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Dielectrophoretic monitoring and interstrain separation of intact Clostridium difficile based on their S(Surface)-layers.

Su YH, Warren CA, Guerrant RL, Swami NS - Anal. Chem. (2014)

Bottom Line: Herein, we demonstrate that morphological differences within the cell wall of particular C. difficile strains with differing S-layer proteins can induce systematic variations in their electrophysiology, due alterations in cell wall capacitance.As a result, dielectrophoretic frequency analysis can enable the independent fingerprinting and label-free separation of intact microbials of each strain type from mixed C. difficile samples.This microfluidic diagnostic platform can assist in the development of therapies for arresting clostridial infections by enabling the isolation of individual strains, optimization of antibiotic treatments and the monitoring of microbiomes.

View Article: PubMed Central - PubMed

Affiliation: Electrical and Computer Engineering, University of Virginia at Thornton Hall , 351 McCormick Road, P.O. Box 400743, Charlottesville, Virginia 22904, United States.

ABSTRACT
Clostridium difficile (C. difficile) infection (CDI) rates have exhibited a steady rise worldwide over the last two decades and the infection poses a global threat due to the emergence of antibiotic resistant strains. Interstrain antagonistic interactions across the host microbiome form an important strategy for controlling the emergence of CDI. The current diagnosis method for CDI, based on immunoassays for toxins produced by pathogenic C. difficile strains, is limited by false negatives due to rapid toxin degradation. Furthermore, simultaneous monitoring of nontoxigenic C. difficile strains is not possible, due to absence of these toxins, thereby limiting its application toward the control of CDI through optimizing antagonistic interstrain interactions. Herein, we demonstrate that morphological differences within the cell wall of particular C. difficile strains with differing S-layer proteins can induce systematic variations in their electrophysiology, due alterations in cell wall capacitance. As a result, dielectrophoretic frequency analysis can enable the independent fingerprinting and label-free separation of intact microbials of each strain type from mixed C. difficile samples. The sensitivity of this contact-less electrophysiological method is benchmarked against the immunoassay and microbial growth rate methods for detecting alterations within both, toxigenic and nontoxigenic C. difficile strains after vancomycin treatment. This microfluidic diagnostic platform can assist in the development of therapies for arresting clostridial infections by enabling the isolation of individual strains, optimization of antibiotic treatments and the monitoring of microbiomes.

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(a–c) Modification of dielectrophoreticspectra (velocityunder DEP) can be used to monitor alterations after vancomycin treatmentof (a) HTCD, (b) LTCD, and (c) NTCD strains. Note that reported velocitiesare averaged over 20 cells, of which an overwhelming majority (95–100%)exhibits the reported velocities, except for vancomycin treated cellsat frequencies close to the DEP crossover, wherein this value dropsto a 50–65% majority of the analyzed cells. (d) Alterationin the magnitude of the DEP response at 1 MHz (velocity under DEP)after vancomycin treatment and (e) change in DEP crossover frequencyafter vancomycin treatment offer information on alterations in cellelectrophysiology.
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fig3: (a–c) Modification of dielectrophoreticspectra (velocityunder DEP) can be used to monitor alterations after vancomycin treatmentof (a) HTCD, (b) LTCD, and (c) NTCD strains. Note that reported velocitiesare averaged over 20 cells, of which an overwhelming majority (95–100%)exhibits the reported velocities, except for vancomycin treated cellsat frequencies close to the DEP crossover, wherein this value dropsto a 50–65% majority of the analyzed cells. (d) Alterationin the magnitude of the DEP response at 1 MHz (velocity under DEP)after vancomycin treatment and (e) change in DEP crossover frequencyafter vancomycin treatment offer information on alterations in cellelectrophysiology.

Mentions: Alterations to the electrophysiologyof cells upon antibiotic treatment, such as distinguishing the degreeof cell wall permeabilization versus cytoplasm disruption, can bequantified by analyzing the dielectrophoretic frequency spectra oftreated versus untreated cells.26,32,42 Herein, we utilize DEP to probe relative differences in the mechanismof microbial disruption for each C. difficile strainafter vancomycin treatment, especially since similar measurementsbased on toxin production and growth rate can only indicate the overallalterations in cell viability, without providing information on thedisruption mechanism. Furthermore, DEP spectra can offer informationon the optimal frequencies for separating vancomycin treated cellsfrom untreated cells of each C. difficile strain,thereby enabling a means for quantifying the efficacy of vancomycintreatment on each strain, especially within heterogeneous C. difficile samples. In general, all the three strainsbecome less polarizable due to functionality alterations to the cellafter 24 h of vancomycin treatment. However, the HTCD strain requiresalmost twice as much vancomycin levels than required for LTCD andNTCD strains to cause alterations to the DEP spectra. As a resultof vancomycin treatment, while the DEP spectra for the HTCD strain(Figure 3a) is shifted toward a higher crossoverfrequency (300 kHz to 600 kHz), the spectra for the LTCD strain (Figure 3b) and the NTCD strain (Figure 3c) are shifted toward successively lower crossover frequencies(500 kHz to 300 kHz for LTCD and 900 kHz to 600 kHz for NTCD). Toquantify the relative alterations after vancomycin treatment, we showthe steady reduction in DEP velocity for each strain at 1 MHz (Figure 3d) and the changes in crossover frequencies (Figure 3e). It is likely that vancomycin treatment altersthe permeability of the cell wall and membrane regions, so that thelowered inverse RC time constant of the system enablesDEP crossover at earlier frequencies, as observed for the NTCD andLTCD strains. For the HTCD strain, on the other hand, the need forhigher vancomycin levels to cause alterations and the up-shiftingof the DEP crossover frequency after vancomycin treatment suggesta relatively sturdier cell wall and membrane that is not easily permeabilized,in comparison to the LTCD and NTCD strains. This is consistent withthe trend of our measurements on minimum inhibitory concentration(0.5 mg/L for NTCD and LTCD and 1 mg/mL for HTCD), indicating theneed for higher antibiotic levels to deactivate HTCD versus otherstrains.43


Dielectrophoretic monitoring and interstrain separation of intact Clostridium difficile based on their S(Surface)-layers.

Su YH, Warren CA, Guerrant RL, Swami NS - Anal. Chem. (2014)

(a–c) Modification of dielectrophoreticspectra (velocityunder DEP) can be used to monitor alterations after vancomycin treatmentof (a) HTCD, (b) LTCD, and (c) NTCD strains. Note that reported velocitiesare averaged over 20 cells, of which an overwhelming majority (95–100%)exhibits the reported velocities, except for vancomycin treated cellsat frequencies close to the DEP crossover, wherein this value dropsto a 50–65% majority of the analyzed cells. (d) Alterationin the magnitude of the DEP response at 1 MHz (velocity under DEP)after vancomycin treatment and (e) change in DEP crossover frequencyafter vancomycin treatment offer information on alterations in cellelectrophysiology.
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Related In: Results  -  Collection

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fig3: (a–c) Modification of dielectrophoreticspectra (velocityunder DEP) can be used to monitor alterations after vancomycin treatmentof (a) HTCD, (b) LTCD, and (c) NTCD strains. Note that reported velocitiesare averaged over 20 cells, of which an overwhelming majority (95–100%)exhibits the reported velocities, except for vancomycin treated cellsat frequencies close to the DEP crossover, wherein this value dropsto a 50–65% majority of the analyzed cells. (d) Alterationin the magnitude of the DEP response at 1 MHz (velocity under DEP)after vancomycin treatment and (e) change in DEP crossover frequencyafter vancomycin treatment offer information on alterations in cellelectrophysiology.
Mentions: Alterations to the electrophysiologyof cells upon antibiotic treatment, such as distinguishing the degreeof cell wall permeabilization versus cytoplasm disruption, can bequantified by analyzing the dielectrophoretic frequency spectra oftreated versus untreated cells.26,32,42 Herein, we utilize DEP to probe relative differences in the mechanismof microbial disruption for each C. difficile strainafter vancomycin treatment, especially since similar measurementsbased on toxin production and growth rate can only indicate the overallalterations in cell viability, without providing information on thedisruption mechanism. Furthermore, DEP spectra can offer informationon the optimal frequencies for separating vancomycin treated cellsfrom untreated cells of each C. difficile strain,thereby enabling a means for quantifying the efficacy of vancomycintreatment on each strain, especially within heterogeneous C. difficile samples. In general, all the three strainsbecome less polarizable due to functionality alterations to the cellafter 24 h of vancomycin treatment. However, the HTCD strain requiresalmost twice as much vancomycin levels than required for LTCD andNTCD strains to cause alterations to the DEP spectra. As a resultof vancomycin treatment, while the DEP spectra for the HTCD strain(Figure 3a) is shifted toward a higher crossoverfrequency (300 kHz to 600 kHz), the spectra for the LTCD strain (Figure 3b) and the NTCD strain (Figure 3c) are shifted toward successively lower crossover frequencies(500 kHz to 300 kHz for LTCD and 900 kHz to 600 kHz for NTCD). Toquantify the relative alterations after vancomycin treatment, we showthe steady reduction in DEP velocity for each strain at 1 MHz (Figure 3d) and the changes in crossover frequencies (Figure 3e). It is likely that vancomycin treatment altersthe permeability of the cell wall and membrane regions, so that thelowered inverse RC time constant of the system enablesDEP crossover at earlier frequencies, as observed for the NTCD andLTCD strains. For the HTCD strain, on the other hand, the need forhigher vancomycin levels to cause alterations and the up-shiftingof the DEP crossover frequency after vancomycin treatment suggesta relatively sturdier cell wall and membrane that is not easily permeabilized,in comparison to the LTCD and NTCD strains. This is consistent withthe trend of our measurements on minimum inhibitory concentration(0.5 mg/L for NTCD and LTCD and 1 mg/mL for HTCD), indicating theneed for higher antibiotic levels to deactivate HTCD versus otherstrains.43

Bottom Line: Herein, we demonstrate that morphological differences within the cell wall of particular C. difficile strains with differing S-layer proteins can induce systematic variations in their electrophysiology, due alterations in cell wall capacitance.As a result, dielectrophoretic frequency analysis can enable the independent fingerprinting and label-free separation of intact microbials of each strain type from mixed C. difficile samples.This microfluidic diagnostic platform can assist in the development of therapies for arresting clostridial infections by enabling the isolation of individual strains, optimization of antibiotic treatments and the monitoring of microbiomes.

View Article: PubMed Central - PubMed

Affiliation: Electrical and Computer Engineering, University of Virginia at Thornton Hall , 351 McCormick Road, P.O. Box 400743, Charlottesville, Virginia 22904, United States.

ABSTRACT
Clostridium difficile (C. difficile) infection (CDI) rates have exhibited a steady rise worldwide over the last two decades and the infection poses a global threat due to the emergence of antibiotic resistant strains. Interstrain antagonistic interactions across the host microbiome form an important strategy for controlling the emergence of CDI. The current diagnosis method for CDI, based on immunoassays for toxins produced by pathogenic C. difficile strains, is limited by false negatives due to rapid toxin degradation. Furthermore, simultaneous monitoring of nontoxigenic C. difficile strains is not possible, due to absence of these toxins, thereby limiting its application toward the control of CDI through optimizing antagonistic interstrain interactions. Herein, we demonstrate that morphological differences within the cell wall of particular C. difficile strains with differing S-layer proteins can induce systematic variations in their electrophysiology, due alterations in cell wall capacitance. As a result, dielectrophoretic frequency analysis can enable the independent fingerprinting and label-free separation of intact microbials of each strain type from mixed C. difficile samples. The sensitivity of this contact-less electrophysiological method is benchmarked against the immunoassay and microbial growth rate methods for detecting alterations within both, toxigenic and nontoxigenic C. difficile strains after vancomycin treatment. This microfluidic diagnostic platform can assist in the development of therapies for arresting clostridial infections by enabling the isolation of individual strains, optimization of antibiotic treatments and the monitoring of microbiomes.

Show MeSH
Related in: MedlinePlus