Electrooptical Analysis of Microbial Cell Suspensions forDetermination of Antibiotic Resistance.

The effects of ampicillin; kanamycin, chloramphenicol, and tetracycline on electrophysical characteristics of cells of sensitive (ampicillin; kanamycin, chloramphenicol) and resistant (ampicillin; kanamycin, chloramphenicol, tetracycline) Escherichia coli strains were studied. Under the action of antibiotics sensitive and resistant E. coli strains acquire different electro-optical properties. Changes in suspension-orientational spectra, that are observed under the action of ampicillin; kanamycin, chloramphenicol, and tetracycline can be used in determination of antibiotic resistance of the studied bacterial strains. In our opinion, the methods of microbial suspension electro-optical analysis can be used in microbiology, mеdicinе, veterinary, and are an effective tool for solving the problems connected with determination of microbial cell antibiotic resistance.

Electro-optical study of the exposure of Azospirillum brasilense carbohydrate epitopes.

The exposure of Azospirillum brasilense carbohydrate epitopes was investigated by electro-optical analysis of bacterial cell suspensions. To study changes in the electro-optical (EO) properties of the suspensions, we used antibodies generated to the complete lipopolysaccharide of A. brasilense type strain Sp7 and also antibodies to the smooth and rough O polysaccharides of Sp7. After 18 hr of culture growth, the EO signal of the suspension treated with antibodies to smooth O polysaccharide was approximately 20% lower than that of the suspension treated with antibodies to complete lipopolysaccharide (control). After 72 hr of culture growth, the strongest EO signal was observed for the cells treated with antibodies to rough O polysaccharide (approximately 46% greater than the control), whereas for the cells treated with antibodies to smooth O polysaccharide, it was much lower (approximately 23% of the control). These data were confirmed by electron microscopy. The results of the study may have importance for the rapid evaluation of changes in lipopolysaccharide form in microbial biotechnology, when the antigenic composition of the bacterial surface requires close control.

Biological sensor based on a lateral electric field-excited resonator.

This paper describes a biological sensor based on a lateral electric field-excited resonator using an X-cut lithium niobate plate. Its potential was shown through the example of biological interaction between bacterial cells and specific bacteriophages. The detection was based on the analysis of the measured real and imaginary parts of electrical impedance for a resonator loaded by the biological suspension under study. It has been shown that the sensor is sensitive to specific interactions between bacterial cells and specific bacteriophages in a pure state as well as in the presence of extraneous microflora. The degree of electrical impedance variation resulting from the biological interaction depends on the numbers of phage particles and bacteria cells. The sensor may be used not only for the qualitative analysis of bacteria but also for their quantitative detection.

Preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens and their use for bacterial detection.

This article reports the first preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens by using a combinatorial phage library of sheep antibodies. The prepared phage antibodies were used for the first time for lipopolysaccharide and flagellin detection by dot assay, electro-optical analysis of cell suspensions, and transmission electron microscopy. Interaction of A. brasilense Sp245 with antilipopolysaccharide and antiflagellin phage-displayed miniantibodies caused the magnitude of the electro-optical signal to change considerably. The electro-optical results were in good agreement with the electron microscopic data. This is the first reported possibility of employing phage-displayed miniantibodies in bacterial detection aided by electro-optical analysis of cell suspensions.

A new electro-optical approach to rapid assay of cell viability.

A new electro-optical (EO) approach was developed and applied to rapidly assay cell viability by using phage M13K07. Since phage M13K07 can replicate only in living bacteria and cannot replicate in the presence of inhibitors, the difference between the EO signals obtained in the presence and absence of the phage can be used as an important factor for evaluating cell viability. Variation in the electrophysical parameters of Escherichia coli XL-1 during its interaction with phage M13K07 was studied under exposure of the cells to various inhibitors of cellular metabolism. Significant changes in the EO signal were found during incubation of living E. coli cells with phage M13K07. At the same time, no changes were recorded during cell incubation with the phage after pretreatment of E. coli XL-1 cells with sodium azide, carbonyl cyanide 3-chlorophenyl hydrazone, chloramphenicol, and kanamycin. This finding can be explained by the decrease in the number of living cells in the culture after preliminary incubation with the chemical agents, and it was confirmed by colony counts by conventional plating onto solid LB medium before and after treatment of the cells with the inhibitors. The EO approach can be used as a rapid method for evaluation of the inhibitory effects of various chemical agents and drugs, and it has the potential for the study of the molecular mechanisms underlying cell death.

Electrophysical characteristics of Azospirillum brasilense Sp245 during interaction with antibodies to various cell surface epitopes.

This work was undertaken to examine the electrooptical characteristics of cells of Azospirillum brasilense Sp245 during their interaction with antibodies developed to various cell surface epitopes. We used the dependences of the cell suspension optical density changes induced by electroorientation on the orienting field frequency (740, 1000, 1450, 2000, and 2800kHz). Cell interactions with homologous strain-specific antibodies to the A. brasilense Sp245 O antigen and with homologous antibodies to whole bacterial cells brought about considerable changes in the electrooptical properties of the bacterial suspension. When genus-specific antibodies to the flagellin of the Azospirillum sheathed flagellum and antibodies to the serologically distinct O antigen of A. brasilense Sp7 were included in the A. brasilense Sp245 suspension, the changes caused in the electrooptical signal were slight and had values close to those for the above changes. These findings agree well with the immunochemical characteristics of the Azospirillum O antigens and with the data on the topographical distribution of the Azospirillum major cell surface antigens. The obtained results can serve as a basis for the development of a rapid test for the intraspecies detection of microorganisms.

Studies of Listeria monocytogenes-antibody binding using electro-orientation.

An electro-optical (EO) approach has been used for studies of Listeria monocytogenes-antibody binding. The EO analyzer, which has been developed at the State Research Center for Applied Microbiology, Obolensk, was used as a basic instrument for EO measurements. AC electro-kinetic effects depend on dielectric properties of bioparticles, their composition, morphology, the medium, and the frequency of applied electrical field. Electro-orientational spectra were used for discrimination of bacteria before and after selective binding with 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 EO signal reaches its maximum at 100-200 kHz. It has been shown that the biospecific interactions of L. monocytogenes cells with anti-Listeria antibody in the presence of Escherichia coli K-12, and Azospirillum brasilense Sp7 change the EO signals significantly. 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 spectra of bacterium in the presence and in the absence of specific antibody.

Electrooptical analysis of the Escherichia coli-phage interaction.

This article describes electrooptical (EO) characterization of biospecific binding between the bacterium Escherichia coli XL-1 and the phage M13K07. The electrooptical analyzer (ELUS EO), which has been developed at the State Research Center for Applied Microbiology, Obolensk, Russia, was used as the basic instrument for EO measurements. The operating principle of the analyzer is based on the polarizability of microorganisms, which depends strongly on their composition, morphology, and phenotype. The principle of analysis of the interaction of E. coli with the phage M13K07 is based on registration of changes of optical parameters of bacterial suspensions. The phage-cell interaction includes the following stages: phage adsorption on the cell surface, entry of viral DNA into the bacterial cell, amplification of phage within infected host, and phage ejection from the cell. In this work, we used M13K07, a filamentous phage of the family Inoviridae. Preliminary study had shown that combination of the EO approach with a phage as a recognition element has an excellent potential for mediator-less detection of phage-bacteria complex formation. The interaction of E. coli with phage M13K07 induces a strong and specific EO signal as a result of substantial changes of the EO properties of the E. coli XL-1 suspension infected by the phage M13K07. The signal was specific in the presence of foreign microflora (E. coli K-12 and Azospirillum brasilense Sp7). Integration of the EO approach with a phage has the following advantages: (1) bacteria from biological samples need not be purified, (2) the infection of phage to bacteria is specific, (3) exogenous substrates and mediators are not required for detection, and (4) it is suitable for any phage-bacterium system when bacteria-specific phages are available.

Effect of p-nitrophenol metabolites on microbial cell electro-optical characteristics.

The effect of cellular p-nitrophenol (PNP) metabolism on the electro-optical (EO) characteristics of Pseudomonas putida C-11, P. putida BA-11, and Acinetobacter calcoaceticum A-122 was studied. When P. putida C-11 was incubated with hydroquinone, the orientational spectra of the cell suspensions changed considerably. When P. putida BA-11 and A. calcoaceticum A-122 were incubated with hydroquinone, no orientational spectrum changes were noted, possibly attesting to the operation of different PNP-metabolic pathways. In C-11, the initial metabolism of PNP may occur via the production of hydroquinone, an intermediate for PNP metabolism; in BA-11 and A. calcoaceticum A-122, via the production of 4-nitropyrocatechin, followed by a rupture of the aromatic ring. The respiratory activity of the strains toward hydroquinone was investigated concurrently. The results suggest that EO analysis is a good candidate for the study of cellular metabolism.