Abstract
The most common method for phage quantitation is the plaque assay, which relies on phage ability to infect bacteria. However, non-infective phage particles may preserve other biological properties; specifically, they may enter interactions with the immune system of animals and humans. Here, we demonstrate real-time quantitative polymerase chain reaction (qPCR) detection of bacteriophages as an alternative to the plaque assay. The closely related staphylococcal bacteriophages A3R and 676Z and the coliphage T4 were used as model phages. They were tested in vivo in mice, ex vivo in human sera, and on plastic surfaces designed for ELISAs. T4 phage was injected intravenously into pre-immunized mice. The phage was completely neutralized by specific antibodies within 5 h (0 pfu/ml of serum, as determined by the plaque assay), but it was still detected by qPCR in the amount of approximately 107 pfu/ml of serum. This demonstrates a substantial timelapse between “microbiological disappearance” and true clearance of phage particles from the circulation. In human sera ex vivo, qPCR was also able to detect neutralized phage particles that were not detected by the standard plaque assay. The investigated bacteriophages differed considerably in their ability to immobilize on plastic surfaces: this difference was greater than one order of magnitude, as shown by qPCR of phage recovered from plastic plates. The ELISA did not detect differences in phage binding to plates. Major limitations of qPCR are possible inhibitors of the PCR reaction or free phage DNA, which need to be considered in procedures of phage sample preparation for qPCR testing. We propose that phage pharmacokinetic and pharmacodynamic studies should not rely merely on detection of antibacterial activity of a phage. Real-time qPCR can be an alternative for phage detection, especially in immunological studies of bacteriophages. It can also be useful for studies of phage-based drug nanocarriers or biosensors.
Highlights
In this work we investigated quantitative polymerase chain reaction (qPCR) as a useful alternative for phage detection, in comparison to standard methods based on phage cultures with bacteria
We demonstrated that the phage, even when neutralized in terms of antibacterial activity, can still be present in the circulation in high amounts and it can be detected and quantitatively assessed by qPCR (Figure 3)
This study demonstrates a substantial time lapse between inactivation of antibacterial activity and true clearance of phage particles from the circulation of pre-immunized individuals. qPCR allows for detection of inactivated bacteriophages that cannot be detected by plaque assay
Summary
Bacteriophages can be used in multiple medical applications (Miedzybrodzki et al, 2012; Kutter et al, 2015; Vandenheuvel et al, 2015; Karimi et al, 2016; Saeed et al, 2017), veterinary (Grant et al, 2016), biotechnology (Oslizło et al, 2011; Lee et al, 2012; O’Sullivan et al, 2016), agriculture (Zaczek et al, 2015), or food processing (Endersen et al, 2014) The most important one is the fact that microbiological detection of phage activity does not meet the real count of bacteriophage particles in samples; it allows for testing how many phages were able to infect their host effectively. Phage cannot be detected by the plaque assay, which makes quantitation of real phage particle content very difficult or even impossible
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