Dissecting Colistin Resistance Mechanisms in Extensively Drug-Resistant Acinetobacter baumannii Clinical Isolates

Abstract

Nosocomial infections with Acinetobacter baumannii are a global problem in intensive care units with high mortality rates. Increasing resistance to first- and second-line antibiotics has forced the use of colistin as last-resort treatment, and increasing development of colistin resistance in A. baumannii has been reported. We evaluated the transcriptional regulator PmrA as potential drug target to restore colistin efficacy in A. baumannii Deletion of pmrA restored colistin susceptibility in 10 of the 12 extensively drug-resistant A. baumannii clinical isolates studied, indicating the importance of PmrA in the drug resistance phenotype. However, two strains remained highly resistant, indicating that PmrA-mediated overexpression of the phosphoethanolamine (PetN) transferase PmrC is not the exclusive colistin resistance mechanism in A. baumannii A detailed genetic characterization revealed a new colistin resistance mechanism mediated by genetic integration of the insertion element ISAbaI upstream of the PmrC homolog EptA (93% identity), leading to its overexpression. We found that eptA was ubiquitously present in clinical strains belonging to the international clone 2, and ISAbaI integration upstream of eptA was required to mediate the colistin-resistant phenotype. In addition, we found a duplicated ISAbaI-eptA cassette in one isolate, indicating that this colistin resistance determinant may be embedded in a mobile genetic element. Our data disprove PmrA as a drug target for adjuvant therapy but highlight the importance of PetN transferase-mediated colistin resistance in clinical strains. We suggest that direct targeting of the homologous PetN transferases PmrC/EptA may have the potential to overcome colistin resistance in A. baumanniiIMPORTANCE The discovery of antibiotics revolutionized modern medicine and enabled us to cure previously deadly bacterial infections. However, a progressive increase in antibiotic resistance rates is a major and global threat for our health care system. Colistin represents one of our last-resort antibiotics that is still active against most Gram-negative bacterial pathogens, but increasing resistance is reported worldwide, in particular due to the plasmid-encoded protein MCR-1 present in pathogens such as Escherichia coli and Klebsiella pneumoniae Here, we showed that colistin resistance in A. baumannii, a top-priority pathogen causing deadly nosocomial infections, is mediated through different avenues that result in increased activity of homologous phosphoethanolamine (PetN) transferases. Considering that MCR-1 is also a PetN transferase, our findings indicate that PetN transferases might be the Achilles heel of superbugs and that direct targeting of them may have the potential to preserve the activity of polymyxin antibiotics.

Keywords: Acinetobacter baumannii; antibiotic resistance; colistin; eptA; ethanolamine transferase; mcr-1; pmrA.

FIG 1

Quantification of pmrC expression levels in colistin-resistant A. baumannii clinical isolates and their ΔpmrA mutants. Expression levels of pmrC were quantified by qRT-PCR in colistin-resistant A. baumannii isolates (white bars) and their ΔpmrA mutants (black bars). The expression levels were normalized to the pmrC expression in the ATCC 17978 reference strain.

FIG 2

Discrimination and quantification of pmrC and eptA. (A) Schematic representation of differences in the pmrC, eptA-1, and eptA-2 coding sequence. Primers marked by black (oVT162/oVT163) and red (oVT164/oVT165) arrows were used to detect pmrC and eptA in qRT-PCR experiments, respectively. The primers marked by green arrows (oVT152/oVT153) were used to genotype the eptA isoforms. Primers marked by blue (oVT198/oVT199) and orange (oVT201/oVT202) arrows were used to discriminate eptA-1 and eptA-2, respectively. (B) Expression levels of eptA were quantified by qRT-PCR in colistin-resistant A. baumannii isolates BV94 and BV189 (white bars) and their ΔpmrA mutants (black bars). The expression levels were normalized to the pmrC expression in the ATCC 17978 reference strain.

FIG 3

Representation of the different ISAbaI-eptA genomic regions present in BV94 and BV189. The nucleic acid sequence of the ISAbaI inverted repeats right and left (IRR and IRL, respectively) and P_out promoter are shown until the eptA start codon. The 9-bp target site duplications (TSD) up- and downstream of ISAbaI are not present for eptA-3, which is consistent with an ISAbaI-eptA-2 duplication. The junction between ABK1_3144 and ISAbaI-eptA-3 has been sequenced, while the sequence downstream of ABK1_2603 could not be resolved. The ABK1 gene annotation is shown according to the genomic sequence of A. baumannii strain 1656-2 (GenBank accession number NC_017162).

FIG 4

Quantification of pmrC and naxD expression levels in A. baumannii BV191 and its ΔpmrA mutants. The expression of pmrC (white bars) and naxD (black bars) was quantified by qRT-PCR and normalized to the gene expression level in the reference strain ATCC 17978.

FIG 5

Schematic representation of A. baumannii colistin resistance mechanisms. The two pathways leading to phosphoethanolamine (PetN) transferase overexpression and colistin resistance are represented. The major A. baumannii PetN transferase overexpression pathway results from pmrC expression, which is activated by the transcriptional regulator PmrA previously phosphorylated (activated) by a mutated variant of the sensor kinase PmrB (PmrB*). Alternatively, A. baumannii PetN transferase overexpression can result from the integration of the ISAbaI insertion element upstream of an eptA isoform. PetN transferase enzymes decorate the outer membrane lipid A with PetN, thereby lowering the negative charge and preventing colistin binding. Potential PmrA inhibitors would only block the pmrC pathway (dark blue cross), while PetN transferase inhibitors would block lipid A modification (red cross) and restore colistin efficacy against A. baumannii.