Preparation of film specimens
The test film was composed by a smooth three-layer film made of two 25 µm external PP layers (Sabic grade, PP-2 Sabic® PP 520L), and a 50 µm central layer of an olefin based thermoplastic elastomer (Zelas™ 7025) produced at Propagroup (Torino, Italy). The inner layer was ready to function as a reservoir for the progressive release of encapsulated essential oils or other compounds with antimicrobial activity. In this work, only structured films without antimicrobial compounds were tested to exclusively check the antimicrobial activity of patterned surfaces.
A topography containing ordered micro- and nano-patterns was imprinted on the surface of the PP film by means of nanoimprint lithography. A nickel stamp was used as a stamp, which was copied from an original silicon master 14. Non-structured smooth films were used in the different tests as control.
Bacterial strains and growth conditions
The bacteria used in this study were the Gram-negative Escherichia coli (ATCC 25922) and the Gram-positive Staphylococcus aureus (ATCC 25923). Bacteria from the frozen stock culture were transferred to Trypcase Soy Agar (TSA) supplemented with 5% sheep blood plates (TSA, bioMèrieux, France) and incubated at 37 °C for 18–24 h. Bacteria were transferred to 50 mL of sterile Tryptic Soy Broth (TSB) medium and grown at 37 °C, 80 rpm, 18–24 h. Prior to inoculation, strains were subcultured into fresh TSB at 1:50 and incubated for 2 h at 37 °C, 80 rpm. Bacteria were collected by centrifugation (2000 rpm, 10 min) and resuspended in phosphate buffered saline (PBS). The number of bacteria was spectrophotometrically adjusted to O.D.600nm = 0.5 (equivalent to 5 × 108 CFUs/mL).
The patterned films evaluated in this study were highly hydrophobic so it was difficult to homogeneously spread the aqueous inoculum over the surface (Figs. 1a and 1b). In order to resolve this problem, the bacterial inocula were applied using glycerol to reduce the surface tension of the suspension. Three different final concentrations of glycerol were tested (5%, 10% and 40%). For all experiments it was decided to use the lowest concentration of glycerol (5%) as the distribution of the inoculum was as homogenous as with higher concentrations of glycerol (data not shown).
Antibacterial activity tests
a) ISO 22196 standard method
The ISO 22196 was performed with slight modifications 11. Pieces of 50 mm x 50 mm of the film and of non-structured smooth control films were placed in sterile 90 mm Petri dishes. Serial dilutions of the bacteria inoculum were made to reach a final bacterial concentration of 6 × 105 CFUs/mL of which 400 µL were pipetted onto the test surfaces. Bacterial inoculum was then covered with 40 mm x 40 mm piece of Stomacher® bag in order to homogeneously spread the inoculum over the surface. The specimens were incubated for 24 h at 35 °C and a relative humidity > 90%. To recover the bacteria from the surfaces of the plastic samples, 10 mL of Soybean Casein Lecithin Polysorbate (SCDLP) broth was added to the Petri dish. The SCDLP was collected and released at least four times to ensure that the specimens were completely washed. Then, 10-fold serial dilutions of the SCDLP were made in PBS and 50 µL of 10− 4, 10− 5, 10− 6 and 10− 7 dilutions were cultured on TSA with 5% sheep blood plates. Plates were incubated at 35 °C for 24 h. The number of colonies per plate was recorded and used to determine the number of viable bacteria per cm2 in accordance with the equation N = (100 x C x D x V)/A, where C is the average of CFU count, D is the dilution factor, V is the volume in mL of SCDLP and A is the surface (cm2) of the film.
b) Immersion inoculation assay
Immersion inoculation assay 7 evaluated the attachment and survival of the bacteria directly from the surface of the film. Serial dilutions of the bacteria inoculum were made in PBS to reach a bacterial concentration of 6 × 104 CFUs/mL. Film samples (50 mm x 50 mm) were placed in a 90 mm sterile Petri dish and were completely covered with 20 mL of bacterial inoculum for 1 h at room temperature without shaking. The films were then washed 3 times with 20 mL of PBS for 10 s while rotating at 80 rpm and allowed to dry under ambient conditions for 1 h before being sampled.
c) Touch-transfer inoculation assay
The Touch-transfer inoculation assay was described by Mann et al.7 and assessed the attachment (transference) and survival of the bacteria (persistence) directly on the surface of the film. In this study, the protocol described by Mann et al.7 was used with minor changes. Briefly, serial dilutions of the bacteria inoculum were made in PBS to reach a bacterial concentration of 6 × 104 CFUs/mL. This inoculum was experimentally established on 50 mm x 50 mm smooth control films, because it provided an adequate number of CFUs (around 50 well defined CFUs per plate using the agar contact method described in the Sampling and colony counting section) to have statistical difference between surfaces with or without antimicrobial activity. As described above, one of the modifications made was the addition of glycerol 5% to the bacterial suspension to reduce the surface tension of the PBS on the film, which allows a more homogeneous distribution of the bacterial suspension on the surface. A sterile velveteen cloth was placed on a replica plating tool (Bel-Art Products, Wayne, NJ) and flooded into 10 mL of the bacterial suspension by direct contact during 1 min. The excess of bacterial suspension was eliminated by placing the inoculated cloth for 10 s onto another dry sterile velveteen cloth. To inoculate the 50 mm x 50 mm film specimens, the inoculated velveteen cloth was pressed for 10 s contact time against the surface of the films. The surfaces were allowed to dry under ambient conditions (± 24 °C and humidity of ± 30%) for 0 min (transference) or 90 min (persistence). Films were sampled and colonies counted as described in the Sampling and colony counting section.
d) Swab inoculation assay
The Swab inoculation assay or Swab assay was designed for this study based in the Touch-transfer inoculation assay in order to obtain more reproducible results. This assay also assessed the attachment and survival of the bacteria on the surface of the film. Serial dilutions of the bacteria inoculum were made in PBS supplemented with glycerol 5% to reach a bacterial concentration of 6 × 104 CFUs/mL. Fifty microliters of the bacterial suspension were pipetted on the films (50 mm x 50 mm) and spread homogenously by rolling a cotton swab over their surface. Films were allowed to dry under ambient conditions (± 24 °C and humidity of ± 35%) for 0 min (transference) or 90 min (persistence) before being sampled.
Sampling and colony counting
To quantify the bacteria on the film surface, the agar contact method was used 14 to directly transfer the bacteria of the surface of the films to the culture agar plates (Count-Tact® plates, bioMèrieux, France). Contact plates were pressed for 10 s onto the surfaces of the films with the help of a Count-Tact® applicator (bioMèrieux, France) that applies a uniform pressure of 0.5 kg on the surface of the contact agar plate. After inoculation, plates were incubated for 18–24 h at 35 °C. Colony counting was performed manually.
Data reporting and statistical analysis
For each assay, 3 samples of each plastic film with micro- and nano-structures and 3 smooth control samples were analysed. Mean colony counts and standard deviation (SD) were calculated for each film and controls. Results were expressed as logarithm of CFUs/cm2. A single student’s t-test was used to compare the bacterial counts between the patterned and the smooth surfaces. A p-value lower than 0.05 was considered statistically significant. Statistical analyses were performed using Prism GraphPad version 8 software (GraphPad Software, Inc., La Jolla, CA, USA).