Immunodetection of bacteriophages by a piezoelectric resonator with lateral electric field

It has been demonstrated that electroacoustic analysis with polyclonal antibodies can be used for bacteriophage detection. The frequency dependences of the real and imaginary parts of electrical impedance of a resonator with a viral suspension with antibodies were shown to be essentially different from the dependences of a resonator with control viral suspension without antibodies. It was shown that ΦAl-Sp59b bacteriophages were detected with the use of antibodies in the presence of foreign virus particles. The ΦAl-Sp59b bacteriophage content in the analyzed suspension was ~10¹⁰–10⁶ phages/mL; the time of analysis was no more than 5 min. The optimally informative parameter for obtaining reliable information was the change in the real or imaginary part of electrical impedance at a fixed frequency near the resonance upon the addition of specific antibodies to the analyzed suspension. It was demonstrated that the interaction between bacteriophages and antibodies can be recorded, offering good prospects for the development of a biological sensor for liquid-phase identification and virus detection.     

Obtaining the phage mini-antibodies and their use for detection of microbial cells by using an electro-acoustic sensor

The phage mini-antibodies to bacterial cells of strain Azospirillum brasilense Sp245 were obtained and the possibility of using them for detection of microbial cells by means of a lateral field excited piezoelectric resonator was studied. It has been found that the frequency dependencies of the real and imaginary parts of the electrical impedance of the resonator loaded by the cell suspension A. brasilense Sp245 with the mini-antibodies, significantly differ from those of the resonator with the control cell suspension without mini-antibodies. The concentration limit of possible determination of the microbial cells in their interaction with the mini-antibodies is equal to 10(3) cells/ml. It has been ascertained that detection of A. brasilense Sp245 cells using the mini-antibodies is possible even in the presence of other cultures, for example, E. coli BL-Ril and A. brasilense Sp7 cells. Therefore, it has been shown for the first time that detection of microbial cells by an electro-acoustic sensor is feasible.     

Obtaining phage mini-antibodies and using them for detection of microbial cells with an electroacoustic sensor

Phage mini-antibodies to bacterial cells of strain Azospirillum brasilense Sp245 were obtained, and the possibility of using them for detection of microbial cells with a lateral field excited piezoelectric resonator was studied. It has been found that the frequency dependences of the real and imaginary parts of electrical impedance of such a resonator loaded with a suspension of A. brasilense Sp245 cells with the mini-antibodies differ significantly from the dependences of the resonator with a control cell suspension without mini-anti-bodies. The limit of possible determination of the concentration of microbial cells is found to be 10³ cells/mL upon interaction with mini-antibodies. It has been ascertained that detection of A. brasilense Sp245 cells with the aid of mini-antibodies is possible even in the presence of other cultures, for example, E. coli BL-Ril and A. brasilense Sp7. Therefore, it has been shown for the first time that detection of microbial cells with an electroacoustic sensor is feasible.     

Application of the method of electro-acoustical analysis for the detection of bacteriophages in a liquid phase

The possibility of the application of electro-acoustic analysis for the detection of bacteriophages was demonstrated for the first time based on the example of the interaction of the FA1-Sp59b bacteriophage with bacterial cells of the strain Azospirillum lipoferum Sp59b. Piezoelectric cross-field resonators with a 1-mL chamber for analyzed liquid were used as the biological sensor. It was revealed that the dependences of the real and imaginary parts of the electrical impedance of the resonator loaded with a suspension of viruses and microbial cells on the frequency was significantly different from those dependences of the resonator that contained a control cell suspension without the virus. It was shown that detection of the FA1-Sp59b bacteriophage using microbial cells was possible with both extraneous viral particles and extraneous microbial cells. The proposed method allows one to accurately determine the type of identified virus after a 5-minute interaction with indicating bacterial culture. As well, the minimum concentration of viruses is five virus particles per cell. These results as a whole demonstrate the possibility of detecting specific interactions of bacteriophages with microbial cells and provide a basis for the development of a biological sensor for the quantitative detection of viruses directly in the liquid phase.     

An acoustic method for the analysis of bacterial cells

The application of a biological electroacoustic sensor based on a lateral electric-field-excited piezoelectric resonator for the study of bacterial cells that interact with specific bacteriophages, mini-antibodies, and polyclonal antibodies was successfully demonstrated. The determined lower limit of microbialcell detection was approximately of 10³ to 10⁴ cells/mL for the duration of the assay of 10 min. The possibility of bacterial-cell detection via interaction with specific agents in the presence of extraneous microbiota was shown. The method allowed us to determine the spectrum of lytic activity of bacteriophages and the sensitivity of microbial cells to bacteriophages. The results of the study showed that application of a sensor piezoelectric lateral-field resonator is a promising technique for the detection and identification of microbial cells and determination of their phage resistance in microbiology, medicine, and veterinary medicine. Furthermore, the results of the experiments made it possible to understand the mechanisms of the processes that occur in a suspension of bacterial cells in the presence of various biological agents. The method also may provide useful information regarding biophysical mechanisms of interactions that occur in microbial populations.     

An Electroacoustic Analysis for Determining the Effect of Amoxicillin on Microbial Cells

The probability of determining the effects of amoxicillin, which is one of β-lactam antibiotics, on microbial cells of Escherichia coli by the electroacoustic analysis method was shown for the first time. A piezoelectric resonator with a lateral electric field with a 1-mL liquid container was used as a biological sensor. It has been established that in the presence of amoxicillin the frequency dependence of the real and imaginary parts of the electrical impedance of a resonator loaded with a suspension of sensitive cells differs significantly from those of the resonator with a control of a microbial cell suspension without an antibiotic. When the resonator is loaded with the amoxicillin-resistant cell suspension, these dependencies are virtually the same. These results open prospects for the use of electroacoustic analysis methods to register the effect of β-lactam antibiotics on microbial cells and evaluate their antibacterial activity.     

Investigation of specific interactions between microbial cells and polyclonal antibodies using a resonator with lateral electric field

The interaction between polyclonal antibodies and Azospirillum brasilense Sp7 cells was studied using a resonator with lateral electric field. To this end, specific polyclonal rabbit antibodies against the O-antigen epitopes of the strain A. brasilense Sp7 were obtained and the possibility of their application for detection of microbial cells using a piezoelectric resonator with lateral electric field was shown. It was established that frequency dependences of the real and imaginary parts of electrical impedance of such a resonator loaded with the suspension of A. brasilense Sp7 cells and antibodies substantially differed from those of the resonator with the control suspension of cells without antibodies. It was shown that the obtained antibodies interacted with azospirilla cells, and the marker was accumulated all over the cell surface. The limit of possible detection of microbial cells during their interaction with antibodies was found to be 10⁴ cells/mL. Detection of A. brasilense Sp7 cells using antibodies proved to be possible in the presence of foreign bacteria. The presented results demonstrate the possibility of recording the interaction between microbial cells and antibodies and developing a biosensor for quantitative detection of microbial cells.     

Biosensor for the detection of bacteriophages based on a super-high-frequency resonator

Azospirillum lipoferum Sp59b microbial cells were immobilized on the surface of thin polystyrene films modified in plasma from a high-frequency discharge of argon (13.56 MHz). The optimal conditions for immobilization, under which cell activity was maintained with respect to specific bacteriophages, were established. It was shown that it is possible to record the interaction of immobilized microbial cells and bacteriophages with a microwave-based resonant system (5–8.5 GHz). It was found that the biosensor made it possible to distinguish the interaction of bacterial cells with specific bacteriophages from the control, in which such interaction was absent. With the obtained super-high-frequency sensor, it was possible to determine the content of ΦAl-Sp59b bacteriophages in a suspension containing ~106 phages/mL. The analysis time was about 10 min. The viability of microbial cells after immobilization was also determined with this sensor. The results obtained with the use of the sensor on the basis of a super-high-frequency resonator have shown that it is promising for the development of methods to determine viral particles and the viability of microbial cells.     

Evaluation of a Cocktail of Three Bacteriophages for Biocontrol of Escherichia coli O157:H7

Escherichia coli O157:H7 is an endemic pathogen causing a variety of human diseases including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. This study concerns the exploitation of bacteriophages as biocontrol agents to eliminate the pathogen E. coli O157:H7. Two distinct lytic phages (e11/2 and e4/1c) isolated against a human strain of E. coli O157:H7, a previously isolated lytic phage (pp01), and a cocktail of all three phages were evaluated for their ability to lyse the bacterium in vivo and in vitro. Phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5-log-unit reduction of pathogen numbers in 1 h at 37°C. However, bacteriophage-insensitive mutants (BIMs) emerged following the challenge. All tested BIMs had a growth rate which approximated that of the parental O157 strain, although many of these BIMs had a smaller, more coccoid cellular morphology. The frequency of BIM formation (10⁻⁶ CFU) was similar for e11/2, pp01, and the phage cocktail, while BIMs insensitive to e4/1c occurred at the higher frequency (10⁻⁴ CFU). In addition, BIMs commonly reverted to phage sensitivity within 50 generations. In an initial meat trial experiment, the phage cocktail completely eliminated E. coli O157:H7 from the beef meat surface in seven of nine cases. Given that the frequency of BIM formation is low (10⁻⁶ CFU) for two of the phages, allied to the propensity of these mutants to revert to phage sensitivity, we expect that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment.     

Bacteriophage Therapy To Reduce Salmonella Colonization of Broiler Chickens

Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by ≥4.2 log₁₀ CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by ≥2.19 log₁₀ CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.     

Assessment of cytotoxicity by impedance spectroscopy

This paper describes a simple and convenient method to monitor on-line cell adhesion by electrical impedance measurements. Immortalized mouse fibroblasts, BALB/3T3, were cultured onto interdigitated electrode structures integrated into the bottom of an in-house fabricated device. Impedance modulus, phase, real and imaginary parts were considered separately and plotted as function of frequency and time to better understand and select the component giving more information on cell adhesion changes. For cytotoxicity assessment, the cells were treated with different concentrations of sodium arsenite used as model toxicant and their responses were monitored on-line. The half inhibition concentration, the required concentration to achieve 50% inhibition, derived from the measurements fall between the results obtained using standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test and colony forming efficiency assay confirming the good sensitivity of the system. In term of impedance signal, the modulus results was found to be the most sensitive of the considered components for cytotoxicity testing of chemicals.     

Diffusion properties of bacteriophages through agarose gel membrane

A simple two‐chamber diffusion method was developed to study the diffusion properties of bacteriophages (phages). The apparent diffusion coefficients (D_app) of Myoviridae phage T4 and filamentous phage fNEL were investigated, and the diffusion of the phages was found to be much slower than the diffusion of three antibiotics, ciprofloxacin, penicillin G, and tetracycline. D_app of T4 and fNEL in water through filter paper were calculated to be 2.8 × 10^?11 m²/s and 6.8 × 10^?12 m²/s, respectively, and D_app of fNEL through agarose gel membrane, an artificial biofilm, was also calculated to be smaller than that of T4. In addition, D_app of phages through agarose gel was dependent on agarose concentration due to the similar size of phage and agarose gel mesh. We concluded that D_app of phages through an artificial biofilm is dependent on both phage morphology and biofilm density, and suggest the use of this method to study diffusion properties through real biofilms. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Effect of low voltage on electrical properties of eye lens

In the present study, the Cole-cole plot of lens tissue has been drawn using AC impedance system (EG and G PARC Model 318) in the frequency range 10 mHz to 10 Hz at low voltage stress. The impedus locus between real part (Z') and imaginary part (Z") of complex impedance of lens was examined. Results showed that the extracellular resistance (Re), distribution factor (alpha) and depressed angle (theta) were significantly varied at experimental low voltages. An attempt has been made to explain the electrical data of voltage-tissue interaction on the basis of solid state biophysics.     

Bacteriophage therapy to reduce salmonella colonization of broiler chickens

Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by > or = 4.2 log10 CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by > or = 2.19 log10 CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.     

Reduction in Exopolysaccharide Viscosity as an Aid to Bacteriophage Penetration through Pseudomonas aeruginosa Biofilms

To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n = 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 × 10⁸ purified phage per ml for 24 h at 37°C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10¹⁰ and 10¹² phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time- and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself.     

The Effect of Static Magnetic Field on Electrical Properties of Lens

We investigated the effect of a static magnetic field (0.1 T) on various electrical parameters of goat eye lens using a computer-aided AC impedance system (EG&G 278) at 30°C. Results of measurements on voltage-current characteristics showed that the static magnetic field alters the current flow in the tissue. The complex impedance plane drawn between real (Z') and imaginary (Z) parts in the form of a Cole-Cole plot demonstrates that under magnetic field, the distribution factor a decreases from 0.3 ± 0.006 to 0.24 ± 0.005 and the extracellular resistance (Re) increases from 52 ± 1.3 to 60 ± 1.5 K ohms in the lens tissue. An attempt has been made to explain the interactive behavior of the magnetic field with the electrical changes in the lens.     

Isolation,growth and genome of the Rhodothermus RM378 thermophilic bacteriophage

Several bacteriophages that infect different strains of the thermophilic bacterium Rhodothermus marinus were isolated and their infection pattern was studied. One phage, named RM378 was cultivated and characterized. The RM378 genome was also sequenced and analyzed. The phage was grouped as a member of the Myoviridae family with A2 morphology. It had a moderately elongated head, with dimensions of 85 and 95 nm between opposite apices and a 150 nm long tail, attached with a connector to the head. RM378 showed a virulent behavior that followed a lytic cycle of infection. It routinely gave lysates with 10¹¹ pfu/ml, and sometimes reached titers as high as 10¹³ pfu/ml. The titer remained stable up to 65 °C but the phage lost viability when incubated at higher temperatures. Heating for 30 min at 96 °C lowered the titer by 10⁴. The RM378 genome consisted of ds DNA of 129.908 bp with a GC ratio of 42.0 % and contained about 120 ORFs. A few structural proteins, such as the major head protein corresponding to the gp23 in T4, could be identified. Only 29 gene products as probable homologs to other proteins of known function could be predicted, with most showing only low similarity to known proteins in other bacteriophages. These and other studies based on sequence analysis of a large number of phage genomes showed RM378 to be distantly related to all other known T4-like phages.     

Environmental Factors Influencing Numbers of Rhizobium leguminosarum biovar trifolii and Its Bacteriophages in Two Field Soils

Fluctuations in numbers of Rhizobium leguminosarum biovar trifolii and its bacteriophages in two fields with different soil types were followed during a 17-month period in 1981 and 1982. Mean levels of both phage and rhizobia varied significantly (P < 0.05) on different occasions, with rhizobial levels varying from 1.6 × 10² to 2.0 × 10⁴ cell per g of soil and phage from 0 to 1.7 × 10⁴ PFU/g of soil. Multivariate regression analysis showed rhizobial levels to be significantly and positively related to vegetation height and solar radiation, but not to mean temperature, precipitation, soil matric potential, or soil type. Rhizobiophage concentrations were significantly and positively related to soil matric potential and vegetation height. They were reduced in the silty clay loam soil, although the presence of 34% clay did not prevent phage multiplication and the occurrence of high phage levels.     

BVPaP-3, a T7-Like Lytic Phage of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>: Its Isolation and Characterisation

The increasing emergence of antibiotic-resistant bacteria has produced a growing interest among scientists in bacteriophages as alternative antimicrobial agents. This article reports a lytic phage against an antibiotic-resistant strain of Pseudomonas aeruginosa. Phage BVPaP-3 is a member of the Podoviridae family and morphologically similar to the T7-like phage gh-1. The phage has a hexagonal head of 58–59 nm in diameter and a short tail of 10 × 8 nm. It is stable at a wide range of pH (6–10) and temperatures (4–40°C). Its optimal growth temperature is 37°C and the adsorption rate constant is 1.19 × 10⁻⁹. Latent and eclipse periods are 20 and 15 min, respectively, and the burst size is 44 after 35 min at 37°C. The phage has a DNA size of 41.31 kb and a proteome of 11 proteins. The major protein is 33 kDa in size.     

Modeling error and stability of endothelial cytoskeletal membrane parameters based on modeling transendothelial impedance as resistor and capacitor in series

Transendothelial impedance across an endothelial monolayer grown on a microelectrode has previously been modeled as a repeating pattern of disks in which the electrical circuit consists of a resistor and capacitor in series. Although this numerical model breaks down barrier function into measurements of cell-cell adhesion, cell-matrix adhesion, and membrane capacitance, such solution parameters can be inaccurate without understanding model stability and error. In this study, we have evaluated modeling stability and error by using a ² evaluation and Levenberg-Marquardt nonlinear least-squares (LM-NLS) method of the real and/or imaginary data in which the experimental measurement is compared with the calculated measurement derived by the model. Modeling stability and error were dependent on current frequency and the type of experimental data modeled. Solution parameters of cell-matrix adhesion were most susceptible to modeling instability. Furthermore, the LM-NLS method displayed frequency-dependent instability of the solution parameters, regardless of whether the real or imaginary data were analyzed. However, the LM-NLS method identified stable and reproducible solution parameters between all types of experimental data when a defined frequency spectrum of the entire data set was selected on the basis of a criterion of minimizing error. The frequency bandwidth that produced stable solution parameters varied greatly among different data types. Thus a numerical model based on characterizing transendothelial impedance as a resistor and capacitor in series and as a repeating pattern of disks is not sufficient to characterize the entire frequency spectrum of experimental transendothelial impedance. cell-cell adhesion; cell-matrix adhesion; cell membrane capacitance; mathematical computation