Effects of Carbon Dioxide Aerosols on the Viability of Escherichia coli during Biofilm Dispersal.

Renu Singh,Jaesung Jang,Ajay K Monnappa,Robert J Mitchell,Seongkyeol Hong

doi:10.1038/srep13766

Abstract

A periodic jet of carbon dioxide (CO2) aerosols is a very quick and effective mechanical technique to remove biofilms from various substrate surfaces. However, the impact of the aerosols on the viability of bacteria during treatment has never been evaluated. In this study, the effects of high-speed CO2 aerosols, a mixture of solid and gaseous CO2, on bacteria viability was studied. It was found that when CO2 aerosols were used to disperse biofilms of Escherichia coli, they led to a significant loss of viability, with approximately 50% of the dispersed bacteria killed in the process. By comparison, 75.6% of the biofilm-associated bacteria were viable when gently dispersed using Proteinase K and DNase I. Indirect proof that the aerosols are damaging the bacteria was found using a recombinant E. coli expressing the cyan fluorescent protein, as nearly half of the fluorescence was found in the supernatant after CO2 aerosol treatment, while the rest was associated with the bacterial pellet. In comparison, the supernatant fluorescence was only 9% when the enzymes were used to disperse the biofilm. As such, these CO2 aerosols not only remove biofilm-associated bacteria effectively but also significantly impact their viability by disrupting membrane integrity.

Highlights

A periodic jet of carbon dioxide (CO2) aerosols is a very quick and effective mechanical technique to remove biofilms from various substrate surfaces
Our group presented a novel CO2 aerosol technique to remove Escherichia coli biofilms[19]. This technique employed periodic jets of carbon dioxide aerosols, a mixture of solid and gaseous CO2, which were generated by the adiabatic expansion of high-pressure CO2 gas through a nozzle. We demonstrated that this CO2 aerosol technique was very effective at removing biofilms from several surface materials with removal efficiencies ranging from 93.2% to 99.9% when treatment times of 40–90 s were employed[20]
Using multiple analytical tools, including confocal microscopy and scanning electron microscopy (SEM) to visualize the biofilms before and after the treatment and flow cytometry combined with colony forming units (CFU) measurements to assess the bacterial viability, we show that these carbon dioxide aerosols are effective at removing bacterial biofilms but they significantly reduce their viability

Summary

Results and Discussion

SEM analysis was initially used to analyze E. coli biofilms grown for one day on silicon chips before and after treating them with CO2 aerosols or hydrolytic enzymes (Fig. 1). This activity of the CO2 aerosols can be attributed to the large momentum (mechanical impact) of the solid CO2 particles and high shear near the silicon surfaces due to the high-speed gas flows, which will be discussed below. The consistency between the different techniques employed in this study helps to substantiate that the CO2 aerosols inflict a significant amount of damage to the biofilm-associated bacterial population during treatment and that this leads to losing the viability It suggests that the “injured” bacteria are viable and capable of producing colonies. The net result of this is a dramatic loss in viability, as demonstrated here by the CFU enumeration and flow cytometry analyses, with nearly 50% of the aerosol dispersed E. coli dying as a direct result of this treatment

Methods

Author Contributions

Additional Information