colistin and 4-amino-4-deoxyarabinose

The Enterobacter cloacae complex (ECC) consists of closely related bacteria commonly associated with the human microbiota. ECC are increasingly isolated from healthcare-associated infections, demonstrating that these Enterobacteriaceae are emerging nosocomial pathogens. ECC can rapidly acquire multidrug resistance to conventional antibiotics. Cationic antimicrobial peptides (CAMPs) have served as therapeutic alternatives because they target the highly conserved lipid A component of the Gram-negative outer membrane. Many Enterobacteriaceae fortify their outer membrane with cationic amine-containing moieties to prevent CAMP binding, which can lead to cell lysis. The PmrAB two-component system (TCS) directly activates 4-amino-4-deoxy-l-arabinose (l-Ara4N) biosynthesis to result in cationic amine moiety addition to lipid A in many Enterobacteriaceae such as E. coli and Salmonella. In contrast, PmrAB is dispensable for CAMP resistance in E. cloacae. Interestingly, some ECC clusters exhibit colistin heteroresistance, where a subpopulation of cells exhibit clinically significant resistance levels compared to the majority population. We demonstrate that E. cloacae lipid A is modified with l-Ara4N to induce CAMP heteroresistance and the regulatory mechanism is independent of the PmrAB

Topics: Amino Sugars; Anti-Bacterial Agents; Bacterial Proteins; Base Sequence; Colistin; Drug Resistance, Bacterial; Enterobacter cloacae; Gene Expression Regulation, Bacterial; Lipid A; Promoter Regions, Genetic

It is known that the arnB (or pmrH) gene encoding uridine 5'-(beta-1-threo-pentapyranosyl-4-ulose diphosphate) aminotransferase plays a critical role in colistin resistance in Pseudomonas aeruginosa through the addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A. In this study, we attempted to obtain a colistin-resistant mutant from an arnB-deleted mutant through exposure to colistin.. We constructed an arnB deletion mutant (P5ΔarnB :: nptIII) from a colistin-susceptible strain (P5) by allelic replacement mutagenesis, and colistin-resistant mutants were selected in vitro using P5 and P5ΔarnB :: nptIII. The growth rate, lipid A structure, biofilm-forming activity and cell viability in diverse stressful conditions (osmotic, oxidative, acidic and heat stress) were investigated. Expression of phoP, pmrA, parR, and cprR was evaluated by qRT-PCR.. An arnB deletion mutant was shown to develop colistin resistance through the addition of l-Ara4N to lipid A, despite a low survival rate (over 1000-fold lower than that of the wild-type strain) in the media with colistin. Two colistin-resistant mutants showed higher survival rates than colistin-susceptible strains against 5 % NaCl. In the presence of acidic and heat stress, P5ΔarnB :: nptIII-CstR exhibited higher survival rates during conditions of 1 % HCl and 42 °C than the other strains. Both phoP and pmrA genes were overexpressed significantly in both colistin-resistant mutants, but parR and cprR genes were not.. We revealed that colistin resistance could be developed despite arnB deletion in P. aeruginosa through the addition of l-Ara4N to lipid A, which was accompanied by diverse physiological changes.

Topics: Amino Sugars; Anti-Bacterial Agents; Bacterial Proteins; Colistin; Drug Resistance, Bacterial; Genes, Bacterial; Lipid A; Microbial Sensitivity Tests; Microbial Viability; Mutation; Pseudomonas aeruginosa; Stress, Physiological

Colistin resistance in Klebsiella pneumoniae typically involves inactivation or mutations of chromosomal genes mgrB, pmrAB or phoPQ, but data regarding consequent modifications of LPS are limited.. To examine the sequences of chromosomal loci implicated in colistin resistance and the respective LPS-derived lipid A profiles using 11 pairs of colistin-susceptible and -resistant KPC-producing K. pneumoniae clinical strains.. The strains were subjected to high-throughput sequencing with Illumina HiSeq. The mgrB gene was amplified by PCR and sequenced. Lipid profiles were determined using MALDI-TOF MS.. All patients were treated with colistimethate prior to the isolation of colistin-resistant strains (MIC >2 mg/L). Seven of 11 colistin-resistant strains had deletion or insertional inactivation of mgrB. Three strains, including one with an mgrB deletion, had non-synonymous pmrB mutations associated with colistin resistance. When analysed by MALDI-TOF MS, all colistin-resistant strains generated mass spectra containing ions at m/z 1955 and 1971, consistent with addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A, whereas only one of the susceptible strains displayed this lipid A phenotype.. The pathway to colistin resistance in K. pneumoniae primarily involves lipid A modification with Ara4N in clinical settings.

Topics: Adult; Aged; Amino Sugars; Anti-Bacterial Agents; Bacterial Proteins; beta-Lactamases; Chromosomes, Bacterial; Colistin; Drug Resistance, Bacterial; Female; Humans; Klebsiella Infections; Klebsiella pneumoniae; Lipid A; Lipopolysaccharides; Male; Membrane Proteins; Microbial Sensitivity Tests; Middle Aged; Mutagenesis, Insertional

The influence of type of bacterial culture media on antibiotic resistance of Proteus mirabilis R and S forms, was tested. P. mirabilis S1959 (S form), R45 and R110 strains (Re and Ra mutant, respectively) cultivated in media supplemented with 10% heat inactivated bovine serum were resistant to ampicillin, amoxicillin, nalidixic acid and nitroxoline. Proteus strains cultivated in media without serum were sensitive to these antibacterial agents. The presence of serum did not change the polymyxin E (colistin) resistance of there Proteus strains tested. The effects of the presence of colistin (1000 U/ml) in culture media on Proteus lipopolysaccharide composition was studied. The content of uronic acids and phosphate residues in lipopolysaccharides isolated from bacteria cultivated in the presence of colistin (LPS-col), were lower than in control LPSs. The contents of 4-amino-4-deoxy-L-arabinose decreases in S1959 LPS-col, increases in R110 LPS-col and remains unchanged in R45 LPS-col. These results indicate that the presence of colistin in cultivation media exerts an influence on the contents of charged components of LPSs isolated from polymyxin E-resistant Proteus R and S strains.

Topics: Amino Sugars; Anti-Bacterial Agents; Blood; Colistin; Culture Media; Drug Resistance, Multiple; Lipopolysaccharides; Phosphates; Proteus mirabilis; Uronic Acids