Bioterrorism Agent Research Articles

Innovative Ricin Toxin Detection: Unraveling Apurinic/Apyrimidinic Lyase Activity and Developing Fluorescence Sensors.

Ricin toxin (RT) is a potential bioterrorism agent because of its high potency, extremely small lethal dose, ease of preparation, and notable stability. Therefore, a portable method is urgently required to efficiently detect and determine the presence of toxicity of RT and evaluate its potency for public health monitoring and counter-bioterrorism responses. Currently, enzyme-based assays for detecting RT mainly focus on its N-glycosidase activity. In this study, we demonstrated that RT exhibits apurinic/apyrimidinic (AP) lyase activity using several methods. Characterization of the enzyme reaction and kinetics revealed that AP lyase activity is optimal at 59 °C and pH 4.0. This activity is highly pH-sensitive, remaining active between pH 3.0 and pH 4.6. Furthermore, we developed a portable fluorescence-based lateral flow assay (FLFA) that detects RT much faster than existing assays based on its N-glycosidase activity. Moreover, this assay can efficiently detect RT at nanogram levels from complex matrix samples within 1.5 h while simultaneously determining its biological activity. In conclusion, the discovery of the AP lyase activity of RT and the development of FLFA represent novel approaches for studying the enzymatic profiles of other ribosome-inactivating proteins.

Breaking the cellular defense: the role of autophagy evasion in Francisella virulence.

Many pathogens have evolved sophisticated strategies to evade autophagy, a crucial cellular defense mechanism that typically targets and degrades invading microorganisms. By subverting or inhibiting autophagy, these pathogens can create a more favorable environment for their replication and survival within the host. For instance, some bacteria secrete factors that block autophagosome formation, while others might escape from autophagosomes before degradation. These evasion tactics are critical for the pathogens' ability to establish and maintain infections. Understanding the mechanisms by which pathogens avoid autophagy is crucial for developing new therapeutic strategies, as enhancing autophagy could bolster the host's immune response and aid in the elimination of pathogenic bacteria. Francisella tularensis can manipulate host cell pathways to prevent its detection and destruction by autophagy, thereby enhancing its virulence. Given the potential for F. tularensis to be used as a bioterrorism agent due to its high infectivity and ability to cause severe disease, research into how this pathogen evades autophagy is of critical importance. By unraveling these mechanisms, new therapeutic approaches could be developed to enhance autophagic responses and strengthen host defense against this and other similarly evasive pathogens.

Disease Caused by Filoviruses: An Update

The Marburg and Ebola viruses belong to the Filoviridae family and are known to cause emerging zoonotic diseases. These viruses have a high case fatality rate and are easily transmissible from person to person, which makes them capable of triggering outbreaks, including in non-endemic regions, and are also considered agents of bioterrorism. Fruit bats are the natural reservoirs of these filoviruses. Transmission to humans occurs through direct contact with bodily fluids or tissues from infected animals or humans. The most severe form of filovirus disease manifests as mucocutaneous hemorrhage, often accompanied by multiorgan failure, which is the main cause of death. Traditionally, these diseases are classified in the group of viral hemorrhagic fevers, although this term is being abandoned, as there are not always hemorrhagic manifestations or fever in the patient's clinical history. Currently, no specific antiviral treatment for filovirus disease exists, and the therapeutic approach consists of supportive measures. However, for the Zaire Ebola virus (EBOV), monoclonal antibodies have already been licensed for treatment and post-exposure prophylaxis, in addition to three vaccines available. Due to the public health importance and the possibility of cases outside Africa, this review aims to improve clinical knowledge and the approach to suspected cases of FD. Improved surveillance and preparedness for potential global outbreaks are essential measures to effectively respond to these public health threats and to ensure that healthcare professionals are well-informed and prepared to deal with these diseases.

A Synthetic Oligosaccharide Resembling Francisella tularensis Strain 15 O-Antigen Capsular Polysaccharide as a Lead for Tularemia Diagnostics and Therapeutics.

Francisella tularensis, a category A bioterrorism agent, causes tularemia in many animal species. F. tularensis subspecies tularensis (type A) and holarctica (type B) are mainly responsible for human tularemia. The high mortality rate of 30-60 % caused by F. tularensis subspecies tularensis if left untreated and the aerosol dispersal renders this pathogen a dangerous bioagent. While a live attenuated vaccine strain (LVS) of F. tularensis type B does not provide sufficient protection against all forms of tularemia infections, a significant level of protection against F. tularensis has been observed for both passive and active immunization of mice with isolated O-antigen capsular polysaccharide. Well-defined, synthetic oligosaccharides offer an alternative approach towards the development of glycoconjugate vaccines. To identify diagnostics and therapeutics leads against tularemia, a collection of F. tularensis strain 15 O-antigen capsular polysaccharide epitopes were chemically synthesized. Glycan microarrays containing synthetic glycans were used to analyze the sera of tularemia-infected and non-infected animals and revealed the presence of IgG antibodies against the glycans. Two disaccharide (13 and 18), both bearing a unique formamido moiety, were identified as minimal glycan epitopes for antibody binding. These epitopes are the starting point for the development of diagnostics and therapeutics against tularemia.

A Synthetic Oligosaccharide Resembling Francisella tularensis Strain 15 O‐Antigen Capsular Polysaccharide as a Lead for Tularemia Diagnostics and Therapeutics

AbstractFrancisella tularensis, a category A bioterrorism agent, causes tularemia in many animal species. F. tularensis subspecies tularensis (type A) and holarctica (type B) are mainly responsible for human tularemia. The high mortality rate of 30–60 % caused by F. tularensis subspecies tularensis if left untreated and the aerosol dispersal renders this pathogen a dangerous bioagent. While a live attenuated vaccine strain (LVS) of F. tularensis type B does not provide sufficient protection against all forms of tularemia infections, a significant level of protection against F. tularensis has been observed for both passive and active immunization of mice with isolated O‐antigen capsular polysaccharide. Well‐defined, synthetic oligosaccharides offer an alternative approach towards the development of glycoconjugate vaccines. To identify diagnostics and therapeutics leads against tularemia, a collection of F. tularensis strain 15 O‐antigen capsular polysaccharide epitopes were chemically synthesized. Glycan microarrays containing synthetic glycans were used to analyze the sera of tularemia‐infected and non‐infected animals and revealed the presence of IgG antibodies against the glycans. Two disaccharide (13 and 18), both bearing a unique formamido moiety, were identified as minimal glycan epitopes for antibody binding. These epitopes are the starting point for the development of diagnostics and therapeutics against tularemia.

A Novel PCR-Free Ultrasensitive GQD-Based Label-Free Electrochemical DNA Sensor for Sensitive and Rapid Detection of Francisella tularensis.

Biological warfare agents are infectious microorganisms or toxins capable of harming or killing humans. Francisella tularensis is a potential bioterrorism agent that is highly infectious, even at very low doses. Biosensors for biological warfare agents are simple yet reliable point-of-care analytical tools. Developing highly sensitive, reliable, and cost-effective label-free DNA biosensors poses significant challenges, particularly when utilizing traditional techniques such as fluorescence, electrochemical methods, and others. These challenges arise primarily due to the need for labeling, enzymes, or complex modifications, which can complicate the design and implementation of biosensors. In this study, we fabricated Graphene Quantum dot (GQD)-functionalized biosensors for highly sensitive label-free DNA detection. GQDs were immobilized on the surface of screen-printed gold electrodes via mercaptoacetic acid with a thiol group. The single-stranded DNA (ssDNA) probe was also immobilized on GQDs through strong π-π interactions. The ssDNA probe can hybridize with the ssDNA target and form double-stranded DNA, leading to a decrease in the effect of GQD but a positive shift associated with the increase in DNA concentration. The specificity of the developed system was observed with different microorganism target DNAs and up to three-base mismatches in the target DNA, effectively distinguishing the target DNA. The response time for the target DNA molecule is approximately 1010 s (17 min). Experimental steps were monitored using UV/Vis spectroscopy, Atomic Force Microscopy (AFM), and electrochemical techniques to confirm the successful fabrication of the biosensor. The detection limit can reach 0.1 nM, which is two-five orders of magnitude lower than previously reported methods. The biosensor also exhibits a good linear range from 105 to 0.01 nM and has good specificity. The biosensor's detection limit (LOD) was evaluated as 0.1 nM from the standard calibration curve, with a correlation coefficient of R2 = 0.9712, showing a good linear range and specificity. Here, we demonstrate a cost-effective, GQD-based SPGE/F. tularensis DNA test suitable for portable electrochemical devices. This application provides good perspectives for point-of-care portable electrochemical devices that integrate sample processing and detection into a single cartridge without requiring a PCR before detection. Based on these results, it can be concluded that this is the first enzyme-free electrochemical DNA biosensor developed for the rapid and sensitive detection of F. tularensis, leveraging the nanoenzyme and catalytic properties of GQDs.

Neutralizing chimeric heavy-chain antibody targeting the L-HN domain of Clostridium botulinum neurotoxin type F.

Botulinum toxin (BoNT) from Clostridium botulinum is the most toxic biotoxin known and is also an important bioterrorism agent. After poisoning, the only effective treatment is injection of antitoxin. However, neutralizing nanoantibodies are safer and more effective, representing a promising therapeutic approach. Therefore, it is important to obtain effective neutralizing nanoantibodies. Hence, the present study aimed to construct a phage antibody library by immunizing a camel and screening specific clones that bind to the L-HN domain of BoNT/F and constructing chimeric heavy-chain antibodies by fusing them with a human Fc fragment. The antibodies' affinity and in vivo neutralizing activities were evaluated. The results showed that 2µg of F20 antibody could completely neutralize 20 × the median lethal dose (LD50) of BoNT/F in vitro. Injection of 5mg/kg F20 at 1h, 2h, 3h, and 4h into mice after BoNT/F challenge resulted in complete survival in vivo. Overall, the antibody might be a candidate for the development of new drugs to treat botulism.

Development of a novel sandwich immunoassay based on targeting recombinant Francisella outer membrane protein A for the diagnosis of tularemia.

Tularemia, caused by the bacterium Francisella tularensis, poses health risks to humans and can spread through a variety of routes. It has also been classified as a Tier 1 Select agent by the CDC, highlighting its potential as a bioterrorism agent. Moreover, it is difficult to diagnose in a timely fashion, owing to the non-specific nature of tularemia infections. Rapid, sensitive, and accurate detection methods are required to reduce mortality rates. We aimed to develop antibodies directed against the outer membrane protein A of F. tularensis (FopA) for rapid and accurate diagnosis of tularemia. We used a baculovirus insect cell expression vector system to produce the FopA antigen and generate anti-FopA antibodies through immunization of BALB/c mice. We then employed hybridoma and phage display technologies to screen for antibodies that could recognize unique epitopes on FopA. Two monoclonal antibodies, 6B12 and 3C1, identified through phage display screening specifically bound to recombinant FopA in a dose-dependent manner. The binding affinity of the anti-FopA 6B12 and 3C1 antibodies was observed to have an equilibrium dissociation constant of 1.76 × 10-10 M and 1.32 × 10-9 M, respectively. These antibodies were used to develop a sandwich ELISA system for the diagnosis of tularemia. This assay was found to be highly specific and sensitive, with detection limits ranging from 0.062 ng/mL in PBS to 0.064 ng/mL in skim milk matrices. Our findings demonstrate the feasibility of a novel diagnostic approach for detecting F. tularensis based on targeting FopA, as opposed to existing tests that target the bacterial lipopolysaccharide.

Multiprobe Amplification (MPA) with Melting Curve Analysis: A Highly Stable and Cost-Effective Platform for the Simultaneous Detection of Eight Potential Bacterial Bioterrorism Agents in Complex Samples.

We aimed to develop an efficient detection platform that can identify a larger number of suspicious samples in a single test, saving time, manpower, and material costs, and providing vital support to the public health system in coping with the current challenging and dynamic bioterrorism threat landscape, particularly in regions of turmoil and conflict. We have successfully developed a high-throughput, multitarget fluorescent array detection platform by effectively combining integrating multiprobe amplification (MPA) with melting curve analysis. Specifically, we have established reliable laboratory testing methods for eight highly pathogenic bacteria, including Bacillus anthracis, Yersinia pestis, Brucella spp., Burkholderia pseudomallei, Francisella tularensis, Vibrio cholerae, Salmonella typhi, and Staphylococcus aureus. Our method achieves sensitive and specific simultaneous detection of eight target bacteria in one well by optimizing the reaction conditions of MPA. In the assessment of 192 simulated environmental samples, both positive and negative coincidence rates were 100.00%. Among 48 simulated clinical samples, the positive coincidence rate reached 97.73%, while maintaining a perfect negative coincidence rate of 100.00%. Moreover, the detection platform holds immense potential for attaining a more comprehensive bioterrorism screening, and its high cost-effectiveness enables the provision of diverse and adaptable diagnostic methods for public health quarantine in underdeveloped countries and regions.

An overview of bacillus anthracis bacteria, one of the most important biological agents

<p>So far, more than 1400 human pathogens have been identified. Of this number, more than 60% have a common origin between humans and animals, which infect humans through animal reservoirs. These diseases include viruses such as influenza, less common (but fatal) diseases such as rabies, and neglected parasites such as <em>echinococcosis</em> and <em>cysticercosis</em>. The subcategory of bacterial zoonoses also varies, with pathogens common in industrial settings (including <em>salmonella</em> and <em>bartonella henselae</em>, the causative agent of cat-scratch disease) and diseases more associated with impoverished tropical regions (such as melioidosis and leptospirosis). In this article, we will examine the <em>bacillus anthracis</em> bacteria that causes anthrax, which is one of the most important bacteria (the cause of zoonotic diseases) and is also considered one of the agents of bioterrorism.<strong></strong></p>

Analysis of Bioterrorism Studies on Lung Health: A Meta-Analysis

Background: Bioterrorism, the use of biological agents to cause mass harm, poses a significant threat to lung health. This meta-analysis aims to evaluate the impact of bioterrorism on lung health, identifying the most frequently used agents, clinical manifestations, and policy implications. Methods: A comprehensive literature search was conducted on PubMed, Scopus, and Web of Science databases from 2018 to 2024. Studies reporting the impact of bioterrorist attacks on lung health were included. Epidemiological, clinical, and interventional data were extracted and analyzed using random effects models. Results: Twenty studies met the inclusion criteria, covering a total of 15,482 participants. The most common bioterrorism agent was Bacillus anthracis (43.5%), followed by Yersinia pestis (21.7%) and Francisella tularensis (17.4%). The most frequently reported clinical manifestations were pneumonia (78.3%), acute respiratory failure (39.1%), and sepsis (26.1%). Mortality rates vary from 5% to 35%, depending on the agent and intervention administered. Conclusion: Bioterrorism poses a serious threat to lung health, causing significant morbidity and mortality. Pneumonia, acute respiratory failure, and sepsis are the most common clinical manifestations. It is important to improve preparedness, early detection, and clinical management to reduce the impact of bioterrorist attacks.

Anthrax the Most Lethal Bioterror: Synthesizing Knowledge and Insights for Researchers and Public Health Professionals

Anthrax, caused by Bacillus anthracis, poses a significant threat as both, a naturally occurring disease and a potent bioterrorism agent. This article comprehensively reviews anthrax, synthesizing historical context, biological characteristics, past bioterror incidents, and implications for public health and national security. From its ancient origins to modern-day weaponization, anthrax has been a persistent challenge. Despite advancements in diagnosis and treatment, the disease's lethal potential underscores the importance of ongoing research, surveillance, and preparedness efforts. With coordinated action among researchers, policymakers, and healthcare professionals, the global community can better mitigate the risks associated with anthrax and safeguard public health and national security.

A glucan-particle based tularemia subunit vaccine induces T-cell immunity and affords partial protection in an inhalation rat infection model.

Tularemia is a zoonotic disease caused by the facultative intracellular gram-negative bacterium Francisella tularensis. F. tularensis has a very low infection dose by the aerosol route which can result in an acute, and potentially lethal, infection in humans. Consequently, it is classified as a Category A bioterrorism agent by the US Centers for Disease Control (CDC) and is a pathogen of concern for the International Biodefence community. There are currently no licenced tularemia vaccines. In this study we report on the continued assessment of a tularemia subunit vaccine utilising β-glucan particles (GPs) as a vaccine delivery platform for immunogenic F. tularensis antigens. Using a Fischer 344 rat infection model, we demonstrate that a GP based vaccine comprising the F. tularensis lipopolysaccharide antigen together with the protein antigen FTT0814 provided partial protection of F344 rats against an aerosol challenge with a high virulence strain of F. tularensis, SCHU S4. Inclusion of imiquimod as an adjuvant failed to enhance protective efficacy. Moreover, the level of protection afforded was dependant on the challenge dose. Immunological characterisation of this vaccine demonstrated that it induced strong antibody immunoglobulin responses to both polysaccharide and protein antigens. Furthermore, we demonstrate that the FTT0814 component of the GP vaccine primed CD4+ and CD8+ T-cells from immunised F344 rats to express interferon-γ, and CD4+ cells to express interleukin-17, in an antigen specific manner. These data demonstrate the development potential of this tularemia subunit vaccine and builds on a body of work highlighting GPs as a promising vaccine platform for difficult to treat pathogens including those of concern to the bio-defence community.

Ebola Haemorrhagic Fever: An Overview

Ebola virus disease is a rare but severe, often fatal illness in humans. Fruit bats are the natural reservoirs of Ebola virus, and it is transmitted to humans from wild animals and spreads between humans. Ebola virus is a class A bioterrorism agent, known to cause highly lethal haemorrhagic fever. Clinical symptoms include fever, myalgia, headache followed by vomiting, diarrhea, hemorrhagic rash, bleeding, and multi-organ failure. Vaccines to protect against Ebola have been developed and used to control the spread of Ebola virus disease. Treatment is mainly early supportive care with rehydration and symptomatic treatment. Ebola virus is a neglected pathogen and the knowledge and scientific information on Ebola virus disease is relatively limited and received little attention. Better understanding of ebolavirus disease mechanisms is needed to guide development of drugs, vaccines, and treatment strategies. Hence this comprehensive review on Ebola virus is undertaken to provide an overview of its transmission, pathogenesis, clinical symptoms, differential diagnosis, laboratory diagnosis, treatment, vaccines and preventive aspects and to highlight its importance, and impact on public health and further research.

Strategies for tularemia pathogen survival, spread and virulence

The bacterium Francisella tularensis is the etiological agent of tularemia, a natural focal, especially dangerous infection, which, “thanks to” its low infectious dose and ability to be transmitted to humans via all possible routes, is a potential bioterrorism agent. This pathogen has been known to mankind for over a hundred years, but it is still impossible to prevent massive human disease outbreaks and sporadic incidence cases, whereas tularemia diagnosis may be verified within several days-to-weeks. The basis for tularemia causative agent virulence is based on its ability to disrupt phagocyte function. In animals and humans, the various Francisella tularensis systems work together to bypass host immune system, attach to and enter eukaryotic cells, block phagosome-lysosome fusion, multiply in various host cells without being detected, inhibit their destruction and cause host cell death to release bacteria and infect neighboring tissue cells, thus developing an infectious disease in different organs. This is achieved through a unique complement-dependent penetration process into host cell, called loop phagocytosis, and an unusual inert endotoxin as well as variation in diverse forms of “free” lipid A modifications and lipid A in the LPS composition, its dynamic acyl chain length regulation, and specifically combined regulatory factors to induce the “pathogenicity island” protein synthesis. Accumulated point mutations, intragenomic rearrangements, deletions, insertions, duplications, transpositions, gene degradation, variation in the number of copies in repeated DNA sequences, as well as homologous and non-homologous recombinations underlie a markedly expanded potential for existence of the tularemia causative agent: they contribute to the holarctic subspecies strain survival in varying conditions, including osmotic shock, to form multiple resistance to various toxic substances and alter F. tularensis subspecies virulence. Analyzing a whole body of publications on the abovementioned aspects for tularemia causative agent life activity attempts to combine the differences, structural features and “tricks” of the F. tularensis species cells allowing them to be a powerful pathogen, with a high potential to adapt upon low pathogen variability and a limited genome length compared with other specially dangerous bacteria.

Web-accessible critical limits and critical values for urgent clinician notification.

To survey the World Wide Web for critical limits/critical values, assess changes in quantitative low/high thresholds since 1990-93, streamline urgent notification practices, and promote global accessibility. We identified Web-posted lists of critical limits/values at university hospitals. We compared 2023 to 1990-93 archived notification thresholds. We found critical notification lists for 26 university hospitals. Laboratory disciplines ranged widely (1-10). The median number of tests was 62 (range 21-116); several posted policies. The breadth of listings increased. Statistically significant differences in 2023 vs. 1990 critical limits were observed for blood gas (pO2, pCO2), chemistry (glucose, calcium, magnesium), and hematology (hemoglobin, platelets, PTT, WBC) tests, and for newborn glucose, potassium, pO2, and hematocrit. Twenty hospitals listed ionized calcium critical limits, which have not changed. Fourteen listed troponin (6), troponin I (3), hs-TnI (3), or troponin T (2). Qualitative critical values expanded across disciplines, encompassing anatomic/surgical pathology. Bioterrorism agents were listed frequently, as were contagious pathogens, although only three hospitals listed COVID-19. Only one notification list detailed point-of-care tests. Two children's hospital lists were Web-accessible. Urgent notifications should focus on life-threatening conditions. We recommend that hospital staff evaluate changes over the past three decades for clinical impact. Notification lists expanded, especially qualitative tests, suggesting that automation might improve efficiency. Sharing notification lists and policies on the Web will improve accessibility. If not dependent on the limited scope of secondary sources, artificial intelligence could enhance knowledge of urgent notification and critical care practices in the 21st Century.

1H, 13C, and 15N backbone and methyl group resonance assignments of ricin toxin A subunit

Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA. As a category-B agent for bioterrorism it is a prime target for therapeutic intervention with antibodies and enzyme blocking inhibitors since no effective therapy exists for ricin. Ricin toxin A subunit (RTA) depurinates the SRL by binding to the P-stalk proteins at a remote site. Stimulation of the N-glycosidase activity of RTA by the P-stalk proteins has been studied extensively by biochemical methods and by X-ray crystallography. The current understanding of RTA’s depurination mechanism relies exclusively on X-ray structures of the enzyme in the free state and complexed with transition state analogues. To date we have sparse evidence of conformational dynamics and allosteric regulation of RTA activity that can be exploited in the rational design of inhibitors. Thus, our primary goal here is to apply solution NMR techniques to probe the residue specific structural and dynamic coupling active in RTA as a prerequisite to understand the functional implications of an allosteric network. In this report we present de novo sequence specific amide and sidechain methyl chemical shift assignments of the 267 residue RTA in the free state and in complex with an 11-residue peptide (P11) representing the identical C-terminal sequence of the ribosomal P-stalk proteins. These assignments will facilitate future studies detailing the propagation of binding induced conformational changes in RTA complexed with inhibitors, antibodies, and biologically relevant targets.

A molecular biology approach to identify Francisella tularensis proteins that interact with Band 3 of human erythrocytes.

Francisella tularensis is a pathogenic gram-negative bacterium that causes the zoonotic disease Tularemia. In addition, F. tularensis is classified as a Class A Bioterrorism agent by the CDC due to the ease of aerosolization and the ability of this bacterium to cause fatal infection in low doses. Previous studies suggest that invasion of mammalian erythrocytes by F. tularensis helps to increase colonization of ticks, an arthropod vector of this pathogen. Our laboratory previously found that the erythrocyte membrane glycoprotein, Band 3 is required by F. tularensis for invasion of red blood cells. Therefore, we predict that bacterial proteins interact with Band 3 to facilitate invasion. To identify these proteins, we will express codon-optimized Band 3 linked to a Glutathione-S transferase tag in F. tularensis LVS. Subsequent pull-down assays may reveal F. tularensis protein binding partners to Band 3 responsible for facilitating erythrocyte invasion.

Role of LpnA in Francisella tularensis Susceptibility to Resazurin

Francisella tularensis is classified by the Centers for Disease Control and Prevention as a Category A bioterrorism agent due to its low infectious dose, ease of aerosolization, and high mortality rate. Given there is no licensed tularemia vaccine and the possibility of antibiotic-resistant F. tularensis strains, there is an urgent need to develop new treatments against this bacterium. Resazurin (Rz) has bactericidal activity against F. tularensis and other gram-negative bacteria including the human pathogens Neisseria gonorrhoeae and Helicobacter pylori. However, the mechanism of action for Rz has yet to be determined. To identify targets of Rz, we screened for Rz-resistant (Rzr) mutants of F. tularensis LVS. Whole genome sequencing was performed on 42 Rzr isolates and all contained mutations within the coding regions of FTL_0421 and FTL_1504. FTL_0421 encodes for LpnA, the major lipoprotein in F. tularensis. The FTL_0421 mutation in Rzr1 resulted in loss of LpnA expression which correlated with increased sensitivity to osmotic stress. To investigate the individual role of lpnA in Rz-resistance, a deletion mutant (ΔlpnA) was generated using standard molecular genetics techniques. An agar dilution assay was then performed to determine the Rz susceptibility of the ΔlpnA strain. A minimum inhibitory concentration (MIC) of 2.75μg/mL was achieved for the ΔlpnA and the LVS wild-type strain. This data suggests mutation of lpnA alone is not sufficient to confer resistance to resazurin. In the future, we will investigate whether lpnA in combination with other genes like FTL_1504 play a role in resazurin susceptibility in F. tularensis. (Supported by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence & the NASA Space Grant Consortium)

Evaluation of sponge wipe surface sampling for collection of potential surrogates for non-spore-forming bioterrorism agents.

Evaluate the efficacy of sponge wipe sampling at recovering potential bacterial surrogates for Category A and B non-spore-forming bacterial bioterrorism agents from hard, nonporous surfaces. A literature survey identified seven nonpathogenic bacteria as potential surrogates for selected Category A and B non-spore-forming bacterial agents. Small (2 × 4 cm) and large (35.6 × 35.6 cm) coupons made from either stainless steel, plastic, or glass, were inoculated and utilized to assess persistence and surface sampling efficiency, respectively. Three commercially available premoistened sponge wipes (3M™, Sani-Stick®, and Solar-Cult®) were evaluated. Mean recoveries from persistence testing indicated that three microorganisms (Yersinia ruckeri, Escherichia coli, and Serratia marcescens) demonstrated sufficient persistence across all tested material types. Sampling of large inoculated (≥107 CFU per sample) coupons resulted in mean recoveries ranging from 6.6 to 3.4 Log10 CFU per sample. Mean recoveries for the Solar-Cult®, 3M™ sponge wipes, and Sani-Sticks® across all test organisms and all material types were≥5.7, ≥3.7, and≥3.4 Log10 CFU per sample, respectively. Mean recoveries for glass, stainless steel, and ABS plastic across all test organisms and all sponge types were≥3.8, ≥3.7, and≥3.4 Log10 CFU per sample, respectively. Recovery results suggest that sponge wipe sampling can effectively be used to recover non-spore-forming bacterial cells from hard, nonporous surfaces such as stainless steel, ABS plastic, and glass.