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

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 IS; AbaI; 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 IS; AbaI; integration upstream of; eptA; was required to mediate the colistin-resistant phenotype. In addition, we found a duplicated IS; AbaI; -; 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. baumannii; IMPORTANCE; 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

A novel genome editing platform for drug resistant Acinetobacter baumannii revealed an AdeR-unrelated tigecycline resistance mechanism

Infections with the Gram-negative coccobacillus Acinetobacter baumannii are a major threat in hospital settings. The progressing emergence of multidrug resistant clinical strains significantly reduces the treatment options for clinicians to fight A. baumannii infections. The current lack of robust methods to genetically manipulate drug resistant A. baumannii isolates impedes research on resistance and virulence mechanisms in clinically relevant strains. In this study we developed a highly efficient and versatile genome editing platform enabling the markerless modification of the genome of A. baumannii clinical and laboratory strains, regardless of their resistance profile.We applied this method for the deletion of AdeR, a transcription factor that regulates the expression of the AdeABC efflux pump in tigecycline resistant A. baumannii, to evaluate its function as a putative drug target. Loss of adeR reduced the MIC90 of tigecycline from 25 μg/ml in the parental strains to 3.1 μg/ml in the ΔadeR mutants indicating its importance in the drug resistant phenotype. However, 60% of the clinical isolates remained non-susceptible to tigecycline after adeR deletion. Evolution of artificial tigecycline resistance in two strains followed by whole genome sequencing revealed loss of function mutations in trm, suggesting its role in an alternative AdeABC-independent tigecycline resistance mechanism. This finding was strengthened by the confirmation of trm disruption in the majority of the tigecycline resistant clinical isolates. This study highlights the development and application of a powerful genome editing platform for A. baumannii enabling future research on drug resistance and virulence pathways in clinical relevant strains

A molecular mechanism for the procentriole recruitment of Ana2

During centriole duplication, a preprocentriole forms at a single site on the mother centriole through a process that includes the hierarchical recruitment of a conserved set of proteins, including the Polo-like kinase 4 (Plk4), Ana2/STIL, and the cartwheel protein Sas6. Ana2/STIL is critical for procentriole assembly, and its recruitment is controlled by the kinase activity of Plk4, but how this works remains poorly understood. A structural motif called the G-box in the centriole outer wall protein Sas4 interacts with a short region in the N terminus of Ana2/STIL. Here, we show that binding of Ana2 to the Sas4 G-box enables hyperphosphorylation of the Ana2 N terminus by Plk4. Hyperphosphorylation increases the affinity of the Ana2-G-box interaction, and, consequently, promotes the accumulation of Ana2 at the procentriole to induce daughter centriole formation.Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]