Microfluidic device for dielectrophoresis manipulation and electrodisruption of respiratory pathogen Bordetella pertussis.

A miniaturized microfluidic device was developed to facilitate electromanipulation of bacterial respiratory pathogens. The device comprises a microchip with circular aluminum electrodes patterned on glass, which is housed in a microfluidic system fabricated utilizing polydimethylsiloxane. The device provides sample preparation capability by exploiting positive dielectrophoresis (DEP) in conjunction with pulsed voltage for manipulation and disruption of Bordetella pertussis bacterial cells. Positive DEP capture of B. pertussis was successfully demonstrated utilizing 10 Vrms and 1 MHz ac fields. Application of dc pulses (300 V amplitude and 50 micros pulsewidth applied 1 s apart) across the aluminum electrodes resulted in electrodisruption and lysis of B. pertussis bacterial cells. Real-time polymerase chain reaction, a 2(3) factorial experimental design and transmission electron microscopy were used to evaluate bacterial cell manipulation and factors affecting bacterial cell disruption. The main factors affecting bacterial cell disruption were electric field strength, the electrical conductivity of the cell suspension sample, and the combined effect of number of pulses and sample conductivity. The bacterial deoxyribonucleic acid target remained undamaged as a result of DEP and cell lysis experimentation. Our findings suggest that a simple miniaturized microfluidic device can achieve important steps in sample preparation on-chip involving respiratory bacterial pathogens.

Newly secreted adenylate cyclase toxin is responsible for intoxication of target cells by Bordetella pertussis.

Adenylate cyclase (AC) toxin is present on the surface of Bordetella pertussis organisms and their addition to eukaryotic cells results in increases in intracellular cAMP. To test the hypothesis that surface-bound toxin is the source for intoxication of cells when incubated with B. pertussis, we characterized the requirements of intoxication from intact bacteria and found that this process is calcium-dependent and blocked by monoclonal antibody to AC toxin or antibody against CD11b, a surface glycoprotein receptor for the toxin. Increases in intracellular cAMP correlate with the number of adherent bacteria, not the total number present in the medium, suggesting that interaction of bacteria with target cells is important for efficient delivery of AC toxin. A filamentous haemagglutinin-deficient mutant (BP353) and a clinical isolate (GMT1), both of which have a marked reduction in AC toxin on their surface, and wild-type B. pertussis (BP338) from which surface AC toxin has been removed by trypsin, were fully competent for intoxicating target cells, demonstrating that surface-bound AC toxin is not responsible for intoxication. B. pertussis killed by gentamicin or gamma irradiation were unable to intoxicate, illustrating that toxin delivery requires viable bacteria. Furthermore, CCCP, a protonophore that disrupts the proton gradient necessary for the secretion of related RTX toxins, blocked intoxication by whole bacteria. These data establish that delivery of this toxin by intact B. pertussis is not dependent on the surface-associated AC toxin, but requires close association of live bacteria with target cells and the active secretion of AC toxin.

[Effect of radiation sterilization on the immunobiological properties of the sorbed protective fraction of Bordetella pertussis]. / Izuchenie vliianiia radiatsionnoi sterilizatsii na immunobiologicheskie svoistva sorbirovannoi protektivnoi fraktsii kokliushnykh mikrobov.

The possibility of using radiation for the sterilization of the dried preparation of B. pertussis adsorbed protective fraction has been studied. The use of gamma irradiation in a dose of 1.5 X 10(3) J/kg has been found to permit obtaining sterile preparations while preserving their initial protective properties.