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State-of-the-art detection of carbapenemases and colistin resistance

Rapid detection of colistin resistance using a newly developed protocol for MALDI-TOF Mass Spectrometry

April 19 • O0535

N. Perrot1, D. Dechaume1, F. Javerliat1, K. Pinkston2, V. Girard1, G. Zambardi1, JP. Charrier3

1) R&D Microbiology, bioMérieux, La Balme-les-Grottes, France
2) bioMérieux Inc., Hazelwood, MO United States
3) bioMérieux, Marcy l'Étoile, France

Background: In the last several years, colistin resistance has increased and its emergence has caused global concern. MALDI-TOF MS can be used as a rapid complementary method to conventional antimicrobial susceptibility testing methods. It allows the detection of the drug target, i.e. the lipid A (LpA) moiety of the lipopolysaccharide, which generates resistance when modified by addition of 4-amino-4-deoxy-L-arabinose and/or phosphoethanolamine.

Materials/methods: A rapid MALDI-TOF sample preparation was developed using weak hydrolysis in acetic acid solution. Modified and unmodified LpA were detected using VITEK®MS Plus mass spectrometer in negative ionization mode, norharmane matrix and a m/z 1000-4000 mass window. A panel of 200 collection isolates from 20 different bacterial species (Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae) were tested. This panel of isolates included susceptible and resistant bacteria harbouring either chromosomal- or plasmid-mediated resistance genes. The MALDI-TOF assay was compared to the colistin susceptibility status given by broth microdilution (BMD), growth on CHROMID® Colistin R agar, PCR testing of mcr genes and whole genome sequencing (WGS).

Results: Detection of mcr genes by PCR was a good predictor of colistin resistance, but the significance of mutations observed by WGS in pmrA, pmrB, phoP, and phoQ genes was frequently uncertain. Some mutations appeared to be associated with resistance whereas others were clearly not.
Prediction of colistin resistance using the MALDI-TOF LpA assay was in agreement with the BMD method and CHROMID® Colistin R agar for a large majority of isolates and species, e.g. LpA versus BMD agreement was 100% (36/36), 98% (41/42), and 100% (6/6) in Escherichia coli, Klebsiella pneumoniae, and Klebsiella aerogenes, respectively. However, for a few species, inconsistent results were observed for some strains, e.g. LpA versus BMD agreement was 83% (33/40) and 87% (27/31) in A. baumannii, and P. aeruginosa, respectively. This observation may be due to colistin hetero-resistance, a still poorly understood phenomenon, making it challenging to diagnose in clinical settings.

Conclusions: The usefulness of a short sample preparation for LpA extraction, associated with a VITEK®MS Plus reading, was demonstrated as a rapid alternative to conventional methods for colistin resistance detection on a large panel of bacterial species and strains.



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