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MSACL 2018 EU : Nicolas

MSACL 2018 EU Abstract

Topic: Microbiology

Characterization of the Virulome of Staphylococcus Aureus by a Highly Multiplex Approach with Scout-MRM

Mouton Nicolas (Presenter)
University de Lyon Claude Bernard

Authors: Nicolas Mouton (1), Mariane Pivard (2), Romain Carrière (1), Sophie Ayciriex (1), Florence Couzon (2), Karen Moreau (2), Fabien Chirot (1), Francois Vandenesch (2), and Jerôme Lemoine (1).
(1) Institut des Sciences Analytiques (ISA), Ecole Normale Supérieure - Lyon, Université Claude Bernard Lyon 1, Université de Lyon, Centre National de la Recherche Scientifique/ UMR5280 - 5 rue de la Doua, 69100 Villeurbanne, France (2) Centre International de Recherche en Infectiologie (CIRI), Universite Claude Bernard - Lyon I, Inserm : U1111, Faculté de médecine Lyon Est - Site Laennec - 7 rue Guillaume Paradin 69372 Lyon Cedex 08, France

Short Abstract

The emergence of antibiotic-resistant bacteria strains is seriously threatening human life, as recently underlined by the World Health Organization. The case of Staphylococcus aureus, S.a. is of particular concern, whose forms resisting to vancomycin or methicillin caused numerous patients death in Hospitals. It is also crucial to better understand how mutations of the bacterial genome induce the emergence of new resistant strains. The sensitivity and multiplexing possibilities of mass spectrometric (MS) approaches appears particularly relevant to tackle such problems. We thus developed a targeted assay covering 90 key proteins involved in S.a. virulence and antibiotic resistance using a novel and a highly multiplexed targeted-MS approach, Scout-MRM. The virulome assay has been implemented for deciphering the proteogenomic links across a clinical collection of 230 S.a. strains.

Long Abstract

Introduction

As recently underlined by the World Health Organization, the emergence of antibiotic-resistant bacteria strains is seriously threatening human life [1, 2]. Staphylococcus aureus (S.a.) is of particular concern as strains resisting to vancomycin or methicillin caused numerous deaths [3-6]. Currently, the choice of the right antibiotic care is still based on susceptibility of microbial growth to the presence of antibiotics, which requires a 48- hour delay after a positive infection detection. Hence, a delay of one to three days occurs between empirical antimicrobial therapy and the antibiotic susceptibility testing result. Beside the clinical need of shortening infection diagnosis, it is also of interest to decipher the influence of the bacterial genome variability on the emergence of new clinical strains with increased antibiotic resistance or virulence profiles. The sensitivity and multiplexing capability of targeted mass spectrometry appears particularly relevant to quantify the proteins associated with these profiles [7, 8]. We thus developed and validated a Scout-Multiple Reaction Monitoring (Scout-MRM) [9] method targeting 90 key proteins involved in the virulence and antibiotic resistance of S.a. and deployed this assay across a clinical strain collection involved in endocarditis and pneumonia.

Methods

The highly multiplexed Scout-MRM assay was validated from 20 S.a. strains of reference then used on strains isolated from patients with well-classified episodes of infective endocarditis or pneumonia. Strains were grown in CCY media for 8 h growth. Bacterial cells and supernatants were subjected to sonication (30 min.) then tryptic digestion (1h) without reduction and alkylation. Scout-MRM assay was performed on a QTRAP® 6500 system (Sciex) equipped with a Turbo ion V TM source and coupled to an Agilent 1290 Infinity LC system. A new function in Analyst® software 1.6.2 enables Scout peptide signal to trigger the monitoring of transition lists released from retention time scheduling [9]. Tryptic peptides were on-line desalted with a solid phase extraction system (XBridge HLB phase) and separated on a C18 column (Acquity C18, 1.7 µm, 2.1x100 mm) with a 20 min. gradient. Data analysis was performed using an in-house developed pipeline under Knime software.

Results

With the aim of implementing targeted mass spectrometry assay in a clinical environment, we first drastically shortened the analysis time to one hour thanks to the combination of concomitant cell lysis and trypsin digestion, and on-line SPE coupled to LC-Scout-MRM analysis. A preliminary study was then conducted on 20 genotyped S.a. strains of reference to validate the Scout-MRM assay towards the specific and sensitive detection of key proteins involved in the virulence and antibiotic resistance mechanisms. A 95% correlation was obtained between the genotype and the proteome when considering proteins involved in antibiotic resistance MecA and toxins like PVL, hemolysin (alpha, beta, and delta), and the main enterotoxins (A, B, C). This correlation was reduced to 82% when considering the whole set of toxins, which may be explained by the lack of expression of some of them in an artificial growth medium and because of some competitions occurring between enterotoxins expression. Moreover, this study shows that despite identical genomic profiles, the level of expression for the actors of the virulence could be tremendously different between S.a. strains, with for instance factors of 100 to 1000 between the lowest and highest expression levels of Panton Valentine leucocidin (PVL) toxins for strains possessing Luk S and Luk F genes.

After the validation of our method with the training set, the whole virulome assay targeting 90 proteins with 270 peptides (900 transitions monitored) has been subsequently used for deciphering the proteogenomic links across a clinical collection of 230 S.a. clinical isolates associated either with endocarditis (n=125) or pneumonia (n=105). A preliminary analysis highlights correlation between the severity of the clinical pattern, pathogenicity and different key toxins such as Panton Valentine leucocidin (PVL) toxins and hemolysins (alpha, beta, delta).

Conclusions & Discussion

Our Scout-MRM approach provided in-depth insight-in the virulence and antibiotic resistance mechanisms in S. aureus via screening of antibiotic resistance determinants. 90 key proteins were measured targeting 270 peptides consisting of 900 Scout-MRM transitions. The method was deployed across a clinical strain collection involved in endocarditis and pneumonia infection. We observed good correlation between genomic data and archetypal virulence factors (PVL, enterotoxins, hemolysins) or methicillin resistance determinant MecA. Scout-MRM assays are currently under development for targeting the virulome of other bacterial species of strong clinical interest (ESKAPE group). Scout-MRM combined with an optimized sample workflow hence demonstrates the ability of targeted mass spectrometry to provide faster microbial identification and extended virulence characterization in an infection diagnosis platform


References & Acknowledgements:

[1] WHO. report, Antimicrobial resistance. 2018.

[2] O’Neill. J, Antimicrobial Resistance: Tackling a Crisis for the Health and Wealth of Nations. (UK Government & Wellcome Trust, 2014) 2014.

[3] Chambers, H. F., Deleo, F. R., Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol 2009, 7, 629-641.

[4] Foster, T. J., Antibiotic resistance in Staphylococcus aureus. Current status and future prospects. FEMS Microbiol Rev 2017, 41, 430-449.

[5] Haaber, J., Penades, J. R., Ingmer, H., Transfer of Antibiotic Resistance in Staphylococcus aureus. Trends Microbiol 2017, 25, 893-905.

[6] Hiramatsu, K., Katayama, Y., Matsuo, M., Sasaki, T., et al., Multi-drug-resistant Staphylococcus aureus and future chemotherapy. J Infect Chemother 2014, 20, 593-601.

[7] Charretier, Y., Dauwalder, O., Franceschi, C., Degout-Charmette, E., et al., Rapid Bacterial Identification, Resistance, Virulence and Type Profiling using Selected Reaction Monitoring Mass Spectrometry. Sci Rep 2015, 5, 13944.

[8] Charretier, Y., Schrenzel, J., Mass spectrometry methods for predicting antibiotic resistance. Proteomics Clin Appl 2016, 10, 964-981.

[9] Rougemont, B., Bontemps Gallo, S., Ayciriex, S., Carriere, R., et al., Scout-MRM: Multiplexed Targeted Mass Spectrometry-Based Assay without Retention Time Scheduling Exemplified by Dickeya dadantii Proteomic Analysis during Plant Infection. Anal Chem 2017, 89, 1421-1426.

The PhD position of N.M. was funded by a doctoral contract accredited by the French Ministry of Education, Higher Education and Research (MENESR). This work was supported by CNRS funding. We thank the Infectious diseases department (Hospices civils de Lyon, Lyon, France) and the CIRI - Centre International de Recherche en Infectiologie (INSERM U1111, CNRS UMR 5308, University Claude Bernard Lyon 1, Ecole Normale Supérieure de Lyon) for helpful discussions and technical support.


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