应用改良庆大霉素保护试验检测肠炎沙门氏菌侵袭力表型

A modified gentamicin protection assay for detecting invasive phenotype of Salmonella enterica serovar Enteritidis

摘要:

肠炎血清型沙门氏菌 (Salmonella enterica serovar Enteritidis，SE) 是引起肠炎和全身感染重要的沙门氏菌血清型之一，了解沙门氏菌侵袭力表型对阐明其感染致病机制至关重要。传统庆大霉素保护试验 (GPA) 存在通量低、重复性差等缺点。文中利用96孔细胞培养和多孔道移液的高通量优势，结合菌落分区微量滴板计数法改良了传统GPA试验方案。应用改良的GPA方法检测了16株SE菌株对非吞噬细胞 (HT-29) 的入侵表型和43株SE菌株对吞噬细胞 (RAW264.7) 的胞内复制表型。通过比较分析SE强、弱菌株JL228和LN248对吞噬细胞 (RAW264.7) 的侵袭力表型发现，改良的GPA得出的数据组内和组间变异系数低、数据重复性强，胞内复制表型也与显微观察结果相符。通过实践应用发现，改良的GPA方法具有通量高、重复性强、结果可靠兼具省时、省力等优点，可作为沙门氏菌菌株侵袭力表型检测的更新方案，为进一步阐明其致病机制提供了更科学有效的方法。

Abstract:

Salmonella enterica serovar Enteritidis (SE) is one of the most important zoonotic pathogens that cause enteritis and systemic infection in animals and human. Understanding invasive capacities of SE isolates is of vital importance to elucidate pathogenesis of Salmonella infection. To improve the throughput capacity and repeatability of classical gentamicin protection assay (GPA), a modified PGA was developed by taking high-throughput advantage of 96-well cell plates and multichannel pipettes. In addition, drop plate technique rather than spread plate method was applied in the modified GPA protocol for bacterial enumeration. The modified GPA protocol was evaluated by phenotyping intracellular replication of a high virulent and a low virulent SE isolates, JL228 and LN248, in a phagocytic cell line RAW264.7. The protocol was then applied in invasive phenotype determination of 16 SE strains to non-phagocytes (HT-29) and the intracellular replication of 43 SE strains to phagocytes (RAW264.7). Significant lower intra-group and inter-group coefficient of variations of the modified GPA was observed, implying good repeatability and reproducibility over traditional protocol. Further, replication phenotypes were also correlated with those from direct observation by confocal microscopy. Collectively, the improved GPA protocol had advantages of high throughput capacity, good repeatability and reliability, it was also noticed that the protocol also represented a fast and labor-saving alternative scheme for the invasive phenotype determination of Salmonella Enteritidis, and providing reliable phenotype profiles for Salmonella-host interplay interpretation.