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

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.

Download Full-text

A Rapid Antimicrobial Susceptibility Test for Klebsiella pneumoniae Using a Broth Micro-Dilution Combined with MALDI TOF MS

Infection and Drug Resistance ◽

10.2147/idr.s305280 ◽

2021 ◽

Vol Volume 14 ◽

pp. 1823-1831

Author(s):

Gang Wang ◽

Guobin Song ◽

Yuanhong Xu

Keyword(s):

Klebsiella Pneumoniae ◽

Antimicrobial Susceptibility ◽

Susceptibility Test ◽

Antimicrobial Susceptibility Test ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

Download Full-text

2155. Accelerated Confirmation of Porin Loss in Carbapenem-Resistant Enterobacterales: A MALDI-TOF Mass Spectrometry-Based Approach

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.1835 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S731-S731

Author(s):

Carlos Correa-Martinez ◽

Evgeny A Idelevich ◽

Karsten Becker

Keyword(s):

Mass Spectrometry ◽

Molecular Weight ◽

Gold Standard ◽

Sodium Dodecylsulfate ◽

Resistant Bacteria ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Carbapenem Resistant ◽

Sds Page ◽

Tof Ms

Abstract Background The accurate identification of carbapenem resistance mechanisms is decisive for the appropriate selection of antibiotic regimens. Numerous methods can detect carbapenemase-producing carbapenem-resistant bacteria (CPCR). However, non-CPCR (NCPCR) are routinely assumed to display porin loss as a diagnosis of exclusion. No further confirmatory tests are performed since the gold standard (sodium dodecylsulfate polyacrylamide gel electrophoresis, SDS–PAGE) is laborious and time consuming. We propose a test for rapid and easy detection of porin loss by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods Clinical meropenem-resistant Enterobacterales strains (10 CPCR, 10 NCPCR) and control strains recommended by EUCAST (5 carbapenemase-producing, one with porin loss, one-negative control) were analyzed. Membrane proteins were extracted by successive centrifugation of bacterial suspensions (McFarland 0.5) and addition of ethanol, formic acid and acetonitrile. MALDI-TOF MS of the protein extracts was performed on a 96-spot target (Bruker Daltonics, Germany). Peaks between 35 and 40 kDa were analyzed for the presence of porins and compared with the bands observed in the SDS–PAGE of the protein extracts. Results Within the molecular weight range of 35–40 kDa, the MALDI-TOF MS-based method revealed peaks in all CPCR isolates corresponding to those observed in the carbapenemase-producing control strains. In contrast, the control strain with porin loss as well as all CNCR isolates showed a lower quantity of peaks in this range. All peaks observed correlated with the bands observed in the SDS–PAGE of the protein extracts at the corresponding molecular weight (Figure 1). Conclusion Yielding results that reliably correspond to the current gold standard, we propose a method for accelerated detection of porin loss as an alternative to the diagnosis of exclusion usually made in routine settings. With a processing time of approximately 20 minutes, the method can be easily implemented in the clinical setting. Applying this MALDI-TOF MS-based approach, valuable information will be provided about a resistance mechanism that otherwise remains unexplained. Disclosures All authors: No reported disclosures.

Download Full-text

Determination of Drug Efflux Pump Efficiency in Drug-Resistant Bacteria Using MALDI-TOF MS

Antibiotics ◽

10.3390/antibiotics9100639 ◽

2020 ◽

Vol 9 (10) ◽

pp. 639 ◽

Cited By ~ 1

Author(s):

Wen-Jung Lu ◽

Hsuan-Ju Lin ◽

Pang-Hung Hsu ◽

Hong-Ting Victor Lin

Keyword(s):

Minimum Inhibitory Concentration ◽

Inhibitory Concentration ◽

Resistant Bacteria ◽

Pump Efficiency ◽

Drug Efflux ◽

Maldi Tof Ms ◽

E Coli ◽

Maldi Tof ◽

Conventional Methods ◽

Tof Ms

Multidrug efflux pumps play an essential role in antibiotic resistance. The conventional methods, including minimum inhibitory concentration and fluorescent assays, to monitor transporter efflux activity might have some drawbacks, such as indirect evidence or interference from color molecules. In this study, MALDI-TOF MS use was explored for monitoring drug efflux by a multidrug transporter, and the results were compared for validation with the data from conventional methods. Minimum inhibitory concentration was used first to evaluate the activity of Escherichia coli drug transporter AcrB, and this analysis showed that the E. coli overexpressing AcrB exhibited elevated resistance to various antibiotics and dyes. Fluorescence-based studies indicated that AcrB in E. coli could decrease the accumulation of intracellular dyes and display various efflux rate constants for different dyes, suggesting AcrB’s efflux activity. The MALDI-TOF MS analysis parameters were optimized to maintain a detection accuracy for AcrB’s substrates; furthermore, the MS data showed that E. coli overexpressing AcrB led to increased ions abundancy of various dyes and drugs in the extracellular space at different rates over time, illustrating continuous substrate efflux by AcrB. This study concluded that MALDI-TOF MS is a reliable method that can rapidly determine the drug pump efflux activity for various substrates.

Download Full-text

Rapid identification of clinical isolates of Klebsiella pneumoniae using MALDI-TOF MS from North India

Bulletin of Pure & Applied Sciences- Zoology ◽

10.5958/2320-3188.2020.00022.4 ◽

2020 ◽

Vol 39a (1) ◽

pp. 194

Author(s):

Sunil Kumar ◽

Zainab Saifi ◽

Anil Kumar Sharma ◽

Sushil Kumar Upadhyay

Keyword(s):

Klebsiella Pneumoniae ◽

Rapid Identification ◽

Clinical Isolates ◽

North India ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

Download Full-text

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

Journal of Clinical Microbiology ◽

10.1128/jcm.00238-21 ◽

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.

Download Full-text