Introduction
Colistin is a last-resort antibiotic for treatment of multiple drug resistant Gram-negative bacterial infections. However, its use is hampered by the requirements of antimicrobial susceptibility testing. There is no reliable disk diffusion method, validated rapid antimicrobial susceptibility methods are scarce and only broth microdilution (BMD) is accepted by regulating bodies as EUCAST. Also, the reliability of BMD has been questioned, especially for species other than Escherichia coli. Here, we validated a rapid MALDI-TOF MS-based assay for lipid A characterisation in Klebsiella pneumoniae, using BMD and whole genome sequencing as reference.

Methods
Lipid A profiling using the MBT Lipid Xtract Kit on the MALDI Biotyper Sirius System (Bruker Daltonics, Germany) was performed on 183 K. pneumoniae strains with previously characterised geno- and phenotype. Acquired spectra were manually examined for peaks at 1971 m/z and/or 1963 m/z, corresponding to 4-amino-4-deoxy-L-arabinose and/or phosphoethanolamine modified lipid A. If present, strains were interpreted as colistin resistant. Also, spectra were processed automated by using the prototype MBT HT LipidART Module (Bruker Daltonics). Results from both approaches were compared to BMD, and (on a subset of strains) to the presence (n=69) or absence (n=54) of confirmed mechanisms of resistance.

Results
Spectra were available for 179 isolates. Of these, 93% (manual) and 89% (automated) were S/R classified in agreement with BMD. Sensitivity for the presence of confirmed genetic resistance mechanisms was 94%, 88% and 96% for manual/automated lipid A analysis and BMD, respectively (Fig. 1).

Summary
MBT Lipid Xtract Kit is a useful and rapid screening tool for colistin resistance in K. pneumoniae. In the full strain collection containing 91 resistant and 92 susceptible isolates categorical agreement was 93% (automated: 88%), indicating the need for BMD confirmation. However, sensitivity for the presence of confirmed mechanisms of resistance was in line with BMD, suggesting a complex geno/pheno association behind colistin resistance in K. pneumoniae. This should be further investigated.

Figure 1: Colistin resistance prediction using lipid A analysis or the reference method (BMD) in K. pneumoniae with (n=69) or without (n=54) genetic markers consistent with colistin resistance.