Comparative Analysis of UV Irradiation Effects on Escherichia coli and Pseudomonas aeruginosa Bacterial Cells Utilizing Biological and Computational Approaches

Abstract

Microorganisms have a large number of tools to withstand different, and sometimes strong, environmental stresses, including irradiation, but this ability should be further evaluated for certain applications. Growth inhibition and morphological alterations of Escherichia coli M-17 and Pseudomonas aeruginosa GRP3 wild-type cells caused by UV-A irradiation have been detected in the present study. Comparative analysis was carried out using well-established microbiological methods (determination of specific growth rate, growth lag phase duration, and colony-forming unit number—CFU) and computational approaches, employing light microscopy and digital image analysis to evaluate bacterial cell morphology. Decreases in the specific growth rate, prolonged lag-phases, and lowered CFUs were observed after 5 and 10 min of UV irradiation (approx. 40 Gy) compared to the control (nonirradiated) cells. Accordingly, two computational parameters—the average bacterial cell surface area and the bacterial cell perimeter (i.e., of the 2D projection of bacterial cells in microscopy image)—were reduced. The ratio of bacterial cell surface area (S) to the square of the perimeter (p ²) was reduced after 5 min of irradiation, but after 10 min of irradiation the studied bacterial cells became flat cylinders. The revealed findings are concluded to be highly useful in developing new, rapid analysis methods to monitor environmental and UV irradiation effects on bacteria and to detect bacterial cell morphology alterations.

Appendix

The calculation of the UV dose was carried out according to the lamp parameters (see Materials and methods) and the distance from the target (15 cm). During 5 min of irradiation, the UV dose (I*₁) was 15 J/cm². The dose of UV rays was decreased to 7.5 J/cm² during 10-min radiation (I*₂) by reducing the lamp power from 250 W to 125 W. The length of the UV reflection (a) in a 15 cm distance was 45 cm.

The intensity of the rays falling on the bacterial layer (I₀₁ and I₀₂ for 5- and 10-min UV exposure, correspondingly) was calculated using the following equation:

$$ {\text{I}}_{01} = \frac{{{\text{I*}}_{1} }}{{{\text{a}}^{2} }} = \frac{15}{2025} = 7. 4 1 {\text{ mW}}/{\text{cm}}^{ 2} ;{\text{I}}_{02} = \frac{{{\text{I*}}_{2} }}{{{\text{a}}^{2} }} = \frac{7.5}{2025} = 3. 7 {\text{ mW}}/{\text{cm}}^{ 2.} $$

The intensity of the rays passing through the bacterial layer (I₁ = 7.3 mW/cm² and I₂ = 3.6 mW/cm² for 5 and 10 min UV exposure, respectively) was measured using a UV detector (UFI-25, Russia), detecting 250-400 nm rays and doses of 0.1-2000 mW/cm².

The differences between the values of the UV falling and passing intensities (I_1k and I_2k for 5 (t₁) and 10 min (t₂) UV exposure, respectively), the surface area of the Petri dish ($\Delta S = \pi r^{2} = 3.14 \times 4^{2} = 50.24$) and the bacterial mass (m = 10 g = 10⁻⁴ kg) were used for the UV-absorbed dose (D) calculations:

$$ D = \frac{{I_{k} \times \Delta S \times t}}{m} $$

Therefore, the calculations of the UV-absorbed doses during 5- and 10-min irradiation (D₁ and D₂, respectively) were

$$ D_{1} = \frac{{26.7 \times 10^{ - 6} {\text{W}}/cm^{2} \times 50.24\, cm^{2} \times 300 {\text{s}}}}{{10^{ - 4} }}{ = 40} . 2 4 {\text{ Ws/kg (Gy)}} $$

$$ D_{2} = \frac{{13.2 \times 10^{ - 6} {\text{W}}/cm^{2} \times 50.24\, cm^{2} \times 600 {\text{s}}}}{{10^{ - 4} }}{ = 39} . 8 {\text{ Ws/kg (Gy)}} $$