Rapid Identification of Carbapenem-Resistant Klebsiella pneumoniae and Carbapenemase Genes via PTR-MS

Xuan Teng,1,2 Kexue Yu,1,2 Qi Zhang,3 Chengcheng Ma,1,2 Wenwen Chu,2 Chengyin Shen,3 Zhou Liu1 1Department of Clinical Laboratory Center, Anhui Chest Hospital, Hefei, 230031, People’s Republic of China; 2Department of Clinical Laboratory, the Second Affiliated Hospital of Anhui Medical University, Hefei, 230601, People’s Republic of China; 3Anhui Province Key Laboratory of Medical Physics and Technology, Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, People’s Republic of ChinaCorrespondence: Zhou Liu, Email [email protected] Chengyin Shen, Email [email protected]: To systematically analyse and identify volatile organic compounds (VOCs) released by clinical Klebsiella pneumoniae (K. pneumoniae) during growth via proton transfer reaction–mass spectrometry (PTR-MS), aiming to establish a rapid and accurate method for differentiating and identifying carbapenem-resistant Klebsiella pneumoniae (CRKP) and KPC and NDM producers.Methods: Nonrepetitive clinical strains isolated from patient specimens were collected from September 2021 to May 2025. The strains were subjected to drug susceptibility testing and carbapenemase genotype identification via the VITEK2 system and polymerase chain reaction (PCR). The clinical strains were incubated in a closed system under the combined pressure of meropenem (MEM) and carbapenemase inhibitors for 3 h. PTR-MS was used to monitor the inhibition rate of the characteristic VOC ion signal intensity to obtain drug susceptibility information of KP and carbapenemase type. Characteristic ions were characterized via fast gas chromatography (FGC)-PTR-MS.Results: A total of 105 clinical isolates, including 53 carbapenem-susceptible Klebsiella pneumoniae (CSKP) isolates and 52 CRKP (43 KPC-positive and 9 NDM-positive) isolates, were collected. With MEM, the sensitivity and specificity of PTR-MS for monitoring CRKP were 98.08% and 100.00%, respectively. In the case of MEM combined with different carbapenemase inhibitors, the assay was evaluated using a subset of isolates (n=31), comprising 22 KPC-positive and 9 NDM-positive strains. The sensitivity and specificity of PTR-MS for monitoring single KPC producers were 90.91% and 100.00%, respectively, and those for single NDM producers were 88.89% and 100.00%, respectively (kappa= 0.853 and 0.919 for KPC- and NDM-positive strains, respectively). FGC–PTR-MS analysis indicated that the VOCs corresponding to these characteristic ions were acetaldehyde, ethanol and acetic acid.Conclusion: Real-time monitoring by PTR-MS of the dynamic release characteristics of specific VOC ions in the headspace of CRKP within 3 h under the combined stress of antibiotics and carbapenemase inhibitors can provide important information for rapidly identifying CRKP and the main clinical carbapenemase types.Keywords: proton transfer reaction-mass spectrometry, volatile organic compounds, K. pneumoniae, meropenem, carbapenemase inhibitors