Bacteriophage therapy presents a promising avenue for combating antibiotic-resistant bacterial infections. Yet, challenges exist, particularly, the lack of a straightforward purification pipeline suitable for widespread application to many phage types, as some phages are known to undergo significant titer loss when purified via current techniques. Electrokinetic methods offer a potential solution to this hurdle, with nonlinear electrophoresis emerging as a particularly appealing approach due to its ability to discern both the size and shape of the target phage particles. Presented herein is the electrokinetic characterization of the mobility of nonlinear electrophoresis for two phages (SPN3US and ϕKZ) and three types of polystyrene nanoparticles. The latter served as controls and were selected based on their sizes and surface charge magnitude. Particle tracking velocimetry experiments were conducted to characterize the mobility of all five particles included in this study. The results indicated that the selected nanoparticles effectively replicate the migration behavior of the two phages under electric fields. Further, it was found that there is a significant difference in the nonlinear electrophoretic response of phages and that of host cells, as first characterized in a previous report, illustrating that electrokinetic-based separations are feasible. The findings from this work are the first characterization of the behavior of phages under nonlinear electrophoresis effects and illustrate the potential for the development of electrokinetic-based phage purification techniques that could aid the advancement of bacteriophage therapy.
On the potential of microscale electrokinetic cascade devices
Phages used for phage therapy of multidrug resistant bacteria must be highly purified prior to use. There are limited purification approaches that are broadly applicable to many phage types. Electrokinetics has shown great potential to manipulate phages, but obstructions from the cell debris produced during phage propagation can severely diminish the capacity of an electrokinetic device to concentrate and purify phage samples. A multipart insulator‐based electrokinetic device is proposed here to remove the larger, undesirable components of mixtures from phage preparations while transferring the freshly purified and concentrated sample to a second stage for downstream analysis. By combining the large debris prescreen and analysis stages in a streamlined system, this approach simultaneously reduces the impact of clogging and minimizes the sample loss observed during manual transferring of purified samples. Polystyrene particles were used to demonstrate a diminished sample loss of approximately one order of magnitude when using the cascade device as opposed to a manual transfer scheme. The purification and concentration of three different phage samples were demonstrated using the first stage of the cascade device as a prescreen. This design provides a simple method of purifying and concentrating valuable samples from a complex mixture that might impede separation capacity in a single channel.
more » « less- Award ID(s):
- 1705895
- PAR ID:
- 10360556
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- ELECTROPHORESIS
- Volume:
- 42
- Issue:
- 23
- ISSN:
- 0173-0835
- Page Range / eLocation ID:
- p. 2474-2482
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
-
Bacterial viruses or phages have great potential in the medical and agricultural fields as alternatives to antibiotics to control nuisance populations of pathogenic bacteria. However, current analysis and purification protocols for phages tend to be resource intensive and have numbers of limitations, such as impacting phage viability. The present study explores the potential of employing the electrokinetic technique of insulator-based dielectrophoresis (iDEP) for virus assessment, separation and enrichment. In particular, the application of the parameter “trapping value” (Tv) is explored as a standardized iDEP signature for each phage species. The present study includes mathematical modeling with COMSOL Multiphysics and extensive experimentation. Three related, but genetically and structurally distinct, phages were studied: Salmonella enterica phage SPN3US, Pseudomonas aeruginosa phage ϕKZ and P. chlororaphis phage 201ϕ2-1. This is the first iDEP study on bacteriophages with large and complex virions and the results illustrate their virions can be successfully enriched with iDEP systems and still retain infectivity. In addition, our results indicate that characterization of the negative dielectrophoretic response of a phage in terms of Tv could be used for predicting individual virus behavior in iDEP systems. The findings reported here can contribute to the establishment of protocols to analyze, purify and/or enrich samples of known and unknown phages.more » « less
-
ABSTRACT We present here the complete genomes of eight phages that infect Paenibacillus larvae , the causative agent of American foulbrood in honeybees. Phage PBL1c was originally isolated in 1984 from a P. larvae lysogen, while the remaining phages were isolated in 2014 from bee debris, honeycomb, and lysogens from three states in the USA.more » « less
-
Dudley, Edward G. (Ed.)ABSTRACT Bacteriophages (phages) are currently available for use by the food industry to control the foodborne pathogen Listeria monocytogenes . Although phage biocontrols are effective under specific conditions, their use can select for phage-resistant bacteria that repopulate phage-treated environments. Here, we performed short-term coevolution experiments to investigate the impact of single phages and a two-phage cocktail on the regrowth of phage-resistant L. monocytogenes and the adaptation of the phages to overcome this resistance. We used whole-genome sequencing to identify mutations in the target host that confer phage resistance and in the phages that alter host range. We found that infections with Listeria phages LP-048, LP-125, or a combination of both select for different populations of phage-resistant L. monocytogenes bacteria with different regrowth times. Phages isolated from the end of the coevolution experiments were found to have gained the ability to infect phage-resistant mutants of L. monocytogenes and L. monocytogenes strains previously found to be broadly resistant to phage infection. Phages isolated from coinfected cultures were identified as recombinants of LP-048 and LP-125. Interestingly, recombination events occurred twice independently in a locus encoding two proteins putatively involved in DNA binding. We show that short-term coevolution of phages and their hosts can be utilized to obtain mutant and recombinant phages with adapted host ranges. These laboratory-evolved phages may be useful for limiting the emergence of phage resistance and for targeting strains that show general resistance to wild-type (WT) phages. IMPORTANCE Listeria monocytogenes is a life-threatening bacterial foodborne pathogen that can persist in food processing facilities for years. Phages can be used to control L. monocytogenes in food production, but phage-resistant bacterial subpopulations can regrow in phage-treated environments. Coevolution experiments were conducted on a Listeria phage-host system to provide insight into the genetic variation that emerges in both the phage and bacterial host under reciprocal selective pressure. As expected, mutations were identified in both phage and host, but additionally, recombination events were shown to have repeatedly occurred between closely related phages that coinfected L. monocytogenes . This study demonstrates that in vitro evolution of phages can be utilized to expand the host range and improve the long-term efficacy of phage-based control of L. monocytogenes . This approach may also be applied to other phage-host systems for applications in biocontrol, detection, and phage therapy.more » « less
-
A radiochemical method for producing 82Se sources with an ultra-low level of contamination of natural radionuclides (40K, decay products of 232Th and 238U) has been developed based on cation-exchange chromatographic purification with reverse removal of impurities. It includes chromatographic separation (purification), reduction, conditioning (which includes decantation, centrifugation, washing, grinding, and drying), and 82Se foil production. The conditioning stage, during which highly dispersed elemental selenium is obtained by the reduction of purified selenious acid (H2SeO3) with sulfur dioxide (SO2) represents the crucial step in the preparation of radiopure 82Se samples. The natural selenium (600 g) was first produced in this procedure in order to refine the method. The technique developed was then used to produce 2.5 kg of radiopure enriched selenium (82Se). The produced 82Se samples were wrapped in polyethylene (12 microm thick) and radionuclides present in the sample were analyzed with the BiPo-3 detector. The radiopurity of the plastic materials (chromatographic column material and polypropylene chemical vessels), which were used at all stages, was determined by instrumental neutron activation analysis. The radiopurity of the 82Se foils was checked by measurements with the BiPo-3 spectrometer, which confirmed the high purity of the final product. The measured contamination level for 208Tl was 8–54 microBq/kg, and for 214Bi the detection limit of 600 microBq/kg has been reached.more » « less
