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Communication Dans Un Congrès Année : 2022

Fast and accurate identification and antibiotic resistance profiling of micro-organisms in blood cultures by SWATH-MS proteomics

Résumé

Introduction Today, approximately 1.3 million people die each year worldwide from bacterial antimicrobial resistance (AMR). AMR is of great concern in the context of bloodstream infections (BSIs) when it evolves into sepsis. If MALDI-TOF has markedly shortened the delay of the pathogen identification step, AMR evaluation is still based on growth tests in the presence of antibiotics. The latter, negatively impacts the diagnosis turnaround time, thus the administration to the patient of active antimicrobial agents. With the aim of streamlining this turnaround time to less than an hour from a positive blood culture, the LC/SWATH-MS sensitivity and specificity performances were evaluated to concomitantly provide the identity and antibiotic resistance profile of pathogens related to BSIs based on peptide surrogates. Methods Our methodology consists in a 10-min sample preparation procedure for pelleting the micro-organisms from a positive blood culture aliquot followed by 10-min concomitant ultrasonic bacterial lysis and tryptic digestion step. All LC/SWATH-MS runs were conducted on a Waters M-Class UPLC system connected to a 7600 ZenoTOF mass spectrometer. The peptides were separated on a C18 micro-LC column (300 µm × 150 mm) at a flow rate of 6 µL/min; 25min total chromatographic time). LC/SWATH-MS data was processed with DIA-NN (1.8) in library-free mode. Sensitivity and specificity performances were assessed with a collection of representative sensitive and resistant clinical strains inoculated in blood culture bottles. Preliminary data DIA-NN’s deep-learning algorithm was used to generate an in silico spectral and retention time profiling library from a FASTA file containing the complete genus and species-specific protein sequences. This library was then used to analyze the raw LC/SWATH-MS data. A comprehensive quality by design approach has been selected to evaluate the key LC-SWATH-MS parameters having the most influence on the overall performance detection of peptide surrogates used for the identification of the pathogen and the detection of antibiotic resistance-related proteins. All optimization designs have been conducted on the same Staphylococcus aureus strain with the objective of identifying the optimal operating conditions for the detection of 7 proteotypic peptides of the Penicillin Binding Protein 2a (PBP2a). The latter, mediated through the mecA gene, confers resistance to methicillin. Initially, a screening design allowed us to determine which of the six parameters evaluated (gradient length, TOF accumulation time, number of SWATH windows, fixed/variable windows, MS m/z range, and MS/MS m/z range) had the highest impact on the number of PBP2a peptides detected. Subsequently, the two most critical parameters (number of SWATH windows and MS m/z range) have been thoroughly optimized using a response surface methodology. The other non-critical parameters were set at the level that produced the best response. Taking a step further, the sensitivity and specificity performances of the optimized LC-SWATH-MS method have been evaluated by analyzing a collection of epidemiologically relevant clinical strains of Gram-negative and Gram-positive bacteria inoculated in blood culture bottles. This study allowed the detection of some of the most common protein effectors involved in bacterial resistance mechanisms such as PBP2a, different types of β-lactamases (AmpC; TEM; SHV; GES; PER; OXA; CTX-M; KPC; NDM), porin and efflux pumps (OprD; MexA/MexB/OprM; MexX/MexY/OprM) as well as vancomycin resistance proteins (VanA; VanB; VanD) to mention a few. Novel aspect Concomitant identification and AMR profiling of pathogens in positive blood cultures by SWATH with a turnaround time of one hour.
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Dates et versions

hal-04000257 , version 1 (22-02-2023)

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  • HAL Id : hal-04000257 , version 1

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Iulia Macavei, Eva Peyrache, Maud Gregson, Romain Carrière, Roxane Prat, et al.. Fast and accurate identification and antibiotic resistance profiling of micro-organisms in blood cultures by SWATH-MS proteomics. 70th Conference on Mass Spectrometry and Allied Topics, American Society for Mass Spectrometry; SCIEX, Jun 2022, Minneapolis, United States. ⟨hal-04000257⟩
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