Ruiz-Pérez de Pipaón Maite, Torres-Sánchez María José, Arroyo-Pedrero Luis Antonio, Prados-Blanco Trinidad, Palomares-Folía José Carlos, Aznar-Martín Javier
Servicio de Microbiología, Hospital Universitario Virgen del Rocío, Sevilla, Spain.
Enferm Infecc Microbiol Clin. 2005 Apr;23(4):208-12. doi: 10.1157/13073146.
The aim of this study was to evaluate the feasibility of detecting Staphylococcus aureus and coagulase-negative staphylococci (CoNS) and of identifying methicillin resistance directly in positive BACTEC blood culture bottles using the LightCycler system.
One hundred thirty-one positive blood culture bottles in which Gram-positive cocci in cluster were observed after Gram staining and 40 positive bottles with microorganisms other than staphylococci were studied. A molecular assay based on an automated DNA extraction protocol with a MagNA Pure LC instrument was used. Oligonucleotide primers and fluorescence-labeled hybridization probes were designed for amplification and sequence-specific detection of both a 408-pb fragment within the mecA gene and a 279-pb fragment within the S. aureus-specific nucA gene.
All the bottles that yielded methicillin-resistant S. aureus (MRSA), methicillin-sensitive S. aureus (MSSA) or methicillin-resistant CoNS (MRCoNS) strains were correctly identified by the nucA and mecA PCR assays. One bottle that yielded a mixed culture of MSSA and MRCoNS gave positive results for both genes. In the 21 bottles with methicillin-susceptible CoNS (MSCoNS), nucA PCR were negative, but two of these bottles gave positive results for the mecA gene. The sensitivity and specificity of the nucA gene assay were 100%. The sensitivity and specificity of the PCR assay for detection of methicillin resistance with the mecA gene were 100% and 97.5%, respectively.
This is a sensitive and highly specific method for identifying staphylococci in positive blood cultures, allowing discrimination between methicillin-susceptible and -resistant strains in less than 3 hours after Gram stain.
本研究的目的是评估使用LightCycler系统直接在阳性BACTEC血培养瓶中检测金黄色葡萄球菌和凝固酶阴性葡萄球菌（CoNS）以及鉴定耐甲氧西林情况的可行性。
对131瓶革兰氏染色后观察到呈葡萄串状革兰氏阳性球菌的阳性血培养瓶以及40瓶含有除葡萄球菌以外微生物的阳性瓶进行了研究。使用了基于MagNA Pure LC仪器自动DNA提取方案的分子检测方法。设计了寡核苷酸引物和荧光标记杂交探针，用于扩增和序列特异性检测mecA基因内的408 pb片段以及金黄色葡萄球菌特异性nucA基因内的279 pb片段。
所有培养出耐甲氧西林金黄色葡萄球菌（MRSA）、甲氧西林敏感金黄色葡萄球菌（MSSA）或耐甲氧西林CoNS（MRCoNS）菌株的培养瓶均通过nucA和mecA PCR检测得到正确鉴定。一瓶培养出MSSA和MRCoNS混合培养物的培养瓶对两个基因均呈阳性结果。在21瓶培养出甲氧西林敏感CoNS（MSCoNS）的培养瓶中，nucA PCR为阴性，但其中两瓶mecA基因呈阳性结果。nucA基因检测的敏感性和特异性均为100%。用于检测mecA基因介导的甲氧西林耐药性的PCR检测的敏感性和特异性分别为100%和97.5%。
这是一种用于鉴定阳性血培养中葡萄球菌的灵敏且高度特异的方法，能够在革兰氏染色后不到3小时区分甲氧西林敏感和耐药菌株。
