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

Singh R, Monnappa AK, Hong S, Mitchell RJ, Jang J - Sci Rep (2015)

Bottom Line: A periodic jet of carbon dioxide (CO2) aerosols is a very quick and effective mechanical technique to remove biofilms from various substrate surfaces.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.

View Article: PubMed Central - PubMed

Affiliation: School of Mechanical and Nuclear Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, S. Korea.

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.

No MeSH data available.


Related in: MedlinePlus

Analysis of the bacteria dispersed during the CO2 aerosol and enzymatic treatments by flow cytometry.The samples are the same as in Fig. 3. (a) Actual number of particles counted within the FSC gated region when using a fixed time of 10 seconds for each sample. (b) Relative populations of the cells (live, dead and injured) in each sample. (c) Relative populations of the cells (live, dead and injured) were dispersed by enzymatic treatment (control), N2 gas, and CO2 aerosol treatments. In these N2 gas and CO2 aerosol treatments, the dispersed bacteria were added to the bacteria present on the chip surfaces. The classification of bacterial cells into each population was performed by staining the samples with the BacLight stain (SYTO9 and propidium iodide) prior to FACS analysis. A total of 5,000 cells were analyzed for each sample, and the results show the average from three independent tests. Statistical analysis was performed using one-way ANOVA followed by the Tukey post hoc test. Statistically significant results are identified with asterisks (*, **, or *** = P values < 0.05, 0.01 or 0.0001, respectively; ns – not significant).
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f4: Analysis of the bacteria dispersed during the CO2 aerosol and enzymatic treatments by flow cytometry.The samples are the same as in Fig. 3. (a) Actual number of particles counted within the FSC gated region when using a fixed time of 10 seconds for each sample. (b) Relative populations of the cells (live, dead and injured) in each sample. (c) Relative populations of the cells (live, dead and injured) were dispersed by enzymatic treatment (control), N2 gas, and CO2 aerosol treatments. In these N2 gas and CO2 aerosol treatments, the dispersed bacteria were added to the bacteria present on the chip surfaces. The classification of bacterial cells into each population was performed by staining the samples with the BacLight stain (SYTO9 and propidium iodide) prior to FACS analysis. A total of 5,000 cells were analyzed for each sample, and the results show the average from three independent tests. Statistical analysis was performed using one-way ANOVA followed by the Tukey post hoc test. Statistically significant results are identified with asterisks (*, **, or *** = P values < 0.05, 0.01 or 0.0001, respectively; ns – not significant).

Mentions: Although the above fluorescence results indicate that a large number of the bacteria are being injured or killed by the aerosol treatment, it still remained uncertain if the decrease in the viable counts (Fig. 3a) is due to cell death or if a portion of the bacterial population was lost due to aerosolization of the biofilm. To address this, we analyzed each of the collections (Control, Aerosol and Aero + Chip) using flow cytometry. As shown in Fig. 4a, when the same volume of sample was analyzed the particle number in the Aerosol sample was approximately 40% lower than that of the Control. However, treatment of the undispersed biofilm still present on the chip with Proteinase K and DNase I (Aero + Chip) increased this to 73%. These values show that a significant number of bacteria are not being captured, and although this contributes to the lower viability seen in Fig. 3a it does not fully account for this discrepancy.


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

Singh R, Monnappa AK, Hong S, Mitchell RJ, Jang J - Sci Rep (2015)

Analysis of the bacteria dispersed during the CO2 aerosol and enzymatic treatments by flow cytometry.The samples are the same as in Fig. 3. (a) Actual number of particles counted within the FSC gated region when using a fixed time of 10 seconds for each sample. (b) Relative populations of the cells (live, dead and injured) in each sample. (c) Relative populations of the cells (live, dead and injured) were dispersed by enzymatic treatment (control), N2 gas, and CO2 aerosol treatments. In these N2 gas and CO2 aerosol treatments, the dispersed bacteria were added to the bacteria present on the chip surfaces. The classification of bacterial cells into each population was performed by staining the samples with the BacLight stain (SYTO9 and propidium iodide) prior to FACS analysis. A total of 5,000 cells were analyzed for each sample, and the results show the average from three independent tests. Statistical analysis was performed using one-way ANOVA followed by the Tukey post hoc test. Statistically significant results are identified with asterisks (*, **, or *** = P values < 0.05, 0.01 or 0.0001, respectively; ns – not significant).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561891&req=5

f4: Analysis of the bacteria dispersed during the CO2 aerosol and enzymatic treatments by flow cytometry.The samples are the same as in Fig. 3. (a) Actual number of particles counted within the FSC gated region when using a fixed time of 10 seconds for each sample. (b) Relative populations of the cells (live, dead and injured) in each sample. (c) Relative populations of the cells (live, dead and injured) were dispersed by enzymatic treatment (control), N2 gas, and CO2 aerosol treatments. In these N2 gas and CO2 aerosol treatments, the dispersed bacteria were added to the bacteria present on the chip surfaces. The classification of bacterial cells into each population was performed by staining the samples with the BacLight stain (SYTO9 and propidium iodide) prior to FACS analysis. A total of 5,000 cells were analyzed for each sample, and the results show the average from three independent tests. Statistical analysis was performed using one-way ANOVA followed by the Tukey post hoc test. Statistically significant results are identified with asterisks (*, **, or *** = P values < 0.05, 0.01 or 0.0001, respectively; ns – not significant).
Mentions: Although the above fluorescence results indicate that a large number of the bacteria are being injured or killed by the aerosol treatment, it still remained uncertain if the decrease in the viable counts (Fig. 3a) is due to cell death or if a portion of the bacterial population was lost due to aerosolization of the biofilm. To address this, we analyzed each of the collections (Control, Aerosol and Aero + Chip) using flow cytometry. As shown in Fig. 4a, when the same volume of sample was analyzed the particle number in the Aerosol sample was approximately 40% lower than that of the Control. However, treatment of the undispersed biofilm still present on the chip with Proteinase K and DNase I (Aero + Chip) increased this to 73%. These values show that a significant number of bacteria are not being captured, and although this contributes to the lower viability seen in Fig. 3a it does not fully account for this discrepancy.

Bottom Line: A periodic jet of carbon dioxide (CO2) aerosols is a very quick and effective mechanical technique to remove biofilms from various substrate surfaces.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.

View Article: PubMed Central - PubMed

Affiliation: School of Mechanical and Nuclear Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, S. Korea.

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.

No MeSH data available.


Related in: MedlinePlus