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

#### 2.1.1. Surfaces and Microorganisms

Polycarbonate resin (Lexan, Fedele s.r.l., Rome, Italy) was the surface chosen for the adhesion experiments. Before each experiment, the chips (2.5 × 5.0 × 0.05 cm) were prepared by washing in acetone for a minimum of 30 min, rinsing in distilled water, and then soaking in 1 N NaOH for 1 h. After a final rinse in distilled water, the chips were allowed to air dry. This cleansing procedure was required to remove fingerprints, oils grease, and other soils that may have been on the materials. The cleaned chips were finally autoclaved at 121 ◦C for 15 min prior to use.

The probiotic strains used for this study were *Bifidobacterium longum* subsp. *infantis* DSM20088 and *Lactobacillus reuteri* DSM20016, both purchased from Leibniz-Institut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen) and stored at −20 ◦C in MRS broth (Oxoid, Milan, Italy).

Before each assay, they were grown in their optimal media at their optimal conditions, until late exponential phase was attained; namely, MRS broth added with cysteine 0.05% (w/v) (Sigma-Aldrich, Milan, Italy) incubated at 37 ◦C for 24 to 48 h, under anaerobic conditions, and MRS broth (Oxoid) incubated at 30 ◦C for 24 to 48 h, under anaerobic conditions, were used for *B. infantis* DSM20088 and *L. reuteri* DSM20016, respectively.

Cells cultures were successively harvested by centrifugation for 10 min at 4500 rpm (4 ◦C) and the pellets were washed twice with sterile saline solution (0.9% NaCl) at 4 ◦C and finally resuspended in the same solution at a cell concentration of 1 <sup>×</sup> 108 CFU/mL.

As pathogen targets were chosen, four strains belonging to the Culture Collection of the Laboratory of Predictive Microbiology (Department of the Science of Agriculture, Food and Environment, Foggia University), and microorganisms with the media and growth conditions used, have been listed in Table 1. The organisms were transferred to fresh Nutrient Agar (NA, Oxoid) periodically to maintain viability and, prior to use, they were activated by two successive 24-h transfers of cells in Nutrient broth (NB, Oxoid) at 37 ◦C. Inocula for experiments were prepared by centrifugation of the 24-h microbial cultures at 3000× *g* for 15 min at 4 ◦C. After centrifugation, the obtained pellets were resuspended in sterile saline solution at 4 ◦C to obtain approximately 108 CFU/mL for each microorganism.


**Table 1.** Pathogen strains used in the study with the indication of their source and optimal media and growth conditions adopted.

\* The strain was isolated from fish products and identified by sequencing the 16SrDNA.

#### 2.1.2. Experiment

Biofilm formation was favoured by simultaneously inoculating the cocktail of identified probiotics (*B. infantis* DSM20088 and *L. reuteri* DSM20016, about ~10<sup>8</sup> CFU/mL) and the pathogenic target (~10<sup>7</sup> CFU/mL) on polycarbonate surfaces (Lexan® tiles, 25 mm <sup>×</sup> 75 mm, 0.5 mm thick) left at room temperature (20 ◦C) for 2 h. After this time interval, the tiles were transferred to aliquots of peptone water (1% bacteriological peptone) and incubated at 15 ◦C for 48 h [18,19]. Specifically, for each pathogen, two samples were prepared: an ACTIVE sample (ACT), containing a chip where probiotics were left to form biofilm; a CONTROL sample (CNT), containing a chip without probiotics. The pathogen sessile cell load was determined after 0, 4, 24, 30 and 48 h after inoculation. At these times, chips were aseptically removed and rinsed with sterile distilled water, in order to eliminate the unattached cells. As suggested in literature [29], sessile cells were detached from chips in a sterile test tube containing 45 mL of sterile saline with a 20 Hz "Vibra Cell" sonicator (SONICS, Newcastle, Conn., USA) for 3 min. Viable and cultivable cells were enumerated by serial dilutions in 0.9% NaCl solution and plating on appropriate media (Table 1).
