Antibacterial photokilling: how sample absorption can alter inter-experiment comparison?

In antibacterial photokilling experiments, the efficacy is generally represented by the correlation between the decrease in the (alive) bacterial number (ΔN) and the light dose (D). In the experimental practise where a λexc -peaked radiation is used (photosensitizer main absorption peak), ΔN is counted starting from the whole irradiated sample, regardless of its optical density OD at λ = λexc. This can be seen as a definition of the “cause” (the impinging energy per unit surface, D) and “the effect” (ΔN), but could not be the best choice to compare experiments where the main difference is represented by the sample OD at λexc. In fact, the decrease in light power going deep into the sample is associated to a decrease in what we could define as a local dose (energy per unit time and surface at a given depth), associated in turn to a local decrease in the photokilling efficacy. Let us imagine two irradiated biofilms differing only for their OD(λexc). Overall, for the same dose D, we will count a smaller killing percentage for the optically thicker sample, having the impression of a less effective response to light. Again, we could argue we are facing a definition problem: does the “photokilling efficacy” include the optical absorption of that specific biofilm or is it generally referred to that bacterial strain in the biofilm form? No issue would arise with two optically thin samples, where all bacteria “receive the same dose” (fluent energy per unit surface), which corresponded to have both optically thin biofilms at λ = λexc. To better inquire this point, a plane biofilm irradiation model was defined and studied theoretically [1], considering: (i) a given biofilm absorption coefficient and constant bacteria density; the biofilm was represented by a layered model, each layer being optically thin (ODlayer (λEXC) << 1); (ii) a monochromatic radiation impinging vertically at λ = λexc corresponding to the photosensitizer main excitation peak; a local dose was defined as the time integral of the light power per unit surface at a given depth; (iii) an exponentially decreasing light power inside the biofilm. The final aim was to undertand the relationship between ΔN and D and its possible dependance on ODBF (λexc) in the 3 cases: ODBF < 1, ODBF ∼ 1 and ODBF > 1. This was obtained by modulating the local photokilling rate layer by layer, starting from an empirical relationship ΔN(D) for the single layer, based on literature results. The preliminary results indicate non-negligible variations in ΔN(D), dependent on the OD difference between the compared cases. This could lead to a better comparison between experiments where the main difference lies in the biofilm optical thickness.