Detection of amino acid substitutions in the GyrA protein of fluoroquinolone-resistant typhoidal Salmonella isolates using high-resolution mass spectrometry

Hassing, Robert‐Jan; Goessens, W. H. F.; Zeneyedpour, Lona; Sultan, Sadaf; Kampen, Jeroen J. A. van; Verbon, Annelies; Genderen, Perry J.J. van; Hays, John P.; Luider, Theo M.; Dekker, Lennard J. M.

doi:10.1016/j.ijantimicag.2016.01.018

Cited by 13 publications

(16 citation statements)

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“…In addition to carbapenemases, other resistance mechanisms of Enterobacterales have been detected by LC-MS such as the ESBL CTX-M by Fleurbaaij et al (2017). But also the detection of different amino-acid substitutions in the GyrA protein resulting in quinolone resistance of Salmonella enterica isolates was demonstrated in a paper of Hassing et al (2016). These studies indicate that LC-MS is universally applicable for protein detection of different resistance mechanisms.…”

Section: Discussionmentioning

confidence: 98%

“…By rapidly detecting specific peptides of many prevalent resistance mechanisms in a single assay, resistance to several representative antibiotics could be predicted for frequently isolated pathogens. So far, several studies have demonstrated direct detection of different resistance mechanisms using these targeted approaches (Charretier et al, 2015; Charretier and Schrenzel, 2016; Hassing et al, 2016). Furthermore, detection by high-resolution MS of each carbapenemase separately i.e., KPC (Wang et al, 2017), NDM (Cecchini et al, 2018), and OXA-48-like (Strich et al, 2019) has been demonstrated in different studies.…”

Section: Introductionmentioning

confidence: 99%

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Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry

et al. 2019

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BackgroundAt present, phenotypic growth inhibition techniques are used in routine diagnostic microbiology to determine antimicrobial resistance of bacteria. Molecular techniques such as PCR are often used for confirmation but are indirect as they detect particular resistance genes. A direct technique would be able to detect the proteins of the resistance mechanism itself. In the present study targeted high resolution mass spectrometry assay was developed for the simultaneous detection of KPC, OXA-48-like, NDM, and VIM carbapenemases.MethodsCarbapenemase specific target peptides were defined by comparing available sequences in GenBank. Selected peptide sequences were validated using 62 Klebsiella pneumoniae and Escherichia coli isolates containing: 16 KPC, 21 OXA-48-like, 16 NDM, 13 VIM genes, and 21 carbapenemase negative isolates.ResultsFor each carbapenemase, two candidate peptides were validated. Method validation was performed in a blinded manner for all 83 isolates. All carbapenemases were detected. The majority was detected by both target peptides. All target peptides were 100% specific in the tested isolates and no peptide carry-over was detected.ConclusionThe applied targeted bottom-up mass spectrometry technique is able to accurately detect the four most prevalent carbapenemases in a single analysis.

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Section: Discussionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry

et al. 2019

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“…Ciprofloxacin resistance in the studied isolates could be explained by mutations in gyrA and parC , and by the presence of AAC(6′)-Ib-cr enzymes, OqxAB efflux pumps and qnr genes. In the current study, we did not correlate mutations at the DNA level to the detected peptides as a targeted MS approach is more suitable to detect these key amino acid substitutions as demonstrated by Hassing et al 26 .…”

Section: Discussionmentioning

confidence: 62%

Exploring antimicrobial resistance to beta-lactams, aminoglycosides and fluoroquinolones in E. coli and K. pneumoniae using proteogenomics

Foudraine

Strepis

Stingl

et al. 2021

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Antimicrobial resistance is mostly studied by means of phenotypic growth inhibition determinations, in combination with PCR confirmations or further characterization by means of whole genome sequencing (WGS). However, the actual proteins that cause resistance such as enzymes and a lack of porins cannot be detected by these methods. Improvements in liquid chromatography (LC) and mass spectrometry (MS) enabled easier and more comprehensive proteome analysis. In the current study, susceptibility testing, WGS and MS are combined into a multi-omics approach to analyze resistance against frequently used antibiotics within the beta-lactam, aminoglycoside and fluoroquinolone group in E. coli and K. pneumoniae. Our aim was to study which currently known mechanisms of resistance can be detected at the protein level using liquid chromatography–mass spectrometry (LC–MS/MS) and to assess whether these could explain beta-lactam, aminoglycoside, and fluoroquinolone resistance in the studied isolates. Furthermore, we aimed to identify significant protein to resistance correlations which have not yet been described before and to correlate the abundance of different porins in relation to resistance to different classes of antibiotics. Whole genome sequencing, high-resolution LC–MS/MS and antimicrobial susceptibility testing by broth microdilution were performed for 187 clinical E. coli and K. pneumoniae isolates. Resistance genes and proteins were identified using the Comprehensive Antibiotic Resistance Database (CARD). All proteins were annotated using the NCBI RefSeq database and Prokka. Proteins of small spectrum beta-lactamases, extended spectrum beta-lactamases, AmpC beta-lactamases, carbapenemases, and proteins of 16S ribosomal RNA methyltransferases and aminoglycoside acetyltransferases can be detected in E. coli and K. pneumoniae by LC–MS/MS. The detected mechanisms matched with the phenotype in the majority of isolates. Differences in the abundance and the primary structure of other proteins such as porins also correlated with resistance. LC–MS/MS is a different and complementary method which can be used to characterize antimicrobial resistance in detail as not only the primary resistance causing mechanisms are detected, but also secondary enhancing resistance mechanisms.

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“…Next, by using a targeted approach (SRM mass spectrometry) was possible to detect methicillin resistance in S. aureus and virulence factors directly from positive blood cultures [34]. Furthermore, by the detection of amino acid substitutions in the GyrA protein using SRM it was possible to detect decreased quinolone susceptibility in typhoidal Salmonella isolates [35]. The combination of identification with antibiotic resistance determination will directly benefit clinical management.…”

Section: Research Articlementioning

confidence: 99%

Identification of Microorganisms Grown in Blood Culture Flasks using Liquid Chromatography–Tandem Mass Spectrometry

et al. 2017

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Aim:Bloodstream infections are a common cause of disease and a fast and accurate identification of the causative agent or agents of bloodstream infections would aid the start of adequate treatment. Materials & methods: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics method was developed for the identification of bacterial species directly from blood cultures that were simulated by inoculating blood culture bottles with single or multiple clinically relevant microorganisms. Results: Using LC-MS/MS, the single species were correctly identified in 100% of the blood cultures, whereas for polymicrobial infections, 78% of both species were correctly identified in blood cultures. Conclusion: The LC-MS/MS method allows for the identification of the causative agent of positive blood cultures.

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Detection of amino acid substitutions in the GyrA protein of fluoroquinolone-resistant typhoidal Salmonella isolates using high-resolution mass spectrometry

Cited by 13 publications

References 16 publications

Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry

Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry

Exploring antimicrobial resistance to beta-lactams, aminoglycosides and fluoroquinolones in E. coli and K. pneumoniae using proteogenomics

Identification of Microorganisms Grown in Blood Culture Flasks using Liquid Chromatography–Tandem Mass Spectrometry

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