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

Abstract

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.

Keywords: MRSA; S. aureus; animals; antimicrobial resistance; biofilms; genetic linages.

Figure 1

Biofilm formation capacity of S. aureus strains isolated from different animal species. The symbols represent the biomass mean of the biofilm formed in independent tests of the individual isolates. The red lines represent the average of biofilm mass formed by all isolates. Statistical significance was determined using Tukey’s multiple comparison test (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 2

The images (126.8 µm × 126.8 µm) correspond to three-dimensional reconstructions obtained by CLSM and processed with the IMARIS 9.1 software, including the virtual projections of the shadows on the right. D: donkey; Dg: dog; C: camel; Rb: rabbit; Rt: rodent; Pi: pig; B: bovine; H: hare; Po: poultry; O owl.

Figure 3

Biovolume in the observation field of 16,078.2 μm² (a), maximum height (b), percentage of surface area covered (c) and roughness (d) of biofilms formed de 12 selected S. aureus isolates. Statistical significance was determined using Tukey’s multiple comparison test. The values marked with the same letter are not statistically significant as determined by the Tukey’s post hoc test (p < 0.05). D: donkey; Dg: dog; C: camel; Rb: rabbit; Rt: rodent; Pi: pig; B: bovine; H: hare; Po: poultry; O owl.

Figure 4

(a) Percentage isolates susceptible to all antimicrobials, resistant to one or two classes of antimicrobials and MDR. (b) Mean of biofilm formation among isolates susceptible to antimicrobials, resistant to one/two classes and MDR isolates. The red lines represent the average of biofilm mass formed by all isolates. Statistical significance was determined using Tukey’s multiple comparison test (* p < 0.05).

Figure 5

Biofilm formation among isolates grouped into different clonal lineages. Relationship between biofilm formation and: (a) STs; (b) spa-types and (c) agr types. The red lines represent the average of biofilm mass formed by all isolates. Statistical significance was determined using Tukey’s multiple comparison test (* p < 0.05; ** p < 0.01; *** p < 0.001).

Figure 6

Effect of tetracycline on the biofilm biomass reduction of 23 isolates at MIC and 10× MIC. Data are presented as mean ± standard deviation for four independent replicates. Statistical significance was determined using Dunnett’s multiple comparison test (* p < 0.05; ** p < 0.01; ***p < 0.001). D: donkey; Dg: dog; C: camel; Rb: rabbit; Rt: rodent; Pi: pig; B: bovine; H: hare; Po: poultry; O owl.

Figure 7

Effect of amikacin on the biofilm biomass reduction of 23 isolates at MIC and 10× MIC. Data are presented as mean ± standard deviation for four independent replicates. Statistical significance was determined using Dunnett’s multiple comparison test (* p < 0.05; ** p < 0.01; *** p < 0.001). D: donkey; Dg: dog; C: camel; Rb: rabbit; Rt: rodent; Pi: pig; B: bovine; H: hare; Po: poultry; O owl.

Figure 8

Metabolic activity of S. aureus biofilms before and after treated with tetracycline at concentrations of MIC and 10× MIC. The results are expressed as percentage of metabolic activity. Statistical significance was determined using Dunnett’s multiple comparison test (* p < 0.05; ** p < 0.01; *** p < 0.001). D: donkey; Dg: dog; C: camel; Rb: rabbit; Rt: rodent; Pi: pig; B: bovine; H: hare; Po: poultry; O owl.

Figure 9

Metabolic activity of S. aureus biofilms before and after treated with amikacin at concentrations of MIC and 10× MIC. The results are expressed as percentage of metabolic activity. Statistical significance was determined using Dunnett’s multiple comparison test (* p < 0.05; ** p < 0.01; *** p < 0.001). D: donkey; Dg: dog; C: camel; Rb: rabbit; Rt: rodent; Pi: pig; B: bovine; H: hare; Po: poultry; O owl.