Induction of MRSA Biofilm by Low-Dose β-Lactam Antibiotics: Specificity, Prevalence and Dose-Response Effects

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of hospital- and community-associated infections. The formation of adherent clusters of cells known as biofilms is an important virulence factor in MRSA pathogenesis. Previous studies showed that subminimal inhibitory (sub-MIC) concentrations of methicillin induce biofilm formation in the community-associated MRSA strain LAC. In this study we measured the ability sub-MIC concentrations of eight other β-lactam antibiotics and six non-β-lactam antibiotics to induce LAC biofilm. All eight β-lactam antibiotics, but none of the non-β-lactam antibiotics, induced LAC biofilm. The dose-response effects of the eight β-lactam antibiotics on LAC biofilm varied from biphasic and bimodal to near-linear. We also found that sub-MIC methicillin induced biofilm in 33 out of 39 additional MRSA clinical isolates, which also exhibited biphasic, bimodal and linear dose-response curves. The amount of biofilm formation induced by sub-MIC methicillin was inversely proportional to the susceptibility of each strain to methicillin. Our results demonstrate that induction of biofilm by sub-MIC antibiotics is a common phenotype among MRSA clinical strains and is specific for β-lactam antibiotics. These findings may have relevance to the use of β-lactam antibiotics in clinical and agricultural settings.

Keywords: MRSA; Staphylococcus aureus; antibiotic; bimodal; biofilm; biphasic; subminimal inhibitory.

FIGURE 1.

Bacterial growth and biofilm formation by S. aureus MRSA strain LAC in the presence of sub-MIC concentrations of eight different β-lactam antibiotics. Bacterial growth (A_{490 nm}) is indicated along the left-hand y axes, biofilm formation (A_{620 nm}) is indicated along the right-hand y axes, and antibiotic concentration is indicated along the x axes. Values show mean absorbance values for duplicate wells. Error bars were omitted for clarity.

FIGURE 2.

Bacterial growth and biofilm formation by S. aureus MRSA strain LAC in the presence of sub-MIC concentrations of six different non-β-lactam antibiotics. Bacterial growth (A_{490 nm}) is indicated along the left-hand y axes, biofilm formation (A_{620 nm}) is indicated along the right-hand y axes, and antibiotic concentration is indicated along the x axes. Values show mean absorbance values for duplicate wells. Error bars were omitted for clarity.

FIGURE 3.

Bacterial growth and biofilm formation six clinical MRSA strains in the presence of sub-MIC concentrations of sub-MIC methicillin. Bacterial growth (A_{490 nm}) is indicated along the left-hand y axes, biofilm formation (A_{620 nm}) is indicated along the right-hand y axes, and antibiotic concentration is indicated along the x axes. Values show mean absorbance values for duplicate wells and error bars indicate range.

FIGURE 4.

Relationship between methicillin sensitivity and biofilm induction among 39 MRSA clinical strains. (A) Relationship between methicillin sensitivity (x axis) and the methicillin concentration that induced maximum biofilm induction (y axis). (B) Relationship between methicillin sensitivity (x axis) and the maximum amount of biofilm induced by sub-MIC methicillin (y axis).