2.1.

Ultraviolet Treatments

The UV LEDs that were used to deliver the irradiation to the biofilms were purchased from Sensor Electronic Technology, Inc. (SETi, Columbia, South Carolina; TO3 package, hemispherical lens window). The diodes were operated in constant current mode. The spectral power distribution of the LEDs is shown in Fig. 1. The setup for performing the irradiations is shown in Fig. 2. The irradiation of the UV-LEDs was measured by an external optical probe (EOP-146, Instrument Systems GmbH, Munich, Germany) and a monochromator. The spectrometer coupled to the monochromator was a SPECTRO 320 (D) Release 5 (Instrument Systems GmbH). Details about the irradiation measurements protocol can be found in Barnkob et al.¹⁸ The irradiance delivered on the biofilms was also cross confirmed with a portable radiometer (NIST-certified UV radiometer); the distances the irradiance measurements were taken ($1.5\pm 0.1\mathrm{cm}$) were the same distances that the exposures were conducted.

Fig. 1

Spectral irradiance of the UVB (black curve) and UVC LEDs (red curve) used for the irradiation of the biofilms.

Fig. 2

Schematic sketch of the UV irradiation setup. The agar plate carrying the biofilm is illuminated by the UV LED in a UV-opaque box.

In all exposures, the distance between the LED and the biofilm (circular entity with diameter $1.0\pm 0.2\mathrm{cm}$) was kept constant at $1.5\pm 0.2\mathrm{cm}$ (the error originates from the agar height in the Petri dish). All biofilms, before and after treatments, were stored in a UV-free environment. As a standard, all treatments were performed on three different biological replicates to test the reproducibility.

2.2.

Biofilm Preparation

A cellulose nitrate membrane filter (pore size: $0.2\text{-}\mu \mathrm{m}$ diameter: 25 mm, Whatman GmbH, Germany) was placed directly on the agar plate. The plate and filter were dried for 30 min at 37°C before the bacterial overnight (ON) culture was added in a single spot ($20\text{-}\mu \mathrm{L}$ bacterial suspension) on the membrane filter. The bacteria were spot inoculated onto the filter directly from the ON culture. The P. aeruginosa used for the experiments was obtained from the Pseudomonas Genetic Stock Center (strain PAO1¹⁹). The biofilms were grown on AB-trace glucose (0.5%) (ABTG) plates and incubated for 24, 48, or 72 h at 37°C. A more detailed description for the ON cultures and filter biofilms methodology (“the micropore assay”) can be found in Bjarnsholt et al.²⁰ For a mature biofilm (either grown for 48 or 72 h) to develop, the membrane filter containing the growing biofilm was transferred to a fresh, dried ABTG plate every 24 h. In this way, the fresh media available to the biofilm facilitated growth.

2.3.

Colony-Forming Unit Determination

Filters with the biofilms growing on top were transferred with sterile forceps to a 15 mL Falcon tube containing 5 mL of saline (0.9% NaCl). Samples were mixed for 15 s, degassed for 5 min, and sonicated for 5 min in an ultrasonication bath to release the bacteria from the filter. Sonication fluid was serial diluted from ${10}^{-1}$ to ${10}^{-8}$ and $20\mu \mathrm{L}$ of all dilutions were spotted once on LB agar plates followed by ON incubation at 37°C. The colony-forming units (CFUs) were counted after 24 h of incubation in the dark at 37°C. Note that CFUs are per initial volume and were calculated with the weighted average method.²¹ An example of how the CFUs looked after counting is shown in Fig. 3. Control samples, i.e., biofilms that were not exposed to UV irradiation, were plated every hour and included in the study as a reference for growth.

Fig. 3

Typical picture of a labeled agar plate after regrowth (control sample). At higher dilutions, countable and well-separated CFUs are formed, as indicated by the black circle. The yellowish spots are the bacterial colonies, the back spots are the marks made by the technician (at the other side of the plate) while counting. The next aggregation of colonies (move counterclockwise) is from one order lower dilution; and the colonies (yellowish spots) are occasionally overlapping.

2.4.

Experimental Design

The objective was to determine how the selected UV region, namely UVB versus UVC, and radiant exposure will influence the viability of P. aeruginosa biofilms. The radiant exposure for less mature biofilms was investigated in the range (approximately) from 70 to $\mathrm{10,000}\mathrm{J}/{\mathrm{m}}^2$. The irradiance was varied between $18.6\pm 0.2\mathrm{W}/{\mathrm{m}}^2$ and $108\pm 11\mathrm{W}/{\mathrm{m}}^2$ with the UVC diode. For the UVB diode, the irradiance was varied between $1\pm 0.1\mathrm{W}/{\mathrm{m}}^2$ and $14.8\pm 0.2\mathrm{W}/{\mathrm{m}}^2$. In all treatments, the exposure time was $<12\min$.

Mature biofilms (48 and 72 h grown) were treated with radiant exposure around $\mathrm{20,000}\mathrm{J}/{\mathrm{m}}^2$, and effectiveness of the treatments (UVB and UVC) was compared to control (no treatment) and less mature biofilms (24 h grown). For testing the effect of maturity on the effectiveness of the treatments, the exposures were performed on three independent biological replicates and executed twice (two technical replicates). Pairwise $t$-tests were used to detect inactivation efficiency differences among treatments ($\mathrm{20,000}\mathrm{J}/{\mathrm{m}}^2$) for the different growth stages.

2.5.

Modeling of Biofilm Survival

GInaFiT¹⁷ a freeware add-in for Microsoft^® Excel, was used to model the biofilm survival curves. Modeling of bacterial survival can provide some insight into the mechanisms behind inactivation and provide guidelines for prediction of required doses to succeed a specific level of inactivation. Microbial inactivation has traditionally been described by first-order kinetics. The GInaFiT tool supports testing of nine types of microbial survival models, and five statistical measures (i.e., sum of squared errors, mean sum of squared errors and its root, $R^2$, and adjusted $R^2$) are provided to monitor the best fit ($f$ function). Here, a choice of five suitable models^22–26 was applied to the mean values (from three technical replicates) of log survival obtained from one biological replicate (replicate 3)

3.1.

Ultraviolet B Inactivation Rate (Less Mature Biofilms)

The log survival values of P. aeruginosa biofilms, after being exposed to UVB irradiation with a central wavelength at 296 nm, as a function of UV-radiant exposure (dose) are presented in Fig. 4. Most models demonstrated a good fit to the experimental data ($R^2\ge 0.9$), except the linear model.²² The “biphasic” model²⁶ fit best to the data (Fig. 4). However, this model requests minimal 10 points to ensure validity. The statistical measures of the models applied are presented in Table 1. If we use the concept of reliability engineering, the reliable dose $d_R$²⁷ (dose needed to reduce number of microorganisms by a factor of 10) is calculated to be $638\mathrm{J}/{\mathrm{m}}^2$ (from the “Weibull model,” the hazard rate was found to be $\alpha =54.86$ and the shape parameter $\beta =0.34$).

Fig. 4

UVB-radiant exposure-dependent antibacterial action on P. aeruginosa biofilms. “Biphasic model” fit to the experimental data. This model requires a minimal 10 points to ensure validity. The error bars for biological replicate 3 represent propagation of uncertainty for the function of log survival, originating from three technical replicates.

Table 1

Statistical measures of the models applied to the experimental data for log reduction of P. aeruginosa by UVB (and UVC). In cases where modeling was not applicable, the indication NA is registered.

The GInaFiT tool predicts that the required dose for achieving a 4-log reduction in the CFUs is $1900\mathrm{J}/{\mathrm{m}}^2$, in accordance with the experimentally observed value (at $2000\mathrm{J}/{\mathrm{m}}^2$, 4.1-log reduction). No CFU was observed when a dose of $\mathrm{10,000}\mathrm{J}/{\mathrm{m}}^2$ was delivered to the biofilm (independently of biological replicate), indicating total inactivation; potential presence of bacteria could not be detected since the treatment challenged the detection limit of the method used (drop plate technique²⁸), nor were CFUs observed for biological replicate two, already at a radiant exposure of $7200\mathrm{J}/{\mathrm{m}}^2$.

3.2.

Ultraviolet C Inactivation Rate (Less Mature Biofilms)

The antibacterial action of UVC irradiation, with central wavelength at 266 nm is shown in Fig. 5 with a “Weibull model fit.²⁴” With $\alpha =9870$ and $\beta =0.44$, the reliable dose $d_R$²⁷ was calculated to be $\mathrm{65,722}\mathrm{J}/{\mathrm{m}}^2$. The statistical measures of the models applied are shown in Table 1. The GInaFiT tool in this case was unable to predict the required dose for achieving a 4-log reduction (maximum log reduction achieved experimentally was 1).

Fig. 5

UVC-radiant exposure-dependent antibacterial action on P. aeruginosa biofilms (experimental data and Weibull fit). The error bars for biological replicate 3 represent propagation of uncertainty for the function of log survival, originating from three technical replicates.

3.3.

Mature Biofilms

The effect of the growth stage of the biofilm to the observed efficacy of the applied treatments is summarized in Fig. 6. No inactivation effect was observed with UVC (radiant exposure around $\mathrm{20,000}\mathrm{J}/{\mathrm{m}}^2$) on 48 h mature biofilms; CFU counts were similar to the nontreated samples. Therefore, the treatment was not attempted on biofilms grown for 72 h. Only a modest inactivation was achieved on the less mature biofilms around 1-log reduction ($0.8\pm 0.3\log$ reductions). To the contrary, UVB (radiant exposure around $\mathrm{20,000}\mathrm{J}/{\mathrm{m}}^2$) was able to inactivate effectively mature biofilms grown for 48 h ($3.2\pm 1.3\log$ reduction) and for 72 h ($2.9\pm 0.7\log$ reduction). However, the efficiency of inactivation decreased with maturity from zero counts for less mature biofilms to around 3-log reduction for mature biofilms. It is interesting to observe that the log reduction achieved by the UVB treatment varies when the biofilms are mature but are “straightforward” for the less mature biofilms. According to the pairwise $t$-tests, there was a significant increase in counts for the control from less mature to mature biofilms ($p=0.003$). UVB treatment was significantly more effective than UVC, both for biofilms grown for 24 and 48 h ($p\le 0.004$). The UVB treatment was equally efficient for killing 48 and 72 h mature biofilms ($p=0.6$).

Fig. 6

CFU regrowth after different treatments are applied (UVB 20,000, UVC $20,000\mathrm{J}/{\mathrm{m}}^2$, and control) on biofilms at different maturity stages (24, 48, and 72 h grown). The error bars represent the standard deviation. No counts were observed for all replicates after the UVB treatment was applied on 24 h grown biofilms. The UVC treatment was not performed on the 72 h grown biofilms since only modest inactivation was observed for biofilms grown for 48 h (missing green column for the UVC treatment). CFUs are per initial volume.

3.4.

Transmission Properties of Biofilms in the Ultraviolet Region

Transmission properties of biofilms in the UV region were measured with a Cary 50 spectrophotometer. The membrane filters on which the original biofilms were grown were opaque in the UV region. Therefore, biofilms were grown on (UV transparent) quartz surfaces (three samples). To grow these biofilms, quartz surfaces were embedded in bacterial culture for 28 h. It cannot be expected that a biofilm grown on quartz will attain the same structure, thickness, and properties as a biofilm grown on a membrane filter. Moreover, the biofilm thickness is not expected to be the same over the whole quartz surface, confirmed by observation of the samples by bare eye. The thickness variation of the biofilm grown at different locations on one quartz plate (“biofilm spot”) is attributed to the biological aspects of biofilm formation. Therefore, large variation for the absolute values of the transmission was expected; the location on the biofilm where the transmission measurement was taking place was not controllable.

The transmission properties of biofilms grown on quartz (Fig. 7) and, more specifically, the ratio of transmission properties at different wavelengths could assist in obtaining a better understanding of the UV wavelength-dependent penetration ability into a thin layer of bacteria existing in a “biofilm state.”

It was observed that on average (three different spots, each on a different biofilm quartz sample, were measured) 10% less UV light was transmitted at a wavelength of 266 nm than at 296 nm, the transmission curve slope was found to be $0.33\pm 0.07$. In the case of biofilm location 2 (obviously the thickest among the “biofilm spots” measured), only 37% was transmitted at a wavelength of 266 nm (Fig. 7).

Therefore, it is believed that, in the case of the biofilms grown on filters, which were much thicker, the UVC irradiation did not penetrate through the whole biofilm volume.

Fig. 7

Transmission spectra of biofilms grown on quartz under the exact same conditions. It is observed that on average 10% more UV light is transmitted at a wavelength of 296 nm than at 266 nm. Only 37% is transmitted at 266 nm for biofilm location 2.