Session: Parallel session 5 - Forensic and cultural heritage

Distinguiching resistance phenotype in Enterobacteriaceae with Ion Pair Chromatography

Penelope BOURMAUD-CADIOT¹, Romain CARRIÈRE¹, Roxane PRAT², Cholé DESBIOLLES², François VANDENESCH², Jérôme LEMOINE¹

¹Institut des Sciences Analytiques, UMR 5280, CNRS, Université Claude Bernard Lyon 1, Villeurbanne, France, Villeurbanne, France
²Institut des Agents Infectieux, Hospices Civils de Lyon, Lyon, France, Lyon, France

Introduction

In the treatment of patients positive to bacteria detection in their blood, it is important to provide the best treatment, in the shortest possible time. Indeed, the patient will need an antibiotic treatment tailored to the bacteria detected in his blood. This is where proteomics comes in: depending on the resistance phenotype of the detected protein, it may belong to different classes of antibiotic action. This abstract will focus on two class A-beta-lactamases: TEM and SHV, which may have a penicillin-like action (PASE) or an Extended Spectrum Beta-Lactamase (ESBL). This action is determined using an LC-MS method for the detection of specific peptides with 5 or 6 amino acids. This not only enables more appropriate patient management, but also avoids the spread of uncontrolled resistance.

Methodology

Analyses were performed by double-injection liquid chromatography in reverse phase coupled with targeted mass spectrometry (6500+). The samples chosen are Enterobacterales whose strains have been isolated and spiked in blood cultures. Once positive, cultures are prepared by lysis and digestion by ultrasound. The first injection allows to detect resistance protein by scout-MRM method and the second one consists of injecting an acid solution (0.4% HFBA) to increase retention on the column to study peptide sequences of 5 or 6 amino acids.

Results

787 strains were analyzed. We were able to detect TEM in 287 of these: 221 TEM-PASE and 38 TEM-ESBL and the specific method for these proteins confirmed whether the variant was a PASE or a ESBL in 112 samples. For the SHV protein, we detected it in 82 strains and were able to confirm the mechanism of action with our complementary method for 23 SHV-PASE and 36 SHV-BLSE.

Conclusions

Double injection is the method of choice for determining a resistance phenotype, enabling appropriate patient management and antibiotic treatment.