Antimicrobial Activities of Bacteriocins E 50-52 and B 602 Against Antibiotic-Resistant Strains Involved in Nosocomial Infections.

The antimicrobial spectra of previously published bacteriocin E 50-52 (39 a.a.; 3,932 Da; pI = 8.5) and bacteriocin B 602 (29 a.a.; 3,864 Da; pI = 7.2) were determined. Named peptides were related to class IIa (pediocin-like) bacteriocins. Minimal inhibitory concentrations (MICs) of bacteriocins have been determined for bacterial isolates that were causative agents of nosocomial infections collected from Russian hospitals in 2003-2007, namely methicillin-resistant Staphylococcus aureus (MRSA) (n = 10); Acinetobacter baumannii (n = 11); Citrobacter freundii (n = 8); Escherichia coli (n = 9); Klebsiella pneumoniae (n = 10); Proteus spp. (n = 6); and Pseudomonas aeruginosa (n = 10). The majority of these tested isolates have been shown to be multidrug resistant and carry genetic determinants of antimicrobial resistance that were detected using polymerase chain reaction (PCR). The MICs of bacteriocin B 602 ranged from ≤0.025-1.56 µg/ml, and for bacteriocin E 50-52 from 0.05 to 6.25 µg/ml for all of 64 bacterial clinical isolates tested. Interestingly, the bacteriocins studied demonstrate activity on both Gram-positive and Gram-negative bacteria. Bacteriocins E 50-52 and B 602 show good activity against nosocomial bacterial agents resistant to many classes of modern antibacterials used in clinical practice. These bacteriocins should be examined as an alternative in treating infections caused by such agents.

Isolation and purification of enterocin E-760 with broad antimicrobial activity against gram-positive and gram-negative bacteria.

Strain NRRL B-30745, isolated from chicken ceca and identified as Enterococcus durans, Enterococcus faecium, or Enterococcus hirae, was initially identified as antagonistic to Campylobacter jejuni. The isolate produced a 5,362-Da bacteriocin (enterocin) that inhibits the growth of Salmonella enterica serovar Enteritidis, S. enterica serovar Choleraesuis, S. enterica serovar Typhimurium, S. enterica serovar Gallinarum, Escherichia coli O157:H7, Yersinia enterocolitica, Citrobacter freundii, Klebsiella pneumoniae, Shigella dysenteriae, Pseudomonas aeruginosa, Proteus mirabilis, Morganella morganii, Staphylococcus aureus, Staphylococcus epidermidis, Listeria monocytogenes, Campylobacter jejuni, and 20 other Campylobacter species isolates. The enterocin, E-760, was isolated and purified by cation-exchange and hydrophobic-interaction chromatographies. The proteinaceous nature of purified enterocin E-760 was demonstrated upon treatment with various proteolytic enzymes. Specifically, the antimicrobial peptide was found to be sensitive to beta-chymotrypsin, proteinase K, and papain, while it was resistant to lysozyme and lipase. The enterocin demonstrated thermostability by retaining activity after 5 min at 100 degrees C and was stable at pH values between 5.0 and 8.7. However, activity was lost below pH 3.0 and above pH 9.5. Administration of enterocin E-760-treated feed significantly (P < 0.05) reduced the colonization of young broiler chicks experimentally challenged and colonized with two strains of C. jejuni by more than 8 log(10) CFU. Enterocin E-760 also significantly (P < 0.05) reduced the colonization of naturally acquired Campylobacter species in market age broiler chickens when administered in treated feed 4 days prior to analysis.

Bacteriocins reduce Campylobacter colonization and alter gut morphology in turkey poults.

Campylobacter is a leading cause of food-borne illness in the United States. Recent evidence has demonstrated that bacteriocins produced by Bacillus circulans and Paenibacillus polymyxa reduce cecal Campylobacter colonization in broiler chickens infected with Campylobacter jejuni. As Campylobacter coli is the most prevalent Campylobacter isolate recovered in turkeys, the objectives of the present study were to evaluate the efficacy of these bacteriocins against C. coli colonization and their influence on the gastrointestinal architecture of young turkeys. In 3 separate trials, a total of 135 day-of-hatch poults (n = 45/trial) were orally challenged on d 3 with approximately 10(6) cfu of a mixture of 3 C. coli isolates. Immediately before bacteriocin treatment (d 10), cecal Campylobacter concentrations averaged 1.1 x 10(7) cfu/ g of cecal contents (n = 15/trial). On d 10 to 12 posthatch, 2 bacteriocin treatment groups were given free access to feed supplemented with purified, microencapsulated bacteriocins, whereas the positive control treatment group had access to untreated feed (n = 10/treatment group per trial). At the end of the 3-d dosing period, ceca and duodenal loops were collected for analysis. In each of the 3 separate trials, treatment with bacteriocin eliminated detectable ceca Campylobacter concentrations (detection limit, 1 x 10(2) cfu/g of cecal contents) vs. controls (1.0 x 106 cfu of Campylobacter/g of cecal contents). Duodenum crypt depth and goblet cell numbers were also reduced in turkeys treated with either bacteriocin vs. controls (P < 0.05). The dynamic reduction in crypt depth and goblet cell density in turkeys dosed with bacteriocin may provide clues to how bacteriocins inhibit enteric Campylobacter.

Isolation of a Lactobacillus salivarius strain and purification of its bacteriocin, which is inhibitory to Campylobacter jejuni in the chicken gastrointestinal system.

We evaluated anti-Campylobacter jejuni activity among >1,200 isolates of different lactic acid bacteria. Lactobacillus salivarius strain NRRL B-30514 was selected for further study. The cell-free, ammonium sulfate precipitate from the broth culture was termed the crude antimicrobial preparation. Ten microliters of the crude preparation created a zone of C. jejuni growth inhibition, and growth within the zone resumed when the crude preparation was preincubated with proteolytic enzymes. Bacteriocin OR-7, derived from this crude preparation, was further purified using ion-exchange and hydrophobic-interaction chromatography. The determined amino acid sequence was consistent with class IIa bacteriocins. Interestingly, OR-7 had sequence similarity, even in the C-terminal region, to acidocin A, which was previously identified from L. acidophilus and had activity only to gram-positive bacteria, whereas OR-7 had activity to a gram-negative bacterium. Bacteriocin activity was stable following exposure to 90 degrees C for 15 min, also consistent with these types of antibacterial peptides. The purified protein was encapsulated in polyvinylpyrrolidone and added to chicken feed. Ten day-of-hatch chicks were placed in each of nine isolation units; two groups of birds were challenged with each of four C. jejuni isolates (one isolate per unit). At 7 days of age, one group of birds was treated with bacteriocin-emended feed for 3 days, and one group was left untreated. At 10 days of age, the birds were sacrificed and the challenge strain was enumerated from the bird cecal content. Bacteriocin treatment consistently reduced colonization at least one millionfold compared with levels found in the untreated groups. Nonchallenged birds were never colonized by C. jejuni. Bacteriocin from L. salivarius NRRL B-30514 appears potentially very useful to reduce C. jejuni in poultry prior to processing.

Hydrogenase electrodes for fuel cells.

Considering crucial problems that limit use of platinum-based fuel cells, i.e. cost and availability, poisoning by fuel impurities and low selectivity, we propose electrocatalysis by enzymes as a valuable alternative to noble metals. Hydrogenase electrodes in neutral media achieve hydrogen equilibrium potential (providing 100% energy conversion), and display high activity in H(2) electrooxidation, which is similar to that of Pt-based electrodes in sulphuric acid. In contrast with platinum, enzyme electrodes are highly selective for their substrates, and are not poisoned by fuel impurities. Hydrogenase electrodes are capable of consuming hydrogen directly from microbial media, which ensures their use as fuel electrodes in treatment of organic wastes.

Distribution and characterization of Campylobacter spp. from Russian poultry.

The distribution of Campylobacter spp. on 13 poultry farms (broiler chicken, quail, pheasant, peacock, and turkey) from eight regions (Vladimir, Vologda, Voronezh, Kaluga, Liptsk, Moscow, Orenburg, and Orel) in Russia was surveyed. Intestinal materials were plated onto Campylobacter-selective medium and plates were incubated microaerobically at 42 degrees C for 24 or 48 h. Identification was based on colonial morphology, microscopic examination, and biochemical tests; latex agglutination assays were used for confirmation. In total, 116 isolates were derived from 370 samples. Isolation rates were similar, regardless of whether the birds were from small or large broiler production farms. Susceptibility of 48 representative (from these production sources) strains of Campylobacter spp. to 38 antimicrobial compounds was determined by disk diffusion assays. All strains tested were sensitive to amikacin, gentamycin, sisomycin, chloramphenicol, imipenem, oleandomycin, erythromycin, azitromycin, and ampicillin. The strains were also sensitive to 100 microg/disk of carbenicillin, fluoroquinolones, and to nitrofurans. Fluoroquinolone sensitivity was most notable and may be related to its limited application in poultry production within Russia. Hippurate and ribosomal RNA gene primers were developed and used to distinguish Campylobacter jejuni and Campylobacter coli and to provide a measure of strain discrimination. The combination of PCR analysis and randomly amplified polymorphic DNA (RAPD) typing were conducted for selected isolates. The various poultry species and the different locations yielded Campylobacter isolates with discrete randomly amplified polymorphic DNA patterns. The distribution and substantial diversity of Campylobacter spp. isolates appears similar to that previously reported in other countries.

[Microbial degraders of some organochlorine compounds]. / Mikrobnye destruktory nekotorykh khlororganicheskikh soedinenii.

A possibility of isolation of microorganisms, potential destructors of chlorinated organics from aged Vietnamese soils polluted with dioxine-containing defoliants was demonstrated. As an example, the ability of one isolated strain to metabolize pentachlorophenol and 2,4-dichlorophenoxyacetic acid was shown under laboratory conditions. An attempt was made to identify intermediates of pentachlorophenol metabolism using HPLC.

[Microorganisms as possible indicators of soil pollution by dioxin-containing defoliants]. / Mikroorganizmy kak vozmozhnoe indikatory integral'noi zagriaznennosti pochv dioksinsoderzhashchimi defoliantami.

The diversity of microorganisms from soils treated in the past with various dosages of dioxin-containing defoliants was studied. Population alterations dependent on dioxin concentrations were elucidated. Soil fungi and, to a smaller extent, actinomycetes were found to be the most sensitive to dioxins.

[Specific electric resistance of a narrow gap between two lipid bilayer membranes]. / Udel'noe élektricheskoe soprotivlenie uzkoi shcheli mezhdu dvumia bisloinymi lipidnymi membranami.

The study deals with measurements of specific electric resistance of a narrow gap between two bilayer lipid membranes (BLM). The technique is based upon the influence of the outer electric field on the distance between two BLM which has been revealed earlier. The curve of specific resistance/solution ionic strength obtained suggests that surface conductivity is significant in similar systems. In gaps narrower than 40 nm possible increase of water viscosity must be also taken into consideration. A hypothetic mechanism of electric connection between cells is proposed.

[Electro-osmotic processes in the region of bilayer lipid membrane contact]. / Elektroosmoticheskie protsessy v oblasti kontakta bisloinykh lipidnykh membran.

The influence of outer voltage on a contact of two bilayer lipid membranes has been revealed and investigated. The effect was due to the origination of an electro-osmotic water flow in the contact region.

[Repair of membrane damage caused by low temperature freezing of E. coli cells]. / Izuchenie reparatsii povrezhdenii membrannogo apparata, vyzvannykh nizkotemperaturnym zamorazhivaniem kletok E. coli.

Low temperature freezing of E. coli cells causes an almost complete cell damage. A transfer of the frozen cells to nutritional media results in a repair of some of the damages, i.e. in reconstitution of the barrier stability of the E. coli outer membrane detected by a decrease in sensitivity of the frozen cells to the detergent and lysozyme action and in a change of the cell membrane potential measured by the penetrating ion method. The repair of the cytoplasmic membrane damage is followed by the changes in the permeability barrier for H+ and endogenous substrates, which results in restoration of ATP synthesis as a response to the artificial proton motive force and in an induction of beta-galactosidase synthesis. At the same time the synthesis of the periplasmic protein, alkaline phosphatase, in the cells after repair remains suppressed. An analysis of various biosynthetic processes demonstrated that the inhibition of lipid synthesis completely suppresses the reduction processes, while protein synthesis is not necessary for the repair. The importance of the transmembrane electrochemical proton gradient for the repair processed in E. coli cells was established; the ATP biosynthesis essential for the repair occurs, in all probability, via the glycolytic pathway and not via oxidative phosphorylation.

[Functional and structural changes of E. coli membranes induced by low temperature freezing]. / Issledobanie funktsional'nykh i strukturnykh izmenenii membrannogo apparata Escherichia coli posle nizkotemperaturnogo zamorazhivaniia.

Low temperature freezing of E. coli cells causes a fall in endogenous respiration and stimulation of respiration by the non-penetrant substrate NADH. This decrease is not due to disturbances in the function of electron transport chain, since the dehydrogenase and oxidase activities and cytochrome content in the membranes of intact and frozen cells are practically the same. Frozen E. coli cells are incapable of ATP synthesis by artificial proton motive force, although the ATPase activity of isolated membranes is not changed. The disturbances in the penetrability barrier of protons after freezing can be revealed from changes in pH of cell suspensions after rapid acidification. It is assumed that the cell penetrability barrier undergoes alterations causing a loss of respiration substrates and, probably, oxidative phosphorylation uncoupling. A correlation between constant damages of the membrane penetrability barrier and cell survival was established. Using spin labelling of different localization, the changes in the state of membrane surface at the intact hydrophobic lipid zone were demonstrated. It was found that freezing does not induce lipid peroxidation.