A novel mechanism of colistin resistance in acinetobacter baumannii

The Gram-negative bacterium Acinetobacter baumannii is a major cause of hospital-acquired infections, including meningitis, bacteremia, ventilator-associated pneumonia and burn infections. Treatment of A. baumannii infections has been significantly hampered in recent years due to the increased prevalence of strains that are resistant to the most commonly used antibiotics. A frequently used salvage therapy for patients infected with multidrug-resistant A. baumannii is the antimicrobial peptide colistin, a polymyxin antibiotic that is structurally similar to polymyxin B. Colistin initially binds to the lipid A moiety of lipopolysaccharide (LPS), resulting in destabilization of the Gram-negative outer membrane. Alarmingly, as the usage of colistin has increased, so to have reports of colistin-resistant A. baumannii strains. The aim of this project was to determine how this emerging pathogen can become resistant to colistin, one of the few remaining treatment options. Chapter 1 describes how colistin resistance in A. baumannii can be due to the loss of lipopolysaccharide. Thirteen independent colistin-resistant derivatives of the A. baumannii type strain ATCC 19606 were characterized, and sequencing of the lipid A biosynthesis pathway genes revealed that all strains contained a mutation within one of the first three genes of the pathway; lpxA, lpxC and lpxD. These mutations ranged from single base changes, resulting in amino acid changes, to large deletions. All mutations resulted in the complete loss of LPS production, culminating in high-level resistance to colistin (>128 μg/ml). Furthermore, similar mutations were identified in colistin-resistant A. baumannii clinical isolates, highlighting the significance of this unique mechanism of colistin resistance. Chapter 2 describes how the movement of an insertion sequence (IS) element in the A. baumannii genome can result in colistin resistance and loss of LPS via insertion into either lpxA or lpxC. ISAba11 had previously been shown to be part of the transposon Tn6021 in the ATCC 19606 genome. This work demonstrates that ISAba11 is both mobile and replicative in the ATCC 19606 genome and that movement of this novel element can result in high-level colistin resistance. Finally, chapter 3 describes how LPS-deficient A. baumannii interact with various components of the innate immune system. Using a series of Toll- like receptor (TLR)-knock-out murine macrophages, it was determined that LPS-deficient A. baumannii stimulated TNFα secretion and NF-κB activation at levels 2-4-fold lower than wild type A. baumannii, but were still able to elicit TNFα secretion (at low levels) via a Toll-like receptor 2- dependent mechanism. Furthermore, this work also determined that while LPS-deficient A. baumannii is not altered in its resistance to human serum, it shows increased susceptibility to the human antimicrobial peptide LL-37. In summary, this study shows that loss of LPS is a novel mechanism of colistin resistance used by A. baumannii, and that the loss of this major outer membrane structural component significantly alters the innate immune response to these cells.