Our aim was to evaluate the performance of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), routinely used in the microbiology laboratory for bacterial identification, for bacterial typing in the setting of extended spectrum beta-lactamase producing Klebsiella pneumoniae (ESBL-KP) outbreak in the neonatal intensive care unit (NICU). Isolates from a 2011 outbreak in the NICU were retrieved from frozen stocks and analyzed by MALDI-TOF. The MALDI typing was compared with core genome multilocus sequence typing (cg-MLST). MALDI typing divided the 33 outbreak isolates into 2 clones: sequence type (ST)-290 and 405. These results were in complete agreement with cg-MLST results. The differentiation of the outbreak isolates into two clones correlated with the patients' location in the NICU, but also with their place of residence. Conclusion: Here, we show that MALDI-TOF MS, which has been integrated into the microbiology laboratory workflow for microbial species identification, can be secondarily used for epidemiological typing at no added cost. What is Known: • Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now routinely used in the microbiology laboratory for bacterial identification What is New: • MALDI typing was used for outbreak investigation in the NICU and divided the outbreak isolates into two clones • MALDI-TOF MS may be secondarily used for epidemiological typing at no added cost.

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