Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.Acinetobacter baumannii is an emerging, opportunistic, Gram-negative bacterial pathogen (19). It is associated with a range of nosocomial infections, including bacteremia, pneumonia, meningitis, and urinary tract infections. Outbreaks, especially in intensive care unit settings, have been identified in numerous countries around the world (23). The treatment of these infections is hampered by the rapid rise in prevalence of A. baumannii strains that are resistant to almost all available antibiotics, including ␤-lactams, fluoroquinolones, tetracyclines, and aminoglycosides (23). In these multidrug-resistant (MDR) strains, colistin (also known as polymyxin E) is often the only remaining treatment (15), although colistin-resistant clinical isolates have already been reported (7,10,21). Intriguingly, some A. baumannii isolates have been shown to display heteroresistance to colistin, where an apparently colistin-susceptible strain (based upon the MIC) harbors a small proportion of colistin-resistant cells (9, 16). Under selective pressure both in vitro (33) and in vivo (10), heteroresistant A. baumannii strains can rapidly give rise to strains with high-level colistin resistance.Colistin is a cationic polypeptide antibiotic that is composed of a cyclic decapeptide linked by an ␣-amide linkage to a fatty acyl chain (15). Its structure differs from that of polymyxin B by only a single amino acid; the two antibiotics demonstrate comparable activities against a range of Gram-negative bacteria (6). Polymyxins are proposed to exert their antibacterial effect on Gram-negative bacteria via a two-step mechanism comprising initial binding to and permeabilization of the outer membrane, followed by destabilization of the cytoplasmic membrane (37). While the exact mechanism of bacterial killing is not clearly defined, a critical first step in the action of polymyxins is the electrostatic interaction between the positively charged peptide and the negatively charged ...

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The structure of the lipid A and core region of the lipopolysaccharide (LPS) from Francisella tularensis (ATCC 29684) was analysed using NMR, mass spectrometry and chemical methods. The LPS contains a β‐GlcN‐(1–6)‐GlcN lipid A backbone, but has a number of unusual structural features; it apparently has no substituent at O‐1 of the reducing end GlcN residue in the lipid part in the major part of the population, no substituents at O‐3 and O‐4 of β‐GlcN, and no substituent at O‐4 of the Kdo residue. The largest oligosaccharide, isolated after strong alkaline deacylation of NaBH4 reduced LPS had the following structure: where Δ‐GalNA‐(1–3)‐β‐QuiNAc represents a modified fragment of the O‐chain repeating unit. Two shorter oligosaccharides lacking the O‐chain fragment were also identified. A minor amount of the disaccharide β‐GlcN‐(1–6)‐α‐GlcN‐1‐P was isolated from the same reaction mixture, indicating the presence of free lipid A, unsubstituted by Kdo and with phosphate at the reducing end. The lipid A, isolated from the products of mild acid hydrolysis, had the structure 2‐N‐(3‐O‐acyl4‐acyl2)‐β‐GlcN‐(1–6)‐2‐N‐acyl1−3‐O‐acyl3‐GlcN where acyl1, acyl2 and acyl3 are 3‐hydroxyhexadecanoic or 3‐hydroxyoctadecanoic acids, acyl4 is tetradecanoic or (minor) hexadecanoic acids. No phosphate substituents were found in this compound. OH‐1 of the reducing end glucosamine, and OH‐3 and OH‐4 of the nonreducing end glucosamine residues were not substituted. LPS of F. tularensis exhibits unusual biological properties, including low endoxicity, which may be related to its unusual lipid A structure.

Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening “superbugs” for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics.

Glycomics, the study of microbial polysaccharides and genes responsible for their formation, requires the continuous development of rapid and sensitive methods for the identification of glycan structures. In this study, methods for the direct analysis of sugars from 10 8 to 10