McGee William M, Verma Arvind, Viirtola Marjaana, Kronewitter Scott R, Neil Jason R, Stephenson James L
Thermo Fisher Scientific, Cambridge, MA, USA.
Thermo Fisher Scientific, Vantaa, Finland.
J Mass Spectrom Adv Clin Lab. 2021 May 29;20:25-34. doi: 10.1016/j.jmsacl.2021.05.001. eCollection 2021 Apr.
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.
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.
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).
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.
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.
耐抗生素革兰氏阴性菌在全球范围内日益受到关注，尤其是那些产生耐药性酶的细菌。OXA-48样碳青霉烯酶可水解大多数β-内酰胺抗生素，对碳青霉烯类抗生素的水解作用通常较弱，但对广谱头孢菌素的作用有限。这些酶常与超广谱β-内酰胺酶共同表达，尤其是CTX-M-15，它通常对广谱头孢菌素表现出高水平耐药性，但对碳青霉烯类敏感。这种联合耐药模式使得成功检测这些特定耐药决定因素对于有效的抗生素治疗至关重要。
本研究的目的是使用质谱法检测和鉴定OXA-48样和CTX-M-15酶，并随后开发一种结合液相色谱法检测这两种酶的方法。
收集在肉汤或琼脂上生长的细胞，进行裂解，在某些情况下进行缓冲液置换。细菌细胞产生的裂解物通过液相色谱-质谱联用（LC-MS）和串联质谱（LC-MS/MS）进行分离和分析。
对OXA-48、OXA-181和OXA-232（本文统称为OXA-48样）以及CTX-M-15的完整蛋白进行了表征和检测。基于每个蛋白组的序列信息片段建立了验收标准，作为蛋白存在的确认标记。总共对25株分离株成功检测了OXA-48样（2株）、CTX-M-15（3株）或两种酶的表达（7株）。13株分离株作为阴性对照。
在此，我们提出了一种使用LC-MS/MS直接独立检测OXA-48样碳青霉烯酶和CTX-M-15β-内酰胺酶的方法。MS/MS增加的灵敏度允许从单个质谱图中同时检测至少两种共洗脱、共分离和共裂解的蛋白。
