The Promise of Disease Detection Dogs in Pandemic Response: Lessons Learned From COVID-19.

One of the lessons learned from the coronavirus disease 2019 (COVID-19) pandemic is the utility of an early, flexible, and rapidly deployable disease screening and detection response. The largely uncontrolled spread of the pandemic in the United States exposed a range of planning and implementation shortcomings, which, if they had been in place before the pandemic emerged, may have changed the trajectory. Disease screening by detection dogs show great promise as a noninvasive, efficient, and cost-effective screening method for COVID-19 infection. We explore evidence of their use in infectious and chronic diseases; the training, oversight, and resources required for implementation; and potential uses in various settings. Disease detection dogs may contribute to the current and future public health pandemics; however, further research is needed to extend our knowledge and measurement of their effectiveness and feasibility as a public health intervention tool, and efforts are needed to ensure public and political support.

Design, organocatalytic synthesis, and bioactivity evaluation of enantiopure fluorinated LpxC inhibitors.

Enantiopure compounds with a strategically incorporated fluorine atom intended to enhance LpxC inhibition have been synthesized using an organocascade fluorination reaction as the key step. These are the first low molecular weight LpxC inhibitors to contain a fluorine atom on a critically important chiral center that is substituted with two pharmacophoric moieties, and were thusly designed to provide new SAR data for this class of compounds. Fluorinated compounds were evaluated against ESKAPE pathogens and exhibited MICs of ≤12.5 µg mL-1 against Pseudomonas aeruginosa.

Discovery of Ubonodin, an Antimicrobial Lasso Peptide Active against Members of the Burkholderia cepacia Complex.

We report the heterologous expression, structure, and antimicrobial activity of a lasso peptide, ubonodin, encoded in the genome of Burkholderia ubonensis. The topology of ubonodin is unprecedented amongst lasso peptides, with 18 of its 28 amino acids found in the mechanically bonded loop segment. Ubonodin inhibits RNA polymerase in vitro and has potent antimicrobial activity against several pathogenic members of the Burkholderia genus, most notably B. cepacia and B. multivorans, causative agents of lung infections in cystic fibrosis patients.

Cyberbiosecurity: A Call for Cooperation in a New Threat Landscape.

The life sciences now interface broadly with information technology (IT) and cybersecurity. This convergence is a key driver in the explosion of biotechnology research and its industrial applications in health care, agriculture, manufacturing, automation, artificial intelligence, and synthetic biology. As the information and handling mechanisms for biological materials have become increasingly digitized, many market sectors are now vulnerable to threats at the digital interface. This growing landscape will be addressed by cyberbiosecurity, the emerging field at the convergence of both the life sciences and IT disciplines. This manuscript summarizes the current cyberbiosecurity landscape, identifies existing vulnerabilities, and calls for formalized collaboration across a swath of disciplines to develop frameworks for early response systems to anticipate, identify, and mitigate threats in this emerging domain.

Susceptibility of Virulent Yersinia pestis Bacteria to Predator Bacteria in the Lungs of Mice.

Multi-drug resistant bacterial infections are a serious threat to global public health. Changes in treatment modalities and prudent use of antibiotics can assist in reducing the threat, but new approaches are also required for untreatable cases. The use of predatory bacteria, such as Bdellovibrio bacteriovorus, is among the novel approaches being considered as possible therapeutics for antibiotic resistant and/or unidentified bacterial infections. Previous studies have examined the feasibility of using predatory bacteria to reduce colony-forming units (CFUs) in the lungs of rats exposed to lethal doses of Klebsiella pneumoniae; here we apply the approach to the Tier 1 select agent Yersinia pestis, and show that three doses of B. bacteriovorus introduced every six hours reduces the number of CFUs of Y. pestis in the lungs of inoculated mice by 86% after 24 h of infection. These experiments further demonstrate that predatory bacteria may serve to combat Gram negative bacterial infections, including those considered potential bioweapon agents, in the future.

Multistate Infestation with the Exotic Disease-Vector Tick Haemaphysalis longicornis - United States, August 2017-September 2018.

Haemaphysalis longicornis is a tick indigenous to eastern Asia and an important vector of human and animal disease agents, resulting in such outcomes as human hemorrhagic fever and reduction of production in dairy cattle by 25%. H. longicornis was discovered on a sheep in New Jersey in August 2017 (1). This was the first detection in the United States outside of quarantine. In the spring of 2018, the tick was again detected at the index site, and later, in other counties in New Jersey, in seven other states in the eastern United States, and in Arkansas. The hosts included six species of domestic animals, six species of wildlife, and humans. To forestall adverse consequences in humans, pets, livestock, and wildlife, several critical actions are indicated, including expanded surveillance to determine the evolving distribution of H. longicornis, detection of pathogens that H. longicornis currently harbors, determination of the capacity of H. longicornis to serve as a vector for a range of potential pathogens, and evaluation of effective agents and methods for the control of H. longicornis.

Keeping it together: absence of genetic variation and DNA incorporation by the predatory bacteria Micavibrio aeruginosavorus and Bdellovibrio bacteriovorus during predation.

The use of predatory bacteria as a potential live therapeutic to control human infection is gaining increased attention. Earlier work with Micavibrio spp. and Bdellovibrio spp. has demonstrated the ability of these predators to control drug-resistant Gram-negative pathogens, Tier-1 select agents and biofilms. Additional studies also confirmed that introducing high doses of the predators into animals does not negatively impact animal well-being and might assist in reducing bacterial burden in vivo. The survival of predators requires extreme proximity to the prey cell, which might bring about horizontal transfer of genetic material, such as genes encoding for pathogenic genetic islands that would indirectly facilitate the spread of genetic material to other organisms. In this study, we examined the genetic makeup of several lab isolates of the predators Bdellovibriobacteriovorus and Micavibrioaeruginosavorus that were cultured repeatedly and stored over a course of 13 years. We also conducted controlled experiments in which the predators were sequentially co-cultured on Klebsiella pneumoniae followed by genetic analysis of the predator. In both cases, we saw little genetic variation and no evidence of horizontally transferred chromosomal DNA from the prey during predator-prey interaction. Culturing the predators repeatedly did not cause any change in predation efficacy.

Examining the efficacy of intravenous administration of predatory bacteria in rats.

The proteobacteria Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are obligate predators of Gram-negative bacteria, and have been proposed to be used to treat multidrug-resistant bacterial infections. The ability of predatory bacteria to reduce bacterial burden in vivo within the lungs of rats has been demonstrated, but it was unknown if predatory bacteria can attenuate systemic bacterial burden administered intravenously. In this study, we first assessed the safety of intravenous inoculation of predatory bacteria in rats. No rat morbidity or adverse histopathology of various organs due to predatory bacteria administration was observed. An increase in proinflammatory cytokines (TNFα and KC/GRO) was observed at two hours post-inoculation; however, cytokines returned to baseline levels by 18 hours. Furthermore, bacterial dissemination analysis demonstrated that predatory bacteria were efficiently cleared from the host by 20 days post-injection. To determine whether predatory bacteria could reduce bacterial burden in vivo, Klebsiella pneumoniae was injected into the tail veins of rats and followed with multiple doses of predatory bacteria over 16 or 24 hours. Predatory bacteria were unable to significantly reduce K. pneumoniae burden in the blood or prevent dissemination to other organs. The results suggest that predatory bacteria may not be effective for treatment of acute blood infections.

Effect of predatory bacteria on the gut bacterial microbiota in rats.

Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are Gram-negative proteobacteria that are obligate predators of other Gram-negative bacteria and are considered potential alternatives to antibiotics. Most studies focusing on predatory bacteria have been performed in vitro, thus the effect of predatory bacteria on a live host, including the impact on the ecology of the native microbiota, has yet to be fully examined. In this study, intrarectal inoculations of Sprague-Dawley rats with predatory bacteria were performed. Additionally, feces were collected for seven days post-inoculation to determine the effect on gut bacterial diversity. Rat colonic tissue exhibited no abnormal histopathological effects due to predatory bacteria. A modest increase in pro-inflammatory cytokines was measured in the colons of rats inoculated with predatory bacteria by 24 and 48 hours, with all but IL-13 returning to baseline by seven days. V4 16S rRNA gene sequencing of fecal DNA demonstrated minimal shifts in taxonomic representation over the week due to predatory bacteria. Changes in bacterial populations due to exposure to B. bacteriovorus are predicted to contribute to health, however, an overgrowth of Prevotella was observed due to exposure to M. aeruginosavorus. This study further addresses safety concerns associated with the potential use of predatory bacteria to treat infections.

Predatory Bacteria Attenuate Klebsiella pneumoniae Burden in Rat Lungs.

Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are predatory bacteria that naturally-and obligately-prey on other Gram-negative bacteria, and their use has been proposed as a potential new approach to control microbial infection. The ability of predatory bacteria to prey on Gram-negative human pathogens in vitro is well documented; however, the in vivo safety and efficacy of predatory bacteria have yet to be fully assessed. In this study, we examined whether predatory bacteria can reduce bacterial burden in the lungs in an in vivo mammalian system. Initial safety studies were performed by intranasal inoculation of rats with predatory bacteria. No adverse effects or lung pathology were observed in rats exposed to high concentrations of predatory bacteria at up to 10 days postinoculation. Enzyme-linked immunosorbent assay (ELISA) of the immune response revealed a slight increase in inflammatory cytokine levels at 1 h postinoculation that was not sustained by 48 h. Additionally, dissemination experiments showed that predators were efficiently cleared from the host by 10 days postinoculation. To measure the ability of predatory bacteria to reduce microbial burden in vivo, we introduced sublethal concentrations of Klebsiella pneumoniae into the lungs of rats via intranasal inoculation and followed with multiple doses of predatory bacteria over 24 h. Predatory bacteria were able to reduce K. pneumoniae bacterial burden, on average, by more than 3.0 log10 in the lungs of most rats as measured by CFU plating. The work presented here provides further support for the idea of developing predatory bacteria as a novel biocontrol agent. IMPORTANCE: A widely held notion is that antibiotics are the greatest medical advance of the last 50 years. However, the rise of multidrug-resistant (MDR) bacterial infections has become a global health crisis over the last decade. As we enter the postantibiotic era, it is crucial that we begin to develop new strategies to combat bacterial infection. Here, we report one such new approach: the use of predatory bacteria (Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus) that naturally-and obligately-prey on other Gram-negative bacteria. To our knowledge, this is the first study that demonstrated the ability of predatory bacteria to attenuate the bacterial burden of a key human pathogen in an in vivo mammalian system. As the prevalence of MDR infections continues to rise each year, our results may represent a shift in how we approach treating microbial infections in the future.

A Course on Terror Medicine: Content and Evaluations.

INTRODUCTION: The development of medical school courses on medical responses for disaster victims has been deemed largely inadequate. To address this gap, a 2-week elective course on Terror Medicine (a field related to Disaster and Emergency Medicine) has been designed for fourth year students at Rutgers New Jersey Medical School in Newark, New Jersey (USA). This elective is part of an overall curricular plan to broaden exposure to topics related to Terror Medicine throughout the undergraduate medical education. RATIONALE: A course on Terror Medicine necessarily includes key aspects of Disaster and Emergency Medicine, though the converse is not the case. Courses on Disaster Medicine may not address features distinctively associated with a terror attack. Thus, a terror-related focus not only assures attention to this important subject but to accidental or naturally occurring incidents as well. METHODS: The course, implemented in 2014, uses a variety of teaching modalities including lectures, videos, and tabletop and hands-on simulation exercises. The subject matter includes biological and chemical terrorism, disaster management, mechanisms of injury, and psychiatry. This report outlines the elective's goals and objectives, describes the course syllabus, and presents outcomes based on student evaluations of the initial iterations of the elective offering. RESULTS: All students rated the course as "excellent" or "very good." Evaluations included enthusiastic comments about the content, methods of instruction, and especially the value of the simulation exercises. Students also reported finding the course novel and engaging. CONCLUSION: An elective course on Terror Medicine, as described, is shown to be feasible and successful. The student participants found the content relevant to their education and the manner of instruction effective. This course may serve as a model for other medical schools contemplating the expansion or inclusion of Terror Medicine-related topics in their curriculum.

Avalanche for shape and feature-based virtual screening with 3D alignment.

This report introduces a new ligand-based virtual screening tool called Avalanche that incorporates both shape- and feature-based comparison with three-dimensional (3D) alignment between the query molecule and test compounds residing in a chemical database. Avalanche proceeds in two steps. The first step is an extremely rapid shape/feature based comparison which is used to narrow the focus from potentially millions or billions of candidate molecules and conformations to a more manageable number that are then passed to the second step. The second step is a detailed yet still rapid 3D alignment of the remaining candidate conformations to the query conformation. Using the 3D alignment, these remaining candidate conformations are scored, re-ranked and presented to the user as the top hits for further visualization and evaluation. To provide further insight into the method, the results from two prospective virtual screens are presented which show the ability of Avalanche to identify hits from chemical databases that would likely be missed by common substructure-based or fingerprint-based search methods. The Avalanche method is extended to enable patent landscaping, i.e., structural refinements to improve the patentability of hits for deployment in drug discovery campaigns.

Examining the safety of respiratory and intravenous inoculation of Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus in a mouse model.

Bdellovibrio spp. and Micavibrio spp. are Gram-negative predators that feed on other Gram-negative bacteria, making predatory bacteria potential alternatives to antibiotics for treating multi-drug resistant infections. While the ability of predatory bacteria to control bacterial infections in vitro is well documented, the in vivo effect of predators on a living host has yet to be extensively examined. In this study, respiratory and intravenous inoculations were used to determine the effects of predatory bacteria in mice. We found no reduction in mouse viability after intranasal or intravenous inoculation of B. bacteriovorus 109J, HD100 or M. aeruginosavorus. Introducing predators into the respiratory tract of mice provoked a modest inflammatory response at 1 hour post-exposure, but was not sustained at 24 hours, as measured by RT-qPCR and ELISA. Intravenous injection caused an increase of IL-6 in the kidney and spleen, TNF in the liver and CXCL-1/KC in the blood at 3 hours post-exposure, returning to baseline levels by 18 hours. Histological analysis of tissues showed no pathological changes due to predatory bacteria. Furthermore, qPCR detected predators were cleared from the host quickly and efficiently. This work addresses some of the safety concerns regarding the potential use of predatory bacteria as a live antibiotic.

Susceptibility of Select Agents to Predation by Predatory Bacteria.

Select Agents are microorganisms and toxins considered to be exploitable as biological weapons. Although infections by many Select Agents can be treated by conventional antibiotics, the risk of an emerging or engineered drug resistant strain is of great concern. One group of microorganisms that is showing potential to control drug resistant Gram-negative bacteria are the predatory bacteria from the genera Bdellovibrio spp. and Micavibrio spp. In this study, we have examined the ability of Bdellovibrio bacteriovorus (B. bacteriovorus) strain 109J, HD100 and Micavibrio aeruginosavorus (M. aeruginosavorus) ARL-13 to prey on a variety of Select Agents. Our findings demonstrate that B. bacteriovorus and M. aeruginosavorus are able to prey efficiently on Yersinia pestis and Burkholderia mallei. Modest predation was also measured in co-cultures of B. bacteriovorus and Francisella tularensis. However, neither of the predators showed predation when Burkholderia pseudomallei and Brucella melitensis were used as prey.

Terror medicine as part of the medical school curriculum.

Terror medicine, a field related to emergency and disaster medicine, focuses on medical issues ranging from preparedness to psychological manifestations specifically associated with terrorist attacks. Calls to teach aspects of the subject in American medical schools surged after the 2001 jetliner and anthrax attacks. Although the threat of terrorism persists, terror medicine is still addressed erratically if at all in most medical schools. This paper suggests a template for incorporating the subject throughout a 4-year medical curriculum. The instructional framework culminates in a short course for fourth year students, such as one recently introduced at Rutgers New Jersey Medical School, Newark, NJ, USA. The proposed 4-year Rutgers curriculum serves as a model that could assist other medical schools contemplating the inclusion of terror medicine in pre-clerkship and clerkship training.

Validation of high throughput sequencing and microbial forensics applications.

High throughput sequencing (HTS) generates large amounts of high quality sequence data for microbial genomics. The value of HTS for microbial forensics is the speed at which evidence can be collected and the power to characterize microbial-related evidence to solve biocrimes and bioterrorist events. As HTS technologies continue to improve, they provide increasingly powerful sets of tools to support the entire field of microbial forensics. Accurate, credible results allow analysis and interpretation, significantly influencing the course and/or focus of an investigation, and can impact the response of the government to an attack having individual, political, economic or military consequences. Interpretation of the results of microbial forensic analyses relies on understanding the performance and limitations of HTS methods, including analytical processes, assays and data interpretation. The utility of HTS must be defined carefully within established operating conditions and tolerances. Validation is essential in the development and implementation of microbial forensics methods used for formulating investigative leads attribution. HTS strategies vary, requiring guiding principles for HTS system validation. Three initial aspects of HTS, irrespective of chemistry, instrumentation or software are: 1) sample preparation, 2) sequencing, and 3) data analysis. Criteria that should be considered for HTS validation for microbial forensics are presented here. Validation should be defined in terms of specific application and the criteria described here comprise a foundation for investigators to establish, validate and implement HTS as a tool in microbial forensics, enhancing public safety and national security.

Engaging actively with issues in the responsible conduct of science: lessons from international efforts are relevant for undergraduate education in the United States.

Numerous studies are demonstrating that engaging undergraduate students in original research can improve their achievement in the science, technology, engineering, and mathematics (STEM) fields and increase the likelihood that some of them will decide to pursue careers in these disciplines. Associated with this increased prominence of research in the undergraduate curriculum are greater expectations from funders, colleges, and universities that faculty mentors will help those students, along with their graduate students and postdoctoral fellows, develop an understanding and sense of personal and collective obligation for responsible conduct of science (RCS). This Feature describes an ongoing National Research Council (NRC) project and a recent report about educating faculty members in culturally diverse settings (Middle East/North Africa and Asia) to employ active-learning strategies to engage their students and colleagues deeply in issues related to RCS. The NRC report describes the first phase of this project, which took place in Aqaba and Amman, Jordan, in September 2012 and April 2013, respectively. Here we highlight the findings from that report and our subsequent experience with a similar interactive institute in Kuala Lumpur, Malaysia. Our work provides insights and perspectives for faculty members in the United States as they engage undergraduate and graduate students, as well as postdoctoral fellows, to help them better understand the intricacies of and connections among various components of RCS. Further, our experiences can provide insights for those who may wish to establish "train-the-trainer" programs at their home institutions.

Effects by anthrax toxins on hematopoiesis: a key role for cytokines as mediators.

An understanding of anthrax toxins on the emerging immune system and blood production are significant to medicine. This study examined the effects of anthrax toxin on hematopoiesis and determined roles for cytokines. Anthrax holotoxin toxin is three components: protective antigen (PA) binds to the target cell and mediates the entry of lethal factor (LF) and edema factor (EF). Anthrax toxin dramatically inhibits signaling in immune cells. We first identified the cell subsets that interacted with the protective antigen (PA) and then studied the effects on hematopoietic progenitors in clonogenic assays: granulocytic-monocytic (CFU-GM) and late erythroid (CFU-E). Multi-color immunofluorescence with FITC-PA indicated its interaction with early and late myeloid cells. Clonogenic assays, in the presence or absence of holotoxin and individual toxin proteins resulted in significant suppression by hologenic toxic alone, despite the presence of growth-promoting cytokines. Antibodies to anthrax receptor (ATR1) reversed the suppressive effects, indicating specificity. Monomeric proteins showed different effects on myeloid and erythroid progenitors. Suppression was not due to cell death, based on undetectable active caspase 3. Cytokine array analyses with supernatants from toxin-stimulated stroma showed an increase in the hematopoietic suppressor, MIP-1α. This finding, in addition to our previous studies, showing an increase in IL-10, suggested indirect roles for cytokines in toxin-mediated hematopoietic suppression. The chemokine, SDF-1α was increased. Since SDF-1 is involved in the mobilization of hematopoietic cells, it is likely that anthrax holotoxin could induce cell exit from BM. In summary, anthrax holotoxin, but not individual toxins, exerted hematopoietic effects on myeloid and erythroid progenitors via specific receptor, partly through the induction of cytokines.