The data parameters involved in UV sterilization discussed in this paper mainly included wavelength, irradiance, UV dose, spot size and irradiation time.
(1) Ultraviolet is the invisible band. The visible part of the light source was not used as the evaluation of the ultraviolet performance when it was lit. Its performance was the measurement and evaluation of the energy of the ultraviolet band, meaning that the ultraviolet flux (energy density) per unit time in a certain plane, referred to as irradiance, expressed in terms of the total energy in a period of time, and the ultraviolet flux was expressed in terms of the following formula:
Where Ε is UV dosage (mJ/cm²), Ι is the irradiance detected on the surface of targets (mW/cm²), and t is the irradiation time (s).
In this experiment, the type of uvc-led was UV-LED (BRT-B35CD7A1CSD, Taiwan High Power Lighting, China) and the wavelength was 265–278 nm, and the beam angle was 100°. The irradiance was measured by the UVC Light Meter (LS125-UVCLED, Shenzhen Linksun Technology, China). It measured the spectrum of 220 nm to 320 nm. The power resolution was 0.1 µW/cm2 and its measurement accuracy was ± 10%. And a digital timer (DH48S-1Z, ECNKO, China) was used to control the time. There was a time error that the time for bacterial exposure was a bit less than the irradiation time and that in which the time from start-up to output was negligible.
At a certain distance from the lamp bead, all areas covered by the light spot were called irradiation areas. The distribution of irradiance of the entire area at the same distance was uneven. The irradiation area of different lamp beads was affected by its divergence angle. The visible light spot distribution at different distances of LED was used as the reference of UVC distribution (Fig. 8). The farther the distance from the lamp bead, the smaller the irradiance and the larger the irradiation area. In the irradiation area at the same distance, the irradiance of the light spot of the lamp bead was uneven. From the figure, the irradiance in the center area of the spot was the highest. In the process of reaching the edge, it decreased gradually with a small increase which reflected in the inactivation effect on bacteria.
The sterilization experiments were conducted in the spot area with relatively uniform irradiance, and the ultraviolet dose was determined to evaluate the sterilization rate. The irradiance in the experiment was measured on the surface of the irradiation target and the irradiation dose was obtained from this, which was the energy value in this paper. In the experiment, the heat dissipation of the lamp beads was good, and the heat of the lamp beads was negligible. The experimental defect was that no filter was used to ensure a single wavelength.
(2) Bacterial strains, culture conditions and irradiations
To evaluate the UV effect on bacteria, ten pathogenic bacteria, A. baumannii, E. faecalis, E. aerogenes, Klebsiella oxytoca, Serratia marcescens, S. aureus, Klebsiella Pneumoniae, E. faecium, Escherichia coli and P. aeruginosa, were selected for this experiment. All strains were separated from the patients and were drug-resistance except A. baumannii 5116, a sensitive strain. The bacteria were from Xi’an Jiaotong University. They were preserved at minus eighty degrees. To obtain enrichment solution, a single colony were picked up by loop and was inoculated into 5 mL Luria-Bertani broth and was set the glass bottle with suspension in a shaking incubator (37℃; 200 rpm). After 14 to 16 hours, the concentration of bacterial solution was evaluated by the optical density at 600 nm measured by an ultraviolet spectrophotometer. The bacteria were collected by centrifugation (2 min; 10,000 rpm), resuspended them in broth and adjusted them to an optical density at 600 nm of 0.2.
To obtain certain quantities of bacteria suitable for irradiation, the bacterial suspension and carried out ten-fold serial dilutions. Approximately 100 µL in multiple concentrations was spread on agar media until they dried out.
Limited by the penetration of ultraviolet C, the thickness of the bacterial layer must be considered. The distribution density of bacteria on the agar medium is the ratio of the number of colonies to the area of the medium. The unit is cells/cm2, and the radius is r (petri dish, r = 4.3 cm). Ideally, the solution of bacteria was spread evenly on agar medium. The size of the bacterium was too small to observe directly and the number of viable bacteria after irradiation was counted by colonies cultured on agar medium for about 12 to 16 hours. When the initial distribution density at 1.36E + 06 and 1.36E + 05 cells/πr2 were single layer which was appropriate for general exposure experiments. All experiments were independent, and each experiment had one or two repetitions.
For the inactivation of multilayer bacteria, the control group was not irradiated, and the irradiation time was half an hour to two hours. After irradiation, the colonies were inoculated and then suspended in LB broth. In the experiment, the volume of scraped bacteria was different. The optical density of bacterial solution at 600 nm were adjusted to 0.2. The solution was applied to count the colonies on the plate for sterilization statistics.
All plates were placed in a thermostatic incubator at 37°C, and growth was observed after 14 to18 hours. The plate colony-counting method was adopted to assess bacterial quantities. Those covered properly were prepared for irradiation experiments. Irradiated immediately after coating. The petri dishes were placed on the tray, and the center of the plates was aligned to the LED directly; it was not totally in the center at 13 mm. After exposure, the medium was placed in an incubator (37℃) for 12–18 hours. Colonies were counted, and images were recorded by an Umax2000 scanner. All bacteria were cultured under the same conditions.
(3) Agarose gel electrophoresis
To explore the UV effect on DNA, the genome of the sensitive strain of A. baumannii was extracted for electrophoretic experiments44. Bacteria untreated were used as control. And the bacteria heated at 100°C for about 10 minutes were the positive control. The bacteria were exposed to UV radiation at 46 mm for one min (18 mJ/cm2) and 10 min (180 mJ/cm2) separately to examine the state of the genome. The genome was extracted by a Bacteria DNA kit (Tian Gen, China).
The gel-making process was carried out in a biosafety cabinet. First, agarose powder (about 0.3 g) was weighed and dissolved in 30 mL 1xTAE solution. After heated 3 minutes in microwave, the 4 µL EB (ethidium bromide) was added and mixed well, the gel was poured into the plastic box, the ruler comb was inserted. The gel was cooled to room temperature and then put the gel board placed in a solution filled with 1x TAE, which was higher than the gel. A marker (5 µL 1000 bp, TianGen, China) was added, and the extracted genome samples were mixed with loading buffer to the aperture in sequence. The volume of samples added was calculated according to the concentration to ensure that the total amount of the genome was consistent. Covered the electrophoresis tank, connected the circuit, turned on the electrophoresis instrument, adjusted the voltage to 100 V. After about 40 min electrophoresis, took out the gel and put it under the gel image machine (Sage creation, China) to observe and record.
(4) Transmission electron microscope44
To investigate the UV effect on bacteria, the transmission electron microscopy was used to observe the inner structure. The sample of control group were taken from bacteria suspension. The bacteria sample B and C were taken from colonies on the agar medium which were irradiated for three hours at 8.2 cm and two hours at 1.3 cm and the doses were 648 mJ/cm2 and 7200 mJ/cm2 separately. The bacteria of sample D were collected from liquid which were irradiated at 1.3 cm for two hours. The bacteria on agar medium were transferred to tubes with PBS and then the samples were centrifuged at 5000 rpm for 5 minutes. And the bacteria in liquid were centrifuged at 5000 rpm for 10 min. The supernatant fluid was removed and glutaraldehyde (2.5%, SCRC, China) was added in 60 sec. The colonies were not blown up. The samples were stored at room temperature.
The following procedures were taken performed by an electron microscope laboratory. The fixed liquid was taken off. The samples were washed by PBS two times. Osmic acid solution (1%, Johnson Matthey, UK) was used to fix samples for two hours at 4°C. The bacterial samples were dehydrated with ethanol gradients (30–100%) and then embedded them in the Epon 812 epoxy resin (Structure Probe, USA) at 60°C for 24 hours. The sample was sliced into 70 to 90 nm slices in ultra-thin slicing machine (LKB, Sweden). The slices were stained with lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 15 minutes, respectively. The thin-section samples were recorded on a transmission electron microscope (Hitachi H-7650, Japan).
(5) Statistical Analysis
The data were examined by Normality and Lognormality Tests. The values were taken from descriptive statistics, 95%CI. The significance of columns was analyzed by one sample t test and Brown-Forsythe and Welch ANOVA tests. XY was computed by Welch's t test. The multiple comparisons were analyzed by the Kruskal-Wallis test. For the theoretical data, Tukey's multiple comparisons test were used to evaluate the significance of rows, two columns, and the main column effect. The experiment had one replicate for each result. All statistical tests were performed by Prism 8 Version 8.2.1 (279).