Colistin Resistance in Acinetobacter baumannii Is Mediated by Complete Loss of Lipopolysaccharide Production

ABSTRACT 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.

[1]  W. Hillen,et al.  Analysis and nucleotide sequence of an origin of DNA replication in Acinetobacter calcoaceticus and its use for Escherichia coli shuttle plasmids. , 1990, Gene.

[2]  R. Jones,et al.  Global assessment of the antimicrobial activity of polymyxin B against 54 731 clinical isolates of Gram-negative bacilli: report from the SENTRY antimicrobial surveillance programme (2001-2004). , 2006, Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases.

[3]  M. Ferraro.  Performance standards for antimicrobial susceptibility testing , 2001 .

[4]  R. Hancock,et al.  Use of the fluorescent probe 1-N-phenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa , 1984, Antimicrobial Agents and Chemotherapy.

[5]  K. Ko,et al.  Two distinct clones of carbapenem-resistant Acinetobacter baumannii isolates from Korean hospitals. , 2009, Diagnostic microbiology and infectious disease.

[6]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[7]  C. Vay,et al.  Selection of colistin-resistant Acinetobacter baumannii isolates in postneurosurgical meningitis in an intensive care unit with high presence of heteroresistance to colistin. , 2009, Diagnostic microbiology and infectious disease.

[8]  C. Murray,et al.  Colistin Heteroresistance in Acinetobacter and Its Association with Previous Colistin Therapy , 2007, Antimicrobial Agents and Chemotherapy.

[9]  P. Nordmann,et al.  Functional Analysis of Insertion Sequence ISAba1, Responsible for Genomic Plasticity of Acinetobacter baumannii , 2009, Journal of bacteriology.

[10]  O. Holst,et al.  The structure of the carbohydrate backbone of the lipopolysaccharide from Acinetobacter baumannii strain ATCC 19606. , 2002, European journal of biochemistry.

[11]  J. Boyce,et al.  Identification of Novel Glycosyltransferases Required for Assembly of the Pasteurella multocida A:1 Lipopolysaccharide and Their Involvement in Virulence , 2009, Infection and Immunity.

[12]  A. Williams,et al.  Structural basis for the acyl chain selectivity and mechanism of UDP-N-acetylglucosamine acyltransferase , 2007, Proceedings of the National Academy of Sciences.

[13]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[14]  Jianjun Li,et al.  Structural analysis of the lipopolysaccharide of Pasteurella multocida strain VP161: identification of both Kdo-P and Kdo-Kdo species in the lipopolysaccharide. , 2005, Carbohydrate research.

[15]  K. Ko,et al.  Independent emergence of colistin-resistant Acinetobacter spp. isolates from Korea. , 2009, Diagnostic microbiology and infectious disease.

[16]  Harald Seifert,et al.  Acinetobacter baumannii: Emergence of a Successful Pathogen , 2008, Clinical Microbiology Reviews.

[17]  J. Turnidge,et al.  Colistin: the re-emerging antibiotic for multidrug-resistant Gram-negative bacterial infections. , 2006, The Lancet. Infectious diseases.

[18]  C. Raetz,et al.  Lipid A modification systems in gram-negative bacteria. , 2007, Annual review of biochemistry.

[19]  Michele Iacono,et al.  Whole-Genome Pyrosequencing of an Epidemic Multidrug-Resistant Acinetobacter baumannii Strain Belonging to the European Clone II Group , 2008, Antimicrobial Agents and Chemotherapy.

[20]  S. Galloway,et al.  A mutant of Escherichia coli defective in the first step of endotoxin biosynthesis. , 1990, The Journal of biological chemistry.

[21]  L. Actis,et al.  Genetic and Phenotypic Analysis of Acinetobacter baumannii Insertion Derivatives Generated with a Transposome System , 2002, Applied and Environmental Microbiology.

[22]  R. Nation,et al.  Antibiograms of multidrug-resistant clinical Acinetobacter baumannii: promising therapeutic options for treatment of infection with colistin-resistant strains. , 2007, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[23]  M. Adams,et al.  Resistance to Colistin in Acinetobacter baumannii Associated with Mutations in the PmrAB Two-Component System , 2009, Antimicrobial Agents and Chemotherapy.

[24]  J. Li,et al.  Heteroresistance to Colistin in Multidrug-Resistant Acinetobacter baumannii , 2006, Antimicrobial Agents and Chemotherapy.

[25]  Michael Brudno,et al.  SHRiMP: Accurate Mapping of Short Color-space Reads , 2009, PLoS Comput. Biol..

[26]  E. Birney,et al.  Velvet: algorithms for de novo short read assembly using de Bruijn graphs. , 2008, Genome research.

[27]  W. Hunter,et al.  Nucleotide substrate recognition by UDP-N-acetylglucosamine acyltransferase (LpxA) in the first step of lipid A biosynthesis. , 2007, Journal of molecular biology.

[28]  Mark Gerstein,et al.  New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis. , 2007, Genes & development.

[29]  B. Zomer,et al.  Meningitis bacterium is viable without endotoxin , 1998, Nature.

[30]  R. Weinstein,et al.  Acinetobacter infection. , 2008, The New England journal of medicine.

[31]  H. Nikaido.  Molecular Basis of Bacterial Outer Membrane Permeability Revisited , 2003, Microbiology and Molecular Biology Reviews.

[32]  J. Li,et al.  Activity of Colistin against Heteroresistant Acinetobacter baumannii and Emergence of Resistance in an In Vitro Pharmacokinetic/Pharmacodynamic Model , 2007, Antimicrobial Agents and Chemotherapy.

[33]  J. Boyce,et al.  Fis Is Essential for Capsule Production in Pasteurella multocida and Regulates Expression of Other Important Virulence Factors , 2010, PLoS pathogens.

[34]  D. Feola,et al.  Polymyxin B Sulfate and Colistin: Old Antibiotics for Emerging Multiresistant Gram-Negative Bacteria , 1999, The Annals of pharmacotherapy.

[35]  D. Peng,et al.  Moraxella catarrhalis Bacterium without Endotoxin, a Potential Vaccine Candidate , 2005, Infection and Immunity.

[36]  P. Wayne.  PERFORMANCE STANDARDS FOR ANTIMICROBIAL SUSCEPTIBILITY TESTING, NINTH INFORMATIONAL SUPPLEMENT , 2008 .