Bacterial Membrane-Derived Vesicles Attenuate Vancomycin Activity against Methicillin-Resistant Staphylococcus aureus

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved numerous antimicrobial resistance mechanisms and is identified as a serious public health threat by the World Health Organization and U.S. Centers for Disease Control and Prevention. The glycopeptide vancomycin (VAN) remains a cornerstone of therapy for severe MRSA infections despite increasing reports of therapeutic failure in hospitalized patients with bacteremia or pneumonia. Recently, the role of released bacterial-derived membrane vesicles (MVs) in antibiotic resistance has garnered attention. Here we examined the effect of exogenous MRSA-derived MVs on VAN activity against MRSA in vitro, using minimum inhibitory concentration and checkerboard assays, and ex vivo, incorporating components of host innate immunity such as neutrophils and serum complement present in blood. Additionally, the proteome of MVs from VAN-exposed MRSA was characterized to determine if protein expression was altered. The presence of MVs increased the VAN MIC against MRSA to values where clinical failure is commonly observed. Furthermore, the presence of MVs increased survival of MRSA pre-treated with sub-MIC concentrations of VAN in whole blood and upon exposure to human neutrophils but not human serum. Unbiased proteomic analysis also showed an elevated expression of MV proteins associated with antibiotic resistance (e.g., marR) or proteins that are functionally linked to cell membrane/wall metabolism. Together, our findings indicate MRSA-derived MVs are capable of lowering susceptibility of the pathogen to VAN, whole-blood- and neutrophil-mediated killing, a new pharmacodynamic consideration for a drug increasingly linked to clinical treatment failures.

Gram-positive bacterial membrane lipids at the host–pathogen interface

“…Multiple factors such as membrane fluidity, proteins, and large genetic networks are shown to influence MV production [ 24 ]. Even though MV production is ubiquitous; factors like membrane stress by antimicrobial peptides, growth phase changes, and temperature have been shown to alter the amount of MVs produced and the type of cargo packaged, thus improving the bacteria’s ability to survive in harsh conditions and aiding disease progression [ 24 , 26 – 33 ].…”

The role of bacterial membrane vesicles in antibiotic resistance

“…Subinhibitory exposure to vancomycin increases MV production in S. aureus 50 . Interestingly, the coincubation of MVs from S. aureus with vancomycin resulted in a two‐ to four‐fold increase in S. aureus resistance 48 . The exact mechanism for this increased resistance is unclear.…”

“…However, MVs from Gram-positive bacteria have been shown to suppress the activity of the antibiotic vancomycin. 48 The spread of methicillin-resistant S. aureus (MRSA) has made vancomycin a critical therapeutic option to treat multidrugresistant Gram-positive infections. Vancomycin inhibits growth by binding to the terminal D-ala-D-ala portion of the cell wall during synthesis, preventing PG crosslinking, which eventually causes cell death.…”

“…50 Interestingly, the coincubation of MVs from S. aureus with vancomycin resulted in a two-to four-fold increase in S. aureus resistance. 48 The exact mechanism for this increased resistance is unclear. However, Gram-positive MVs can contain upward of 50% PG, 51 and vancomycin is known to bind PG.…”

“…Gram‐positive bacteria are naturally resistant to outer membrane targeting compounds, and it is unlikely that MVs would suppress antibiotic activity due to the lack of LPS. However, MVs from Gram‐positive bacteria have been shown to suppress the activity of the antibiotic vancomycin 48 . The spread of methicillin‐resistant S. aureus (MRSA) has made vancomycin a critical therapeutic option to treat multidrug‐resistant Gram‐positive infections.…”