Direct Detection of Carbapenemase-associated Proteins of Acinetobacter Baumannii Using Nanodiamonds and Mass Spectrometry
Kai-Chih Chang2,3, Chin-Yi Chung1, Chen-Hsing Yeh2, Kuo-Hsiu Hsu1, Ya-Ching Chin1, Sin-Siang Huang1, Bo-Rong Liu1, Hsi-An Chen1, Anren Hu2, Po-Chi Soo2, Wen-Ping Peng1*
1Department of Physics, National Dong Hwa University, Hualien, Taiwan
2Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien, Taiwan
3Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan
* Presenter:Wen-Ping Peng,
The appearance and spread of carbapenem-resistant Acinetobacter baumannii (CRAB) pose a challenge for optimization of antibiotic therapies and outbreak preventions. Traditional phenotypic assays such as the Modified Hodge Test (MHT) or polymerase chain reaction-based detection of the carbapenemase genes are time-consuming and complicated. Therefore, new approaches to detect carbapenemase-producing A. baumannii are of great importance. Here, we have developed a rapid and novel method using detonation nanodiamonds (DNDs) as a platform for concentration and extraction of A. baumannii carbapenemase-associated proteins prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF-MS) analysis. To concentrate and extract the A. baumannii carbapenemase-associated proteins, we tested several protein precipitation conditions and found a 0.5% trifluoroacetic acid (TFA) solution within the bacterial suspension could result in good ion signals with DNDs. A total of 66 A. baumannii clinical-isolates including 51 carbapenem-resistant strains and 15 carbapenem-susceptible strains were tested. Our result showed that among the 51 carbapenem-resistant strains 49 strains had a signal at m/z ~ 40347; among the 15 carbapenem-susceptible strains, 4 strains showed a signal at m/z ~ 40347. With on-diamond digestion, we confirmed the captured protein at m/z ~ 40347 was related to ADC family extended-spectrum class C beta-lactamase, from A. baumannii. Using this ADC family protein as a biomarker (m/z ~ 40347) for carbapenem susceptibility test of A. baumannii, the sensitivity and the specificity could reach 96% and 73% as compared to traditional imipenem susceptibility testing (MIC results). However, the sensitivity and specificity of this method could reach 100% as compared to polymerase chain reaction (PCR) result. Our approach could directly detect the carbapenemase-associated proteins of A. baumannii within 90 minutes and did not need to add any carbapenemase substrate which was required in the MHT or other mass spectrometric methods. For future applications, our method could be efficiently used in the detection of other carbapenemase-producing bacteria.

Keywords: Carbapenemase-associated Proteins, Acinetobacter baumannii, Nanodiamond, MALDI-TOF MS, biomarker