Evaluation of rapid carbapenemase producers detection method based on MALDI-TOF MS spectra analysis, and molecular and epidemiological characterization of multidrug-resistant Enterobacteriaceae

The occurrence and rapid spreading of multi-resistant bacteria is an important issue of public health, which is increasing worldwide over the last two decades. β-lactams-resistant Enterobacteriaceae, with particular reference to carbapenems, are actually one of the main concerns at international level, as their resistance is often plasmid-mediated, thus rapidly spread and often associated with hospital outbreaks with high rates of mortality. Clinical microbiology labs, especially in hospital settings, require to quickly identifying patients who carry multi-resistant bacterial strains, especially carbapenemase producers, in order to contain their spreading, preventing epidemic outbreaks and rapidly address proper pharmacological therapy. Research is moving towards development of new diagnostic tools to speed up identification of multi-resistant strains in clinical specimens, thus drastically shortening the time necessary to obtain analysis reports. Rapid identification of colonized patients, in fact, is the only valid strategy to curb epidemic spreading, mainly when is due to plasmid-mediated resistant determinants. The objective of this study is to validate the use of Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) for quick identification of carbapenemase-producing Enterobacteria strains, thus confirming that a characteristic MS peak (11,109Da±8) is related to Klebsiella pneumoniae carbapenemase (KPC) production. Moreover, the presence of other peaks related to NDM- (New Delhi metallo-β-lactamase) and VIM- (Verona integron–encoded metallo-β-lactamase) carbapenemases as well as the evaluation of MALDI-TOF MS as a tool to quickly identify epidemic hotbeds, were investigated. At this aim, selected clinical samples KPC-producing and control samples carbapenem-susceptible or carbapenem-resistant through other resistance mechanisms or producing hydrolytic enzymes other than KPC (NDM and VIM) were analyzed. The presence of the 11,109Da peak in 98% (225/230) of the KPC-producing strains compared to the controls, which lacked the peak, confirmed strong correlation between KPC production and the presence of the 11,109Da peak in MALDI-TOF MS spectrum. The reliability of the method was also verified by searching for this specific peak during routine workflow, analysing the MALDI-TOF MS spectra of 183 patient samples isolated during multidrug-resistant (MDR) screening. The positive correlation between the presence of 11,109Da peak and Carba NP test confirmed the reliability of MALDI-TOF MS analysis as rapid and inexpensive screening method for carbapenemase-producing strains, apt to investigate a high number of samples in KPC-endemic context. In this way, the molecular methods more expensive and difficult to perform during routine workflow, can be used to resolve discrepant strains only. We did not find, instead, any correlation between other peaks and carbapenemases other than KPC, namely NDM and VIM, through the analysis of the spectra of 15 NDM-producing Enterobacteria strains and 23 Enterobacteria strains and 13 P. aeruginosa strains VIM producers. This result is in line with the high number of plasmids harboring the genes codifying for these enzymes. Finally, the use of MALDI-TOF MS was found not particularly promising as tool to identify clonal relationship between bacterial strains as compared to Pulsed-Field Gel Electrophoresis (PFGE) using 44 K. pneumoniae strains isolated during an epidemic outbreak. Drawbacks could be related to the current software. We therefore confirmed MALDI-TOF MS system is a good tool to quickly and accurate identifying carbapenemase-producing bacterial strains, that represents effective advancement in clinical diagnostic.