Exploring the Biofilm Formation Capacity in S. pseudintermedius and Coagulase-Negative Staphylococci Species.

The ability of biofilm formation seems to play an important role in the virulence of staphylococci. However, studies reporting biofilm formation of coagulase-negative staphylococci isolated from animals are still very scarce. Thus, we aimed to evaluate the biofilm-forming capacity of CoNS and S. pseudintermedius isolated from several animal species and to investigate the effect of conventional antimicrobials on biofilm reduction. A total of 35 S. pseudintermedius and 192 CoNS were included. Biofilm formation was accessed by the microtiter plate assay and the biofilms were stained by crystal violet. Association between biofilm formation and staphylococci species and antimicrobial resistance was also performed. Biofilm susceptibility testing was performed with tetracycline and amikacin at the minimum inhibitory concentration (MIC) and 10 × MIC. The metabolic activity of the biofilm cells after antimicrobial treatment was accessed by the XTT assay. All isolates formed biofilm, with S. urealyticus producing the most biofilm biomass and S. pseudintermedius producing the least biomass. There was a positive association between biofilm formation and multidrug resistance as well as resistance to individual antimicrobials. Neither tetracycline nor amikacin were able to eradicate the biofilm, not even at the highest concentration used. This study provides new insights into biofilm formation and the effects of antimicrobials on CoNS species.

Biofilm Formation of Staphylococcus aureus from Pets, Livestock, and Wild Animals: Relationship with Clonal Lineages and Antimicrobial Resistance.

This study aimed to compare the biofilm formation ability of Staphylococcus aureus isolated from a wide range of animals and study the association between biofilm formation and antimicrobial resistance and genetic lineages. A total of 214 S. aureus strains isolated from pets, livestock, and wild animals were evaluated regarding their ability to form biofilms by the microtiter biofilm assay and their structure via confocal scanning laser microscopy. Statistical analysis was used to find an association between biofilm formation and antimicrobial resistance, multidrug resistance, sequence types (STs), spa and agr-types of the isolates. The antimicrobial susceptibility of 24 h-old biofilms was assessed against minimum inhibitory concentrations (MIC) and 10× MIC of amikacin and tetracycline, and the biomass reduction was measured. The metabolic activity of biofilms after antimicrobial treatment was evaluated by the XTT assay. All isolates were had the ability to form biofilms. Yet, significant differences in biofilm biomass production were detected among animal species. Multidrug resistance had a positive association with biofilm formation as well as methicillin-resistance. Significant differences were also detected among the clonal lineages of the isolates. Both tetracycline and amikacin were able to significantly reduce the biofilm mass. However, none of the antimicrobials were able to eradicate the biofilm at the maximum concentration used. Our results provide important information on the biofilm-forming capacity of animal-adapted S. aureus isolates, which may have potential implications for the development of new biofilm-targeted therapeutics.

Antibiotic Resistance and Biofilm-Forming Ability in Enterococcal Isolates from Red Meat and Poultry Preparations.

This study investigated the resistance to antibiotics and the capacity to form a biofilm of 200 isolates of enterococci isolated from raw preparations of beef (51 strains), pork (47), chicken (50), and turkey (52) acquired in north-western Spain. Fifteen antimicrobials of clinical importance were tested by the disc diffusion method. The average number of resistances per strain was 4.48 ± 1.59. If resistant strains were taken together with those showing reduced susceptibility, the total number of resistances per strain was 6.97 ± 2.02. Two isolates (1.0% of strains) were resistant to a single antibiotic, twenty-two isolates (11.0%) presented resistance to two, one strain (0.5%) was resistant to three, and 175 isolates (87.5%) showed a multiple drug-resistant phenotype (MDR; defined as no susceptibility to at least one agent from each of three or more antimicrobial categories). The prevalence of resistance varied between 0.5% (gentamicin) and 100% (kanamycin). All strains produced biofilm on polystyrene microwell plates, determined using crystal violet assay. Isolates were classified as having a weak (51 strains; average optical density at 580 nanometers -OD580- = 0.206 ± 0.033), moderate (78 strains; average OD580 = 0.374 ± 0.068), or strong (71 strains; average OD580 = 1.167 ± 0.621) ability to produce biofilm (p < 0.05). Isolates from beef preparations produced the most substantial (p < 0.05) biofilms. The results of this study indicate that meat and poultry preparations are major reservoirs of antibiotic-resistant enterococcal strains capable of forming a biofilm. In order for food-borne infections to be prevented, the importance of careful handling of these foodstuffs during preparation, avoiding cross-contamination, and ensuring thorough cooking, is stressed.

Characterization of Biofilms Formed by Foodborne Methicillin-Resistant Staphylococcus aureus.

The objective of this study was to evaluate the capacity of 49 methicillin resistant Staphylococcus aureus (MRSA) from foods of animal origin (42 from dairy products and 7 from meat and meat products) to form biofilms. Overall, a higher biofilm biomass was observed for those MRSA strains harboring SCCmec type IV, while 8 MRSA strains (5 from dairy products and 3 from meat and meat products) were classified as strong biofilm formers in standard Tryptic Soy Broth medium. When a prolonged incubation period (48 h) was applied for those 8 MRSA strains, an increased biofilm biomass accumulation was observed during the time course, whereas the number of viable cells within the biofilms decreased as the biomass increased. The capacity of biofilm production correlated pretty well between the experiments using polystyrene microtiter plates and stainless steel micro-well plates, and significant higher values were observed in stainless steel when glucose was added to TSB during the enrichment. Biofilms were further characterized by confocal laser scanning microscope (CLSM), confirming that proteins and α-polysaccharides were the predominant components inside the extracellular polymeric matrix of biofilms formed by MRSA strains. In conclusion, our results confirm that MRSA isolates from foods of animal origin have significant capacity for forming biofilms with a high protein content, which can play a key role for the successful dissemination of MRSA lineages via food. Knowledge of the capacity of MRSA strains to produce biofilms, as well as characterization of the main MRSA biofilms matrix components, can help both to counteract the mechanisms involved in biofilm formation and resistance and to define more rational control strategies by using tailor-made cleaning agents.

Visualization and quantification of the cellular and extracellular components of Salmonella Agona biofilms at different stages of development.

Salmonella is a major food-borne pathogen able to persist in food processing environments because of its ability to form biofilms. A Salmonella enterica serotype Agona isolate from poultry (S24) was grown at 37°C in biofilms for up to 144 hours (H144) in attachment to polystyrene surfaces. Biofilm structures were examined at different stages in their development (H3, H24, H48, H72, H96 and H144) using confocal laser scanning microscopy (CLSM) in conjunction with fluorescent dyes for live cells (SYTO 9), dead cells (propidium iodide), proteins (fluorescein isothiocyanate isomer I), lipids (DiD'oil), α-polysaccharides (concanavalin A, tetramethylrhodamine conjugate), and ß-polysaccharides (calcofluor white M2R). Strain S24 developed a robust biofilm at H72 (biovolume of 166,852.5 ± 13,681.8 µm3 in the observation field of 16,078.2 µm2). The largest biovolume of live cells was also detected at H72 (128,110.3 ± 4,969.1 µm3), decreasing thereafter, which was probably owing to the detachment of cells prior to a new phase of colonization. The percentage of dead cells with regard to total cells in the biofilms increased throughout the incubation, ranging from 2.3 ± 1.1% (H24) to 44.2 ± 11.0% (H144). Proteins showed the greatest biovolume among the extracellular components within the biofilms, with values ranging from 1,295.1 ± 1,294.9 µm3 (H3) to 19,186.2 ± 8,536.0 µm3 (H96). Maximum biovolume values of 15,171.9 ± 660.7 µm3 (H48), 7,055.3 ± 4,415.2 µm3 (H144), and 2,548.6 ± 1,597.5 µm3 (H72) were observed for ß-polysaccharides, α-polysaccharides and lipids, respectively. A strong (P < 0.01) positive correlation was found between the total biovolume of biofilm and the biovolume of live cells, proteins and ß-polysaccharides, which may serve as useful markers of biofilm formation. The present work provides new insights into the formation of S. Agona biofilms. Our findings may contribute to the designing of reliable strategies for preventing and removing these bacterial communities.