Use of an electro-optical sensor and phage antibodies for immunodetection of Herbaspirillum.

A sheep single-chain antibody-fragment library (Griffin.1, UK) was used to obtain miniantibodies to the lipopolysaccharide of Herbaspirillum seropedicae Z78. Using electro-optical analysis and electron microscopy, we recorded a biospecific interaction of antigenic determinants on the cell surface with phage antibodies against the LPS of H. seropedicae Z78 (mini-AbsLPS). Control experiments were run to rule out nonspecific binding of the mini-AbsLPS to cells of Azospirillum brasilense Sp245. Use of the highly specific mini-AbsLPS enabled the lipopolysaccharide of H. seropedicae Z78 to be detected in a mixture of bacterial cells by electro-optical means (analysis time, ∼5 min). This report is the first to show the possibility of rapid detection of Herbaspirillum on the basis of electro-optical analysis coupled with the use of mini-AbsLPS. The results are promising for the development of biosensor-based methods to detect potentially human-harmful prokaryotes whose structures either have not been studied or are absent from commercial databases.

[Change of Electrophysical Properties of Escherichia coli Cells Due to Levomycetin and Tetracycline Action].

The effect of chloramphenicol and tetracycline, as inhibitors of protein synthesis, on electrophysical properties of Escherichia coli K-12 cells was investigated. Significant changes in the orientation spectra (OS) of the cell suspensions incubated with various concentrations of chloramphenicol were observed only at the first five frequencies of the electric field (10-1000 kHz). When the cells were exposed to chloramphenicol (1.5 mcg/ml) or tetracycline (1.7 mcg/ml), no changes in the OS were recorded. Significant changes in the electrooptic signal were observed, when the K-12 cells were simultaneously incubated with chloramphenicol (1.5 mcg/ml) and tetracycline (1.7 mcg/ml), that could be due to the synergistic action of the antibiotics. Therefore, the electrooptic analysis provided registration of higher antibacterial effect with the simultaneous use of chloramphenicol and tetracycline. Additional control experiments with the cell culture on the LB nutrient medium containing chloramphenicol and tetracycline were performed. The results suggested that the use of electrophysical methods for investigation of antibiotics effect on microorganisms was rather efficient.

Electrooptical Assay for Record of Antibiotic Action on Microbial Cells.

Development of rapid and sensitive procedures for determination of microbial resistance to antibiotics is one of the most urgent trends in microbiology. The problem is shown to be solved by using electrooptical assay based on change of the electrophysical properties of suspended bacterial cells exposed to antibiotics with different mechanisms of action. Possible determination of the microbial cell susceptibility to antibiotics and their antibacterial activity is demonstrated. The results showed the procedure of electrooptical assay to be prospective in solving the problem of the microbiol cells antibiotic susceptibility in microbiology, medicine and veterinary.

[Determination of Microbial Susceptibility to Sulfanilamides by Electrooptic Analysis].

The effect of sulfanilamides (soluble streptocid as an example) on changing of the electrophysical properties (EP) of microbial cells of Escherichia coli XL-1, BL-Ril, Pseudomonasputida C-11 and BA-11 was studied. It was shown that significant changes in the orientation spectra (OS) of the cell suspensions incubated at various concentrations of the sulfanilamide resulted in changing of the electrooptic (EO) signal of the cell suspension at the first five frequencies of the orientation electric field (10-1000 Hz) with the use of soluble streptocid in a concentration of 0.3 mcg/ml. The dynamics of the drug effect on the microbial cells demonstrated a decrease of the EO signal value 5 minutes after the exposure by -59% vs. the control (the cells not exposed to the drug). During the following exposure the EO signal value practically did not change (within 5%). The changes of the OS of the cell suspensions exposed to soluble streptocid significantly differed for the susceptible and resistant strains. Determination of the activity of sulfanilamides by electrooptic analysis of microbial cell suspensions was considered possible. Changing of the microbial suspencion OS under the effect of sulfanilamides can be used as a test on the microbial cell susceptibility to drugs.

[Determination of Azospirillum Brasilense Cells With Bacteriophages via Electrooptical Analysis of Microbial Suspensions].

The dependence-of changes in the electrooptical properties of Azospirillum brasilense cell suspension Sp7 during interaction with bacteriophage ΦAb-Sp7 on the number and time of interactions was studied. Incubation of cells with bacteriophage significantly changed the electrooptical signal within one minute. The selective effect of bacteriophage ΦAb on 18 strains of bacteria of the genus Azospirillum was studied: A. amazonense Ami4, A. brasilense Sp7, Cd, Sp107, Sp245, Jm6B2, Brl4, KR77, S17, S27, SR55, SR75, A. halopraeferans Au4, A. irakense KBC1, K A3, A. lipoferum Sp59b, SR65 and RG20a. We determined the limit of reliable determination of microbial cells infected with bacteriophage: - 10(4) cells/mL. The presence of foreign cell cultures of E. coli B-878 and E. coli XL-1 did not complicate the detection of A brasilense Sp7 cells with the use of bacteriophage ΦAb-Sp7. The results demonstrated that bacteriophage (ΦAb-Sp7 can be used for the detection of Azospirillum microbial cells via t electrooptical analysis of cell suspensions.

[Electrooptical parameters of kanamycin-treated E. coli cell suspensions].

The effect of kanamycin on the electrophysical parameters of cell suspensions of Escherichia coli K-12 and pMMB33 was investigated. Incubation of the sensitive K-12 strain with kanamycin resulted in significant changes in the orientation spectra (OS) of the cell suspensions; these changes were not revealed in the case of the resistant pMMB33 strain. In the case of the sensitive K-12 strain incubated with different kanamycin concentrations, changes in the OS of the cell suspensions occurred within the 10-1000 kHz frequency range of the orienting electrical field. The most pronounced change in the electrooptical signal was observed at 10 microg/ml of kanamycin. Control experiments were carried out by standard plating on nutrient media. Thus, the OS changes of suspensions in the presence of antibiotics may be used as a test for microbial resistance to such antibiotics.

[Changes in electrophysical characteristics of Azospirillum brasilense Sp7 cells during their interaction with polyclonal antibodies].

Electrooptical characteristics of Azospirillum brasilense Sp7 cells during their specific interaction with polyclonal rabbit antibodies were studied. Dependence of optical density of cell suspension during electroorientation of cells from frequency of orienting field in interval 10, 100, 250, and 500 kHz was evaluated. Itwas shown that electrooptical (EO) characteristics of bacterial suspensions change during interaction of A. brasilense cells with antibodies, and maximal changes occur when frequency of oriented field amounts 100-250 kHz. During interaction of A. brasilense Sp7 with strain-specific polyclonal antibodies in the presence of Escherichia coli K-12 and Pseudomonas putida C-11 decrease of amplitude of analytic signal was observed but detection of A. brasilense Sp7 cells was possible. Possibility of detection of microorganisms by EO analysis during their interaction with antibodies was shown.

[Electrooptic analysis of Escherichia coli cell suspension for assessment of antibacterial activity of levomycetin].

Influence of chloramphenicol on electrophysiologic charateristics of Escherichia coli strains susceptible (K-12 strain) and resistant (pBR-325 strain) to it has been studied. It has been shown that incubation of susceptible bacteria with chloramphenicol leads to significant change of magnitute of electrooptic (EO) signal. Significant changes in orientantional spectra of suspensions of susceptible to chloramphenicol cells incubated with different concentrations of antibiotic were observed only on first five frequencies of orienting electric field (10 - 1000 kHz). Maximal change of EO signal occurred at chloramphenicol concentration 35 mg/ml and it didn't depend on the time of antibiotic exposure. Incubation of resistant strain pBR-325 with chloramphenicol did not lead to change of EO parameters of cell suspension. Potential for use of electrophysical analytic methods for assessment of antibacterial activity of chloramphenicol to control effect of antibiotics on microorganisms has been proposed.

[The electrooptical parameters of suspensions of Escherichia coli XL-1 cells interacting with helper phage M13K07].

The electrophysical properties of Escherichia coli XL-1 cells interacting with helper phage M13K07 were studied as a function of the phage-to-cell ratio and the contact time. The electro-optical signal of bacterial cells changed considerably as soon as 10 min after the onset of their incubation with phage particles, presumably due to phage adsorption on the cell surface. The maximum changes in the orientational spectra of cell suspensions were observed when the phage-to-cell ratio was 20. Selectivity studies showed that E. coli XL-1 cells interacting with the helper phage M13K07 in the presence of foreign microflora, such as E. coli K-12 or Azospirillum brasilense Sp7, can be identified by using their electrophysical properties. Changes in the orientational spectra of cell suspensions are interpreted with the stage of phage-bacterium interaction taken into account. The results obtained can probably be used to devise a new rapid method for identification of microorganisms and to study the particular stages of cell infection by bacteriophages.

[The effect of ampicillin on the electrophysical properties of Escherichia coli cells].

The study of the effect of ampicillin on the electrophysical properties of Escherichia coli cells showed that this antibiotic influences the orientational spectra (OS) of the ampicillin-susceptible E. coli strains K-12 and XL-1 within the frequency range 10-1000 kHz of the orienting electric field and does not affect the OS of the ampicillin-resistant strains K-12(pUC-18) and XL-1(pHEN1). The change in the electrooptical signal of the ampicillin-susceptible cells was maximum at an ampicillin concentration of 50 microg/ml and did not depend on the exposure time. The conclusion is drawn that changes in the OS of cells can be used to evaluate their resistance to ampicillin.

[Investigation of electrophysical properties of Listeria monocytogenes cells during the interaction with monoclonal antibodies].

An electrooptical approach was used in studies of Listeria monocytogenes-antibody binding. An electrooptical analyzer, which has been developed at the State Research Center for Applied Microbiology (Obolensk, Russia), was used as a basic instrument for electrooptical measurements. The analyzer consists of the following modules: a sample preparation module, a mixer, an AC field generator, an EO-flow cell, a microcontroller for transfer of liquid, a thermal system, an operator interface, and an image processor. The sample preparation module includes a unit for an automatic filter changing device and a hydraulic system. Since the AC electrokinetic effects depend on the dielectric properties of bioparticles, their composition, morphology, phenotype, the medium, and the frequency of applied electrical field, the electroorientational spectra were used for discrimination of different types of bacteria, a given type being "controlled" (and identified) by the selective choice of binding agents (antibodies). The measurements were performed using a discrete set of frequencies of the orienting electric field (10, 100, 250, and 500 kHz). During biospecific interactions, an antibody is bound to the microorganism, causing a change in the dielectric properties of the microorganism-antibody complex, and the electrooptic signal reaches its maximum at 100-200 kHz. It was shown that the biospecific interactions of Listeria monocytogenes cells with anti-Listeria antibody in the presence of E. coli K-12, and A. brasilense sp7 significantly change the electrooptical signals. Thus, the determination of the presence of particular bacteria within a mixed sample may be achieved by selection and matching of antibodies specific to individual bacterium types and by comparing the spectra of bacterium in the presence and in the absence of specific binding agent (antibody).

[Changes in electrophysical properties of Escherichia coli K-12 cells under the action of kanamycin and tetracycline].

Essential changes in the orientation spectra (OS) of cell suspensions incubated at different concentrations of kanamycin, were found to occur only in the first 5 frequencies of the orienting electrical field (10-1,000 kHz)). The maximum change in the intensity of the electrooptical signal occurred at a concentration of kanamycin of 10 microg/ml. Antibiotic concentration of 5 microg/ml caused no changes in OS. During the incubation of the cells with tetracycline (1.7, 2.5, 5.0 microg/ml) no changes in OS of the cell suspension were registered. Considerable changes in the intensity of the electrooptical signal occurred during the incubation of the cells with kanamycin (5 microg/ml) and tetracycline (1.7 microg/ml) simultaneously, which was due to the synergic action of these two antibiotics. Thus, as found with the use of the electrooptical analysis, the joint action of kanamycin and tetracycline could increase their antibacterial effect. The results demonstrated the effectiveness of using electrophysical methods for the study of the mechanism of action of antibiotics on bacterial cells and for the control of such action.

[The electro-optical investigation of suspensions of Escherichia coli K-12 cells metabolizing glucose, lactose, and galactose]. / Electroopticheskie issledovahiia kletochnoi suspenzii shtamma Escherichia coli K-12 pri metabolizme gliukozy i laktozy.

The electro-optical characteristics of suspensions of Escherichia coli K-12 cells metabolizing glucose, lactose, and galactose were studied my measuring the suspension turbidity as a function of cell alignment in an orienting electric field whose frequency was varied from 10 kHz to 10 MHz. In a frequency range of 10 kHz to 1 MHz, the orientational spectra of E. coli K-12 cells grown on glucose and lactose considerably changed after their incubation in the presence of the sugars. These changes likely reflect alterations in the polarizability of the cells induced by sugar metabolism.

Comparison of the electrooptical properties and specific respiratory activity of Acinetobacter calcoaceticum A-122.

The electrooptical (EO) properties of a cell suspension and the specific respiratory activity of cells towards p-nitrophenol (PNP) were compared during PNP metabolism in Acinetobacter calcoaceticum strain A-122. The frequency dependence of the suspension's turbidity changes due to cellular orientation (orientational spectra) at frequencies of an orienting electric field of 10-10,000 kHz was determined. Orientational spectral changes observed during PNP incubation of the cells were followed over the range of 10-502 kHz. There were linear relationships between the magnitude of the EO effect at a 502-kHz frequency and the concentration of PNP over the range of 0.1-0.8 mM, and between the specific respiratory activity of the cells and the concentration of PNP over the range of 0.1-1.0 mM. The knowledge gained from these studies suggests a direct relationship between alterations in the cellular EO properties and PNP metabolism.

An investigation into the electro-physical characteristics of microbial cells during the metabolism of toxic compounds under conditions of limited O2 availability.

The purpose of the work reported here was to experimentally clarify the interconnection between changes in the electro-physical characteristics of microbial suspensions and processes of the metabolism of certain toxic compounds (acrylamide and p-nitrophenol (PNP)) in cells containing enzyme systems of the initial metabolism of these compounds. In this work, we used cells of two strains, Acinetobacter calcoaceticum A-122 and Brevibacterium sp. 13PA, which are capable of utilising PNP and acrylamide, respectively, as the sole source of carbon. Suspensions of these cells exhibited appreciable decreases in the magnitude of the electro-optical signal when the microbial metabolism of PNP and acrylamide was inhibited under conditions of limited O2 supply. This attests to a relationship between the electro-physical characteristics of microbial suspensions and cellular metabolic reactions and also to the negligibly small effect that the non-specific interaction of substrate with the cells has on the suspensions' electro-optical properties.

Electrophysical monitoring of culture process of recombinant Escherichia coli strains.

A novel method for monitoring the cell culture process has been developed. The method is based on the measurements of electro-optical characteristics of cell suspension, calculation of cell structure parameters, and the relationship between accumulation of proteins and change of these parameters' employment. Application of the method for the monitoring of a culture process of a recombinant strain is considered. The process of growth of recombinant strains cannot be sufficiently predicted and the direct measurement of cell culture parameters is unlikely to be the most efficient way of solving the problem.Escherichia coli plasmid-free and recombinant strains synthesizing the fusion protein consisting of tumor necrosis factor-alpha (TNF) and thymosin-alpha(1) (T) were studied. It was found that cytoplasmic electroconductivity of the strains investigated increased during the culture process. The accumulation of insoluble recombinant pThy-315-encoded hybrid protein TNF(SINGLEBOND)T in cells resulted in a decrease of the membrane dielectric permeability. To determine variations of membrane dielectric permeability the amount of insoluble recombinant protein TNF(SINGLEBOND)T in the bacterial cells should be calculated.

[Determination, by the electro-optical method, of the number of undamaged bacterial cells after extreme exposures]. / Opredelenie élektroopticheskim metodom chisla nepovrezhdennykh bakterial'nykh kletok posle ékstremal'nykh vozdeistvii.

The results of electro-optical measurements can be used to determine rapidly how the number of intact cells decreases before and after the action of certain extreme factors. The experiments were conducted with suspensions of Escherichia coli and Bacillus thuringiensis cells subjected to the action of heat, toluene, ethanol, and freezing. Electro-optical measurements can make it possible to assay the relative number of cells with both lethal and sublethal damages.

[Analysis of the changes in the electrophysical and morphometric parameters in the growth and sporulation periods of Bacillus thuringiensis]. / Analiz izmenenii élektrofizicheskikh i morfometricheskikh parametrov v period rosta i sporuliatsii u Bacillus thuringiensis.

Changes in the electrophysical and morphometrical parameters of Bacillus thuringiensis cells were studied during their growth and differentiation using the electrophysical procedure with the frequency of an alternating electric field varying within a wide range. The appearing variations were interpreted on the basis of the data about changes in the concentration of vegetative cells and spores, the optical density and pH of the cultural broth, and the assimilation of glucose by the culture. The changes found in the electrophysical and morphometrical parameters were shown to accompany the main physiological processes in the cells. In principle, they can be used to control the process of cultivation.