Sensor system for analysis of biofilm sensitivity to ampicillin.

The resistance of biofilms to antibiotics is a key factor that makes bacterial infections unsusceptible to antimicrobial therapy. The results of classical tests of cell sensitivity to antibiotics cannot be used to predict therapeutic success in infections associated with biofilm formation. We describe a simple and rapid method for the real-time evaluation of bacterial biofilm sensitivity to antibiotics, with Pseudomonas putida and ampicillin as examples. The method uses an electric biosensor to detect the difference between changes in the biofilm electric polarizability, thereby evaluating antibiotic sensitivity. The electric signals showed that P. putida biofilms were susceptible to ampicillin and that at high antibiotic concentrations, the biofilms differed markedly in their susceptibility (dose-dependent effect). The sensor also detected differences between biofilms before and after ampicillin treatment. The electric-signal changes enabled us to describe the physical picture of the processes occurring in bacterial biofilms in the presence of ampicillin. The approach used in this study is promising for evaluating the activity of various compounds against biofilms, because it permits a conclusion about the antibiotic sensitivity of biofilm bacteria to be made in real time and in a short period (analysis time, not longer than 20 min). An added strong point is that analysis can be done directly in liquid, without preliminary sample preparation. KEY POINTS: • Sensor system to analyze biofilm antimicrobial susceptibility is described. • The signal change depended on the ampicillin concentration (dose-dependent effect). • The sensor allows real-time determination of the antibiofilm effect of ampicillin.

Use of Azospirillum baldaniorum cells in quercetin detection.

The possibility of detection and determination of flavonoids by using microbial cells was shown for the first time using the quercetin - Azospirillum baldaniorum Sp245 model system. The activity of the flavonoids quercetin, rutin and naringenin toward A. baldaniorum Sp245 was evaluated. It was found that when the quercetin concentration ranged from 50 to 100 µM, the number of bacterial cells decreased. Rutin and naringenin did not affect bacterial numbers. Quercetin at 100 µM increased bacterial impedance by 60 %. Under the effect of quercetin, the magnitude of the electro-optical signal from cells decreased by 75 %, as compared with the no-quercetin control. Our data show the possibility of developing sensor-based systems for the detection and determination of flavonoids.

Microfluidic bioanalytical system for biofilm formation indication.

The formation of biofilms is a key factor that researchers must consider when they work with bacterial cultures. We describe a new microfluidic bioanalytical sensory system for indicating biofilm formation. The method is demonstrated with Pseudomonas bacteria as an example and is based on the real-time recording of cell-polarizability changes caused by an alternating electric field. Control experiments using phase-contrast microscopy and traditional microbiological plating were done that proved biofilms had formed. The physical picture was described of the sensor-signal changes during cell transition from planktonic to biofilm growth. This transition was indicated by the appearance of a peak-shaped signal at 500 kHz and by an increase in the recorded relaxation time. Phenomena of increase in the signal relaxation time from 2.4 s for planktonic to 25.4 s for biofilm cells. The proposed microfluidic sensor system for indicating biofilm formation holds much promise, because it ensures an analysis time of about 20-30 min. An added bonus is that for this system there is no need to grow bacterial biofilms in a sensor and the flow cell is reusable.

Use of an optical sensor to directly monitor the metabolic activity of growing bacteria.

The metabolic activity of growing bacteria was directly monitored by using an electro-optical (EO) sensor. The sensor enables examination of bacteria in batch and continuous cultures. As examples, we report studies with Еscherichia coli, a bacterium with an aerobic type of metabolism, and Lactobacillus plantarum, a bacterium with an anaerobic type of metabolism. Bacterial growth was accompanied by a simultaneous change in both the hydrodynamic mean size (HMS) of the bacteria and the concentration of ions in the cytoplasm (CIC). Both variables were associated with the regulation of cellular metabolic activity, which can be cyclic during intense bacterial growth. A simultaneous change in metabolic activity and osmotic regulation was also found. For СIC and HMS measurements, we used online results of the EO analysis of cells suspended in water. The measured results for the CIC and HMS can be used to directly monitor bacterial metabolism. The results of this study are of practical importance for the real-time EO monitoring of the metabolic activity of growing bacteria without preliminary sample preparation.

Bacteria-based electro-optical platform for ampicillin detection in aquatic solutions.

We have shown for the first time that it is possible to use a bacteria-based sensory system consisting of the bacterium Pseudomonas putida TSh-18 and an electro-optical sensor to detect ampicillin in the concentration range 0.5-600 µg/mL. Changes in the anisotropy of cell polarizability were detected at 900 and 2100 kHz; these represented the state of the cytoplasm and of the cell membrane, respectively. The changes indicate the quickest cell response to changes in the characteristics of the bacterial culture exposed to ampicillin. We have also shown that it is possible to monitor the ampicillin in the presence of kanamycin. In control experiments, we examined the effects of ampicillin and kanamycin on bacterial cells by phase-contrast microscopy and by standard microbiological tests on solid media. P. putida TSh-18 is recommended as a sensor system for ampicillin detection. Electro-optical analysis ensures detection of ampicillin in aquatic solutions in real-time, takes 10 min, and offers a lower limit of ampicillin detection of 0.5 µg/mL, which is lower than the European Community's maximum residue limit standards for penicillin antibiotics.

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.

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.

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.