Department of Orthopaedic Surgery, Duke University, Durham, NC, USA.
Clin Orthop Relat Res. 2024 Oct 1;482(10):1839-1847. doi: 10.1097/CORR.0000000000003074. Epub 2024 Apr 23.
Recurrent bone and joint infection with Staphylococcus aureus is common. S. aureus can invade and persist in osteoblasts and fibroblasts, but little is known about this mechanism in chondrocytes. If S. aureus were able to invade and persist within chondrocytes, this could be a difficult compartment to treat.
QUESTION/PURPOSE: Can S. aureus infiltrate and persist intracellularly within chondrocytes in vitro?
Cell lines were cultured in vitro and infected with S. aureus. Human chondrocytes (C20A4) were compared with positive controls of human osteoblasts (MG63) and mouse fibroblasts (NIH3T3), which have previously demonstrated S. aureus invasion and persistence (human fibroblasts were not available to us). Six replicates per cell type were followed for 6 days after infection. Cells were treated daily with antibiotic media for extracellular killing. To determine whether S. aureus can infiltrate chondrocytes, fluorescence microscopy was performed to qualitatively assess the presence of intracellular bacteria, and intracellular colony-forming units (CFU) were enumerated 2 hours after infection. To determine whether S. aureus can persist within chondrocytes, intracellular CFUs were enumerated from infected host cells each day postinfection.
S. aureus invaded human chondrocytes (C20A4) at a level (2.8 x 10 5 ± 5.5 x 10 4 CFUs/mL) greater than positive controls of human osteoblasts (MG63) (9.5 x 10 2 ± 2.5 x 10 2 CFUs/mL; p = 0.01) and mouse fibroblasts (NIH3T3) (9.1 x 10 4 ± 2.5 x 10 4 CFUs/mL; p = 0.02). S. aureus also persisted within human chondrocytes (C20A4) for 6 days at a level (1.4 x 10 3 ± 5.3 x 10 2 CFUs/mL) greater than that of human osteoblasts (MG63) (4.3 x 10 2 ± 3.5 x 10 1 CFUs/mL; p = 0.02) and mouse fibroblasts (NIH3T3) (0 CFUs/mL; p < 0.01). S. aureus was undetectable within mouse fibroblasts (NIH3T3) after 4 days. There were 0 CFUs yielded from cell media, confirming extracellular antibiotic treatment was effective.
S. aureus readily invaded human chondrocytes (C20A4) in vitro and persisted viably for 6 days after infection, evading extracellular antibiotics. Chondrocytes demonstrated a greater level of intracellular invasion and persistence by S. aureus than positive control human osteoblast (MG63) and mouse fibroblast (NIH3T3) cell lines.
Chondrocyte invasion and persistence may contribute to recurrent bone and joint infections. Additional research should assess longer periods of persistence and whether this mechanism is present in vivo.
金黄色葡萄球菌引起的复发性骨和关节感染很常见。金黄色葡萄球菌可以侵入并在成骨细胞和纤维母细胞中持续存在，但对于软骨细胞中的这种机制知之甚少。如果金黄色葡萄球菌能够侵入并在软骨细胞内持续存在，那么这将是一个难以治疗的部位。
问题/目的：金黄色葡萄球菌能否在体外渗透并在软骨细胞内持续存在？
体外培养细胞系并感染金黄色葡萄球菌。将人软骨细胞（C20A4）与先前已证明具有金黄色葡萄球菌侵袭和持续存在能力的阳性对照物（人成骨细胞（MG63）和小鼠成纤维细胞（NIH3T3））进行比较。（我们没有获得人成纤维细胞）。在感染后 6 天内，每细胞类型进行 6 次重复。每天用抗生素培养基处理细胞以杀死细胞外的细菌。为了确定金黄色葡萄球菌是否可以渗透到软骨细胞中，通过荧光显微镜定性评估了细胞内细菌的存在，并且在感染后 2 小时测定了细胞内的菌落形成单位（CFU）。为了确定金黄色葡萄球菌是否可以在软骨细胞内持续存在，在感染后的每一天都从感染的宿主细胞中测定细胞内 CFU。
金黄色葡萄球菌侵入人软骨细胞（C20A4）的水平（2.8 x 10 5 ± 5.5 x 10 4 CFUs/mL）高于阳性对照物的人成骨细胞（MG63）（9.5 x 10 2 ± 2.5 x 10 2 CFUs/mL；p = 0.01）和小鼠成纤维细胞（NIH3T3）（9.1 x 10 4 ± 2.5 x 10 4 CFUs/mL；p = 0.02）。金黄色葡萄球菌在人软骨细胞（C20A4）内持续存在 6 天的水平（1.4 x 10 3 ± 5.3 x 10 2 CFUs/mL）高于人成骨细胞（MG63）（4.3 x 10 2 ± 3.5 x 10 1 CFUs/mL；p = 0.02）和小鼠成纤维细胞（NIH3T3）（0 CFUs/mL；p < 0.01）。在第 4 天，在小鼠成纤维细胞（NIH3T3）中未检测到金黄色葡萄球菌。细胞培养基中没有 CFU 产生，这证实了细胞外抗生素治疗是有效的。
金黄色葡萄球菌在体外容易侵入人软骨细胞（C20A4），并在感染后持续存活 6 天，逃避了细胞外抗生素的作用。软骨细胞中金黄色葡萄球菌的细胞内侵袭和持续存在水平高于阳性对照物的人成骨细胞（MG63）和小鼠成纤维细胞（NIH3T3）细胞系。
软骨细胞的侵袭和持续存在可能导致复发性骨和关节感染。应进行进一步研究以评估更长时间的持续存在情况以及该机制是否存在于体内。
