Evaluation of rapid KPC carbapenemase detection method based on MALDI-TOF VITEK MS spectra analysis

Centonze, Anna Rita; Bertoncelli, Anna; Savio, C.; Orza, Pierantonio; Bedenić, Branka; Mazzariol, Annarita

doi:10.1099/jmm.0.000831

Cited by 12 publications

(9 citation statements)

References 19 publications

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“…In a larger set of samples, the consistent presence of the biomarker peak could be verified, but only in isolates that harbor plasmids with a particular transposon (Tn4401a) while absent in KPC producing strains with other transposon types (Youn et al, 2016). This finding was further confirmed to be independent of the sequence type of K. pneumoniae (Gaibani et al, 2016) and isolates resistant due to other carbapenemases (VIM and NDM, for example) were found negative (Centonze et al, 2018). Therefore, the reported specificities and sensitivities are possibly only valid for the tested sample sets.…”

Section: Detection Of Biomarkers Associated With Antibiotic Resistancementioning

confidence: 62%

One System for All: Is Mass Spectrometry a Future Alternative for Conventional Antibiotic Susceptibility Testing?

Welker

Belkum

2019

Front. Microbiol.

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The two main pillars of clinical microbiological diagnostics are the identification of potentially pathogenic microorganisms from patient samples and the testing for antibiotic susceptibility (AST) to allow efficient treatment with active antimicrobial agents. While routine microbial species identification is increasingly performed with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), routine AST still largely relies on conventional and molecular techniques such as broth microdilution or disk and gradient diffusion tests, PCR and automated variants thereof. However, shortly after the introduction of MALDI-TOF MS based routine identification, first attempts to perform AST on the same instruments were reported. Today, a number of different approaches to perform AST with MALDI-TOF MS and other MS techniques have been proposed, some restricted to particular microbial taxa and resistance mechanisms while others being more generic. Further, while some of the methods are in a stage of proof of principles, others are already commercialized. In this review we discuss the different principal approaches of mass spectrometry based AST and evaluate the advantages and disadvantages compared to conventional and molecular techniques. At present, the possibility that MS will soon become a routine tool for AST seems unlikely – still, the same was true for routine microbial identification a mere 15 years ago.

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Section: Detection Of Biomarkers Associated With Antibiotic Resistancementioning

confidence: 62%

One System for All: Is Mass Spectrometry a Future Alternative for Conventional Antibiotic Susceptibility Testing?

Welker

Belkum

2019

Front. Microbiol.

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“…This peak is associated with the protein pO19 inserted into the same region of the transposon tn4401, often harbouring the bla KPC gene in K. pneumoniae carbapenemaseproducing strains. The Tn4401a isoform and pO19 elements associated with bla KPC resistance gene have been detected with a PCR reaction [13].…”

Section: Resultsmentioning

confidence: 99%

“…KPC was detected by 11 109 Da peak analysis of matrix-assisted laser desorption/ionizationetime-of-flight (MALDI-TOF) spectra [12,13]. The presence of Tn4401 and pO19 elements associated with the bla KPC resistance gene were detected with a PCR reaction [13]. A PCR-based replicon typing (PBRT) protocol [14] was performed to check the plasmid profile of the strains.…”

Section: Methodsmentioning

confidence: 99%

Klebsiella pneumoniae carbapenemase (KPC) producer resistant to ceftazidime–avibactam due to a deletion in the blaKPC3 gene

Antinori

Unali

Bertoncelli

et al. 2020

Clinical Microbiology and Infection

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Objectives: Carbapenemase-producing strains of Klebsiella pneumoniae (KPC) are a great health concern, and therapy with ceftazidimeeavibactam represents a choice for the treatment of infections involving these strains. We report a strain resistant to ceftazidimeeavibactam due to a deletion of six nucleotides in the bla KPC gene sequence. Methods: Two strains, namely AMP920 and AMP2009, were isolated from the same patient a month apart. Antimicrobial susceptibility testing was performed both by broth microdilution and by Etest. Immunoenzymatic assay to detect carbapenemase was performed for both strains. The bla KPC gene of both strains was amplified by PCR and sequenced. Enzyme activity towards carbapenems was tested by the CarbaNP test and hydrolysis spectrophotometer assay. Results: The two isolates differed in antimicrobial susceptibility. AMP920 showed meropenem and imipenem resistance (MIC 32 and 32 mg/mL). A month later the carbapenem MIC decreased to 8 and 1 mg/mL respectively, while the ceftazidimeeavibactam MIC increased from 1 to 16 mg/mL. Both isolates showed a positive immunoenzymatic test for the KPC enzyme, but only AMP920 showed a positive CarbaNP test hydrolysing imipenem. The Bla KPC gene was amplified in both strains. After sequencing, the two amplicons showed a KPC3 variant. The gene of the second isolate showed a deletion of six nucleotides at 498e503, resulting in a mutant variant with the deletion of glutamic acid and leucine residues at positions 167 and 168. Conclusions: We detected a new deletion in the bla KPC gene of a clinical strain of K. pneumoniae which resulted in resistance to ceftazidimeeavibactam. The amino acids deleted are in the U loop (amino acids 165e179) of the KPC enzyme, enhancing ceftazidime affinity and preventing avibactam binding.

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“…The detection of proteins from KPC‐producing bacteria using MALDI‐TOF MS has been reported in various studies, which focused on analysing the hydrolysed form of β‐lactam substrates or detecting a hypothetical protein linked to a plasmid (p019 protein, 11109 Da MS peak) [29–32]. Targeted analysis of unique peptides has also been validated using LC‐MS/MS [33].…”

Section: Figurementioning

confidence: 99%

Optimization of a lysis method to isolate periplasmic proteins from Gram‐negative bacteria for clinical mass spectrometry

Cheon¹,

Lee²,

Yang³

et al. 2021

Proteomics Clinical Apps

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Purpose Clinical mass spectrometry requires a simple step process for sample preparation. This study aims to optimize the method for isolating periplasmic protein from Gram‐negative bacteria and apply to clinical mass spectrometry. Experimental Design The Klebsiella pneumoniae carbapenemase (KPC)‐producing E. coli standard cells were used for optimizing the osmotic shock (OS) lysis method. The supernatant from OS lysis was analysed by LC‐MS/MS and MALDI‐TOF MS. The effectiveness of the OS lysis method for KPC‐2‐producing Enterobacteriaceae clinical isolates were then confirmed by MALDI‐TOF MS. Results The optimized OS lysis using KPC‐2 producing E. coli standard cells showed a high yield of KPC‐2 protein and enriches periplasmic proteins. Compared with other lysis methods, the detection sensitivity of KPC‐2 protein significantly increased in MALDI‐TOF MS analysis. Nineteen clinical isolates were validated by MALDI‐TOF MS using the OS method, which also showed higher detection sensitivity compared to other lysis method (e.g., 1.5% n‐octyl‐β‐D‐glucopyranoside) (p < 0.001). Conclusions and Clinical Relevance This study provides a straightforward, rapid, affordable, and detergent‐free method for the analysis of periplasmic proteins from Enterobacteriaceae clinical isolates. This approach may contribute to MS‐based clinical diagnostics.

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Evaluation of rapid KPC carbapenemase detection method based on MALDI-TOF VITEK MS spectra analysis

Cited by 12 publications

References 19 publications

One System for All: Is Mass Spectrometry a Future Alternative for Conventional Antibiotic Susceptibility Testing?

One System for All: Is Mass Spectrometry a Future Alternative for Conventional Antibiotic Susceptibility Testing?

Klebsiella pneumoniae carbapenemase (KPC) producer resistant to ceftazidime–avibactam due to a deletion in the blaKPC3 gene

Optimization of a lysis method to isolate periplasmic proteins from Gram‐negative bacteria for clinical mass spectrometry

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