Biofilm Formation by Methicillin-Resistant and Methicillin-Sensitive Staphylococcus aureus Strains from Hospitalized Patients in Poland

Abstract

Biofilm-mediated infections in the hospital environment have a significant negative impact on patient health. This study aimed to investigate biofilm production in vitro and the presence of icaABCD genes in methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains isolated from hospitalized patients. MRSA (73) and MSSA (57) strains were evaluated for biofilm production by the microtiter plate method. The presence of ica operon was investigated by PCR. Out of 130 strains, 99.2% were biofilm producers. Strong biofilms were formed by 39.7% of MRSA and 36.8% of MSSA strains. The highest percentage of strong biofilm producers was found among the strains isolated from sputum and tracheostomy tube (66.7%), nose and catheter (50%), throat (44.4%), and bronchoalveolar washings (43.8%). The strains isolated from bronchoalveolar washings produced significantly more biofilm than strains isolated from wound and anus. The ability of biofilm forming by fecal strains was significantly lower compared to strains from other materials. MRSA strains had significantly higher ability of biofilm formation than MSSA strains (P = 0.000247). The presence of ica operon in MRSA was detected in all strains. Comparison of strong biofilm biomass of the strains with icaABCD, icaABD, and icaAD revealed that strains with icaABCD and icaABD produced highly significantly more biofilm than strains with icaAD. Biofilm forming by both MRSA and MSSA strains indicates high ability of theses strains to persist in hospital environment which increases the risk of disease development in hospitalized patients.

Figure 1

Biofilm-forming ability of the four most numerous groups of S. aureus strains isolated from blood (B), bronchoalveolar washings (BW), wound (W), and anus (AN). Values of absorbance at 492 nm (A₄₉₂) were compared by using Kruskal-Wallis test. In analysis, multiple comparisons of average ranks at P ≤ 0.05 were used. The designation with different letters indicates that biofilm biomass formed by different groups of strains differs significantly.

Figure 2

Comparison of ability of biofilm forming by MRSA and MSSA strains. Two group comparisons of average values of absorbance at 492 nm (A₄₉₂) were analyzed by using Mann-Whitney U-test. The designation with different letters indicates that biofilm biomass formed by MRSA and MSSA strains differs significantly at P = 0.000247.

Figure 3

Electrophoresis in 1.5% agarose gel PCR products obtained by using specific primers for icaABCD genes. Lines M: molecular weight markers (1000, 900, 800, 700, 600, 500, 400, 300, 200, 100 bp; GenoPlast Biochemicals, Poland), Lines 1–4: products (188 bp) obtained by using specific primers for icaA gene; Lines 5–8: products (900 bp) obtained by using specific primers for icaB gene; Lines 9–12: products (1100 bp) obtained by using specific primers for icaC gene; Lines 13–16: products (198 bp) obtained by using specific primers for icaD gene.

Figure 4

Comparison of ability of strong biofilm forming by S. aureus strains with icaABCD, icaABD, and icaAD genes. Values of absorbance at 492 nm were compared by using Kruskal-Wallis test. In analysis, multiple comparisons of average ranks at P ≤ 0.01 were used. The designation with different letters indicates that biofilm biomass formed by strains with different genes differs significantly.

Figure 5

The relationships between the presence of ica operon genes and susceptibility or resistance to methicillin. ∗ The icaABCD genes were significantly associated with the MRSA strains (P = 0.04) (χ² test).