Abstract
We investigated the drug-resistant mechanisms of intracellular survival of methicillin-resistant S. aureus (MRSA). Our established MRSA clinical strain, OJ-1, with high biofilm-forming ability, and a macrophage cell line, J774A, were used. After ingestion of OJ-1 by J774A, the cells were incubated for ten days with vancomycin at doses 30 times higher than the minimum inhibitory concentration. The number of phagocytosed intracellular OJ-1 gradually decreased during the study but plateaued after day 7. In J774A cells with intracellular OJ-1, the expression of LysoTracker-positive lysosomes increased until day 5 and then declined from day 7. In contrast, LysoTracker-negative and OJ-1-retaining J774A cells became prominent from day 7, and intracellular OJ-1 also escaped from the autophagosome. Electron microscopy also demonstrated that OJ-1 escaped the phagosomes and was localized in the J774A cytoplasm. At the end of incubation, when vancomycin was withdrawn, OJ-1 started to grow vigorously. The present results indicate that intracellular phagocytosed biofilm-forming MRSA could survive for more than ten days by escaping the lysosomes and autophagosomes in macrophages. Intracellular MRSA may survive in macrophages, and accordingly, they could be resistant to antimicrobial drug treatments. However, the mechanisms their escape from the lysosomes are still unknown. Additional studies will be performed to clarify the lysosome-escaping mechanisms of biofilm-forming MRSA.
Highlights
The Dulbecco’s modified Eagle’s medium–low glucose (DMEM)-based growth medium was used for both J774A and OJ-1
We used a biofilm-forming methicillinresistant S. aureus (MRSA) clinical strain OJ-1 and a macrophage cell line J774A to investigate intracellular survival of OJ-1 incubated with overdosed vancomycin
The results suggest that in patients with MRSA wound infection, which could be treated with debridement and antibacterial drugs, bacteria phagocytosed by macrophages in wounds may survive even after antibacterial therapy
Summary
Bacteria that invade tissues are sensed, ingested, and killed by professional phagocytes, including macrophages and neutrophils [1,2]. Bacteria are recognized as a foreign body by the scavenger receptor in macrophages and endocytosed in the endosomes formed by invaginating the cell membrane, namely, phagosomes [2]. NADPH oxidase on the membrane generates radical oxygen species in the phagosome. The phagosome fuses with the lysosome, forming a phagolysosome, and under acidic conditions, hydrogen peroxide and hydroxyl radicals are produced to kill the bacteria. The phagolysosomes possess acid phosphatase and proteases, which participate in digesting the bacteria [3]. The host cells utilize an autophagy system that eliminates surviving intracellular bacteria [4].
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