A New Method to Easily Assess Bacteriostatic and Bactericidal Activity of Ultraviolet Radiation Using Quantitative Image Analysis

Ultraviolet (UV) radiation can elicit both bactericidal and bacteriostatic activity depending on light parameters and targeted bacteria. Current methods based on bacterial growth on solid medium allow measurement of only bactericidal but not bacteriostatic activity, while liquid cultures exhibit low light penetration. Here, we propose a method to quantify both bactericidal and bacteriostatic activity of radiation based on (a) bacterial cultures on solid medium, (b) acquisition and quantitative analysis of photographic images of plates containing bacterial colonies, (c) application of two mathematical equations to evaluate bactericidal and bacteriostatic activity. The proposed method considers the differences in growth on test and control (unexposed) plates. The measurements performed on the plates image are the independent variables of the mathematical equations returning the values of bactericidal and bacteriostatic activity. Experimentally, a test was performed using Escherichia coli grown on a solid medium and exposed to UVA (365 nm) radiation. The standard method allowed quantifying bactericidal activity and evaluating only qualitatively bacteriostatic activity of the radiation. Differently, the new method here proposed allowed quantification of both activities. The proposed method proved to be simple, enabling deep assessment of the antibacterial effects of UV radiation directly on the solid medium through image acquisition and analysis.


INTRODUCTION
The antibacterial activity of an agent (e.g.antibiotic, radiation, etc.) can be distinguished as bacteriostatic activity-meaning that the agent prevents bacterial growth when it is applied-and bactericidal activity-when it kills bacterial cells.Antibacterial agents can combine both actions, depending on various factors like bacterial species, cell number and density, dose and duration of the treatment (1)(2)(3).
The antibacterial activity of ultraviolet (UV) radiation (100-400 nm) has been known for a long time and is used in applications such as sterilization (4)(5)(6)(7)(8)(9)(10)(11).The efficiency of UV effects against bacteria depends on different parameters such as wavelength range, radiation intensity, exposure duration, bacterial concentration and species.It is recognized that UVC radiation (100-280 nm) has a powerful bactericidal effect but also presents severe risks to human health (12).Instead, UVB (280-315 nm) and UVA (315-400 nm) radiations are safer for humans and can provide antibacterial effects-bactericidal and/or bacteriostaticin appropriate conditions (13,14).Although an environmental bactericidal effect may guarantee higher performance in improving health conditions and reducing the risk of many pathologies, the bacteriostatic effect may be sufficient for some applications with sanitization purposes.Indeed, bacteriostatic agents can achieve specific thresholds of bacterial reduction, but require a higher dose or time than bactericidal agents (1,15).Thus, reliable determination of both bacteriostatic and bactericidal activity is necessary to assess the appropriate exposure conditions for UVA or UVB light that provide both safety (for humans) and efficient bacterial inhibition, for example using low intensity for long exposure times or vice versa.
Antibacterial activity of UV light is usually determined by spreading bacteria on a solid medium, exposing them to the radiation for a time interval, then counting colony forming units (CFU) and comparing test results to unexposed control plates (16,17).However, this method enables the measurement of bactericidal but not bacteriostatic activity because all viable bacterial units are considered, without keeping into account whether their growth was inhibited during exposure.Alternatively, liquid cultures can be exposed to UV radiation to evaluate the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC); however, this method is limited by the poor penetration capacity of UV radiation in a liquid medium, especially at high bacterial concentrations (18,19).In fact, the radiation easily reaches the upper layers of bacteria while the bottom layers are protected by the upper layers.Because of this "shadowing effect" (cell-to-cell protection), the evaluation of the antibacterial activity using this method might not be realistic.
Here, we propose a new method based on the different growth rates of bacteria affected by bactericidal or bacteriostatic radiation on a solid medium.The method consists of (i) bacterial cultures on a solid medium exposed to light radiation, (ii) photographic image acquisition of the plates, (iii) incubation of the plates at the proper conditions for bacterial growth and, again, (iv) photographic image acquisition of the plates.The quantitative analysis of images and the application of two equations allow us to directly quantify both bactericidal and bacteriostatic activity of light radiation.

MATERIALS AND METHODS
UVA radiation source.UVA source is a lighting fixture (FORALL, Verona, Italy) equipped with fourteen 365 nm UVA LEDs (Seoul Viosys) and wide beam optics (Khatod) specific for UV applications.The lamp has an emission between 360 and 400 nm (UVA spectrum), with a peak at 365 nm.Two hundred W/m 2 irradiance was measured at a 50 cm distance from the lamp (perpendicular to the axis of the lamp and passing through the center of the lamp itself) where the plates were placed.
Antibacterial activity testing.The antibacterial activity of UVA radiation was tested on Escherichia coli ATCC 8739.Starting from a 0.5 McFarland bacterial suspension in saline solution, six 10-fold dilutions until 10 −6 were prepared.20 μL of the initial suspension and each dilution (total n = 7) were deposited on Luria-Bertani (LB) agar plates in duplicate (14 droplets in total).Each experimental condition (test/control plate) was repeated in triplicate, for a total amount of 42 droplets.The 3 test plates were closed with a transparent lid and exposed to the UVA radiation for 16 h at a 50 cm distance from the light source (perpendicularly; irradiance = 1.1 × 10 7 J/m 2 ), while the 3 control plates were closed with the lid and covered with aluminum foil.At the end of the irradiation, plates were imaged as described below (first time point, t 1 ).Then plates were incubated for 24 h at 37 °C.After incubation, CFU were counted to confirm the eventual presence of bactericidal activity, and images of the plates were acquired again (second time point, t 2 ).
Image acquisition and analysis.Test and control plates were photographed in a light tight chamber with low diffuse illumination using IVIS Spectrum (Perkin Elmer, Massachusetts, USA), equipped with 4 light sources (quartz lamps) and a 2048 × 2048 CCD detector (Fig. 1).The instrument was used in photograph modality and images were acquired with exposure time = 0.2 s, Binning = 4, F/stop = 8.Images were analyzed with Living Image 4.5 Software (Perkin Elmer).Circular regions of interest (ROIs) were drawn on the images corresponding to the 14 drops of bacterial suspension visible on the plates and the total photographic pixel counts (uncalibrated measurement of the photons incident on the CCD camera, hereafter named "counts") measured by the CCD in the ROI were considered.In addition, two ROIs were drawn in empty areas of each plate to measure the background intensity of the medium.For each plate, the mean value of the background was subtracted from the light intensity measured in the drops.

Plate quantification
For each experimental condition, the mean of the backgroundcorrected counts (AEstandard deviation) measured on the ROIs drawn on the 42 droplets (7 dilutions, in duplicate; 3 plates per condition) was calculated to obtain the total counts.Thus, we define C 1 and T 1 as the mean total counts respectively in control and test plates at the first time point (t 1 , measured after 16 h of exposure to UVA radiation; t 1 = 16 h).Analogously, C 2 and T 2 are the mean total counts in control and test plates at the second time point (t 2 , 24 h after t 1 ; t 2 = 40 h) (Fig. 2, Table 1).
The mean total counts obtained in the experimental procedure were T 1 = 1.09 × 10 5 , T 2 = 5.72 × 10 5 , C 1 = 3.45 × 10 5 , C 2 = 9.48 × 10 5 .The results are schematically represented in Fig. 3 and the parameters T 1 , T 2 , C 1 and C 2 are the variables proposed here to evaluate bacteriostatic and bactericidal activity as explained in the following subsection.

Theoretical approach and bacteriostatic and bactericidal activity quantification
To evaluate bacteriostatic and bactericidal activity (BsA and BcA, respectively, in this Section), we can start observing that, at least at the theoretical level (i.e.excluding experimental errors): (hyp1) T 1 , C 1 , T 2 , C 2 ≥ 0 means that the signal coming from the drops is not lower with respect to the ones coming from the medium (i.e.bacteria are observable in at least two drops on the plates); (hyp2) T 1 ≤ C 1 .Just after irradiation (time point t 1 ), treated plates cannot show bacterial growth higher than control plates; (hyp3) T 1 ≤ T 2 .At time t 1 , the bacterial content cannot be higher with respect to time t 2 in treated plates; (hyp4) T 2 ≤ C 2 .At time t 2 , the bacterial content in treated plates cannot be higher with respect to the content in control plates at time t 2 ; it follows that also T 1 ≤ C 2 .
Here, we propose to evaluate the BsA with the following equation: Photochemistry and Photobiology, 2023, 99 1477 where C 1 , T 1 , C 2 and T 2 are the total counts defined in the previous Section.
Analogously, BcA can be evaluated as follow: The expression for BsA is of the form: where the factor A considers the difference between T 2 and T 1 , normalized to the maximum difference at t 2 (C 2 −T 1 ) and F ¼ C1ÀT1 C1 .Instead, the expression for BcA is: where B considers the difference between C 2 and T 2 , normalized on the maximum difference at t 2 (C 2 −T 1 ).Noteworthily, it is possible to observe that: (obs1) The sum A + B = 1; (obs2) The factor F, which compares in both eqs.( 1) and ( 2), considers the difference between C 1 and T 1 normalized on the maximum difference at t 1 (C 1 −0 = C 1 ).Moreover, BsA + BcA = F; (obs3) The previous four hypotheses (hyp1-hyp4) guarantee that all the numerators and denominators of the fractions appearing in eqs.( 1) and ( 2) are non-negative.Thus, BsA and BcA are positive quantities.Moreover, factor F assumes values between 0 and 1; (obs4) The eqs. ( 1) and ( 2) exist only for C 1 ≠ 0 and C 2 ≠ T 1 ; these conditions will be further considered in the Discussion section; (obs5) If T 1 < C 1 and T 2 < C 2 , it means that the viability of bacterial cells is reduced in test plates in comparison with control plates due to BcA; (obs6) If T 1 < C 1 and T 2 > T 1 , the bacterial growth has been inhibited during exposure due to BsA; (obs7) Considering that radiation can present (i) only BsA, (ii) only BcA or (iii) a combination of BsA and BcA, the mathematical expressions of BsA and BcA need to result in values in the range [0, 1], the highest value representing the maximum activity; moreover, when BsA = 1, BcA must be equal to 0, and vice versa.

Particular cases
Figure 4 shows some particular cases and the limits for BsA and BcA.
(case3) If T 1 = 0 and T 2 = C 2 .In this case, BsA reaches the maximum value (BsA = 1) and BcA = 0 (Fig. 3C) meaning that the bactericidal activity is null, and the bacteriostatic activity is maximum.

BsA and BcA as function of T 1 and T 2 values
Considering the approximated experimental values (divided for a factor of 1000) only for didactical purposes (T 1 = 100, C 1 = 350, T 2 = 575, C 2 = 950), here we show the behavior of BsA and BcA by varying one parameter and keeping the other three fixed.We are interested here in the behavior of BsA and BcA as a function of T 1 alone or, alternatively, T 2 alone.Figure 5A shows BsA and BcA when T 1 is in the range [0-350] and T 2 = 575.Both BsA and BcA functions decrease monotonically with increasing T 1 (Fig. 5A) with a behavior almost linear for very low T 1 values and reaching the 0 value when T 1 = C 1 .
Figure 5B shows BsA and BcA behavior when T 2 is in the range [100-950] and T 1 = 100.BsA and BcA are linear functions of T 2 (as visible in eqs. 1 and 2): increasing with T 2 the former, decreasing with T 2 the latter.Considering BsA and BcA as a functions of both variables T 1 e T 2 , Fig. 6 shows the behaviors of BsA and BcA when T 1 is in the range [0-350] and T 2 in the range [0-950] (fixed C 1 = 350, C 2 = 950) (Fig. 5A,B, respectively).The black triangle in the bottom part of both panels is due to the hypothesis that T 2 must be higher than T 1 (hyp3).
Finally, to better understand the behavior of BsA and BcA as a function of both variables T 1 , and T 2 , the 3D representation of their values obtained from eqs. ( 1) and ( 2), varying T 1 in the range [0-350] and T 2 in the range [0-950] (fixed C 1 = 350, C 2 = 575), are shown in Fig. 7 (A e B, respectively).

Experimental results
Considering the measurements obtained in the experimental procedure reported in the previous subsection (T 1 = 1.09 × 10 5 , T 2 = 5.72 × 10 5 , C 1 = 3.45 × 10 5 , C 2 = 9.48 × 10 5 ) and inserting these values in eqs.( 1) and ( 2), BsA and BcA can both be quantified: BsA = 0.377 and BcA = 0.306.At t 2 , CFU visible on the plates were counted and the bacterial load was estimated as follows: 1.5 × 10 8 CFU/mL in the control plates and 1.25 × 10 2 CFU/mL in the UVA-exposed plates.The reduction of CFU in the treated plates in comparison with the control plates indicates that UVA radiation exhibited a bactericidal activity, confirming the result obtained using the proposed method.In addition, we observed that at t 1 , colonies were visible in control plates but not in treated plates; in the latter, colonies became visible at t 2 .This indicates that the UVA radiation had a bacteriostatic activity (i.e.inhibiting bacterial growth, but not killing bacterial cells) that cannot be quantified by CFU count.

DISCUSSION
The distinction between bactericidal and bacteriostatic activity appears to be clear according to the definition, but experimentally it is possible to distinguish the two activities only in certain conditions.While the antimicrobial activity of a compound in a liquid medium can be distinguished as bactericidal or Photochemistry and Photobiology, 2023, 99 1479 bacteriostatic based on the measurement of its MBC and MIC (1), the same approach cannot be applied to UV radiation due to its different physical nature.In fact, UV penetration can be relatively low in the liquid medium due to internal scattering and absorbance (both of liquid medium and solid particles) (20,21), in addition to the shadowing effect of bacterial layers (18).Moreover, bacteria in a liquid medium could also adhere to the container's surface, with a different angle of incident light (nonperpendicular) that could influence the radiation's effect.Irradiation of surfaces or solid media is usually performed to assess the overall antimicrobial activity of radiation (16), but the distinction between bactericidal and bacteriostatic activity is not possible using this method.Only a few, elaborate methods have been proposed in the literature to measure bacteriostatic activity of radiation too, but they require culturing of exposed surfaces in liquid medium or exposure of liquid suspension in complex conditions (non-absorbing medium, stirring, etc.) (22,23).
The approach proposed here is based on the different growth rates of bacteria affected by bactericidal or bacteriostatic radiation on a solid medium.The method requires imaging of the UV-irradiated plates followed by image analysis.Imaging a plate only takes a few seconds.We performed it using the IVIS Spectrum instrument, but a light tight chamber with an inexpensive CMOS camera could also provide images with sufficient quality.Image analysis can be performed with any software written for this purpose and likely only requires a few minutes for expert users.Thus, this method allows us to assess the total antimicrobial activity easily and quickly and to distinguish between its bactericidal and bacteriostatic components.However, the method is limited to the described assumptions, meaning that bacteriostatic and bactericidal activity can be quantified only if there is visible bacterial growth on the control plates (C 1 ≠ 0, C 2 ≠ 0).For example, in the case of particularly slowgrowing bacteria, it is possible that no growth could be  observed at t 1 (C 1 = 0); however, in such case, quantification can easily be performed with the standard method (CFU count in control plates against zero CFU in treated plates) if there is growth at t 2 .The hypotheses (hyp1-hyp4) in Section 3.2 are based on experience and represent usually acceptable conditions when bacteria are exposed to an antimicrobial agent.Analogously, the existence of eqs.( 1) and ( 2) requires C 1 ≠ 0 and C 2 ≠ T 1 (obs4), both experimentally admissible conditions (i.e.growth in control plates and growth over time are expected for the validity of the experiment).
We performed an experimental test by irradiating E. coli cells plated on a solid medium with UVA light.After irradiation (t 1 ), bacterial colonies were visible only in control plates; after additional incubation without irradiation (t 2 ), bacterial colonies became visible in test plates too, indicating-only qualitatively-that the radiation elicited bacteriostatic activity (inhibiting bacterial growth but not killing the bacterial cells).At t 2 , treated plates presented a lower number of CFU in comparison with control plates, due to the bactericidal activity of the radiation.Applying the proposed eqs.( 1) and ( 2), we measured both activities and found BsA = 0.377 and BcA = 0.306.Notably, the results are not intended to be a percentage and the sum BsA + BcA is not necessarily equal to 1, being, in this case, BsA + BcA = 0.71 = F, as noted in obs2.With currently available tools, the evaluation of bacteriostatic activity could have only been qualitative.
We verified that our method could complement the standard procedure, while the latter alone only allows for assessing bactericidal activity.We could not perform validation with the few other methods proposed in the literature that could enable us to measure bacteriostatic activity due to their complexity.Nonetheless, using our method, we could assess both the bacteriostatic and bactericidal activity of UV radiation.The method we propose is an implementation of classical techniques that could be helpful to researchers studying the antimicrobial activity of radiation.

Figure 1 .
Figure 1.Control bacterial culture plate photographed in light-controlled conditions (IVIS Spectrum instrument tight light camera lightened with four light sources-quartz halogen lamps) at t 2 (left panel) and schematic representation of the dilutions (0.5 McFarland and 10-fold dilutions from −1 to −6) and ROIs (14 numbered circles on droplets; 2 background circles) (right panel).Counts scale is shown.Bkg = background.

Figure 2 .
Figure 2. Mean total counts (mean AE standard deviation of the 42 droplets, background-corrected) for the four experimental conditions: control plates at time point t 1 (C 1 ), control plates at time point t 2 (C 2 ), test (exposed) plates at t 1 (T 1 ) and test plates at t 2 (T 2 ).[Color figure can be viewed at wileyonlinelibrary.com]

Figure 3 .
Figure 3. Graphical visualization of C 1 , T 1 , C 2 and T 2 as defined in terms of time points (x-axis) and counts (y-axis).

Figure 5 .
Figure 5. BsA (red line) and BcA (black line) as a function of T1 (A) and T2 (B) separately.T1 and T2 (mean total counts) are visible on the x-axis, BsA and BcA values are visible on the y-axis.

Figure 6 .
Figure 6.BsA (A) and BcA (B) as function of both T1 (x-axis, ranging from 0 to 350) and T2 (y-axis, from 0 to 950).The black triangle is due to the hypothesis that T 2 must be higher than T 1 .The colors represent the values of BsA or BcA according to the scale bars.

Figure 7 .
Figure 7. BsA (A) and BcA (B) as a function of T 1 and T 2 are represented in a 3D space (T 1 on x-axis, T 2 on y-axis and BsA or BcA on z-axis).The colors represent the values of BsA or BcA according to the scale bar.

Table 1 .
Definition of the mean total counts of interest.