Introduction

Colistin antibiotic (i.e., Polymyxin E) is known since the 1950"s, but its use into the clinic was suspended for years due to side effects. Today, it is once again prescribed in humans as last-resort therapy for otherwise untreatable Gram-negative infections (e.g., Pseudomonas aeruginosa infections). Colistin kills efficiently bacteria by disrupting the outer membrane integrity, but unfortunately resistance and heteroresistance (HR) have been described. HR is a poorly characterized phenomenon where subpopulations of apparently isogenic bacteria exhibit a range of antibiotic susceptibilities. Colistin resistance and HR are mainly due to the covalent modification of Lipid A (LpA) membrane by positively charged groups, such as 4-amino-4-desoxy-L-arabinose (L-Ara-4N) or phosphoethanolamine (pEtN). Mass differences between native and modified LpA peaks are observable by MALDI-TOF and allow the detection of colistin resistance but were not demonstrated on possibly HR isolates.

The purpose of this study was to evaluate MALDI-TOF detection of native/modified LpA peaks in clinical isolates of Pseudomonas aeruginosa, a species known for HR prevalence, grown with or without colistin selection pressure.

Materials and methods

Two P. aeruginosa controls (susceptible & resistant) and 5 P. aeruginosa clinical isolates showing variable minimum inhibitory concentration (MIC) and consequently suspected of HR, were characterized according to :

the percentage of resistant cells in a total bacterial population after culture on non-selective versus selective colistin medium;the detection of LpA, LpA-L-Ara-4N, and LpA-pEtN by MALDI-TOF (VITEK®MS);the MIC, measured by broth microdilution (BMD), of the different bacterial subpopulations, resistant or not.

Results

After selection on colistin agar, the possibly HR strains showed a very significant increase in their proportion of resistant cells, accompanied by an increase in MIC and the appearance of modified LpA. However, before selective culture, the low percentage of resistant cells in the initial populations was insufficient to allow the observation of modified LpA characteristic peaks.

Conclusion

MALDI-TOF detection of modified LpA does not seem suitable for the characterization of colistin resistance for strains of P. aeruginosa, suspected of HR, cultivated without selection pressure.