# Effect of Ozonized Water against Pathogenic Bacteria and Filamentous Fungi on Stainless Steel
## 3 Results and Discussion
### Bacteria  
For the tested strains, a marked tailing was observed at all the tested ozone concentrations. This phenomenon was already observed by the same authors when sanitizing agents such as UV-C radiation [46] or the ozone itself [35] were used. Similarly, it has been registered by other authors and related to heterogeneous treatments, to the aggregation of microorganisms, to some intrinsic mechanism of bacterial physiology leading to resistant sub-populations [47; 48; 49]. Together with the tailing, a huge variability in the concentrations of the surviving microorganisms at each of the considered combinations was highlighted, albeit the high number of repetitions for each tested combination. This variability had not been registered in tests carried out on bacterial and fungal suspensions (unpublished data), so it was hypothesized that the multi-layering of the inoculum could have given protection to the lower layers of cells against the oxidative processes exerted by ozone, even considering that variations within the same train of tests have been already registered by other authors who tested aqueous ozone [6; 25].  
For all the considered strains, a progressive reduction in the surviving cells was observed, and the reduction proved more marked as the ozone concentration increased. When ozone concentrations increased from 1.0 to 6.0 mg L\({}^{-1}\), the LCR moved from 2.4 to more than 3.9 for the tested _S. genthemberg_ and from 1.1 to 2.0 for the tested _S. typhimurium_, the former showing a higher sensibility to aqueous ozone compared to the latter. Analogously, considering the same increase in ozone concentration, the LCR moved from 1.3 to 5.2 for the tested Listeria monocytogenes Scott A and from 2.5 to 5.3 for the tested _Listeria monocytogenes ATCC_ 7644, the latter strain proving more sensible to the ozone applied in its liquid form.  
A huge variability was observed considering the 1-D values of the different strains or species tested. At the lowest ozone concentration, the resistance of _L. monocytogenes Scott A_ and _S. typhimurium_ proved sensibly higher (from 6.5 to 9 times) than those registered for the other two tested strains, whereas at higher concentrations the calculated resistance values prove comparable.  
The comparison with previous works on the same topic is not easy since literature data focused on biofilms rather than free cells inoculated on specific surfaces. The only paper concerning inoculated cells was that by [PERSON] et al. [25], where cells of _L. monocytogenes_ attached to SS were subjected to 2 to 3 LCR, yet in the first 2 min of treatment, 1 ppm of aqueous ozone in static conditions was applied. Differently, on biofilms, more papers are available. With regard to _L. monocytogenes_, ozonized water proved active at a concentration of 4 ppm for 3 min on cells attached to SS, whereas a four-fold increase in sanitizer was needed to destroy all biofilm cells [50]. On polystyrene, a reduction of about 0.9, 3.4, and 4.1 Log was reached when ozonized water was applied at 1.0, 2.0, 1 md, and 4.0 ppm, respectively [30]. With regard to _Salmonella_ spp., the effectiveness of ozonized water seemed less marked, a reduction less than 0.8 CFU/cm\({}^{2}\) being obtained on cells adhered to SS after 20 min of exposure [26].