Pestis Cells Research Articles

Adhesiveness of the specific phages to yersiniae cells assessed by optical trapping

In recent years, there has been increasing interest in using bacteriophages in the complex therapy of some infectious diseases. Objectively, it is related to the spread of antibiotic resistance and a small number of side effects triggered by phages applied locally and parenterally. Yersiniae are no exception to this rule, but their interaction with virions has not been studied thoroughly. In three types of lytic Yersiniae bacteriophages are used for diagnostic purposes, which differ in specificity, virulence, and morphological features. We evaluated the interaction force between phages and bacteriae by optical trapping.
 Materials and methods. Live Yersinia pseudotuberculosis and Y. pestis cells with different LPS structure and preparations of three bacteriophages Pokrovskayas phage, dHerelles phage (also named as the pseudotuberculosis phage) and the phage L-413C were used. The Pokrovskayas phage is well studied and widely used in diagnostics, but the mechanism of its adsorption on Y. pestis and Y. pseudotuberculosis cells is not well-understood. The phage L-413C lyses plague bacteria, while dHerrels phage can lyse both pathogens. Virions were linked to the surface of aminated glass. Optically trapped microbial cells were approached to the glass surface and then retracted in the opposite direction. The amplitude of the leap was recalculated into force through calibration coefficients. The primary data were processed in the program R; statistical analysis was performed using Matlab 7.0 and Statistica 12
 Results and discussion. The analysis of histograms revealed the presence of a specific component in the Y. pseudotuberculosisdHerrells phage (Fmean = 7.463.52 pN) system, as well as in all pairs involving plague microbe. In the latter case, the interaction forces were comparable: 8.643.83 pN for dHerelles phage,11.034.22 pN for Pokrovskayas phage, and 10.424.79 pN for phage L-413C. The average detachment force from the substrate treated with bovine serum albumin (BSA) was comparable for both cell types. Our results convincingly prove an opportunity for using the optical trapping to estimate interaction force in other bacteriumphage systems.

Activation of T-helper in vitro under the Influence of Yersinia pestis Antigens in People Vaccinated against the Plague

Relevance. The search for informative markers for assessing the immunological efficacy of a live plague vaccine remains an urgent scientific task. The goal of the studyAims was to make a comparative assessment of the level helper T-cells activation by CD69 and HLA-DR markers, in people, vaccinated against plague, in an in vitro test, using the disintegrated by ultrasonic Y. pestis cells, grown at temperature 28 °C as a specific stimulator.Materials & Methods. A cytofluorimetric analysis of blood samples of 45 individuals vaccinated against the plague was carried out. The dependence of the result of cytological analysis of the two studied cell markers on the immunoregulatory index (IRI) of the vaccinated donor at the time of vaccination was revealed.Results. It was found that for newly vaccinated individuals, the T-helpers were more intensive and prolonged by the early activation marker, while for those revaccinated with the immunoregulatory index, at the time of vaccination more than 1.5, the more intense cellular response was by the late activation marker.Conclusions. Thus, the possibility of quantitative evaluation of the immunological efficacy of vaccination against plague, based on the identification of lymphocyte activation markers when stimulated with a specific antigen, is confirmed.

Force spectroscopy of interactions between Yersinia pseudotuberculosis and Yersinia pestis cells and monoclonal antibodies using optical tweezers.

The interactions of a microbial cell with host cells and humoral factors play an important role in the development of infectious diseases. The study of these mechanisms contributes to the development of effective methods for the treatment of bacterial infections. One of the possible approaches to studying bacterial adhesion to host cells is based on the use of the optical trap method. The aim of this work was to assess the significance of lipopolysaccharide O-antigen on the adhesiveness of Yersinia pseudotuberculosis using a model system including a bacterial cell captured by a laser beam and monoclonal antibodies (mAbs) bound covalently to a glass substrate. Registered interaction forces between Y. pseudotuberculosis cells and complementary antibodies to the O-antigen of lipopolysaccharide (LPS) or the B antigen outer membrane protein were 5.9 ± 3.3 and 2.0 ± 1.8 pN, respectively. Interaction forces between O-antigen deficient Y. pestis cells and the mentioned mAbs were 4.2 ± 2.9 and 9.6 ± 4.9 pN. The results are qualitatively consistent with earlier data obtained by using a model system based on polymer beads sensitized with LPS from Y. pseudotuberculosis and Y. pestis and surfaces coated by the aforementioned antibodies. This indicates that the immunochemical activity of Y. pseudotuberculosis cells is mediated mainly by the lipopolysaccharide. The model described can be used in similar studies of physicochemical and immunochemical mechanisms of bacterial adhesiveness.

Formation of Neutrophil Extracellular Traps when Modeling Plague Infection in Mice Immunized with Yersinia pestis EV NIIEG

The purpose of the study was to determine the effect of Yersinia pestis EV NIIEG on the process of neutrophil extracellular traps formation in vivo when modeling plague infection and assess their contribution to antiplague protection.Materials and methods. BALB/c mice, which were immunized subcutaneously with the Y. pestis EV NIIEG vaccine strain, were used in the study. Animals were infected with a virulent strain Y. pestis 231 at a dose of 20 LD50 (103 CFU). To evaluate the contribution of neutrophil extracellular traps (NETs) to antibacterial protection, an experimental model was used based on fermenting NETs in the abdominal cavity of mice with nuclease. To calculate the number of NETs in peritoneal exudate (PE) fluorescent microscopy was applied. Phagocytic activity of PE cells was determined by flow cytometry. Bactericidal effect of NETs was recorded using bacteriological method.Results and discussion. In pre-immunized mice, the process of NETs formation in response to the reintroduction of plague microbe living cells was 5 times more intense than in intact animals and was accompanied by a significant increase in the killing of Y. pestis cells in PE. The use of micrococcus nuclease in the experiment for fermentation of the NETs, produced in the body of immunized animals, provided evidence of NET participation in conferring anti-infective protection against plague infection. Thus, the established fact of the NET formation in case of Y. pestis infection of mice immunized with Y. pestis EV NIIEG vaccine strain and the influence of this process on the effectiveness of protection against plague is the basis for further clarifying the immunopathogenetic role of neutrophil granulocytes in plague.

Effect of antiplague vaccination on phagocytic activity of human blood granulocytes

Anti-plague vaccination stimulates phagocytosis of Yersinia pestis cells by blood leukocytes of laboratory animals. However its effect on the phagocytic activity of human white blood cells towards plague microbes has not been studied. In this work, flow cytometry was used to study phagocytic activity of blood granulocytes towards Y. pestis, Escherichia coli and Staphylococcus aureus in the people vaccinated, or not vaccinated against plague. These persons inhabited the territories of natural plague foci in Russian Federation. The results of phagocytic activity were evaluated in microvolumes of whole blood. The induced IFNγ production was evaluated in blood by enzyme immunoassay technique, and the titers of specific antibodies to plague-specific F1 antigen were determined in blood serum samples. It was found, that the subjects who have never been vaccinated against plague had 3-fold lower granulocyte phagocytic activity towards Y. pestis than the persons repeatedly vaccinated with live plague vaccine, and 5-fold lower than phagocytosis of E. coli and S. aureus. The next annual revaccination caused additional stimulation of in vitro phagocytic capacity of blood leukocytes with respect to plague microbes. Individual values of phagocytic indices correlated with serum antibody titers (r = 0.65, p = 0.0004), and with levels of IFNγ production (r = 0.73, p = 0.0004). Thus, the obtained data confirmed dependence between phagocytic activity of human blood leukocytes and the pathogen type, thus allowing to demonstrate a connection between activated phagocytic response to Y. pestis cells in live plague-vaccinated individuals with other immune indexes of vaccine efficiency (specific antibodies to F1, induced IFNγ production). Further research in this direction will bring us closer to development of an informative test for assessing the intensity of antiplague immunity.

Development of Anti-Yersinia pestis Human Antibodies with Features Required for Diagnostic and Therapeutic Applications.

BackgroundYersinia pestis is a category A infective agent that causes bubonic, septicemic, and pneumonic plague. Notably, the acquisition of antimicrobial or multidrug resistance through natural or purposed means qualifies Y. pestis as a potential biothreat agent. Therefore, high-quality antibodies designed for accurate and sensitive Y. pestis diagnostics, and therapeutics potentiating or replacing traditional antibiotics are of utmost need for national security and public health preparedness.MethodsHere, we describe a set of human monoclonal immunoglobulins (IgG1s) targeting Y. pestis fraction 1 (F1) antigen, previously derived from in vitro evolution of a phage-display library of single-chain antibodies (scFv). We extensively characterized these antibodies and their effect on bacterial and mammalian cells via: ELISA, flow cytometry, mass spectrometry, spectroscopy, and various metabolic assays.ResultsTwo of our anti-F1 IgG (αF1Ig 2 and αF1Ig 8) stood out for high production yield, specificity, and stability. These two antibodies were additionally attractive in that they displayed picomolar affinity, did not compete when binding Y. pestis, and retained immunoreactivity upon chemical derivatization. Most importantly, these antibodies detected <1,000 Y. pestis cells in sandwich ELISA, did not harm respiratory epithelial cells, induced Y. pestis agglutination at low concentration (350 nM), and caused apparent reduction in cell growth when radiolabeled at a nonagglutinating concentration (34 nM).ConclusionThese antibodies are amenable to the development of accurate and sensitive diagnostics and immuno/radioimmunotherapeutics.

Investigations into Enhancing Yersinia pestis Cells Viability following Environmental Sampling for Forensic Analysis.

Following an intentional or accidental bio-warfare agent (BWA) release, environmental sample analysis is absolutely critical to determine the extent of contamination. When dealing with nonspore forming BWA (e.g., Yersinia pestis), retention of cell viability is central to such analyses. Even though significant advances have been achieved in DNA sequencing technologies, a positive identification of BWAs in environmental samples must be made through the ability of cells to form colony-forming units upon culturing. Inability to revive the cells between collection and analysis renders such studies inconclusive. Commercial kits designed to preserve the viability of pathogens contained within clinical samples are available, but many of them have not been examined for their ability to preserve samples containing suspected BWAs. The study was initiated to examine the applicability of commercial solutions aiding in retention of Y.pestis viability in samples stored under nonpermissive temperatures, that is, 40 and 37°C. While none of the tested solutions sustained cell viability at 40°C, the results show five out of 17 tested preservatives were capable of supporting viability of Y.pestis at 37°C.

Development of a rapid viability polymerase chain reaction method for detection of Yersinia pestis

Due to the occurrence of natural plague outbreaks and its historical usage as a biological weapon, Yersinia pestis is considered one of the high-priority biological threat agents. It can remain viable in certain environments including water for >100 days. Because of its slow-growth characteristic, it usually takes three or more days to detect and confirm the identity of viable Y. pestis cells by PCR, serological, or biochemical assays when using the traditional microbiological plate-culture-based analysis, and that too, assuming faster growing microbes present in a water sample do not mask the Y. pestis colonies and interfere with analysis. Therefore, a rapid-viability Polymerase Chain Reaction (RV-PCR) method was developed for detection of Y. pestis. The RV-PCR method combines 24 h-incubation broth culture in a 48-well plate, and pre- and post-incubation differential PCR analyses, thereby allowing for rapid and high-throughput sample analysis compared with the current plate culture method. One chromosomal and two plasmid gene target-based real-time PCR assays were down-selected, showing ca. 10 genome equivalent detection; the chromosomal assay was then used for RV-PCR method development. A 101-cell level (10–99 cells) sensitivity of detection was demonstrated even with complex sample backgrounds including known PCR inhibitors (ferrous sulfate and humic acid), as well as metal oxides and microbes present in Arizona Test Dust (ATD). The method sensitivity was maintained in the presence of dead Y. pestis cells up to 104 cells per sample. While affording high-throughput and rapid sample analysis, the 48-well plate format used in this method for sample enrichment significantly reduced labor requirements and generation of BioSafety Level-3 (BSL-3) laboratory waste as compared to the usual microbiological plate-culture-based methods. This method may serve as a model for other vegetative bacterial pathogens.

Structural and Functional Characterization of Vitronectin and Ail for Host Cell Invasion by Y. pestis

Ail (attachment invasion locus) is a membrane protein that is highly expressed on the surface of the bacterium Yersinia pestis, which is the causative agent of plague. Ail interacts with various human host factors to promote pathogenesis and bacterial survival including: (1) resistance to lysis by the human complement system, a major effector of innate immunity; (2) adhesion to human cells; (3) suppression of blood clotting; and (4) enhanced fibrinolysis. Recently, we have demonstrated that Ail also recruits Vitronectin (Vn) from human serum to the Y. pestis cell surface and promotes its degradation by the Y. pestis outer membrane protease Plasminogen activator (Pla). Vn is a 459-residue multidomain, multifunctional protein that regulates the complement, coagulation and fibrinolysis pathways. It contains binding sites for a wide range of molecules, including Heparin, Integrins and pathogenic proteins. Despite its importance, little is known about the structure of Vn, or the molecular interaction between Ail and Vn to promote Y. pestis pathogenesis. Here, we demonstrate that Vn fragments can be expressed and purified from E. coli for in vitro binding assays and NMR structural studies. The data provide molecular information about the interaction of Ail with this central human protein and help explain the functional role of Ail in Y. pestis pathogenesis.

Study of Interactions of Microscopic Fungi Isolated from Burrow Microbiotopes of Great Gerbil and Little Souslik with Plague Agent and its Vectors

At present there is strong evidence of the long-term persistence of Yersinia pestis in soil during interepizootic period in nonculturable and L-forms of bacteria. This information helps to reveal the mechanisms of emergence, establishment and extinction of plague foci. Deciphering causes of transformation of plague microbe in rodent burrows is possible only under condition of detailed study of biocenotic relations of plague causative agent with each of the multiple biocenosis components, including microorganisms -permanent inhabitants of the burrow microbiotopes in natural plague foci. Purpose of the study: Study of interactions of microscopic fungi-micromycetes iso-lated from burrow microbiotopes of great gerbil and little souslik in the Ural-Emba interfluve and in Nogai steppe with plague agent and its vectors. Materials and methods: The effect of 74 micromycete species, isolated in natural plague foci, on Yersinia pestis virulent strains was studied in vitro and in vivo (in organisms of X. skrjabini fleas). In the experiments in vitro fungi metabolites were applied on the lawns of Y. pestis test-cultures with subsequent recording of the size of lysed zones. Fleas were challenged in tubes with filtering-paper strips impregnated by fungal cultural liquid and then LT100 value was calculated. In the experiments in vivo fungus conidia were applied on the surface of insect cuticle; flea challenging with plague was carried out on agonizing white mice infected by plague microbe. Insect challenge with plague and fungal conidia was alternated in different succession. Results and discussion: Of 74 micromycete strains 78.4% produced antagonistic effect in relation to 1-5 Y. pestis strains. Ashergillus versicolor, A. clavatus, A. sulfereus, Mucor racematus, Penicillium baarnensens, P. sp. relative to P. charlesii, P. sp. relative to P. martensii, P. insectus, P. chrysogenum fungi induced growth suppression of all five test strains of Y. pestis. High antibiotic activity of fungi metabolites was accompanied by insecticidal activity against Xenopsylla skrjabini fleas. Microscopic fungi P. funiculosum, P. cyclopium, P. chrisogenum, P. charlesii. P. canescens, A. versicolor induced 100% flea death on 4-6 day. As a result of insect pretreatment by conidia of P. funiculosum with subsequent challenging with plague microbe block formation in fleas decreased 14 times, and the number of Y. pestis cells after 12 days was 25-33 times less independent of succession, in which insects were challenged with microorganisms. Conclusion. It is supposed that micromycetes could play an important role in natural plague foci, adjusting epizootic activity of plague vectors and causative agent.

Specific Appurtenance, Numbers, and Dynamics of Interaction of Acanthamoeba from Soils of Gorno-Altai High-Mountain Plague Focus with Yersinia pestis Strains

Objective of the study is to analyze the appurtenance, numbers, and dynamics of interaction of acanthamoeba from soils of GornoAltai plague focus with Yersinia pestis 367 strain, isolated in 2016 in enzootic territory of this focus. Materials and methods. Utilized were soil amoeba from Gorno-Altai high-mountain focus and the strain Y. pestis 367 of the main subspecies of antique biovar, isolated there in 2016. Determination of systematic relation of the isolated amoeba was carried out using PCR with genus specific primers and sequencing of the obtained PCR fragments followed by identification of nucleotide sequences against GenBank database. Localization of Y. pestis cells in acanthamoeba was performed using fluorescent antibody technique by means of Axio Imager Z2 (Carl Zeiss, Germany). Results and conclusions. For the first time ever established has been the presence of Acanthamoeba castellanii in soils of burrows of Marmota altaica in the numbers of up to 300000 cells/gr in Gorno-Altai high-mountain focus. Investigated has been the dynamics of interaction of these microorganisms. Preservation of the agent in vacuoles of endoplasmatic reticulum within 14 days has been revealed. It is an indicative of the possibility of Y. pestis persistence in amoeba of Acanthamoeba subspecies in soil biocoenosis of Gorno-Altai high-mountain plague focus.

THE ROLE OF YERSINIA PESTIS RESIDENT PLASMIDS PMT1, PCD1, AND PPCP1 IN THE PRODUCTION OF LIPOPOLYSACCHARIDE EXTRACELLULAR FORM

Objective of the study is to investigate the role of resident plasmids pMT1, pCD1, and pPCP1 in the production of extracellular form of Yersinia pestis lipopolysaccharide (LPS). Materials and methods . The experiments have been performed using Y. pestis strain EV76 (pMT1, pCD1, pPCP1), carrying the whole plasmid set, as well as plasmid-free Y. pestis variant EV76 (pMT1 - , pCD1 - , pPCP1 - ), and isogenic clones, harbouring only one plasmid: Y. pestis EV76 (pMT1); Y. pestis EV76 (pCD1); Y. pestis EV76 (pPCP1). The presence of extracellular LPS in the incubation medium of Y. pestis EV76 cells has been confirmed by supernatant toxicity for laboratory animals and also by LAL-test reaction. Results and conclusions . It has been established that LPS extracellular form is produced by 37 °C Y. pestis EV76 cultures of the initial strain and its variants, carrying pMT1 or pPCP1 plasmid. Plasmid-free cultures and variant harbouring pCPP1 plasmid are deprived of such ability. The results of LAL-test has shown that the process of LPS separation from cell wall membrane into the environment is associated with translocation of proteins encoded by pMT1 and pCD1 plasmids and constitutes a natural form of existence of Y. pestis cells. The involvement of pCD1 plasmid in realization of the toxic potential of Y. pestis LPS has been established for the first time ever.

Dermal neutrophil, macrophage and dendritic cell responses to Yersinia pestis transmitted by fleas.

Yersinia pestis, the causative agent of plague, is typically transmitted by the bite of an infected flea. Many aspects of mammalian innate immune response early after Y. pestis infection remain poorly understood. A previous study by our lab showed that neutrophils are the most prominent cell type recruited to the injection site after intradermal needle inoculation of Y. pestis, suggesting that neutrophil interactions with Y. pestis may be important in bubonic plague pathogenesis. In the present study, we developed new tools allowing for intravital microscopy of Y. pestis in the dermis of an infected mouse after transmission by its natural route of infection, the bite of an infected flea. We found that uninfected flea bites typically induced minimal neutrophil recruitment. The magnitude of neutrophil response to flea-transmitted Y. pestis varied considerably and appeared to correspond to the number of bacteria deposited at the bite site. Macrophages migrated towards flea bite sites and interacted with small numbers of flea-transmitted bacteria. Consistent with a previous study, we observed minimal interaction between Y. pestis and dendritic cells; however, dendritic cells did consistently migrate towards flea bite sites containing Y. pestis. Interestingly, we often recovered viable Y. pestis from the draining lymph node (dLN) 1 h after flea feeding, indicating that the migration of bacteria from the dermis to the dLN may be more rapid than previously reported. Overall, the innate cellular host responses to flea-transmitted Y. pestis differed from and were more variable than responses to needle-inoculated bacteria. This work highlights the importance of studying the interactions between fleas, Y. pestis and the mammalian host to gain a better understanding of the early events in plague pathogenesis.

Electron-Microscopic Investigation of Interactions between &lt;I&gt;Yersinia pseudotuberculosis&lt;/I&gt;, &lt;I&gt;Yersinia pestis&lt;/I&gt; Cells and Specific Bacteriophages

Carried out has been electron-microscopic investigation of dynamics of interactions between Yersinia pseudotuberculosis, Yersinia pestis cells and specific bacteriophages in vitro. Identified have been peculiarities in morphological manifestation of the interactions. Determined are morphometric characteristics of the bacteriophages utilized in the experiment. It is demonstrated that after 40 minutes of co-incubation of Y. pseudotuberculosis with specific bacteriophage emergence of persisting forms of microbial cells is observed, while in case of EV strain infecting with Pokrovskaya bacteriophage, this phenomenon is not detected.

Inactivation of avirulent pgm+ and Δpgm Yersinia pestis by ultraviolet light (UV-C)

Yersinia pestis is the causative agent of bubonic plague. Though not considered a foodborne pathogen, Y. pestis can survive, and even grow, in some foods, and the foodborne route of transmission is not without precedent. As such, concerns exist over the possible intentional contamination of foods with this deadly pathogen. Here we report the inactivation of avirulent (pYV-minus) strains of Y. pestis by ultraviolet light (UV-C, 254 nm). Two strains of Y. pestis containing an intact pgm virulence locus (pgm+) and strains from which the pgm locus was spontaneously deleted (Δpgm) were tested using cells grown in both logarithmic and stationary phase. The D10 values for inactivation (the UV-C dose required to inactivate one log of bacterial cells) of Y. pestis on the surface of agar plates ranged from 0.69 to 1.09 mJ/cm2. A significant difference was observed between the inactivation of cells of Y. pestis strain Yokohama grown in logarithmic and stationary phases, but no significant difference between growth phase sensitivity to UV-C was observed in Y. pestis strain Kuma. No difference in D10 values was observed between pgm+ and Δpgm strains of Yokohama grown to either logarithmic or stationary phase. A measurable difference was observed between the D10 of Kuma pgm+ and Kuma Δpgm grown in logarithmic phase, but this difference was diminished in the Kuma strains grown to stationary phase. Though strain variations exist, the results showing that UV-C can inactivate Y. pestis cells on agar surfaces suggest that UV-C would be effect in inactivating Y. pestis on food surfaces, particularly foods with a smooth surface.

Evaluation of swabs and transport media for the recovery of Yersinia pestis

The Government Accountability Office report investigating the surface sampling methods used during the 2001 mail contamination with Bacillus anthracis brought to light certain knowledge gaps that existed regarding environmental sampling with biothreat agents. Should a contamination event occur that involves non-spore forming biological select agents, such as Yersinia pestis, surface sample collection and processing protocols specific for these organisms will be needed. Two Y. pestis strains (virulent and avirulent), four swab types (polyester, macrofoam, rayon, and cotton), two pre-moistening solutions, six transport media, three temperatures, two levels of organic load, and four processing methods (vortexing, sonicating, combined sonicating and vortexing, no agitation) were evaluated to determine the conditions that would yield the highest percent of cultivable Y. pestis cells after storage. The optimum pre-moistening agent/transport media combination varied with the Y. pestis strain and swab type. Directly inoculated macrofoam swabs released the highest percent of cells into solution (93.9% recovered by culture) and rayon swabs were considered the second best swab option (77.0% recovered by culture). Storage at 4°C was found to be optimum for all storage times and transport media. In a worst case scenario, where the Y. pestis strain is not known and sample processing and analyses could not occur until 72h after sampling, macrofoam swabs pre-moistened with PBS supplemented with 0.05% Triton X-100 (PBSTX), stored at 4°C in neutralizing buffer (NB) as a transport medium (PBSTX/NB) or pre-moistened with NB and stored in PBSTX as a transport medium (NB/PBSTX), then vortexed 3min in the transport medium, performed significantly better than all other conditions for macrofoam swabs, regardless of strain tested (mean 12 – 72h recovery of 85.9–105.1%, p<0.001). In the same scenario, two combinations of pre-moistening medium/transport medium were found to be optimal for rayon swabs stored at 4°C (p<0.001), then sonicated 3min in the transport medium; PBSTX/PBSTX and NB/PBSTX (mean 12–72h recovery of 83.7–110.1%).

Detection and Isolation of Yersinia pestis Without Fraction 1 Antigen by Monoclonal Antibody in Foods and Water

Most available immunoassays for Yersinia pestis are based on the detection of fraction 1 antigen (F1) when yersiniae are grown at 37°C. A monoclonal antibody (MAb) was developed based on the detection of surface antigens that are not F1. F1-deficient Y. pestis cells were induced and used to immunize BALB/c mice from which MAb (immunoglobulin G1), which specifically recognizes Y. pestis, with or without F1, was obtained. This MAb (6B5) did not cross-react with enteric bacteria, including Yersinia enterocolitica. Enzyme-linked immunosorbent assay results revealed that MAb 6B5 is specific for Y. pestis, with the exception of a minor cross-reaction with Yersinia pseudotuberculosis. Western immunoblot analysis revealed that MAb 6B5 recognizes a Y. pestis outer membrane protein of ca. 30 kDa. Magnetic beads that were coated with MAb 6B5 were compared with beads coated with polyclonal antibody (PAb; rabbit) against Y. pestis for the isolation of Y. pestis in food and water samples by using a PATHATRIX cell concentration apparatus. Enrichment cultures of Y. pestis in different foods by using two different times (6 and 24 h) in brain heart infusion broth at 37°C were evaluated. Results revealed MAb 6B5-coated magnetic beads were equivalent to magnetic beads coated with PAb against Y. pestis A1122 whole cells in concentrating Y. pestis for isolation, especially when samples were enriched for 6 h. However, the selectivity for Y. pestis of the magnetic beads coated with MAb 6B5 was greater than that coated with PAb.

A Yersinia pestis-specific, lytic phage preparation significantly reduces viable Y. pestis on various hard surfaces experimentally contaminated with the bacterium

Five Y. pestis bacteriophages obtained from various sources were characterized to determine their biological properties, including their taxonomic classification, host range and genomic diversity. Four of the phages (YpP-G, Y, R and YpsP-G) belong to the Podoviridae family, and the fifth phage (YpsP-PST) belongs to the Myoviridae family, of the order Caudovirales comprising of double-stranded DNA phages. The genomes of the four Podoviridae phages were fully sequenced and found to be almost identical to each other and to those of two previously characterized Y. pestis phages Yepe2 and φA1122. However, despite their genomic homogeneity, they varied in their ability to lyse Y. pestis and Y. pseudotuberculosis strains. The five phages were combined to yield a “phage cocktail” (tentatively designated “YPP-100”) capable of lysing the 59 Y. pestis strains in our collection. YPP-100 was examined for its ability to decontaminate three different hard surfaces (glass, gypsum board and stainless steel) experimentally contaminated with a mixture of three genetically diverse Y. pestis strains CO92, KIM and 1670G. Five minutes of exposure to YPP-100 preparations containing phage concentrations of ca. 109, 108 and 107 PFU/mL completely eliminated all viable Y. pestis cells from all three surfaces, but a few viable cells were recovered from the stainless steel coupons treated with YPP-100 diluted to contain ca. 106 PFU/mL. However, even that highly diluted preparation significantly (p = < 0.05) reduced Y. pestis levels by ≥ 99.97%. Our data support the idea that Y. pestis phages may be useful for decontaminating various hard surfaces naturally- or intentionally-contaminated with Y. pestis.

Determination of post-culture processing with carbohydrates by MALDI-MS and TMS derivatization GC–MS

Biological materials generally require stabilization to retain activity or viability in a dry form. A number of industrial products, such as vaccines, probiotics and biopesticides have been produced as dry preparations. The same methods and materials used for stabilizing commercial microbial products may be applicable to preserving biothreat pathogens in a dry form. This is a likely step that may be encountered when looking at samples from terrorism attempts since only spores, such as those from Bacillus anthracis, are inherently stable when dried. The stabilizers for microbial preparations generally include one or more small carbohydrates. Different formulations have been reported for different industrial products and are often determined empirically. However sugar alcohols (mannitol and sorbitol) and disaccharides (lactose, sucrose and trehalose) are the common constituents of these formulations. We have developed an analytical method for sample preparation and detection of these simple carbohydrates using two complementary analytical tools, MALDI-MS and GC–MS. The native carbohydrates and other constituents of the formulation are detected by MALDI-MS as a screening tool. A longer and more detailed analysis is then used to specifically identify the carbohydrates by derivatization and GC–MS detection. Both techniques were tested against ten different types of stabilization recipes with Yersinia pestis cell mass cultured on different media types used as the biological component. A number of additional components were included in these formulations including proteins and peptides from serum or milk, polymers (e.g. poly vinyl pyrrolidone – PVP) and detergents (e.g. Tween). The combined method was characterized to determine several figures of merit. The accuracy of the method was 98% for MALDI-MS and 100% for GC–MS. The repeatability for detection of carbohydrates by MALDI-MS was determined to be 96%. The repeatability of compound identification by GC–MS was determined by monitoring variation in retention time, which is vital for identification of isomeric carbohydrates. The figures of merit illustrate an effective and accurate method for mono and disaccharide detection independent of formulation. This meets our primary goal for method development as small carbohydrates are among the most common stabilizers employed.

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively. Although the natural occurrence of both diseases' is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease's inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management. Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant "light-tagged" reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens. For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage ΦA1122 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage Wβ::luxAB. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format. Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.