Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

BACKGROUND: Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics. METHODOLOGY/PRINCIPAL FINDINGS: The objective of this work was to develop an alternative to conventional phage lysis tests--a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages phiA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. phiA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10(3) CFU/ml (equivalent to as few as one Y. pestis cell per 1-microl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, phiA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR. CONCLUSIONS/SIGNIFICANCE: Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria

Determination of lysis speed and burst sizes for bacteriophages φA1122, L-413C, and P2 <i>vir1</i> on <i>Y. pestis</i> CO92 pgm⁻.

<p>Phage burst sizes (an average phage progeny produced by one bacterial cell) correspond to plateaus on the curves.</p

Dynamics of growth of phages φA1122 and L-413C on different concentrations of <i>Y. pestis</i> cells detected by qPCR.

<p>The starting points of phage infection correspond to 100 PFU per 1 µl sample and are normalized to 1. A. The titer rise of φA1122. B. L-413C amplification.</p

Parameters of φA1122- and L-413C-based qPCR tests for phage DNA and live phage particles determined by linear regression method.

<p>A and B, standard curves plotted for DNA concentrations of φA1122 and L-413C, respectively. C and D, standard curves plotted for live phage particles of φA1122 and L-413C, respectively.</p

Temporal Progression of Pneumonic Plague in Blood of Nonhuman Primate: A Transcriptomic Analysis

<div><p>Early identification of impending illness during widespread exposure to a pathogenic agent offers a potential means to initiate treatment during a timeframe when it would be most likely to be effective and has the potential to identify novel therapeutic strategies. The latter could be critical, especially as antibiotic resistance is becoming widespread. In order to examine pre-symptomatic illness, African green monkeys were challenged intranasally with aerosolized <i>Yersinia pestis</i> strain CO92 and blood samples were collected in short intervals from 45 m till 42 h post-exposure. Presenting one of the first genomic investigations of a NHP model challenged by pneumonic plague, whole genome analysis was annotated <i>in silico</i> and validated by qPCR assay. Transcriptomic profiles of blood showed early perturbation with the number of differentially expressed genes increasing until 24 h. By then, <i>Y</i>. <i>pestis</i> had paralyzed the host defense, as suggested by the functional analyses. Early activation of the apoptotic networks possibly facilitated the pathogen to overwhelm the defense mechanisms, despite the activation of the pro-inflammatory mechanism, toll-like receptors and microtubules at the port-of-entry. The overexpressed transcripts encoding an early pro-inflammatory response particularly manifested in active lymphocytes and ubiquitin networks were a potential deviation from the rodent models, which needs further verification. In summary, the present study recognized a pattern of <i>Y</i>. <i>pestis</i> pathogenesis potentially more applicable to the human system. Independent validation using the complementary omics approach with comprehensive evaluation of the organs, such as lungs which showed early bacterial infection, is essential.</p></div

Bacterial strains used and results of phage propagation as detected by a standard lysis procedure and by the described qPCR assay.

<p>Notes: Phage propagation was performed at 28°C unless it is specifically indicated in the column “φA1122 Growth/qPCR”.</p><p>*Serovars of <i>Y. pseudotuberculosis</i> and <i>Y. enterocolitica</i> are shown in parentheses if known.</p><p>**On <i>Y. pseudotuberculosis</i> IB, a very weak propagation of φA1122 was observed at 24°C, six orders of magnitude lower than in <i>Y. pestis</i> CO92.</p