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 ...

We recently demonstrated that colistin resistance in Acinetobacter baumannii can result from mutational inactivation of genes essential for lipid A biosynthesis (Moffatt JH, et al., Antimicrob. Agents Chemother. 54:4971-4977). Consequently, strains harboring these mutations are unable to produce the major Gram-negative bacterial surface component, lipopolysaccharide (LPS). To understand how A. baumannii compensates for the lack of LPS, we compared the transcriptional profile of the A. baumannii type strain ATCC 19606 to that of an isogenic, LPS-deficient, lpxA mutant strain. The analysis of the expression profiles indicated that the LPS-deficient strain showed increased expression of many genes involved in cell envelope and membrane biogenesis. In particular, upregulated genes included those involved in the Lol lipoprotein transport system and the Mla-retrograde phospholipid transport system. In addition, genes involved in the synthesis and transport of poly-␤-1,6-N-acetylglucosamine (PNAG) also were upregulated, and a corresponding increase in PNAG production was observed. The LPS-deficient strain also exhibited the reduced expression of genes predicted to encode the fimbrial subunit FimA and a type VI secretion system (T6SS). The reduced expression of genes involved in T6SS correlated with the detection of the T6SS-effector protein AssC in culture supernatants of the A. baumannii wild-type strain but not in the LPS-deficient strain. Taken together, these data show that, in response to total LPS loss, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface.

Infections caused by Acinetobacter baumannii are of increasing concern, largely due to the multidrug resistance of many strains. Here we show that insertion sequence ISAba11 movement can result in inactivation of the A. baumannii lipid A biosynthesis genes lpxA and lpxC, resulting in the complete loss of lipopolysaccharide production and high-level colistin resistance.

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