Department of Biomedical Sciences and Pathobiology, Life Sciences 1, 970 Washington Street, SW, Virginia Tech, Blacksburg, VA 24061, USA.
Department of Physics, 850 West Campus Drive, Virginia Tech, Blacksburg, VA 24061, USA.
Biosens Bioelectron. 2015 Aug 15;70:433-40. doi: 10.1016/j.bios.2015.03.041. Epub 2015 Mar 30.
Methicillin-resistance among Staphylococcus species is a major health problem in hospitals, communities, and animals. There is a need for culture-free diagnostic assays that can be carried out rapidly, and maintain a high degree of sensitivity and specificity. To address this need an ionic self-assembled multilayer (ISAM) film was deposited on the surface of a long-period grating (LPG) optical fiber by immersion alternately in poly-allylamine hydrochloride and in poly-1-[p-(3'-carboxy-4'-hydroxyphenylazo) benzenesulfonamido]-1,2-ethandiyl (PCBS), resulting in terminal carboxyl groups on the LPG-ISAM. The terminal carboxyl groups were covalently conjugated to monoclonal antibodies (MAb) specific to penicillin-binding-protein 2a of methicillin resistant (MR) staphylococci. After exposure of the LPG-ISAM to 10(2) colony forming units (CFU)/ml of MR S. aureus (MRSA) for 50 min., light transmission was reduced by 19.7%. In contrast, after exposure to 10(6) CFU/ml of methicillin-sensitive S. aureus (MSSA) attenuation of light transmission was less than 1.8%. Exposure of the LPG-ISAM to extracts of liver, lungs, or spleen from mice infected with MRSA attenuated light transmission by 11.7-73.5%. In contrast, exposure of the biosensor to extracts from MSSA-infected mice resulted in 5.6% or less attenuation of light transmission. When the sensor was tested with 36 strains of MR staphylococci, 15 strains of methicillin-sensitive staphylococci, 10 strains of heterologous genera (all at 10(4) CFU/ml), or tissue samples from mice infected with MRSA, there was complete agreement between MR and non-MR bacteria determined by antibiotic susceptibility testing and the biosensor assay when the cutoff value for attenuation of light transmission was 6.3%. Thus, the biosensor described has the potential to detect MR staphylococci in clinical samples with a high degree of sensitivity and specificity.
耐甲氧西林金黄色葡萄球菌是医院、社区和动物中的主要健康问题。需要能够快速进行的无培养诊断检测方法，并且保持高度的灵敏度和特异性。为了满足这一需求，通过在聚烯丙基盐酸盐和聚 1-[p-(3'-羧基-4'-羟基苯偶氮)苯磺酰胺基]-1,2-乙二基（PCBS）中交替浸泡，在长周期光纤（LPG）的表面上沉积了离子自组装多层（ISAM）薄膜，导致 LPG-ISAM 上的末端羧基。末端羧基通过共价键与针对耐甲氧西林金黄色葡萄球菌（MRSA）的青霉素结合蛋白 2a 的单克隆抗体（MAb）偶联。将 LPG-ISAM 暴露于 10（2）个菌落形成单位（CFU）/ml 的耐甲氧西林金黄色葡萄球菌（MRSA）50 分钟后，透光率降低了 19.7%。相比之下，将 LPG-ISAM 暴露于 10（6）个 CFU/ml 的甲氧西林敏感金黄色葡萄球菌（MSSA）后，透光率的衰减小于 1.8%。将 LPG-ISAM 暴露于从感染耐甲氧西林金黄色葡萄球菌的小鼠的肝脏、肺部或脾脏提取的提取物中，透光率降低了 11.7-73.5%。相比之下，将生物传感器暴露于从 MSSA 感染的小鼠提取的提取物中，透光率的衰减小于 5.6%。当该传感器用 36 株耐甲氧西林金黄色葡萄球菌、15 株甲氧西林敏感金黄色葡萄球菌、10 株异源属（均为 10（4）CFU/ml）或感染耐甲氧西林金黄色葡萄球菌的小鼠的组织样本进行测试时，通过抗生素药敏试验和生物传感器检测确定的耐甲氧西林和非耐甲氧西林细菌之间完全一致当透光率衰减的截止值为 6.3%时。因此，该生物传感器具有在临床样本中以高灵敏度和特异性检测耐甲氧西林金黄色葡萄球菌的潜力。
