Abstract：
OBJECTIVE: Staphylococcus aureus is one of the most common pathogens causing bloodstream infection. A rapid characterisation of resistance to methicillin and, occasionally, to aminoglycosides for particular indications, is therefore crucial to quickly adapt the treatment and improve the clinical outcomes of septic patients. Among analytical technologies, targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a promising tool to detect resistance mechanisms in clinical samples.
METHODS: A rapid proteomic method was developed to detect and quantify the most clinically relevant antimicrobial resistance effectors in S. aureus in the context of sepsis: PBP2a, PBP2c, APH(3\')-III, ANT(4\')-I, and AAC(6\')-APH(2\'\'), directly from positive blood cultures and in less than 70 min including a 30-min cefoxitin-induction step. The method was tested on spiked blood culture bottles inoculated with 124 S.aureus, accounting for the known genomic diversity of SCCmec types and the genetic background of the strains.
RESULTS: This method provided 99% agreement for PBP2a (n = 98/99 strains) detection. Agreement was 100% for PBP2c (n = 5/5), APH(3\')-III (n = 16/16), and ANT(4\')-I (n = 20/20), and 94% for AAC(6\')-APH(2\'\') (n = 16/17). Across the entire strain collection, 100% negative agreement was reported for each of the 5 resistance proteins. Additionally, relative quantification of ANT(4\')-I expression allowed to discriminate kanamycin-susceptible and -resistant strains, in all strains harbouring the ant(4\')-Ia gene.
CONCLUSIONS: The LC-MS/MS method presented herein demonstrates its ability to provide a reliable determination of S. aureus resistance mechanisms, directly from positive blood cultures and in a short turnaround time, as required in clinical laboratories.
METHODS: A rapid proteomic method was developed to detect and quantify the most clinically relevant antimicrobial resistance effectors in S. aureus in the context of sepsis: PBP2a, PBP2c, APH(3\')-III, ANT(4\')-I, and AAC(6\')-APH(2\'\'), directly from positive blood cultures and in less than 70 min including a 30-min cefoxitin-induction step. The method was tested on spiked blood culture bottles inoculated with 124 S.aureus, accounting for the known genomic diversity of SCCmec types and the genetic background of the strains.
RESULTS: This method provided 99% agreement for PBP2a (n = 98/99 strains) detection. Agreement was 100% for PBP2c (n = 5/5), APH(3\')-III (n = 16/16), and ANT(4\')-I (n = 20/20), and 94% for AAC(6\')-APH(2\'\') (n = 16/17). Across the entire strain collection, 100% negative agreement was reported for each of the 5 resistance proteins. Additionally, relative quantification of ANT(4\')-I expression allowed to discriminate kanamycin-susceptible and -resistant strains, in all strains harbouring the ant(4\')-Ia gene.
CONCLUSIONS: The LC-MS/MS method presented herein demonstrates its ability to provide a reliable determination of S. aureus resistance mechanisms, directly from positive blood cultures and in a short turnaround time, as required in clinical laboratories.
摘要：
目的：金黄色葡萄球菌是引起血流感染最常见的病原菌之一。快速表征耐甲氧西林，偶尔,用于特定适应症的氨基糖苷类，因此，对于快速适应治疗和改善败血症患者的临床结局至关重要。在分析技术中，靶向液相色谱-串联质谱(LC-MS/MS)已成为检测临床样品耐药机制的有前景的工具.
方法：开发了一种快速蛋白质组学方法来检测和定量脓毒症中金黄色葡萄球菌中最临床相关的抗微生物耐药性效应物：PBP2a，PBP2c,APH(3')-III,ANT(4\')-I,和AAC(6\')-APH(2\'\')，直接从阳性血液培养物中提取，并在不到70分钟的时间内进行，包括30分钟的头孢西丁诱导步骤。该方法在接种124例金黄色葡萄球菌的加标血培养瓶上进行了测试，考虑SCCmec类型的已知基因组多样性和菌株的遗传背景。
结果：该方法为PBP2a（n=98/99菌株）检测提供了99％的一致性。PBP2c的协议为100%(n=5/5)，APH(3')-III(n=16/16)，和ANT(4')-I(n=20/20)，AAC(6\')-APH(2\'\')(n=16/17)为94%。在整个菌株收集中，对于5种抗性蛋白中的每一种都报告了100%的负一致性。此外,ANT（4'）-I表达的相对定量允许区分卡那霉素敏感和耐药菌株，在所有具有蚂蚁（4'）-Ia基因的菌株中。
结论：本文提供的LC-MS/MS方法证明了其提供可靠的金黄色葡萄球菌耐药机制测定的能力，直接来自阳性血液培养，在很短的周转时间内，根据临床实验室的要求。
方法：开发了一种快速蛋白质组学方法来检测和定量脓毒症中金黄色葡萄球菌中最临床相关的抗微生物耐药性效应物：PBP2a，PBP2c,APH(3')-III,ANT(4\')-I,和AAC(6\')-APH(2\'\')，直接从阳性血液培养物中提取，并在不到70分钟的时间内进行，包括30分钟的头孢西丁诱导步骤。该方法在接种124例金黄色葡萄球菌的加标血培养瓶上进行了测试，考虑SCCmec类型的已知基因组多样性和菌株的遗传背景。
结果：该方法为PBP2a（n=98/99菌株）检测提供了99％的一致性。PBP2c的协议为100%(n=5/5)，APH(3')-III(n=16/16)，和ANT(4')-I(n=20/20)，AAC(6\')-APH(2\'\')(n=16/17)为94%。在整个菌株收集中，对于5种抗性蛋白中的每一种都报告了100%的负一致性。此外,ANT（4'）-I表达的相对定量允许区分卡那霉素敏感和耐药菌株，在所有具有蚂蚁（4'）-Ia基因的菌株中。
结论：本文提供的LC-MS/MS方法证明了其提供可靠的金黄色葡萄球菌耐药机制测定的能力，直接来自阳性血液培养，在很短的周转时间内，根据临床实验室的要求。
