ABSTRACT
OBJECTIVES: The rapid dissemination of the mcr-1 gene via plasmid-mediated transfer has raised concerns regarding the efficacy of colistin as a last-resort treatment for multidrug-resistant Gram-negative bacterial infections. Current mcr-1 gene detection methods mainly focus on cultured bacteria, which is a complex and time-consuming process requiring skilled personnel, making it unsuitable for field analysis. METHODS: A rapid detection technique combining recombinase polymerase amplification with a lateral flow dipstick targeting uncultured clinical samples was developed. RESULTS: This new method targeting the mcr-1 gene region (23 232-23 642 bp, no. KP347127.1) achieved a low detection limit of 10 copies/µL. The whole process was carried out with high specificity and was completed within 20 min. The evaluation assay was conducted using 45 human faecal samples; 16 strains yielded a 98% accuracy, closely matching antimicrobial susceptibility outcomes. CONCLUSIONS: The novel method integrates nucleic acid extraction, isothermal amplification, and a test assay, suggesting the potential for timely colistin resistance surveillance in frontline disease control and healthcare settings, supporting future prevention and clinical standardization efforts.
Subject(s)
Bacterial Proteins , Drug Resistance, Bacterial , Enterobacteriaceae , Ethanolaminephosphotransferase , Nucleic Acid Amplification Techniques , Recombinases , Humans , Feces/microbiology , Enterobacteriaceae/drug effects , Enterobacteriaceae/genetics , Enterobacteriaceae/isolation & purification , DNA-Directed DNA Polymerase , Ethanolaminephosphotransferase/analysis , Ethanolaminephosphotransferase/genetics , Bacterial Proteins/analysis , Bacterial Proteins/genetics , Drug Resistance, Bacterial/genetics , Colistin/pharmacology , Anti-Bacterial Agents/pharmacologySubject(s)
Diacylglycerol Cholinephosphotransferase/analysis , Ethanolaminephosphotransferase/analysis , Saccharomyces cerevisiae/enzymology , Cytidine Diphosphate/analogs & derivatives , Diacylglycerol Cholinephosphotransferase/drug effects , Diglycerides/metabolism , Enzyme Activation , Ethanolaminephosphotransferase/drug effects , Phosphatidylcholines/biosynthesis , Phosphatidylethanolamines/biosynthesis , Phospholipids/pharmacologyABSTRACT
Phospholipid base exchange and cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EPT) activities were assessed in the membranes of neutrophils or lymphocytes from patients with various inflammatory disorders. Ethanolamine exchange activity was significantly enhanced in both neutrophils and lymphocytes from patients with active Behçet's disease, active systemic lupus erythematosus (SLE), and severe bacterial infections and slightly enhanced in those from patients with active rheumatoid arthritis (RA), compared with healthy controls. No abnormal findings were found in CPT, EPT, or serine or choline base exchange activities in the leukocytes from any of the diseased groups tested or in the ethanolamine exchange activity of patients with severe viral infections and inactive SLE, RA, and Behçet's disease. The authors have recently demonstrated the enhancement of transmethylation and phospholipase A2 activity in human leukocyte membranes at the height of inflammatory disease states, as well as the activation of leukocyte ethanolamine exchange by bioactive stimulants. These data postulate that phosphatidylethanolamine synthesis by the base exchange reaction may be the precursor of transmethylation and its subsequent activation of phospholipase A2, leading to the induction of arachidonic acid cascade.
Subject(s)
Inflammation/metabolism , Leukocytes/metabolism , Membrane Lipids/metabolism , Phospholipids/metabolism , Adolescent , Adult , Behcet Syndrome/metabolism , Diacylglycerol Cholinephosphotransferase/analysis , Ethanolamine , Ethanolaminephosphotransferase/analysis , Ethanolamines/metabolism , Female , Humans , Lupus Erythematosus, Systemic/metabolism , Male , Methylation , Middle Aged , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/metabolism , Phospholipases A/analysis , Phospholipases A2ABSTRACT
Highly purified rat brain myelin isolated by two different procedures showed appreciable activity for CDP-ethanolamine: 1,2-diacyl-sn-glycerol ethanolaminephosphotransferase (EC 2.7.8.1). Specific activity was close to that of total homogenate and approximately 12-16% that of brain microsomes. Three other lipid-synthesizing enzymes, cerebroside sulfotransferase, lactosylceramide sialyltransferase, and serine phospholipid exchange enzyme, were found to have less than 0.5% the specific activity in myelin compared with microsomes. Washing the myelin with buffered salt or taurocholate did not remove the phosphotransferase, but activity was lost from both myelin and microsomes by treatment with Triton X-100. It resembled the microsomal enzyme in having a pH optimum of 8.5 and a requirement for Mn2+ and detergent, but differed in showing no enhancement with EGTA. The diolein Km was similar for the two membranes (2.5-4 x 10(-4) M), but the CDP-ethanolamine Km was lower for myelin (3-4 x 10(-5) M) than for microsomes (11 - 13 x 10(-5 M). Evidence is reviewed that this enzyme is able to utilize substrate from the axon in situ.
