Elucidating constraints for differentiation of major human Klebsiella pneumoniae clones using MALDI-TOF MS

2016 ◽  
Vol 36 (2) ◽  
pp. 379-386 ◽  
Author(s):  
C. Rodrigues ◽  
Â. Novais ◽  
C. Sousa ◽  
H. Ramos ◽  
T. M. Coque ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

scholarly journals Occurrence of the p019 gene in the blaKPC-harbouring plasmids: adverse clinical impact for direct tracking of KPC-producing Klebsiella pneumoniae by MALDI-TOF MS

2021 ◽  
Author(s):  
Eva Gato ◽  
Ignacio Pedro Constanso ◽  
Bruno Kotska Rodiño-Janeiro ◽  
Paula Guijarro-Sánchez ◽  
Tyler Alioto ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Computational Analysis ◽  
Direct Detection ◽  
Clinical Impact ◽  
Mass Peak ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Direct Tracking ◽  
Geographical Locations ◽  
Tof Ms

MALDI-TOF MS has recently been used for the direct detection of KPC-producing isolates by analysis of the 11,109 Da mass peak representing the P019 protein. In this study we evaluate the presence of the 11,109 Da mass peak in a collection of 435 unduplicated K. pneumoniae clinical isolates. The prevalence of the P019 peak in the blaKPC K. pneumoniae isolates was 49.2% (32/65). The 11,109 Da mass peak was not observed in any of the other carbapenemase (319) or non carbapenemase producers (116). Computational analysis of the presence of the p019 gene was performed in the aforementioned carbapenemase-producing K. pneumoniae isolates fully characterized by WGS and in a further collection of 1,649 K. pneumoniae genomes included in EuSCAPE. Herein, we have demonstrated that the p019 gene is not exclusively linked to the pKpQil plasmid, but it is present in the following plasmids: IncFIB(K)/IncFII(K)/ColRNAI, IncFIB(pQil), IncFIB(pQil)/ColRNAI, IncFIB(pQil)/IncFII(K), IncFIB(K)/IncFII(K) and IncX3. Besides, we have proven the independent movement of the Tn4401 and the ISKpn31, of which the p019 gene is a component. The absence of the p019 gene was obvious in Col440I, Col(pHAD28), IncFIB(K)/IncX3/IncFII(K), IncFIB(K)/IncFII(K) plasmids. In addition, we also observed another plasmid in which neither Tn4401 nor ISKpn31 was found, IncP6. In the EuSCAPE, the occurrence of p019 varied from 0% to 100% among the different geographical locations. The adverse clinical impact of the diminished prevalence of the p019 gene within the plasmid encoding KPC-producing Klebsiella pneumoniae puts forward the need for reconsideration when applying this technique in a clinical setting.

scholarly journals An improved MALDI-TOF MS data analysis pipeline for the identification of carbapenemase-producing Klebsiella pneumoniae

2021 ◽  
Author(s):  
Eva Gato ◽  
Ignacio Pedro Constanso ◽  
Ana Candela ◽  
Fátima Galán ◽  
Bruno Kotska Rodiño-Janeiro ◽  
...  
Keyword(s):  
Data Analysis ◽  
Klebsiella Pneumoniae ◽  
Operating Procedure ◽  
Standard Operating Procedure ◽  
Resistant Bacteria ◽  
Analysis Pipeline ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Data Analysis Pipeline ◽  
Tof Ms

The increasing emergence of carbapenemase-producing Klebsiella pneumoniae (CPK) is a global health alarm. Rapid methods that require minimum sample preparation and rapid data analysis are urgently required. 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 (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 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 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 inter-laboratory reproducibility and maximize the information selected from the bacterial proteome. Using the Random Forest (RF) algorithm, 100% of CPK producing isolates were correctly identified when all the peaks in the spectra were selected as input features and TIC normalization was applied. Thus, we have demonstrated that real-time direct tracking of CPK is possible using MALDI-TOF MS.

scholarly journals Rapidly quantitative antimicrobial susceptibility testing for Klebsiella pneumoniae using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry under optimal conditions

2021 ◽  
Author(s):  
Anran Zhang ◽  
Qianqian Chen ◽  
Yulan Dong ◽  
Bin Tian ◽  
Jing Li ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Incubation Time ◽  
Susceptibility Testing ◽  
Laser Desorption Ionization ◽  
Optimal Conditions ◽  
Ionization Time ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Tof Ms

Abstract Background Klebsiella pneumoniae infections, especially Carbapenem-resistant Klebsiella pneumoniae (CRKP), have become an “Urgent Threats” with high morbidity and mortality. Therefore, rapidly determining of the susceptibility and timely choosing an appropriate antibiotic were the important premises of the treatment of Klebsiella pneumoniae infections. The present study was first to explore the fitness of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in quantitative rapid antimicrobial susceptibility testing (RAST) with optimal conditions. Methods Firstly, we optimized the methodology based on the formic acid extraction method. Taking broth micro-dilution method (BMD) as a reference method, we utilized 25-fold, 50-fold and 100-fold dilutions matching with appropriate incubation time to evaluate the consistency of minimum inhibition concentration (MIC) values derived from BMD and MALDI-TOF MS. Finally, the performances of optimal dilution and incubation time were verified through the reproducibility on different three days. Results Directly incubated in the EP tubes and skipped the washing step can not only simplify the procedure, but reduce the unnecessary loss of bacteria biomass to improve sensitivity. The optimal volumes of 70% formic acid/100% acetonitrile (FA/ACN) and supernatant on the target plate were 3 µL and 2 µL, respectively. Comparing the different combination, 25-fold dilution of the 0.5 McFarland suspension and 2hr incubation time resulted in best performance. The consistency between the MS-MIC and corresponding BMD-MIC values in CRKP and Carbapenem-susceptible Klebsiella pneumoniae (CSKP) strains accounted for 76.67% and 66.67%, respectively, which generated the substantial agreements between MS-MIC and BMD-MIC values for CRKP strains (Kappa=0.643) and the moderate agreements for CSKP strains (Kappa=0.476). Finally, the MALDI-TOF MS-based RAST showed a well repeatability. Conclusion Considerable potentials were demonstrated for universal and mechanism-indepent RAST by MALDI-TOF MS with optimal conditions, which strengthened its application in accelerating reporting time and clinical diagnosis.

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