Session: Parallel session 1 - environmental analysis

Carbapenem resistance in Enterobacteriaceae by targeted mass spectrometry

Romain CARRIERE¹, Pénelope BOURMAUD-CADIOT¹, Gabriel CARVALHO², Roxane PRAT³, Chloé DESBIOLLES³, François VANDENESCH³, Jérôme LEMOINE¹

¹Institut des Sciences Analytiques, UMR 5280, CNRS, Université Claude Bernard Lyon 1, Villeurbanne, France, Lyon, France
²Weezion, Villeurbanne, France, Villeurbanne, France
³Institut des Agents Infectieux, Hospices Civils de Lyon, Lyon, France, Lyon, France

Context

Carbapenem resistance in Enterobacteriaceae is increasing every year. Resistance to these antibiotics of last resort is mainly due to the expression of carbapenemases and/or reduced membrane permeability caused by the decreased expression or mutation of the OmpC and OmpF porins. Characterizing changes in membrane permeability can only be achieved using a quantitative proteomics approach. In this work, we used liquid chromatography coupled to targeted mass spectrometry to determine the expression levels of carbapenemases and OmpC porin directly from positive blood cultures.

Methodology

236 E.coli, 137 K.pneumoniae and 134 Enterobacter spp were selected exhibiting a range of carbapenem resistance phenotypes. These strains were spiked in blood cultures bottles. Soon positivity, bacteria cells were isolated, then submitted to cell disruption and trypsin digestion carried out in a Bioruptor ultrasonicator. The samples were analyzed on a 1290 HPLC system coupled to a 6500+ QTRAP system in Scout-MRM mode. Peptide surrogates of Omp C porin and 8 carbapenemases were targeted and their respective expression levels were calculated relative to ribosomal protein expression.

Results

Among the 236 E.coli strains, 33 were detected to be resistant to at least 1 carbapenem antibiotic. For K.pneumoniae, this ratio was 58/137 strains and 37/134 for the Enterobacter spp strains. For the resistant strains, at least 1 carbapenemase was detected in 27/33 E.coli, 50/58 K.pneumoniae and 21/37 Enterobacter spp. For the strains where no carbapenemase was detected though resistant to carbapenems, 9 strains were tested and OmpC porin was either not detected or detected at a very low expression level or mutated in 9 strains.

Conclusion

Targeted mass spectrometry can be used to evaluate the porin expression level and to characterize and quantify carbapenemases. This technique could contribute to a better understanding of the carbapenem resistance phenotype.