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

Bacteriophage-Resistant Mutants in Yersinia pestis: Identification of Phage Receptors and Attenuation for Mice

Background: Bacteriophages specific for Yersinia pestis are routinely used for plague diagnostics and could be an alternative to antibiotics in case of drug-resistant plague. A major concern of bacteriophage therapy is the emergence of phageresistant mutants. The use of phage cocktails can overcome this problem but only if the phages exploit different receptors. Some phage-resistant mutants lose virulence and therefore should not complicate bacteriophage therapy. Methodology/Principal Findings: The purpose of this work was to identify Y. pestis phage receptors using site-directed mutagenesis and trans-complementation and to determine potential attenuation of phage-resistant mutants for mice. Six receptors for eight phages were found in different parts of the lipopolysaccharide (LPS) inner and outer core. The receptor for R phage was localized beyond the LPS core. Most spontaneous and defined phage-resistant mutants of Y. pestis were attenuated, showing increase in LD 50 and time to death. The loss of different LPS core biosynthesis enzymes resulted in the reduction of Y. pestis virulence and there was a correlation between the degree of core truncation and the impact on virulence. The yrbH and waaA mutants completely lost their virulence. Conclusions/Significance: We identified Y. pestis receptors for eight bacteriophages. Nine phages together use at least seven different Y. pestis receptors that makes some of them promising for formulation of plague therapeutic cocktails. Most phage-resistant Y. pestis mutants become attenuated and thus should not pose a serious problem for bacteriophag

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

Recent Findings Regarding Maintenance of Enzootic Variants of Yersinia pestis in Sylvatic Reservoirs and Their Significance in the Evolution of Epidemic Plague

Despite the widespread presence of bubonic plague in sylvatic reservoirs throughout the world, the causative agent (Yersinia pestis) evolved in its present form within the last 20,000 years from enteropathogenic Yersinia pseudotuberculosis. Comparison of the genomes from the two species revealed that Y. pestis possesses only a few unique plasmid-encoded genes that contribute to acute disease, whereas this organism has lost about 13% of the chromosomal genes that remain active in Y. pseudotuberculosis. These losses reflect readily detectable additions, deletions, transpositions, inversions, and acquisition of about 70 insertion sequence (IS) inserts, none of which are likely to promote increased virulence. In contrast, major enzymes of intermediary metabolism, including glucose 6-phosphate dehydrogenase (Zwf ) and aspartase, are present but not catalytically functional due to the presence of missense mutations. The latter are generally not detectable by the technology of bioinformatics and, in the case of Y. pestis, result in radical changes in the metabolic flow of carbon. As an important consequence, plague bacilli exhibit a stringent low-calcium response characterized by conversion of L-glutamate (and metabolically related amino acids) to L-aspartate with secretion of the latter into supernatant fluid at 37°C in culture media containing Na+ but lacking added Ca2+. This phenomenon also occurs in vivo and likely adversely affects the bioenergetics of host amino acid pools. Curiously, aspartase is functional in all tested enzootic (pestoides) strains of Y. pestis. These isolates are typically restricted to the ancient plague reservoirs of Central Asia and Africa and are fully virulent in members of the rodent Superfamily Muroidea but avirulent in guinea pigs and man. The implications of these findings for the distribution and ecology of Y. pestis could be significant

Russian vaccines against especially dangerous bacterial pathogens

In response to the epidemiological situation, live attenuated or killed vaccines against anthrax, brucellosis, cholera, glanders, plague and tularemia were developed and used for immunization of at-risk populations in the Former Soviet Union. Certain of these vaccines have been updated and currently they are used on a selective basis, mainly for high risk occupations, in the Russian Federation. Except for anthrax and cholera these vaccines currently are the only licensed products available for protection against the most dangerous bacterial pathogens. Development of improved formulations and new products is ongoing

A multiwavelength analysis of a collection of short-duration GRBs observed between 2012 and 2015

We investigate the prompt emission and the afterglow properties of short-duration gamma-ray burst (sGRB) 130603B and another eight sGRB events during 2012-2015, observed by several multiwavelength facilities including the Gran Canarias Telescope 10.4 m telescope. Prompt emission high energy data of the events were obtained by INTEGRAL-SPI-ACS, Swift-BAT, and Fermi-GBM satellites. The prompt emission data by INTEGRAL in the energy range of 0.1-10 MeV for sGRB 130603B, sGRB 140606A, sGRB 140930B, sGRB 141212A, and sGRB 151228A do not show any signature of the extended emission or precursor activity and their spectral and temporal properties are similar to those seen in case of other short bursts. For sGRB 130603B, our new afterglow photometric data constrain the pre-jet-break temporal decay due to denser temporal coverage. For sGRB 130603B, the afterglow light curve, containing both our new and previously published photometric data is broadly consistent with the ISM afterglow model. Modeling of the host galaxies of sGRB 130603B and sGRB 141212A using the LePHARE software supports a scenario in which the environment of the burst is undergoing moderate star formation activity. From the inclusion of our late-time data for eight other sGRBs we are able to: place tight constraints on the non-detection of the afterglow, host galaxy, or any underlying 'kilonova' emission. Our late-time afterglow observations of the sGRB 170817A/GW170817 are also discussed and compared with the sub-set of sGRBs

A multi-wavelength analysis of a collection of short-duration GRBs observed between 2012-2015

We investigate the prompt emission and the afterglow properties of short duration gamma-ray burst (sGRB) 130603B and another eight sGRB events during 2012-2015, observed by several multi-wavelength facilities including the GTC 10.4m telescope. Prompt emission high energy data of the events were obtained by INTEGRAL/SPI/ACS, Swift/BAT and Fermi/GBM satellites. The prompt emission data by INTEGRAL in the energy range of 0.1-10 MeV for sGRB 130603B, sGRB 140606A, sGRB 140930B, sGRB 141212A and sGRB 151228A do not show any signature of the extended emission or precursor activity and their spectral and temporal properties are similar to those seen in case of other short bursts. For sGRB130603B, our new afterglow photometric data constraints the pre jet-break temporal decay due to denser temporal coverage. For sGRB 130603B, the afterglow light curve, containing both our new as well as previously published photometric data is broadly consistent with the ISM afterglow model. Modeling of the host galaxies of sGRB 130603B and sGRB 141212A using the LePHARE software supports a scenario in which the environment of the burst is undergoing moderate star formation activity. From the inclusion of our late-time data for 8 other sGRBs we are able to; place tight constraints on the non-detection of the afterglow, host galaxy or any underlying kilonova emission. Our late-time afterglow observations of the sGRB 170817A/GW170817 are also discussed and compared with the sub-set of sGRBs