背景:耐抗生素革兰氏阴性菌在全球范围内受到越来越多的关注,尤其是那些产生抗性的酶。OXA-48样碳青霉烯酶水解大多数β-内酰胺抗生素,通常碳青霉烯类的低水平水解,但对广谱头孢菌素的作用有限。这些经常与广谱β-内酰胺酶共表达,特别是CTX-M-15,它通常对广谱头孢菌素表现出高水平的耐药性,然而碳青霉烯类易感。组合的抗性概况使得需要成功检测这些特定抗性决定因素对于有效的抗生素治疗是必要的。
目的:本研究的目的是使用质谱检测和鉴定OXA-48样和CTX-M-15酶,并随后开发了一种与液相色谱法结合检测两种酶的方法。
方法:收获在肉汤或琼脂上生长的细胞,lysed,and,在某些情况下,缓冲交换。分离由细菌细胞产生的裂解物,并通过液相色谱-质谱(LC-MS)和串联质谱(LC-MS/MS)进行分析。
结果:表征并检测OXA-48、OXA-181和OXA-232(本文统称为OXA-48样)和CTX-M-15的完整蛋白质。建立基于来自每个蛋白质组的序列信息片段的接受标准作为蛋白质存在的确认标记。共有25个分离株成功检测了OXA-48样(2),CTX-M-15(3),或两种(7)酶的表达。13个分离株作为阴性对照。
结论:在这里,我们提出了一种使用LC-MS/MS直接和独立检测OXA-48样碳青霉烯酶和CTX-M-15β-内酰胺酶的方法。MS/MS的增加的灵敏度允许同时检测至少两种共洗脱,来自单一质谱的共分离和共片段化蛋白质。
BACKGROUND: Antibiotic-resistant Gram-negative bacteria are of a growing concern globally, especially those producing enzymes conferring resistance. OXA-48-like carbapenemases hydrolyze most β-lactam antibiotics, with typically low-level hydrolysis of carbapenems, but have limited effect on broad-spectrum cephalosporins. These are frequently co-expressed with extended spectrum β-lactamases, especially CTX-M-15, which typically shows high level resistance to broad-spectrum cephalosporins, yet is carbapenem susceptible. The combined resistance profile makes the need for successful detection of these specific resistance determinants imperative for effective antibiotic therapy.
OBJECTIVE: The objective of this study is to detect and identify OXA-48-like and CTX-M-15 enzymes using mass spectrometry, and to subsequently develop a method for detection of both enzyme types in combination with liquid chromatography.
METHODS: Cells grown in either broth or on agar were harvested, lysed, and, in some cases buffer-exchanged. Lysates produced from bacterial cells were separated and analyzed via liquid chromatography with mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS).
RESULTS: The intact proteins of OXA-48, OXA-181, and OXA-232 (collectively OXA-48-like herein) and CTX-M-15 were characterized and detected. Acceptance criteria based on sequence-informative fragments from each protein group were established as confirmatory markers for the presence of the protein(s). A total of 25 isolates were successfully tested for OXA-48 like (2), CTX-M-15 (3), or expression of both (7) enzymes. Thirteen isolates served as negative controls.
CONCLUSIONS: Here we present a method for the direct and independent detection of both OXA-48-like carbapenemases and CTX-M-15 β-lactamases using LC-MS/MS. The added sensitivity of MS/MS allows for simultaneous detection of at least two co-eluting, co-isolated and co-fragmented proteins from a single mass spectrum.