Bryan H. Bellaire

Validated inactivation procedures are required for the safe handling and downstream analysis of highly pathogenic organisms, particularly those categorized as biological select agents and toxins (BSATs). TRIzol™-based extraction methods are widely used for nucleic acid and protein isolation, yet their reliability for bacterial inactivation has not been comprehensively evaluated. In this study, we assessed TRIzol-based extraction methods for sample quality and inactivation reliability across a series of mock failure scenarios using five attenuated bacterial isolates: Francisella tularensis holarctica LVS, Bacillus anthracis Sterne, Yersinia enterocolitica, Mycobacterium marinum , and Burkholderia cepacia . Dilution of TRIzol to induce incomplete cell lysis for the initial extraction step, including 0% TRIzol, consistently inactivated all surrogate organisms, suggesting that downstream precipitation and sample washing reagents, including isopropanol and 70% ethanol, were sufficient to inactivate organisms in the absence of TRIzol. Several protocol failure scenarios were then evaluated to simulate human error by omitting extraction, precipitation, and washing steps individually or in combination for the most resistant organism, B. anthracis Sterne strain. Failure-scenario testing demonstrated that reliable inactivation of B. anthracis required strict adherence to the complete protocol due to the spore-forming ability of B. anthracis . Collectively, this work provides a reference with experimental evidence supporting the use of TRIzol™-based extraction as a bacterial inactivation strategy for a wide range of bacterial pathogens.

A myriad of coronaviruses cause diseases from a common cold to severe lung infections and pneumonia. SARS-CoV-2 was discovered to be the etiologic agent of the Coronavirus pandemic and many laboratory techniques were examined for virus culture and basic and applied research. Understanding the replication kinetics and characterizing the effect the virus has on different cell lines is crucial for developing in vitro studies. With the emergence of multiple variants of SARS-CoV-2, a comparison between their infectivity and replication in common cell lines will help give us a clear understanding of their characteristic differences in pathogenicity. In this study we compared the cytopathic effect and replication of Wild-Type (USA/WA1), Omicron (B.1.1.529), and Delta (B.1.617.2) variants on five different cell lines; VeroE6, VeroE6 cells expressing high endogenous ACE2, VeroE6 cells expressing human ACE2 and TMPRSS2, Calu3 cells highly expressing human ACE2 and A549 cells. This data will aid researchers with experimental planning and viral pathogenicity analysis and provide a baseline for testing any future variants.

A myriad of coronaviruses cause diseases from a common cold to severe lung infections and pneumonia. SARS-CoV-2 was discovered to be the etiologic agent of the Coronavirus pandemic and many laboratory techniques were examined for virus culture and basic and applied research. Understanding the replication kinetics and characterizing the effect the virus has on different cell lines is crucial for developing in vitro studies. With the emergence of multiple variants of SARS-CoV-2, a comparison between their infectivity and replication in common cell lines will help give us a clear understanding of their characteristic differences in pathogenicity. In this study we compared the cytopathic effect and replication of Wild Type (WT), Omicron (B.1.1.529), and Delta (B.1.617.2) variants on 5 different cell lines; VeroE6, VeroE6 cells expressing high endogenous ACE2, VeroE6 cells expressing human ACE2 and TMPRSS2, Calu3 cells highly expressing human ACE2 and A549 cells. This data will aid researchers with experimental planning and viral pathogenicity analysis and provide a baseline for testing any future variants.

The potential infectivity of severe acute respiratory syndrome associated coronavirus-2 (SARS-CoV-2) in animals raises a public health and economic concern, particularly the high susceptibility of white-tailed deer (WTD) to SARS-CoV-2. The disparity in the disease outcome between humans and WTD is very intriguing, as the latter are often asymptomatic, subclinical carriers of SARS-CoV-2. To date, no studies have evaluated the innate immune factors responsible for the contrasting SARS-CoV-2-associated disease outcomes in these mammalian species. A comparative transcriptomic analysis in primary respiratory epithelial cells of human (HRECs) and WTD (Deer-RECs) infected with the SARS-CoV-2 WA1/2020 strain was assessed throughout 48 h post inoculation (hpi). Both HRECs and Deer-RECs were susceptible to virus infection, with significantly ( P < 0.001) lower virus replication in Deer-RECs. The number of differentially expressed genes (DEG) gradually increased in Deer-RECs but decreased in HRECs throughout the infection. The ingenuity pathway analysis of DEGs further identified that genes commonly altered during SARS-CoV-2 infection mainly belong to cytokine and chemokine response pathways mediated via interleukin-17 (IL-17) and nuclear factor-κB (NF-κB) signaling pathways. Inhibition of the NF-κB signaling in the Deer-RECs pathway was predicted as early as 6 hpi. The findings from this study could explain the lack of clinical signs reported in WTD in response to SARS-CoV-2 infection as opposed to the severe clinical outcomes reported in humans. IMPORTANCE: This study demonstrated that human and white-tailed deer primary respiratory epithelial cells are susceptible to the SARS-CoV-2 WA1/2020 strain infection. However, the comparative transcriptomic analysis revealed that deer cells could limit viral replication without causing hypercytokinemia by downregulating IL-17 and NF-κB signaling pathways. Identifying differentially expressed genes in human and deer cells that modulate key innate immunity pathways during the early infection will lead to developing targeted therapies toward preventing or mitigating the “cytokine storm” often associated with severe cases of coronavirus disease 19 (COVID-19). Moreover, results from this study will aid in identifying novel prognostic biomarkers in predicting SARS-CoV-2 adaption and transmission in deer and associated cervids.

Antimicrobial resistance is a growing problem in modern healthcare. Most antimicrobial susceptibility tests (AST) require long culture times which delay diagnosis and effective treatment. Our group has previously reported a proof-of-concept demonstration of a rapid AST in Escherichia coli using deuterium labeling and MALDI mass spectrometry. Culturing bacteria in D₂O containing media incorporates deuterium in newly synthesized lipids, resulting in a mass shift that can be easily detected by mass spectrometry. The extent of new growth is measured by the average mass of synthesized lipids that can be correlated with resistance in the presence of antimicrobials. In this work, we adapt this procedure to methicillin-resistant Staphylococcus aureus using the Bruker MALDI-TOF Biotyper, a low-cost instrument commonly available in diagnostic laboratories. The susceptible strain showed a significant decrease in average mass in on-target microdroplet cultures after 3 hours of incubation with 10 µg/mL methicillin, while the resistant strain showed consistent labeling regardless of methicillin concentration. This assay allows us to confidently detect methicillin resistance in S. aureus after only 3 hours of culture time and minimal sample processing, reducing the turn-around-time significantly over conventional assays. The success of this work suggests its potential as a rapid AST widely applicable in many clinical microbiology labs with minimal additional costs.

A myriad of coronaviruses cause diseases from a common cold to severe lung infections and pneumonia. SARS-CoV-2 was discovered to be the etiologic agent of the Coronavirus pandemic and many laboratory techniques were examined for virus culture and basic and applied research. Understanding the replication kinetics and characterizing the effect the virus has on different cell lines is crucial for developing in vitro studies. With the emergence of multiple variants of SARS-CoV-2, a comparison between their infectivity and replication in common cell lines will help give us a clear understanding of their characteristic differences in pathogenicity. In this study we compared the cytopathic effect and replication of Wild Type (WT), Omicron (B.1.1.529), and Delta (B.1.617.2) variants on 5 different cell lines; VeroE6, VeroE6 cells expressing high endogenous ACE2, VeroE6 cells expressing human ACE2 and TMPRSS2, Calu3 cells highly expressing human ACE2 and A549 cells. This data will aid researchers with experimental planning and viral pathogenicity analysis and provide a baseline for testing any future variants.

Mycobacterium marinum is a waterborne pathogen responsible for tuberculosis-like infections in cold-blooded animals and is an opportunistic pathogen in humans. M. marinum is the closest genetic relative of the Mycobacterium tuberculosis complex and is a reliable surrogate for drug susceptibility testing. We synthesized and evaluated two nanoparticle (NP) formulations for compatibility with rifampicin, isoniazid, pyrazinamide, and ethambutol (PIRE), the front-line antimycobacterial drugs used in combination against active tuberculosis infections. Improved in vitro antimicrobial activity was observed with encapsulated rifampicin alone or in a cocktail of drugs formulated through co-encapsulation in amphiphilic polyanhydride NPs. Broth antimicrobial testing revealed that the encapsulation of PIRE in NP resulted in a significant increase in antimicrobial activity, with the benefit over soluble formulations at biologically relevant concentrations ranging from >10 to >3,000 fold. M. marinum-infected human macrophages treated with NP-PIRE were cleared of viable bacteria in 48 h following a single treatment, representing a >4 log reduction in colony-forming units and a >2,000-fold increase in antimicrobial activity. The amphiphilic polyanhydride nanoparticles demonstrated the ability to co-encapsulate PIRE antibiotics and enhance their antimicrobial activity against M. marinum in infected macrophages in culture and in vitro. These data suggest that polyanhydride nanoparticles are a promising nanotherapeutic for combatting Mycobacterium infections through improved intracellular targeting of encapsulated antibiotics.

The potential infectivity of SARS-CoV-2 in animals raises a public health and economic concern, particularly the high susceptibility of white-tailed deer (WTD) to SARS-CoV-2. The disparity in the disease outcome between humans and WTD is very intriguing, as the latter are often asymptomatic, subclinical carriers of SARS-CoV-2. To date, no studies have evaluated the innate immune factors responsible for the contrasting SARS-CoV-2-associated disease outcomes in these mammalian species. A comparative transcriptomic analysis in primary respiratory epithelial cells of human (HRECs) and WTD (Deer-RECs) infected with SARS-CoV-2 was assessed throughout 48 hours post inoculation (hpi). Both HRECs and Deer-RECs were susceptible to SARS-COV-2, with significantly ( P < 0.001) lower virus replication in Deer-RECs. The number of differentially expressed genes (DEG) gradually increased in Deer-RECs but decreased in HRECs throughout the infection. The ingenuity pathway analysis of DEGs further identified that genes commonly altered during SARS-CoV-2 infection mainly belong to cytokine and chemokine response pathways mediated via IL-17 and NF-κB signaling pathways. Inhibition of the NF-κB signaling in the Deer-RECs pathway was predicted as early as 6 hpi. The findings from this study could explain the lack of clinical signs reported in WTD in response to SARS-CoV-2 infection as opposed to the severe clinical outcomes reported in humans. HIGHLIGHTS: White-tailed deer primary respiratory epithelial cells are susceptible to SARS- CoV-2 without causing hyper cytokine gene expression. Downregulation of IL-17 and NF-κB signaling pathways after SARS-CoV-2 infection could be key to the regulated cytokine response in deer cells. Deer innate immune system could play a critical role in early antiviral and tissue repair response following SARS-CoV-2 infection.

Human coronavirus (HCoV)-NL63 is an important contributor to upper and lower respiratory tract infections, mainly in children, while severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, can cause lower respiratory tract infections, and more severe, respiratory and systemic disease, which leads to fatal consequences in many cases. Using microscopy, immunohistochemistry (IHC), virus-binding assay, reverse transcriptase qPCR (RT-qPCR) assay, and flow cytometry, we compared the characteristics of the susceptibility, replication dynamics, and morphogenesis of HCoV-NL63 and SARS-CoV-2 in monolayer cultures of primary human respiratory epithelial cells (HRECs). Less than 10% HRECs expressed ACE2, and SARS-CoV-2 seemed much more efficient than HCoV-NL63 at infecting the very small proportion of HRECs expressing the ACE2 receptors. Furthermore, SARS-CoV-2 replicated more efficiently than HCoV-NL63 in HREC, which correlates with the cumulative evidence of the differences in their transmissibility.

The Coronaviridae family hosts various coronaviruses responsible for many diseases, from the common cold, severe lung infections to pneumonia. SARS-CoV-2 was discovered to be the etiologic agent of the Coronavirus pandemic, and numerous basic and applied laboratory techniques were utilized in virus culture and examination of the disease. Understanding the replication kinetics and characterizing the virus’ effect on different cell lines is crucial for developing in vitro studies. With the emergence of multiple variants of SARS-CoV-2, a comparison between their infectivity and replication in common cell lines will give us a clear understanding of the characteristic differences in pathogenicity. In this study, we compared the cytopathic effect (CPE) and replication of Wild Type (WT), Omicron (B.1.1.529), and Delta (B.1.617.2) variants on 5 different cell lines; VeroE6, VeroE6 expressing high endogenous ACE2, VeroE6 highly expressing human ACE2 (VeroE6/ACE2) and TMPRSS2 (VeroE6/hACE2/ TMPRSS2), Calu3 cells highly expressing human ACE2 and A549 cells. All 3 VeroE6 cell lines were susceptible to WT strain, where CPE and replication were observed. Along with being susceptible to Wild type, VeroE6/hACE2/TMPRSS2 cells were susceptible to both omicron and delta strains, whereas VeroE6/ACE2 cells were only susceptible to omicron in a dose-dependent manner. No CPE was observed in both human lung cell lines, A549 and Calu3/hACE2, but Wild type and omicron replication was observed. As SAR-CoV-2 continues to evolve, this data will benefit researchers in experimental planning, viral pathogenicity analysis, and providing a baseline for testing future variants.