Microbial acoustic sensor test-system based on a piezoelectric resonator with a lateral electric field for kanamycin detection in liquid.

A microbial test-system for real-time determination of low/residual concentrations of kanamycin in a liquid without the need for special labels is presented. The main element of the system was a piezoelectric resonator excited by a lateral electric field based on an X-cut lithium niobate plate 0.5 mm thick with two rectangular electrodes on one side. On the other side of the resonator, there was a 1.5 ml liquid container. As a sensory element we used Escherichia coli B-878 microbial cells, which are sensitive to kanamycin. For measurement 1 ml of this cells suspension was placed in a liquid container and then the test liquid in the amount of 2 µl containing kanamycin was added. The change in the real part of the electrical impedance of the resonator before and after the test liquid addition was used as an analytical signal which indicated the presence of kanamycin. The lower limit of determination of kanamycin turned out to be 1.0 µg/ml with an analysis time of 10 min. The test-system allows to detect kanamycin in the presence of such antibiotic as ampicillin and polymixin.

New approach for determination of antimicrobial susceptibility to antibiotics by an acoustic sensor.

For the first time, a rapid method was proposed to determine the susceptibility of Escherichia coli cells to antibiotics by the example of ampicillin by using a biological sensor based on a slot mode in an acoustic delay line. It has been established that an indicator of the antibiotic activity to microbial cells is the difference between the recorded sensor's signal before and after exposure cells with antibiotic. The depth and frequency of the peaks of resonant absorption in the frequency dependence of the insertion loss of sensor varied after adding an antibiotic with different concentrations to the microbial cells. By using the acoustic sensor based on slot-mode a criterion of E. coli sensitivity to ampicillin was established. The advantages of this method are the ability to carry out the analysis directly in the liquid, the short analysis time (within 10-15 min), and the possibility to reusable sensor.

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.

Sensor for ampicillin based on a microwave electrodynamic resonator.

The paper describes a new biological sensor which represents a resonator based on a segment of a rectangular waveguide of 8 GHz band with shear dimensions of 28.5 × 12.6 mm2. On one side, the resonator is bounded by a metallic short-circuited wall; on the other side, it is bounded by a lithium niobate plate with a porous polystyrene film. This film, applied by centrifugation and modified in high-frequency discharge plasma in argon, was used to immobilize cells of Escherichia coli K-12. This resonator was connected through a coaxial-waveguide adapter to the S parameter meter, by means of which the reflection coefficient S11 in the plane of the lithium niobate plate was measured. The addition of an aqueous solution of ampicillin at 4-50 µg/ml to immobilized cells led to a significant change in the reflection coefficient of S11 from - 10.15 dB to - 15.09 dB. At the same time, the resonance frequency changed insignificantly within the range 8.06-8.068 GHz. The optimal time for modifying the polystyrene film for obtaining the required porosity and the optimal time for the immobilization of the bacterial cells were determined. The immobilized cells retained their activity for 4 months at a temperature of 4 °C. The study showed the promise of such a biosensor to determine ß-lactam antibiotics in aqueous solutions by using ampicillin as an example. The limit of detection of the developed biosensor with respect to ampicillin was established (4 µg/ml).

Analysis of the microbial cell-Ab binding in buffer solution by the piezoelectric resonator.

The possibility of the registration of the interaction of the cells Azospirillum lipoferum Sp59b with the specific antibodies directly in the conducting suspensions by using an acoustic sensor was shown. The main element of the sensor is a piezoelectric resonator with a lateral electric field. The analysis is based on a comparison of the resonator's electrical impedance before and after the specific biological interaction between the cells and antibodies. By using this sensor one can detect and identify the bacterial cells directly in the buffer solution with the conductivity between 2.4 and 20 µS/cm. The minimum detectable concentration of the bacterial cells turned out to be ∼103 cells/ml and for a short time (less than 10 min). Also the possibility of the detection of the cells in the presence of the extraneous microflora was shown. The results provide the opportunities for the development of a new class of the methods for the analysis of the microbial cells in real-time directly in the buffer solution.

[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.

[A STUDY OF THE ISOLATED BACTERIOPHAGE ΦAB-SP7 ADSORPTION ON THE CELL SURFACE OF THE AZOSPIRILLUM BRASILENSE SP7].

The bacteriophage ΦAb-Sp7 was isolated from the cells of the Azospirillum brasilense Sp7. The morphology, size of the gram-negative colonies, and range of lytic activity against other strains and species of the genus Azospirillum was tested. The isolated phage DNA was examined using electrophoretic and restriction analysis, and the size of the genome were established. The electron microscopy. resuIts show that the phage (capsid) has a strand-like form. The electron microscopy study of the bacteriophage ΦAb-Sp7 adsorption on the A. brasilense Sp7 bacterial surface was performed.

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 a Spectrum of Lytic Activity of Bacteriophages by the Method of Acoustic Analysis].

The changes in the electro-acoustic parameters of cell suspension due to the interaction of cells with bacteriophages both in a pure. culture and in the presence of extraneous microflora were investigated. It has been found that the specific changes in the electroacoustic parameters of cell suspension under the action of bacteriophage occur only in microbial cells which are sensitive to the bacteriophage studied. It has been established that a sensor unit allows of distinguishing a situation when the bacterial cells are infected with specific bacteriophages of the control experiments and a situation with no introduction of infection. An approximate criterion of the presence of specific interactions of bacteriophages and cells in suspension was developed. In accordance with this criterion the change in electrical impedance of the sensor unit must not be less than - 1%. In control experiments a standard microbiological technique, plating the cells infected with bacteriophages on solid nutrient medium, was used. For the first time the possibility of using the method of electroacoustic analysis for determination of a spectrum of lytic activity of bacteriophages was shown. The results obtained may be used for development of a new express method for determining the sensitivity to bacteriophages of the microbial cells.

[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 organophosphorus aromatic nitro insecticides and p-nitrophenol by microbial-cell respiratory activity.

We examined the possibility of measuring the organophosphorus aromatic nitro insecticides metaphos and sumithion as well as their hydrolysis product p-nitrophenol (PNP) by the specific respiratory activity (SRA) of Pseudomonas putida C-11, P. putida BA-11, and Acinetobacter calcoaceticum A-122. The plots of cellular SRA against the two insecticides and PNP were linear over the ranges of 0.5-2.5 microM for P. putida C-11 and BA-11 and 0.5-1.0 microM for A. calcoaceticum A-122. P. putida BA-11 showed the greatest respiratory-response selectivity in the determination of the test substrates. We made comparison studies of the SRA of cells immobilised by two methods: carrier-surface adsorption and inclusion in various gels. We discuss the feasibility of developing a microbial sensor system for the determination of metaphos, sumithion, and PNP in aqueous media.