The Cell Membrane as a Major Site of Damage during Aerosolization of Escherichia coli

Abstract

This study aimed to provide data on the survival and site of damage of Escherichia coli cells following aerosolization using two different techniques, nebulization and flow focusing. Four metabolic stains were assessed for their ability to detect respiratory activities and membrane homeostasis in aerosolized E. coli cells. The degree of sublethal injury increased significantly over the 10-min period of aerosolization in E. coli cells aerosolized by using the Collison nebulizer, reaching up to 99.9% of the population. In contrast, a significantly lower proportion of the population was sublethally damaged during aerosolization using the flow-focusing aerosol generator (FFAG). Concomitantly, loss of membrane homeostasis increased at a higher rate in nebulized cells (68 to 71%) than in those aerosolized by using the FFAG (32 to 34%). The activities of respiratory enzymes decreased at increased rates in nebulized cells (27 to 37%) compared to the rates of decrease in cells aerosolized by using the FFAG (59 to 61%). The results indicate that the physiology of an aerosolized bacterium is linked to the method of aerosol generation and may affect the interpretation of a range of aerobiological phenomenon.Aerosolization of bacteria has importance from medical, agricultural, and biodefense perspectives. Bacterial infections of the respiratory tract are transmitted by aerosols produced by coughing and sneezing (26,35,36,37). Insecticidal bacteria, such as Bacillus thuringiensis, may be disseminated by aerosols to control agricultural pests (3). Dissemination of biowarfare agents can occur by the production of an aerosol, and an understanding of survival is important for hazard management (32). In all these examples, for the aerosolized bacteria to exert an effect, they must remain viable in the aerosolized state for a period of time.The physiological status of bacteria changes with time spent in the aerosolized state. Loss of culturability on plating media is often biphasic, being defined by an initial rapid decline over the first few minutes followed by further decline at a lower rate (13,39,41,48). The initial process of aerosolization, for example, nebulization, will impart device-dependent mechanical stress, such as physical shear and wall impaction, resulting from operation of the pressurized system (33,34,38). After aerosolization, a number of parameters will contribute to loss of viability in the airborne bacteria, including temperature, particle size, desiccation (relative humidity), UV radiation, oxidative shock, and toxic pollutants (carbon monoxide, sulfur dioxide, nitrogen dioxide, nitrogen oxide, ozone, and "open air factor") (9,10,13,16,20,36,42,43,46,48). Furthermore, bacteriological factors, such as growth phase or sporulation state, will influence survival (8, 9). Bacteria have been proposed to enter the viable-but-nonculturable (VBNC) state as a result of both aerosolization and impingement. VBNC cells represent a physiological state where bacteria cannot be cultured on nonselective agar yet retain certain...

Release of Free DNA by Membrane-Impaired Bacterial Aerosols Due to Aerosolization and Air Sampling

“…It was found in our earlier study that the viability of P. fluorescens bacteria aerosolized by a Collison nebulizer (BGI Inc., Waltham, MA) decreased by over 50% after 90 min of continuous aerosolization (23). Similarly, Thomas et al indicated that 99.9% of an Escherichia coli population suffered sublethal injury after a 10-min aerosolization by a Collison nebulizer (24). They also concluded that the cell membrane was the major site of damage due to impaction and shear force stress that disturbed membrane homeostasis (24).…”

Aerobiological Stabilities of Different Species of Gram-Negative Bacteria, Including Well-Known Biothreat Simulants, in Single-Cell Particles and Cell Clusters of Different Compositions

“…Although the aerobiological stability characteristics of several species of Gramnegative bacteria already have been investigated, including S. marcescens (7,8,17,18), E. coli (19)(20)(21)(22)(23)(24)(25), and P. agglomerans (6,12,(26)(27)(28)(29)(30), most of these studies were performed more than 3 decades ago; thus, several knowledge gaps still remain. An inherent challenge regarding the aerobiological stability of Gram-negative bacteria is the observation that few generalizations seem to be applicable, suggesting that different genera, species, and even strains of Gram-negative bacteria have to be considered separate entities regarding their stability characteristics (11,15,16).…”

“…It also could be interesting to study several strains of the same bacterial species to specifically address strain-specific aerobiological stability characteristics. The ambition for the future also involves adopting additional characterization methods, including flow cytometry in combination with membrane integrity and metabolic cell staining (20,45,46) and quantitative PCR in combination with ethidium or propidium monoazide live-dead discrimination techniques (47)(48)(49)(50), as well as the study of multiple strains of the same bacterial species to investigate strain-specific aerobiological stability characteristics.…”

Chamber catalogues of optical and fluorescent signatures distinguish bioaerosol classes

“…Direct phase contrast and fluorescence microscopy (Hernandez et al, 1999) showed these bacteria remained intact after inspecting nebulizer reflux immediately following their aerosolization. Based on previous studies with many of the same bacterial models used here, some microbes, particularly Gram negative bacteria harvested in their log growth phase, can experience significant viability losses after being refluxed in Collison nebulizers even after 10 min (Stone and Johnson, 2002;Thomas et al, 2011). To minimize aerosolization stress and viability variance, bacterial cultures were harvested in early stationary phase, and nebulization times were limited to 2 min.…”