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Abstract The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), sparked an international debate on effective ways to prevent and treat the virus. Specifically, there were many varying opinions on the use of ivermectin (IVM) throughout the world, with minimal research to support either side. IVM is an FDA-approved antiparasitic drug that was discovered in the 1970s and was found to show antiviral activity. The objective of this study is to examine the binding behavior and rates of association and dissociation between SARS-CoV-2 receptor binding domain (RBD), IVM, and their combination using aminopropylsilane (APS) biosensors as surrogates for the hydrophobic interaction between the viral protein and human angiotensin-converting enzyme 2 (ACE2) receptors to determine the potential of IVM as a repurposed drug for SARS-CoV-2 prevention and treatment. The IVM, RBD, and combination binding kinetics were analyzed using biolayer interferometry (BLI) and validated with multiple in silico techniques including protein–ligand docking, molecular dynamics simulation, molecular mechanics-generalized Born surface area (MM-GBSA), and principal component analysis (PCA). Our results suggest that with increasing IVM concentrations the association rate with the hydrophobic biosensor increases with a simultaneous decrease in dissociation. Significant kinetic changes to RBD, when combined with IVM, were found only at a concentration a thousand times the approved dosage with minimal changes found over a 35-min time period. Our study suggests that IVM is not an effective preventative or treatment method at the currently approved dosage. 

Abstract While antibiotic resistance poses a threat from both Gram-positive bacteria (GPB) and Gram-negative bacteria (GNB), GNB pose a more imminent public health hazard globally. GNB are a threat to growing antibiotic resistance because of the complex makeup of the membrane. The AcrAB-TolC efflux pump is a known resistance mechanism ofEscherichia coli (E. coli)cells. This study utilized molecular dynamics modeling to visualize some of the changes occurring at a molecular level when airborne bacteria are exposed to stress and antibiotics. This study was conducted to build upon previous experimental research showing that there is an increase in antibiotic resistance and efflux pump activity when exposed to aerosolization. AcrB and AcrAB-TolC proteins were simulated under standard and increased pressure to compare the effect of aerosolization on the binding to the three different antibiotics (puromycin (PUY), ampicillin (AMP) and sulfamethoxazole-trimethoprim (SXT)) to the AcrB binding site. Analysis such as root-mean-square deviation of atomic positions and root-mean-square fluctuation, the opening of TolC, and the significant molecular mechanics with generalized Born and surface area solvation (MM-GBSA) scores associated with specific ligands were recorded. Resistance in experimental data indicated a relationship between the docking scores and some ligand–protein interactions. Results showed that there was more flexibility in the proteins within simulations conducted under standard pressure for the AcrB protein and the full tripartite complex AcrAB-TolC, showing that increased pressure causes more rigidity. MM-GBSA scores, used to calculate the free energy of ligand–protein binding, did not show a significant change, but interestingly, the strongest MM-GBSA scores were for ligands that moved to another binding pocket and did not result in resistance or opening of the efflux pump. However, the ligand moved from the binding site and did not cause the opening of TolC to increase significantly, whereas PUY and AMP were bound to the binding site for the duration of all simulations. AMP ligands under increased pressure showed the largest change in opening of the TolC efflux pump and aligns with experimental data showingE. colicells had the most resistance to AMP after aerosolization. These results, in addition to other real-time changes such as OM proteins and mutations of targets within the cell, could be used to delineate and mitigate antibiotic resistance mechanisms. 

Gram-negative bacteria pose an increased threat to public health because of their ability to evade the effects of many antimicrobials with growing antibiotic resistance globally. One key component of gram-negative bacteria resistance is the functionality and the cells’ ability to repair the outer membrane (OM) which acts as a barrier for the cell to the external environment. The biosynthesis of lipids, particularly lipopolysaccharides, or lipooligosaccharides (LPS/LOS) is essential for OM repair. Here we show the phenotypic and genotypic changes of Escherichia coli MG1655 (E. coli) before and after exposure to short-term aerosolization, 5 min, and long-term indoor aerosolization, 30 min. Short-term aerosolization samples exhibited major damages to the OM and resulted in the elongation of the cells. Long-term aerosolization seemed to lead to cell lysis and aggregation of cell material. Disintegrated OM rendered some of the elongated cells susceptible to cytoplasmic leakage and other damages. Further analysis of the repairs the E. coli cells seemed to enact after short-term aerosolization revealed that the repair molecules were likely lipid-containing droplets that perfectly countered the air pressure impacting the E. coli cells. If lipid biosynthesis to counter the pressure is inhibited in bacteria that are exposed to environmental conditions with high air velocity, the cells would lyse or be exposed to more toxins and thus become more susceptible to antimicrobial treatments. This article is the first to show lipid behavior in response to aerosolization stress in airborne bacteria both genotypically and phenotypically. Understanding the relationship between environmental conditions in ventilated spaces, lipid biosynthesis, and cellular responses is crucial for developing effective strategies to combat bacterial infections and antibiotic resistance. By elucidating the repair mechanisms initiated by E. coli in response to aerosolization, this study contributes to the broader understanding of bacterial adaptation and vulnerability under specific environmental pressures. These insights may pave the way for novel therapeutic approaches that target lipid biosynthesis pathways and exploit vulnerabilities in bacterial defenses, ultimately improving treatment outcomes 

Hydraulic fracturing, or fracking, requires large amounts of water to extract fossil fuel from rock formations. As a result of hydraulic fracturing, the briny wastewater, often termed back-produced fracturing or fracking water (FW), is pumped into holding ponds. One of the biggest challenges with produced water management is controlling microbial activity that could reduce the pond water’s reusable layer and pose a significant environmental hazard. This study focuses on the characterization of back-produced water that has been hydraulically fractured using chemical and biological analysis and the development of a high-throughput screening method to evaluate and predict the antimicrobial effect of four naturally and commercially available acidic inhibitors (edetic acid, boric acid, tannic acid, and lactic acid) on the growth of the FW microbiome. Liquid cultures and biofilms of two laboratory model strains, the vegetative Escherichia coli MG1655, and the spore-forming Bacillus atrophaeus (also known as Bacillus globigii, BG) bacteria, were used as reference microorganisms. Planktonic bacteria in FW were more sensitive to antimicrobials than sessile bacteria in biofilms. Spore-forming BG bacteria exhibited more sensitivity to acidic inhibitors than the vegetative E. coli cells. Organic acids were the most effective bacterial growth inhibitors in liquid culture and biofilm. 

Spreading patterns of the coronavirus disease (COVID-19) showed that infected and asymptotic carriers both played critical role in escalating transmission of virus leading to global pandemic. Indoor environments of res- taurants, classrooms, hospitals, offices, large assemblies, and industrial installations are susceptible to virus outbreak. Industrial facilities such as fabrication rooms of meat processing plants, which are laden with moisture and fat in indoor air are the most sensitive spaces. Fabrication room workers standing next to each other are exposed to the risk of long-range viral droplets transmission within the facility. An asymptomatic carrier may transmit the virus unintentionally to fellow workers through sporadic sneezing leading to community spread. A novel Computational Fluid Dynamics (CFD) model of a fabrication room with typical interior (stationary objects) was prepared and investigated. Study was conducted to identify indoor airflow patterns, droplets spreading patterns, leading droplets removal mechanism, locations causing maximum spread of droplets, and infection index for workers along with stationary objects in reference to seven sneeze locations covering the entire room. The role of condensers, exhaust fans and leakage of indoor air through large and small openings to other rooms was investigated. This comprehensive study presents flow scenarios in the facility and helps identify locations that are potentially at lower or higher risk for exposure to COVID-19. The results presented in this study are suitable for future engineering analyses aimed at redesigning public spaces and common areas to minimize the spread of aerosols and droplets that may contain pathogens. 

A low cutpoint wetted wall bioaerosol sampling cyclone (LCP-WWC), with an aerosol sampling flow rate of 300 L/min at 55″ H2O pressure drop and a continuous liquid outflow rate of about 0.2 mL/min, was developed by upgrading an existing system. The laboratory strain Escherichia coli MG1655 was aerosolized using a six-jet Collison Nebulizer and collected at high velocity using the LCP-WWC for 10 min with different collection liquids. Each sample was quantitated during a 15-day archiving period after aerosolization for culturable counts (CFUs) and gene copy numbers (GCNs) using microbial plating and whole-cell quantitative polymerase chain (qPCR) reaction. The samples were analyzed for protein composition and antimicrobial resistance using protein gel electrophoresis and disc diffusion susceptibility testing. Aerosolization and collection were followed by an initial period of quiescence or dormancy. After 2 days of archiving at 4 °C and RT, the bacteria exhibited increased culturability and antibiotic resistance (ABR), especially to cell wall inhibitors (ampicillin and cephalothin). The number of resistant bacteria on Day 2 increased nearly four-times compared to the number of cells at the initial time of collection. The mechanical stress of aerosolization and high-velocity sampling likely stunned the cells triggering a response of dormancy, though with continued synthesis of vital proteins for survival. This study shows that an increase in intensity in environmental conditions surrounding airborne bacteria affects their ability to grow and their potential to develop antimicrobial resistance. 

Wastewater-based surveillance (WBS) on SARS-CoV-2 has been proved to be an effective approach to estimate the prevalence of COVID-19 in communities and cities. However, its application was overlooked at smaller scale, such as a single facility. Meat processing plants are hotspots for COVID-19 outbreaks due to their unique environment that are favorable for the survival and persistence of SARS-CoV-2. This is the first known WBS study in meat processing plants. The goal was to understand the temporal variation of the SARS-CoV-2 levels in wastewater from a meat processing plant in Canada during a three-month campaign and to find any correlation with clinically confirmed cases in the surrounding city area. Higher SARS-CoV-2 concentrations and detection frequencies were observed in the solid fraction compared to the liquid fraction of the wastewater. The viruses can be preserved in the solid fraction of wastewater for up to 12 days. The wastewater virus level did not correlate to the city-wide COVID-19 cases due to the unmatching scales. WBS on SARS-CoV-2 in meat processing plants can be useful for identifying COVID-19 outbreaks in the facility and serve as an effective alternative when resources for routine individual testing are not available. 

Abstract A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the cause of the COVID-19 pandemic that originated in China in December 2019. Although extensive research has been performed on SARS-CoV-2, the binding behavior of spike (S) protein and receptor binding domain (RBD) of SARS-CoV-2 at different environmental conditions have yet to be studied. The objective of this study is to investigate the effect of temperature, fatty acids, ions, and protein concentration on the binding behavior and rates of association and dissociation between the S protein and RBD of SARS-CoV-2 and the hydrophobic aminopropylsilane (APS) biosensors using biolayer interferometry (BLI) validated with molecular dynamics simulation. Our results suggest three conditions—high ionic concentration, presence of hydrophobic fatty acids, and low temperature—favor the attachment of S protein and RBD to hydrophobic surfaces. Increasing the temperature within an hour from 0 to 25 °C results in S protein detachment, suggesting that freezing can cause structural changes in the S protein, affecting its binding kinetics at higher temperature. At all the conditions, RBD exhibits lower dissociation capabilities than the full-length S trimer protein, indicating that the separated RBD formed stronger attachment to hydrophobic surfaces compared to when it was included in the S protein. 

To understand how SARS-CoV-2 spreads indoors, in this study bovine coronavirus was aerosolized as simulant into a plexiglass chamber with coupons of metal, wood and plastic surfaces. After aerosolization, chamber and coupon surfaces were swiped to quantify the virus concentrations using quantitative polymerase chain reaction (qPCR). Bio-layer interferometry showed stronger virus association on plastic and metal surfaces, however, higher dissociation from wood in 80% relative humidity. Virus aerosols were collected with the 100 L/min wetted wall cyclone and the 50 L/min MD8 air sampler and quantitated by qPCR. To monitor the effect of the ventilation on the virus movement, PRD1 bacteriophages as virus simulants were disseminated in a ¾ scale air-conditioned hospital test room with twelve PM2.5 samplers at 15 L/min. Higher virus concentrations were detected above the patient’s head and near the foot of the bed with the air inlet on the ceiling above, exhaust bottom left on the wall. Based on room layout, air measurements and bioaerosol collections computational flow models were created to visualize the movement of the virus in the room airflow. The addition of air curtain at the door minimized virus concentration while having the inlet and exhaust on the ceiling decreased overall aerosol concentration. Controlled laboratory experiments were conducted in a plexiglass chamber to gain more insight into the fundamental behavior of aerosolized SARS-CoV-2 and understand its fate and transport in the ambient environment of the hospital room.