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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 

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. 

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. 

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.