Mathoera Rejiv B, Kok Dik J, Verduin Cees M, Nijman Rien J M
Subdivision of Pediatric Urology, Department of Urology, Sophia Children's Hospital, Erasmus Medical Center, 3000 DR Rotterdam, The Netherlands.
Infect Immun. 2002 Dec;70(12):7022-32. doi: 10.1128/IAI.70.12.7022-7032.2002.
Proteus mirabilis infection often leads to stone formation. We evaluated how bacterium-mucin adhesion, invasion, and intracellular crystal formation are related to antibiotic sensitivity and may cause frequent stone formation in enterocystoplasties. Five intestinal (Caco-2, HT29, HT29-18N2, HT29-FU, and HT29-MTX) and one ureter cell line (SV-HUC-1) were incubated in artificial urine with five Proteus mirabilis strains. Fluorescence-activated cell sorting (FACS), laser scanning microscopy, and electron microscopy evaluated cellular adhesion and/or invasion, pathologic changes to mitochondria, and P. mirabilis-mucin colocalization (MUC2 and MUC5AC). An MTT (thiazolyl blue tetrazolium bromide) assay and FACS analysis of caspase-3 evaluated the cellular response. Infected cells were incubated with antibiotics at dosages representing the expected urinary concentrations in a 10-year-old, 30-kg child to evaluate bacterial invasion and survival. All cell lines showed colocalization of P. mirabilis with human colonic mucin (i.e., MUC2) and human gastric mucin (i.e., MUC5AC). The correlation between membrane mucin expression and invasion was significant and opposite for SV-HUC-1 and HT29-MTX. Microscopically, invasion by P. mirabilis with intracellular crystal formation and mitochondrial damage was found. Double membranes surrounded bacteria in intestinal cells. Relative resistance to cotrimoxazole and augmentin was found in the presence of epithelial cells. Ciprofloxacin and gentamicin remained effective. Membrane mucin expression was correlated with relative antibiotic resistance. Cell invasion by P. mirabilis and mucin- and cell type-related distribution and response differences indicate bacterial tropism that affects crystal formation and mucosal presence. Bacterial invasion seems to have cell type-dependent mechanisms and prolong bacterial survival in antibiotic therapy, giving a new target for therapeutic optimalization of antibiotic treatment.
奇异变形杆菌感染常导致结石形成。我们评估了细菌与粘蛋白的粘附、侵袭及细胞内晶体形成与抗生素敏感性之间的关系，以及它们如何在肠囊成形术中导致频繁的结石形成。将五种肠道细胞系（Caco-2、HT29、HT29-18N2、HT29-FU和HT29-MTX）和一种输尿管细胞系（SV-HUC-1）与五种奇异变形杆菌菌株在人工尿液中共同孵育。通过荧光激活细胞分选（FACS）、激光扫描显微镜和电子显微镜评估细胞粘附和/或侵袭、线粒体的病理变化以及奇异变形杆菌与粘蛋白的共定位（MUC2和MUC5AC）。采用MTT（噻唑蓝四氮唑溴盐）法和caspase-3的FACS分析评估细胞反应。将感染细胞与代表一名10岁、30公斤儿童预期尿液浓度的抗生素剂量孵育，以评估细菌的侵袭和存活情况。所有细胞系均显示奇异变形杆菌与人结肠粘蛋白（即MUC2）和人胃粘蛋白（即MUC5AC）共定位。SV-HUC-1和HT29-MTX的膜粘蛋白表达与侵袭之间的相关性显著且相反。显微镜下发现奇异变形杆菌侵袭并伴有细胞内晶体形成和线粒体损伤。肠道细胞中的细菌被双层膜包围。在上皮细胞存在的情况下，发现对复方新诺明和阿莫西林/克拉维酸相对耐药。环丙沙星和庆大霉素仍然有效。膜粘蛋白表达与相对抗生素耐药性相关。奇异变形杆菌的细胞侵袭以及与粘蛋白和细胞类型相关的分布及反应差异表明细菌具有嗜性，这会影响晶体形成和黏膜存在。细菌侵袭似乎具有细胞类型依赖性机制，并在抗生素治疗中延长细菌存活时间，为优化抗生素治疗提供了新的靶点。
