*3.1. E*ff*ect of Probiotic Biofilms on Pathogen Sessile Growth*

In order to evaluate the effect of probiotic biofilms on the development of pathogenic microorganisms, evidence was provided on the growth in sessile form of *L. monocytogenes, E. coli* O157:H7, *St. aureus* and *S. enterica*. Table 2 shows the cellular loads in sessile form relating to the targets studied; the data analysis shows how the pathogens studied were able to develop in all samples, even if they exhibited a wide range in their ability to colonize the surface, with the highest initial adhesion recovered for *S. enterica* (about 6 log CFU/cm2) against the lowest one (about 4 log CFU/cm2) recovered for *L. monocytogenes*. However, cellular loads were always lower in ACT samples (presence of probiotic biofilm, about 6.5–7 log CFU/cm2) compared to the CNT samples (absence of probiotic biofilm), highlighting that the studied biofilm was able to control the growth of all inoculated pathogenic targets. To quantify the effectiveness of probiotic biofilms in slowing down the pathogens' adhesion, for each time of analysis the difference between the cellular loads recovered in CNT and ACT samples was calculated. As it can be inferred from Table 2, for *E. coli* O157:H7, there was a significant decrease in cell load compared to control of more than 1 and 2 logarithmic cycles after 4 and 48 h of incubation, respectively, and the biofilm efficacy increased over time. Similar results were observed for *St. aureus*. On the contrary, for *L. monocytogenes* the effectiveness of probiotic biofilm was maximum after 4 h (1.43 ± 0.28), but it decreased over time; this loss of efficacy was also recorded for *S. enterica*, with cell load reductions ranging from 1 to 0.2 logarithmic cycles after 24 and 48 h, respectively. As expected, biofilms were odorless and invisible to the naked eye. The idea to use probiotics into the prevention of infections and other diseases has already been proposed [7], and is also stimulated by the need of new alternative intervention strategies to combat bacteria pathogenesis due to the increasing evidence of antibiotics resistance of many pathogens. Abdelhamid et al. [33] observed that cell-free preparations of different probiotics belonging to *Lactobacillus* and *Bifidobacterium* species were able to reduce the growth of *E. coli*, whereas Kaboosi [34] showed that probiotics from yogurts had antibacterial effects against Gram negative bacteria such as *E. coli*, *Salmonella* Typhi and *Ps. aeruginosa*, and Gram positive bacteria such as *S. aureus*. Similarly, Tejero-Sariñena et al. [35] found that 15 strains of probiotics had antibacterial properties against gram negative *Salmonella* Typhimurium and *Clostridium di*ffi*cile*. However, most of these studies propose the use of compounds (mainly bio-surfactants) with antimicrobial activity produced by probiotics, and contained in their cell-free supernatants [20–27]; on the contrary, this study proposes the use of a probiotic biofilm that exploits

the in vivo metabolism of two bacterial strains (*Lactobacillus* and *Bifidobacterium*) adhering on abiotic surfaces and not the substances secreted by probiotics and subsequently recovered and used. In 2014 Schobitz et al. [36] proposed a biocontroller consisting of the thermally treated fermentate (TTF) from two *Carnobacterium maltaromaticum* strains (ATCC PTA 9380 and ATCC PTA 9381), a strain of *Enterococcus mundtii* (ATCC PTA 9382), plus nisin at a concentration of 1000 IU/mL, with all these components entrapped in an alginate matrix supported by a mesh-type fabric. The strains used in our study are different, and no bacteriocin or polymer is used, but the proposed probiotic biofilm should be formed on different surfaces chosen according the purpose (an active packaging and/or a medical device). Moreover, our solution, thanks to the maintenance of a continuous metabolism, should ensure an uninterrupted and stronger activity of the active substances (mainly bacteriocins and/or other LAB-produced antimicrobial compounds such as hydrogen peroxide, carbon dioxide, diacetyl, organic acids), being the same in loco produced [37]. Similar to our study, Gomez et al. [28] used in situ biofilms formed by potential probiotic LAB strains isolated from Brazilian s foods (*Lactococcus lactis* VB69, *L. lactis* VB94, *Lactobacillus sakei* MBSa1, *Lactobacillus curvatus* MBSa3, *L. lactis* 368, *Lactobacillus helveticus* 354, *Lactobacillus casei* 40, and *Weissela viridescens* 113) to inhibit pathogenic growth: they found the total inhibition in pathogens *E. coli* O157:H7, *L. monocytogenes* and *Salmonella* Typhimurium biofilm formation, in 24, 48, and 72 h of exposure using *L. lactis* 368, *Lactobacillus curvatus* MBSa3 and *Lactobacillus sakei* MBSa1. For the other strains, the inhibition was time-dependent and varied according to the strain and target pathogen; for *L. monocytogenes,* reductions ranged from 4- to 7-log units over 24 and 48 h, and the inhibition was observed only within the first 24–48 h, after which the pathogen was able to grow. In *Salmonella* Typhimurium and *E. coli* O157:H7 experiments, sessile cells were not detected during 24 h of incubation in the presence of most LAB tested; during 48 and 72 h, reductions between 5 and 3 log for *E. coli* O157:H7 and 4 log for *Salmonella* Typhimurium were achieved.


**Table 2.** Cellular loads (Log CFU/cm2) recovered for *Listeria monocytogenes*, *Escherichia coli* O157:H7, *Staphylococcus aureus* and *Salmonella enterica* during their sessile growth with (ACTIVE, ACT) or without (CONTROL, CNT) probiotic biofilms.


**Table 2.** *Cont.*

\* A, B, Values in the same lines with different letters are significantly different (Student s t-test) (*p* < 0.05). \*\* Biofilm Efficacy = CNT–ACT. \*\*\* a, b, c, d, Values in the same columns with different letters are significantly different (one-way ANOVA and Tukey s test) (*p* < 0.05).
