# Prevalence and Characterization of Staphylococcus aureus Isolated from Meat and Milk in Northeastern Italy

Debora Pinamonti, Marisa Manzano, Michela Maifreni, Silvia Bianco, Beki

Domi, Alessia Ferrin, Jamila Anba-Mondoloni, Julien Dechamps, Roman

Briandet, Jasmina Vidic

► To cite this version:

Debora Pinamonti, Marisa Manzano, Michela Maifreni, Silvia Bianco, Beki Domi, et al. Prevalence and Characterization of Staphylococcus aureus Isolated from Meat and Milk in Northeastern Italy. Journal of Food Protection, 2025, 88 (2), pp.100442. 10.1016/j.jfp.2024.100442. hal-05191232

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Research Paper

# Prevalence and Characterization of *Staphylococcus aureus* Isolated from Meat and Milk in Northeastern Italy

<figure><img src="image_2.png" /></figure>

Debora Pinamonti<fnref n="1" />, Marisa Manzano<fnref n="1" />,*, Michela Maifreni<fnref n="1" />, Silvia Bianco<fnref n="1" />, Beki Domi<fnref n="1" />, Alessia Ferrin<fnref n="1" />, Jamila Anba-Mondoloni<fnref n="2" />, Julien Dechamps<fnref n="2" />, Roman Briandet<fnref n="2" />, Jasmina Vidic<fnref n="2" />,*

## ARTICLE INFO

### Keywords:

- Antimicrobial resistance

- Bacterial cell surface

- Biofilm production capability

- Caco-2 cell line

- Food

- *Staphylococcus aureus*

## ABSTRACT

*Staphylococcus aureus* is a pathogenic microorganism often found in animal-derived foods and is known for its ability to readily develop resistance to antibiotic treatments. This study was designed to determine the prevalence of *S. aureus* strains in raw milk and meat in Italy and to evaluate their antibiotic resistance profiles and biofilm production. Among the meat isolates, 41.67% were resistant to ampicillin, and 25% were methicillin-resistant *S. aureus* (MRSA). In milk, 20% of the isolates were resistant to gentamycin, while 5.71% were MRSA. The prevalence of multidrug-resistant strains was higher in meat (16.67%) compared to milk (5.71%). The biofilm formation capability was assessed in most of the isolates (80% in milk and 100% in meat). Representative strains exhibiting different antibiotic resistance profiles were all negative for the enterotoxin genes *sea*, *seb*, *sec*, *sed*, and *see*, but harbored potential virulence factors such as hemolytic activity, high pigmentation, low cell envelop permeability, charged and hydrophobicity. Finally, the interaction of representative strains with human Caco-2 intestinal cell line showed that most strains had an adhesion capacity. Our findings reveal that foodborne isolates of *S. aureus* present a considerable threat to consumers due to their production of virulence factors, which enhance their pathogenicity and increase the likelihood of antibiotic treatment failures.

*Staphylococcus aureus* is a Gram-positive, facultative anaerobic round-shaped bacterium typically arranged in grape-like clusters. It is a commensal organism, commonly colonizing the human skin and nasopharyngeal tract. *S. aureus* can lead to various human diseases ranging from localized infections to severe conditions like sepsis and pneumonia (Tuon et al., 2023). In 2019, *S. aureus* infections were responsible for 1 million deaths worldwide (GBD Antimicrobial Resistance Collaborators, 2022) In Europe, EFSA and ECDC reported that *S. aureus* caused 178,000 zoonoses (EFSA, ECDC, 2021). Due to its adaptability to different environments, *S. aureus* has evolved resistance to antistaphylococcal drugs used in both human and animal treatment, such as ampicillin, erythromycin, tetracycline, and cefoxitin (ECDC et al., 2021; Liang et al., 2023). Currently, methicillin-resistant *S. aureus* (MRSA) strains showing resistance to the β-lactam antibiotic group represent an emergent issue. Among European countries, Italy is of particular interest because of an important prevalence of *S. aureus* in protein-rich foods, such as meat, milk, and dairy products (Johler et al., 2018; Rodríguez-López et al., 2020). Furthermore, a recent meta-analysis indicated that the majority of antibiotic-resistant

*S. aureus* was isolated from meat in Italy (Gunjan et al., 2023). Considering that monitoring and characterizing *S. aureus* in food are crucial for understanding antibiotic resistance spreading in nonhospital settings, it is important to examine the relation between the phenotypes of *S. aureus* food isolates and their susceptibility to antibiotics in clinical use (Filipello et al., 2019; Johler et al., 2018; Léguillier et al., 2024).

The cell envelope, comprising cell membrane and a thick cell wall in *S. aureus*, is a barrier that protects bacterial cells from threatening environments and enables their survival, adherence, biofilm formation, pathogenesis and contributes to antibiotic resistance (Filipello et al., 2019; Khelissa et al., 2017; Krasowska and Sigler, 2014; Nikolic and Mudgil, 2023). The cell wall is the outermost layer and, thus, the most accessible target to antimicrobials and disinfectants used by the food industry (Bridier et al., 2015; Charron et al., 2023; Yuan et al., 2021). It is mainly formed of peptidoglycan and teichoic acid that determine the cell surface charge and hydrophobicity. Hydrophobic interactions favor *S. aureus* attachment to hydrophobic materials used in the food industry (such as Teflon, coated glass, stainless steel) and thus promotes biofilm formation. Hydrophobicity also

<footnote n="1">¹University of Udine, Department of Agricultural, Food, Environmental and Animal Science, 33100 Udine, Italy</footnote>

<footnote n="2">²Université Paris-Saclay, Micalis Institute, INRAE, AgroParisTech, 78352 Jouy en Josas, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: marisa.manzano@uniud.it (M. Manzano), jasmina.vidic@inrae.fr (J. Vidic).</footnote>
contributes to the strain pathogenicity since it is involved in *S. aureus* adherence to human cells (Krasowska and Sigler, 2014). Moreover, surface charge and hydrophobicity modulate the permeability of *S. aureus* envelope and may selectively regulate the passage of ions and nutrients necessary for bacterial growth. It was previously shown that these surface properties can prevent some antibiotics to enter *S. aureus* cells (Nikolic and Mudgil, 2023; Léguillier et al., 2024).

The yellow (golden) pigmentation produced by *S. aureus* has antioxidative properties that protect the bacterial cell from the innate host immune response involving oxidation-based clearance (Siems et al., 2023). In addition, it protects the microbe from antibiotics that produce reactive oxygen species such as some beta-lactams and fluoroquinolones (Randazzo et al., 2020). Finally, the versatility of *S. aureus* pathogenicity also comes from the production of toxins. Cytolytic toxins known as pore and non-pore-forming hemolysins are able to damage a variety of human cells, while exotoxins, mostly enterotoxins, are significant contributors to food intoxication (Ljevaković-Musladin et al., 2023; Zhang et al., 2016).

In this study, we isolated *S. aureus* strains from raw milk at farms, and beef hamburgers and pork sausages sold in retail in Northeastern Italy, in order to determine *S. aureus* prevalence, propensity for biofilm formation, and antibiotic susceptibility. In addition, we performed a comprehensive study of selected *S. aureus* strains in terms of their cell surface characteristics, biofilm-forming ability, virulence factors, and interaction with human intestinal epithelial cells Caco-2, providing important information about the virulence potential of *S. aureus* in foods.

## Materials and methods

**Food sample collection.** A total of 29 raw milk samples were collected from bulk milk tanks in different dairy cow farms in Northeastern Italy. Fresh raw meat samples were purchased from local supermarkets in Udine, Italy. A total of 14 meat samples were tested of which 8 were beef hamburgers from adult cattle slaughtered in Italy, and 6 were pork sausage mixtures. All samples were stored at the temperature of 4 ± 2 °C until analyses. Microbiological analyses were performed within 24 h after milking or purchase.

**Microbial isolation of S. aureus and phenotypical identification.** The UNI EN ISO 6888-1:2023 recommendation with some modifications was used to isolate *Staphylococcus* strains (Da Silva et al., 2018). The protocol was applied directly on 10 ml from the 150 ml of milk collected at factories, while 100 g of meat collected at supermarkets was added with 100 ml of physiological saline solution (0.9% NaCl, Sigma, Italy), homogenized in a stomacher lab blender (Astori Tecnica, Italy), and then, 10 ml of the solution were used for the analysis. Plates of Baird Parker (BP) Agar medium supplemented with Egg Yolk Tellurite Emulsion Ø 150 mm (Oxoid, Milan, Italy) were incubated with 0.5 ml of bacterial suspension at 37 °C in aerobic conditions for 48 h. Colony-forming units (CFUs) with good growth, and black, shiny colonies with white and clear zones were counted and up to 5 colonies of presumed *S. aureus* were picked up from the selective medium and streaked on Plate Count Agar, PCA (Oxoid, Milan, Italy) to obtain pure cultures. A total of 99 coagulase-positive *S. aureus* strains were isolated and subjected to catalase (3% H₂O₂, Sigma-Aldrich, Italy), oxidase test (Microbact Oxidase Strips, Oxoid, Italy), and Gram staining (Liofilchem Diagnostics, Italy). The isolates were stored in Brain Heart Infusion (BHI) broth (Oxoid, Italy) with 20% glycerol (Sigma-Aldrich, Italy) at -80 °C for further analyses. The total bacterial count was determined for all samples (Table S1) using the drop plate method. Samples were serially diluted in a physiological solution in tenfold steps, and five 10 µL drops from each dilution were plated onto PCA plates, followed by incubation at 30 °C for 24 h.

**S. aureus species confirmation.** The isolates were grown on BHI Agar (Oxoid, Italy) plates at 37 °C for 48 h and DNA was extracted following the phenol-chloroform method described by Vizzini et al. (2021). The concentration, purity, and quality of the genomic DNA were evaluated with NanoDrop One Microvolume UV-Vis Spectrophotometers (Thermo Fisher Scientific, USA).

The DNAs were used for species confirmation by conventional PCR with specific primers S. aur_nuc-Fw (5'-GCGATTGATGGTGA TACGGTT-3') and S. aur_nuc-Rv (5'-AGCCAAGCCTTGACGAAC TAAAGC-3') reported in Oliveira et al. (2022) for the species-specific thermonuclease (nuc) gene with an expected amplicon of 270 bp. The PCR amplification was performed in a 50 µl reaction with 1X Green GoTaq Reaction Buffer and 1.5 mM MgCl₂ (Promega, USA), 0.2 mM each dNTP (Applied Biosystems, United Kingdom), 0.2 µM each primer (Eurofins Genomics, Germany), 1.25 U GoTaq G2 DNA Polymerase (Promega, USA), and 2 µl of the template DNA at 100 ng/µl. The amplification was performed in a thermal cycler (Applied Biosystems, United Kingdom), and the protocol was 94 °C for 5 min, 35 cycles of 94 °C for 1 min, 55 °C for 30 sec, and 72 °C for 1 min, and a final extension at 72 °C for 7 min. The amplicons underwent electrophoresis at 5 V/cm in a 2% (w/v) agarose gel in TBE 1X stained with 0.5 µg/ml ethidium bromide (Sigma-Aldrich, Italy) and then examined under UV light with bio-imaging system Gen-eGenius (SynGene, Cambridge, England).

**Antibiotic susceptibility test.** Confirmed isolates of *S. aureus* were tested for antimicrobial susceptibility following the EUCAST Disk Diffusion test instructions (EUCAST, 2022). Briefly, the inoculum suspension in physiological solution from an overnight culture to the density of 0.5 McFarland turbidity was spread on Mueller-Hinton (MH) agar plates (Oxoid, Italy). Antibiotic disks were applied on the inoculum, and the plates were incubated at 37 °C in aerobic conditions for 18 h. The quality control strain *S. aureus* DSM 2569 was also used (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures). The following antibiotics were tested: Cefoxitin (30 µg/disk), Levofloxacin (5 µg/disk), Netilmicin (10 µg/disk), Tetracycline (30 µg/disk), Erythromycin (15 µg/disk), Gentamicin (10 µg/disk), Rifampicin (5 µg/disk), Linezolid (10 µg/disk), Ampicillin (10 µg/disk), and Ciprofloxacin (5 µg/disk) (Liofilchem, Italy). The halo diameters were measured and interpreted into susceptibility categories according to the EUCAST breakpoint tables (EUCAST, 2021).

**Quantitative biofilm assay.** Overnight precultures of *S. aureus* were diluted in fresh Luria Bertani (Oxoid, Italy) broth to 10⁷ CFU/ml. Samples of 210 µl of the diluted cultures were inoculated in a 96-well polystyrene (PS) microtiter plate (Cellstar, Italy) and incubated at 37 °C for 72 h. Biofilm amount was measured by discarding the medium, rinsing twice the wells with sterile saline solution, fixing the biofilm at 60 °C for 1 h with hot air, and staining with a 2% crystal violet solution (Merck, Germany) at room temperature for 15 min. Wells were then washed with sterile bidistilled water. The dye was solubilized with 95% ethanol (VWR, France) at room temperature for 30 min, and absorbance at 570 nm was determined using a microtiter plate reader (Sunrise Tecan, Italy). For each experiment, the cut-off value was calculated as explained by Stepanović et al. (2007). For each assay, eight wells were analyzed for each sample and the mean was calculated.

**Electrophoretic mobility and hydrophobicity.** The electrophoretic mobility of *S. aureus* strains was analyzed by zeta potential measurements using a Zetasizer Pro (Malvern Panalytical Instrumentation, United Kingdom) and the disposable capillary cell DTS1070. At least five measurements were performed on each sample containing 10⁸ cells/ml, and the results obtained were presented as mean ± SD.

The hydrophobicity of the bacterial cell surface was assessed through the microbial adhesion to solvents method by comparing the cell affinity to the polar (PBS) and the nonpolar solvent (xylene) as previously described Shaffique et al. (2022) with some modifications. Xylene (300 µl) was added to the test tubes containing 2 ml of washed bacterial cells (10⁸ CFU/ml) in PBS. Following 10 min
incubation at 30 °C upon agitation on vortex for 120 sec, the suspensions were allowed to separate the polar and nonpolar phases. The PBS polar phase was carefully removed with a Pasteur pipette and transferred to an UV–Vis cuvette. Optical density was measured at 400 nm using a Novaspect II spectrophotometer (France). The experiments were performed with three biological replicates.

**Cell membrane permeability.** To determine the permeability of *S. aureus* envelop, strains were grown in BHI at 37 °C overnight. Cultures were pelleted by centrifugation and washed in PBS. The OD~600~ was adjusted to 0.5 with PBS, and 200 µl aliquots were distributed to a 96-well cell culture plate. Then, ethidium bromide was added to 1 µg/ml final concentration in wells (Rodrigues et al., 2011). Fluorescence was measured using the Tecan Spake multimodal reader (France) with the 530 nm band-pass and the 600 nm high-pass filters as the excitation and detection wavelengths, respectively. Fluorescence was acquired every 60 sec for 120 min at 37 °C. Each assay was performed at least in three independent experiments.

**Bacterial cytotoxicity and adhesion to epithelial cell Caco-2.**
Human epithelial cell line Caco-2 (HTB-37 ATCC) was used to simulate the intestinal epithelial cells to assay the pathogenicity of the *S. aureus* isolates. Caco-2 cells were thawed from –80 °C and recovered by adding Dulbecco's Modified Eagle Medium, DMEM (Gibco, France) supplemented with 20% Fetal Calf Serum (Gibco, France) and 1% Penicillin-Streptomycin (Sigma-Aldrich, USA) and cultivated in T25 flasks (Sarstedt, USA) at 37 °C in a 5% CO~2~ humidified incubator.

To compare the adhesion abilities of representative *S. aureus* isolates, Caco-2 cells were detached from the flasks with 1 ml of Trypsin-EDTA (Gibco, France) at 37 °C for 5 min upon softly shaking. The enzymatic reaction was stopped by adding 5 ml of completed DMEM, and the cell pellet was resuspended with the medium. Caco-2 were seeded at 10⁵ cells per well into a 24-well culture plate and incubated at 37 °C in a 5% CO₂ humidified incubator for 4 days. The medium was refreshed every 2 days. The bacterial overnight cultures in BHI broth were pelleted, washed with PBS, and resuspended in infection medium (DMEM without serum and antibiotic) to the multiplicity of infection (MOI) of 10³. To evaluate the number of adhered bacterial cells, the infected Caco-2 monolayers were incubated at 37 °C in a humidified 5% CO₂ incubator for 1 h, washed with PBS five times, and lysed with 0.1% Triton X-100 (Labo-Si, France) at room temperature for 30 min. Lysates were plated on BHI agar plates. Bacteria were counted after plate incubation at 37 °C for 48 h. The experiments were performed in quadruplicates.

The assessment of cellular metabolic activity through the 3-(4,5-d imethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test was employed to access the cytotoxicity of *S. aureus* supernatants toward Caco-2 cells, using the test previously described Vidic et al. (2016). *S. aureus* sterile-filtered supernatants were prepared from overnight cultures in BHI. Caco-2 were seeded at a density of 30,000 cells per well on 96-well plates in 100 µl DMEM medium. After 24 h, the completed medium was exchanged with 100 µl of medium containing 20% of bacterial supernatant. After 24 h incubation, a freshly prepared 0.8 mg/ml MTT solution in PBS was added to the cells and incubated for 1 h. Subsequently, the cell layer was dried and formed MTT formazan was suspended in 100 µl of dimethyl sulfoxide (DMSO). The MTT-formazan absorption was quantified using a microplate reader (Tecan, France), measuring the absorbance at 560 nm and corrected for a background signal by subtracting the signal measured at 670 nm as described previously (Vidic et al., 2016). Cell survival was quantified and expressed as % of cells treated only with PBS. The experiments were performed at least in triplicate.

**Confocal laser scanning microscope image acquisition of biofilms.** To perform Confocal Laser Scanning Microscope (CLSM) biofilms observation, *S. aureus* strains were grown in 96-well plates (Microclear, Greiner Bio-one, France) in BHI. After 1 h at 37 °C, the wells were rinsed with BHI to remove the planktonic cells. After 72 h, the biomass was stained with LIVE/DEAD BacLight bacterial Viability kit (Themo Fisher Scientific, France). The CLSM imaging and analysis was performed on Z-stacks of horizontal plane images that were acquired in 1 µm steps using a Leica SP8 AOBS inverted laser scanning microscope (CLSM, LEICA Microsystems, Wetzlar, Germany) at the INRAE MIMA2 platform in Jouy en Josas, France. At least two stacks of images were acquired randomly on three independent samples at 800 Hz with a ×63 water objective (N.A. = 1.2). Semi-quantitative descriptors (bio-volume, thickness, and roughness) were extracted using BiofilmQ software.

**Cell pigmentation.** *S. aureus* overnight cultures (2 ml) were streaked on PCA plates and incubated at 37 °C for 48 h. The photos of the cell pigmentation were taken using a camera (Canon EOS). The color of the pellet was compared to estimate triterpene staphyloanthin carotenoid production in different strains.

**Hemolytic activity.** Hemolytic activity of *S. aureus* isolates was tested by spotting 10 µl aliquots of *S. aureus* overnight cultures on 5% sheep blood agar plates (BioMérieux SA, France). After drying, plates were incubated at 37 °C overnight and photographed using a camera (Canon EOS).

**Detection of enterotoxin genes.** The PCR targets were classical *sea*, *seb*, *sec*, *sed*, and *see* enterotoxin genes. PCR amplification was conducted in 0.2 ml PCR reaction tubes with a 50-µl reaction volume. The master mix consisted of 1X Phusion HF Buffer (BioLabs, New England), 200 µM PCR Nucleotide Mix (Applied Biosystems, United Kingdom), 0.5 µM forward and reverse primer specific for the enterotoxin gene target (Eurofins Genomics, Germany), 1 U/50 µl Phusion High-Fidelity DNA Polymerase (BioLabs, New England). One µl of the template DNA at 100 ng/µl was added. Detailed information on primer sequences, PCR product sizes, and amplification conditions are reported in Supplementary information (Table S1, Supplementary). The generated amplicons were assessed through electrophoresis in a 2% (w/v) agarose gel (Sigma-Aldrich, Italy) prepared in 0.5X TAE buffer with 0.5 µg/ml ethidium bromide, after mixing with Gel Loading Dye 1X. Electrophoresis was performed at 5 V/cm, and DNA visualization occurred under UV light using the E-box VX5 (Vilber, France).

**Statistics.** The results (quantitative analysis on biofilm, cell surface characteristics, and virulence toward Caco-2) were reported as mean values with respective standard errors. Data analysis was conducted utilizing ordinary one-way ANOVA followed by posthoc test, Tukey's multiple comparison test to assess the significance of observed differences (GraphPad Prism 8.0). Significance was established at a *P* value of <0.05.

## Results

**Prevalence of *Staphylococcus* spp. in raw milk and meat samples.** Meat and milk samples were screened for the occurrence of *Staphylococcus* spp. Based on the classical microbiological growth on BP plates, the presumed *Staphylococcus* count was up to $7 \times 10^3$ CFU/ml in raw cow milk, $1 \times 10^3$ CFU/g in pork sausages, $3.9 \times 10^1$ CFU/g in minced beef meat. Meat and milk samples were screened for the total bacterial count and occurrence of *Staphylococcus* spp. (Table S1). The highest prevalence of presumed *S. aureus* was found in beef hamburgers (62%), followed by pork sausages (50%), and raw cow milk (41.4%) (Table 1).

Among the isolated colonies, a total of 99 coagulase-positive *S. aureus* strains were randomly selected and subjected to biochemical reaction tests (positive catalase, negative oxidase, and positive Gram staining) for confirmation. Samples were randomly collected in different areas of the Friuli Venezia Giulia (FVG) Region to have a real idea of the microbial contamination of food samples. Then, 47 *S. aureus* strains were additionally confirmed with PCR test, amplifying the *nuc* gene encoding *S. aureus* species-specific thermonuclease enzyme. *S. aureus* was mostly confirmed in pork sausages (12/14, 85.7%), followed by raw cow milk (35/66, 53.0%). No strain was confirmed
<table><caption>**Table 1**
*Staphylococcus aureus* occurrence in food and PCR species confirmation</caption><thead><tr><th>Food matrices</th><th>Number of samples</th><th>Number of positive samples</th><th>% of positive samples</th><th>Number of isolated and PCR<sup>1</sup> confirmed S. aureus</th></tr></thead><tbody><tr><td>Cow milk</td><td>29</td><td>12</td><td>41.4</td><td>35</td></tr><tr><td>Pork sausage</td><td>6</td><td>3</td><td>50</td><td>12</td></tr><tr><td>Beef hamburger</td><td>8</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Total</td><td>43</td><td>15</td><td>34.9</td><td>47</td></tr></tbody></table>

among the isolates from beef hamburgers. The discrepancy between the presence and absence of *S. aureus* in pork and beef may be explained by matrix complexity and on-plate interference of other microorganisms, such as *Staphylococcus saprophyticus* and *Staphylococcus epidermidis* that can grow on BP plates.

**Antibiotic resistance profile of S. aureus isolates.** The antibiotic resistance profiling of *S. aureus* isolates was determined with the disk diffusion testing of 10 antibiotics representative of the main antibiotic classes used for the treatment of *S. aureus* infections both in clinical and animal healthcare (Fig. 1). The strains isolated from raw cow milk reveal resistance against gentamycin (20%, 7/35), followed by tetracycline, cefoxitin, erythromycin, ciprofloxacin, and ampicillin (5.7%, 2/35) (Table S2, Supplementary). Among the strains isolated from pork sausages, the most frequent resistance was against ampicillin (41.7%, 5/12), followed by cefoxitin (25%, 3/12), tetracycline, levofloxacin (16.7%, 2/12), rifampicin, and linezolid (8.3%, 1/12). No resistance against netilmicin was found. The strains resistant to three or more antibiotics were considered multidrug-resistant. In raw pork sausages, a higher percentage of multidrug-resistant strains (16.7%, 2/12) was observed than in milk (5.7%, 2/35).

**Quantitative biofilm assay.** All the confirmed *S. aureus* strains were tested for the biofilm production capability. The results were expressed in mean OD values of eight replicates for each strain and

compared with the cut-off value (ODc). Strains that showed OD ≤ ODc were categorized as nonadherent *S. aureus*, while strains with OD > ODc were considered able to adhere to the surface and produce biofilm. A total of 80% (28/35) of *S. aureus* strains isolated from raw milk were biofilm producers, while 100% (12/12) strains isolated from pork sausages were able to form biofilm. The biofilm producer strains were grouped into three different classes based on the ODc value: weakly adherent (ODc < OD < 2xODc), moderately adherent (2xODc < OD < 4xODc), and strongly adherent (4xODc < OD) as shown in Figure 2. The 42.9% of the *S. aureus* isolated from raw cow milk and 66.7% of fresh pork sausages were strongly adherent.

**Selection of representative S. aureus isolates.** To further characterize *S. aureus* isolates for their biofilm formation efficiency, phenotypes, and virulence, representative strains were chosen using the protocol illustrated in Figure 3. In summary, among 47 confirmed *S. aureus* strains isolated from milk and meat matrices, 8 strains were selected because they were able to produce strong biofilm but differed in their antibiotic susceptibilities: 2 were not antibiotic-resistant strains (219, 220), 2 were resistant to ampicillin and tetracycline (301, 302), 2 were resistant to ampicillin and cefoxitin (303, 304), and 2 were multidrug-resistant strains (227, 228).

**Bio-volume, thickness, and roughness of the biofilm.** To determine the architectures of the biofilms of selected *S. aureus* strains on a hydrophobic polystyrene substrate, each biofilm was visualized using 3D projections from CLSM images. To avoid the interference of planktonic cells, the inocula were replaced by the medium 90 min after seeding. The biofilm-forming cells were then allowed to grow for 72 h. CLSM image acquisition showed that all the strains were able to form three-dimensional structures (Fig. 4A). The *S. aureus* strains

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Antibiotic resistance profile of *S. aureus* isolates. Prevalence of antibiotic-resistance of the confirmed *S. aureus* strains was tested against ampicillin (10 µg/disk), ciprofloxacin (5 µg/disk), gentamicin (10 µg/disk), erythromycin (15 µg/disk), cefoxitin (30 µg/disk); levofloxacin (5 µg/disk), linezolid (10 µg/disk), netilmicin (10 µg/disk), rifampicin (5 µg/disk), and tetracycline (30 µg/disk).</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Quantitative biofilm production. Different adherence capability of the confirmed *S. aureus* strains isolated from raw cow milk and pork sausage was calculated from biofilm quantification in microtiter plates.</figcaption></figure>

<footnote n="1">¹ nuc gene was used as target for *S. aureus* species confirmation.</footnote>
<figure><img src="image_5.png" /><figcaption>**Figure 3.** A schematic illustration of the selection process of representative *S. aureus*. A total of 99 *Staphylococcus* spp. was isolated from raw cow milk and raw meat. 47 isolates were confirmed by species-specific PCR and tested for antibiotic resistance and biofilm formation capability. Based on their resistance profiles and biofilm production, 8 isolates were chosen for bacterial cell surface characterization, cytotoxicity, adhesion to epithelial Caco-2 intestinal cell line, and confocal laser scanning microscope image acquisition of biofilms.</figcaption></figure>

isolated from pork sausages (301, 302, 303, 304) developed a thick and packed biofilm, highly condensed, whereas the strains isolated from raw cow milk (219, 220, 227, 228) developed biofilms of flatter, less compact, and nonhomogeneous structures containing micro holes. Diversity in the 3D structure of biofilms was particularly evident between meat and milk isolates when considering the cell vitality: biofilms of the strains isolated from milk contained more dead cells than biofilms formed by the strains isolated from meat. Especially, the multidrug-resistant strain 227 isolated from milk produced biofilm with many dead cells, at the given condition. Multidrug-resistant strains 227 and 228 had minimal biofilm biovolumes, and thickness (Fig. 4B and Table S3, Supplementary).

**Hemolytic activity, enterotoxin production, and pigmentation of selected strains.** The representative strains showed different hemolysis type: strains 303 and 304 had no hemolysis activity, while strains 301 and 302 presented α hemolysis and all the other strains β hemolysis (219, 220, 227, 228). The strains isolated from raw meat produced more yellow-pigmented pellets (301, 302, 303, 304) than the strains isolated from milk (219, 220, 227, 228). No classical enterotoxin gene (*sea*, *seb*, *sec*, *sed*, *see*) was detected among the representative strains (Table S4, Supplementary).

**Cell surface characterization of representative S. aureus strains.** Characterization of bacterial envelope was performed to assess bacterial surface charge and hydrophobicity because both are

<figure><img src="image_6.png" /><figcaption>**Figure 4.** Biofilm characteristics of representative strains. (A) Visualization through the confocal microscope and (B) Quantitative analyses on biovolume, thickness, and roughness of the biofilm. \*, *P* < 0.05 control comparison with 227 and 228 multidrug resistant strains.</figcaption></figure>
involved in antibiotic resistance and biofilm formation. Besides, the production of the natural pigment in *S. aureus*, staphyloxanthin, was compared in selected strains, because staphyloxanthin is a hallmark phenotype in the bacterial virulence and it regulates membrane permeability to cationic antimicrobial peptides (Elmesseri et al., 2022). The surface charge was estimated by measuring the bacterial electrophoretic mobility in solutions of pH 4.8 and pH 7.4 that simulated foods of acidic and neutral pH. All strains were negatively charged at both pHs (Fig. 5A and B). At pH 4.8, all strains were of similar surface charge (Fig. 5B), while at pH 7.4, only strains isolated from milk were of similar surface charge (Fig. 5A). Among the strains isolated from pork sausages, strain 302 was significantly more negatively charged than 304 (P < 0.05). Although both strains were highly pigmented (Fig. S1, Supplementary) and resistant to ampicillin, strain 302 was resistant to tetracycline and had no hemolytic activity while 304 was resistant to cefoxitin and showed hemolytic activity (Fig. 5). These phenotypes suggest the differences between cell walls in strains 302 and 304.

Next, we examined the bacterial adhesion to xylene to investigate the bacterial hydrophobicity (Fig. 5C). A high variability was observed between strains isolated from the same food matrix (P < 0.05). Among the strains from raw pork sausages, a significant difference was again observed between strain 302 (resistant to ampicillin, and tetracycline, with hemolytic activity) and strains 303 and 304 (resistant to ampicillin and cefoxitin, without hemolytic activity). This confirms that cell walls differed between 302 and 303/304 strains. Interestingly, among the strains isolated from raw cow milk, there was a significant difference in hydrophobicity between two multidrug-resistant strains 227 and 228 (Fig. 5C). Strain 227 had a low hydrophobicity, while strain 228 had similar hydrophobicity to strains 219 and 220 that were susceptible to all antibiotics tested.

In addition, we compared the cellular permeability of representative strains because a low permeability of the bacterial cell wall has been identified as a challenging barrier for effective antibiotic therapy (Ghai & Ghai, 2018). As expected, the ethidium bromide assay showed that the permeability of the multidrug-resistant strains 227 and 228 was lower than that of other strains (Fig. 5D).

**Virulence of representative *S. aureus* strains toward Caco-2 cells.** The capability of selected *S. aureus* isolates to adhere to mammalian cells was tested using Caco-2 intestinal epithelial cell line (Fig. 6A). All the isolates adhered to Caco-2 cells, showing adhesion values >6 log₁₀ CFU/ml, similar to the reference *S. aureus* strain DSM 2569, with the exception of the multiresistant strain 219 isolated from raw cow milk which showed a slightly lower value of 0.55 log₁₀ CFU/ml.

To test whether selected *S. aureus* isolates secrete cytotoxic molecules, the effect on bacterial secretomes on Caco-2 intestinal cell line was analyzed using the MTT assay. In this assay, the reduction of the tetrazolium dye MTT is an indicator of cellular viability. As shown in Fig. 6B, the addition of secretomes inducted no significant MTT reduction.

## Discussion

The health risks of foodborne *S. aureus* are associated with its resistance to antimicrobials, particularly to β-lactam antibiotics, production of enterotoxins, adhesion, and biofilm formation which promote the bacterium colonization of food environments. The present study provides information about the prevalence of *S. aureus* in the milk and meat purchased in Northeastern Italy. *S. aureus* was recovered from 41.8% of milk samples. The found prevalence of *S. aureus* in milk is slightly higher than the reported trend of cow's milk contamination in Europe between 2000 and 2016 (Ou et al., 2018). We found that 50% of pork sausages and no beef hamburgers were contaminated by *S. aureus*. Importantly, no milk or meat isolate had *sea*, *seb*, *sec*, *sed*, and *see* enterotoxin genes.

*S. aureus* strains isolated in this study were resistant to at least one of the tested antibiotics, except for netilmicin. Related to β-lactam antibiotic class resistance, MRSA is nowadays the most emergent issue. Most of the meat isolates showed resistance to ampicillin (41.7%). This is not surprising given the high frequency of β-lactam prescriptions in veterinary. Besides, we found 25% MRSA among strains isolated from meat and 5.7% from milk. Some MRSA strains additionally exhibited a high resistance to tetracycline (25% in meat and 5.7% in milk). Tetracycline is also extensively used in veterinary medicine, which can explain the prevalence of strains resistant to this antibiotic. Interestingly, to treat mastitis β-lactam is nowadays replaced by erythromycin (Nossair et al., 2018) for which the resistance was found in milk

<figure><img src="image_7.png" /><figcaption>**Figure 5.** Haemolytic activity and cell surface characteristics of representative strains. Electrophoretic mobilities measured by zeta potential at pH 7.4 (A) and pH 4.8 (B). (C) Cell surface hydrophobicity based on the affinity between bacteria and xylene solvent. (D) Bacterial cell wall permeability measured with the ethidium bromide influx assay. *, P < 0.05.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Figure 6.** Virulence of representative strains toward Caco-2 intestinal cell line. (A) Adhesion to Caco-2 cell line after 1-h incubation at 37 °C. Adhesion was calculated by counting the number of bacteria in the eukaryotic cell suspension after lysis with 0.1% Triton X-100. The results are expressed as log [CFU/ml]. (B) MTT assay for cell toxicity of bacterial supernatant on Caco-2 cells. Absorbance was measured at 560 nm and 670 nm after 24-h incubation with the infected solution.</figcaption></figure>

isolates. Other antibiotics, commonly used to treat *S. aureus* infections, are aminoglycosides and fluoroquinolones, against which the resistant strains were found in both milk and meat. A notable remark in the current study is related to linezolid, a new synthetic antibacterial agent that belongs to oxazolidinones. Despite linezolid being a recent antibiotic, we identified resistant strains in meat samples (8.3%). This contrasts with the study from 2022 when no resistance to this drug was found in meat and milk isolates (Oliveira et al., 2022).

*S. aureus* can form biofilms on abiotic and biotic surfaces which is a considerable problem for the food industry. Most of the strains isolated from raw milk (80%) and pork sausages (100%) were biofilm producers. Nevertheless, the 3D structure of biofilm formed by representative strain showed that meat isolates form a biofilm of important volume and thickness, while biofilms of milk isolates contained many dead cells.

The surface charge, hydrophobicity, and membrane permeability of representative strains carrying different antibiotic resistances were characterized for their significant roles in bacterial pathogenicity. Gram-positive bacteria display a negative surface charge due to the presence of negatively charged functional moieties in the envelope (teichuronic acid, peptidoglycan, and teichoic acid) (Ferreyra Maillard et al., 2021). Indeed, all tested isolates were of negative surface charge. Their electrophoretic mobility was a function of the pH, reflecting possible strain adaptation to grow in different foods. For instance, fresh raw meat has a pH between 5.4 and 6, while nontreated raw milk has a pH ~ 6.7 (Luong et al., 2022; Sun et al., 2022).

The surface charge can modify bacterial susceptibility to some antibiotics directly through the electrostatic interactions or indirectly by modifying the permeability of the bacterial envelope. Indeed, we found that multidrug-resistant strains had lower permeability than other strains isolated from the same food matrix. The low permeability of the bacterial cells contributes to antibiotic resistance by preventing drugs to bind and enter the bacterial cell.

*S. aureus* surface has a characteristically hydrophobic nature which is attributed to *S. aureus* adaptation strategy. Lather et al. (2016) found out that *S. aureus* can modify the natural hydrophobicity to survive antimicrobials. We found higher negatively charged, lower hydrophobicity and higher permeability in *S. aureus* isolates resistant to ampicillin and tetracycline compared to isolates resistant to ampicillin and cefoxitin. These differences may be correlated to modes of action of antibiotics. For instance, tetracycline passively diffuses in bacterial cells to block protein synthesis (Ferreyra Maillard et al., 2021), while cefoxitin reduces the permeability and the charge by modifying the cell wall composition (Uddin et al., 2021).

Bacterial surface characteristics are crucial in determining its adherence to host cells (Mergani et al., 2021). Typically, bacteria with elevated hydrophobicity tend to easily adhere to eukaryotic cells. Nev-

ertheless, we found no relation between the hydrophobic characteristics of the tested strains and their adhesion capability. Additionally, we tested the cytotoxicity of bacterial secretomes because most of the *S. aureus* isolates showed hemolytic activity. No significant toxicity of secretomes toward Caco-2 cells was found. However, this finding cannot exclude that tested isolates can invade intestinal cells. For instance, previously was found that low pathogenic *S. aureus* Newman strain was able to invade the Caco-2 cells without causing significant cytotoxic effects (Mergani et al., 2021).

## Conclusions

This study highlights the contamination of raw food by *S. aureus*, with a notable prevalence observed in raw milk and meat, presenting a significant risk. Although the strains tested negative for entero-toxin genes and showed no direct toxicity to Caco-2 cells, they demonstrated robust biofilm formation, hemolysin and staphylovan-thin production, and resistance to both conventional and emerging antibiotics. Our findings suggest that *S. aureus* adapts by modifying its surface properties, such as reducing permeability to limit antibiotic entry or altering surface charge to enhance resistance. Further research is needed to fully understand the risks posed by circulating foodborne *S. aureus*, particularly its mechanisms of antibiotic resistance.

## CRediT authorship contribution statement

**Debora Pinamonti:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Formal analysis. **Marisa Manzano:** Writing – review & editing, Writing – original draft, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization. **Michela Maifreni:** Writing – review & editing, Methodology. **Silvia Bianco:** Formal analysis. **Beki Domi:** Formal analysis. **Alessia Ferrin:** Formal analysis. **Jamila Anba-Mondoloni:** Writing – review & editing, Formal analysis. **Julien Dechamps:** Writing – review & editing, Formal analysis. **Roman Briandet:** Writing – review & editing. **Jasmina Vidic:** Writing – review & editing, Writing – original draft, Supervision, Resources, Methodology, Investigation, Funding acquisition, Conceptualization.

## Funding

This work was supported in a part by the French National Agency for Research (ANR-21-CE21-009 "Siena" and ANR-21-CE42-0008 "Elise").
# Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

# Appendix A. Supplementary material

Supplementary material to this article can be found online at https://doi.org/10.1016/j.jfp.2024.100442.

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