MSACL 2023 Abstract

Introduction
Ultra-fast and accurate identification (ID) of sepsis causative micro-organisms directly from positive blood culture (PBC) is a key step in patient treatment. Even if MALDI-TOF MS is the standard of care, it has some limitations regarding ID performance for polymicrobial samples and differentiation of some species complex (typically Enterobacter complex).

Objectives
We developed a very fast SCOUT-triggered MRM (stMRM) based proteomics assay targeting ribosomal protein-deriving peptides covering the 114 most prevalent species in blood infections. We challenged it against artificial PBC and clinical routine samples. We aimed to develop a new sepsis diagnostic tool ready to be implemented in routine hospital services.

Methods
Samples collection
1059 bacterial strains were obtained from different French National Reference Centers (CNR): Staphylococcus aureus (n=124), Pseudomonas aeruginosa (n=309) and Acinetobacter baumannii (n=181) and pittii (n=7), Enterococcus faecium (n=108) and faecalis (n=11), Citrobacter amaloniticus (n=4), Citrobacter farmeri (n=2), Citrobacter freundii (n=19), Citrobacter koseri (n=9), Citrobacter portucalensis (n=2), Enterobacter asburiae (n=4), Enterobacter cloacae (n=8), Enterobacter hormachei (n=38), Enterobacter roggenkampii (n=7), Escherichia coli (n=115), Hafnia alvei (n=4), Klebsiella aerogenes (n=13), Klebsiella oxytoca (n=16), Klebsiella pneumoniae (n=10), Klebsiella variicola (n=2), Morganella morganii (n=17), Pantoea agglomerans (n=2), Pantoea septica (n=3), Proteus mirabilis (n=20), Proteus vulgaris (n=1), Providencia rettgeri (n=2), Providencia stuartii (n=1), Salmonella enterica (n=6), Serratia marcescens (n=14).

The strains listed above were spiked both in aerobic (AE) and anaerobic (ANA) (when relevant) blood culture bottles with human sterile blood to mimic blood infection and incubated in a BACT/ALERT® VIRTUO® (bioMérieux). A total of 1617 samples were analyzed. Species identification was obtained from Whole Genome Sequencing and compared to the result of the developed method.

A second step with a cohort of 264 PBC from routine patient samples was blindly selected. Species identification was obtained by MALDI-TOF mass spectrometry (VITEK-MS) after a subculture on agar plate and was compared to the result obtained with stMRM assay.


Materials
Genus and species-specific best-flyer peptides deriving from ribosomal proteins have been identified with conventional shotgun proteomics applied to 114 bacteria and yeast species representative of the sepsis epidemiology.
Upon BC positivity, bacteria were first isolated from blood, then lysed and digested with trypsin under ultrasounds. Peptide mixtures were analysed using reversed phase liquid chromatography coupled with tandem mass spectrometry LC-MS/MS with a SCOUT-triggered MRM (stMRM) method.

LC separation was carried out on a XBridge BEH C18 column (100 * 1 mm, particle size 3.5 µm, porosity 130Å) using a multi-step gradient going from 2% to 50% acetonitrile containing 0.1% (v/v) formic acid in 4.7 min at a flow rate of 100 µL/min. The total analysis run time lasts 7,8 min.
A SCOUT-triggered MRM method was built for the screening of about 230 peptides homogenously located in 7 groups of transitions successively triggered by 3 peptides derived from trypsin auto digestion and 3 spiked-in synthetic peptides. In-house dedicated database with reference peptides was developed and algorithms were set up for automatic processing of the signal integrated by Sciex OS software.

The total workflow lasts less than 45 minutes.

Results
From spiked PBC, a rate of 100 % (1617/1617) in terms of sensitivity and specificity was reached both in AE and ANA PBC. Some species including Enterobacter cloacae, Enterobacter hormachei, Enterobacter asburiae, Acinetobacter baumannii, Acinetobacter pittii, Klebsiella variicola not differentiated with MALDI-TOF MS have been differentiated with the developed LC-MS/MS method.
From patient PBC, successful identification was obtained for 93% (245/264) of the samples on Day 0. For these samples, 100% agreement was obtained with MALDI-TOF MS identification (results obtained on Day 1). To note, for 6/11 polymicrobial samples characterized at Day 1, both pathogens were correctly identified at Day 0. For 2/11 polymicrobial samples the most abundant micro-organism was identified.

Conclusions
The SCOUT-triggered MRM-MS method provides in less than 45 min the ID of sepsis-causative micro-organisms directly from PBC in mono or polymicrobial samples. The sensitivity and specificity performances equal or exceed those of MALDI-TOF MS. This ID method can be combined with rapid genus-specific SCOUT-triggered MRM-MS assay for the quantitative detection of proteins involved in antibiotics resistance.
This method could also be applied to different matrices like urines.