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An Improved Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Data Analysis Pipeline for the Identification of Carbapenemase-Producing Klebsiella pneumoniae.

ABSTRACT: The increasing emergence of carbapenemase-producing Klebsiella pneumoniae (CPK) isolates is a global health alarm. Rapid methods that require minimum sample preparation and rapid data analysis are urgently required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been used by clinical laboratories for identification of antibiotic-resistant bacteria; however, discrepancies have arisen regarding biological and technical issues. The aim of this study was to standardize an operating procedure and data analysis for identification of CPK by MALDI-TOF MS. To evaluate this approach, a series of 162 K. pneumoniae isolates (112 CPK and 50 non-CPK) were processed in the MALDI BioTyper system (Bruker Daltonik, Germany) following a standard operating procedure. The study was conducted in two stages; the first is denominated the "reproducibility stage" and the second "CPK identification." The first stage was designed to evaluate the biological and technical variation associated with the entire analysis of CPK and the second stage to assess the final accuracy of MALDI-TOF MS for the identification of CPK. Therefore, we present an improved MALDI-TOF MS data analysis pipeline using neural network analysis implemented in Clover MS Data Analysis Software (Clover Biosoft, Spain) that is designed to reduce variability, guarantee interlaboratory reproducibility, and maximize the information selected from the bacterial proteome. Using the random forest (RF) algorithm, 100% of CPK isolates were correctly identified when all the peaks in the spectra were selected as input features and total ion current (TIC) normalization was applied. Thus, we have demonstrated that real-time direct tracking of CPK is possible using MALDI-TOF MS.

SUBMITTER: Gato E

PROVIDER: S-EPMC8218777 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Streptococcus suis, particularly S. suis serotype 2 (SS2), is an important zoonotic pathogen causing meningitis in humans worldwide. Although the proper classification of the causative and pathogenic serotype is salutary for the clinical diagnosis, cross-reactions leading to the indistinguishability of serotypes by the current serotyping methods are significant limitations. In the present study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of extracted peptides was developed to improve the classification of serotype of S. suis. The peptide mass fingerprint (PMFs) database of S. suis was generated from the whole-cell peptides of 32 reference strains of S. suis isolates obtained from pigs. Thirty-two human S. suis isolates from clinical cases in Thailand were used to validate this alternative serotyping method in direct comparison to the multiplex (m)PCR approach. All reference strains, representing 32 serotypes of S. suis, exhibited their individual PMFs patterns, thus allowing differentiation from one another. Highly pathogenic SS2 and SS14 were clearly differentiated from the otherwise serologically closely related SS1/2 and SS1, respectively. The developed MALDI-TOF-MS serotyping method correctly classified the serotype in 68.8% (22/32) of the same serotype isolates generated from the PMFs database; while the validity for the clinical human isolates was 62.5% (20/32). The agreement between the MALDI-TOF-MS and mPCR serotyping was moderate with a Kappa score of 0.522, considering that mPCR could correctly serotype up to 75%. The present study demonstrated that PMFs from the developed MALDI-TOF-MS-based method could successfully discriminate the previously indistinguishable highly pathogenic SS2 and SS14 from SS1/2 and SS1, respectively. Moreover, this serotyping method distinguished pathogenic SS6, and so is an alternative approach of choice to rapidly and reliably serotype clinically pathogenic S. suis isolates.

Dataset Information

An Improved Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Data Analysis Pipeline for the Identification of Carbapenemase-Producing Klebsiella pneumoniae.

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