Isolation and characterization of Shiga toxigenic Escherichia coli of animal and bird origin by multiplex polymerase chain reaction.

AIM: The purpose of this study was to determine the virulence genes and serotype of Shiga toxin producing Escherichia coli (STEC) strains isolated from animals and birds. MATERIALS AND METHODS: A total of 226 different samples viz., fecal, intestinal content, rectal swab and heart blood were collected from different clinically affected/healthy animals and birds and were streaked on McConkeys' lactose agar and eosin methylene blue agar for isolation of E. coli, confirmed by staining characteristics and biochemical tests. By polymerase chain reaction (PCR) all the E. coli isolates were screened for certain virulence genes, viz., Shiga toxin 1 (stx1), stx2 and eae and enterohemolytic (Ehly) phenotype was observed in washed sheep blood agar plate. All the isolated E. coli strains were forwarded to the National Salmonella and Escherichia Centre, Central Research Institute, Kasauli (Himachal Pradesh) for serotyping. RESULTS: Out of 226 samples 138 yielded E. coli. All the isolates were screened for molecular detection of different virulent genes, viz. stx1, stx2 and eae, based on which 36 (26.08%) were identified as STEC. Among those STEC isolates, 15 (41.67%), 14 (38.89%), 1 (2.78%) exhibited eae, stx2, stx1 alone, respectively, whereas 4 (11.11%) and 2 (5.56%) carried both stx1 and stx2, stx2 and eae, respectively. Among the STEC isolates 22 were belonged to 15 different sero-groups, viz., O2, O20, O22, O25, O43, O60, O69, O90, O91, O95, O106, O118, O130, O162 and O170 and others were untypable. Ehly phenotype was observed in 10 (27.78%) the STEC isolates. CONCLUSION: The present study concluded that STEC could be isolated from both clinically affected as well as healthy animals and birds. Regular monitoring of more samples from animal and bird origin is important to identify natural reservoir of STEC to prevent zoonotic infection.

Specific bio-recognition reactions observed with an integrated Mach-Zehnder interferometer.

The combination of an integrated Mach-Zehnder-interferometer (iMZI) at the bottom of a fluidic microchannel system with supramolecular interfacial binding layers optimized for biosensing purposes is described. The model system used is based on the highly specific interaction of streptavidin to its 'ligand' biotin: a single monolayer of a correspondingly derivatized silane-compound is formed by a self-assembly procedure on top of the channel rib guiding the light through the channels. Injection of a streptavidin solution which leads to the formation of a protein monolayer of d = 2.8 nm in effective thickness results in a phase shift of the sample light relative to the reference channel of delta phi = 6 pi, in good agreement with the theoretical sensitivity of delta phi/delta df = 2.9 pi/nm for a protein layer (n = 1.45) calculated for the device.

Translocation of alkali metal cations by lipophilic cyclodextrin derivatives through black lipid membranes.

Lipophilic cyclodextrin (CD) derivatives, synthetic ionophores, were prepared to transport alkali metal cations across a black lipid membrane (BLM). The purpose of this study is to develop a new class of an artificial transportation system of alkali metal cations via bilayer lipid membranes, by using CD derivatives as a cation carrier. A lipophilic CD derivative incorporated into a BLM forms a complex with an alkali metal cation at one surface of the membrane. This charged complex migrates to the opposite side of the membrane and then releases the cation into the subphase. CD derivatives have various types of acyl groups as a complexing site and formed a 1:1 complex with the alkali metal cation. The complex formation was interpreted by an induced-fit mechanism. It is found that the ability of CD derivative for forming a complex and/or transporting cations across the BLM depends on the bulkiness of acyl groups. The conductivities of heptakis (2,6-di-O-propyl-3-O-propionyl)-beta-CD were higher than those of valinomycin regardless of sizes of cations. The order of the conductivity in all derivatives is Li+ < Na+ < K+ approximately = Rb+ approximately = Cs+, regardless of the types of acyl groups in the derivatives. The effects of alkali metal cation concentration in the aqueous phase and CD concentration in the membrane on the translocation are also discussed.

Design and test of a simple high-temperature laser microrefractometer.

The design of a laser microrefractometer that is suitable for temperature-dependent measurements is described. The refractive index of methylene iodide is measured in the temperature range of 22-92 °C for laser wavelengths covering almost the entire visible range of the spectrum: 442, 488, 515, 543, 594, and 633 nm. A detailed analysis of the temperature-related experimental error is made.

Polarization-dependent photothermal beam deflection for the determination of the order parameter C(2) in a laser-oriented polymer system.

We demonstrate polarization-dependent photothermal beam deflection as a powerful tool for analyzing quasi-two-dimensional molecular orientation. As examples we used two laser-beam-oriented polymer systems: DR1 in poly(methyl methacrylate) (PMMA) and PMMA with covalently bound DR1 and for comparison Phenol Blue in PMMA. Different order parameters C(2), for these systems have been found. It was also possible to orient by laser beam the DR1 chromophores cyclically by changing the polarization direction of the orienting laser beam and following these chromophore reorientations. The long-term stability of the orientation was investigated as well. The irreversible bleaching that is due to this laser treatment could be determined. Angular hole burning could easily be detected in these systems.

Electron microscopic investigations on free-standing mixed lipid Langmuir-Blodgett-Kuhn monolayers: phase separation and aging process.

Lipid monolayers were prepared by the Langmuir-Blodgett-Kuhn technique (LBK) as free-standing films spanning a diameter of up to 1 micron. These films were investigated by electron spectroscopic imaging (ESI) and electron energy loss spectroscopy (EELS). The free-standing monolayer is shown to be in a transient state in which an aging process is proceeding: after storage for two weeks in air at room temperature the films tear off the edges of the perforated supporting film. Ca2+ ions induced lateral phase separation in these films prepared from a 50:50 mixture of lecithin/glycerol could be visualized by means of ESI, i.e., by comparing micrographs below and above the Ca absorption edge in the EEL spectrum. The domain sizes of the demixed phases were determined to vary between 30 and 60 nm. In addition it was shown that the counter ion of the negatively charged glycerol in these films is Ca2+ and not Na+.

Ca(2+)-induced lateral phase separation in black lipid membranes and its coupling to the ion translocation by gramicidin.

We analyze the single-channel current fluctuations of gramicidin incorporated into biomolecular lipid membranes (BLM) of binary mixtures of phosphatidylcholine (PC) and phosphatidylglycerol (PG) as a function of the Ca2+ concentration in the electrolyte (0.5 M CsCl, pH 6) solution. At low Ca2+ levels (cCa2+ < 10(-6) M) a monomodal conductance histogram and a single average lifetime suggests a homogeneous mixture over the full range of composition (PG(1-x)PCx, 0 < or = x < or = 1). At higher Ca2+ concentrations phase separation processes are inferred from the appearance of bimodal conductance histograms. The two channel populations (in the two coexisting phases) can also be distinguished through their different average lifetimes. By a systematic variation of the mole fractions of the two lipid components we derive the respective phase boundaries and thus the full Ca2+ concentration-composition phase diagram.

Lateral order in binary lipid alloys and its coupling to membrane functions.

Densitometry, Raman spectroscopy and small angle neutron scattering are employed to elucidate the miscibility behavior of lipid mixtures organized as liposomal dispersions. First, temperature-composition-phase diagrams for several binary alloys of dialkyl-lecithins differing in chain lengths by an increasing number of CH2-groups are derived. A mixture of dimyristoyllecithin and distearoyllecithin (delta CH2 = 4) shows a peritectic phase behavior with a miscibility gap in the gel state. In the fluid phase, at high enough temperatures, homogeneous mixtures of the two components are formed at all molar ratios. However, upon approaching a critical point by either lowering the temperature or increasing the hydrostatic pressure, critical concentration fluctuations are observed. If one component of a binary mixture is charged, electrostatic interactions can be used to induce phase separation at constant temperature. This is demonstrated for Ca2(+)-driven demixing in alloys of lecithin and negatively charged phosphatidylglycerol. The influence of the various concepts for the induction of lateral structure formation in lipid membranes on integral functional units like ionophores is demonstrated by analysing the single channel current fluctuations of gramicidin in bimolecular lipid membranes. Ca2+, as well as polyelectrolyte-induced phase separations are shown and discussed as examples for the important (lateral) order-function relationship in biomembranes.

pH-control of the miscibility properties of a binary lipid alloy and its influence on the ion transport by gramicidin.

We studied the coupling of a membrane function (the transport of ions by the pore forming polypeptide gramicidin) to chemically driven phase changes in black membranes of binary lipid mixtures. In particular, we investigated the influence of the aqueous pH value on the fluid-fluid demixing effect of Ca2+ to phosphatidylcholine/phosphatidylglycerol bilayers. It is found that one can switch, under certain conditions, between a homogeneously mixed and a phase separated membrane by changing the pH. We interpret this as being caused by the change in the degree of dissociation of one of the lipid components.

Phase separation in bimolecular mixed lipid membranes induced by polylysine.

We demonstrate, for the first time, polylysine-induced phase separation in a bimolecular lipid membrane of a lecithin/phosphatidylglycerol-mixture by analysing the single channel current fluctuations of gramicidin. The bimodal conductance histograms are direct evidence for the incorporation of the transport system into the two coexisting phases of different composition.