Membrane Vesicles Are the Dominant Structural Components of Ceftazidime-Induced Biofilm Formation in an Oxacillin-Sensitive MRSA

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) has received increasing attention in recent years. However, the characteristics and relevant mechanisms of biofilm formation in oxacillin-sensitive MRSA (OS-MRSA) are poorly understood. This study was designed to characterize biofilm formation in OS-MRSA BWSA15 in response to ceftazidime (TZ) by comparing the methicillin-sensitive S. aureus (MSSA) strain BWSA23 and the oxacillin-resistant MRSA (OR-MRSA) strain BWSA11. The biofilms and biofilm-forming cells were observed by electron microscopy. Biofilms grown on microtiter plates were chemically decomposed and analyzed by Fourier transform infrared spectroscopy. The transcriptional regulation of genes associated with methicillin resistance, surface adhesion, fatty acid biosynthesis, and global regulation (sigma B) was investigated. A significant increase in biofilm formation ability (10.21-fold) and aggregation ability (2.56-fold) was observed in BWSA15 upon the treatment with TZ (16 μg/ml). The TZ-induced biofilm formation in BWSA15 was characterized by a disappearance of polysaccharide-like extracellular substances and an appearance of a large number of intercellular MVs from extracellular matrix. Few MVs were identified in the biofilms formed by BWSA11 and BWSA23. There was a significant upregulation of mecA, sigB, and fatty acid biosynthesis-associated genes and downregulation of icaA, icaD, clfA, clfB, and fnaA in BWSA15 upon the treatment with TZ. The formation of intracellular junctions of MVs in the biofilms of BWSA15 was mediated by a significant increase in the proportion of proteins as well as by an increase in the proportion of non-ionized carboxyl groups in fatty acids. This study demonstrated that beta-lactam antibiotics can induce biofilm formation in OS-MRSA, and the biofilm induction in OS-MRSA can mainly be attributed to exposed MVs with increased hydrophobicity rather than polysaccharide intercellular adhesins, cell wall-anchored surface proteins, and extracellular DNA.

Keywords: MRSA; antibiotic resistance; biofilm; membrane vesicle; methicillin-resistant Staphylococcus aureus.

FIGURE 1

Relative biofilm formation index of Staphylococcus aureus. ^TZ, in the presence of ceftazidime (TZ) (16 μg/ml). Error bars represent the standard deviations (n = 3). ^∗P < 0.05 (Student’s t-test) indicate significant differences compared to BWSA15.

FIGURE 2

Surface adhesion of S. aureus. ^TZ, in the presence of TZ (16 μg/ml). Error bars represent the standard deviations (n = 3). ^∗P < 0.05 (Student’s t-test) indicate significant differences compared to BWSA15.

FIGURE 3

Intercellular aggregation of S. aureus. ^TZ, in the presence of TZ (16 μg/ml). Error bars represent the standard deviations (n = 3). ^∗P < 0.05 (Student’s t-test) indicate significant differences compared to BWSA15.

FIGURE 4

Scanning electron microscopy images of MVs formed by S. aureus BWSA15 (A), S. aureus BWSA15 in the presence of TZ (B), S. aureus BWSA11 (C), and S. aureus BWSA23 (D). ^TZ, biofilm formed under TZ stress (16 μg/ml); white arrows indicate MVs; black arrows indicate mucous-like extracellular substances.

FIGURE 5

Secretion and production of MVs by S. aureus. TEM images of thin sections of biofilm-forming S. aureus BWSA15 in the presence of TZ (A,B) and S. aureus BWSA23 (C) showing the extracellular MVs (arrow 1) and intracellular MV-like structures (arrow 2–5). PM, plasma membrane.

FIGURE 6

FTIR spectra of biofilms formed by BWSA15, BWSA11, and BWSA23. ^TZ, in the presence of TZ (16 μg/ml).

FIGURE 7

Fold change in the transcriptional expression of genes associated with (A) the biosynthesis of the PBP2a protein (mecA), (B) the surface adhesins including the surface proteins (clfA, clfB, and fnbA) and the exopolysaccharides PIA/PNAG (icaA and icaD), (C) the membrane fatty acids (fabD, fabF, fabG, and fabH), and (D) sigma factor B (sigB) in S. aureus. ND, not detectable. ^TZ, in the presence of TZ (16 μg/ml). Error bars represent the standard deviations (n = 3). ^∗P < 0.05 (Student’s t-test) indicate significant differences compared to BWSA15.

FIGURE 8

Schematic representation of the cross section of the MV precursor, septum, and mesosome in S. aureus. The MV precursor is characterized by fusion between the MV-like structure and the PM (A); the septum is characterized by cell wall extension along with a non-coiled invagination in the PM (B); and the mesosome is characterized by cell wall infiltration in a folded and coiled invagination in the PM (C). PM, plasma membrane.