the principal kinase effector downstream of the mammalian target of rapamycin complex 1 (mTORC1). S6K1 is sensitive to a wide range of signaling inputs, including growth factors, amino acids, energy levels and hypoxia. S6K1 relays these signals to regulate a growing list of substrates and interacting proteins in control of oncogenic processes, such as cell growth and proliferation, cell survival and apoptosis and cell migration and invasion. Several lines of evidence suggest an important role for S6K1 in estrogen receptor (ER)-positive breast cancer. S6K1 directly phosphorylates and activates ERα. Furthermore, S6K1 expression is estrogenically regulated. Therefore, hyperactivation of mTORC1/S6K1 signaling may be closely related to ER-positive status in breast cancer and may be utilized as a marker for prognosis and a therapeutic target.

  15. Synaptic activation of ribosomal protein S6 phosphorylation occurs locally in activated dendritic domains.

    PubMed

    Pirbhoy, Patricia Salgado; Farris, Shannon; Steward, Oswald

    2016-06-01

    Previous studies have shown that induction of long-term potentiation (LTP) induces phosphorylation of ribosomal protein S6 (rpS6) in postsynaptic neurons, but the functional significance of rpS6 phosphorylation is poorly understood. Here, we show that synaptic stimulation that induces perforant path LTP triggers phosphorylation of rpS6 (p-rpS6) locally near active synapses. Using antibodies specific for phosphorylation at different sites (ser235/236 versus ser240/244), we show that strong synaptic activation led to dramatic increases in immunostaining throughout postsynaptic neurons with selectively higher staining for p-ser235/236 in the activated dendritic lamina. Following LTP induction, phosphorylation at ser235/236 was detectable by 5 min, peaked at 30 min, and was maintained for hours. Phosphorylation at both sites was completely blocked by local infusion of the NMDA receptor antagonist, APV. Despite robust induction of p-rpS6 following high frequency stimulation, assessment of protein synthesis by autoradiography revealed no detectable increases. Exploration of a novel environment led to increases in the number of p-rpS6-positive neurons throughout the forebrain in a pattern reminiscent of immediate early gene induction and many individual neurons that were p-rpS6-positive coexpressed Arc protein. Our results constrain hypotheses about the possible role of rpS6 phosphorylation in regulating postsynaptic protein synthesis during induction of synaptic plasticity.

  16. S6 kinase 1 is required for rapamycin-sensitive liver proliferation after mouse hepatectomy.

    PubMed

    Espeillac, Catherine; Mitchell, Claudia; Celton-Morizur, Séverine; Chauvin, Céline; Koka, Vonda; Gillet, Cynthia; Albrecht, Jeffrey H; Desdouets, Chantal; Pende, Mario

    2011-07-01

    Rapamycin is an antibiotic inhibiting eukaryotic cell growth and proliferation by acting on target of rapamycin (TOR) kinase. Mammalian TOR (mTOR) is thought to work through 2 independent complexes to regulate cell size and cell replication, and these 2 complexes show differential sensitivity to rapamycin. Here we combine functional genetics and pharmacological treatments to analyze rapamycin-sensitive mTOR substrates that are involved in cell proliferation and tissue regeneration after partial hepatectomy in mice. After hepatectomy, hepatocytes proliferated rapidly, correlating with increased S6 kinase phosphorylation, while treatment with rapamycin derivatives impaired regeneration and blocked S6 kinase activation. In addition, genetic deletion of S6 kinase 1 (S6K1) caused a delay in S phase entry in hepatocytes after hepatectomy. The proliferative defect of S6K1-deficient hepatocytes was cell autonomous, as it was also observed in primary cultures and hepatic overexpression of S6K1-rescued proliferation. We found that S6K1 controlled steady-state levels of cyclin D1 (Ccnd1) mRNA in liver, and cyclin D1 expression was required to promote hepatocyte cell cycle. Notably, in vivo overexpression of cyclin D1 was sufficient to restore the proliferative capacity of S6K-null livers. The identification of an S6K1-dependent mechanism participating in cell proliferation in vivo may be relevant for cancer cells displaying high mTOR complex 1 activity and cyclin D1 accumulation.

  17. Evaluation of Enterococcus faecalis adhesion, penetration, and method to prevent the penetration of Enterococcus faecalis into root cementum: Confocal laser scanning microscope and scanning electron microscope analysis

    PubMed Central

    Halkai, Rahul S.; Hegde, Mithra N.; Halkai, Kiran R.

    2016-01-01

    Aim: To ascertain the role of Enterococcus faecalis in persistent infection and a possible method to prevent the penetration of E. faecalis into root cementum. Methodology: One hundred and twenty human single-rooted extracted teeth divided into five groups. Group I (control): intact teeth, Group II: no apical treatment done, Group III divided into two subgroups. In Groups IIIa and IIIb, root apex treated with lactic acid of acidic and neutral pH, respectively. Group IV: apical root cementum exposed to lactic acid and roughened to mimic the apical resorption. Group V: apical treatment done same as Group IV and root-end filling done using mineral trioxide aggregate (MTA). Apical one-third of all samples immersed in E. faecalis broth for 8 weeks followed by bone morphogenetic protein and obturation and again immersed into broth for 8 weeks. Teeth split into two halves and observed under confocal laser scanning microscope and scanning electron microscope, organism identified by culture and polymerase chain reaction techniques. Results: Adhesion and penetration was observed in Group IIIa and Group IV. Only adhesion in Group II and IIIB and no adhesion and penetration in Group I and V. Conclusion: Adhesion and penetration of E. faecalis into root cementum providing a long-term nidus for subsequent infection are the possible reason for persistent infection and root-end filling with MTA prevents the adhesion and penetration. PMID:27994316

  18. Development of a genomic site for gene integration and expression in Enterococcus faecalis

    PubMed Central

    DebRoy, Sruti; van der Hoeven, Ransome; Singh, Kavindra V.; Gao, Peng; Harvey, Barrett R.; Murray, Barbara E.; Garsin, Danielle A.

    2012-01-01

    Enterococcus faecalis, a gram-positive opportunistic pathogen, has become one of the leading causes of nosocomial infections. Normally a resident of the gastrointestinal tract, extensive use of antibiotics has resulted in the rise of E. faecalis strains that are resistant to multiple antibiotics. This, compounded with the ability to easily exchange antibiotic determinants with other bacteria, has made certain E. faecalis infections difficult to treat medically. The genetic toolbox for the study of E. faecalis has expanded greatly in recent years, but has lacked methodology to stably introduce a gene in single copy in a non-disruptive manner for complementation or expression of non-native genes. In this study, we identified a specific site in the genome of E. faecalis OG1RF that can serve as an expression site for a gene of interest. This site is well conserved in most of the sequenced E. faecalis genomes. A vector has also been developed to integrate genes into this site by allelic exchange. Using this system, we complemented an in-frame deletion in eutV, demonstrating that the mutation does not cause polar effects. We also generated an E. faecalis OG1RF strain that stably expresses the green fluorescent protein and is comparable to the parent strain in terms of in vitro growth and pathogenicity in C. elegans and mice. Another major advantage of this new methodology is the ability to express integrated genes without the need for maintaining antibiotic selection, making this an ideal tool for functional studies of genes in infection models and co-culture systems. PMID:22542850

  19. Transfer of tetracycline resistance genes with aggregation substance in food-borne Enterococcus faecalis.

    PubMed

    Choi, Jong-Mi; Woo, Gun-Jo

    2015-04-01

    Enterococcus faecalis has the ability to conjugate with the aid of aggregation substance (AS) and inducible sex pheromones to exchange genetic elements in food matrix. To evaluate the food safety condition and the transferable factor, 250 tetracycline-resistant food-borne E. faecalis were collected in Korea. Among the isolates, a majority of tetracycline-resistant isolates (49.6 %) harbored both the tet(M) and tet(L) genes together, followed by tet(M) (19.6 %), and tet(L) (6.8 %) alone. Also, we found the combination of tet(L)/tet(M)/tet(O) or tet(M)/tet(O). We identified two tet(S) genes including the isolate carrying tet(M) + tet(S) genes. Additionally, most E. faecalis were positive for cpd and ccf (both 96.8 %) followed by cob (57.2 %). Through mating experiments, we confirmed E. faecalis possessing the Int-Tn gene and/or any AS gene successfully transferred tet genes to JH2-2 E. faecalis, whereas neither E. faecalis carrying AS genes nor the Int-Tn gene showed the conjugation. Pulsed-field gel electrophoresis results supported a distinct pattern, implying transfer of genetic information. Our study revealed a high occurrence of tetracycline resistance genes in E. faecalis from various foods. The widespread dissemination of tetracycline resistance genes would be promoted to transfer tetracycline resistance genes by pheromone-mediated conjugation systems.

  20. Collagen degradation and MMP9 activation by Enterococcus faecalis contributes to intestinal anastomotic leak

    PubMed Central

    Shogan, B. D.; Belogortseva, N.; Luong, P. M.; Zaborin, A.; Lax, S.; Bethel, Cindy; Ward, M.; Muldoon, J. P.; Singer, M.; An, G.; Umanskiy, K.; Konda, V.; Shakhsheer, B.; Luo, J.; Klabbers, R.; Hancock, L. E.; Gilbert, J.; Zaborina, O.; Alverdy, J. C.

    2016-01-01

    Even under the most expert care, a properly constructed intestinal anastomosis can fail to heal resulting in leakage of its contents, peritonitis and sepsis. The cause of anastomotic leak remains unknown and its incidence has not changed in decades. Here, we demonstrate that the commensal bacterium Enterococcus faecalis contributes to the pathogenesis of anastomotic leak through its capacity to degrade collagen and to activate tissue matrix metalloprotease-9 (MMP9) in host intestinal tissues. We demonstrate in rats that leaking anastomotic tissues were colonized by E. faecalis strains that showed an increased collagen-degrading activity and also an increased ability to activate host MMP9, both of which contributed to anastomotic leakage. We demonstrate that the E. faecalis genes gelE and sprE were required for E. faecalis-mediated MMP9 activation. Either elimination of E. faecalis strains through direct topical antibiotics applied to rat intestinal tissues or pharmacological suppression of intestinal MMP9 activation prevented anastomotic leak in rats. In contrast, the standard recommended intravenous antibiotics used in patients undergoing colorectal surgery did not eliminate E. faecalis at anastomotic tissues nor did they prevent leak in our rat model. Finally, we show in humans undergoing colon surgery and treated with the standard recommended intravenous antibiotics, that their anastomotic tissues still contained E. faecalis and other bacterial strains with collagen-degrading/MMP9 activity. We suggest that intestinal microbes with the capacity to produce collagenases and to activate host metalloproteinase MMP9 may break down collagen in the gut tissue contributing to anastomotic leak. PMID:25947163

  1. Enterococcus faecalis infection activates phosphatidylinositol 3-kinase signaling to block apoptotic cell death in macrophages.

    PubMed

    Zou, Jun; Shankar, Nathan

    2014-12-01

    Apoptosis is an intrinsic immune defense mechanism in the host response to microbial infection. Not surprisingly, many pathogens have evolved various strategies to manipulate this important pathway to benefit their own survival and dissemination in the host during infection. To our knowledge, no attempts have been made to explore the host cell survival signals modulated by the bacterium Enterococcus faecalis. Here, we show for the first time that during early stages of infection, internalized enterococci can prevent host cell (RAW264.7 cells, primary macrophages, and mouse embryonic fibroblasts [MEFs]) apoptosis induced by a wide spectrum of proapoptotic stimuli. Activation of caspase 3 and cleavage of the caspase 3 substrate poly(ADP-ribose) polymerase were inhibited in E. faecalis-infected cells, indicating that E. faecalis protects macrophages from apoptosis by inhibiting caspase 3 activation. This antiapoptotic activity in E. faecalis-infected cells was dependent on the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which resulted in the increased expression of the antiapoptotic factor Bcl-2 and decreased expression of the proapoptotic factor Bax. Further analysis revealed that active E. faecalis physiology was important for inhibition of host cell apoptosis, and this feature seemed to be a strain-independent trait among E. faecalis isolates. Employing a mouse peritonitis model, we also determined that cells collected from the peritoneal lavage fluid of E. faecalis-infected mice showed reduced levels of apoptosis compared to cells from uninfected mice. These results show early modulation of apoptosis during infection and have important implications for enterococcal pathogenesis.

  2. Rapid Kill—Novel Endodontic Sealer and Enterococcus faecalis

    PubMed Central

    Zaltsman, Nathan; Houri-Haddad, Yael; Abramovitz, Itzhak; Davidi, Michael Perez; Weiss, Ervin I.

    2013-01-01

    With growing concern over bacterial resistance, the identification of new antimicrobial means is paramount. In the oral cavity microorganisms are essential to the development of periradicular diseases and are the major causative factors associated with endodontic treatment failure. As quaternary ammonium compounds have the ability to kill a wide array of bacteria through electrostatic interactions with multiple anionic targets on the bacterial surface, it is likely that they can overcome bacterial resistance. Melding these ideas, we investigated the potency of a novel endodontic sealer in limiting Enterococcus faecalis growth. We used a polyethyleneimine scaffold to synthesize nano-sized particles, optimized for incorporation into an epoxy-based endodontic sealer. The novel endodontic sealer was tested for its antimicrobial efficacy and evaluated for biocompatibility and physical eligibility. Our results show that the novel sealer foundation affixes the nanoparticles, achieving surface bactericidal properties, but at the same time impeding nanoparticle penetration into eukaryotic cells and thereby mitigating a possible toxic effect. Moreover, adequate physical properties are maintained. The nanosized quaternary amine particles interact within minutes with bacteria, triggering cell death across wide pH values. Throughout this study we demonstrate a new antibacterial perspective for endodontic sealers; a novel antibacterial, effective and safe antimicrobial means. PMID:24223159

  3. Structural proteins of Enterococcus faecalis bacteriophage ϕEf11

    PubMed Central

    Stevens, Roy H.; Zhang, Hongming; Hsiao, Chaiwing; Kachlany, Scott; Tinoco, Eduardo M. B.; DePew, Jessica; Fouts, Derrick E.

    2016-01-01

    ABSTRACT ϕEf11, a temperate Siphoviridae bacteriophage, was isolated by induction from a root canal isolate of Enterococcus faecalis. Sequence analysis suggested that the ϕEf11 genome included a contiguous 8 gene module whose function was related to head structure assembly and another module of 10 contiguous genes whose products were responsible for tail structure assembly. SDS-PAGE analysis of virions of a ϕEf11 derivative revealed 11 well-resolved protein bands. To unify the deduced functional gene assignments emanating from the DNA sequence data, with the structural protein analysis of the purified virus, 6 of the SDS-PAGE bands were subjected to mass spectrometry analysis. 5 of the 6 protein bands analyzed by mass spectrometry displayed identical amino acid sequences to those predicted to be specified by 4 of the ORFs identified in the ϕEf11 genome. These included: ORF8 (predicted scaffold protein), ORF10 (predicted major head protein), ORF15 (predicted major tail protein), and ORF23 (presumptive antireceptor). PMID:28090386

  4. Kinetic studies on enzymatic acetylation of chloramphenicol in Streptococcus faecalis.

    PubMed Central

    Nakagawa, Y; Nitahara, Y; Miyamura, S

    1979-01-01

    The kinetics of chloramphenicol (CP) acetylation by CP acetyltransferase from Streptococcus faecalis was studied. CP was shown to be acetylated enzymatically to its 3-O-acetyl derivative (3-AcCP) in the presence of acetyl coenzyme A, after which 3-AcCP was converted nonenzymatically to its 1-O-acetyl isomer, 1-O-acetyl CP (1-AcCP). At equilibrium, the 1-AcCP and 3-AcCP were present in a 1:4 ratio. Subsequently the diacetylated product, 1,3-O-O-diacetyl CP [1,3-(Ac)2CP], was enzymatically produced from 1-AcCP by the same enzyme. Theoretical calculation of rate constants (k1, k2, k3) for each successive reaction is as follows: (Formula: see text). This calculation gave k1 = 0.4 min-1, k2 = 0.002 min-1, and k3 = 0.016 min-1. Experimental results agreed closely with these calculated values. Images PMID:119483

  5. [High-level production of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by feb-batch culture of Alcaligenes eutrophus].

    PubMed

    Cai, Y B; Liu, M Q; Yi, Z H; Chen, Q; Weng, W Q

    2001-09-01

    Fermentation strategies for production P (3HB-co-3HV) from glucose and propionic (or valeric) acid by Alcaligenes eutrophus were studied. During the culture, we controlled pH of the broth by feeding precusors of 3HV- propionic or valeric acid after Ammonia feeding stopped. When propionic acid were used as the precusor, for 50 hours, we obtained a cell dry weight, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content and a 3HV fraction of 149.9 g/L, 124.9 g/L, 83.3% and 12.4 mol%, respectively, with a PHA productivity of 2.50 g h-1 L-1. When valeric acid were used as the precusor, for 45 hours, we obtained a cell dry weight, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content and a 3HV fraction of 160.2 g/L, 119.0 g/L, 74.2% and 17.7 mol%, respectively, with a PHA productivity of 2.64 g h-1 L-1. Prior to this study, it hasn't been reported to obtain such high level productivity and 3HV fraction at the same time by Alcaligenes eutrophus.

  6. [Influence of yeast extract on the fermentation of glucose by the demulsifying strain Alcaligenes sp. S-XJ-1].

    PubMed

    Huang, Xiang-Feng; Wang, Kai; Li, Ming-Xia; Wang, Cai-Lin; Lu, Li-Jun; Liu, Jia

    2013-04-01

    The demulsifying strain Alcaligenes sp. S-XJ-1, isolated from oil contaminated soil, was cultivated with glucose as the carbon source. The influences of yeast extract on the growth, demulsifying ability and the element composition of the strain were investigated. The results showed that the yeast extract could increase the biomass and enhance the glucose utilization of Alcaligenes sp. S-XJ-1. When the concentration of the yeast extract was 5 g x L(-1), the biomass was increased up to 3.0 g x L(-1), and the glucose utilization achieved 58%. The demulsifying ability of the strain was improved with increasing yeast extract concentration. When the concentration of the yeast extract was 10 g x L(-1), the demulsification ratio of the obtained cell was 76%. While the C/N ratio of the cells decreased with the increasing concentration of yeast extract. The proteins of cells were extracted and measured. The results showed that the proteins of the obtained cell increased with the increasing concentration of yeast extract, in accordance with the increased concentrations of proteins on the surface of the cells as measured by FTIR. It is estimated that the increase of the proteins leads to the improvement of the demulsifying ability of the demulsifying strain and theses proteins play essential roles in the demulsifying process.

  7. Arsenite oxidation by Alcaligenes sp. strain RS-19 isolated from arsenic-contaminated mines in the Republic of Korea.

    PubMed

    Yoon, In-Ho; Chang, Jin-Soo; Lee, Ji-Hoon; Kim, Kyoung-Woong

    2009-02-01

    Arsenite [As(III)]-oxidizing bacteria play important roles in reducing arsenic [As] toxicity and mobility in As-contaminated areas. As-resistant bacteria were isolated from the soils of two abandoned mines in the Republic of Korea. The isolated bacteria showed relatively high resistances to As(III) up to 26 mM. The PCR-based 16S rRNA analysis revealed that the isolated As-resistant bacteria were close relatives to Serratia marcescensa, Pseudomonas putida, Pantoea agglomerans, and Alcaligenes sp. Among the five As-resistant bacterial isolates, Alcaligenes sp. strain RS-19 showed the highest As(III)-oxidizing activity in batch tests, completely oxidizing 1 mM of As(III) to As(V) within 40 h during heterotrophic growth. This study suggests that the indigenous bacteria have evolved to retain the ability to resist toxic As in the As-contaminated environments and moreover to convert the species to a less toxic form [e.g., from As(III) to As(V)] and also contribute the biogeochemical cycling of As by being involved in speciation of As.

  8. Thermostable Alkaline Phytase from Alcaligenes sp. in Improving Bioavailability of Phosphorus in Animal Feed: In Vitro Analysis

    PubMed Central

    Vijayaraghavan, Ponnuswamy; Primiya, R. Raja; Prakash Vincent, Samuel Gnana

    2013-01-01

    A bacterial isolate, Alcaligenes sp. secreting phytase (EC 3.1.3.8), was isolated and characterized. The optimum conditions for the production of phytase included a fermentation period of 96 h, pH 8.0, and the addition of 1% (w/v) maltose and 1% (w/v) beef extract to the culture medium. This enzyme was purified to homogeneity and had an apparent molecular mass of 41 kDa. The optimum pH range and temperature for the activity of phytase were found to be 7.0-8.0 and 60°C, respectively. This enzyme was strongly inhibited by 0.005 M of Mn2+, Mg2+, and Zn2+. In vitro studies revealed that the phytase from Alcaligenes sp. released inorganic phosphate from plant phytates. Phytase released 1930 ± 28, 1740 ± 13, 1050 ± 31, 845 ± 7, 1935 ± 32, and 1655 ± 21 mg inorganic phosphate/kg plant phytates, namely, chick pea, corn, green pea, groundnut, pearl pea, and chick feed, respectively. PMID:25969790

  9. Rhizosphere colonization and arsenic translocation in sunflower (Helianthus annuus L.) by arsenate reducing Alcaligenes sp. strain Dhal-L.

    PubMed

    Cavalca, Lucia; Corsini, Anna; Bachate, Sachin Prabhakar; Andreoni, Vincenza

    2013-10-01

    In the present study, six arsenic-resistant strains previously isolated were tested for their plant growth promoting characteristics and heavy metal resistance, in order to choose one model strain as an inoculum for sunflower plants in pot experiments. The aim was to investigate the effect of arsenic-resistant strain on sunflower growth and on arsenic uptake from arsenic contaminated soil. Based on plant growth promoting characteristics and heavy metal resistance, Alcaligenes sp. strain Dhal-L was chosen as an inoculum. Beside the ability to reduce arsenate to arsenite via an Ars operon, the strain exhibited 1-amino-cyclopropane-1-carboxylic acid deaminase activity and it was also able to produce siderophore and indole acetic acid. Pot experiments were conducted with an agricultural soil contaminated with arsenic (214 mg kg⁻¹). A real time PCR method was set up based on the quantification of ACR3(2) type of arsenite efflux pump carried by Alcaligenes sp. strain Dhal-L, in order to monitor presence and colonisation of the strain in the bulk and rhizospheric soil. As a result of strain inoculation, arsenic uptake by plants was increased by 53 %, whereas ACR3(2) gene copy number in rhizospheric soil was 100 times higher in inoculated than in control pots, indicating the colonisation of strain. The results indicated that the presence of arsenate reducing strains in the rhizosphere of sunflower influences arsenic mobilization and promotes arsenic uptake by plant.

  10. The ribosomal protein S6 in renal cell carcinoma: functional relevance and potential as biomarker

    PubMed Central

    Knoll, Maximilian; Macher-Goeppinger, Stephan; Kopitz, Jürgen; Duensing, Stefan; Pahernik, Sascha; Hohenfellner, Markus; Schirmacher, Peter; Roth, Wilfried

    2016-01-01

    Inhibitors of the mTOR pathway, such as everolimus, are promising compounds to treat patients with renal cell carcinomas (RCCs). However, the precise mechanisms of action are far from clear, and biomarkers predicting the response to mTOR inhibitors are still missing. Here, we provide evidence that in RCCs the rpS6 protein is the major mediator of anti-tumoral effects exerted by everolimus. Inhibition of mTOR signaling results in substantially decreased clonogenicity and proliferation of RCC cells, but did not significantly induce apoptosis. Everolimus effectively blocked protein biosynthesis both in vitro and in a novel ex vivo tissue slice model using fresh vital human RCC tissue. Compared to other components of the mTOR pathway, phosphorylation of rpS6 was most effectively downregulated by everolimus. Importantly, siRNA-mediated downregulation of rpS6, but not of 4ebp1 or p27, abolished the inhibitory effects of everolimus on proliferation and protein synthesis. Moreover, we analyzed the tissue expression of phosphorylated rpS6 (p-rpS6) and non-phosphorylated rpS6 in a large collection of patients with RCCs (n=598 and n=548, respectively). Expression of both proteins qualified as independent negative prognostic markers with a substantially shorter survival of patients with RCCs exhibiting high levels of rpS6 and p-rpS6. Taken together, our functional studies identified rpS6 as a main mediator of the anti-tumoral activity of Everolimus. Therefore, further (pre-)clinical evaluations of rpS6 as a predictive marker for everolimus-based treatment for RCC patients are warranted. Finally, the combined detection of phosphorylated and non-phosphorylated rpS6 could represent a robust prognostic marker to identify patients with high risk RCCs. PMID:26506236

  11. Cytochromes c-552 from two strains of the hydrogenotrophic bacterium Alcaligenes eutrophus are sequence homologs of the cytochromes c8 from the denitrifying pseudomonads.

    PubMed

    Klarskov, K; Bartsch, R G; Meyer, T E; Cusanovich, M A; Van Beeumen, J J

    1997-12-05

    Soluble cytochromes c-552 were purified from two strains of the hydrogenothrophic species Alcaligenes eutrophus and their amino acid sequences determined. The two cytochromes were found to have 5 differences out of a total of 89 residues. The proteins are clearly related to the cytochromes c8 (formerly called Pseudomonas cytochromes c-551), but require a single residue insertion after the methionine sixth heme ligand relative to the Pseudomonas aeruginosa protein. The consensus residues Trp56 and Trp77, characteristic for the c8 family, are also present in the Alcaligenes proteins. Overall, the Alcaligenes cytochromes are only 43% identical to the Pseudomonas proteins which average 68% identity to one another. They are also only 45% identical to cytochrome c8 from Hydrogenobacter thermophilus, another hydrogenothrophic species, which indicates that the hydrogen utilizing bacteria are not more closely related to one another than they are to other species. The finding of cytochrome c8 in Alcaligenes eutrophus completes the recent characterization of a cytochrome cd1-nitrite reductase from this bacterial species and suggests the existence of the same denitrification pathway as in Pseudomonas where these two proteins are reaction partners.

  12. Atypical protein kinase Clambda binds and regulates p70 S6 kinase.

    PubMed Central

    Akimoto, K; Nakaya, M; Yamanaka, T; Tanaka, J; Matsuda, S; Weng, Q P; Avruch, J; Ohno, S

    1998-01-01

    p70 S6 kinase (p70 S6K) has been implicated in the regulation of cell cycle progression. However, the mechanism of its activation is not fully understood. In the present work, evidence is provided that an atypical protein kinase C (PKC) isotype, PKClambda, is indispensable, but not sufficient, for the activation of p70 S6K. Both the regulatory and kinase domains of PKClambda associate directly with p70 S6K. Overexpression of the kinase domain without kinase activity or the regulatory domain of PKClambda results in the suppression of the serum-induced activation of p70 S6K. In addition, two types of dominant-negative mutants of PKClambda, as well as a kinase-deficient mutant of p70 S6K, suppress serum-induced DNA synthesis and E2F activation. The overexpresion of the active form of PKClambda, however, fails to activate p70 S6K. These results suggest that PKClambda is a mediator in the regulation of p70 S6K activity and plays an important role in cell cycle progression. PMID:9761742

  13. 8 CFR 1236.4 - Removal of S-5, S-6, and S-7 nonimmigrants.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 8 Aliens and Nationality 1 2011-01-01 2011-01-01 false Removal of S-5, S-6, and S-7 nonimmigrants... OF ALIENS ORDERED REMOVED Detention of Aliens Prior to Order of Removal § 1236.4 Removal of S-5, S-6, and S-7 nonimmigrants. (a) Condition of classification. As a condition of classification and...

  14. Regulation of anti-apoptotic Bcl-2 family protein Mcl-1 by S6 kinase 2

    PubMed Central

    Sridharan, Savitha

    2017-01-01

    The anti-apoptotic Bcl-2 family protein myeloid cell leukemia-1 (Mcl-1) plays an important role in breast cancer cell survival and chemoresistance. We have previously shown that knockdown of the 40S ribosomal protein S6 kinase-2 (S6K2), which acts downstream of the mechanistic target of rapamycin complex 1 (mTORC1), enhanced breast cancer cell death by apoptotic stimuli. The increase in cell death by S6K2 depletion was partly due to inactivation of Akt. In the present study, we investigated if S6K2 regulates Mcl-1, which acts downstream of Akt. Silencing of S6K2 but not S6K1 in T47D cells decreased Mcl-1 level, and potentiated apoptosis induced by TRAIL and doxorubicin. Knockdown of S6K2 also decreased the level of anti-apoptotic Bcl-xl. Depletion of the tumor suppressor protein PDCD4 (programmed cell death 4), which regulates translation of several anti-apoptotic proteins, reversed downregulation of Bcl-xl but not Mcl-1 and failed to reverse the effect of S6K2 knockdown on potentiation of doxorubicin-induced apoptosis. Downregulation of Mcl-1 by S6K2 knockdown was partly restored by the proteasome inhibitor MG132. Overexpression of catalytically-active Akt or knockdown of glycogen synthase kinase-3 (GSK3)-β, a substrate for Akt, had little effect on Mcl-1 downregulation caused by S6K2 deficiency. Silencing of S6K2 increased the level of c-Jun N-terminal kinase (JNK) and knockdown of JNK1 increased basal Mcl-1 level and partly reversed the effect of S6K2 knockdown on Mcl-1 downregulation. JNK1 knockdown also had a modest effect in attenuating the increase in doxorubicin-induced apoptosis caused by S6K2 deficiency. These results suggest that S6K2 regulates apoptosis via multiple mechanisms, and involves both Akt and JNK. PMID:28301598

  15. Ribosomal Protein S6 Phosphorylation in the Nervous System: From Regulation to Function

    PubMed Central

    Biever, Anne; Valjent, Emmanuel; Puighermanal, Emma

    2015-01-01

    Since the discovery of the phosphorylation of the 40S ribosomal protein S6 (rpS6) about four decades ago, much effort has been made to uncover the molecular mechanisms underlying the regulation of this post-translational modification. In the field of neuroscience, rpS6 phosphorylation is commonly used as a readout of the mammalian target of rapamycin complex 1 signaling activation or as a marker for neuronal activity. Nevertheless, its biological role in neurons still remains puzzling. Here we review the pharmacological and physiological stimuli regulating this modification in the nervous system as well as the pathways that transduce these signals into rpS6 phosphorylation. Altered rpS6 phosphorylation observed in various genetic and pathophysiological mouse models is also discussed. Finally, we examine the current state of knowledge on the physiological role of this post-translational modification and highlight the questions that remain to be addressed. PMID:26733799

  16. Virulence factors in vancomycin-resistant and vancomycin- susceptible Enterococcus faecalis from Brazil

    PubMed Central

    Camargo, I. L. B. C.; Zanella, R. C.; Gilmore, M. S.; Darini, A. L. C.

    2008-01-01

    Enterococci are members of commensal flora of animals and insects, but are also important opportunistic pathogens. Our objective was to observe if there was any difference of virulence in several groups of E. faecalis, mainly between vancomycin-resistant E. faecalis (VREFS) of colonization and infection. VREFS and vancomycin-sensitive E. faecalis from Brazil were screened for the presence of virulence factor genes. Phenotypic assays were used to assess in vitro expression, to understand the pathogenic potential of these isolates and to determine whether a correlation exists between virulence and antibiotic resistance. Different virulence profiles were found suggesting that the disseminating clone may have generated several variations. However, our study showed that one constellation of traits appeared most commonly: gelatinase, aggregation substance and esp (GEA). These factors are important because they have been implicated in cell aggregation and biofilm formation. Biofilm formation may promote the conjugation of plasmids harboring resistance and virulence genes, enhancing the probability of entry of new resistance genes into species. Curiously, the profile GEA was not exclusive to VREFS, it was the second most observed in VSEFS isolates from colonization and infection in hospitalized patients and also from rectal swabs of healthy volunteers. Such strains appear to represent the entry gateway to new resistance genes into E. faecalis and may contribute to the spreading of E. faecalis mainly in hospitals. PMID:24031215

  17. High Frequency of Vancomycin-Resistant Enterococcus Faecalis in an Iranian Referral Children Medical Hospital

    PubMed Central

    POURAKBARI, Babak; AGHDAM, Mojtaba Kamali; MAHMOUDI, Shima; ASHTIANI, Mohammad Taghi Haghi; SABOUNI, Farah; MOVAHEDI, Zahra; ALYARI, Amir Esmael; SADEGHI, Reihane Hosseinpour; MAMISHI, Setareh

    2012-01-01

    ABSTRACT Background: Enterococci have emerged in recent years as important nosocomial pathogens. Although most enterococcal human infections are caused by Enterococcus faecalis, studies on vancomycin resistance are usually limited to Enterococcus faecium isolates and a little is known about E. faecalis. Therefore we undertook this study to obtain information about the prevalence of vancomycin -resistant E. faecalis (VREF) and genes responsible for resistance. Material and methods: Ninety-one E. faecalis isolates of different patients admitted at Children's Medical Center from August 2009 to June 2010 were included in this cross-sectional study. Antimicrobial testing was performed by Kirby-Bauer disk diffusion method according to Clinical Laboratories Standards Institute (CLSI). Results: Among all isolates, 15 (16%) were identified as VR E. faecalis. PCR analysis revealed that all VREF isolates were positive for the vanA gene. Conclusion: The present study reports the highest range of VREF in Iran. The increased frequency of VREF, as seen with rapid rise in the number of VanA isolates should be considered in infection control practices. PMID:23400108

  18. Genetic diversity of Enterococcus faecalis isolated from environmental, animal and clinical sources in Malaysia.

    PubMed

    Daniel, Diane S; Lee, Sui M; Gan, Han M; Dykes, Gary A; Rahman, Sadequr

    2017-02-18

    Enterococcus faecalis ranks as one of the leading causes of nosocomial infections. A strong epidemiological link has been reported between E. faecalis inhabiting animals and environmental sources. This study investigates the genetic diversity, antibiotic resistance and virulence determinants in E. faecalis from three sources in Malaysia. A total of 250 E. faecalis isolates were obtained consisting of 120 isolates from farm animals, 100 isolates from water sources and 30 isolates from hospitalized patients. Pulse-field gel electrophoresis-typing yielded 63 pulsotypes, with high diversity observed in all sources (D=≥0.901). No pulsotype was common to all the three sources. Each patient room had its own unique PFGE pattern which persisted after six months. Minimum inhibitory concentrations of Vancomycin, Gentamicin, Penicillin, Tetracycline, Nitrofurantoin, Levofloxacin, Ciprofloxacin and Fosfomycin were evaluated. Resistance to Tetracycline was most prevalent in isolates from farm animals (62%) and water sources (49%). Water isolates (86%) had a higher prevalence of the asa1 gene, which encodes for aggregation substance, whereas clinical (78%) and farm animal isolates (87%) had a higher prevalence of the esp gene, encoding a surface exposed protein. This study generates knowledge on the genetic diversity of E. faecalis with antibiotic resistance and virulence characteristics from various sources in Malaysia.

  19. The in vitro Effect of Irrigants with Low Surface Tension on Enterococcus faecalis

    PubMed Central

    Giardino, Luciano; Estrela, Carlos; Generali, Luigi; Mohammadi, Zahed; Asgary, Saeed

    2015-01-01

    Introduction: Due to the complex anatomy of the root canal system and high surface tension of common root canal irrigants (RCI), conducting an investigation on RCIs containing surfactants is a priority. The aim of this in vitro study was to verify the antibacterial potential of RCI with low surface tension in root canals infected with Enterococcus faecalis (E. faecalis). Methods and Materials: Thirty-five extracted human maxillary anterior teeth were prepared and inoculated with E. faecalis for 60 days. After root canal preparation, the teeth were randomly divided to one positive and one negative control groups and 5 experimental groups: Hypoclean/Tetraclean NA, Hypoclean, Tetraclean, NaOCl/Tetraclean and NaOCl. Bacterial growth was observed by turbidity of culture medium and then measured using a UV spectrophotometer. Data were analyzed in three time intervals (pre-instrumentation and, 20 min and 72 h after canal preparation) using the ANOVA and post hoc Tukey’s tests. The level of significance was set at 0.05. Results: The results indicated the presence of E. faecalis in all post-irrigation samples irrespective of the RCI. However, the optical densities in both post-irrigation periods showed bacterial reduction and significant differences between groups. Conclusion: RCI with low surface tension showed antibacterial potential in E. faecalis infected roots. PMID:26229541

  20. Proteolytic activity of Enterococcus faecalis VB63F for reduction of allergenicity of bovine milk proteins.

    PubMed

    Biscola, V; Tulini, F L; Choiset, Y; Rabesona, H; Ivanova, I; Chobert, J-M; Todorov, S D; Haertlé, T; Franco, B D G M

    2016-07-01

    With the aim of screening proteolytic strains of lactic acid bacteria to evaluate their potential for the reduction of allergenicity of the major bovine milk proteins, we isolated a new proteolytic strain of Enterococcus faecalis (Ent. faecalis VB63F) from raw bovine milk. The proteases produced by this strain had strong activity against caseins (αS1-, αS2-, and β-casein), in both skim milk and sodium caseinate. However, only partial hydrolysis of whey proteins was observed. Proteolysis of Na-caseinate and whey proteins, observed after sodium dodecyl sulfate-PAGE, was confirmed by analysis of peptide profiles by reversed-phase HPLC. Inhibition of proteolysis with EDTA indicated that the proteases produced by Ent. faecalis VB63F belonged to the group of metalloproteases. The optimal conditions for their activity were 42°C and pH 6.5. The majority of assessed virulence genes were absent in Ent. faecalis VB63F. The obtained results suggest that Ent. faecalis VB63F could be efficient in reducing the immunoreactivity of bovine milk proteins.

  1. Screening of Bacteriocin-producing Enterococcus faecalis Strains for Antagonistic Activities against Clostridium perfringens

    PubMed Central

    Kim, So-Young

    2014-01-01

    This study was conducted to isolate and characterize bacteriocin-producing bacteria against Clostridium perfringens (C. perfringens) from domestic animals to determine their usefulness as probiotics. Bacteriocin-producing bacteria were isolated from pig feces by the spot-on-lawn method. A total of 1,370 bacterial stains were isolated, and six were tentatively selected after identifying the inhibitory activity against the pathogenic indicator C. perfringens KCTC 3269 and KCTC 5100. The selected strains were identified as Enterococcus faecalis (E. faecalis) by 16s rRNA sequencing. Most of the isolated bacterial strains were resistant to 0.5% bile salts for 48 h and remained viable after 2 h at pH 3.0. Some E. faecalis also showed strong inhibitory activity against Listeria monocytogenes KCTC 3569, KCTC 3586 and KCTC 3710. In the present study, we finally selected E. faecalis AP 216 and AP 45 strain based on probiotic selection criteria such as antimicrobial activity against C. perfringens and tolerance to acid and bile salts. The bacteriocins of E. faecalis AP 216 and AP 45 strains were highly thermostable, showing anticlostridial activities even after incubation at 121℃ for 15 min. These bacteriocinproducing bacteria and/or bacteriocins could be used in feed manufacturing as probiotics as an alternative to antibiotics in the livestock industry. PMID:26761495

  2. Multi-drug-resistant Enterococcus faecalis among Egyptian patients with urinary tract infection.

    PubMed

    Abdelkareem, Mohammad Z; Sayed, Mohamed; Hassuna, Noha A; Mahmoud, Mahmoud S; Abdelwahab, Sayed F

    2017-04-01

    The prevalence of Enterococcus faecalis (E. faecalis) infections among Egyptians with urinary tract infection (UTI), their antimicrobial susceptibility and mechanisms of resistance are under investigated. In this study, 300 urine samples were collected from UTI patients to identify E. faecalis. Antimicrobial susceptibility to 18 antimicrobial agents was tested. The presence of aac(6)-Ie-aph(2)Ia, erm(B) and mef(A/E) genes was examined by PCR. Fifty-seven (19%) isolates were identified as E. faecalis. All isolates were sensitive to teicoplanin and were completely resistant to nalidixic acid, cefotaxime and cefadroxil. Multi-drug-resistant (MDR) was found to be 100% with 45 different antibiotypes. The aac(6)Ia-aph(2)Ia gene was found in 100 and 90% of the isolates resistant to gentamicin at concentrations of 120 and 10 μg, respectively. erm(B) and mef(A/E) genes were present in 92.5% (37/40) and 2.5% (1/40) of erythromycin-resistant isolates, respectively. We conclude that there is a high prevalence of E. faecalis in UTI cases with a 100% MDR rate indicating a serious problem in treating infections by this organism in Egypt.

  3. Confocal microscopy evaluation of the effect of irrigants on Enterococcus faecalis biofilm: An in vitro study.

    PubMed

    Flach, Nicole; Böttcher, Daiana Elisabeth; Parolo, Clarissa Cavalcanti Fatturi; Firmino, Luciana Bitello; Malt, Marisa; Lammers, Marcelo Lazzaron; Grecca, Fabiana Soares

    2016-01-01

    The purpose of this study was to evaluate in vitro the effectiveness of two endodontic irrigants and their association against Enterococcus faecalis (E. faecalis) by confocal laser scanning microscope (CLSM). Twenty-four bovine incisors were inoculated in a monoculture of E. faecalis for 21 days. After this period, the teeth were divided into three test groups (n = 5) according to the chemical used. Group 1: 2.5% sodium hypochlorite (NaOCl), group 2: 2% chlorhexidine gel (CHX), group 3: 2.5% NaOCl + 2% CHX gel, and two control groups (n = 3): negative control group (NCG)-sterile and without root canals preparation and positive control group (PCG)-saline. Then, the samples were stained with SYTO9 and propidium iodide and subjected to analysis by CLSM. Bacterial viability was quantitatively analyzed by the proportions of dead and live bacteria in the biofilm remnants. Statistical analysis was performed by the One-way ANOVA test (p = 0.05). No statistical differences were observed to bacterial viability. According to CLSM analysis, none of the tested substances could completely eliminate E. faecalis from the root canal space. Until now, there are no irrigant solutions able to completely eliminate E. faecalis from the root canal. In this regard, the search for irrigants able to intensify the antimicrobial action is of paramount importance.

  4. Genetic relationships among Enterococcus faecalis isolates from different sources as revealed by multilocus sequence typing.

    PubMed

    Chen, X; Song, Y Q; Xu, H Y; Menghe, B L G; Zhang, H P; Sun, Z H

    2015-08-01

    Enterococcus faecalis is part of the natural gut flora of humans and other mammals; some isolates are also used in food production. So, it is important to evaluate the genetic diversity and phylogenetic relationships among E. faecalis isolates from different sources. Multilocus sequence typing protocol was used to compare 39 E. faecalis isolates from Chinese traditional food products (including dairy products, acidic gruel) and 4 published E. faecalis isolates from other sources including human-derived isolates employing 5 housekeeping genes (groEL, clpX, recA, rpoB, and pepC). A total of 23 unique sequence types were identified, which were grouped into 5 clonal complexes and 10 singletons. The value of standardized index of association of the alleles (IA(S)=0.1465) and network structure indicated a high frequency of intraspecies recombination across these isolates. Enterococcus faecalis lineages also exhibited clearly source-clustered distributions. The isolates from dairy source were clustered together. However, the relationship between isolates from acidic gruel and one isolate from a human source was close. The MLST scheme presented in this study provides a sharable and continuously growing sequence database enabling global comparison of strains from different sources, and will further advance our understanding of the microbial ecology of this important species.

  5. Molecular characterization of Rifr mutations in Enterococcus faecalis and Enterococcus faecium.

    PubMed

    Du, Xiaoxing; Hua, Xiaoting; Qu, Tingting; Jiang, Yan; Zhou, Zhihui; Yu, Yunsong

    2014-08-01

    Mutation rate is an important factor affecting the appearance and spread of acquired antibiotic resistance. The frequencies and types of enterococci mutations were determined in this study. The MICs of rifampicin in enterococci and their rifampicin-resistant mutants were determined by the Clinical and Laboratory Standards Institute (CLSI) agar dilution method. The Enterococcus faecalis isolates A15 and 18165 showed no significant differences in mutation frequencies or mutation rates. In Enterococcus faecium, the mutation frequency and mutation rate were both 6·4-fold lower than in E. faecalis. The spectrum of mutations characterized in E. faecium B42 differed significantly from that of E. faecalis. The types and rate of mutations indicated that E. faecalis had a higher potential to develop linezolid resistance. Rifampicin resistance was associated with mutations in the rpoB gene. Rifampicin MICs for the E. faecalis mutant were 2048 mg/l, but rifampicin MICs for E. faecium mutants ranged from 64 to 1024 mg/l.

  6. Detection of the macrolide-efflux protein A gene mef(A) in Enterococcus faecalis.

    PubMed

    Schwaiger, Karin; Hölzel, Christina; Bauer, Johann

    2011-09-01

    The mef(A) gene codes for an efflux protein that conveys resistance to 14- and 15-membered macrolides. Enterococci are emerging pathogens, as well as indicator and reservoir bacteria that are known to have a strong tendency to acquire resistance genes. A total of 485 Enterococcus faecalis strains of porcine (n = 239) and human origin (n = 246) were screened for the presence of the mef(A) gene by using polymerase chain reaction. In total, 29 E. faecalis of porcine (n = 10) and human (n = 19) origin were positive for the presence of the mef(A) gene. Most of the mef(A)-containing strains were isolated from fecal samples of healthy individuals; only one strain originated from a stool sample of a diseased pig. To our knowledge, this is the first report on the occurrence of the mef(A) gene in E. faecalis apart from mating experiments. The main clinical relevance of this study is that donor E. faecalis might transfer the mef(A) gene to recipients that are usually combated with macrolides. Hence, the role of E. faecalis as a resistance reservoir with respect to limited treatment options are a cause for concern.

  7. Assessment of pheromone response in biofilm forming clinical isolates of high level gentamicin resistant Enterococcus faecalis.

    PubMed

    Jayanthi, S; Ananthasubramanian, M; Appalaraju, B

    2008-01-01

    Twenty five clinical isolates of high level gentamicin resistant Enterococcus faecalis were tested for their biofilm formation and pheromone responsiveness. The biofilm assay was carried out using microtiter plate method. Two isolates out of the 25 (8%) were high biofilm formers and 19 (76%) and four (16%) isolates were moderate and weak biofilm formers respectively. All the isolates responded to pheromones of E. faecalis FA2-2 strain. On addition of pheromone producing E. faecalis FA2-2 strain to these isolates, seven of 19 (37%) moderate biofilm formers developed into high biofilm formers. Similarly one of the 4 (25%) weak biofilm formers developed into high level biofilm former. Twelve (48%) of the 25 isolates were transconjugated by cross streak method using gentamicin as selective marker. This proves that the genetic factor for gentamicin resistance is present in the pheromone responsive plasmid. Among these twelve transaconjugants, seven isolates and one isolate were high biofilm formers on addition of E. faecalis FA2-2 and prior to its addition respectively. Out of the total 25 isolates, eight transconjugants for gentamicin resistance could turn to high biofilm formers on addition of the pheromone producing strain. All the isolates were resistant to more than two antibiotics tested. All the isolates were sensitive to vancomycin. The results indicate the significance of this nosocomial pathogen in biofilm formation and the role of pheromone responding clinical isolates of E. faecalis in spread of multidrug resistance genes.

  8. Identification of Polyketide Inhibitors Targeting 3-Dehydroquinate Dehydratase in the Shikimate Pathway of Enterococcus faecalis

    PubMed Central

    Hernandez-Valladares, Maria; Go, Maybelle Kho; Tung, Alvin; Aguda, Adeleke H.; Robinson, Robert C.; Yew, Wen Shan

    2014-01-01

    Due to the emergence of resistance toward current antibiotics, there is a pressing need to develop the next generation of antibiotics as therapeutics against infectious and opportunistic diseases of microbial origins. The shikimate pathway is exclusive to microbes, plants and fungi, and hence is an attractive and logical target for development of antimicrobial therapeutics. The Gram-positive commensal microbe, Enterococcus faecalis, is a major human pathogen associated with nosocomial infections and resistance to vancomycin, the “drug of last resort”. Here, we report the identification of several polyketide-based inhibitors against the E. faecalis shikimate pathway enzyme, 3-dehydroquinate dehydratase (DHQase). In particular, marein, a flavonoid polyketide, both inhibited DHQase and retarded the growth of Enterococcus faecalis. The purification, crystallization and structural resolution of recombinant DHQase from E. faecalis (at 2.2 Å resolution) are also reported. This study provides a route in the development of polyketide-based antimicrobial inhibitors targeting the shikimate pathway of the human pathogen E. faecalis. PMID:25072253

  9. Indole production provides limited benefit to Escherichia coli during co-culture with Enterococcus faecalis.

    PubMed

    Pringle, Shelly L; Palmer, Kelli L; McLean, Robert J C

    2017-01-01

    Escherichia coli lives in the gastrointestinal tract and elsewhere, where it coexists within a mixed population. Indole production enables E. coli to grow with other gram-negative bacteria as indole inhibits N-acyl-homoserine lactone (AHL) quorum regulation. We investigated whether E. coli indole production enhanced competition with gram-positive Enterococcus faecalis, wherein quorum signaling is mediated by small peptides. During planktonic co-culture with E. faecalis, the fitness and population density of E. coli tnaA mutants (unable to produce indole) equaled or surpassed that of E. coli wt. During biofilm growth, the fitness of both populations of E. coli stabilized around 100 %, whereas the fitness of E. faecalis declined over time to 85-90 %, suggesting that biofilm and planktonic populations have different competition strategies. Media supplementation with indole removed the competitive advantage of E. coli tnaA in planktonic populations but enhanced it in biofilm populations. E. coli wt and tnaA showed similar growth in Luria-Bertani (LB) broth. However, E. coli growth was inhibited in the presence of filter-sterilized spent LB from E. faecalis, with inhibition being enhanced by indole. Similarly, there was also an inhibition of E. faecalis growth by proteinaceous components (likely bacteriocins) from spent culture media from both E. coli strains. We conclude that E. coli indole production is not a universal competition strategy, but rather works against gram-negative, AHL-producing bacteria.

  10. Vitality of Enterococcus faecalis inside dentinal tubules after five root canal disinfection methods

    PubMed Central

    Vatkar, Niranjan Ashok; Hegde, Vivek; Sathe, Sucheta

    2016-01-01

    Aim: To compare the vitality of Enterococcus faecalis within dentinal tubules after subjected to five root canal disinfection methods. Materials and Methods: Dentin blocks (n = 60) were colonized with E. faecalis. After 4 weeks of incubation, the dentin blocks were divided into one control and five test groups (n = 10 each). The root canals of test groups were subjected to one of the disinfection methods, namely, normal saline (NS), sodium hypochlorite (NaOCl), chlorhexidine digluconate (CHX), neodymium-doped yttrium aluminum garnet (Nd: YAG) laser, and diode laser. The effect of disinfection methods was assessed by LIVE/DEAD BacLight stain under the confocal laser scanning microscopy to determine the “zone of dead bacteria” (ZDB). Mean values were calculated for ZDB and the difference between groups was established. Results: Penetration of E. faecalis was seen to a depth of >1000 μm. Viable bacteria were detected with NS irrigation. NaOCl and CHX showed partial ZDB. When the root canals were disinfected with Nd: YAG and diode lasers, no viable bacteria were found. Conclusion: E. faecalis has the ability to colonize inside dentinal tubules to a depth of >1000 μm. In contrast to conventional irrigants, both Nd: YAG and diode lasers were effective in eliminating the vitality of E. faecalis. NS, NaOCl, and CHX showed viable bacteria remaining in dentinal tubules. PMID:27656064

  11. Co-immobilization of Pseudomonas stutzeri YHA-13 and Alcaligenes sp. ZGED-12 with polyvinyl alcohol-alginate for removal of nitrogen and phosphorus from synthetic wastewater.

    PubMed

    Han, Yonghe; Zhang, Wenxian; Lu, Wenxian; Zhou, Zhihua; Zhuang, Zhigang; Li, Min

    2014-01-01

    Nitrogen (N) and phosphorus (P) are the two main factors causing water eutrophication. Immobilized micro-organisms have been widely studied in N and P removal. However, the effects of various immobilizing conditions on the removal efficiency of N and P using immobilized micro-organism beads (IMOBs) remain unclear. Polyvinyl alcohol (PVA) and alginate, as the two frequently immobilizing-used matrixes, were used for co-immobilizing Pseudomonas stutzeri YHA-13 and Alcaligenes sp. ZGED-12. PVA, alginate and CaCl₂contents, immobilization time and different wet biomass ratios of P. stutzeri to Alcaligenes sp. were conducted to elucidate their roles in and influences on the removal efficiency of N and P from synthetic wastewater. The application potential of IMOBs was estimated as well. Results showed that IMOBs prepared by cross-link of 4% PVA and 2-3% alginate with 5% CaCl₂and saturated boric acid solution for 10-15 min are the best ones in removal of N and P. Though IMOBs containing P. stutzeri and/or Alcaligenes sp. were capable of removal of the two nutrients, the highest removal efficiency was observed when the wet biomass ratio of P. stutzeri to Alcaligenes sp. was adjusted to 2:2. In addition, the IMOBs were of good ability to remove chemical oxygen demand (COD), NO(3)(-), NO(2)(-), NH(4)(+)- N, total nitrogen (TN) and total phosphorus (TP) from artificial wastewater. Of which, micro-organisms immobilized in matrixes were mainly responsible for NO(3)(-) and TP removal. Therefore, P. stutzeri YHA-13 and Alcaligenes sp. ZGED-12 are reliable bioresources to remove N and P from wastewater.

  12. Involvement of S6K1 in mitochondria function and structure in HeLa cells.

    PubMed

    Park, Jisoo; Tran, Quangdon; Mun, Kisun; Masuda, Kouhei; Kwon, So Hee; Kim, Seon-Hwan; Kim, Dong-Hoon; Thomas, George; Park, Jongsun

    2016-12-01

    The major biological function of mitochondria is to generate cellular energy through oxidative phosphorylation. Apart from cellular respiration, mitochondria also play a key role in signaling processes, including aging and cancer metabolism. It has been shown that S6K1-knockout mice are resistant to obesity due to enhanced beta-oxidation, with an increased number of large mitochondria. Therefore, in this report, the possible involvement of S6K1 in regulating mitochondria dynamics and function has been investigated in stable lenti-shS6K1-HeLa cells. Interestingly, S6K1-stably depleted HeLa cells showed phenotypical changes in mitochondria morphology. This observation was further confirmed by detailed image analysis of mitochondria shape. Corresponding molecular changes were also observed in these cells, such as the induction of mitochondrial fission proteins (Drp1 and Fis1). Oxygen consumption is elevated in S6K1-depeleted HeLa cells and FL5.12 cells. In addition, S6K1 depletion leads to enhancement of ATP production in cytoplasm and mitochondria. However, the relative ratio of mitochondrial ATP to cytoplasmic ATP is actually decreased in lenti-shS6K1-HeLa cells compared to control cells. Lastly, induction of mitophagy was found in lenti-shS6K1-HeLa cells with corresponding changes of mitochondria shape on electron microscope analysis. Taken together, our results indicate that S6K1 is involved in the regulation of mitochondria morphology and function in HeLa cells. This study will provide novel insights into S6K1 function in mitochondria-mediated cellular signaling.

  13. Identification of a Dual Inhibitor of Janus Kinase 2 (JAK2) and p70 Ribosomal S6 Kinase1 (S6K1) Pathways*

    PubMed Central

    Byun, Sanguine; Lim, Semi; Mun, Ji Young; Kim, Ki Hyun; Ramadhar, Timothy R.; Farrand, Lee; Shin, Seung Ho; Thimmegowda, N. R.; Lee, Hyong Joo; Frank, David A.; Clardy, Jon; Lee, Sam W.; Lee, Ki Won

    2015-01-01

    Bioactive phytochemicals can suppress the growth of malignant cells, and investigation of the mechanisms responsible can assist in the identification of novel therapeutic strategies for cancer therapy. Ginger has been reported to exhibit potent anti-cancer effects, although previous reports have often focused on a narrow range of specific compounds. Through a direct comparison of various ginger compounds, we determined that gingerenone A selectively kills cancer cells while exhibiting minimal toxicity toward normal cells. Kinase array screening revealed JAK2 and S6K1 as the molecular targets primarily responsible for gingerenone A-induced cancer cell death. The effect of gingerenone A was strongly associated with relative phosphorylation levels of JAK2 and S6K1, and administration of gingerenone A significantly suppressed tumor growth in vivo. More importantly, the combined inhibition of JAK2 and S6K1 by commercial inhibitors selectively induced apoptosis in cancer cells, whereas treatment with either agent alone did not. These findings provide rationale for dual targeting of JAK2 and S6K1 in cancer for a combinatorial therapeutic approach. PMID:26242912

  14. In vitro activity of Amazon plant extracts against Enterococcus faecalis

    PubMed Central

    de Castilho, Adriana Lígia; da Silva, Juliana Paola Correa; Saraceni, Cintia Helena Coury; Díaz, Ingrit Elida Collantes; Paciencia, Mateus Luís Barradas; Varella, Antonio Drauzio; Suffredini, Ivana Barbosa

    2014-01-01

    Previous studies analyzing 2,200 plant extracts indicated anti-enterococcal activity in 25 extracts obtained from Brazilian forests’ plants. In the present study, these extracts were subjected to microdilution broth assay (MDBA) and disk diffusion assay (DDA) using planktonic Enterococcus faecalis ATCC® 29212™ and were submitted to phytochemical analysis in TLC and HPLC. Three extracts obtained from Ipomoea alba (MIC < 40 μg/mL), Diclinanona calycina (MIC ≤ 40 μg/mL) and Moronobea coccinea (40 < MIC < 80 μg/mL; MBC = 80 μg/mL) showed significant bactericidal activity in the MDBA and four extracts obtained from I. alba (14.04 ± 0.55 mm diameter) S. globulifera (14.43 ± 0.33 mm and 12.18 ± 0.28 mm diameter) and Connarus ruber var. ruber (13.13 ± 0.18 mm diameter) were active in DDA. Residues H2O obtained from Psidium densicomum (mean of 16.78 mm diameter) and from Stryphnodendron pulcherrimum (mean of 15.97 mm diameter) have shown an improved antibacterial activity after fractionation if compared to that obtained from the respective crude extracts. Antioxidant activity was observed in some residues of the active extracts. TLC analysis showed that phenolic compounds are likely to be found in active extracts. Three molecules were isolated from S. globulifera and were identified by 13C NMR lupeol, α-amyrin and 3β-hydroxyglutin-5-ene. The present chemical and biological findings suggest that these extracts are a potential source of new anti-Enterococcus compounds to be introduced in endodontic therapy. PMID:25477906

  15. Interference in Pheromone-Responsive Conjugation of a High-Level Bacitracin Resistant Enterococcus faecalis Plasmid of Poultry Origin

    PubMed Central

    Tremblay, Cindy-Love; Archambault, Marie

    2013-01-01

    The current study reports on contact interference of a high-level bacitracin- resistant pheromone-responsive plasmid of Enterococcus faecalis strain 543 of poultry origin during conjugative transfer of bcr antimicrobial resistance genes using a polyclonal antiserum aggregation substance44–560 (AS). After induction with pheromones produced by the recipient strain E. faecalis JH2-2, clumping of the donor E. faecalis strain 543 was observed as well as high transfer frequencies of bcr in short time broth mating. Filter mating assays from donor strain E. faecalis 543 to the recipient strain E. faecalis JH2-2 revealed conjugative transfer of asa1 (AS), bcrRAB and traB (negative regulator pheromone response) genes. The presence of these genes in transconjugants was confirmed by antimicrobial susceptibility testing, PCR, Southern hybridization and sequencing. A significant reduction in formation of aggregates was observed when the polyclonal anti-AS44–560 was added in the pheromone-responsive conjugation experiments as compared to the induced state. Moreover, interference of anti-AS44–560 antibodies in pheromone-responsive conjugation was demonstrated by a reduction in horizontal transfer of asa1 and bcr genes between E. faecalis strain 543 and E. faecalis JH2-2. Reducing the pheromone-responsive conjugation of E. faecalis is of interest because of its clinical importance in the horizontal transfer of antimicrobial resistance. PMID:24030654

  16. Biofilm formation on polystyrene under different temperatures by antibiotic resistant Enterococcus faecalis and Enterococcus faecium isolated from food.

    PubMed

    Marinho, A R; Martins, P D; Ditmer, E M; d'Azevedo, P A; Frazzon, J; Van Der Sand, S T; Frazzon, A P G

    2013-01-01

    The ability of antibiotic resistant E. faecalis and E. faecium isolated from food to form biofilm at different temperatures in the absence or presence of 0.75% glucose was evaluated. A synergistic effect on biofilm at 10 °C, 28 °C, 37 °C and 45 °C and glucose was observed for E. faecalis and E. faecium.

  17. In vitro studies of plasmid-mediated penicillinase from Streptococcus faecalis suggest a staphylococcal origin.

    PubMed Central

    Murray, B E; Mederski-Samoraj, B; Foster, S K; Brunton, J L; Harford, P

    1986-01-01

    A strain of Streptococcus faecalis with plasmid-mediated penicillinase production was studied further. Partially purified penicillinase from the S. faecalis strain hydrolyzed penicillin, ampicillin, and ureido-penicillins but not penicillinase-resistant semisynthetic penicillins, cephalosporins, or imipenem; hydrolysis was inhibited by clavulanic acid. Hydrolysis of a given antibiotic correlated with a marked increase in the minimal inhibitory concentration (MIC) of that drug when a high inoculum was used. As with most enterococci, the MICs of cephalosporins and penicillinase-resistant semisynthetic penicillins were too high for clinical usefulness, although these agents did not show an inoculum effect. Based upon hybridization under stringent conditions of plasmid DNA from the S. faecalis strain to cloned penicillinase genes from Staphylococcus aureus, it appears that these resistance determinants are highly homologous and suggests that this enzyme was introduced into streptococci from staphylococci. Images PMID:3080475

  18. Effect of NaCl Treatments on Tyramine Biosynthesis of Enterococcus faecalis.

    PubMed

    Liu, Fang; Wang, Xinxin; Du, Lihui; Wang, Daoying; Zhu, Yongzhi; Geng, Zhiming; Xu, Xiaoxi; Xu, Weimin

    2015-05-01

    The effect of NaCl stress (0 to 8%, wt/vol) on the growth and tyramine production in two Enterococcus faecalis strains was examined during culture time. The growth of E. faecalis was inhibited by the increase in NaCl concentration, but tyramine production was unaffected. Tyramine accumulated rapidly during the logarithmic phase of the strains, and the final tyramine levels were approximately 800 μg/ml. Relative gene expression of four genes in the tyrosine decarboxylase locus, namely, tyrRS, tyrDC, tyrP, and nhaC, was evaluated at different incubation times. The results showed that NaCl stress could upregulate the expression of tyrDC and tyrP to improve the tyramine production of a single E. faecalis strain under certain conditions, and TyrS could act as a negative regulator on the genetic regulation of the tyramine cluster.

  19. Ribosomal protein S6 phosphorylation is controlled by TOR and modulated by PKA in Candida albicans.

    PubMed

    Chowdhury, Tahmeena; Köhler, Julia R

    2015-10-01

    TOR and PKA signaling pathways control eukaryotic cell growth and proliferation. TOR activity in model fungi, such as Saccharomyces cerevisiae, responds principally to nutrients, e.g., nitrogen and phosphate sources, which are incorporated into the growing cell mass; PKA signaling responds to the availability of the cells' major energy source, glucose. In the fungal commensal and pathogen, Candida albicans, little is known of how these pathways interact. Here, the signal from phosphorylated ribosomal protein S6 (P-S6) was defined as a surrogate marker for TOR-dependent anabolic activity in C. albicans. Nutritional, pharmacologic and genetic modulation of TOR activity elicited corresponding changes in P-S6 levels. The P-S6 signal corresponded to translational activity of a GFP reporter protein. Contributions of four PKA pathway components to anabolic activation were then examined. In high glucose concentrations, only Tpk2 was required to upregulate P-S6 to physiologic levels, whereas all four tested components were required to downregulate P-S6 in low glucose. TOR was epistatic to PKA components with respect to P-S6. In many host niches inhabited by C. albicans, glucose is scarce, with protein being available as a nitrogen source. We speculate that PKA may modulate TOR-dependent cell growth to a rate sustainable by available energy sources, when monomers of anabolic processes, such as amino acids, are abundant.

  20. Characterization of a phorbol ester-stimulated S6 kinase from MDCK renal epithelial cells

    SciTech Connect

    Meier, K.E.; Krebs, E.G.

    1987-05-01

    Increased phosphorylation of S6, a 40S ribosomal subunit protein, is observed in mammalian cells in response to growth factors and phorbol esters. The goal of this study was to identify the S6 kinase that is stimulated by phorbol ester treatment of MDCK cells. MDCK clone D1 cells express high levels of protein kinase C(PKC). PKC and S6 kinase activities were measured following DEAE-Sephacel fractionation of cytosol; this procedure separated the two kinase activities. When confluent MDCK-D1 cells were exposed to 100 nM phorbol 12-myristate 13-acetate (PMA), 95% of the total cellular PKC activity became associated with the particulate fraction within 1 hour. Cytosolic S6 kinase activity was maximal by 1 hour and then declined thereafter, preceding any detectable loss of total cellular PKC. The PMA-responsive S6 kinase was partially purified from MDCK-D1 cytosol by consecutive steps of DEAE-Sephacel, ammonium sulfate precipitation, Ultrogel AcA 34, heparin-agarose, and Ultrogel AcA 34. The partially-purified enzyme had an apparent molecular size of approximately 80 kDa. In addition to S6, the enzyme phosphorylated synthetic peptides based on the carboxyl terminal sequence of S6. S6 kinase activity utilized ATP but not GTP, and was inhibited by heparin, NaCl, and ..beta..-glycerophosphate. In conclusion, a phorbol ester-stimulated S6 kinase has been partially purified from an epithelial cell line. This kinase is distinct from PKC.

  1. Biotyping, serotyping and phage typing of Streptococcus faecalis isolated from dental plaque in the human mouth.

    PubMed

    Smyth, C J; Matthews, H; Halpenny, M K; Brandis, H; Colman, G

    1987-02-01

    Thirty Streptococcus faecalis isolates from mixed dental plaque samples were classified into four groups on the basis of biotype, tetracycline susceptibility, phage type and serotype combinations. The organisms were from patients on haemodialysis, from staff of the dialysis unit, and from controls. Three biotypes were distinguished by seven biochemical tests: production of acid from inositol, sucrose and xylose; rapid or delayed production of acid from sorbitol; gelatin liquefaction; and production of alkaline phosphatase and beta-galactosidase. With a set of eight typing antisera for S. faecalis, 15 strains were non-typable, 12 were serotype 1 and three were serotype 19. With a set of 17 bacteriophages specific for S. faecalis, all of the oral isolates were typable; 40% were lysotype I1 and the remainder lysotype V6b. On the basis of biotype-serotype-phage-type combinations, indications of possible spread of strains between haemodialysis patients and dialysis unit staff were obtained. Biotyping and serotyping of 13 German isolates of S. faecalis of phage type I1 from four clinical sources and tripartite typing of three control strains provided additional evidence for the potential of biotyping in distinguishing between strains of identical serotype and phage type. One oral isolate of S. faecium was of phage type XX. None of the oral isolates of S. faecalis, of which 14 exhibited delayed sorbitol fermentation, reacted with group-G streptococcal grouping reagents or antiserum. Slow sorbitol fermentation does not appear to be a definitive phenotypic marker for S. faecalis strains possessing antigens that react with both group-D and group-G grouping reagents.

  2. Effects of iron and phytic acid on production of extracellular radicals by Enterococcus faecalis.

    PubMed

    Moore, Danny R; Kotake, Yashige; Huycke, Mark M

    2004-12-01

    Enterococcus faecalis is a human intestinal commensal that produces extracellular superoxide, hydrogen peroxide, and hydroxyl radical while colonizing the intestinal tract. To determine whether dietary factors implicated in colorectal cancer affect oxidant production by E. faecalis, radicals were measured in rats colonized with this microorganism while on diets supplemented with iron or phytic acid. Hydroxyl radical activity was measured by assaying for aromatic hydroxylation products of D-phenylalanine using reverse-phase high-performance liquid chromatography and electrochemical detection. In vitro, as expected, iron enhanced, and phytic acid decreased, hydroxyl radical formation by E. faecalis. For rats colonized with E. faecalis given supplemental dietary iron (740 mg elemental iron as ferric phosphate per kg diet) or phytic acid (1.2% w/w), no differences were found in concentrations of urinary ortho- or meta- isomers of D-phenylalanine compared to rats on a basal diet. Aqueous radicals in colonic contents were further assessed ex vivo by electron spin resonance using 5,5-dimethyl-1-pyrroline-N-oxide as a spin trap. Mixtures of thiyl (sulfur-centered) and oxygen-centered radicals were detected across all diets. In vitro, similar spectra were observed when E. faecalis was incubated with hydrogen sulfide, air-oxidized cysteine, or an alkylsulfide, as typical sulfur-containing compounds that might occur in colonic contents. In conclusion, intestinal colonization with E. faecalis in a rat model generates both thiyl and oxygen-centered radicals in colonic contents. Radical formation, however, was not significantly altered by short-term dietary supplementation with iron or phytic acid.

  3. Enterococcal surface protein Esp does not facilitate intestinal colonization or translocation of Enterococcus faecalis in clindamycin-treated mice.

    PubMed

    Pultz, Nicole J; Shankar, Nathan; Baghdayan, Arto S; Donskey, Curtis J

    2005-01-15

    Enterococcal surface protein (Esp) is a cell wall-associated protein of Enterococcus faecalis that has been identified as a potential virulence factor. We used a mouse model to examine whether Esp facilitates intestinal colonization or translocation of E. faecalis to mesenteric lymph nodes. After clindamycin treatment, similar levels of high-density colonization were established after orogastric inoculation of an E. faecalis isolate containing the esp gene within a large pathogenicity island and an isogenic mutant created by allelic replacement of the esp gene with a chloramphenicol resistance cassette (P=0.7); translocation to mesenteric lymph nodes was detected in 3 of 12 (25%) mice in both groups. Isogenic mutants of FA2-2 (a plasmid-free derivative of E. faecalis strain JH2) with or without the esp gene failed to establish colonization of clindamycin-treated mice. These results suggest that Esp does not facilitate intestinal colonization or translocation of E. faecalis.

  4. Detection of the esp gene in high-level gentamicin resistant Enterococcus faecalis strains from pet animals in Japan.

    PubMed

    Harada, Tetsuya; Tsuji, Noboru; Otsuki, Koichi; Murase, Toshiyuki

    2005-03-20

    We investigated the prevalence of the esp gene and the susceptibility to gentamicin in Enterococcus faecalis and E. faecium strains obtained from pet animals. Nine of 30 E. faecalis and 2 of 38 E. faecium strains from the pet animals had the esp gene. Three esp-positive E. faecalis strains, which were isolated from two dogs and a cat, showed gentamicin MICs of > or =256 microg/ml and harbored the high-level gentamicin resistance (HLGR) gene, aac(6')-Ie-aph(2'')-Ia. Of the nine esp-positive E. faecalis strains, five, including the three strains with the HLGR gene, were closely related by numerical analysis of PFGE patterns. Longitudinal investigation needs to elucidate whether the HLGR gene was incorporated into a subpopulation of the esp-positive E. faecalis.

  5. Long Periodic Helimagnetic Ordering in CrM 3S6 (M = Nb and Ta)

    NASA Astrophysics Data System (ADS)

    Kousaka, Y.; Ogura, T.; Zhang, J.; Miao, P.; Lee, S.; Torii, S.; Kamiyama, T.; Campo, J.; Inoue, K.; Akimitsu, J.

    2016-09-01

    We report long periodic chiral helimagnetic orderings in ferromagnetic inorganic compounds CrM 3S6 (M = Nb and Ta) with a chiral space group of P6322. Magnetization in polycrystalline samples and high resolution powder neutron diffraction were measured. Our powder neutron diffraction measurements in CrM 3S6 successfully separated nuclear and magnetic satellite peaks, having the period of hundreds of angstroms along the c— axis. Therefore, we propose that the magnetic ordering in ferromagnetic CrM3S6 is not ferromagnetic, but long periodic chiral helimagnetic ordering.

  6. Efficacy of Ceftobiprole Medocaril against Enterococcus faecalis in a Murine Urinary Tract Infection Model

    PubMed Central

    Murray, Barbara E.

    2012-01-01

    We evaluated ceftobiprole against the well-characterized Enterococcus faecalis strain OG1RF (with and without the β-lactamase [Bla] plasmid pBEM10) in a murine urinary tract infection (UTI) model. Ceftobiprole was equally effective for Bla+ and Bla− OG1 strains, while ampicillin was moderately to markedly (depending on the inoculum) less effective against Bla+ than Bla− OG1 strains. These data illustrate an in vivo effect on ampicillin of Bla production by E. faecalis and the stability and efficacy of ceftobiprole in experimental UTI. PMID:22450988

  7. Evaluation of Rutter Sigma S6 Ice Navigation Radar on USCGC Healy during Arctic Shield 2014

    DTIC Science & Technology

    2015-03-01

    determine if the Sigma S6 processor is an improvement upon current radar processing capabilities. Data were collected simultaneously from both radar...system was evaluated. The Sigma S6 radar processor in the Ice Navigation radar system is unique in that it processes a full dynamic range of data (12...Sigma 6 processor and a hand-held camera to record CG radar display data . Photographs, videos and other environmental data will be collected through

  8. Skeletal myocyte hypertrophy requires mTOR kinase activity and S6K1

    SciTech Connect

    Park, In-Hyun . E-mail: ihpark@uiuc.edu; Erbay, Ebru; Nuzzi, Paul; Chen Jie

    2005-09-10

    The protein kinase mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and growth, with the ribosomal subunit S6 kinase 1 (S6K1) as one of the key downstream signaling effectors. A critical role of mTOR signaling in skeletal muscle differentiation has been identified recently, and an unusual regulatory mechanism independent of mTOR kinase activity and S6K1 is revealed. An mTOR pathway has also been reported to regulate skeletal muscle hypertrophy, but the regulatory mechanism is not completely understood. Here, we report the investigation of mTOR's function in insulin growth factor I (IGF-I)-induced C2C12 myotube hypertrophy. Added at a later stage when rapamycin no longer had any effect on normal myocyte differentiation, rapamycin completely blocked myocyte hypertrophy as measured by myotube diameter. Importantly, a concerted increase of average myonuclei per myotube was observed in IGF-I-stimulated myotubes, which was also inhibited by rapamycin added at a time when it no longer affected normal differentiation. The mTOR protein level, its catalytic activity, its phosphorylation on Ser2448, and the activity of S6K1 were all found increased in IGF-I-stimulated myotubes compared to unstimulated myotubes. Using C2C12 cells stably expressing rapamycin-resistant forms of mTOR and S6K1, we provide genetic evidence for the requirement of mTOR and its downstream effector S6K1 in the regulation of myotube hypertrophy. Our results suggest distinct mTOR signaling mechanisms in different stages of skeletal muscle development: While mTOR regulates the initial myoblast differentiation in a kinase-independent and S6K1-independent manner, the hypertrophic function of mTOR requires its kinase activity and employs S6K1 as a downstream effector.

  9. 8 CFR 236.4 - Removal of S-5, S-6, and S-7 nonimmigrants.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 8 Aliens and Nationality 1 2011-01-01 2011-01-01 false Removal of S-5, S-6, and S-7 nonimmigrants... of Aliens Prior to Order of Removal § 236.4 Removal of S-5, S-6, and S-7 nonimmigrants. (a) Condition... section 101(a)(15)(S) of the Act, nonimmigrants in S classification must have executed Form I-854, Part...

  10. Genome sequence of Corynebacterium nuruki S6-4 T, isolated from alcohol fermentation starter.

    PubMed

    Shin, Na-Ri; Whon, Tae Woong; Roh, Seong Woon; Kim, Min-Soo; Jung, Mi-Ja; Lee, Jina; Bae, Jin-Woo

    2011-08-01

    Corynebacterium nuruki S6-4(T), isolated from Korean alcohol fermentation starter, is a strictly aerobic, nonmotile, Gram-positive, and rod-shaped bacterium belonging to the genus Corynebacterium and the actinomycete group. We report here the draft genome sequence of C. nuruki strain S6-4(T) (3,106,595 bp, with a G+C content of 69.5%).

  11. Genome Sequence of Corynebacterium nuruki S6-4T, Isolated from Alcohol Fermentation Starter▿

    PubMed Central

    Shin, Na-Ri; Whon, Tae Woong; Roh, Seong Woon; Kim, Min-Soo; Jung, Mi-Ja; Lee, Jina; Bae, Jin-Woo

    2011-01-01

    Corynebacterium nuruki S6-4T, isolated from Korean alcohol fermentation starter, is a strictly aerobic, nonmotile, Gram-positive, and rod-shaped bacterium belonging to the genus Corynebacterium and the actinomycete group. We report here the draft genome sequence of C. nuruki strain S6-4T (3,106,595 bp, with a G+C content of 69.5%). PMID:21685278

  12. Ribosomal protein S6 associates with alphavirus nonstructural protein 2 and mediates expression from alphavirus messages.

    PubMed

    Montgomery, Stephanie A; Berglund, Peter; Beard, Clayton W; Johnston, Robert E

    2006-08-01

    Although alphaviruses dramatically alter cellular function within hours of infection, interactions between alphaviruses and specific host cellular proteins are poorly understood. Although the alphavirus nonstructural protein 2 (nsP2) is an essential component of the viral replication complex, it also has critical auxiliary functions that determine the outcome of infection in the host. To gain a better understanding of nsP2 function, we sought to identify cellular proteins with which Venezuelan equine encephalitis virus nsP2 interacted. We demonstrate here that nsP2 associates with ribosomal protein S6 (RpS6) and that nsP2 is present in the ribosome-containing fractions of a polysome gradient, suggesting that nsP2 associates with RpS6 in the context of the whole ribosome. This result was noteworthy, since viral replicase proteins have seldom been described in direct association with components of the ribosome. The association of RpS6 with nsP2 was detected throughout the course of infection, and neither the synthesis of the viral structural proteins nor the presence of the other nonstructural proteins was required for RpS6 interaction with nsP2. nsP1 also was associated with RpS6, but other nonstructural proteins were not. RpS6 phosphorylation was dramatically diminished within hours after infection with alphaviruses. Furthermore, a reduction in the level of RpS6 protein expression led to diminished expression from alphavirus subgenomic messages, whereas no dramatic diminution in cellular translation was observed. Taken together, these data suggest that alphaviruses alter the ribosome during infection and that this alteration may contribute to differential translation of host and viral messages.

  13. Identification of amino acid residues essential for catalytic activity of gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIB 9867.

    PubMed

    Chua, C H; Feng, Y; Yeo, C C; Khoo, H E; Poh, C L

    2001-10-16

    Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) is a ring cleavage enzyme that utilizes gentisate as a substrate yielding maleylpyruvate as the ring fission product. Mutant GDOs were generated by both random mutagenesis and site-directed mutagenesis of the gene cloned from Pseudomonas alcaligenes NCIB 9867. Alignment of known GDO sequences indicated the presence of a conserved central core region. Mutations generated within this central core resulted in the complete loss of enzyme activity whereas mutations in the flanking regions yielded GDOs with enzyme activities that were reduced by up to 78%. Site-directed mutagenesis was also performed on a pair of highly conserved HRH and HXH motifs found within this core region. Conversion of these His residues to Asp resulted in the complete loss of catalytic activity. Mutagenesis within the core region could have affected quaternary structure formation as well as cofactor binding. A mutant enzyme with increased catalytic activities was also characterized.

  14. Amphoteric surfactant N-oleoyl-N-methyltaurine utilized by Pseudomonas alcaligenes with excretion of N-methyltaurine.

    PubMed

    Denger, Karin; Mayer, Jutta; Hollemeyer, Klaus; Cook, Alasdair M

    2008-11-01

    The amphoteric surfactant N-oleoyl-N-methyltaurine, which is in use in skin-care products, was utilized by aerobic bacteria as the sole source of carbon or of nitrogen in enrichment cultures. One isolate, which was identified as Pseudomonas alcaligenes, grew with the xenobiotic compound as the sole source of carbon and energy. The sulfonate moiety, N-methyltaurine, was excreted quantitatively during growth, while the fatty acid was dissimilated. The initial degradative reaction was shown to be hydrolytic and inducible. This amidase reaction could be demonstrated with crude cell extracts. The excreted N-methyltaurine could be utilized by other bacteria in cocultures. Complete degradation of similar natural compounds in bacterial communities seems likely.

  15. [Introduction of mutator phage D3112 of Pseudomonas aeruginosa into Alcaligenes eutrophus var. metallotollerans (Strain CH34)].

    PubMed

    Krylov, V N

    1996-01-01

    It is demonstrated that the intact genome of a D3112 tranposable phage (TP) of Pseudomonas aeruginosa, integrated into a recombinant plasmid RP4 :: D3112, can be transferred by means of conjugation from P. putida PpG1 (RP4npt :: D3112) donor cells into Alcaligenes eutrophus var. metallotollerans cells. P. aeruginosa strains are unacceptable as donors because they have a bactericidal effect on A. eutrophus. RP4npt :: D3112 plasmid is stably inherited by A. eutrophus with D3112 being expressed and successfully reproduced. However, TP loses the induction ability after UV irradiation or mitomycin C treatment. It is suggested that D3112 TP and its miniderivatives could be used in manipulations with A. eutrophus var. metallotolerans.

  16. Tn5563, a transposon encoding putative mercuric ion transport proteins located on plasmid pRA2 of Pseudomonas alcaligenes.

    PubMed

    Yeo, C C; Tham, J M; Kwong, S M; Yiin, S; Poh, C L

    1998-08-15

    Sequence analysis of pRA2, an endogenous 33-kb plasmid from Pseudomonas alcaligenes NCIB 9867 (strain P25X), revealed the presence of a 6256-bp transposon of the Tn3 family, designated Tn5563. Tn5563, which is flanked by two 39-bp inverted repeats, encodes a transposase, a resolvase, and two open reading frames which share amino acid sequence similarities with the mercuric ion transport proteins MerT and MerP encoded by several mer operons. However, no other mer operon genes were found on Tn5563. Sequencing of a RP4::XIn hybrid plasmid indicates possible interactions between pRA2 and the P25X chromosome mediated by Tn5563.

  17. New Biocatalysts: Essential Tools for a Sustainable 21st Century Chemical Industry

    DTIC Science & Technology

    2005-01-01

    sp. Candida parapsilosis Fusarium oxysporum Pseudomonas putida Saccharomyces sake Alcaligenes faecalis Enterobacter aerogenes Erwinia carotovora...Rhodococcus rhodochrous Serratia marcescens Xanthobacter agilis Nocardia corallina Beauveria bassiana Rhodococcus erythropolis Alcaligenes sp., Pseudomonas

  18. Characterization of an extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Pseudomonas alcaligenes LB19.

    PubMed

    Kim, Do Young; Nam, Jin Sik; Rhee, Young Ha

    2002-01-01

    An extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase from an isolate, Pseudomonas alcaligenes LB19, was purified to electrophoretic homogeneity by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and gel permeation chromatography using Sephadex G-150. The molecular mass of the enzyme, which consisted of a single polypeptide chain, was approximately 27.6 kDa. The pI value of the enzyme was estimated to be 5.7, and its maximum activity was observed at pH 9.0 and 45 degreesC. The enzyme was significantly inactivated by EDTA and phenylmethylsulfonyl fluoride (PMSF) but insensitive to dithiothreitol. It was also markedly inhibited by 0.1% Tween 80 and 0.05% Triton X-100. The purified enzyme could hydrolyze various types of bacterial aliphatic and aromatic MCL-PHAs but not poly(3-hydroxybutyrate), polycaprolactone, and poly(L-lactide). Biodegradation rates of the aromatic MCL-PHAs were significantly lower than those of the aliphatic MCL-PHAs, regardless of the compositions and types of aromatic substituents. It was able to hydrolyze medium-chain-length p-nitrophenylalkanoates more efficiently than the shorter-chain forms. The main hydrolysis products of poly(3-hydroxynonanoate) were identified as monomer units. The results demonstrated in this study suggest that the MCL-PHA depolymerase from P. alcaligenes LB19 is a distinct enzyme, which are different from those of other MCL-PHA degrading bacteria in its quaternary structure, pI value, sensitivity to EDTA and PMSF, and hydrolysis products of MCL-PHA.

  19. Draft Genome Sequence of Enterococcus faecalis Strain UCD-PD3

    PubMed Central

    De Vries, Dana R.; Martin, Alexandra L.; Coil, David A.

    2016-01-01

    Here, we present the draft genome sequence of Enterococcus faecalis strain UCD-PD3. The assembly contains 2,861,314 bp in 73 contigs. This strain was isolated from a feral domestic cat (Felis catus) anal sac secretion sample, as part of a project on isolating and characterizing the microbes present in feline anal sacs. PMID:27979940

  20. Production of tyramine by Enterococcus faecalis strains in water-boiled salted duck.

    PubMed

    Liu, Fang; Du, Lihui; Xu, Weiyan; Wang, Daoying; Zhang, Muhan; Zhu, Yongzhi; Xu, Weimin

    2013-05-01

    The potential to produce biogenic amines was investigated with 15 Lactococcus lactis and 15 Enterococcus faecalis strains isolated from water-boiled salted duck. The production of biogenic amines from the isolated strains grown in de Man Rogosa Sharpe broth containing precursor amino acids was determined by thin-layer chromatography and high-performance liquid chromatography. None of the L. lactis strains produced any biogenic amines, whereas 12 strains of E. faecalis produced tyramine and b -phenylethylamine. PCR assays were used to detect the presence of tyrosine decarboxylase genes in all of the isolated strains. Only the 12 biogenic amine-producing Enterococcus strains had a 924-bp fragment characteristic for the tyrosine decarboxylase gene. The comparison of the amplified partial tyrDC gene sequences of the 12 positive Enterococcus strains revealed 99% similarity within the same species. The tyramine production of the sterilized water-boiled salted duck inoculated with E. faecalis R612Z1 increased significantly during storage. This study reveals that the isolated E. faecalis strains can produce tyramine and β-phenylethylamine in the medium; however, they can only produce tyramine in water-boiled salted duck.

  1. Peptidoglycan O Acetylation and Autolysin Profile of Enterococcus faecalis in the Viable but Nonculturable State

    PubMed Central

    Pfeffer, John M.; Strating, Hendrik; Weadge, Joel T.; Clarke, Anthony J.

    2006-01-01

    The O acetylation of peptidoglycan occurs specifically at the C-6 hydroxyl group of muramoyl residues. Using a combination of high-performance liquid chromatography-based organic acid analysis and carbohydrate analysis by high-pH anion-exchange chromatography, we determined that strains of Entercoccus durans, E. faecalis, E. faecium, and E. hirae produce O-acetylated peptidoglycan. The levels of O acetylation ranged from 19% to 72% relative to the muramic acid content, and they were found to vary with the growth phase of the culture. Increases of 10 to 40% in O acetylation were observed with cultures entering the stationary phase. Cells of E. faecalis in the viable but nonculturable (VBNC) state had the highest levels of peptidoglycan O acetylation. The presence of this modification to peptidoglycan was shown to inhibit the action of hen egg white lysozyme in a concentration-dependent manner. Zymography using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing either O-acetylated or chemically de-O-acetylated peptidoglycan was used to monitor the production of specific autolysins in E. faecalis. Differences in the expression of specific autolysins were observed with the age of the culture, and VBNC E. faecalis produced the highest levels of these enzymes. This technique also permitted classification of the enterococcal autolysins into enzymes that preferentially hydrolyze either O-acetylated or non-O-acetylated peptidoglycan and enzymes that show no apparent preference for either substrate type. PMID:16428393

  2. Antimicrobial activity of essential oils and chloroform alone and combinated with cetrimide against Enterococcus faecalis biofilm

    PubMed Central

    Ferrer Luque, Carmen Maria; González-Rodríguez, Maria Paloma; Arias-Moliz, Maria Teresa; Baca, Pilar

    2013-01-01

    Abstract The Enterococcus faecalis bacteria have been identified as the most commonly recovered species from teeth with persistent endodontic infections. The antimicrobial activity of essential oils and chloroform (CHL), alone and in association with various concentrations of cetrimide (CTR), against biofilm of Enterococcus faecalis was investigated. Solutions of CHL, eucalyptus oil (EO) and orange oil (OO) associated with CTR at 0.3%, 0.2%, 0.1%, and 0.05% were used to determine antimicrobial activity by exposing treated bovine dentine blocks to E. faecalis. Biofilms grown in the dentine blocks for 7 days were exposed to solutions for 2 and 5 min. Biofilm reduction between OO and EO at 2 min did not show any significant differences; however, OO had a higher kill percentage of biofilms than did the eucalyptus oil at 5 min (p < 0.01). Combinations with CTR at all concentrations achieved a 100% kill rate at 2 and 5 min. The association of CTR with solvent agents achieved the maximum antimicrobial activity against E. faecalis biofilms in dentine. PMID:24265917

  3. An indigenous gut bacterium, Enterococcus faecalis (Lactobacillales: Enterococcaceae), increases seed consumption by Harpalus pensylvanicus (Coleoptera: Carabidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Harpalus pensylvanicus is a beneficial beetle contributing to insect control and seed predation in North American cropland. The bacterial endosymbiont Enterococcus faecalis is found in the intestinal tract of H. pensylvanicus and is thought to contribute to the digestion of the insect's seed diet. W...

  4. The antimicrobial effect of chloroform on Enterococcus faecalis after gutta-percha removal.

    PubMed

    Edgar, Scott W; Marshall, J Gordon; Baumgartner, J Craig

    2006-12-01

    The purpose of this in vitro study was to evaluate the antimicrobial effectiveness of chloroform on Enterococcus faecalis when used as a gutta-percha solvent during endodontic retreatment. Bilaterally matched human teeth were instrumented, infected with E. faecalis, and obturated. The gutta-percha was then removed using either chloroform or saline. Bacterial samples were collected after gutta-percha removal and following additional apical enlargement. A significant difference was seen (p<0.05) between the number of colony forming units (CFU) of E. faecalis for teeth retreated using chloroform (mean 21+56 CFU/ml) versus saline (mean 280+480 CFU/ml). Negative cultures were obtained in 11 of 17 chloroform samples and none of the saline samples. Samples taken after apical enlargement two sizes larger than the original master apical file showed a significant difference (p<0.05) between teeth retreated using chloroform versus saline. Negative cultures were seen in 9 of 17 chloroform samples and 1 of 17 saline samples. This study demonstrated that the use of chloroform during endodontic retreatment significantly reduced intracanal levels of cultivatable E. faecalis.

  5. Phage endolysins with broad antimicrobial activity against Enterococcus faecalis clinical strains.

    PubMed

    Proença, Daniela; Fernandes, Sofia; Leandro, Clara; Silva, Filipa Antunes; Santos, Sofia; Lopes, Fátima; Mato, Rosario; Cavaco-Silva, Patrícia; Pimentel, Madalena; São-José, Carlos

    2012-06-01

    Increasing antibiotic resistance of bacterial pathogens has drawn the attention to the potential use of bacteriophage endolysins as alternative antibacterial agents. Here we have identified, characterized, and studied the lytic potential of two endolysins, Lys168 and Lys170, from phages infecting Enterococcus faecalis. Lys168 and Lys170 belong to the cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) and amidase-2 protein families, respectively. Lys168 is quite a unique enterococcal phage endolysin. It shares 95% amino acidic identity with the endolysin of Staphylococcus aureus phage SAP6, which in turn is distantly related to all known CHAP endolysins of S. aureus phages. Lys170 seems to be a natural chimera assembling catalytic and cell-wall-binding domains of different origin. Both endolysins showed a clear preference to act against E. faecalis and they were able to lyse a high proportion of clinical isolates of this species. Specifically, Lys168 and Lys170 lysed more than 70% and 90% of the tested isolates, respectively, which included a panel of diverse and typed strains representative of highly prevalent clonal complexes. Lys170 was active against all tested E. faecalis VRE strains. The quasi specificity toward E. faecalis is discussed considering the nature of the enzymes' functional domains and the structure of the cell wall peptidoglycan.

  6. Antibacterial Effect of All-in-one Self-etch Adhesives on Enterococcus faecalis

    PubMed Central

    Ebrahimi Chaharom, Mohammad Esmaeel; Ajami, Amir Ahmad; Abed Kahnamouei, Mehdi; Jafari Navimipour, Elmira; Tehranchi, Pardis; Zand, Vahid; Sadeghi, Mohammad Reza; Sohrabi, Aydin

    2014-01-01

    Background and aims. The aim of this study was to evaluate the antibacterial activity of one-step self-etch adhesives on Enterococcus faecalis on days 1, 7 and 14 with the use of modified direct contact test. Materials and methods. The modified directcontact test was used to evaluate the antibacterial effect of Adper Easy One, Bond Force, Clearfil S3 Bond, Futurabond M, G-Bond, iBond and OptiBond All-in-one adhesives on Enterococcus faecalis after aging the samples in phosphate-buffered saline for one, seven and fourteen days. Data were analyzed using one-way ANOVA and post hoc Tukey tests. Aging effect of each adhesive was evaluated by paired-sample test. In this study, P<0.05 was considered significant. Results. All the tested adhesives exhibited antibacterial activity after one day and had significant differences with the positive control group (P<0.05). After one week, OptiBond All-in-one, iBond and Futurabond M exhibited significant differences in bacterial growth from other groups (P<0.05). There were no significant differences between the groups in two weeks (P>0.05). Conclusion. iBond exhibited the highest antibacterial effect on E. faecalis after one week. Futurabond and OptiBond All-in-one exhibited antibacterial effects against E. faecalis for one week. PMID:25587384

  7. Antimicrobial Activity of Calcium Hydroxide and Betamethasone on Enterococcus faecalis; An in vitro Assessment

    PubMed Central

    Tabrizizadeh, Mahdi; Rasti, Mojtaba; Ayatollahi, Fatemeh; Mossadegh, Mohammad Hossein; Zandi, Hengameh; Dehghan, Farzad; Mousavi, Zohreh

    2015-01-01

    Introduction: Calcium hydroxide (CH) is one of the most common intracanal medications. Corticosteroids (CS) are used in endodontics because of their anti-inflammatory activity. This study aimed to evaluate the antimicrobial effect of CH+betamethasone and CH+saline against Enterococcus faecalis (E. faecalis) using agar diffusion test and measuring the microbial zone of inhibition (ZOI). Methods and Materials: Four plates containing Mueller-Hinton broth and E. faecalis culture media, were prepared. In each plate, 5 holes (5×3 mm) were created and a creamy mixture of CH+betamethasone was inserted into the holes (10 holes for each material). Two holes with ampicillin disks and two empty holes were used as negative and positive controls, respectively. Plates were incubated for 24 h and then the diameter of microbial ZOI was measured. The pH of each mixture was measured by pH meter. Data were analyzed using the Mann-Whitney U test. Results: The mean diameter of ZOI for CH+betamethasone and CH+saline was 3.4 and 3 mm, respectively. The difference was not significant (P=0.143). The pH was 12.5 for CH+saline and 12.3 CH+betamethasone, respectively. Conclusion: The mixture of CH+betamethasone had good antimicrobial effects against E. faecalis. Further studies are needed to confirm the value of this mixture in clinical settings. PMID:26213541

  8. Effects of ionophores on Enterococcus faecalis and E. faecium growth in pure and mixed ruminal culture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enterococcus faecalis and faecium are Gram-positive human pathogens that can live in the gastrointestinal tract of food animals. Vancomycin-resistant enterococci (VRE) are an increasing threat to humans as a nosocomial infection, as well as a reservoir of antibiotic resistance genes. Ionophores ar...

  9. Antimicrobial Susceptibility Patterns of Enterococcus faecalis and Enterococcus faecium Isolated from Poultry Flocks in Germany.

    PubMed

    Maasjost, J; Mühldorfer, K; Cortez de Jäckel S; Hafez, H M

    2015-03-01

    Between 2010 and 2011, 145 Enterococcus isolates (Enterococcus faecalis, n = 127; Enterococcus faecium, n = 18) were collected during routine bacteriologic diagnostics from broilers, layers, and fattening turkeys in Germany showing various clinical signs. The susceptibility to 24 antimicrobial agents was investigated by broth microdilution test to determine minimum inhibitory concentrations (MICs). All E. faecalis isolates (n = 127) were susceptible to the beta-lactam antibiotics ampicillin, amoxicillin-clavulanic acid, and penicillin. Corresponding MIC with 50% inhibition (MIC50) and MIC with 90% inhibition (MIC90) values of these antimicrobial agents were at the lower end of the test range (≤ 4 μg/ml). In addition, no vancomycin-resistant enterococci (VRE) were found. High resistance rates were identified in both Enterococcus species for lincomycin (72%-99%) and tetracycline (67%-82%). Half or more than half of Enterococcus isolates were resistant to gentamicin (54%-72%) and the macrolide antibiotics erythromycin (44%-61%) and tylosin-tartate (44%-56%). Enterococcus faecalis isolated from fattening turkeys showed the highest prevalence of antimicrobial resistance compared to other poultry production systems. Eighty-nine out of 145 Enterococcus isolates were resistant to three or more antimicrobial classes. Again, turkeys stood out with 42 (8 1%) multiresistant isolates. The most-frequent resistance patterns of E. faecalis were gentamicin, lincomycin, and tetracycline in all poultry production systems.

  10. Investigation of mechanism and molecular epidemiology of linezolid-resistant Enterococcus faecalis in China.

    PubMed

    Wang, Lipeng; He, Yunyan; Xia, Yun; Wang, Huijuan; Liang, Shumei

    2014-08-01

    Enterococcus is a major cause of important nosocomial infections. Linezolid, the first member of an entirely new class of antibiotics (oxazolidinones), is effective against serious infections caused by Enterococcus. However, resistance to linezolid has been discovered throughout the world rapidly. From 2011 to 2013, nine linezolid-resistant E. faecalis isolates were collected and the possible mechanisms of linezolid resistance, including mutations in domain V of 23S rRNA genes and in ribosomal proteins L3 and L4, and the multiresistance gene cfr, were investigated. Furthermore, an epidemiological survey of the nine linezolid-resistant E. faecalis isolates was performed by pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and DiversiLab. The three methods were compared to evaluate their merits and demerits, respectively. We failed to find the resistance mechanisms that have been revealed in recent years by PCR and sequencing analysis in the linezolid-resistant E. faecalis. Epidemiological investigation suggested that a small-scale outbreak of linezolid-resistant E. faecalis emerged in neurosurgery ICU from March to May of 2013. DiversiLab was a reliable typing tool and a suitable alternative to PFGE because it was as discriminatory as PFGE and better than MLST.

  11. Biochemical and Genetic Characterization of the Enterococcus faecalis Oxaloacetate Decarboxylase Complex

    PubMed Central

    Repizo, Guillermo D.; Blancato, Víctor S.; Mortera, Pablo; Lolkema, Juke S.

    2013-01-01

    Enterococcus faecalis encodes a biotin-dependent oxaloacetate decarboxylase (OAD), which is constituted by four subunits: E. faecalis carboxyltransferase subunit OadA (termed Ef-A), membrane pump Ef-B, biotin acceptor protein Ef-D, and the novel subunit Ef-H. Our results show that in E. faecalis, subunits Ef-A, Ef-D, and Ef-H form a cytoplasmic soluble complex (termed Ef-AHD) which is also associated with the membrane. In order to characterize the role of the novel Ef-H subunit, coexpression of oad genes was performed in Escherichia coli, showing that this subunit is vital for Ef-A and Ef-D interaction. Diminished growth of the oadA and oadD single deletion mutants in citrate-supplemented medium indicated that the activity of the complex is essential for citrate utilization. Remarkably, the oadB-deficient strain was still capable of growing to wild-type levels but with a delay during the citrate-consuming phase, suggesting that the soluble Ef-AHD complex is functional in E. faecalis. These results suggest that the Ef-AHD complex is active in its soluble form, and that it is capable of interacting in a dynamic way with the membrane-bound Ef-B subunit to achieve its maximal alkalinization capacity during citrate fermentation. PMID:23435880

  12. Complete Genome Sequence of Enterococcus faecalis Strain W11 Isolated from an Algal Food Product

    PubMed Central

    Takizawa, Noboru

    2016-01-01

    Here, we report the complete genome sequence of Enterococcus faecalis strain W11 isolated from an algal food product in Japan. This study should facilitate the identification of a novel mechanism of glycerol metabolic control in lactic acid bacteria. PMID:27688337

  13. Survival and activity of Streptococcus faecalis and Escherichia coli in petroleum-contaminated tropical marine waters

    SciTech Connect

    Santo Domingo, J.W.; Fuentes, F.A.; Hazen, T.C.

    1987-12-31

    The in situ survival and activity of Streptococcus faecalis and Escherichia coli were studied using membrane diffusion chambers in tropical marine waters receiving oil refinery effluents. Protein synthesis, DNA synthesis, respiration or fermentation, INT reduced per cell, and ATP per cell were used to measure physiological activity. Cell densities decreased significantly over time at both sites for both S. faecalis and E. coli; however, no significant differences in survival pattern were observed between S. faecalis and E.coli. Differences in protein synthesis between the two were only observed at a study site which was not heavily oiled. Although fecal streptococci have been suggested as a better indicator of fecal contamination than fecal coliforms in marine waters, in this study both E. coli and S. faecalis survived and remained physiologically active for extended periods of time. These results suggest that the fecal streptococci group is not a better indicator of fecal contamination in tropical marine waters than the fecal coliform group, especially when that environment is high in long-chained hydrocarbons.

  14. Transcriptomic and functional analysis of NaCl-induced stress in Enterococcus faecalis.

    PubMed

    Solheim, Margrete; La Rosa, Sabina Leanti; Mathisen, Thomas; Snipen, Lars G; Nes, Ingolf F; Brede, Dag Anders

    2014-01-01

    The robust physiology of Enterococcus faecalis facilitates tolerance to various stresses. We here report the transcriptional response of E. faecalis V583 to growth in the presence of 6.5% NaCl. Among the early responses observed was an immediate down-regulation of mscL, accompanied by an up-regulation of genes predicted to be involved in uptake of extracellular potassium and glycine betaine. The high NaCl concentration also induced expression of chaperons and cell envelope related traits, such as the enterococcal polysaccharide antigen (epa) locus. Functional genetic analysis revealed reduced salt stress resistance in both epaB and epaE mutants. The reduced salt resistance phenotype associated with the epaB mutant was restored by complementation, hence demonstrating a role of Epa in the physiological robustness of E. faecalis. Furthermore, we demonstrate that Epa confers increased resistance towards multiple cell envelope stress-inducing factors. Accordingly, these findings delineate a potential link between the robust nature of E. faecalis and its ability to perform as a human pathogen, and provide a new perspective on the mechanisms by which Epa contributes to virulence. Notably, the high NaCl concentration also resulted in strict repression of the gelE-sprE operon and impaired gelatinase activity. We demonstrate that NaCl antagonize the GBAP-pheromone dependent induction in a concentration dependent manner.

  15. Clinical and molecular epidemiology of hospital Enterococcus faecalis isolates in eastern France.

    PubMed

    Mulin, Blandine; Bailly, Pascale; Thouverez, Michelle; Cailleaux, Vincent; Cornette, Christian; Dupont, Marie-Jeanne; Talon, Daniel

    1999-03-01

    OBJECTIVE: To report on the occurrence of Enterococcus faecalis hospital isolates obtained during 1 year in hospitals in the Franche-Comté region of France. METHODS: Clinical isolates of E. faecalis of different antibiotic susceptibility phenotypes from hospitalized patients were characterized by pulsed-field gel electrophoresis. Patients with positive cultures were investigated by three case-control studies to identify risk factors for colonization/infection. RESULTS: The crude incidence of colonization/infection was 2.37%, and 4-day and 7-day colonization rates after admission were 10.0% and 6.36%, respectively. The rates of high-level resistance to kanamycin (HLKR) and to gentamicin (HLGR) were 47.1% and 7.1%, respectively. No isolate was resistant to glycopeptides or produced beta-lactamase. The 209 hospital isolates obtained during the study yielded 98 major DNA patterns, of which two were major epidemic patterns including HLKR isolates. No single factor was significantly associated with colonization/infection by HLKR isolates. The length of hospitalization before isolation was associated with colonization by HLGR isolates. CONCLUSIONS: The isolation frequency of E. faecalis strains with acquired resistance to aminoglycoside antibiotics, and the wide dissemination of resistant strains with characteristics that allow them to persist and spread, argue for further large prospective surveys of clinical isolates of E. faecalis in hospitals.

  16. Enterococcus faecalis Bearing Aggregation Substance Is Resistant to Killing by Human Neutrophils despite Phagocytosis and Neutrophil Activation

    PubMed Central

    Rakita, Robert M.; Vanek, Natalie N.; Jacques-Palaz, Karen; Mee, Mee; Mariscalco, M. Michele; Dunny, Gary M.; Snuggs, Mark; Van Winkle, W. Barry; Simon, Scott I.

    1999-01-01

    Enterococcus faecalis aggregation substance (AS) mediates efficient bacterium-bacterium contact to facilitate plasmid exchange as part of a bacterial sex pheromone system. We have previously determined that AS promotes direct, opsonin-independent binding of E. faecalis to human neutrophils (PMNs) via complement receptor type 3 and other receptors on the PMN surface. We have now examined the functional consequences of this bacterium-host cell interaction. AS-bearing E. faecalis was phagocytosed and internalized by PMNs, as determined by deconvolution fluorescence microscopy. However, these bacteria were not killed by PMNs, and internalized bacteria excluded propidium iodide, indicating intact bacterial membranes. Resistance to killing occurred despite activation of PMNs, as indicated by an increase in both functional and total surface Mac-1 expression, shedding of l-selectin, and an increase in PMN extracellular superoxide and phagosomal oxidant production. Deconvolution fluorescence microscopy also revealed that phagosomes containing AS-bearing bacteria were markedly larger than phagosomes containing opsonized E. faecalis, suggesting that some modification of phagosomal maturation may be involved in AS-induced resistance to killing. PMN phagosomal pH was significantly higher after ingestion of nonopsonized AS-bearing E. faecalis than after that of opsonized bacteria. The novel ability of AS to promote intracellular survival of E. faecalis inside PMNs suggests that AS may be a virulence factor used by strains of E. faecalis. PMID:10531268

  17. Bacteriocin protein BacL1 of Enterococcus faecalis is a peptidoglycan D-isoglutamyl-L-lysine endopeptidase.

    PubMed

    Kurushima, Jun; Hayashi, Ikue; Sugai, Motoyuki; Tomita, Haruyoshi

    2013-12-27

    Enterococcus faecalis strains are commensal bacteria in humans and other animals, and they are also the causative agent of opportunistic infectious diseases. Bacteriocin 41 (Bac41) is produced by certain E. faecalis clinical isolates, and it is active against other E. faecalis strains. Our genetic analyses demonstrated that the extracellular products of the bacL1 and bacA genes, which are encoded in the Bac41 operon, coordinately express the bacteriocin activity against E. faecalis. In this study, we investigated the molecular functions of the BacL1 and BacA proteins. Immunoblotting and N-terminal amino acid sequence analysis revealed that BacL1 and BacA are secreted without any processing. The coincidental treatment with the recombinant BacL1 and BacA showed complete bacteriocin activity against E. faecalis, but neither BacL1 nor BacA protein alone showed the bacteriocin activity. Interestingly, BacL1 alone demonstrated substantial degrading activity against the cell wall fraction of E. faecalis in the absence of BacA. Furthermore, MALDI-TOF MS analysis revealed that BacL1 has a peptidoglycan D-isoglutamyl-L-lysine endopeptidase activity via a NlpC/P60 homology domain. These results collectively suggest that BacL1 serves as a peptidoglycan hydrolase and, when BacA is present, results in the lysis of viable E. faecalis cells.

  18. In vivo broiler experiments to assess anti-Campylobacter jejuni activity of a live Enterococcus faecalis strain.

    PubMed

    Robyn, J; Rasschaert, G; Hermans, D; Pasmans, F; Heyndrickx, M

    2013-01-01

    Bacterial gastroenteritis caused by thermotolerant Campylobacter species, mainly Campylobacter jejuni, has been the most reported zoonotic disease in many developed countries in recent years. Reducing Campylobacter shedding on the farm could result in a reduction of the number of campylobacteriosis cases. In 2 independent broiler seeder experiments, in which broiler chickens were orally inoculated with 2 amounts of Enterococcus faecalis MB 5259, we established whether a live E. faecalis strain was capable of reducing cecal Campylobacter colonization in broiler chickens. In previous in vitro experiments it has been demonstrated that this E. faecalis MB 5259 displays anti-Campylobacter activity. The effect of pH and bile salts on E. faecalis MB 5259 showed that growth and survival of E. faecalis MB 5259 can be impaired during passage through the gastrointestinal tract of broiler chickens. Despite these results E. faecalis MB 5259 was capable of colonizing the broiler ceca. Contrary to the in vitro experiments, in which E. faecalis MB 5259 inhibited C. jejuni MB 4185 growth, no inhibition was observed in the in vivo experiments independent of the inoculum size.

  19. Microbial shifts in the porcine distal gut in response to diets supplemented with Enterococcus Faecalis as alternatives to antibiotics

    PubMed Central

    Li, Pinghua; Niu, Qing; Wei, Qingtian; Zhang, Yeqiu; Ma, Xiang; Kim, Sung Woo; Lin, Mingxin; Huang, Ruihua

    2017-01-01

    Gut microbiota plays an important role in host health and nutrient digestion of animals. Probiotics have become one of effective alternatives to antibiotics enhancing animal health and performance through modulating gut microbiota. Previously, our research demonstrated that dietary Enterococcus Faecalis UC-100 substituting antibiotics enhanced growth and health of weaned pigs. To investigate the alterations of microbiota in the distal gut of pigs fed E. faecalis UC-100 substituting antibiotics, this study assessed fecal microbiota in pigs from different dietary treatments: the basal diet group, the E. faecalis group, and the antibiotic group on d 0, 14, and 28 of feeding through 16 S rRNA sequencing. Twenty-one phyla and 137 genera were shared by all pigs, whereas 12 genera were uniquely identified in the E. faecalis group on d 14 and 28. Bacterial abundance and diversity in the E. faecalis group, bacterial diversity in the antibiotic group, especially abundances of Fibrobacteres phylum and 12 genera in the E. faecalis group and antibiotics group were lower than that in the basal diet group on d 28. These results showed that microbial shifts in the porcine gut in response to diets containing E. faecalis were similar to the response to which containing antibiotics. PMID:28165001

  20. The nuclear receptor DHR3 modulates dS6 kinase-dependent growth in Drosophila.

    PubMed

    Montagne, Jacques; Lecerf, Caroline; Parvy, Jean-Philippe; Bennion, Janis M; Radimerski, Thomas; Ruhf, Marie-Laure; Zilbermann, Frederic; Vouilloz, Nicole; Stocker, Hugo; Hafen, Ernst; Kozma, Sara C; Thomas, George

    2010-05-06

    S6 kinases (S6Ks) act to integrate nutrient and insulin signaling pathways and, as such, function as positive effectors in cell growth and organismal development. However, they also have been shown to play a key role in limiting insulin signaling and in mediating the autophagic response. To identify novel regulators of S6K signaling, we have used a Drosophila-based, sensitized, gain-of-function genetic screen. Unexpectedly, one of the strongest enhancers to emerge from this screen was the nuclear receptor (NR), Drosophila hormone receptor 3 (DHR3), a critical constituent in the coordination of Drosophila metamorphosis. Here we demonstrate that DHR3, through dS6K, also acts to regulate cell-autonomous growth. Moreover, we show that the ligand-binding domain (LBD) of DHR3 is essential for mediating this response. Consistent with these findings, we have identified an endogenous DHR3 isoform that lacks the DBD. These results provide the first molecular link between the dS6K pathway, critical in controlling nutrient-dependent growth, and that of DHR3, a major mediator of ecdysone signaling, which, acting together, coordinate metamorphosis.

  1. Crystal structures of the S6K1 kinase domain in complexes with inhibitors.

    PubMed

    Niwa, Hideaki; Mikuni, Junko; Sasaki, Shunta; Tomabechi, Yuri; Honda, Keiko; Ikeda, Mariko; Ohsawa, Noboru; Wakiyama, Motoaki; Handa, Noriko; Shirouzu, Mikako; Honma, Teruki; Tanaka, Akiko; Yokoyama, Shigeyuki

    2014-09-01

    Ribosomal protein S6 kinase 1 (S6K1) is a serine/threonine protein kinase that plays an important role in the PIK3/mTOR signaling pathway, and is implicated in diseases including diabetes, obesity, and cancer. The crystal structures of the S6K1 kinase domain in complexes with staurosporine and the S6K1-specific inhibitor PF-4708671 have been reported. In the present study, five compounds (F108, F109, F176, F177, and F179) were newly identified by in silico screening of a chemical library and kinase assay. The crystal structures of the five inhibitors in complexes with the S6K1 kinase domain were determined at resolutions between 1.85 and 2.10 Å. All of the inhibitors bound to the ATP binding site, lying along the P-loop, while the activation loop stayed in the inactive form. Compound F179, with a carbonyl group in the middle of the molecule, altered the αC helix conformation by interacting with the invariant Lys123. Compounds F176 and F177 bound slightly distant from the hinge region, and their sulfoamide groups formed polar interactions with the protein. The structural features required for the specific binding of inhibitors are discussed.

  2. Gene transfer of vancomycin and tetracycline resistances among Enterococcus faecalis during cheese and sausage fermentations.

    PubMed

    Cocconcelli, Pier Sandro; Cattivelli, Daniela; Gazzola, Simona

    2003-12-01

    This study assessed the frequency of transfer of two mobile genetic elements coding for virulence determinants and antibiotic resistance factors, into food associated enterococci during fermentation processes. First, the transfer of the pheromone-inducible pCF10 plasmid, carrying tetracycline resistance and aggregation substance (AS) as virulence factor, between clinical and food strains of Enterococcus faecalis, was investigated in models of cheese and fermented sausage. The experiments demonstrated that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from E. faecalis OG1rf cells to food strain E. faecalis BF3098c and that the plasmid subsequently persisted in these environments. Very high frequency of transfer was observed in sausage (10(-3)/recipient) if compared to cheese (10(-6)) and plate mating (10(-4)). Transconjugants were subsequently verified by PCR. The second transmissible element was the plasmid harbouring the vancomycin resistance (VanA phenotype) from E. faecalis A256. The transfer of this antibiotic resistance to a food strain of E. faecalis was studied in vitro and in food models. Although the transfer of vancomycin resistance was achieved in all the environments, the highest conjugation frequencies were observed during the ripening of fermented sausages, reaching 10(-3) transconjugants/recipient cell. PCR confirmed the transfer of the VanA genotype into a food associated Enterococcus strain. This study showed that even in the absence of selective pressure, mobile genetic elements carrying antibiotic resistance and virulence determinants can be transferred at high frequency to food associated enterococci during cheese and sausage fermentation.

  3. Antibacterial Efficacy of Different Concentrations of Sodium Hypochlorite Gel and Solution on Enterococcus faecalis Biofilm

    PubMed Central

    Zand, Vahid; Lotfi, Mehrdad; Soroush, Mohammad Hosein; Abdollahi, Amir Ardalan; Sadeghi, Mehdi; Mojadadi, Ali

    2016-01-01

    Introduction: This in vitro study compared the antibacterial efficacy of 2.5% sodium hypochlorite gel and 2.5% and 5.25% sodium hypochlorite solutions on Enterococcus faecalis (E. faecalis) biofilm. Methods and Materials: The root canals of 60 extracted human single-rooted teeth were contaminated with E. faecalis and incubated for 6 weeks. The samples were randomly assigned to three experimental groups and one control group (n=15). The study protocol in the experimental groups consisted of injection of 5 mL of each irrigant into the root canals. Samples were collected from the root canal walls and 1:10 serial dilutions were prepared and added to Muller Hinton Agar (MHA) plates and incubated at 37°C for 48 h. A classic colony counting technique was used for determining vital E. faecalis bacterial counts in MHA plates. The Kruskal-Wallis test was used for statistical analysis of the data. The level of significance was set at 0.05. Results: The antibacterial effect of the irrigants in all three experimental groups was significantly greater than the control group (P<0.05), with no significant difference between 2.5% and 5.25% NaOCl solutions (P>0.05). The effect of 2.5% and 5.25% NaOCl solutions were significantly superior to 2.5% NaOCl gel (P<0.05). Conclusion: Under the limitations of this study, 2.5% NaOCl gel was effective in reducing E. faecalis counts; however this effect was less than that of NaOCl solutions. PMID:27790262

  4. Large-Scale Screening of a Targeted Enterococcus faecalis Mutant Library Identifies Envelope Fitness Factors

    PubMed Central

    Rigottier-Gois, Lionel; Alberti, Adriana; Houel, Armel; Taly, Jean-François; Palcy, Philippe; Manson, Janet; Pinto, Daniela; Matos, Renata C.; Carrilero, Laura; Montero, Natalia; Tariq, Muhammad; Karsens, Harma; Repp, Christian; Kropec, Andrea; Budin-Verneuil, Aurélie; Benachour, Abdellah; Sauvageot, Nicolas; Bizzini, Alain; Gilmore, Michael S.; Bessières, Philippe; Kok, Jan; Huebner, Johannes; Lopes, Fatima; Gonzalez-Zorn, Bruno; Hartke, Axel; Serror, Pascale

    2011-01-01

    Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% Gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence. PMID:22194979

  5. Antibacterial effect of urushiol on E. faecalis as a root canal irrigant

    PubMed Central

    Kim, Sang-Wan

    2017-01-01

    Objectives The purpose of this study was to compare the antibacterial activity of urushiol against Enterococcus faecalis (E. faecalis) to that of NaOCl. Materials and Methods The canals of thirty two single rooted human teeth were instrumented with Ni-Ti files (ProTaper Next X1, X2, X3, Dentsply). A pure culture of E. faecalis ATCC 19433 was prepared in sterile brain heart infusion (BHI) broth. The teeth were submerged in the suspension of E. faecalis and were incubated at 37℃ for 7 days to allow biofilm formation. The teeth were randomly divided into three experimental groups according to the irrigant used, and a negative control group where no irrigant was used (n = 8). Group 1 used physiologic normal saline, group 2 used 6% NaOCl, and group 3 used 10 wt% urushiol solution. After canal irrigation, each sample was collected by the sequential placement of 2 sterile paper points (ProTaper NEXT paper points, size X3, Dentsply). Ten-fold serial dilutions on each vials, and 100 µL were cultured on a BHI agar plate for 8 hours, and colony forming unit (CFU) analysis was done. The data were statistically analyzed using Kruskal-Wallis and Mann-whitney U tests. Results Saline group exhibited no difference in the CFU counts with control group, while NaOCl and urushiol groups showed significantly less CFU counts than saline and control groups (p < 0.05). Conclusions The result of this study suggests 10% urushiol and 6% NaOCl solution had powerful antibacterial activity against E. faecalis when they were used as root canal irrigants. PMID:28194365

  6. Synergistic activities of macrolide antibiotics against Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans isolated from patients with cystic fibrosis.

    PubMed

    Saiman, Lisa; Chen, Yunhua; Gabriel, Pablo San; Knirsch, Charles

    2002-04-01

    Azithromycin and clarithromycin were paired with other antibiotics to test synergistic activity against 300 multidrug-resistant pathogens isolated from cystic fibrosis (CF) patients. Clarithromycin-tobramycin was most active against Pseudomonas aeruginosa and inhibited 58% of strains. Azithromycin-trimethoprim-sulfamethoxazole, azithromycin-ceftazidime, and azithromycin-doxycycline or azithromycin-trimethoprim-sulfamethoxazole inhibited 40, 20, and 22% of Stenotrophomonas maltophilia, Burkholderia cepacia complex, and Achromobacter (Alcaligenes) xylosoxidans strains, respectively.

  7. Early post-hatching starvation delays p70 S6 kinase activation in the muscle of neonatal chicks.

    PubMed

    Bigot, Karine; Taouis, Mohammed; Picard, Michel; Tesseraud, Sophie

    2003-12-01

    Chicken muscle ribosomal protein S6 kinase (S6K1) has been recently characterised and its enzymic activity is regulated by the nutritional and hormonal (insulin) status in vivo. The regulation of S6K1 is still unknown in neonatal chicks. The present study aimed to compare the activation of S6K1 in early-feeding (EF) and 48 h-delayed-feeding (DF) chicks from hatching to 4 d of age. During post-hatching starvation, S6K1 activity remained at the basal level measured in the control-hatched chicks. The maximum S6K1 activity was recorded on the first day of feeding with an increase of about 2.5-fold in the EF and DF chicks (P<0.01). S6K1 activity was correlated with plasma insulin level, suggesting a probable insulin-dependent S6K1 activation. The feeding-induced increase in S6K1 activity was related to its Thr389 residue phosphorylation. A similar pattern for protein kinase B phosphorylation was observed, upstream from S6K1. The S6K1 pathway was stimulated to the same extent in the EF and DF chicks, which indicates that post-hatching starvation did not increase S6K1 activation. It is concluded that muscle S6K1 is activated as soon as food is available without improvement in the response of the S6K1 pathway after post-hatching starvation.

  8. The Circadian Protein BMAL1 Regulates Translation in Response to S6K1-Mediated Phosphorylation.

    PubMed

    Lipton, Jonathan O; Yuan, Elizabeth D; Boyle, Lara M; Ebrahimi-Fakhari, Darius; Kwiatkowski, Erica; Nathan, Ashwin; Güttler, Thomas; Davis, Fred; Asara, John M; Sahin, Mustafa

    2015-05-21

    The circadian timing system synchronizes cellular function by coordinating rhythmic transcription via a transcription-translational feedback loop. How the circadian system regulates gene expression at the translational level remains a mystery. Here, we show that the key circadian transcription factor BMAL1 associates with the translational machinery in the cytosol and promotes protein synthesis. The mTOR-effector kinase, ribosomal S6 protein kinase 1 (S6K1), an important regulator of translation, rhythmically phosphorylates BMAL1 at an evolutionarily conserved site. S6K1-mediated phosphorylation is critical for BMAL1 to both associate with the translational machinery and stimulate protein synthesis. Protein synthesis rates demonstrate circadian oscillations dependent on BMAL1. Thus, in addition to its critical role in circadian transcription, BMAL1 is a translation factor that links circadian timing and the mTOR signaling pathway. More broadly, these results expand the role of the circadian clock to the regulation of protein synthesis.

  9. JPRS Report, Science & Technology, China.

    DTIC Science & Technology

    2007-11-02

    1992 2 China The Construction of Genomic Library Alcaligenes faecalis and Cloning of nifH Gene Sequence Homologous to Klebsiella pneumoniae [Hai...The Construction of Genomic Library Alcaligenes faecalis and Cloning of nifÜ Gene Sequence Homologous to Klebsiella pneumoniae 40091002K Beijing...3769 2430] of the Institute of Genetics, CAS, Beijing] [Text] A genomic library of Alcaligenes faecalis A-15 HI which possesses rather high

  10. Mutations in the Drosophila gene encoding ribosomal protein S6 cause tissue overgrowth.

    PubMed Central

    Stewart, M J; Denell, R

    1993-01-01

    We have characterized two P-element-induced, lethal mutations in Drosophila melanogaster which affect the larval hemocytes, mediators of the insect immune response. Each mutant displays larval melanotic tumors characteristic of mutations affecting the insect cellular immune system, and the moribund animals develop grossly hypertrophied hematopoietic organs because of increased cell proliferation and extra rounds of endoreduplication in some hematopoietic cells. Surprisingly, these mutations are due to P element insertions in the 5' regulatory region of the Drosophila gene encoding ribosomal protein S6 and cause a reduction of S6 transcript abundance in mutant larvae. Images PMID:8384310

  11. Cross-talk between sirtuin and mammalian target of rapamycin complex 1 (mTORC1) signaling in the regulation of S6 kinase 1 (S6K1) phosphorylation.

    PubMed

    Hong, Sungki; Zhao, Bin; Lombard, David B; Fingar, Diane C; Inoki, Ken

    2014-05-09

    p70 ribosomal S6 kinase (S6K1), a major substrate of the mammalian target of rapamycin (mTOR) kinase, regulates diverse cellular processes including protein synthesis, cell growth, and survival. Although it is well known that the activity of S6K1 is tightly coupled to its phosphorylation status, the regulation of S6K1 activity by other post-translational modifications such as acetylation has not been well understood. Here we show that the acetylation of the C-terminal region (CTR) of S6K1 blocks mTORC1-dependent Thr-389 phosphorylation, an essential phosphorylation site for S6K1 activity. The acetylation of the CTR of S6K1 is inhibited by the class III histone deacetylases, SIRT1 and SIRT2. An S6K1 mutant lacking acetylation sites in its CTR shows enhanced Thr-389 phosphorylation and kinase activity, whereas the acetylation-mimetic S6K1 mutant exhibits decreased Thr-389 phosphorylation and kinase activity. Interestingly, relative to the acetylation-mimetic S6K1 mutant, the acetylation-defective mutant displays higher affinity toward Raptor, an essential scaffolding component of mTORC1 that recruits mTORC1 substrates. These observations indicate that sirtuin-mediated regulation of S6K1 acetylation is an additional important regulatory modification that impinges on the mechanisms underlying mTORC1-dependent S6K1 activation.

  12. Ribosomal Protein Mutations Induce Autophagy through S6 Kinase Inhibition of the Insulin Pathway

    PubMed Central

    Pereboom, Tamara C.; Goos, Yvonne J.; Seinen, Cor W.; van Oirschot, Brigitte A.; van Dooren, Rowie; Gastou, Marc; Giles, Rachel H.; van Solinge, Wouter; Kuijpers, Taco W.; Gazda, Hanna T.; Bierings, Marc B.; Da Costa, Lydie; MacInnes, Alyson W.

    2014-01-01

    Mutations affecting the ribosome lead to several diseases known as ribosomopathies, with phenotypes that include growth defects, cytopenia, and bone marrow failure. Diamond-Blackfan anemia (DBA), for example, is a pure red cell aplasia linked to the mutation of ribosomal protein (RP) genes. Here we show the knock-down of the DBA-linked RPS19 gene induces the cellular self-digestion process of autophagy, a pathway critical for proper hematopoiesis. We also observe an increase of autophagy in cells derived from DBA patients, in CD34+ erythrocyte progenitor cells with RPS19 knock down, in the red blood cells of zebrafish embryos with RP-deficiency, and in cells from patients with Shwachman-Diamond syndrome (SDS). The loss of RPs in all these models results in a marked increase in S6 kinase phosphorylation that we find is triggered by an increase in reactive oxygen species (ROS). We show that this increase in S6 kinase phosphorylation inhibits the insulin pathway and AKT phosphorylation activity through a mechanism reminiscent of insulin resistance. While stimulating RP-deficient cells with insulin reduces autophagy, antioxidant treatment reduces S6 kinase phosphorylation, autophagy, and stabilization of the p53 tumor suppressor. Our data suggest that RP loss promotes the aberrant activation of both S6 kinase and p53 by increasing intracellular ROS levels. The deregulation of these signaling pathways is likely playing a major role in the pathophysiology of ribosomopathies. PMID:24875531

  13. Identification and characterization of a mitogen-activated S6 kinase.

    PubMed Central

    Jenö, P; Ballou, L M; Novak-Hofer, I; Thomas, G

    1988-01-01

    Treatment of Swiss mouse 3T3 cells with epidermal growth factor, orthovanadate, or serum results in the activation of a kinase that phosphorylates protein S6 of the 40S ribosomal subunit in vitro. This kinase is eluted as a single peak of activity from either a Mono Q anion-exchange column at 0.34 M NaCl or a Mono S cation-exchange column at 0.20 M NaCl. Treatment of the peak fraction from the Mono S column with phosphatase 2A completely abolishes the activity of the enzyme. The kinase appears to be distinct from protein kinase C, cAMP-dependent protein kinase, and two protease-activated kinases, PAK II and H4P. The kinase has been purified to apparent homogeneity and migrates as a single band at Mr 70,000 in NaDodSO4/polyacrylamide gels. The kinase exhibits the ability to autophosphorylate, and this activity directly parallels S6 phosphorylation activity on the final step of purification. In vitro, the kinase incorporates up to 5 mol of phosphate into S6, and the tryptic phosphopeptide maps obtained are equivalent to those from S6 phosphorylated in vivo. Most important, treatment of the purified kinase with phosphatase 2A results in complete inactivation of the enzyme, arguing that the activity of the kinase is directly controlled by phosphorylation. Images PMID:3257566

  14. Highly efficient acousto-optic diffraction in Sn2P2S6 crystals.

    PubMed

    Martynyuk-Lototska, I Yu; Mys, O G; Grabar, A A; Stoika, I M; Vysochanskii, Yu M; Vlokh, R O

    2008-01-01

    We have studied the acousto-optic (AO) diffraction in Sn2P2S6 crystals and found that they manifest high values of an AO figure of merit. The above crystals may therefore be used as highly efficient materials in different AO applications.

  15. Regulation of Adipogenesis by Quinine through the ERK/S6 Pathway

    PubMed Central

    Ning, Xiaomin; He, Jingjing; Shi, Xin’e; Yang, Gongshe

    2016-01-01

    Quinine is a bitter tasting compound that is involved in the regulation of body weight as demonstrated in in vivo animal models and in vitro models of the adipogenic system. Arguments exist over the positive or negative roles of quinine in both in vivo animal models and in vitro cell models, which motivates us to further investigate the functions of quinine in the in vitro adipogenic system. To clarify the regulatory functions of quinine in adipogenesis, mouse primary preadipocytes were induced for differentiation with quinine supplementation. The results showed that quinine enhanced adipogenesis in a dose dependent manner without affecting lipolysis. The pro-adipogenic effect of quinine was specific, as other bitter tasting agonists had no effect on adipogenesis. Moreover, the pro-adipogenic effect of quinine was mediated by activation of ERK/S6 (extracellular-signal-regulated kinase/Ribosomal protein S6) signaling. Knockdown of bitter taste receptor T2R106 (taste receptor, type 2, member 106) impaired the pro-adipogenic effect of quinine and suppressed the activation of ERK/S6 signaling. Taken together, quinine stimulates adipogenesis through ERK/S6 signaling, which at least partly functions via T2R106. PMID:27089323

  16. Pheromone-responsive conjugative vancomycin resistance plasmids in Enterococcus faecalis isolates from humans and chicken feces.

    PubMed

    Lim, Suk-Kyung; Tanimoto, Koichi; Tomita, Haruyoshi; Ike, Yasuyoshi

    2006-10-01

    The drug resistances and plasmid contents of a total of 85 vancomycin-resistant enterococcus (VRE) strains that had been isolated in Korea were examined. Fifty-four of the strains originated from samples of chicken feces, and 31 were isolated from hospital patients in Korea. Enterococcus faecalis KV1 and KV2, which had been isolated from a patient and a sample of chicken feces, respectively, were found to carry the plasmids pSL1 and pSL2, respectively. The plasmids transferred resistances to vancomycin, gentamicin, kanamycin, streptomycin, and erythromycin to E. faecalis strains at a high frequency of about 10(-3) per donor cell during 4 hours of broth mating. E. faecalis strains containing each of the pSL plasmids formed clumps after 2 hours of incubation in broth containing E. faecalis FA2-2 culture filtrate (i.e., the E. faecalis sex pheromone), and the plasmid subsequently transferred to the recipient strain in a 10-min short mating in broth, indicating that the plasmids are responsive to E. faecalis pheromones. The pSL plasmids did not respond to any of synthetic pheromones for the previously characterized plasmids. The pheromone specific for pSL plasmids has been designated cSL1. Southern hybridization analysis showed that specific FspI fragments from each of the pSL plasmids hybridized with the aggregation substance gene (asa1) of the pheromone-responsive plasmid pAD1, indicating that the plasmids had a gene homologous to asa1. The restriction maps of the plasmids were identical, and the size of the plasmids was estimated to be 128.1 kb. The plasmids carried five drug resistance determinants for vanA, ermB, aph(3'), aph(6'), and aac(6')/aph(2'), which encode resistance to vancomycin, erythromycin, kanamycin, streptomycin, and gentamicin/kanamycin, respectively. Nucleotide sequence analyses of the drug resistance determinants and their flanking regions are described in this report. The results described provide evidence for the exchange of genetic information

  17. 21 CFR 172.809 - Curdlan.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ...) produced by pure culture fermentation from the nonpathogenic and nontoxicogenic bacterium Alcaligenes faecalis var. myxogenes. (b) Curdlan meets the following specifications when it is tested according to...

  18. 21 CFR 172.809 - Curdlan.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ...) produced by pure culture fermentation from the nonpathogenic and nontoxicogenic bacterium Alcaligenes faecalis var. myxogenes. (b) Curdlan meets the following specifications when it is tested according to...

  19. 21 CFR 172.809 - Curdlan.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ...) produced by pure culture fermentation from the nonpathogenic and nontoxicogenic bacterium Alcaligenes faecalis var. myxogenes. (b) Curdlan meets the following specifications when it is tested according to...

  20. 21 CFR 172.809 - Curdlan.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... from the nonpathogenic and nontoxicogenic bacterium Alcaligenes faecalis var. myxogenes. (b) Curdlan... per square centimeter. (10) Aerobic plate count, not more than 103 per gram. (11) Coliform...

  1. Preclinical validation of a novel compound targeting p70S6 kinase in breast cancer

    PubMed Central

    Segatto, Ilenia; Massarut, Samuele; Boyle, Robert; Baldassarre, Gustavo; Walker, David; Belletti, Barbara

    2016-01-01

    Breast cancer is a frequent and treatable disease. However, when recurrent, breast cancer often becomes refractory to therapy and progresses into metastatic forms that are typically incurable. Thus, understanding and targeting the critical pathways underlying breast cancer recurrence is urgently needed to eradicate primary disease and achieve better prognosis. Recently, we have demonstrated that the ribosomal protein p70S6K is activated in residual breast cancer cells as a result of post-surgical inflammation and that interfering with its activity in the peri-operative setting strongly suppresses recurrence in a mouse model. In order to develop clinically-exploitable treatments targeting p70S6K, we have tested a newly generated compound, called FS-115. FS-115 potently inhibited p70S6K1 (IC50 35nM) with high selectivity over other AGC kinases or PI3K pathway kinases. In vitro, treatment with FS-115 efficiently blocked p70S6K activity in breast cancer cell lines and impaired colony formation and anchorage independent growth. Pharmacokinetic profiling showed that FS-115 exhibited high oral bioavailability, optimal plasma distribution and high brain penetrance. In nude mice, FS-115 strongly suppressed tumor take-rate and primary tumor growth. Oral dosing with FS-115 in a peri-operative schedule was effective in decreasing local recurrence of breast cancer and a long-term treatment schedule was well tolerated and efficiently suppressed distant metastasis formation. Altogether, we propose that FS-115 might be a good candidate for the treatment of breast cancer patients at high risk to relapse. Summary Statement Our results confirm that inhibition of p70S6K represents a valuable opportunity for restraining loco-regional relapse and metastasis in breast cancer and identify in FS-115 a promising candidate-inhibitor to move from preclinical to clinical treatments. PMID:27155197

  2. Characteristics of an environmental strain, Enterococcus faecalis CECT7121, and its effects as additive on craft dry-fermented sausages.

    PubMed

    Sparo, M; Nuñez, G G; Castro, M; Calcagno, M L; García Allende, M A; Ceci, M; Najle, R; Manghi, M

    2008-06-01

    Lactic acid bacteria are the most adequate microorganisms for natural preservation of food. In the present work, the strain of Enterococcus faecalis CECT7121 was employed in the manufacture of craft dry-fermented sausages and its performance as a biopreservative was analysed. This strain is devoid of the genes for haemolysin and gelatinase and does not produce biogenic amines. It is sensitive to almost all the antibiotics tested and opsonophagocytic assays showed that it is devoid of a capsule. This strain had a high LD50 (10(11)CFU ml(-1)) in mice. No statistical differences were found between control and sausages inoculated with E. faecalis CECT7121 regarding the production of lactic acid, pH variation over time, reaching a minimum pH value of 5.1, and sensory analysis in both series. Sausages inoculated with E. faecalis CECT7121 had lower viable counts of Enterobacteriaceae, Staphylococcus aureus and other Gram-positive cocci at the end of fermentation and 7 days and no viable enterobacteria and S. aureus were recovered at the end of drying. E. faecalis CECT7121 did not affect the growth of Lactobacillus spp. but it displaced the autochthonous populations of enterococci. E. faecalis CECT7121 was recovered in each time point as assessed by its inhibitory activity on Listeria monocytogenes and S. aureus. These results would indicate that the addition of E. faecalis CECT7121 during the manufacture of craft dry-fermented sausages offers an interesting alternative for biopreservation.

  3. Substantivity of Ag-Ca-Si mesoporous nanoparticles on dentin and its ability to inhibit Enterococcus faecalis.

    PubMed

    Fan, Wei; Wu, Yujie; Ma, Tengjiao; Li, Yanyun; Fan, Bing

    2016-01-01

    The main purpose of this study was to investigate the substantivity of Ag-Ca-Si mesoporous nanoparticles (Ag-MCSNs) on dentin and its residual antibacterial effects against Enterococcus faecalis. Ag-MCSNs were fabricated and characterized, ion release profile and pH were tested, and the ability to inhibit planktonic E. faecalis as well as the cytotoxicity was evaluated. Dentin slices were medicated with Ca(OH)2 paste, 2 % chlorhexidine gel and Ag-MCSNs paste for 7 days and then irrigated. Dentin slices were then immersed in E. faecalis suspension for 6 days and then transferred to fresh brain heart infusion solution. The optical density value within 10 h after immersing and transferring were measured and compared among groups. Results indicated that Ag-MCSNs showed high pH, sustained Ag(+)-Ca(2+)-SiO3 (2-) ion release, and high substantivity on dentin. The Ag-MCSNs exhibited strong antibacterial effects against planktonic E. faecalis and much better residual inhibition effects against E. faecalis growth on dentin than Ca(OH)2 paste (P < 0.05). The Ag-MCSNs showed excellent antibacterial ability against E. faecalis and high substantivity on dentin, which might be developed to a new effective intra-canal medicament for human teeth.

  4. Cloning, purification, crystallization and preliminary crystallographic analysis of SecA from Enterococcus faecalis

    SciTech Connect

    Meining, Winfried; Scheuring, Johannes; Fischer, Markus; Weinkauf, Sevil

    2006-06-01

    SecA ATPase from E. faecalis has been cloned, overexpressed, purified and crystallized. Crystals belong to space group C2 and diffract to 2.4 Å resolution. The gene coding for SecA from Enterococcus faecalis was cloned and overexpressed in Escherichia coli. In this protein, the lysine at position 6 was replaced by an asparagine in order to reduce sensitivity towards proteases. The modified protein was purified and crystallized. Crystals diffracting to 2.4 Å resolution were obtained using the vapour-diffusion technique. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 203.4, b = 49.8, c = 100.8 Å, α = γ = 90.0, β = 119.1°. A selenomethionine derivative was prepared and is currently being tested in crystallization trials.

  5. Detection of opsonic antibodies against Enterococcus faecalis cell wall carbohydrates in immune globulin preparations.

    PubMed

    Hufnagel, M; Sixel, K; Hammer, F; Kropec, A; Sava, I G; Theilacker, C; Berner, R; Huebner, J

    2014-08-01

    Three different commercially available polyvalent immune globulins (IG) were investigated for the existence of antibodies against cell wall carbohydrates of four different E. faecalis serotypes (using a cell wall carbohydrate-enzyme-linked immunosorbent assay), and whether these antibodies mediated opsonic killing (using an opsonic-killing assay). All three IG preparations contained antibodies against all four serotypes (CPS-A to CPS-D). However, only one of the three IG preparations showed opsonic killing against all four serotypes. Average killing was higher against serotypes A and B (72 and 79 %, respectively) than against serotypes C and D (30 and 37 %, respectively). Such IG preparations could play a role as an adjuvant therapeutic option in life-threatening infections with E. faecalis, particularly when resistant strains are involved.

  6. In vitro antimicrobial effect of bacteriophages on human dentin infected with Enterococcus faecalis ATCC 29212.

    PubMed

    Paisano, A F; Spira, B; Cai, S; Bombana, A C

    2004-10-01

    This study assessed the effect of bacteriophages on the viability of Enterococcus faecalis. Human dental roots were inoculated with a suspension of E. faecalis at three different multiplicities of infection - 0.1, 1.0 and 10.0. The phage lysate was able to significantly inhibit bacteria growth when incubated at the multiplicities of infection of 1.0, 10.0 and 0.1. The dental roots were also inoculated with bacteria for 6 days to allow bacterial penetration into the teeth tubules. Addition of the phage lysate to the roots following the 6-day incubation period led to a substantial reduction in bacteria viability. Phage therapy may be an important alternative for the treatment of root canal infections refractory to conventional endodontic therapy.

  7. Survival and activity of Streptococcus faecalis and Escherichia coli in tropical freshwater

    SciTech Connect

    Muniz, I.; Jimenez, L.; Toranzos, G.A.; Hazen, T.C.

    1988-12-31

    The survival of Streptococcus facecalis and Escherichia coli was studied in situ in a tropical rain forest watershed using membrane diffusion chambers. Densities were determined by acridine orange direct count and Coulter Counter. Population activity was determined by microautoradiography, cell respiration, and by nucleic acid composition. Densities of S. facecalis and E. coli decreased less than 1 log unit after 105 h as measured by direct count methods. Activity as measured by respiration, acridine orange activity, and microautoradiography indicated that both bacteria remained moderately active during the entire study. After 12 h, E. coli was more active than S. faecalis as measured by nucleic acid composition. E. coli and S. faecalis survived and remained active for more than 5 days. Consequently, both would seem to be unsuitable as indicators of recent fecal contamination in tropical waters.

  8. Continuous Drip Flow System to Develop Biofilm of E. faecalis under Anaerobic Conditions

    PubMed Central

    Gonzalez, Ana Maria; Corpus, Erika; Silva-Herzog, Daniel; Aragon-Piña, Antonio; Cohenca, Nestor

    2014-01-01

    Purpose. To evaluate a structurally mature E. faecalis biofilm developed under anaerobic/dynamic conditions in an in vitro system. Methods. An experimental device was developed using a continuous drip flow system designed to develop biofilm under anaerobic conditions. The inoculum was replaced every 24 hours with a fresh growth medium for up to 10 days to feed the system. Gram staining was done every 24 hours to control the microorganism purity. Biofilms developed under the system were evaluated under the scanning electron microscope (SEM). Results. SEM micrographs demonstrated mushroom-shaped structures, corresponding to a mature E. faecalis biofilm. In the mature biofilm bacterial cells are totally encased in a polymeric extracellular matrix. Conclusions. The proposed in vitro system model provides an additional useful tool to study the biofilm concept in endodontic microbiology, allowing for a better understanding of persistent root canal infections. PMID:25371913

  9. [In vitro activity of ampicillin-ceftriaxone against Enterococcus faecalis isolates recovered from invasive infections].

    PubMed

    Burguer Moreira, Noelia; Nastro, Marcela; Vay, Carlos; Famiglietti, Ángela; Rodríguez, Carlos Hernán

    2016-01-01

    In vitro activity of the combination of ampicillin- ceftriaxone against 30 Enterococcus faecalis isolates recovered from invasive infections in patients admitted to Hospital de Clínicas José de San Martin in the city of Buenos Aires was assessed. Ampicillin- ceftriaxone synergies were determined by microdilution in Müeller-Hinton (MH) broth with and without subinhibitory concentrations of ceftriaxone. Synergy was detected in 22/30 isolates. A decrease in both minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) was observed in 14/30 isolates, whereas in 6/30 isolates the decrease was observed in the MIC value and only in the MBC value in the 2 remaining isolates. The bactericidal activity of the combination showed to be higher at low concentrations of ampicillin (< 1 μg/ml). We detected in vitro synergy using the ampicillin-ceftriaxone combination and thus, its efficacy was confirmed in the treatment of severe infections by E. faecalis.

  10. Transcriptomic response of Enterococcus faecalis V583 to low hydrogen peroxide levels.

    PubMed

    Yan, Xue; Budin-Verneuil, Aurélie; Verneuil, Nicolas; Gilmore, Michael S; Artigaud, Sébastien; Auffray, Yanick; Pichereau, Vianney

    2015-02-01

    Enterococcus faecalis is a Gram-positive commensal bacterium inhabiting the gastrointestinal tracts of human and other mammals, but is also increasingly recognized as an opportunistic human pathogen. Oxidative stress is one of the major challenges encountered by enterococci, both in their natural environment and during infection. In this paper, we evaluated the transcriptomic response of E. faecalis to oxidative stress, and showed that transcript abundance was reduced for 93 genes and increased for 39 genes during growth in medium containing 1.75 mM H2O2. The presence of hydrogen peroxide affected several metabolic pathways, including a large decrease in ethanolamine utilization and methylglyoxal metabolism, and an increase in transcript abundance for several transport systems. In particular, four operons encoding iron transporters appeared highly induced. By contrast, in our experimental conditions, the expression of most of the genes known to be involved in the enterococcal response to oxidative stress, did not appear significantly altered.

  11. CRISPR-Cas and Restriction-Modification Act Additively against Conjugative Antibiotic Resistance Plasmid Transfer in Enterococcus faecalis

    PubMed Central

    Price, Valerie J.; Huo, Wenwen; Sharifi, Ardalan

    2016-01-01

    ABSTRACT Enterococcus faecalis is an opportunistic pathogen and a leading cause of nosocomial infections. Conjugative pheromone-responsive plasmids are narrow-host-range mobile genetic elements (MGEs) that are rapid disseminators of antibiotic resistance in the faecalis species. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification confer acquired and innate immunity, respectively, against MGE acquisition in bacteria. Most multidrug-resistant E. faecalis isolates lack CRISPR-Cas and possess an orphan locus lacking cas genes, CRISPR2, that is of unknown function. Little is known about restriction-modification defense in E. faecalis. Here, we explore the hypothesis that multidrug-resistant E. faecalis strains are immunocompromised. We assessed MGE acquisition by E. faecalis T11, a strain closely related to the multidrug-resistant hospital isolate V583 but which lacks the ~620 kb of horizontally acquired genome content that characterizes V583. T11 possesses the E. faecalis CRISPR3-cas locus and a predicted restriction-modification system, neither of which occurs in V583. We demonstrate that CRISPR-Cas and restriction-modification together confer a 4-log reduction in acquisition of the pheromone-responsive plasmid pAM714 in biofilm matings. Additionally, we show that the orphan CRISPR2 locus is functional for genome defense against another pheromone-responsive plasmid, pCF10, only in the presence of cas9 derived from the E. faecalis CRISPR1-cas locus, which most multidrug-resistant E. faecalis isolates lack. Overall, our work demonstrated that the loss of only two loci led to a dramatic reduction in genome defense against a clinically relevant MGE, highlighting the critical importance of the E. faecalis accessory genome in modulating horizontal gene transfer. Our results rationalize the development of antimicrobial strategies that capitalize upon the immunocompromised status of multidrug-resistant E. faecalis

  12. The Heterodimeric ABC Transporter EfrCD Mediates Multidrug Efflux in Enterococcus faecalis

    PubMed Central

    Hürlimann, Lea M.; Corradi, Valentina; Hohl, Michael; Bloemberg, Guido V.; Tieleman, D. Peter

    2016-01-01

    Nosocomial infections with Enterococcus faecalis are an emerging health problem. However, drug efflux pumps contributing to intrinsic drug resistance are poorly studied in this Gram-positive pathogen. In this study, we functionally investigated seven heterodimeric ABC transporters of E. faecalis that are annotated as drug efflux pumps. Deletion of ef0789-ef0790 on the chromosome of E. faecalis resulted in increased susceptibility to daunorubicin, doxorubicin, ethidium, and Hoechst 33342, and the corresponding transporter was named EfrCD. Unexpectedly, the previously described heterodimeric multidrug ABC transporter EfrAB contributes marginally to drug efflux in the endogenous context of E. faecalis. In contrast, heterologous expression in Lactococcus lactis revealed that EfrAB, EfrCD, and the product of ef2226-ef2227 (EfrEF) mediate the efflux of fluorescent substrates and confer resistance to multiple dyes and drugs, including fluoroquinolones. Four of seven transporters failed to exhibit drug efflux activity for the set of drugs and dyes tested, even upon overexpression in L. lactis. Since all seven transporters were purified as heterodimers after overexpression in L. lactis, a lack of drug efflux activity is not attributed to poor expression or protein aggregation. Reconstitution of the purified multidrug transporters EfrAB, EfrCD, and EfrEF in proteoliposomes revealed functional coupling between ATP hydrolysis and drug binding. Our analysis creates an experimental basis for the accurate prediction of drug efflux transporters and indicates that many annotated multidrug efflux pumps might be incapable of drug transport and thus might fulfill other physiological functions in the cell. PMID:27381387

  13. Opsonic Antibodies to Enterococcus faecalis Strain 12030 Are Directed against Lipoteichoic Acid

    PubMed Central

    Theilacker, Christian; Kaczynski, Zbigniew; Kropec, Andrea; Fabretti, Francesca; Sange, Tatjana; Holst, Otto; Huebner, Johannes

    2006-01-01

    A teichoic acid (TA)-like polysaccharide in Enterococcus faecalis has previously been shown to induce opsonic antibodies that protect against bacteremia after active and passive immunization. Here we present new data providing a corrected structure of the antigen and the epitope against which the opsonic antibodies are directed. Capsular polysaccharide isolated from E. faecalis strain 12030 by enzymatic digestion of peptidoglycan and chromatography (enzyme-TA) was compared with lipoteichoic acid (LTA) extracted using butanol and purified by hydrophobic-interaction chromatography (BuOH-LTA). Structural determinations were carried out by chemical analysis and nuclear magnetic resonance spectroscopy. Antibody specificity was assessed by enzyme-linked immunosorbent assay and the opsonophagocytosis assay. After alanine ester hydrolysis, there was structural identity between enzyme-TA and BuOH-LTA of the TA-parts of the two molecules. The basic enterococcal LTA structure was confirmed: 1,3-poly(glycerol phosphate) nonstoichiometrically substituted at position C-2 of the glycerol residues with d-Ala and kojibiose. We also detected a novel substituent at position C-2, [d-Ala→6]-α-d-Glcp-(1→2-[d-Ala→6]-α-d-Glcp-1→). Antiserum raised against enzyme-TA bound equally well to BuOH-LTA and dealanylated BuOH-LTA as to the originally described enzyme-TA antigen. BuOH-LTA was a potent inhibitor of opsonophagocytic killing by the antiserum to enzyme-TA. Immunization with antibiotic-killed whole bacterial cells did not induce a significant proportion of antibodies directed against alanylated epitopes on the TA, and opsonic activity was inhibited completely by both alanylated and dealanylated BuOH-LTA. In summary, the E. faecalis strain 12030 enzyme-TA is structurally and immunologically identical to dealanylated LTA. Opsonic antibodies to E. faecalis 12030 are directed predominantly to nonalanylated epitopes on the LTA molecule. PMID:16988246

  14. A rare case of Enterococcus faecalis-induced orbital cellulitis and myositis

    PubMed Central

    Kohli, Piyush; Ichhpujani, Parul; Bansal, Rakesh Kumar; Kumar, Suresh

    2016-01-01

    Orbital cellulitis is an infection of soft tissue behind the orbital septum. Common pathogens isolated include Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pneumoniae. It is a straightforward diagnosis and usually responds to empirical treatment without any sequela. We report a case of orbital cellulitis caused by Enterococcus faecalis, which was complicated by myositis of levator palpebrae superioris. To the best of our knowledge, only one case report exists dating way back to 1986. PMID:27688288

  15. Global Regulation of Gene Expression by the MafR Protein of Enterococcus faecalis

    PubMed Central

    Ruiz-Cruz, Sofía; Espinosa, Manuel; Goldmann, Oliver; Bravo, Alicia

    2016-01-01

    Enterococcus faecalis is a natural inhabitant of the human gastrointestinal tract. However, as an opportunistic pathogen, it is able to colonize other host niches and cause life-threatening infections. Its adaptation to new environments involves global changes in gene expression. The EF3013 gene (here named mafR) of E. faecalis strain V583 encodes a protein (MafR, 482 residues) that has sequence similarity to global response regulators of the Mga/AtxA family. The enterococcal OG1RF genome also encodes the MafR protein (gene OG1RF_12293). In this work, we have identified the promoter of the mafR gene using several in vivo approaches. Moreover, we show that MafR influences positively the transcription of many genes on a genome-wide scale. The most significant target genes encode components of PTS-type membrane transporters, components of ABC-type membrane transporters, and proteins involved in the metabolism of carbon sources. Some of these genes were previously reported to be up-regulated during the growth of E. faecalis in blood and/or in human urine. Furthermore, we show that a mafR deletion mutant strain induces a significant lower degree of inflammation in the peritoneal cavity of mice, suggesting that enterococcal cells deficient in MafR are less virulent. Our work indicates that MafR is a global transcriptional regulator. It might facilitate the adaptation of E. faecalis to particular host niches and, therefore, contribute to its potential virulence. PMID:26793169

  16. [Antimicrobial susceptibility of Enterococcus faecalis isolated from patients in Córdoba (Spain)].

    PubMed

    Causse, M; Franco-Alvarez de Luna, F; García-Mayorgas, A D; Rodríguez, F C; Casal, M

    2006-06-01

    Enterococcus faecalis is a pathogenic microorganism. The aim of this investigation was to study the antibiotic susceptibility of the strains isolated in Cordoba in a 20-month period (January 2004 to August 2005). Susceptibility rates to betalactamics were 98% to ampicillin and 99% to amoxicillin/clavulanic acid; high-dose aminoglycosides (streptomycin 1000 microg and gentamycin 500 microg) obtained 56% and 76%, respectively. We found no strains resistant to glycopeptides (vancomycin and teicoplanin) or to linezolid.

  17. Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis*

    PubMed Central

    Park, Young-Guen; Jung, Min-Cheol; Song, Heesang; Jeong, Ki-Woong; Bang, Eunjung; Hwang, Geum-Sook; Kim, Yangmee

    2016-01-01

    Enterococcus faecalis is a Gram-positive, commensal bacterium that lives in the gastrointestinal tracts of humans and other mammals. It causes severe infections because of high antibiotic resistance. E. faecalis can endure extremes of temperature and pH. Acyl carrier protein (ACP) is a key element in the biosynthesis of fatty acids responsible for acyl group shuttling and delivery. In this study, to understand the origin of high thermal stabilities of E. faecalis ACP (Ef-ACP), its solution structure was investigated for the first time. CD experiments showed that the melting temperature of Ef-ACP is 78.8 °C, which is much higher than that of Escherichia coli ACP (67.2 °C). The overall structure of Ef-ACP shows the common ACP folding pattern consisting of four α-helices (helix I (residues 3–17), helix II (residues 39–53), helix III (residues 60–64), and helix IV (residues 68–78)) connected by three loops. Unique Ef-ACP structural features include a hydrophobic interaction between Phe45 in helix II and Phe18 in the α1α2 loop and a hydrogen bonding between Ser15 in helix I and Ile20 in the α1α2 loop, resulting in its high thermal stability. Phe45-mediated hydrophobic packing may block acyl chain binding subpocket II entry. Furthermore, Ser58 in the α2α3 loop in Ef-ACP, which usually constitutes a proline in other ACPs, exhibited slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. These results might provide insights into the development of antibiotics against pathogenic drug-resistant E. faecalis strains. PMID:26631734

  18. Chitosan-propolis nanoparticle formulation demonstrates anti-bacterial activity against Enterococcus faecalis biofilms.

    PubMed

    Ong, Teik Hwa; Chitra, Ebenezer; Ramamurthy, Srinivasan; Siddalingam, Rajinikanth Paruvathanahalli; Yuen, Kah Hay; Ambu, Stephen Periathamby; Davamani, Fabian

    2017-01-01

    Propolis obtained from bee hives is a natural substance with antimicrobial properties. It is limited by its insolubility in aqueous solutions; hence ethanol and ethyl acetate extracts of Malaysian propolis were prepared. Both the extracts displayed antimicrobial and anti-biofilm properties against Enterococcus faecalis, a common bacterium associated with hospital-acquired infections. High performance liquid chromatography (HPLC) analysis of propolis revealed the presence of flavonoids like kaempferol and pinocembrin. This study investigated the role of propolis developed into nanoparticles with chitosan for its antimicrobial and anti-biofilm properties against E. faecalis. Bacteria that grow in a slimy layer of biofilm are resistant to penetration by antibacterial agents. The use of nanoparticles in medicine has received attention recently due to better bioavailability, enhanced penetrative capacity and improved efficacy. A chitosan-propolis nanoformulation was chosen based on ideal physicochemical properties such as particle size, zeta potential, polydispersity index, encapsulation efficiency and the rate of release of the active ingredients. This formulation inhibited E. faecalis biofilm formation and reduced the number of bacteria in the biofilm by ~90% at 200 μg/ml concentration. When tested on pre-formed biofilms, the formulation reduced bacterial number in the biofilm by ~40% and ~75% at 200 and 300 μg/ml, respectively. The formulation not only reduced bacterial numbers, but also physically disrupted the biofilm structure as observed by scanning electron microscopy. Treatment of biofilms with chitosan-propolis nanoparticles altered the expression of biofilm-associated genes in E. faecalis. The results of this study revealed that chitosan-propolis nanoformulation can be deemed as a potential anti-biofilm agent in resisting infections involving biofilm formation like chronic wounds and surgical site infections.

  19. Deactivation of Enterococcus Faecalis Bacteria by an Atmospheric Cold Plasma Brush

    NASA Astrophysics Data System (ADS)

    Chen, Wei; Huang, Jun; Du, Ning; Liu, Xiao-Di; Lv, Guo-Hua; Wang, Xing-Quan; Zhang, Guo-Ping; Guo, Li-Hong; Yang, Si-Ze

    2012-07-01

    An atmospheric cold plasma brush suitable for large area and low-temperature plasma-based sterilization is designed and used to treat enterococcus faecalis bacteria. The results show that the efficiency of the inactivation process by helium plasma is dependent on applied power and exposure time. After plasma treatments, the cell structure and morphology changes can be observed by scanning electron microscopy. Optical emission measurements indicate that reactive species such as O and OH play a significant role in the sterilization process.

  20. Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis.

    PubMed

    Park, Young-Guen; Jung, Min-Cheol; Song, Heesang; Jeong, Ki-Woong; Bang, Eunjung; Hwang, Geum-Sook; Kim, Yangmee

    2016-01-22

    Enterococcus faecalis is a Gram-positive, commensal bacterium that lives in the gastrointestinal tracts of humans and other mammals. It causes severe infections because of high antibiotic resistance. E. faecalis can endure extremes of temperature and pH. Acyl carrier protein (ACP) is a key element in the biosynthesis of fatty acids responsible for acyl group shuttling and delivery. In this study, to understand the origin of high thermal stabilities of E. faecalis ACP (Ef-ACP), its solution structure was investigated for the first time. CD experiments showed that the melting temperature of Ef-ACP is 78.8 °C, which is much higher than that of Escherichia coli ACP (67.2 °C). The overall structure of Ef-ACP shows the common ACP folding pattern consisting of four α-helices (helix I (residues 3-17), helix II (residues 39-53), helix III (residues 60-64), and helix IV (residues 68-78)) connected by three loops. Unique Ef-ACP structural features include a hydrophobic interaction between Phe(45) in helix II and Phe(18) in the α1α2 loop and a hydrogen bonding between Ser(15) in helix I and Ile(20) in the α1α2 loop, resulting in its high thermal stability. Phe(45)-mediated hydrophobic packing may block acyl chain binding subpocket II entry. Furthermore, Ser(58) in the α2α3 loop in Ef-ACP, which usually constitutes a proline in other ACPs, exhibited slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. These results might provide insights into the development of antibiotics against pathogenic drug-resistant E. faecalis strains.

  1. Mature biofilms of Enterococcus faecalis and Enterococcus faecium are highly resistant to antibiotics.

    PubMed

    Holmberg, Anna; Rasmussen, Magnus

    2016-01-01

    Enterococcus faecalis and Enterococcus faecium are important nosocomial pathogens that form biofilms on implanted materials. We compare the antibiotic sensitivity of bacteria in new (established during 24 hours) and mature (established during 120 hours) enterococcal biofilms. Mature biofilms contained more bacteria and were much more tolerant to antibiotics, including rifampicin-containing combinations, as judged by determination of minimal biofilm eradication concentrations and by time-kill experiments of bacteria in biofilms formed on beads of bone cement.

  2. Effect of chitosan-ethylenediamine tetraacetic acid on Enterococcus faecalis dentinal biofilm and smear layer removal

    PubMed Central

    Geethapriya, Nagarajan; Subbiya, Arunajatesan; Padmavathy, Kesavaram; Mahalakshmi, Krishnan; Vivekanandan, Paramasivam; Sukumaran, Virudhachalam Ganapathy

    2016-01-01

    Objective: The objective of the study was to evaluate the effectiveness of chitosan and chitosan-ethylenediamine tetraacetic acid (EDTA) (3:1,1:1,1:3) in comparison with 5.2% sodium hypochlorite (NaOCl) in disinfecting Enterococcus faecalis biofilm on root canal dentin and in the removal of smear layer with minimal erosion. Materials and Methods: Seventy single-rooted extracted human mandibular premolars (n = 70) were selected for the study. Forty tooth samples were biomechanically prepared, vertically sectioned, and sterilized by autoclaving. The tooth sections were artificially infected with E. faecalis (ATCC 29212 [n = 35] and clinical isolate [SBEF2, n = 35]) to form mature dentinal biofilm in vitro. The tooth samples were treated with the test solutions: chitosan and chitosan-EDTA (3:1, 1:1, 1:3), and the killing time was determined. The smear layer removal ability of the test solutions (Group A: chitosan-EDTA [1:1], Group B: EDTA, Group C: control) (n = 10 tooth/group) was assessed. Results: Chitosan and chitosan-EDTA (3:1, 1:1, 1:3) exhibited antibacterial activity against both the strains of E. faecalis. Chitosan and chitosan-EDTA caused 3 log reduction in the viable count of the sessile cells of E. faecalis at 15 min while 5.2% NaOCl exhibited 99.98% inhibition at 15 min. Chitosan-EDTA (1:1) was found to be effective in removing the smear layer and showed lesser erosion than EDTA at the coronal and middle portions. Conclusion: Chitosan-EDTA (1:1) is a potential root canal irrigant that performs a dual role – root canal disinfection and smear layer removal. PMID:27656070

  3. Chitosan-propolis nanoparticle formulation demonstrates anti-bacterial activity against Enterococcus faecalis biofilms

    PubMed Central

    Ong, Teik Hwa; Chitra, Ebenezer; Ramamurthy, Srinivasan; Siddalingam, Rajinikanth Paruvathanahalli; Yuen, Kah Hay; Ambu, Stephen Periathamby

    2017-01-01

    Propolis obtained from bee hives is a natural substance with antimicrobial properties. It is limited by its insolubility in aqueous solutions; hence ethanol and ethyl acetate extracts of Malaysian propolis were prepared. Both the extracts displayed antimicrobial and anti-biofilm properties against Enterococcus faecalis, a common bacterium associated with hospital-acquired infections. High performance liquid chromatography (HPLC) analysis of propolis revealed the presence of flavonoids like kaempferol and pinocembrin. This study investigated the role of propolis developed into nanoparticles with chitosan for its antimicrobial and anti-biofilm properties against E. faecalis. Bacteria that grow in a slimy layer of biofilm are resistant to penetration by antibacterial agents. The use of nanoparticles in medicine has received attention recently due to better bioavailability, enhanced penetrative capacity and improved efficacy. A chitosan-propolis nanoformulation was chosen based on ideal physicochemical properties such as particle size, zeta potential, polydispersity index, encapsulation efficiency and the rate of release of the active ingredients. This formulation inhibited E. faecalis biofilm formation and reduced the number of bacteria in the biofilm by ~90% at 200 μg/ml concentration. When tested on pre-formed biofilms, the formulation reduced bacterial number in the biofilm by ~40% and ~75% at 200 and 300 μg/ml, respectively. The formulation not only reduced bacterial numbers, but also physically disrupted the biofilm structure as observed by scanning electron microscopy. Treatment of biofilms with chitosan-propolis nanoparticles altered the expression of biofilm-associated genes in E. faecalis. The results of this study revealed that chitosan-propolis nanoformulation can be deemed as a potential anti-biofilm agent in resisting infections involving biofilm formation like chronic wounds and surgical site infections. PMID:28362873

  4. The effect of different root canal medicaments on the elimination of Enterococcus faecalis ex vivo

    PubMed Central

    Dammaschke, Till; Jung, Nina; Harks, Inga; Schafer, Edgar

    2013-01-01

    Objective: The aim of this study was to evaluate the antimicrobial effect of chlorhexidine gel (CHX-G) 2%, chlorhexidine powder (CHX-P) 1%, povidone-iodine (PVP-I), polyhexanide and camphorated-and-mentholated chlorophenol (ChKM) ex vivo. Materials and Methods: For every medicament group 10 root segments (15 mm long) of extracted human teeth were prepared to ISO-size 45 and sterilized (n = 50). The root segments were then inoculated with Enterococcus faecalis and aerobically incubated at 37°C. After 1 week, ten root canals were filled with one of the medicaments, respectively and aerobically incubated at 37°C for another week. Ten teeth served as positive controls and were filled with sterile saline solution. After 7 days, the medicaments were inactivated and all root canals were instrumented to ISO-size 50. The obtained dentin samples were dispersed in Ringer solution followed by the preparation of serial dilutions. 10 μl per sample were applied to an agar plate and incubated at 37°C for 48 h. The colony forming units were counted and the reduction factors (RFs) were calculated and statistically analyzed. Results: Compared with the positive controls all medicaments exhibited an antibacterial effect against E. faecalis. The RFs for CHX-G, CHX-P and ChKM were significantly higher compared to PVP-I and polyhexanide (P < 0.05). In contrast to PVP-I and polyhexanide, CHX-G, CHX-P and ChKM were able to eliminate E. faecalis from all dentin samples. Conclusions: Within the limitations of this ex vivo investigation, 2% CHX-G and CHX-P were as effective as ChKM against E. faecalis. Thus, when choosing a root canal medicament the better biocompatibility of CHX compared with ChKM should be taken in consideration. PMID:24932119

  5. High Incidence of Virulence Factors Among Clinical Enterococcus faecalis Isolates in Southwestern Iran

    PubMed Central

    Heidari, Hamid; Hasanpour, Somayeh; Ebrahim-Saraie, Hadi Sedigh

    2017-01-01

    Background Over the past two decades, enterococci have emerged as an important agent responsible for hospital acquired infection. Several virulence factors contribute to the adherence, colonization, evasion of the host immune response, and pathogenicity and severity of the infection. Enterococcus faecalis is the most common and virulent species causing infections in hospitalized patients. The aim of the present study was to examine the prevalence of genes encoding virulence factors and antimicrobial resistance patterns of E. faecalis strains isolated from hospitalized patients in Shiraz, south west of Iran. Materials and Methods A total of 51 E. faecalis isolates from the urine, blood, pleural fluid, peritoneal fluid, eye discharge, endotracheal tube (ETT) and transjugular intrahepatic portosystemic shunt (TIPS) specimens of patients were identified by phenotypic and genotypic methods. Antimicrobial sensitivity tests and detection of virulence factors were performed using standard methods. Results The efa and asa1 were the most frequently detected gene (100%) among the isolates, followed by esp (94.1%), ace (90.2%), gelE (80.4%), cylA (64.7%), and hyl (51%). More than half of the isolates (52.9%) were high level gentamicin resistant (HLGR). Vancomycin resistance was observed among 23 (45.1%) isolates. The lowest antimicrobial activity was related to erythromycin (3.9%), tetracycline (5.9%) and ciprofloxacin (9.8%). No isolate was found resistant to fosfomycin and linezolid. Conclusion Our data indicated a high incidence of virulence factors among E. faecalis strains isolated from clinical samples. Colonization of drug resistant virulent isolates in hospital environment may lead to life threatening infection in hospitalized patients. Therefore, infection control procedures should be performed. PMID:28332345

  6. Application of waste frying oils in the biosynthesis of biodemulsifier by a demulsifying strain Alcaligenes sp. S-XJ-1.

    PubMed

    Liu, Jia; Peng, Kaiming; Huang, Xiangfeng; Lu, Lijun; Cheng, Hang; Yang, Dianhai; Zhou, Qi; Deng, Huiping

    2011-01-01

    Exploration of biodemulsifiers has become a new research aspect. Using waste frying oils (WFOs) as carbon source to synthesize biodemulsifiers has a potential prospect to decrease production cost and to improve the application of biodemulsifiers in the oilfield. In this study, a demulsifying strain, Alcaligenes sp. S-XJ-1, was investigated to synthesize a biodemulsifier using waste frying oils as carbon source. It was found that the increase of initial pH of culture medium could increase the biodemulsifier yield but decrease the demulsification ratio compared to that using paraffin as carbon source. In addition, a biodemulsifier produced by waste frying oils and paraffin as mixed carbon source had a lower demulsification capability compared with that produced by paraffin or waste frying oil as sole carbon source. Fed-batch fermentation of biodemulsifier using waste frying oils as supplementary carbon source was found to be a suitable method. Mechanism of waste frying oils utilization was studied by using tripalmitin, olein and tristearin as sole carbon sources to synthesize biodemulsifier. The results showed saturated long-chain fatty acid was difficult for S-XJ-1 to utilize but could effectively enhance the demulsification ability of the produced biodemulsifier. Moreover, FT-IR result showed that the demulsification capability of biodemulsifiers was associated with the content of C=O group and nitrogen element.

  7. Identification of a catabolic transposon, Tn4371, carrying biphenyl and 4-chlorobiphenyl degradation genes in Alcaligenes eutrophus A5.

    PubMed Central

    Springael, D; Kreps, S; Mergeay, M

    1993-01-01

    Alcaligenes eutrophus A5 catabolizes biphenyl to CO2 via benzoate and 4-chlorobiphenyl to 4-chlorobenzoate. In curing and conjugation experiments, the A5 endogenous 51-kb IncP1 plasmid pSS50 was found to be dispensable for biphenyl and 4-chlorobiphenyl catabolism. Transfer of the biphenyl- and 4-chlorobiphenyl-degrading phenotype by means of pSS50 was observed at a frequency of 10(-5) per transferred plasmid in matings of A5 with other A. eutrophus strains. Transconjugants harbor enlarged pSS50 derivatives which contain additional genetic information governing the oxidation of biphenyl and 4-chlorobiphenyl to benzoate and 4-chlorobenzoate and originating from the chromosome of strain A5. The following observations indicate that the catabolic genes reside on a 59-kb large transposon (Tn4371) for which a restriction map is presented. (i) Tn4371 transposes between different replicons and at different locations of the same replicon. (ii) Transposition was observed in a Rec- strain of A. eutrophus. (iii) Tn4371 transposes as a single, contiguous piece of DNA. Although an RP4::Tn4371 plasmid was stably maintained in different hosts, the plasmid conferred growth on biphenyl only when present in strains of A. eutrophus and in an Acinetobacter sp. strain. Images PMID:8383664

  8. Degradation of Chlorophenols by Alcaligenes eutrophus JMP134(pJP4) in Bleached Kraft Mill Effluent

    PubMed Central

    Valenzuela, J.; Bumann, U.; Cespedes, R.; Padilla, L.; Gonzalez, B.

    1997-01-01

    The ability of Alcaligenes eutrophus JMP134(pJP4) to degrade 2,4-dichlorophenoxyacetic acid, 2,4,6-trichlorophenol, and other chlorophenols in a bleached kraft mill effluent was studied. The efficiency of degradation and the survival of strain JMP134 and indigenous microorganisms in short-term batch or long-term semicontinuous incubations performed in microcosms were assessed. After 6 days of incubation, 2,4-dichlorophenoxyacetate (400 ppm) or 2,4,6-trichlorophenol (40 to 100 ppm) were extensively degraded (70 to 100%). In short-term batch incubations, indigenous microorganisms were unable to degrade such of compounds. Degradation of 2,4,6-trichlorophenol by strain JMP134 was significantly lower at 200 to 400 ppm of compound. This strain was also able to degrade 2,4-dichlorophenoxyacetate, 2,4,6-trichlorophenol, 4-chlorophenol, and 2,4,5-trichlorophenol when bleached Kraft mill effluent was amended with mixtures of these compounds. On the other hand, the chlorophenol concentration and the indigenous microorganisms inhibited the growth and survival of the strain in short-term incubations. In long-term (>1-month) incubations, strain JMP134 was unable to maintain a large, stable population, although extensive 2,4,6-trichlorophenol degradation was still observed. The latter is probably due to acclimation of the indigenous microorganisms to degrade 2,4,6-trichlorophenol. Acclimation was observed only in long-term, semicontinuous microcosms. PMID:16535488

  9. Total degradation of pentachloroethane by an engineered Alcaligenes strain expressing a modified camphor monooxygenase and a hybrid dioxygenase.

    PubMed

    Iwakiri, Ryo; Yoshihira, Kunichika; Ngadiman; Futagami, Taiki; Goto, Masatoshi; Furukawa, Kensuke

    2004-06-01

    We engineered biphenyl-degrading Alcaligenes sp. strain KF711 for total degradation of pentachloroethane (PCA), which expresses a modified camphor monooxygenase and a hybrid dioxygenase consisting of TodC1 (a large subunit of toluene dioxygenase of Pseudomonas putida F1) and BphA2-BphA3-pbhA4 (a small subunit, ferredoxin and ferredoxin reductase of biphenyl dioxygenase, respectively, in strain KF707). Modified camphor monooxygenase genes (camCAB) were supplied as a plasmid and the todC1 gene was integrated within the chromosomal bph gene cluster by a single crossover recombination. The resultant strain KF711S-3cam dechlorinated PCA to trichloroethene by the action of the modified camphor monooxygenase under anaerobic conditions. The same strain subsequently degraded trichloroethene formed oxidatively by the action of the Tol-Bph hybrid dioxygenase under aerobic conditions. Thus sequential anaerobic and aerobic treatments of the KF711S-3cam resting cells resulted in efficient and total degradation of PCA.

  10. A combination of site-directed mutagenesis and chemical modification to improve diastereopreference of Pseudomonas alcaligenes lipase.

    PubMed

    Chen, Hui; Wu, Jianping; Yang, Lirong; Xu, Gang

    2013-12-01

    A combination of site-directed mutagenesis and chemical modification was employed to alter protein structure with the objective of improving diastereopreference over that achieved by simple site-directed mutagenesis. Conformational analysis using molecular dynamic (MD) simulation of Pseudomonas alcaligenes lipase (PAL) indicated that stronger steric exclusion and structural rigidity facilitated diastereopreference. A cysteine (Cys) residue was introduced using site-directed mutagenesis to construct variant A272C. The modifier 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) was then reacted with the introduced Cys residue to provide stronger steric exclusion and structural rigidity. The modification was verified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Diastereopreference was improved significantly. The diastereomeric excess (dep) of l-menthol increased from 35% with wild type PAL to 90% with A272C-DTNB modified PAL when the conversion ratio of l-menthyl propionate was nearly 100%. Conformation and kinetic parameter analysis showed that A272C-DTNB modified PAL exhibited stronger steric exclusion and increased structural rigidity around the modification site that inhibited the hydrolysis of non-targeted substrates. The combination of site-directed mutagenesis and chemical modification could be an effective method to alter protein properties and enhance diastereopreference through the combined effect of steric exclusion and structural rigidity.

  11. Metabolism of 2-, 3- and 4-hydroxybenzoates by soil isolates Alcaligenes sp. strain PPH and Pseudomonas sp. strain PPD.

    PubMed

    Deveryshetty, Jaigeeth; Suvekbala, V; Varadamshetty, Gautham; Phale, Prashant S

    2007-03-01

    Pseudomonas sp. strain PPD and Alcaligenes sp. strain PPH isolated from soil by enrichment culture technique utilize 2-, 3- and 4-hydroxybenzoates as the sole source of carbon and energy. The degradation pathways were elucidated by performing whole-cell O(2) uptake, enzyme activity and induction studies. Depending on the mixture of carbon source and the preculture condition, strain PPH was found to degrade 2-hydroxybenzoate either via the catechol or gentisate route and has both salicylate 1-hydroxylase and salicylate 5-hydroxylase. Strain PPD utilizes 2-hydroxybenzoate via gentisate. Both strains degrade 3- and 4-hydroxybenzoate via gentisate and protocatechuate, respectively. Enzymes were induced by respective hydroxybenzoate. Growth pattern, O(2) uptake and enzyme activity profiles on the mixture of three hydroxybenzoates as a carbon source suggest coutilization by both strains. When 3- or 4-hydroxybenzoate grown culture was used as an inoculum, strain PPH failed to utilize 2-hydroxybenzoate via catechol, indicating the modulation of the metabolic pathways, thus generating metabolic diversity.

  12. Upstream process optimization of polyhydroxybutyrate (PHB) by Alcaligenes latus using two-stage batch and fed-batch fermentation strategies.

    PubMed

    Wang, Bingqing; Sharma-Shivappa, Ratna R; Olson, Jonathan W; Khan, Saad A

    2012-11-01

    This research focused on optimizing the upstream process time for production of polyhydroxybutyrate (PHB) from sucrose by two-stage batch and fed-batch fermentation with Alcaligenes latus ATCC 29714. The study included selection of strain, two-stage batch fermentations with different time points for switching to nitrogen limited media (14, 16 or 18 h) and fed-batch fermentations with varied time points (similar to two stage) for introducing nitrogen limited media. The optimal strain to produce PHB using sucrose as carbon source was A. latus ATCC 29714 with maximum-specific growth rate of 0.38 ± 0.01 h(-1) and doubling time of 1.80 ± 0.05 h. Inducing nitrogen limitation at 16 h and ending second stage at 26 h gave optimal performance for PHB production, resulting in a PHB content of 46.7 ± 12.2 % (g PHB per g dry cell weight) at the end of fermentation. This was significantly higher (P ≤ 0.05) (approximately 7 %) than the corresponding fed batch run in which nitrogen limitation was initiated at 16 h.

  13. Production and characterization of poly(3-hydroxybutyrate) generated by Alcaligenes latus using lactose and whey after acid protein precipitation process.

    PubMed

    Berwig, Karina Hammel; Baldasso, Camila; Dettmer, Aline

    2016-10-01

    Whey after acid protein precipitation was used as substrate for PHB production in orbital shaker using Alcaligenes latus. Statistical analysis determined the most appropriate hydroxide for pH neutralization of whey after protein precipitation among NH4OH, KOH and NaOH 10%w/v. The results were compared to those of commercial lactose. A scale-up test in a 4L bioreactor was done at 35°C, 750rpm, 7L/min air flow, and 6.5 pH. The PHB was characterized through Fourier Transform Infrared Spectroscopy, thermogravimetry and differential scanning calorimetry. NH4OH provided the best results for productivity (p), 0.11g/L.h, and for polymer yield, (YP/S), 1.08g/g. The bioreactor experiment resulted in lower p and YP/S. PHB showed maximum degradation temperature (291°C), melting temperature (169°C), and chemical properties similar to those of standard PHB. The use of whey as a substrate for PHB production did not affect significantly the final product quality.

  14. The Spx Regulator Modulates Stress Responses and Virulence in Enterococcus faecalis

    PubMed Central

    Kajfasz, Jessica K.; Mendoza, Jorge E.; Gaca, Anthony O.; Miller, James H.; Koselny, Kristy A.; Giambiagi-deMarval, Marcia; Wellington, Melanie; Abranches, Jacqueline

    2012-01-01

    The ability to cope with endogenous or host-generated reactive oxygen species is considered a key virulence attribute of the opportunistic pathogen Enterococcus faecalis, a leading cause of hospital-acquired infections. In this study, we used in silico and mutational analyses to identify and characterize the role of the Spx global regulator in oxidative stress tolerance and virulence in E. faecalis. While the Δspx strain grew as well as the wild-type strain under anaerobic conditions, the mutant strain exhibited impaired growth under aerobic conditions and was highly sensitive to oxidative stress agents. The spx mutant strain was also sensitive to a variety of other stressful conditions, including antibiotic stress and killing by the mouse-derived macrophage cell line J774. Using a murine model of foreign body-associated peritonitis, we demonstrated that the ability of the Δspx strain to colonize the peritoneum and disseminate in the bloodstream was significantly reduced compared to that of the parent strain. Transcriptional analysis revealed that a large number of known oxidative stress genes are under positive control by Spx. Collectively, our results show that Spx is a major stress gene regulator and is implicated in the pathophysiology of E. faecalis. The relationship of Spx to other oxidative stress regulators is also discussed. PMID:22508863

  15. Enterococcus faecalis reconfigures its gene regulatory network activation under copper exposure

    PubMed Central

    Latorre, Mauricio; Galloway-Peña, Jessica; Roh, Jung Hyeob; Budinich, Marko; Reyes-Jara, Angélica; Murray, Barbara E.; Maass, Alejandro; González, Mauricio

    2014-01-01

    A gene regulatory network was generated in the bacterium Enterococcus faecalis in order to understand how this organism can activate its expression under different copper concentrations. The topological evaluation of the network showed common patterns described in other organisms. Integrating microarray experiments allowed the identification of sub-networks activated under low (0.05 mM CuSO4) and high (0.5 mM CuSO4) copper concentrations. The analysis indicates the presence of specific functionally activated modules induced by copper, highlighting the regulons LysR, ArgR as global regulators and CopY, Fur and LexA as local regulators. Taking advantage of the fact that E. faecalis presented a homeostatic module isolated, we produced an in vivo intervention removing this system from the cell without affecting the connectivity of the global transcriptional network. This strategy led us to find that this bacterium can reconfigure its gene expression to maintain cellular homeostasis, activating new modules principally related to glucose metabolism and transcriptional processes. Finally, these results position E. faecalis as the organism having the most complete and controllable systemic model of copper homeostasis available to date. PMID:24382465

  16. Pheromone killing of multidrug-resistant Enterococcus faecalis V583 by native commensal strains

    PubMed Central

    Gilmore, Michael S.; Rauch, Marcus; Ramsey, Matthew M.; Himes, Paul R.; Varahan, Sriram; Manson, Janet M.; Lebreton, Francois; Hancock, Lynn Ernest

    2015-01-01

    Multidrug-resistant Enterococcus faecalis possess numerous mobile elements that encode virulence and antibiotic resistance traits as well as new metabolic pathways, often constituting over one-quarter of the genome. It was of interest to determine how this large accretion of mobile elements affects competitive growth in the gastrointestinal (GI) tract consortium. We unexpectedly observed that the prototype clinical isolate strain V583 was actively killed by GI tract flora, whereas commensal enterococci flourished. It was found that killing of V583 resulted from lethal cross-talk between accumulated mobile elements and that this cross-talk was induced by a heptapeptide pheromone produced by native E. faecalis present in the fecal consortium. These results highlight two important aspects of the evolution of multidrug-resistant enterococci: (i) the accretion of mobile elements in E. faecalis V583 renders it incompatible with commensal strains, and (ii) because of this incompatibility, multidrug-resistant strains sharing features found in V583 cannot coexist with commensal strains. The accumulation of mobile elements in hospital isolates of enterococci can include those that are inherently incompatible with native flora, highlighting the importance of maintaining commensal populations as means of preventing colonization and subsequent infection by multidrug-resistant strains. PMID:26039987

  17. Transcriptome profiling of TDC cluster deletion mutant of Enterococcus faecalis V583.

    PubMed

    Perez, Marta; Ladero, Victor; Del Rio, Beatriz; Redruello, Begoña; de Jong, Anne; Kuipers, Oscar P; Kok, Jan; Martin, M Cruz; Fernandez, Maria; Alvarez, Miguel A

    2016-09-01

    The species Enterococcus faecalis is able to catabolise the amino acid tyrosine into the biogenic amine tyramine by the tyrosine decarboxilase (TDC) pathway Ladero et al. (2012) [1]. The TDC cluster comprises four genes: tyrS, an aminoacyl-tRNA synthetase-like gene; tdcA, which encodes the tyrosine decarboxylase; tyrP, a tyrosine/tyramine exchanger gene and nhaC-2, which encodes an Na(+)/H(+) antiporter and whose role in the tyramine biosynthesis remains unknown [2]. In E. faecalis V583 the last three genes are co-transcribed as a single polycistronic mRNA forming the catabolic operon, while tyrS is transcribed independently of the catabolic genes as a monocistronic mRNA [2]. The catabolic operon is transcriptionally induced by tyrosine and acidic pH. On the opposite, the tyrS expression is repressed by tyrosine concentrations [2]. In this work we report the transcriptional profiling of the TDC cluster deletion mutant (E. faecalis V583 ΔTDC) [2] compared to the wild-type strain, both grown in M17 medium supplemented with tyrosine. The transcriptional profile data of TDC cluster-regulated genes were deposited in the Gene Expression Omnibus (GEO) database under accession no. GSE77864.

  18. Inhibition of initial adhesion of uropathogenic Enterococcus faecalis by biosurfactants from Lactobacillus isolates.

    PubMed Central

    Velraeds, M M; van der Mei, H C; Reid, G; Busscher, H J

    1996-01-01

    In this study, 15 Lactobacillus isolates were found to produce biosurfactants in the mid-exponential and stationary growth phases. The stationary-phase biosurfactants from lactobacillus casei subsp. rhamnosus 36 and ATCC 7469, Lactobacillus fermentum B54, and Lactobacillus acidophilus RC14 were investigated further to determine their capacity to inhibit the initial adhesion of uropathogenic Enterococcus faecalis 1131 to glass in a parallel-plate flow chamber. The initial deposition rate of E. faecalis to glass with an adsorbed biosurfactant layer from L. acidophilus RC14 or L. fermentum B54 was significantly decreased by approximately 70%, while the number of adhering enterococci after 4 h of adhesion was reduced by an average of 77%. The surface activity of the biosurfactants and their activity inhibiting the initial adhesion of E. faecalis 1131 were retained after dialysis (molecular weight cutoff, 6,000 to 8,000) and freeze-drying. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy revealed that the freeze-dried biosurfactants from L. acidophilus RC14 and L. fermentum B54 were richest in protein, while those from L. casei subsp. rhamnosus 36 and ATCC 7469 had relatively high polysaccharide and phosphate contents. PMID:8787394

  19. Bactericidal effect of plasma jet with helium flowing through 3% hydrogen peroxide against Enterococcus faecalis.

    PubMed

    Zhou, Xin-Cai; Li, Yu-Lan; Liu, De-Xi; Cao, Ying-Guang; Lu, Xin-Pei

    2016-11-01

    The aim of the present study was to assess the antimicrobial activity of plasma jet with helium (He) flowing through 3% hydrogen peroxide in root canals infected with Enterococcus faecalis. A total of 42 single-rooted anterior teeth were prepared, sterilized, inoculated with an E. faecalis suspension and incubated for 7 days. Next, the teeth were randomly divided into six experimental groups (including groups treated by plasma jet with or without He for different time durations) and one control group treated without plasma. The number of surviving bacteria in each canal was determined by counting the colony forming units (CFU)/ml on nutrient agar plates. The results indicated that statistically significant reduction in CFU/ml (P<0.005) existed for all treatment groups relative to the control group. The greatest reductions in CFU/ml were observed for Group 3 (7.027 log unit reduction) and Group 2 (6.237 log unit reduction), which were treated by plasma jet sterilization with He flowing through 3% hydrogen peroxide for 4 min or for 2 min, respectively. In addition, the reduction in Group 3 was significantly greater compared with that in Group 2 or in the groups treated by plasma jet sterilization without He flowing through 3% hydrogen peroxide for 1, 2 or 4 min. In conclusion, plasma jet with or without He flowing through 3% hydrogen peroxide can effectively sterilized root canals infected with E. faecalis and should be considered as an alternative method for root canal disinfection in endodontic treatments.

  20. Genetic Diversity and Antibiotic Resistance of Enterococcus faecalis Isolates from Traditional Korean Fermented Soybean Foods.

    PubMed

    Lee, Jong-Hoon; Shin, Donghun; Lee, Bitnara; Lee, Inhyung; Jeong, Do-Won

    2017-02-24

    Eighty-five Enterococcus faecalis isolates collected from animals (40 isolates), Meju (Korean fermented soybean product; 27 isolates), humans (10 isolates), and various environmental samples (eight isolates) were subjected to multilocus sequence typing (MLST) to identify genetic differences between samples of different origins. MLST analysis resulted in 44 sequence types (STs), and the eBURST algorithm clustered the STs into 21 clonal complexes (CCs) and 17 singletons. The predominant STs, ST695 (21.1%, 18/85) and ST694 (9.4%, 8/85), were singletons, and only contained isolates originating from Meju. None of the STs in the current study belonged to CC2 or CC9, which comprise clinical isolates with high levels of antibiotic resistance. The E. faecalis isolates showed the highest rates of resistance to tetracycline (32.9%), followed by erythromycin (9.4%) and vancomycin (2.4%). All isolates from Meju were sensitive to these three antibiotics. Hence, MLST uncovered genetic diversity within E. faecalis, and clustering of the STs using eBURST revealed a correlation between the genotypes and origins of the isolates.

  1. Enterococcus faecalis zinc-responsive proteins mediate bacterial defence against zinc overload, lysozyme and oxidative stress.

    PubMed

    Abrantes, Marta C; Kok, Jan; Silva Lopes, Maria de Fátima

    2014-12-01

    Two Enterococcus faecalis genes encoding the P-type ATPase EF1400 and the putative SapB protein EF0759 were previously shown to be strongly upregulated in the presence of high concentrations of zinc. In the present work, we showed that a Zn(2+)-responsive DNA-binding motif (zim) is present in the promoter regions of these genes. Both proteins were further studied with respect to their involvement in zinc homeostasis and invasion of the host. EF0759 contributed to intramacrophage survival by an as-yet unknown mechanism(s). EF1400, here renamed ZntAEf, is an ATPase with specificity for zinc and plays a role in dealing with several host defences, i.e. zinc overload, oxidative stress and lysozyme; it provides E. faecalis cells with the ability to survive inside macrophages. As these three host defence mechanisms are important at several sites in the host, i.e. inside macrophages and in saliva, this work suggested that ZntAEf constitutes a crucial E. faecalis defence mechanism that is likely to contribute to the ability of this bacterium to endure life inside its host.

  2. Involvement of PhoP-PhoS homologs in Enterococcus faecalis virulence.

    PubMed

    Teng, Fang; Wang, Ling; Singh, Kavindra V; Murray, Barbara E; Weinstock, George M

    2002-04-01

    Eleven PhoP-PhoS homolog pairs were identified by searching the Enterococcus faecalis V583 genome sequence database at The Institute for Genomic Research with the Bacillus subtilis PhoP-PhoS sequences. Each pair appears to be a potential two-component system composed of a response regulator and a sensor kinase. Seven of the homologs were disrupted in E. faecalis strain OG1RF. TX10293, a mutant disrupted in one of these genes (etaR, the first gene of the gene pair designated etaRS), showed delayed killing and a higher 50% lethal dose in a mouse peritonitis model. The predicted EtaR protein sequence showed greatest similarity to LisR of Listeria monocytogenes (77%) and CsrR of Streptococcus pyogenes (70%); EtaS is 53% similar to LisK and 54% similar to CsrS. When grown in vitro, the TX10293 mutant was more sensitive to low pH (pH 3.4) and more resistant to high temperature (55 degrees C) than wild-type OG1RF. In conclusion, many potential two-component systems are identified for E. faecalis, one of which, EtaRS, was shown to be involved in stress response and virulence.

  3. Genome-based characterization of hospital-adapted Enterococcus faecalis lineages

    PubMed Central

    Raven, Kathy E.; Reuter, Sandra; Gouliouris, Theodore; Reynolds, Rosy; Russell, Julie E.; Brown, Nicholas M.; Török, M. Estée; Parkhill, Julian; Peacock, Sharon J.

    2016-01-01

    Vancomycin-resistant Enterococcus faecalis (VREfs) is an important nosocomial pathogen1,2. We undertook whole genome sequencing of E. faecalis associated with bloodstream infection in the UK and Ireland over more than a decade to determine the population structure and genetic associations with hospital adaptation. Three lineages predominated in the population, two of which (L1 and L2) were nationally distributed, and one (L3) geographically restricted. Genome comparison with a global collection identified that L1 and L3 were also present in the USA, but were genetically distinct. Over 90% of VREfs belonged to L1–L3, with resistance acquired and lost multiple times in L1 and L2, but only once followed by clonal expansion in L3. Putative virulence and antibiotic resistance genes were over-represented in L1, L2 and L3 isolates combined, versus the remainder. Each of the three main lineages contained a mixture of vancomycin-resistant and -susceptible E. faecalis (VSEfs), which has important implications for infection control and antibiotic stewardship. PMID:27213049

  4. Pheromone killing of multidrug-resistant Enterococcus faecalis V583 by native commensal strains.

    PubMed

    Gilmore, Michael S; Rauch, Marcus; Ramsey, Matthew M; Himes, Paul R; Varahan, Sriram; Manson, Janet M; Lebreton, Francois; Hancock, Lynn Ernest

    2015-06-09

    Multidrug-resistant Enterococcus faecalis possess numerous mobile elements that encode virulence and antibiotic resistance traits as well as new metabolic pathways, often constituting over one-quarter of the genome. It was of interest to determine how this large accretion of mobile elements affects competitive growth in the gastrointestinal (GI) tract consortium. We unexpectedly observed that the prototype clinical isolate strain V583 was actively killed by GI tract flora, whereas commensal enterococci flourished. It was found that killing of V583 resulted from lethal cross-talk between accumulated mobile elements and that this cross-talk was induced by a heptapeptide pheromone produced by native E. faecalis present in the fecal consortium. These results highlight two important aspects of the evolution of multidrug-resistant enterococci: (i) the accretion of mobile elements in E. faecalis V583 renders it incompatible with commensal strains, and (ii) because of this incompatibility, multidrug-resistant strains sharing features found in V583 cannot coexist with commensal strains. The accumulation of mobile elements in hospital isolates of enterococci can include those that are inherently incompatible with native flora, highlighting the importance of maintaining commensal populations as means of preventing colonization and subsequent infection by multidrug-resistant strains.

  5. Adherence and intracellular survival within human macrophages of Enterococcus faecalis isolates from coastal marine sediment.

    PubMed

    Sabatino, Raffaella; Di Cesare, Andrea; Pasquaroli, Sonia; Vignaroli, Carla; Citterio, Barbara; Amiri, Mehdi; Rossi, Luigia; Magnani, Mauro; Mauro, Alessandro; Biavasco, Francesca

    2015-09-01

    Enterococcus faecalis is part of the human intestinal microbiota and an important nosocomial pathogen. It can be found in the marine environment, where it is also employed as a fecal indicator. To assess the pathogenic potential of marine E. faecalis, four strains isolated from marine sediment were analyzed for their ability to survive in human macrophages. Escherichia coli DH5α was used as a negative control. The number of adherent and intracellular bacteria was determined 2.5 h after the infection (T0) and after further 24h (T24) by CFU and qPCR counts. At T24 adherent and intracellular enterococcal CFU counts were increased for all strains, the increment in intracellular bacteria being particularly marked. No CFU of E. coli DH5α were detected. In contrast, qPCR counts of intracellular enterococcal and E. coli bacteria were similar at both time points. These findings suggest that whereas E. coli was killed within macrophages (no CFU, positive qPCR), the E. faecalis isolates not only escaped killing, but actually multiplied, as demonstrated by the increase in the viable cell population. These findings support earlier data by our group, further documenting that marine sediment can be a reservoir of pathogenic enterococci.

  6. Comparison of the bile salts and sodium dodecyl sulfate stress responses in Enterococcus faecalis.

    PubMed Central

    Flahaut, S; Frere, J; Boutibonnes, P; Auffray, Y

    1996-01-01

    The resistance to detergents and detergent-induced tolerance of a gastrointestinal organism, Enterococcus faecalis ATCC 19433, were examined. The most remarkable observation was the rapid response of cells in contact with bile salts and sodium dodecyl sulfate (SDS). The killing by high concentrations of detergents was nearly instantaneous. A 5-s adaptation with moderate sublethal concentrations of bile salts or SDS (0.08 or 0.01%, respectively) was sufficient to induce significant adaptation against homologous lethal conditions (0.3% bile salts or 0.017% SDS). However, resistance to a subsequent lethal challenge progressively increased further to a maximum reached after 30 min of adaptation. Furthermore, extremely strong cross-resistances were observed with bile salts- and SDS-adapted cells. However, no relationship seems to exist between levels of tolerance and de novo-synthesized proteins, since blockage of protein synthesis during adaptation had no effect on induction of resistance to bile salts and SDS. We conclude that this induced tolerance to detergent stress is independent of protein synthesis. Nevertheless, the stress-induced protein patterns of E. faecalis ATCC 19433 showed significant modifications. The rates of synthesis of 45 and 34 proteins were enhanced after treatments with bile salts and SDS, respectively. In spite of the overlap of 12 polypeptides, the protein profiles induced by the two detergents were different, suggesting that these detergents trigger different responses in E. faecalis. Therefore, bile salts cannot be substituted for SDS in biochemical detergent shock experiments with bacteria. PMID:8779581

  7. Implication of hypR in the virulence and oxidative stress response of Enterococcus faecalis.

    PubMed

    Verneuil, Nicolas; Rincé, Alain; Sanguinetti, Maurizio; Auffray, Yanick; Hartke, Axel; Giard, Jean-Christophe

    2005-11-01

    HypR has recently been described as the first transcriptional regulator involved in the oxidative stress response and in the intracellular survival of Enterococcus faecalis within macrophages. In order to characterize the HypR regulon, real-time quantitative RT-PCR experiments were performed. The expression of four genes involved in the oxidative stress response encoding catalase, glutathione reductase, and the two subunits of alkyl hydroperoxide reductase were down regulated in the hypR background under H(2)O(2) condition. These findings show that HypR acts as a transcriptional activator, especially during oxidative stress. In addition, DNAse I footprinting assays allowed us to identify the HypR-protected DNA regions corresponding to the "HypR box" in the hypR promoter. Moreover, the effect of the hypR mutation on the virulence of E. faecalis was evaluated in comparison with the wild-type JH2-2 strain using a mouse peritonitis model. Our results revealed that HypR appears to be an important virulence factor in E. faecalis.

  8. Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin

    PubMed Central

    Comerlato, Carolina Baldisserotto; de Resende, Mariah Costa Carvalho; Caierão, Juliana; d'Azevedo, Pedro Alves

    2013-01-01

    Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution. PMID:23903974

  9. Antimicrobial activity of tetraacetylethylenediamine-sodium perborate versus sodium hypochlorite against Enterococcus faecalis

    PubMed Central

    Shakouie, Sahar; Salem Milani, Amin; Eskandarnejad, Mahsa; Rahimi, Saeed; Froughreyhani, Mohammad; Galedar, Saeede; Ranjbar, Ehsan

    2016-01-01

    Background. This study evaluated the antimicrobial activity of Tetraacetylethylenediamine-sodium perborate (TAED-SP) in comparison to 2.5% and 5% sodium hypochlorite (NaOCl) against Enterococcus faecalis. Methods. A standard suspension of E. faecalis was inoculated on 60 plates containing Mueller-Hinton agar culture medium. Four sterile disks of Beckman filtration paper were placed on each plate. TAED-SP, 5% and 2.5% NaOCl were placed on three disks. Sterile physiologic saline was placed on the fourth disk as negative control. After 24-hour incubation, the diameter of the inhibition zone around the disks was measured using a transparent ruler. One-way Analysis of Variance (ANOVA) was used to compare the mean zone of microbial growth in the groups. P-values less than 0.05 were considered statistically significant. Results. There was a significant difference in the diameter of the inhibition zones between groups (P < 0.05). The Tukey post hoc test showed a higher diameter of the inhibitory zone with TAED-SP than that of 2.5% NaOCl. However, there were no significant differences between the inhibitory zones of TAED-SP and 5% NaOCl. Conclusion. TAED-SP and 5% NaOCl have similar antibacterial activity against E. faecalis; however, TAED-SP has a greater antibacterial effect compared to 2.5% NaOCl. PMID:27092214

  10. Crystal structure of an aerobic FMN-dependent azoreductase (AzoA) from Enterococcus faecalis.

    PubMed

    Liu, Zhi-Jie; Chen, Huizhong; Shaw, Neil; Hopper, Sherryll L; Chen, Lirong; Chen, Siwei; Cerniglia, Carl E; Wang, Bi-Cheng

    2007-07-01

    The initial critical step of reduction of the azo bond during the metabolism of azo dyes is catalyzed by a group of NAD(P)H dependant enzymes called azoreductases. Although several azoreductases have been identified from microorganisms and partially characterized, very little is known about the structural basis for substrate specificity and the nature of catalysis. Enterococcus faecalis azoreductase A (AzoA) is a highly active azoreductase with a broad spectrum of substrate specificity and is capable of degrading a wide variety of azo dyes. Here, we report the crystal structure of the AzoA from E. faecalis determined at 2.07 A resolution with bound FMN ligand. Phases were obtained by single wavelength anomalous scattering of selenomethionine labeled protein crystals. The asymmetric unit consisted of two dimers with one FMN molecule bound to each monomer. The AzoA monomer takes a typical NAD(P)-binding Rossmann fold with a highly conserved FMN binding pocket. A salt bridge between Arg18 and Asp184 restricts the size of the flavin binding pocket such that only FMN can bind. A putative NADH binding site could be identified and a plausible mechanism for substrate reduction is proposed. Expression studies revealed azoA gene to be expressed constitutively in E. faecalis.

  11. Optimization of Thixoforging Parameters for C70S6 Steel Connecting Rods

    NASA Astrophysics Data System (ADS)

    Özkara, İsa Metin; Baydoğan, Murat

    2016-11-01

    A microalloyed steel, C70S6, with a solidification interval of 1390-1479 °C, was thixoforged in the semisolid state in a closed die at temperatures in the range 1400-1475 °C to form a 1/7 scaled-down model of a passenger vehicle connecting rod. Die design and an optimized thixoforging temperature eliminated the excessive flash and other problems during forging. Tension test samples from connecting rods thixoforged at the optimum temperature of 1440 °C exhibited nearly the same hardness, yield strength, and ultimate tensile strength as conventional hot forged samples but ductility decreased by about 45% due to grain boundary ferrite network formed during cooling from the thixoforging temperature. Thus, C70S6-grade steel can be thixoforged at 1440 °C to form flash-free connecting rods. This conclusion was also validated using FEA analysis.

  12. Magnetic Resonance in the Chiral Helimagnet CrNb3S6

    NASA Astrophysics Data System (ADS)

    Yoshizawa, Daichi; Kishine, Jun-ichiro; Kousaka, Yusuke; Togawa, Yoshihiko; Mito, Masaki; Akimitsu, Jun; Inoue, Katsuya; Hagiwara, Masayuki

    Recently, magnetic substances with chirality, namely the handedness of the magnetic structure, have attracted considerable attention because of the anomalous phenomena which appear in magnetic fields. CrNb3S6 is one of the chiral magnets formed by exchange and Dzyaloshinsky-Moriya (DM) interactions. Electron spin resonance (ESR) measurements of CrNb3S6 in magnetic fields parallel to the c-axis (helical axis) have been performed to evaluate the exchange and the DM constants that determine the helical structure. Fitting the ESR data to a calculated mode based on a spin wave theory yields values for the ferromagnetic inter-plane exchange constant J/kB = 16.2 K, the DM constant D/kB = 1.29 K, and the single-ion anisotropy constant

  13. Temperature retrieval error in Rayleigh-Brillouin scattering using Tenti's S6 kinetic model

    NASA Astrophysics Data System (ADS)

    Wu, Lei; Reese, Jason M.; Zhang, Yonghao

    2016-11-01

    The Rayleigh-Brillouin spectrum of light scattered by gas density fluctuations contains information about the gas temperature. This information can be retrieved by comparing experimentally measured spectra with theoretical line shapes determined from the linearized Boltzmann equation. However, the linearized Boltzmann equation is difficult to solve so Tenti's S6 kinetic model has been widely used for several decades because of its simplicity. In this paper, the linearized Boltzmann equation is solved by the efficient fast spectral method, and the temperature retrieval error associated with the Tenti's S6 model is systematically investigated for both spontaneous and coherent Rayleigh-Brillouin scattering, for different gas rarefaction and intermolecular potentials. Our results indicate useful calibrations for laser technologies that use Rayleigh-Brillouin scattering to profile for gas temperatures with high accuracy.

  14. Sucralose activates an ERK1/2-ribosomal protein S6 signaling axis.

    PubMed

    Guerra, Marcy L; Kalwat, Michael A; McGlynn, Kathleen; Cobb, Melanie H

    2017-02-01

    The sweetener sucralose can signal through its GPCR receptor to induce insulin secretion from pancreatic β cells, but the downstream signaling pathways involved are not well-understood. Here we measure responses to sucralose, glucagon-like peptide 1, and amino acids in MIN6 β cells. Our data suggest a signaling axis, whereby sucralose induces calcium and cAMP, activation of ERK1/2, and site-specific phosphorylation of ribosomal protein S6. Interestingly, sucralose acted independently of mTORC1 or ribosomal S6 kinase (RSK). These results suggest that sweeteners like sucralose can influence β-cell responses to secretagogues like glucose through metabolic as well as GPCR-mediated pathways. Future investigation of novel sweet taste receptor signaling pathways in β cells will have implications for diabetes and other emergent fields involving these receptors.

  15. Room-Temperature Synthesis of Thiostannates from {[Ni(tren)]2[Sn2S6]}n.

    PubMed

    Hilbert, Jessica; Näther, Christian; Weihrich, Richard; Bensch, Wolfgang

    2016-08-15

    The compound {[Ni(tren)]2[Sn2S6]}n (1) (tren = tris(2-aminoethyl)amine, C6H18N4) was successfully applied as source for the room-temperature synthesis of the new thiostannates [Ni(tren)(ma)(H2O)]2[Sn2S6]·4H2O (2) (ma = methylamine, CH5N) and [Ni(tren)(1,2-dap)]2[Sn2S6]·2H2O (3) (1,2-dap = 1,2-diaminopropane, C3H10N2). The Ni-S bonds in the Ni2S2N8 bioctahedron in the structure of 1 are analyzed with density functional theory calculations demonstrating significantly differing Ni-S bond strengths. Because of this asymmetry they are easily broken in the presence of an excess of ma or 1,2-dap immediately followed by Ni-N bond formation to N donor atoms of the amine ligands thus generating [Ni(tren)(amine)](2+) complexes. The chemical reactions are fast, and compounds 2 and 3 are formed within 1 h. The synthesis concept presented here opens hitherto unknown possibilities for preparation of new thiostannates.

  16. High in Vitro Anti-Tumor Efficacy of Dimeric Rituximab/Saporin-S6 Immunotoxin

    PubMed Central

    Bortolotti, Massimo; Bolognesi, Andrea; Battelli, Maria Giulia; Polito, Letizia

    2016-01-01

    The anti-CD20 mAb Rituximab has revolutionized lymphoma therapy, in spite of a number of unresponsive or relapsing patients. Immunotoxins, consisting of toxins coupled to antibodies, are being investigated for their potential ability to augment Rituximab efficacy. Here, we compare the anti-tumor effect of high- and low-molecular-weight Rituximab/saporin-S6 immunotoxins, named HMW-IT and LMW-IT, respectively. Saporin-S6 is a potent and stable plant enzyme belonging to ribosome-inactivating proteins that causes protein synthesis arrest and consequent cell death. Saporin-S6 was conjugated to Rituximab through an artificial disulfide bond. The inhibitory activity of HMW-IT and LMW-IT was evaluated on cell-free protein synthesis and in two CD20+ lymphoma cell lines, Raji and D430B. Two different conjugates were separated on the basis of their molecular weight and further characterized. Both HMW-IT (dimeric) and LMW-IT (monomeric) maintained a high level of enzymatic activity in a cell-free system. HMW-IT, thanks to a higher toxin payload and more efficient antigen capping, showed stronger in vitro anti-tumor efficacy than LMW-IT against lymphoma cells. Dimeric HMW-IT can be used for lymphoma therapy at least for ex vivo treatments. The possibility of using HMW-IT augments the yield in immunotoxin preparation and allows the targeting of antigens with low internalization rates. PMID:27338475

  17. Final report of APMP.QM-S6: clenbuterol in porcine meat

    NASA Astrophysics Data System (ADS)

    Sin, D. W.-M.; Ho, C.; Yip, Y.-C.

    2016-01-01

    At the CCQM Organic Analysis Working Group (OAWG) Meeting held in April 2012 and the APMP TCQM Meeting held in November 2012, an APMP supplementary comparison (APMP.QM-S6) on the determination of clenbuterol in porcine meat was supported by the OAWG and APMP TCQM. This comparison was organized by the Government Laboratory, Hong Kong. In order to accommodate a wider participation, a pilot study (APMP.QM-P22) was run in parallel to APMP.QM-S6. This study provided the means for assessing the measurement capabilities for determination of low-polarity measurands in a procedure that requires extraction, clean-up, analytical separation, and selective detection in a food matrix. A total of 7 institutes registered for the supplementary comparison and 6 of them submitted their results. 4 results were included for SCRV calculation. All participating laboratories applied Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry (ID-LCMS/MS) technique with clenbuterol-d9 as internal standard spiked for quantitation in this programme. KEY WORDS FOR SEARCH APMP.QM-S6 and Clenbuterol Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).

  18. Serodiversity of Opsonic Antibodies against Enterococcus faecalis —Glycans of the Cell Wall Revisited

    PubMed Central

    Theilacker, Christian; Kaczyński, Zbigniew; Kropec, Andrea; Sava, Irina; Ye, Libin; Bychowska, Anna; Holst, Otto; Huebner, Johannes

    2011-01-01

    In a typing system based on opsonic antibodies against carbohydrate antigens of the cell envelope, 60% of Enterococcus faecalis strains can be assigned to one of four serotypes (CPS-A to CPS-D). The structural basis for enterococcal serotypes, however, is still incompletely understood. Here we demonstrate that antibodies raised against lipoteichoic acid (LTA) from a CPS-A strain are opsonic to both CPS-A and CPS-B strains. LTA-specific antibodies also bind to LTA of CPS-C and CPS-D strains, but fail to opsonize them. From CPS-C and CPS-D strains resistant to opsonization by anti-LTA, we purified a novel diheteroglycan with a repeating unit of →6)-β-Galf-(1→3)- β-D-Glcp-(1→ with O-acetylation in position 5 and lactic acid substitution at position 3 of the Galf residue. The purified diheteroglycan, but not LTA absorbed opsonic antibodies from whole cell antiserum against E. faecalis type 2 (a CPS-C strain) and type 5 (CPS-D). Rabbit antiserum raised against purified diheteroglycan opsonized CPS-C and CPS-D strains and passive protection with diheteroglycan-specific antiserum reduced bacterial counts by 1.4 – 3.4 logs in mice infected with E. faecalis strains of the CPS-C and CPS-D serotype. Diheteroglycan-specific opsonic antibodies were absorbed by whole bacterial cells of E. faecalis FA2-2 (CPS-C) but not by its isogenic acapsular cpsI-mutant and on native PAGE purified diheteroglycan co-migrated with the gene product of the cps-locus, suggesting that it is synthesized by this locus. In summary, two polysaccharide antigens, LTA and a novel diheteroglycan, are targets of opsonic antibodies against typeable E. faecalis strains. These cell-wall associated polymers are promising candidates for active and passive vaccination and add to our armamentarium to fight this important nosocomial pathogen. PMID:21437253

  19. Transcriptome Analysis of Enterococcus faecalis during Mammalian Infection Shows Cells Undergo Adaptation and Exist in a Stringent Response State

    PubMed Central

    Frank, Kristi L.; Colomer-Winter, Cristina; Grindle, Suzanne M.; Lemos, José A.; Schlievert, Patrick M.; Dunny, Gary M.

    2014-01-01

    As both a commensal and a major cause of healthcare-associated infections in humans, Enterococcus faecalis is a remarkably adaptable organism. We investigated how E. faecalis adapts in a mammalian host as a pathogen by characterizing changes in the transcriptome during infection in a rabbit model of subdermal abscess formation using transcriptional microarrays. The microarray experiments detected 222 and 291 differentially regulated genes in E. faecalis OG1RF at two and eight hours after subdermal chamber inoculation, respectively. The profile of significantly regulated genes at two hours post-inoculation included genes involved in stress response, metabolism, nutrient acquisition, and cell surface components, suggesting genome-wide adaptation to growth in an altered environment. At eight hours post-inoculation, 88% of the differentially expressed genes were down-regulated and matched a transcriptional profile consistent with a (p)ppGpp-mediated stringent response. Subsequent subdermal abscess infections with E. faecalis mutants lacking the (p)ppGpp synthetase/hydrolase RSH, the small synthetase RelQ, or both enzymes, suggest that intracellular (p)ppGpp levels, but not stringent response activation, influence persistence in the model. The ability of cells to synthesize (p)ppGpp was also found to be important for growth in human serum and whole blood. The data presented in this report provide the first genome-wide insights on E. faecalis in vivo gene expression and regulation measured by transcriptional profiling during infection in a mammalian host and show that (p)ppGpp levels affect viability of E. faecalis in multiple conditions relevant to mammalian infection. The subdermal abscess model can serve as a novel experimental system for studying the E. faecalis stringent response in the context of the mammalian immune system. PMID:25545155

  20. Ribosomal protein S6 kinase1 coordinates with TOR-Raptor2 to regulate thylakoid membrane biosynthesis in rice.

    PubMed

    Sun, Linxiao; Yu, Yonghua; Hu, Weiqin; Min, Qiming; Kang, Huiling; Li, Yilu; Hong, Yue; Wang, Xuemin; Hong, Yueyun

    2016-07-01

    Ribosomal protein S6 kinase (S6K) functions as a key component in the target of rapamycin (TOR) pathway involved in multiple processes in eukaryotes. The role and regulation of TOR-S6K in lipid metabolism remained unknown in plants. Here we provide genetic and pharmacological evidence that TOR-Raptor2-S6K1 is important for thylakoid galactolipid biosynthesis and thylakoid grana modeling in rice (Oryza sativa L.). Genetic suppression of S6K1 caused pale yellow-green leaves, defective thylakoid grana architecture. S6K1 directly interacts with Raptor2, a core component in TOR signaling, and S6K1 activity is regulated by Raptor2 and TOR. Plants with suppressed Raptor2 expression or reduced TOR activity by inhibitors mimicked the S6K1-deficient phenotype. A significant reduction in galactolipid content was found in the s6k1, raptor2 mutant or TOR-inhibited plants, which was accompanied by decreased transcript levels of the set of genes such as lipid phosphate phosphatase α5 (LPPα5), MGDG synthase 1 (MGD1), and DGDG synthase 1 (DGD1) involved in galactolipid synthesis, compared to the control plants. Moreover, loss of LPPα5 exhibited a similar phenotype with pale yellow-green leaves. These results suggest that TOR-Raptor2-S6K1 is important for modulating thylakoid membrane lipid biosynthesis, homeostasis, thus enhancing thylakoid grana architecture and normal photosynthesis ability in rice.

  1. Attenuation of ribosomal protein S6 phosphatase activity in chicken embryo fibroblasts transformed by Rous sarcoma virus.

    PubMed Central

    Belandia, B; Brautigan, D; Martín-Pérez, J

    1994-01-01

    In chicken embryo fibroblasts, phosphorylation of the 40S ribosomal protein S6 increases during G1 but returns to basal level by mitosis. In contrast, in Rous sarcoma virus (RSV)-transformed fibroblasts, S6 remains highly phosphorylated throughout mitosis. This study investigated the mechanism by which RSV alters the pattern of S6 phosphorylation. Pulse-chase experiments demonstrate that phosphate turnover in S6 is rapid in normal cells and in cells infected with an RSV transformation-defective virus. In contrast, phosphate turnover in S6 is severely reduced in cells infected with temperature-sensitive RSV at a temperature permissive for transformation, indicating a diminished S6 phosphatase activity. Fractionation of cell lysates by DEAE chromatography showed an almost threefold lower S6 phosphatase activity in RSV-transformed versus normal cells. The S6 phosphatase was sensitive to inhibitor 2 and specifically recognized by an antibody to type 1 phosphatase (PP1). The S6 phosphatase activity recovered by immunoprecipitation of PP1 was threefold lower in transformed cells, but the steady-state level of expression and the rate of synthesis of PP1 were not altered by oncogenic transformation. Together, the results show that transformation by RSV reduced the S6-PP1 activity. Images PMID:8264587

  2. Replacement of tyrosine 181 by phenylalanine in gentisate 1,2-dioxygenase I from Pseudomonas alcaligenes NCIMB 9867 enhances catalytic activities.

    PubMed

    Tan, Chew Ling; Yeo, Chew Chieng; Khoo, Hoon Eng; Poh, Chit Laa

    2005-11-01

    xlnE, encoding gentisate 1,2-dioxygenase (EC 1.13.11.4), from Pseudomonas alcaligenes (P25X) was mutagenized by site-directed mutagenesis. The mutant enzyme, Y181F, demonstrated 4-, 3-, 6-, and 16-fold increases in relative activity towards gentisate and 3-fluoro-, 4-methyl-, and 3-methylgentisate, respectively. The specific mutation conferred a 13-fold higher catalytic efficiency (k(cat)/Km) on Y181F towards 3-methylgentisate than that of the wild-type enzyme.

  3. Acoustic and elastic properties of Sn(2)P(2)S(6) crystals.

    PubMed

    Mys, O; Martynyuk-Lototska, I; Grabar, A; Vlokh, R

    2009-07-01

    We present the results concerned with acoustic and elastic properties of Sn(2)P(2)S(6) crystals. The complete matrices of elastic stiffness and compliance coefficients are determined in both the crystallographic coordinate system and the system associated with eigenvectors of the elastic stiffness tensor. The acoustic slowness surfaces are constructed and the propagation and polarization directions of the slowest acoustic waves promising for acousto-optic interactions are determined on this basis. The acoustic obliquity angle and the deviation of polarization of the acoustic waves from purely transverse or longitudinal states are quantitatively analysed.

  4. Phenotypic and molecular antibiotic resistance profile of Enterococcus faecalis and Enterococcus faecium isolated from different traditional fermented foods.

    PubMed

    Sánchez Valenzuela, Antonio; Lavilla Lerma, Leyre; Benomar, Nabil; Gálvez, Antonio; Pérez Pulido, Rubén; Abriouel, Hikmate

    2013-02-01

    A collection of 55 enterococci (41 Enterococcus faecium and 14 E. faecalis strains) isolated from various traditional fermented foodstuffs of both animal and vegetable origins, and water was evaluated for resistance against 15 antibiotics. Lower incidence of resistance was observed with gentamicin, ampicillin, penicillin and teicoplanin. However, a high incidence of antibiotic resistance was detected for rifampicin (12 out of 14 of isolates), ciprofloxacin (9/14), and quinupristin/dalfopristin (8/14) in E. faecalis strains. Enterococcus faecium isolates were resistant to rifampicin (25/41), ciprofloxacin (23/41), erythromycin (18/41), levofloxacin (16/41), and nitrofurantoin (15/41). One Enterococcus faecalis and two E. faecium strains were resistant to vancomycin (MIC>16 μg/mL). Among 55 isolates, 27 (19 E. faecium and eight E. faecalis) were resistant to at least three antibiotics. High level of multidrug resistance to clinically important antibiotics was detected in E. faecalis strains (57% of E. faecalis versus 46% of E. faecium), which showed resistance to six to seven antibiotics, especially those isolated from foods of animal origin. So, it is necessary to re-evaluate the use of therapeutic antibiotics in stock farms at both regional and international levels due to the high number of multiple resistant (MR) bacteria. Fifty-six MR E. faecalis and E. faecium strains selected from this and previous studies (Valenzuela et al., 2008, 2010) were screened by polymerase chain reaction for antibiotic resistance genes, revealing the presence of tet(L), tet(M), ermB, cat, efrA, efrB, mphA, or msrA/B genes. The ABC Multidrug Efflux Pump EfrAB was detected in 96% of E. faecalis strains and also in 13% of E. faecium strains; this is the first report describing EfrAB in this enterococcal species. The efflux pump-associated msrA/B gene was detected in 66.66% of E. faecium strains, but not in E. faecalis strains.

  5. Optical Sensitizing of Photorefractive Sn2P2S6 With CW and Pulsed Pre-Exposure (Preprint)

    DTIC Science & Technology

    2015-06-16

    time delayed transient photoinduced absorption ( photo -chromism) [1], and transient photoinduced scattering (transient beam fanning) [2]. The second...smaller effect of sensitizing with considerably different lifetimes of secondary centers was observed in nominally undoped Sn2P2S6, tellurium doped ...Sn2P2S6:Te 1%, and co- doped Sn2P2S6:Sb:Te 0.5%, Sb 0.5% samples. Well below the saturation level, the light induced absorption increases linearly

  6. p70 ribosomal S6 kinase regulates subpleural fibrosis following transforming growth factor-α expression in the lung

    PubMed Central

    Madala, Satish K.; Thomas, George; Edukulla, Ramakrishna; Davidson, Cynthia; Schmidt, Stephanie; Schehr, Angelica

    2015-01-01

    The p70 ribosomal S6 kinase (S6K) is a downstream substrate that is phosphorylated and activated by the mammalian target of rapamycin complex and regulates multiple cellular processes associated with fibrogenesis. Recent studies demonstrate that aberrant mTORC1-S6K signaling contributes to various pathological conditions, but a direct role in pulmonary fibroproliferation has not been established. Increased phosphorylation of the S6K pathway is detected immediately following transforming growth factor-α (TGF-α) expression in a transgenic model of progressive lung fibrosis. To test the hypothesis that the S6K directly regulates pulmonary fibroproliferative disease we determined the cellular sites of S6K phosphorylation during the induction of fibrosis in the TGF-α model and tested the efficacy of specific pharmacological inhibition of the S6K pathway to prevent and reverse fibrotic disease. Following TGF-α expression increased phosphorylation of the S6K was detected in the airway and alveolar epithelium and the mesenchyme of advanced subpleural fibrotic regions. Specific inhibition of the S6K with the small molecule inhibitor LY-2584702 decreased TGF-α and platelet-derived growth factor-β-induced proliferation of lung fibroblasts in vitro. Administration of S6K inhibitors to TGF-α mice prevented the development of extensive subpleural fibrosis and alterations in lung mechanics, and attenuated the increase in total lung hydroxyproline. S6K inhibition after fibrosis was established attenuated the progression of subpleural fibrosis. Together these studies demonstrate targeting the S6K pathway selectively modifies the progression of pulmonary fibrosis in the subpleural compartment of the lung. PMID:26566903

  7. Anti-peptide antibodies for examining the conformation, molecular assembly and localization of an intracellular protein, ribosomal protein S6, in vivo.

    PubMed

    Nakagawa, Masatoshi; Ohmido, Nobuko; Ishikawa, Katsumi; Uchiyama, Susumu; Fukui, Kiichi; Azuma, Takachika

    2008-03-01

    Ribosomal protein S6 (rpS6) is known to relate to cell proliferation. Our recent proteome analysis of human metaphase chromosomes revealed the enrichment of rpS6 during mitosis. Here, structure, localization and molecular assembly in vitro and in vivo of a human rpS6, were examined using antibodies (Abs) prepared by immunizing rabbits with synthetic peptides. Five peptides, Ser6-Asp20 (S6-1), Ile52-Gly66 (S6-2), Asp103-Gly117 (S6-3), Asn146-Lys160 (S6-4) and Arg178-Ile192 (S6-5) were chosen as epitopes of human rpS6. These peptides except for S6-3 induced strong Ab production, and with an enzyme-linked immunosorbent assay, anti-S6-2, anti-S6-4 and anti-S6-5, showed high reactivity to recombinant rpS6 (r-rpS6), while anti-S6-1 did not, suggesting that S6-2, S6-4 and S6-5 were exposed on the r-rpS6 surface, while S6-1 was less exposed or possessed a different conformation. The immunostaining of HeLa cells as well as isolated chromosomes suggested that rpS6 occurs in endoplasmic reticulum (ER) but less possible on chromosomes since no Abs showed localization of rpS6 to chromosomes. In addition, the immunostaining suggested that only S6-4 is exposed on the protein surface, while S6-2 and S6-5 are buried by the interaction with other macromolecules in HeLa cells. Present our result shows new possibility of antibodies as tools for structure-oriented cell biology.

  8. Differences in Antibiotic Resistance Patterns of Enterococcus faecalis and Enterococcus faecium Strains Isolated from Farm and Pet Animals

    PubMed Central

    Butaye, Patrick; Devriese, Luc A.; Haesebrouck, Freddy

    2001-01-01

    The prevalence of acquired resistance in 146 Enterococcus faecium and 166 Enterococcus faecalis strains from farm and pet animals, isolated in 1998 and 1999 in Belgium, against antibiotics used for growth promotion and for therapy was determined. Acquired resistance against flavomycin and monensin, two antibiotics used solely for growth promotion, was not detected. Avoparcin (glycopeptide) resistance was found sporadically in E. faecium only. Avilamycin resistance was almost exclusively seen in strains from farm animals. Resistance rates were higher in E. faecium strains from broiler chickens than in strains from other animal groups with tylosin and virginiamycin and in E. faecalis as well as in E. faecium strains with narasin and bacitracin. Resistance against ampicillin was mainly found among E. faecium strains from pets and was absent in E. faecalis. Tetracycline resistance occurred most often in strains from farm animals, while enrofloxacin resistance, only found in E. faecalis, occurred equally among strains from all origins. Resistance against gentamicin was very rare in broiler strains, whereas resistance rates were high in strains from other origins. It can be concluded that resistance against antibiotics used solely for growth promotion was more prevalent in E. faecium strains than in E. faecalis strains. With few exceptions, resistance against the different categories of antibiotics was more prevalent in strains from farm animals than in those from pets. PMID:11302798

  9. Prevalence of Virulence Factors and Vancomycin-resistant Genes among Enterococcus faecalis and E. faecium Isolated from Clinical Specimens

    PubMed Central

    NASAJ, Mona; MOUSAVI, Seyed Masoud; HOSSEINI, Seyed Mostafa; ARABESTANI, Mohammad Reza

    2016-01-01

    Background: The aim of this study was to determine the occurrence of virulence determinants and vancomycin-resistant genes among Enterococcus faecalis and E. faecium obtained from various clinical sources. Methods: The study was performed on the 280 enterococcal isolated from clinical specimens in Hamadan hospitals, western Iran in 2012–14. Antibiotic susceptibility testing was performed using disk diffusion and Minimal Inhibitory Concentration (MIC) methods. The presence of vancomycin-resistant genes and virulence genes was investigated using PCR. Results: Totally 280 enterococcal isolates were identified as follows: E. faecalis (62.5%), E. faecium (24%) and Enterococcus spp (13.5%). The results of antibiotic susceptibility testing showed that resistance rates to vancomycin and teicoplanin in E. faecalis and E. faecium isolates were 5% and 73%, respectively. Of Sixty vancomycin-resistant Enterococci strains, fifty-one isolates were identified as E. faecium (VREfm) and nine as E. faecalis (VREfs). Prevalence of esp, hyl, and asa1 genes were determined as 82%, 71.6%, and 100%, respectively in E. faecium strains; and 78%, 56/6%, and 97%, respectively in E. faecalis strains. Conclusion: The increased frequency of VREF, as seen with rapid rise in the number of vanA isolates should be considered in infection control practices. PMID:27648425

  10. Effects of storage temperature on tyramine production by Enterococcus faecalis R612Z1 in water-boiled salted ducks.

    PubMed

    Liu, Fang; Du, Lihui; Wu, Haihong; Wang, Daoying; Zhu, Yongzhi; Geng, Zhiming; Zhang, Muhan; Xu, Weimin

    2014-10-01

    Tyramine production by Enterococcus faecalis R612Z1 in water-boiled salted ducks was evaluated during storage at different temperatures. The results showed that E. faecalis R612Z1 could produce tyramine in meat samples when the storage temperature was no less than 4°C. The E. faecalis R612Z1 counts of the meat samples reached 10(8) CFU/g on day 7 at 4°C and on day 4 at 10°C. However, the tyramine content of the meat samples stored at 10°C increased to 23.73 μg/g (on day 10), which was greater than the level in the samples stored at 4°C (7.56 μg/g). Reverse transcription quantitative PCR detection of the expression level of the tyrDC gene in E. faecalis R612Z1 in the meat samples revealed no significant changes at different storage temperatures. Thus, the changes in tyramine production of E. faecalis R612Z1 may be due to the different enzymatic activities at different storage temperatures.

  11. Characterization of lead-resistant river isolate Enterococcus faecalis and assessment of its multiple metal and antibiotic resistance.

    PubMed

    Aktan, Yasin; Tan, Sema; Icgen, Bulent

    2013-06-01

    Contamination of surface waters has a direct impact on the public health of entire communities. Microorganisms inhabiting contaminated surface waters have developed mechanisms of coping with a variety of toxic metals and drugs. Investigations were carried out to isolate and identify lead-resistant bacteria from the river Kızılırmak along the city of Kırıkkale, Turkey. Of the 33 lead-resistant isolates, one isolate with a minimal inhibitory concentration of 1,200 mg L(-1) was isolated and identified as Enterococcus faecalis by using biochemical tests and 16S rRNA sequencing. Lead-resistant E. faecalis isolate was found out to be resistant to other heavy metals like aluminum, lithium, barium, chromium, iron, silver, tin, nickel, zinc, and strontium and to drugs like amikacin, aztreonam, and gentamicin. E. faecalis harbored four plasmids with the molecular sizes of 1.58, 3.06, 22.76, and 28.95 kb. Plasmid profile analyses of cured derivatives revealed that the lead resistance ability of E. faecalis was still existing despite the elimination of all the plasmids. Moreover, the antibiotic resistance pattern of the cured derivatives did not demonstrate any change from the parental strain. Our findings indicated that the lead resistance genes of E. faecalis were located on the chromosomal DNA rather than the plasmid.

  12. An In Vitro Study on the Effects of Nisin on the Antibacterial Activities of 18 Antibiotics against Enterococcus faecalis

    PubMed Central

    Ling, Junqi; Ma, Jinglei; Huang, Lijia; Zhang, Luodan

    2014-01-01

    Enterococcus faecalis rank among the leading causes of nosocomial infections worldwide and possesses both intrinsic and acquired resistance to a variety of antibiotics. Development of new antibiotics is limited, and pathogens continually generate new antibiotic resistance. Many researchers aim to identify strategies to effectively kill this drug-resistant pathogen. Here, we evaluated the effect of the antimicrobial peptide nisin on the antibacterial activities of 18 antibiotics against E. faecalis. The MIC and MBC results showed that the antibacterial activities of 18 antibiotics against E. faecalis OG1RF, ATCC 29212, and strain E were significantly improved in the presence of 200 U/ml nisin. Statistically significant differences were observed between the results with and without 200 U/ml nisin at the same concentrations of penicillin or chloramphenicol (p<0.05). The checkerboard assay showed that the combination of nisin and penicillin or chloramphenicol had a synergetic effect against the three tested E. faecalis strains. The transmission electron microscope images showed that E. faecalis was not obviously destroyed by penicillin or chloramphenicol alone but was severely disrupted by either antibiotic in combination with nisin. Furthermore, assessing biofilms by a confocal laser scanning microscope showed that penicillin, ciprofloxacin, and chloramphenicol all showed stronger antibiofilm actions in combination with nisin than when these antibiotics were administered alone. Therefore, nisin can significantly improve the antibacterial and antibiofilm activities of many antibiotics, and certain antibiotics in combination with nisin have considerable potential for use as inhibitors of this drug-resistant pathogen. PMID:24586598

  13. Antibacterial and residual antimicrobial activities against Enterococcus faecalis biofilm: A comparison between EDTA, chlorhexidine, cetrimide, MTAD and QMix

    NASA Astrophysics Data System (ADS)

    Zhang, Rui; Chen, Min; Lu, Yan; Guo, Xiangjun; Qiao, Feng; Wu, Ligeng

    2015-08-01

    We compared the antibacterial and residual antimicrobial activities of five root canal irrigants (17% EDTA,2% chlorhexidine,0.2% cetrimide, MTAD, and QMix) in a model of Enterococcus faecalis biofilm formation. Sixty dentin blocks with 3-week E. faecalis biofilm were divided into six equal groups and flushed with irrigant for 2 min or left untreated. A blank control group was also established. Antibacterial activities of the irrigants were evaluated by counting colony forming units. To test residual antimicrobial activities, 280 dentin blocks were divided into seven equal groups and flushed with irrigant for 2 min or left untreated and then incubated with E. faecalis suspension for 48 h, or used as a blank. No bacteria were observed in the blank control group. The number of viable E. faecalis was significantly fewer in the irrigant-treated groups compared with the untreated control (P < 0.05). Among the five irrigants, QMix had the strongest antibacterial activity. Residual antimicrobial activities of CHX were significantly higher at 12 h, 24 h and 36 h compared to untreated control (P < 0.05). All five root canal irrigants were effective to some extent against E. faecalis, but QMix and CHX had the strongest, and CHX the longest (up to 36 h), antimicrobial activity.

  14. Antimicrobial activity of some essential oils against oral multidrug-resistant Enterococcus faecalis in both planktonic and biofilm state

    PubMed Central

    Benbelaïd, Fethi; Khadir, Abdelmounaïm; Abdoune, Mohamed Amine; Bendahou, Mourad; Muselli, Alain; Costa, Jean

    2014-01-01

    Objective To evaluate some essential oils in treatment of intractable oral infections, principally caused by biofilm of multidrug-resistant Enterococcus faecalis (E. faecalis), such as persistent endodontic infections in which their treatment exhibits a real challenge for dentists. Methods Ten chemically analyzed essential oils by gas chromatography-mass spectrometry were evaluated for antimicrobial activity against sensitive and resistant clinical strains of E. faecalis in both planktonic and biofilm state using two methods, disk diffusion and broth micro-dilution. Results Studied essential oils showed a good antimicrobial activity and high ability in E. faecalis biofilm eradication, whether for sensitive or multidrug-resistant strains, especially those of Origanum glandulosum and Thymbra capitata with interesting minimum inhibitory concentration, biofilm inhibitory concentration, and biofilm eradication concentration values which doesn't exceed 0.063%, 0.75%, and 1.5%, respectively. Conclusions Findings of this study indicate that essential oils extracted from aromatic plants can be used in treatment of intractable oral infections, especially caused by biofilm of multidrug-resistant E. faecalis. PMID:25182948

  15. Differences in antibiotic resistance patterns of Enterococcus faecalis and Enterococcus faecium strains isolated from farm and pet animals.

    PubMed

    Butaye, P; Devriese, L A; Haesebrouck, F

    2001-05-01

    The prevalence of acquired resistance in 146 Enterococcus faecium and 166 Enterococcus faecalis strains from farm and pet animals, isolated in 1998 and 1999 in Belgium, against antibiotics used for growth promotion and for therapy was determined. Acquired resistance against flavomycin and monensin, two antibiotics used solely for growth promotion, was not detected. Avoparcin (glycopeptide) resistance was found sporadically in E. faecium only. Avilamycin resistance was almost exclusively seen in strains from farm animals. Resistance rates were higher in E. faecium strains from broiler chickens than in strains from other animal groups with tylosin and virginiamycin and in E. faecalis as well as in E. faecium strains with narasin and bacitracin. Resistance against ampicillin was mainly found among E. faecium strains from pets and was absent in E. faecalis. Tetracycline resistance occurred most often in strains from farm animals, while enrofloxacin resistance, only found in E. faecalis, occurred equally among strains from all origins. Resistance against gentamicin was very rare in broiler strains, whereas resistance rates were high in strains from other origins. It can be concluded that resistance against antibiotics used solely for growth promotion was more prevalent in E. faecium strains than in E. faecalis strains. With few exceptions, resistance against the different categories of antibiotics was more prevalent in strains from farm animals than in those from pets.

  16. [Photoreactivation of Escherichia coli and Enterococcus faecalis in the secondary effluent disinfected by UV-TiO2].

    PubMed

    Wang, Xi-Feng; Gong, Xin; Hu, Xiao-Lian; Ren, Bo-Zhi

    2014-04-01

    Effects of photoreactivating light intensity (0-41 microW x cm(-2)) on photoreactivation of Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) in the secondary effluent after UV and UV-TiO2 disinfection were investigated. The results indicated that the disinfection efficiency of UV-TiO2 was much higher than that of UV disinfection. The photoreactivation rate of E. coli was much higher in UV disinfection than that in UV-TiO2 disinfection. Under high light intensity in UV-TiO2 disinfection, high resurrection rate can be induced. However, a higher resurrection rate can be introduced even under low light intensity in the UV disinfection. Meanwhile, UV-TiO2 disinfection had a strong inhibition effect on E. faecalis photoreactivation, when the light intensity was lower than 21 microW x cm(-2), three was no resurrection occurred on E. faecalis after 72 h resurrection irradiation, only under a strong photoreactivating light intensity, the resurrection rate of E. faecalis was observed.

  17. The TIR Domain Containing Locus of Enterococcus faecalis Is Predominant among Urinary Tract Infection Isolates and Downregulates Host Inflammatory Response

    PubMed Central

    Kraemer, Thomas Daniel; Quintanar Haro, Orlando Daniel; Domann, Eugen; Chakraborty, Trinad; Tchatalbachev, Svetlin

    2014-01-01

    Based on Toll/interleukin-1 receptor (TIR) domain structure homology, we detected a previously uncharacterized gene encoding for a TIR domain containing protein (Tcp) in the genome of Enterococcus faecalis. We assigned this gene the name tcpF (as in Tcp of E. faecalis). Screening of E. faecalis samples revealed that tcpF is more common in isolates from urinary tract infections (UTIs) than in human faecal flora. tcpF alleles showed moderate single nucleotide polymorphism (SNP) among UTI isolates. Infection of mouse RAW264.7 macrophages with a tcpF knock-out mutant led to elevated cytokine response compared to the isogenic wild type E. faecalis strain. In silico analysis predicted significant tertiary structure homology to the TIR domain of human TLR1 (TLR1-TIR). When transiently expressed in cultured eukaryotic cells, TcpF caused suppression of TLR2-dependent NF-κB activation suggesting for TcpF a role as a factor in E. faecalis that benefits colonization by modulating the host's immune responses. PMID:25147569

  18. The effect of sodium hypochlorite on Enterococcus faecalis when grown on dentine as a single- and multi-species biofilm.

    PubMed

    Yap, Benlee; Zilm, Peter S; Briggs, Nancy; Rogers, Anthony H; Cathro, Peter C

    2014-12-01

    Enterococcus faecalis is often involved in the aetiology of apical periodontitis after endodontic treatment. This project aimed to establish, on dentine in vitro, a multi-species biofilm containing E. faecalis, and to determine if the organism had an increased resistance to sodium hypochlorite compared with an axenic biofilm. Biofilms were established on dentine discs in flow cells with either E. faecalis alone (axenic) or together with Fusobacterium nucleatum and Streptococcus sanguinis. Following treatment with either 0.9% sodium hypochlorite or saline, the viability of E. faecalis was determined by serial plating and qualitative analysis was performed by scanning electron microscopy and confocal laser scanning microscopy. Viable counts indicated that 0.9% NaOCl is highly effective against E. faecalis grown alone and as part of a multi-species biofilm (P = 0.0005 and P = 0.001, respectively). No significant difference in its survival in the two biofilm types was found (P = 0.8276).

  19. The Polyamine N-Acetyltransferase-Like Enzyme PmvE Plays a Role in the Virulence of Enterococcus faecalis

    PubMed Central

    Martini, Cecilia; Michaux, Charlotte; Bugli, Francesca; Arcovito, Alessandro; Iavarone, Federica; Cacaci, Margherita; Sterbini, Francesco Paroni; Hartke, Axel; Sauvageot, Nicolas; Sanguinetti, Maurizio; Posteraro, Brunella

    2014-01-01

    We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyA operon, ef_3001, renamed pmvE (for polyamine metabolism and virulence of E. faecalis). PmvE shares strong homologies with N1-spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalis strain carrying the recombinant plasmid pMSP3535-pmvE (V19/p3535-pmvE), which allows the induction of pmvE by addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalis in the Galleria mellonella infection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N-acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis, likely through its involvement in the polyamine metabolism. PMID:25385793

  20. Expression of Alzheimer-Type Neurofibrillary Epitopes in Primary Rat Cortical Neurons Following Infection with Enterococcus faecalis

    PubMed Central

    Underly, Robert; Song, Mee-Sook; Dunbar, Gary L.; Weaver, Charles L.

    2016-01-01

    The neurofibrillary tau pathology and amyloid deposits seen in Alzheimer’s disease (AD) also have been seen in bacteria-infected brains. However, few studies have examined the role of these bacteria in the generation of tau pathology. One suggested link between infection and AD is edentulism, the complete loss of teeth. Edentulism can result from chronic periodontal disease due to infection by Enterococcus faecalis. The current study assessed the ability to generate early Alzheimer-like neurofibrillary epitopes in primary rat cortical neurons through bacterial infection by E. faecalis. Seven-day old cultured neurons were infected with E. faecalis for 24 and 48 h. An upward molecular weight shift in tau by Western blotting (WB) and increased appearance of tau reactivity in cell bodies and degenerating neurites was found in the 48 h infection group for the antibody CP13 (phospho-Serine 202). A substantial increase in reactivity of Alz-50 was seen at 24 and 48 h after infection. Furthermore, extensive microtubule-associated protein 2 (MAP2) reactivity also was seen at 24 and 48 h post-infection. Our preliminary findings suggest a potential link between E. faecalis infection and intracellular changes that may help facilitate early AD-like neurofibrillary pathology. HighlightsEnterococcus faecalis used in the generation of AD neurofibrillary epitopes in rat.Infection increases Alz-50, phospho-Serine 202 tau, and MAP2 expression.Infection by Enterococcus may play a role in early Alzheimer neurofibrillary changes. PMID:26834627

  1. Downregulation of p70S6K Enhances Cell Sensitivity to Rapamycin in Esophageal Squamous Cell Carcinoma

    PubMed Central

    Lu, Zhaoming; Peng, Kezheng; Wang, Ning; Liu, Hong-Min

    2016-01-01

    It has been demonstrated that mTOR/p70S6K pathway was abnormally activated in many cancers and rapamycin and its analogs can restrain tumor growth through inhibiting this pathway, but some tumors including esophageal squamous cell carcinoma (ESCC) appear to be insensitive to rapamycin in recent studies. In the present study, we explored the measures to improve the sensitivity of ESCC cells to rapamycin and identified the clinical significance of the expression of phosphorylated p70S6K (p-p70S6K). The results showed that, after downregulating the expression of p70S6K and p-p70S6K by p70S6K siRNA, the inhibitory effects of rapamycin on cell proliferation, cell cycle, and tumor growth were significantly enhanced in vitro and in vivo. Furthermore, p-p70S6K had strong positive expression in ESCC tissues and its expression was closely related to lymph node metastasis and the TNM staging. These results indicated that p-p70S6K may participate in the invasion and metastasis in the development of ESCC and downregulation of the expression of p-p70S6K could improve the sensitivity of cells to rapamycin in ESCC. PMID:27595116

  2. Obesity-induced CerS6-dependent C16:0 ceramide production promotes weight gain and glucose intolerance.

    PubMed

    Turpin, Sarah M; Nicholls, Hayley T; Willmes, Diana M; Mourier, Arnaud; Brodesser, Susanne; Wunderlich, Claudia M; Mauer, Jan; Xu, Elaine; Hammerschmidt, Philipp; Brönneke, Hella S; Trifunovic, Aleksandra; LoSasso, Giuseppe; Wunderlich, F Thomas; Kornfeld, Jan-Wilhelm; Blüher, Matthias; Krönke, Martin; Brüning, Jens C

    2014-10-07

    Ceramides increase during obesity and promote insulin resistance. Ceramides vary in acyl-chain lengths from C14:0 to C30:0 and are synthesized by six ceramide synthase enzymes (CerS1-6). It remains unresolved whether obesity-associated alterations of specific CerSs and their defined acyl-chain length ceramides contribute to the manifestation of metabolic diseases. Here we reveal that CERS6 mRNA expression and C16:0 ceramides are elevated in adipose tissue of obese humans, and increased CERS6 expression correlates with insulin resistance. Conversely, CerS6-deficient (CerS6(Δ/Δ)) mice exhibit reduced C16:0 ceramides and are protected from high-fat-diet-induced obesity and glucose intolerance. CerS6 deletion increases energy expenditure and improves glucose tolerance, not only in CerS6(Δ/Δ) mice, but also in brown adipose tissue- (CerS6(ΔBAT)) and liver-specific (CerS6(ΔLIVER)) CerS6 knockout mice. CerS6 deficiency increases lipid utilization in BAT and liver. These experiments highlight CerS6 inhibition as a specific approach for the treatment of obesity and type 2 diabetes mellitus, circumventing the side effects of global ceramide synthesis inhibition.

  3. Characterization of the duplicate ribulose-1,5-bisphosphate carboxylase genes and cbb promoters of Alcaligenes eutrophus.

    PubMed

    Kusian, B; Bednarski, R; Husemann, M; Bowien, B

    1995-08-01

    Autotrophic CO2 fixation via the Calvin carbon reduction cycle in Alcaligenes eutrophus H16 is genetically determined by two highly homologous cbb operons, one of which is located on the chromosome and the other on megaplasmid pHG1 of the organism. An activator gene, cbbR, lies in divergent orientation only 167 bp upstream of the chromosomal operon and controls the expression of both cbb operons. The two 5'-terminal genes of the operons, cbbLS, coding for ribulose-1,5-bisphosphate carboxylase/oxygenase, were sequenced. Mapping of the 5' termini of the 2.1-kb cbbLS transcripts by primer extension and by nuclease S1 treatment revealed a single transcriptional start point at the same relative position for the chromosomal and plasmid-borne cbb operons. The derived cbb operon promoter showed similarity to sigma 70-dependent promoters of Escherichia coli. For the 1.4-kb transcripts of cbbR, the transcriptional start points were different in autotrophic and heterotrophic cells. The two corresponding cbbR promoters overlapped the cbb operon promoter and also displayed similarities to sigma 70-dependent promoters. The deficient cbbR gene located on pHG1 was transcribed as well. A newly constructed double operon fusion vector was used to determine the activities of the cbb promoters. Fusions with fragments carrying the cbb intergenic control regions demonstrated that the cbb operon promoters were strongly regulated in response to autotrophic versus heterotrophic growth conditions. In contrast, the cbbR promoters displayed low constitutive activities. The data suggest that the chromosomal and plasmid-borne cbb promoters of A. eutrophus H16 are functionally equivalent despite minor structural differences.

  4. Molecular Characterization of a Novel ortho-Nitrophenol Catabolic Gene Cluster in Alcaligenes sp. Strain NyZ215▿

    PubMed Central

    Xiao, Yi; Zhang, Jun-Jie; Liu, Hong; Zhou, Ning-Yi

    2007-01-01

    Alcaligenes sp. strain NyZ215 was isolated for its ability to grow on ortho-nitrophenol (ONP) as the sole source of carbon, nitrogen, and energy and was shown to degrade ONP via a catechol ortho-cleavage pathway. A 10,152-bp DNA fragment extending from a conserved region of the catechol 1,2-dioxygenase gene was obtained by genome walking. Of seven complete open reading frames deduced from this fragment, three (onpABC) have been shown to encode the enzymes involved in the initial reactions of ONP catabolism in this strain. OnpA, which shares 26% identity with salicylate 1-monooxygenase of Pseudomonas stutzeri AN10, is an ONP 2-monooxygenase (EC 1.14.13.31) which converts ONP to catechol in the presence of NADPH, with concomitant nitrite release. OnpC is a catechol 1,2-dioxygenase catalyzing the oxidation of catechol to cis,cis-muconic acid. OnpB exhibits 54% identity with the reductase subunit of vanillate O-demethylase in Pseudomonas fluorescens BF13. OnpAB (but not OnpA alone) conferred on the catechol utilizer Pseudomonas putida PaW340 the ability to grow on ONP. This suggests that OnpB may also be involved in ONP degradation in vivo as an o-benzoquinone reductase converting o-benzoquinone to catechol. This is analogous to the reduction of tetrachlorobenzoquinone to tetrachlorohydroquinone by a tetrachlorobenzoquinone reductase (PcpD, 38% identity with OnpB) in the pentachlorophenol degrader Sphingobium chlorophenolicum ATCC 39723. PMID:17616586

  5. Class 1 integrons and tetracycline resistance genes in alcaligenes, arthrobacter, and Pseudomonas spp. isolated from pigsties and manured soil.

    PubMed

    Agersø, Yvonne; Sandvang, Dorthe

    2005-12-01

    The presence of tetracycline resistance (Tc(r)) genes and class I integrons (in-1), and their ability to cotransfer were investigated in Tc(r) gram-negative (185 strains) and gram-positive (72 strains) bacteria from Danish farmland and pigsties. The isolates belonged to the groups or species Escherichia coli, Enterobacter spp., Arthrobacter spp., Alcaligenes spp., Pseudomonas spp., and Corynebacterium glutamicum. The 257 isolates were screened for in-1. Eighty-one of the gram-negative isolates were also screened for the Tc(r) genes tet(A), tet(B), and tet(C), and all (n = 72) gram-positive isolates were screened for tet(33). Fourteen (7%) of the soil isolates and eleven (25%) of the pigsty isolates contained in-1. All isolates that contained tet genes also contained in-1, except one gram-negative isolate from a pigsty that contained tet(B). All gram-positive isolates with in-1 also contained tet(33). No isolates contained more than one tet gene. The in-1-positive isolates were tested for resistance to selected antimicrobial agents and showed resistance to three to nine drugs. Filter-mating experiments showed cotransfer of Tc(r) and class I integrons from soil isolates to Escherichia coli and/or Pseudomonas putida. We conclude that soil bacteria in close contact to manure or pigsty environment may thus have an important role in horizontal spread of resistance. Use of tetracyclines in food animal production may increase not only Tc(r) but also multidrug resistance (caused by the presence tet genes and in-1) in bacteria.

  6. Molecular characterization of an inducible gentisate 1,2-dioxygenase gene, xlnE, from Pseudomonas alcaligenes NCIMB 9867.

    PubMed

    Yeo, Chew Chieng; Wong, Mark Vee-Meng; Feng, Yongmei; Song, Keang Peng; Poh, Chit Laa

    2003-07-17

    Pseudomonas alcaligenes NCIMB 9867 (strain P25X) produces isofunctional enzymes of the gentisate pathway that enables the degradation of xylenols and cresols via gentisate. Previous reports had indicated that one set of enzymes is constitutively expressed whereas the other set is strictly inducible by aromatic hydrocarbon substrates. The gene encoding gentisate 1,2-dioxygenase (GDO), the enzyme that catalyzes the cleavage of the gentisate aromatic ring, was cloned from strain P25X. The GDO gene, designated xlnE, is 1,044 bp, and is part of a 5.4 kb operon which consists of six genes, xlnEFGHID. Transcription of this operon was driven by a sigma 70-type promoter, PxlnE, located 123 bp upstream of the xlnE start codon. Primer extension analysis showed that the xlnE transcription start point is located at the -87 adenine residue. In a P25X xlnE knockout mutant, GDO activity could only be detected when cells were grown in the presence of aromatic substrates, suggesting that xlnE encodes for the constitutive copy of GDO. This was verified by constructing a P25X strain with xlnE transcriptionally fused to a promoterless catechol 2,3-dioxygenase gene. In this strain, catechol 2,3-dioxygenase activity was detected in cells that were grown in the absence of aromatic inducers. However, catechol 2,3-dioxygenase activity increased up to four fold when these cells were grown in the presence of aromatic substrates, in particular 3-hydroxybenzoate. Thus, xlnE is in fact, inducible and the constitutive activity observed under non-inducing conditions was due to its relatively high basal levels of expression.

  7. Molecular characterization of a novel ortho-nitrophenol catabolic gene cluster in Alcaligenes sp. strain NyZ215.

    PubMed

    Xiao, Yi; Zhang, Jun-Jie; Liu, Hong; Zhou, Ning-Yi

    2007-09-01

    Alcaligenes sp. strain NyZ215 was isolated for its ability to grow on ortho-nitrophenol (ONP) as the sole source of carbon, nitrogen, and energy and was shown to degrade ONP via a catechol ortho-cleavage pathway. A 10,152-bp DNA fragment extending from a conserved region of the catechol 1,2-dioxygenase gene was obtained by genome walking. Of seven complete open reading frames deduced from this fragment, three (onpABC) have been shown to encode the enzymes involved in the initial reactions of ONP catabolism in this strain. OnpA, which shares 26% identity with salicylate 1-monooxygenase of Pseudomonas stutzeri AN10, is an ONP 2-monooxygenase (EC 1.14.13.31) which converts ONP to catechol in the presence of NADPH, with concomitant nitrite release. OnpC is a catechol 1,2-dioxygenase catalyzing the oxidation of catechol to cis,cis-muconic acid. OnpB exhibits 54% identity with the reductase subunit of vanillate O-demethylase in Pseudomonas fluorescens BF13. OnpAB (but not OnpA alone) conferred on the catechol utilizer Pseudomonas putida PaW340 the ability to grow on ONP. This suggests that OnpB may also be involved in ONP degradation in vivo as an o-benzoquinone reductase converting o-benzoquinone to catechol. This is analogous to the reduction of tetrachlorobenzoquinone to tetrachlorohydroquinone by a tetrachlorobenzoquinone reductase (PcpD, 38% identity with OnpB) in the pentachlorophenol degrader Sphingobium chlorophenolicum ATCC 39723.

  8. Microbial degradation of alkylbenzenesulphonates. Metabolism of homologues of short alkyl-chain length by an Alcaligenes sp

    PubMed Central

    Bird, J. Anthony; Cain, Ronald B.

    1974-01-01

    1. An organism isolated from sewage and identified as an Alcaligenes sp. utilized benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate as sole source of carbon and energy for growth. Higher alkylbenzenesulphonate homologues and the hydrocarbons, benzene, toluene, phenylethane and 1-phenyldodecane were not utilized. 2. 2-Phenylpropanesulphonate was metabolized to 4-isopropylcatechol. 3. 1-Phenylpropanesulphonate was metabolized to an ortho-diol, which was tentatively identified, in the absence of an authentic specimen, as 4-n-propylcatechol. 4. In the presence of 4-isopropylcatechol, which inhibited catechol 2,3-dioxygenase, 4-ethylcatechol accumulated in cultures growing on phenylethane-p-sulphonate. 5. Authentic samples of catechol, 3-methylcatechol, 4-methylcatechol, 4-ethylcatechol and 3-isopropylcatechol were oxidized by heat-treated extracts to the corresponding 2-hydroxyalkylmuconic semialdehydes. Ring cleavage occurred between C-2 and C-3. 6. The catechol derived from 1-phenylpropanesulphonate was oxygenated by catechol 2,3-dioxygenase to a compound with all the properties of a 2-hydroxyalkylmuconic semialdehyde, but it was not rigorously identified. 7. The catechol 2,3-dioxygenase induced by growth on benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate showed a constant ratio of specific activities with catechol, 3-methylcatechol, 4-methylcatechol and 4-ethylcatechol that was independent of the growth substrate. At 60°C, activity towards these substrates declined at an identical first-order rate. 8. Enzymes of the `ortho' pathway of catechol metabolism were present in small amounts in cells grown on benzenesulphonate, toluene-p-sulphonate or phenylethane-p-sulphonate. 9. The catechol 1,2-dioxygenase oxidized the alkylcatechols, but the rates and the total extents of oxidation were less than for catechol itself. The oxidation products of these alkylcatechols were not further metabolized. PMID:4375955

  9. Optimizing ZnS/6LiF scintillators for wavelength-shifting-fiber neutron detectors

    SciTech Connect

    Crow, Lowell; Funk, Loren L; Hannan, Bruce W; Hodges, Jason P; Riedel, Richard A; Wang, Cai-Lin

    2016-01-01

    In this paper we compare the performance of grooved and flat ZnS/6LiF scintillators in a wavelength shifting-fiber (WLSF) detector. Flat ZnS/6LiF scintillators with the thickness L=0.2-0.8 mm were characterized using photon counting and pulse-height analysis and compared to a grooved scintillator of approximately 0.8 mm thick. While a grooved scintillator considerably increases the apparent thickness of the scintillator to neutrons for a given coating thickness, we find that the flat scintillators perform better than the grooved scintillators in terms of both light yield and neutron detection efficiency. The flat 0.8-mm-thick scintillator has the highest light output, and it is 52% higher compared with a grooved scintillator of same thickness. The lower light output of the grooved scintillator as compared to the flat scintillator is consistent with the greater scintillator-WLSF separation and the much larger average emission angle of the grooved scintillator. We also find that the average light cone width, or photon travel-length as measured using time-of-flight powder diffraction of diamond and vanadium, decreases with increasing L in the range of L=0.6-0.8 mm. This result contrasts with the traditional Swank diffusion model for micro-composite scintillators, and could be explained by a decrease in photon diffusion-coefficient or an increase in micro-particle content in the flat scintillator matrix for the thicker scintillators.

  10. Magnetization reversal phenomena in (Cr0.70Ti0.30)5S6

    NASA Astrophysics Data System (ADS)

    Hashimoto, Satoshi; Matsuda, Yuji; Sato, Tetsuya; Anzai, Shuichiro

    2005-12-01

    Magnetization reversal phenomena (MRP) along magnetic order-order transitions have recently been reported on impurity-doped magnetic systems. Because imperfect long-range magnetic order exists in these systems, it is expected that a systematic investigation of MRP will give physical information on thermomagnetic processes of magnetic systems in the range from the micro- to nanoscales. As a typical order-order transition (a state doubly modulated by helical and canting orders to a collinear ferrimagnetic state) has been known to occur on Cr5S6 at a transition temperature Tt, we investigate the magnetizations of (Cr0.70Ti0.30)5S6 on heating and cooling runs in various magnetic fields. At 20Oe, the field-cooled magnetization just below the Curie temperature has a positive sign; the sign turns negative below the compensation temperature TCM (first step) and finally returns to positive below Tt (second step). The first-step MRP observed in this system is explained by the potential barriers resulting from anisotropy energy when the preferred direction of collinear ferrimagnetic moment reverses. The proposed mechanism for second-step MRP is the pinning effect caused by the impurity atoms (Ti) in the helical long-range-order chains. Comparing other examples of MRPs, we discuss the roles of local impurity centers in the thermomagnetic process in magnetic order-order transitions.

  11. Enterococcus faecalis strains from food, environmental, and clinical origin produce ACE-inhibitory peptides and other bioactive peptides during growth in bovine skim milk.

    PubMed

    Gútiez, Loreto; Gómez-Sala, Beatriz; Recio, Isidra; del Campo, Rosa; Cintas, Luis M; Herranz, Carmen; Hernández, Pablo E

    2013-08-16

    Enterococcus faecalis isolates from food and environmental origin were evaluated for their angiotensin-converting enzyme (ACE)-inhibitory activity (ACE-IA) after growth in bovine skim milk (BSM). Most (90% active) but not all (10% inactive) E. faecalis strains produced BSM-derived hydrolysates with high ACE-IA. Known ACE-inhibitory peptides (ACE-IP) and an antioxidant peptide were identified in the E. faecalis hydrolysates by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (RP-HPLC-MS/MS). Antimicrobial activity against Pediococcus damnosus CECT4797 and Listeria ivanovii CECT913 was also observed in the E. faecalis hydrolysates. The incidence of virulence factors in the E. faecalis strains with ACE-IA and producers of ACE-IP was variable but less virulence factors were observed in the food and environmental strains than in the clinical reference strains. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) based analysis demonstrated that food and environmental E. faecalis strains were genetically different from those of clinical origin. When evaluated, most E. faecalis strains of clinical origin also originated BSM-derived hydrolysates with high ACE-IA due to the production of ACE-IP. Accordingly, the results of this work suggest that most E. faecalis strains of food, environmental and clinical origin produce BSM-derived bioactive peptides with human health connotations and potential biotechnological applications.

  12. Targeted sampling protocol as prelude to bacterial source tracking with Enterococcus faecalis.

    PubMed

    Kuntz, Robin L; Hartel, Peter G; Godfrey, Dominique G; McDonald, Jennifer L; Gates, Keith W; Segars, William I

    2003-01-01

    Recent studies suggest that host origin databases for bacterial source tracking (BST) must contain a large number of isolates because bacterial subspecies change with geography and time. A new targeted sampling protocol was developed as a prelude to BST to minimize these changes. The research was conducted on the Sapelo River, a tidal river on the Georgia coast. A general sampling of the river showed fecal enterococcal numbers ranging from <10 (below the limit of detection) to 990 colony-forming units (CFU) per 100 mL. Locations with high enterococcal numbers were combined with local knowledge to determine targeted sampling sites. Fecal enterococcal numbers around one site ranged from <10 to 24,000 CFU per 100 mL. Bacterial source tracking was conducted to determine if a wastewater treatment facility at the site was responsible for this contamination. The fecal indicator bacterium was Enterococcus faecalis. Ribotyping, automated with a RiboPrinter (DuPont Qualicon, Wilmington, DE), was the BST method. Thirty-seven ribotypes were observed among 83 Ent. faecalis isolates obtained from the Sapelo River and the wastewater lagoon. Sixteen ribotypes were associated with either the river or the lagoon, and only five ribotypes (14%) were shared. Nevertheless, these five ribotypes represented 39 of the 83 Ent. faecalis isolates, almost a majority (47%). These results suggest that the fecal contamination in the river came from the wastewater treatment facility. As a prelude to BST, targeted sampling minimized subspecies changes with geography and time, and eliminated the need for a permanent host origin database by restricting BST to a small geographic area and requiring sampling to be completed in one day.

  13. Probing surface adhesion forces of Enterococcus faecalis to medical-grade polymers using atomic force microscopy.

    PubMed

    Sénéchal, Annie; Carrigan, Shawn D; Tabrizian, Maryam

    2004-05-11

    The aim of this study was to compare the initial adhesion forces of the uropathogen Enterococcus faecalis with the medical-grade polymers polyurethane (PU), polyamide (PA), and poly(tetrafluoroethylene) (PTFE). To quantify the cell-substrate adhesion forces, a method was developed using atomic force microscopy (AFM) in liquid that allows for the detachment of individual live cells from a polymeric surface through the application of increasing force using unmodified cantilever tips. Results show that the lateral force required to detach E. faecalis cells from a substrate differed depending on the nature of the polymeric surface: a force of 19 +/- 4 nN was required to detach cells from PU, 6 +/- 4 nN from PA, and 0.7 +/- 0.3 nN from PTFE. Among the unfluorinated polymers (PU and PA), surface wettability was inversely proportional to the strength of adhesion. AFM images also demonstrated qualitative differences in bacterial adhesion; PU was covered by clusters of cells with few cell singlets present, whereas PA was predominantly covered by individual cells. Moreover, extracellular material could be observed on some clusters of PU-adhered cells as well as in the adjacent region surrounding cells adhered on PA. E. faecalis adhesion to the fluorinated polymer (PTFE) showed different characteristics; only a few individual cells were found, and bacteria were easily damaged, and thus detached, by the tip. This work demonstrates the utility of AFM for measurement of cell-substrate lateral adhesion forces and the contribution these forces make toward understanding the initial stages of bacterial adhesion. Further, it suggests that initial adhesion can be controlled, through appropriate biomaterial design, to prevent subsequent formation of aggregates and biofilms.

  14. Teicoplanin versus vancomycin for prophylaxis of experimental Enterococcus faecalis endocarditis in rats.

    PubMed Central

    Entenza, J M; Calandra, T; Moosmann, Y; Malinverni, R; Glauser, M P

    1992-01-01

    Teicoplanin was compared with vancomycin for the prophylaxis of experimental Enterococcus faecalis endocarditis in rats. Single intravenous doses of teicoplanin (7 mg/kg of body weight) or vancomycin (15 mg/kg) were given 30 min before bacterial challenge. Two strains of E. faecalis (309 and 1209) isolated from patients with endocarditis were tested. Bacterial inocula ranged from 10(4) (i.e., the inoculum infecting 90% of the control rats [ID90]) to 10(7) CFU/ml. The MICs and MBCs of teicoplanin and vancomycin were, respectively, 0.25 to greater than 128 mg/liter and 2 to greater than 128 mg/liter for strain 309 and 0.5 to greater than 128 mg/liter and 0.5 to greater than 128 mg/liter for strain 1209. Vancomycin prevented endocarditis only in 60% (strain 309) and in 87% (strain 1209) of rats challenged with the smallest bacterial-inoculum size (ID90), whereas teicoplanin prevented endocarditis in 100% of rats challenged with the same inoculum (strain 309; P = 0.05), in 87% of rats challenged with 10 times the ID90 (strain 309; P = 0.02), and in 95% of rats challenged with 100 times the ID90 (strain 1209; P = 0.0003). The combination of teicoplanin plus gentamicin (4 mg/kg) extended the protection to inocula 100 times the ID90 (strain 309; 96% of sterile animals) and 1,000 times the ID90 (strain 1209; 100% of sterile animals). Prevention of endocarditis was likely to be due to a prolonged inhibition of bacterial growth by sustained levels of teicoplanin in serum and not to bacterial killing. Indeed, teicoplanin did not exhibit any bactericidal activity either in vitro (time-kill curves) or in vivo (serum bactericidal activity). Teicoplanin proved to be superior to vancomycin in the prophylaxis of experimental E. faecalis endocarditis in rats. PMID:1416824

  15. Basal Levels of (p)ppGpp in Enterococcus faecalis: the Magic beyond the Stringent Response

    PubMed Central

    Gaca, Anthony O.; Kajfasz, Jessica K.; Miller, James H.; Liu, Kuanqing; Wang, Jue D.; Abranches, Jacqueline; Lemos, José A.

    2013-01-01

    ABSTRACT The stringent response (SR), mediated by the alarmone (p)ppGpp, is a conserved bacterial adaptation system controlling broad metabolic alterations necessary for survival under adverse conditions. In Enterococcus faecalis, production of (p)ppGpp is controlled by the bifunctional protein RSH (for “Rel SpoT homologue”; also known as RelA) and by the monofunctional synthetase RelQ. Previous characterization of E. faecalis strains lacking rsh, relQ, or both revealed that RSH is responsible for activation of the SR and that alterations in (p)ppGpp production negatively impact bacterial stress survival and virulence. Despite its well-characterized role as the effector of the SR, the significance of (p)ppGpp during balanced growth remains poorly understood. Microarrays of E. faecalis strains producing different basal amounts of (p)ppGpp identified several genes and pathways regulated by modest changes in (p)ppGpp. Notably, expression of numerous genes involved in energy generation were induced in the ∆rsh ∆relQ [(p)ppGpp0] strain, suggesting that a lack of basal (p)ppGpp places the cell in a “transcriptionally relaxed” state. Alterations in the fermentation profile and increased production of H2O2 in the (p)ppGpp0 strain substantiate the observed transcriptional changes. We confirm that, similar to what is seen in Bacillus subtilis, (p)ppGpp directly inhibits the activity of enzymes involved in GTP biosynthesis, and complete loss of (p)ppGpp leads to dysregulation of GTP homeostasis. Finally, we show that the association of (p)ppGpp with antibiotic survival does not relate to the SR but rather relates to basal (p)ppGpp pools. Collectively, this study highlights the critical but still underappreciated role of basal (p)ppGpp pools under balanced growth conditions. PMID:24065631

  16. Antibacterial Efficacy of Super-Oxidized Water on Enterococcus faecalis Biofilms in Root Canal

    PubMed Central

    Zan, Recai; Alacam, Tayfun; Hubbezoglu, Ihsan; Tunc, Tutku; Sumer, Zeynep; Alici, Oguzhan

    2016-01-01

    Background The success of endodontic treatment depends on a few crucial factors. One of these factors is the complete chemomechanic preparation of root canal against various bacteria. In particular, the effect of resistant bacteria may cause intense pain with flare-up and formation of periapical lesions. Therefore, the strong effect of irrigants plays an important role in terms of the complete elimination of these bacteria to achieve long-term successful treatment. Objectives The aim of this study was to investigate the antibacterial effects of super-oxidized water (SPO) in root canals infected with Enterococcus faecalis biofilms. Methods One hundred twenty single-root, premolar teeth were selected. Initially, the teeth were prepared and then disinfected. E. faecalis were inoculated and kept at 37°C for 24 hours in the root canals. The re-inoculation procedure was repeated on the first, fourth, seventh, and tenth days. The infected root canals were divided into one negative (saline) and one positive (sodium hypochlorite) control group and four experimental groups (super-oxidized water: 1, 2, 3, or 5 minutes) (n = 20). Paper points were placed in the root canals to control and evaluate the biofilm formation. Biofilms were counted on blood agar plates, and data was evaluated and statistically analyzed using one-way ANOVA and Tukey’s test. Results Although sodium hypochlorite (NaOCl) showed no statistically significant difference when compared with three and five minutes of SPO irrigation (P > 0.05), NaOCl showed statistically significant differences among all other groups (P < 0.05). Conclusions Super-oxidized water indicated a remarkable and similar bactericidal effect to that of traditional NaOCl against E. faecalis biofilms. In terms of successful endodontic treatment approaches, super-oxidized water may be used as an effective irrigation solution in clinics. PMID:27800142

  17. Bactericidal effect of plasma jet with helium flowing through 3% hydrogen peroxide against Enterococcus faecalis

    PubMed Central

    Zhou, Xin-Cai; Li, Yu-Lan; Liu, De-Xi; Cao, Ying-Guang; Lu, Xin-Pei

    2016-01-01

    The aim of the present study was to assess the antimicrobial activity of plasma jet with helium (He) flowing through 3% hydrogen peroxide in root canals infected with Enterococcus faecalis. A total of 42 single-rooted anterior teeth were prepared, sterilized, inoculated with an E. faecalis suspension and incubated for 7 days. Next, the teeth were randomly divided into six experimental groups (including groups treated by plasma jet with or without He for different time durations) and one control group treated without plasma. The number of surviving bacteria in each canal was determined by counting the colony forming units (CFU)/ml on nutrient agar plates. The results indicated that statistically significant reduction in CFU/ml (P<0.005) existed for all treatment groups relative to the control group. The greatest reductions in CFU/ml were observed for Group 3 (7.027 log unit reduction) and Group 2 (6.237 log unit reduction), which were treated by plasma jet sterilization with He flowing through 3% hydrogen peroxide for 4 min or for 2 min, respectively. In addition, the reduction in Group 3 was significantly greater compared with that in Group 2 or in the groups treated by plasma jet sterilization without He flowing through 3% hydrogen peroxide for 1, 2 or 4 min. In conclusion, plasma jet with or without He flowing through 3% hydrogen peroxide can effectively sterilized root canals infected with E. faecalis and should be considered as an alternative method for root canal disinfection in endodontic treatments. PMID:27882119

  18. Genome-Wide Identification of Small RNAs in the Opportunistic Pathogen Enterococcus faecalis V583

    PubMed Central

    Shioya, Kouki; Michaux, Charlotte; Kuenne, Carsten; Hain, Torsten; Verneuil, Nicolas; Budin-Verneuil, Aurélie; Hartsch, Thomas; Hartke, Axel; Giard, Jean-Christophe

    2011-01-01

    Small RNA molecules (sRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sRNAs in the Gram positive opportunistic pathogen Enterococcus faecalis. We characterized 11 sRNAs by tiling microarray analysis, 5′ and 3′ RACE-PCR, and Northern blot analysis. Six sRNAs were specifically expressed at exponential phase, two sRNAs were observed at stationary phase, and three were detected during both phases. Searches of putative functions revealed that three of them (EFA0080_EFA0081 and EFB0062_EFB0063 on pTF1 and pTF2 plasmids, respectively, and EF0408_EF04092 located on the chromosome) are similar to antisense RNA involved in plasmid addiction modules. Moreover, EF1097_EF1098 shares strong homologies with tmRNA (bi-functional RNA acting as both a tRNA and an mRNA) and EF2205_EF2206 appears homologous to 4.5S RNA member of the Signal Recognition Particle (SRP) ribonucleoprotein complex. In addition, proteomic analysis of the ΔEF3314_EF3315 sRNA mutant suggests that it may be involved in the turnover of some abundant proteins. The expression patterns of these transcripts were evaluated by tiling array hybridizations performed with samples from cells grown under eleven different conditions some of which may be encountered during infection. Finally, distribution of these sRNAs among genome sequences of 54 E. faecalis strains was assessed. This is the first experimental genome-wide identification of sRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen. PMID:21912655

  19. Genome-wide identification of small RNAs in the opportunistic pathogen Enterococcus faecalis V583.

    PubMed

    Shioya, Kouki; Michaux, Charlotte; Kuenne, Carsten; Hain, Torsten; Verneuil, Nicolas; Budin-Verneuil, Aurélie; Hartsch, Thomas; Hartke, Axel; Giard, Jean-Christophe

    2011-01-01

    Small RNA molecules (sRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sRNAs in the gram positive opportunistic pathogen Enterococcus faecalis. We characterized 11 sRNAs by tiling microarray analysis, 5' and 3' RACE-PCR, and Northern blot analysis. Six sRNAs were specifically expressed at exponential phase, two sRNAs were observed at stationary phase, and three were detected during both phases. Searches of putative functions revealed that three of them (EFA0080_EFA0081 and EFB0062_EFB0063 on pTF1 and pTF2 plasmids, respectively, and EF0408_EF04092 located on the chromosome) are similar to antisense RNA involved in plasmid addiction modules. Moreover, EF1097_EF1098 shares strong homologies with tmRNA (bi-functional RNA acting as both a tRNA and an mRNA) and EF2205_EF2206 appears homologous to 4.5S RNA member of the Signal Recognition Particle (SRP) ribonucleoprotein complex. In addition, proteomic analysis of the ΔEF3314_EF3315 sRNA mutant suggests that it may be involved in the turnover of some abundant proteins. The expression patterns of these transcripts were evaluated by tiling array hybridizations performed with samples from cells grown under eleven different conditions some of which may be encountered during infection. Finally, distribution of these sRNAs among genome sequences of 54 E. faecalis strains was assessed. This is the first experimental genome-wide identification of sRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen.

  20. Evaluation of the Antibacterial Efficacy of Azadirachta Indica, Commiphora Myrrha, Glycyrrhiza Glabra Against Enterococcus Faecalis using Real Time PCR

    PubMed Central

    Anand, Suresh; Rajan, Mathan; Venkateshbabu, Nagendrababu; Kandaswamy, Deivanayagam; Shravya, Yarramreddy; Rajeswari, Kalaiselvam

    2016-01-01

    Aim: To compare the antibacterial efficacy of Azadirachta indica (Neem), Commiphora myrrha (Myrrh), Glycyrrhiza glabra (Liquorice) with 2% Chlorhexidine (CHX) against E. faecalis by using Real Time PCR Materials and Methods: A total of fifty teeth specimens (n=50) were inoculated with E. faecalis for 21 days. Specimens were divided into five groups (Group 1: Myrrh, Group 2: Neem, Group 3: Liquorice, Group 4: 2% CHX and Group 5: Saline (negative control)). The intracanal medicaments were packed inside the tooth. After 5 days, the remaining microbial load was determined by using real time PCR Results: Threshold cycle (Ct) values of Myrrh extract, Neem extract, Liquorice Extract, 2% CHX and saline were found to be 30.94, 23.85, 21.38, 30.93 and 17.8 respectively Conclusion: Myrrh extract showed inhibition of E.faecalis equal to that of 2% CHX followed by Neem, Liquorice and Saline PMID:27386000

  1. Epidemiological alteration in pathogens found in ground meat in Iran: unexpected predominance of vancomycin-resistant Enterococcus faecalis.

    PubMed

    Sadeghifard, Nourkhoda; Kazemian, Hossein; Mohebi, Reza; Sekawi, Zamberi; Khosravi, Afra; Valizadeh, Nasrin; Ghafourian, Sobhan

    2015-01-01

    Colonization of the human and animal intestinal tract with potential pathogenic bacteria is correlated with the risk of contamination of food products. The current study analyzed the prevalence of Enterococcus faecalis and Escherichia coli O157H7 in ground meat in Ilam, Iran. Both index organisms were identified following standard food microbiological methods. For E. faecalis, the susceptibility to vancomycin was tested, and PCR was used to check for the vanA gene. E. faecalis was present in all 24 ground meat samples, with no E. coli O157H7 detected in samples. The analysis showed the presence of the vanA gene in 5/24 vancomycin resistant enterococci. In conclusion, this study for the first time demonstrates the presence of vancomycin-resistant enterococci in ground meat in Iran. This observation warrants further epidemiologic investigation and should be followed up in the future.

  2. First Report of the Multidrug Resistance Gene cfr in Enterococcus faecalis of Animal Origin

    PubMed Central

    Liu, Yang; Wang, Yang; Wu, Congming; Shen, Zhangqi; Schwarz, Stefan; Du, Xiang-Dang; Dai, Lei; Zhang, Wanjiang

    2012-01-01

    The multiresistance gene cfr was identified for the first time in an Enterococcus faecalis isolate of animal origin. The 32,388-bp plasmid pEF-01, which carried the cfr gene, was sequenced completely. Three copies of the insertion sequence IS1216 were identified in pEF-01, and the detection of a cfr- and IS1216-containing amplicon by inverse PCR suggests that IS1216 may play a role in the dissemination of cfr by a recombination process. PMID:22203597

  3. VanE-type vancomycin-resistant Enterococcus faecalis clinical isolates from Australia.

    PubMed

    Abadía-Patiño, Lorena; Christiansen, Keryn; Bell, Jan; Courvalin, Patrice; Périchon, Bruno

    2004-12-01

    Three distinct Enterococcus faecalis VanE-type isolates-BM4574, BM4575, and BM4576-obtained in Australia were studied. Expression of the resistance genes was constitutive in BM4575, probably due to a 2-bp deletion into the vanSE gene, and inducible in BM4574 and BM4576. Transcription analysis of the vanE operons suggested that the five genes were cotranscribed from an initiation site located 25 bp upstream from the ATG start codon of vanE.

  4. Biochemical characterization of an anti-Candida factor produced by Enterococcus faecalis

    PubMed Central

    2012-01-01

    Background Because Candida albicans is resistant to several antifungal antibiotics, there is a need to identify other less toxic natural products, particularly antimicrobial proteins, peptides or bacteriocin like inhibitory substances. An attempt has been made to purify and characterise an anti-Candida compound produced by Enterococcus faecalis. Results An anti-Candida protein (ACP) produced by E. faecalis active against 8 C. albicans strains was characterised and partially purified. The ACP showed a broad-spectrum activity against multidrug resistant C. albicans MTCC 183, MTCC 7315, MTCC 3958, NCIM 3557, NCIM 3471 and DI. It was completely inactivated by treatment with proteinase K and partially by pronase E. The ACP retained biological stability after heat-treatment at 90°C for 20 min, maintained activity over a pH range 6–10, and remained active after treatment with α-amylase, lipase, organic solvents, and detergents. The antimicrobial activity of the E. faecalis strain was found exclusively in the extracellular filtrate produced in the late logarithmic growth phase. The highest activity (1600 AU mL-1) against C. albicans MTCC 183 was recorded at 48 h of incubation, and activity decreased thereafter. The peptide showed very low haemagglutination and haemolytic activities against human red blood cells. The antimicrobial substance was purified by salt-fractionation and chromatography. Partially purified ACP had a molecular weight of approximately 43 KDa in Tricine-PAGE analysis. The 12 amino acid N terminal sequence was obtained by Edman degradation. The peptide was de novo sequenced by ESI-MS, and the deduced combined sequence when compared to other bacteriocins and antimicrobial peptide had no significant sequence similarity. Conclusions The inhibitory activity of the test strain is due to the synthesis of an antimicrobial protein. To our knowledge, this is the first report on the isolation of a promising non-haemolytic anti-Candida protein from E

  5. Intrathecal/Intraventricular Linezolid in Multidrug-Resistant Enterococcus faecalis Ventriculitis

    PubMed Central

    Lich, Brian F.; Conner, Andrew K.; Burks, Joshua D.; Glenn, Chad A.; Sughrue, Michael E.

    2016-01-01

    Background The use of intrathecal antibiotic therapy for the treatment of ventriculitis and/or meningitis has demonstrated efficacy especially when sterilization of the cerebrospinal fluid is not possible with intravenous antibiotics alone. Case Description We describe the successful treatment of Enterococcus faecalis ventriculitis utilizing intrathecal linezolid in a 32-year-old female patient with severe allergy to vancomycin, prohibitive bacterial susceptibilities, and failure of previous attempts to sterilize the cerebrospinal fluid despite multimodal treatment. Conclusion Intrathecal linezolid is a useful treatment in the setting of multidrug-resistant bacterial ventriculitis. We present a useful dosing regimen for the administration of intrathecal linezolid. PMID:27867829

  6. Surface-Associated Lipoproteins Link Enterococcus faecalis Virulence to Colitogenic Activity in IL-10-Deficient Mice Independent of Their Expression Levels.

    PubMed

    Ocvirk, Soeren; Sava, Irina G; Lengfelder, Isabella; Lagkouvardos, Ilias; Steck, Natalie; Roh, Jung H; Tchaptchet, Sandrine; Bao, Yinyin; Hansen, Jonathan J; Huebner, Johannes; Carroll, Ian M; Murray, Barbara E; Sartor, R Balfour; Haller, Dirk

    2015-06-01

    The commensal Enterococcus faecalis is among the most common causes of nosocomial infections. Recent findings regarding increased abundance of enterococci in the intestinal microbiota of patients with inflammatory bowel diseases and induction of colitis in IL-10-deficient (IL-10-/-) mice put a new perspective on the contribution of E. faecalis to chronic intestinal inflammation. Based on the expression of virulence-related genes in the inflammatory milieu of IL-10-/- mice using RNA-sequencing analysis, we characterized the colitogenic role of two bacterial structures that substantially impact on E. faecalis virulence by different mechanisms: the enterococcal polysaccharide antigen and cell surface-associated lipoproteins. Germ-free wild type and IL-10-/- mice were monoassociated with E. faecalis wild type OG1RF or the respective isogenic mutants for 16 weeks. Intestinal tissue and mesenteric lymph nodes (MLN) were collected to characterize tissue pathology, loss of intestinal barrier function, bacterial adhesion to intestinal epithelium and immune cell activation. Bone marrow-derived dendritic cells (BMDC) were stimulated with bacterial lysates and E. faecalis virulence was additionally investigated in three invertebrate models. Colitogenic activity of wild type E. faecalis (OG1RF score: 7.2±1.2) in monoassociated IL-10-/- mice was partially impaired in E. faecalis lacking enterococcal polysaccharide antigen (ΔepaB score: 4.7±2.3; p<0.05) and was almost completely abrogated in E. faecalis deficient for lipoproteins (Δlgt score: 2.3±2.3; p<0.0001). Consistently both E. faecalis mutants showed significantly impaired virulence in Galleria mellonella and Caenorhabditis elegans. Loss of E-cadherin in the epithelium was shown for all bacterial strains in inflamed IL-10-/- but not wild type mice. Inactivation of epaB in E. faecalis reduced microcolony and biofilm formation in vitro, altered bacterial adhesion to intestinal epithelium of germ-free Manduca sexta larvae

  7. Surface-Associated Lipoproteins Link Enterococcus faecalis Virulence to Colitogenic Activity in IL-10-Deficient Mice Independent of Their Expression Levels

    PubMed Central

    Lengfelder, Isabella; Lagkouvardos, Ilias; Steck, Natalie; Roh, Jung H.; Tchaptchet, Sandrine; Bao, Yinyin; Hansen, Jonathan J.; Huebner, Johannes; Carroll, Ian M.; Murray, Barbara E.; Sartor, R. Balfour; Haller, Dirk

    2015-01-01

    The commensal Enterococcus faecalis is among the most common causes of nosocomial infections. Recent findings regarding increased abundance of enterococci in the intestinal microbiota of patients with inflammatory bowel diseases and induction of colitis in IL-10-deficient (IL-10-/-) mice put a new perspective on the contribution of E. faecalis to chronic intestinal inflammation. Based on the expression of virulence-related genes in the inflammatory milieu of IL-10-/- mice using RNA-sequencing analysis, we characterized the colitogenic role of two bacterial structures that substantially impact on E. faecalis virulence by different mechanisms: the enterococcal polysaccharide antigen and cell surface-associated lipoproteins. Germ-free wild type and IL-10-/- mice were monoassociated with E. faecalis wild type OG1RF or the respective isogenic mutants for 16 weeks. Intestinal tissue and mesenteric lymph nodes (MLN) were collected to characterize tissue pathology, loss of intestinal barrier function, bacterial adhesion to intestinal epithelium and immune cell activation. Bone marrow-derived dendritic cells (BMDC) were stimulated with bacterial lysates and E. faecalis virulence was additionally investigated in three invertebrate models. Colitogenic activity of wild type E. faecalis (OG1RF score: 7.2±1.2) in monoassociated IL-10-/- mice was partially impaired in E. faecalis lacking enterococcal polysaccharide antigen (ΔepaB score: 4.7±2.3; p<0.05) and was almost completely abrogated in E. faecalis deficient for lipoproteins (Δlgt score: 2.3±2.3; p<0.0001). Consistently both E. faecalis mutants showed significantly impaired virulence in Galleria mellonella and Caenorhabditis elegans. Loss of E-cadherin in the epithelium was shown for all bacterial strains in inflamed IL-10-/- but not wild type mice. Inactivation of epaB in E. faecalis reduced microcolony and biofilm formation in vitro, altered bacterial adhesion to intestinal epithelium of germ-free Manduca sexta larvae

  8. Separation and characterization of effective demulsifying substances from surface of Alcaligenes sp. S-XJ-1 and its application in water-in-kerosene emulsion.

    PubMed

    Huang, Xiangfeng; Peng, Kaiming; Feng, Yi; Liu, Jia; Lu, Lijun

    2013-07-01

    The main goal of this work was to analyze the effect of surface substances on demulsifying capability of the demulsifying strain Alcaligenes sp. S-XJ-1. The demulsifying substances were successfully separated from the cell surface with dichloromethane-alkali treatment, and exhibited 67.5% of the demulsification ratio for water-in-kerosene emulsions at a dosage of 356mg/L. FT-IR, TLC and ESI-MS analysis confirmed the presence of a carbohydrate-protein-lipid complex in the demulsifying substances with the major molecular ions from mass-to-charge ratio (m/z) 165 to 814. After the substances separated, the cell morphology changed from aggregated to dispersed, and the concentration of cell surface functional groups decreased. Cell surface hydrophobicity and the ability of cell adhesion to hydrophobic surface of the treated cells was also reduced compared with original cell. It was proved that the demulsifying substances had a significant effect on cell surface properties and accordingly with demulsifying capability of Alcaligenes sp. S-XJ-1.

  9. Nerve growth factor-sensitive S6 kinase in cell-free extracts from PC12 cells

    SciTech Connect

    Matsuda, Y.; Nakanishi, N.; Dickens, G.; Guroff, G.

    1986-05-01

    Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a 2-10 fold increase in the ability to phosphorylate the ribosomal protein S6. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of (/sup 32/P) from (/sup 32/P)ATP into S6 occurred after 4-7 minutes of nerve growth factor treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000 and is not inhibited by the inhibitor of cAMP-dependent protein kinase, NaCl, or trifluoperazine, nor is it activated by the addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase, protein kinase C, nor the result of proteolytic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cyclic AMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. But the effect of NGF in increasing S6 kinase activity cannot be mimicked by treatment of control extract with cAMP-dependent protein kinase in vitro. Thus, it is unlikely to result from the phosphorylation of a less active form of the S6 kinase by a cAMP-dependent protein kinase.

  10. Longer Intestinal Persistence of Enterococcus faecalis Compared to Enterococcus faecium Clones in Intensive-Care-Unit Patients▿

    PubMed Central

    Ruiz-Garbajosa, Patricia; del Campo, Rosa; Coque, Teresa M.; Asensio, Angel; Bonten, Marc; Willems, Rob; Baquero, Fernando; Cantón, Rafael

    2009-01-01

    The dynamics of intestinal colonization with enterococcal clones in intensive-care-unit (ICU) patients was evaluated. Eight patients admitted directly to the neurosurgical ICU at the Ramón y Cajal University Hospital (Madrid, Spain) from the community and with no overlapping stay during a 10-month period in 2006 were studied. Rectal swab specimens were collected on admission and daily until the patients were discharged. Clonality was determined by pulsed-field gel electrophoresis and multilocus sequence typing. Clonal colonization dynamics were estimated by using two new parameters: the clonal diversity per patient per day (CDPD) and the clonal persistence ratio (CPR). Enterococcus faecalis isolates (n = 123) and Enterococcus faecium isolates (n = 66) were resolved into 13 and 15 clones, respectively. The CDPD of E. faecalis steadily increased during admission, and E. faecalis showed a higher (P = 0.001) CPR value than E. faecium (0.86 and 0.42, respectively). E. faecium, with the exception of an ampicillin-resistant clone belonging to clonal complex 17, frequently appeared as a short-term colonizer, even though the E. faecalis clones had significantly (P = 0.03) more days under antibiotic exposure than E. faecium (77.5 and 65 days/100 colonization days, respectively). E. faecalis had a longer persistence than E. faecium, except for the CC17 ampicillin-resistant clone, and E. faecalis showed a cumulative increase in CDPD, whereas E. faecium did not. CDPD and CPR were useful for measuring the dynamics of intestinal colonization with enterococcal clones. PMID:19052172

  11. Antimicrobial efficacy of different concentration of sodium hypochlorite on the biofilm of Enterococcus faecalis at different stages of development

    PubMed Central

    Frough-Reyhani, Mohammad; Soroush-Barhaghi, Mohammadhosien; Amini, Mahsa; Gholizadeh, Yousefreza

    2016-01-01

    Background Persistent infection of the root canal due to the presence of resistance bacterial species, such as Enterococcus faecalis, has always been one of the most important reasons for endodontic treatment failure. This study investigated the antimicrobial efficacy of 1%, 2.5 % and 5% sodium hypochlorite in eliminating E. faecalis biofilms at different stages of development. Material and Methods In this study 4-, 6- and 10-week-old E. faecalis biofilms were subjected to one of the following approaches: phosphate-buffered saline solution (PBS) or 1%, 2.5% and 5% NaOCl. Dentin chip suspensions were used for colony forming unit (CFU) counting to estimate remaining E. faecalis counts. Statistical comparison of the means was carried out with Kruskal-Wallis test, and pair-wise comparisons were made by Mann-Whitney U test, at a significance level of P<0.05. Results The results showed that 2.5% and 5% NaOCl completely eliminated E. faecalis biofilms in three stages of biofilm development, whereas 1% NaOCl resulted in 85.73%, 81.88% and 78.62% decreases in bacterial counts in 4-, 6- and 10-week-old biofilms, respectively, which was significantly more than those with PBS (p<0.05). Conclusions The bacteria in mature and old biofilms were more resistant to 1% NaOCl than were the bacteria in young biofilms. Overall survival rate and residual bacteria increased with biofilm aging. Key words:Antibacterial, biofilm, E. faecalis, sodium hypochlorite. PMID:27957257

  12. Phosphorylated S6K1 (Thr389) is a molecular adipose tissue marker of altered glucose tolerance.

    PubMed

    Moreno-Navarrete, José María; Ortega, Francisco; Sánchez-Garrido, Miguel Ángel; Sabater, Mònica; Ricart, Wifredo; Zorzano, Antonio; Tena-Sempere, Manuel; Fernández-Real, José Manuel

    2013-01-01

    Molecular tissue markers of altered glucose metabolism will be useful as potential targets for antidiabetic drugs. S6K1 is a downstream signal of insulin action. We aimed to evaluate (pThr389)S6K1 and total S6K1 levels in human and rat fat depots as candidate markers of altered glucose metabolism. (pThr389)S6K1 and total S6K1 levels were measured using enzyme linked immune sorbent assay (ELISA) in 49 adipose tissue samples from subjects with morbid obesity and in 18 peri-renal white adipose tissue samples from rats. The effects of high glucose and rosiglitazone have been explored in human preadipocytes. (pThr389)S6K1/(total)S6K1 in subcutaneous adipose tissue was significantly increased subjects with Type 2 diabetes (0.78 ± 0.26 vs. 0.55 ± 0.14, P=.02) and associated with fasting glucose (r=0.46, P=.04) and glycated hemoglobin (r=0.63, P=.02) in SAT. Similar associations with fasting glucose (r=0.43, P=.03) and IRS1 (r=-0.41, P=.04) gene expression were found in visceral adipose tissue. In addition, rat experiments confirmed the higher (pThr389)S6K1/totalS6K1 levels in adipose tissue in association with obesity-associated metabolic disturbances. (pThr389)S6K1/totalS6K1 was validated using western blot in rat adipose tissue. Both ELISA and western blot data significantly correlated (r=0.85, P=.005). In human preadipocytes, high glucose medium led to increased (pThr389)S6K1/total S6K1 levels in comparison with normal glucose medium, which was significantly decreased under rosiglitazone administration. In conclusion, in human and rat adipose tissue, phosphorylated S6K1 is a marker for increased glucose levels.

  13. Room-temperature ferroelectricity in CuInP2S6 ultrathin flakes

    DOE PAGES

    Liu, Fucai; You, Lu; Seyler, Kyle L.; ...

    2016-08-11

    In this study, two-dimensional (2D) materials have emerged as promising candidates for various optoelectronic applications based on their diverse electronic properties, ranging from insulating to superconducting. However, cooperative phenomena such as ferroelectricity in the 2D limit have not been well explored. Here, we report room-temperature ferroelectricity in 2D CuInP2S6 (CIPS) with a transition temperature of ~320 K. Switchable polarization is