Sepsis by Enterobacteria: A MALDI Affair Markus Kostrzewa (Presenter) Bruker Daltonik GmbH Presenter Bio: Study of biology at the Justus-Liebig-University, Giessen, Germany. Diploma (1990) and Ph.D. thesis(1993) about molecular evolution of plastids. 1993 - 1997 Postdoc at the Institute of Human Genetics, JLU Giessen (AG Prof. U. Müller). • Diagnostic of hereditary human diseases • Research in the framework of the Human Genome Project Joined Bruker in 1998 Head of the new R&D group “Bioanalytical Development“, establishment of molecular biology and biology related development at Bruker Daltonik. Development of DNA analysis by MALDI-TOF mass spectrometry, “Clincal Proteomics” - methods, consumables and software for mass spectrometry profiling of body fluids and tissues Development of microorganism identification by MALDI-TOF mass spectrometry (“MALDI Biotyper” system). 2005 Director Molecular Biology, R&D 2012 Vice President Clinical Mass Spectrometry R&D 2017 Vice President Microbiology & Diagnostics R&D Heading the the microbiology and clinical research and development; application-, consumable-, and software- development. Strategic planning of innovative products in the field of clinical microbiology and diagnostics. Driving regulatory approval process (IVD-CE labelling, FDA clearance, ISO/AOAC certification for Food market) Safety officer for medical products More than 100 peer reviewed publications Member of the German Society of Neurogenetics Member of the German Society of Hygiene and Microbiology Member of European Society of Clinical Microbiology Member of the American Society for Microbiology

Authors: Markus Kostrzewa (1), Miriam Cordovana
(1) Bruker Dalltonik GmbH, Bremen, Germany (2) University Hospital Sant'Orsola-Malpighi, Bologna, Italy

Enterobacteria are the most frequent causative agents of sepsis. The spread of cephalosporinase- and carbapenemase-production in these species is a worrying threat for the effectiveness of the antibiotic therapy. Every hour saved in the detection of such strains can be crucial for the patients’ clinical outcome.
In this study, we investigate an innovative full MALDI-TOF MS based approach to quickly detect cephalosporinase- and carbapenemase-producing enterobacteria directly from the positive blood cultures bottles, applying the novel tools of the Biotyper system (Bruker Daltonik). The bacterial pellet extracted by Sepsityper was used for the species identification, for the detection of KPC-producing strains by subtyping, and for evaluation of cephalosporinase and carbapenemase activity by STAR-Cepha and STAR-Carba hydrolysis assays.

Introduction

Enterobacteria are the most frequent causative agents of sepsis. Empiric treatment of sepsis often includes a beta-lactam antibiotic (β-lactam+β-lactamase inhibitor, cephalosporins, carbapenems) as a wide spectrum drug, but bacterial strains that produce cephalosporinase (ESβL, AmpC) or carbapenemase enzymes are spreading worldwide, undermining the effectiveness of the antibiotic treatment with beta-lactam drugs.

The detection of these resistant strains as fast as possible is crucial for the patients’ clinical outcome, since mortality increases significantly for every hour of delay in adopting the proper therapy. Laboratory methods for the verification of cephalosporinase production are laborious and slow, further not applicable directly on positive blood cultures bottle, requiring a subculturing step. Although several non-mass spec commercial methods are available to confirm carbapenemase production, none of them has the features of universality that are strongly demanded to be applied on positive blood cultures.

In this study, we investigated a full MALDI based approach to quickly detect cephalosporinase- and carbapenemase-producing enterobacteria directly from positive blood cultures bottles, using a combination of the most recent applications of the MALDI Biotyper system (Bruker Daltonik GmbH, Germany). The bacterial pellet extracted by Sepsityper was used first for species identification, including the simultaneous subtyping for KPC-harboring Klebsiella pneumoniae, then for the evaluation of carbapenemase- and cephalosporinase-production by STAR-Carba and STAR-Cepha hydrolysis assays.

Methods

N=92 blood cultures positive at routine testing for enterobacteria were analyzed with this novel full MALDI-TOF MS based approach. Several species of several genera were included.

The samples underwent direct identification by Sepsityper (Bruker Daltonik). In case of K. pneumoniae, during the identification process the strains were further subtyped to detect KPC-producing isolates by the specific automated algorithm implemented into the Biotyper software.

The same pellet used for species identification was used to detect carbapenemase- and cephalosporinase-production performing STAR-Carba (IVD) and STAR-Cepha (RUO) hydrolysis assays, respectively (Bruker Daltonik). Results of MALDI assay were compared with results of the reference phenotypical method to detect cephalosporinase activity (disk diffusion test with inhibitor), and with results of disk-diffusion synergy test to detect carbapenemase-production.

Results

92/92 samples were identified by MALDI Biotyper at high confidence level.

KPC subtyping detected 11/12 (91.7%) KPC-producing strains. The KPC specific peak was detected in none of the other strains.

STAR-Carba hydrolysis assay resulted positive for 16/16 carbapenemase producing strains (n=12 KPC-K. pneumoniae, n=1 KPC-E. coli, and n=3 MβL-producing K. pneumoniae), and negative for all the other n= 76 strains.

STAR-Cepha hydrolysis assay resulted positive for 16/16 ESβL-producing strains, for 3/3 high level AmpC-producing-strains, and for 15 of the 16 carbapenemase-producing strains but was negative for 1 MβL-K. pneumoniae. It resulted negative for all the other n= 57 strains (wild-type, penicillinase- or constitutive AmpC-producers).

Conclusions & Discussion

In this study, the full MALDI based approach proved to be a reliable, accurate and extremely rapid method to investigate the most worrisome and life-threatening bacterial resistances to beta-lactam agents. It enabled to deliver a conclusive report in a time frame between 30 min (when a KPC-producing K. pneumoniae was detected by subtyping) and 2 h hours (in all the other cases) from the harvest of the positive blood culture.

Moreover, the combination approach showed to be very user-friendly. All single tests are performed on the same hardware platform, thus this method results suitable for the implementation in a routine workflows.