Rapid Determination of Colistin Resistance in Clinical Strains of Acinetobacter baumannii by Use of the Micromax Assay

Abstract

Colistin is an old antibiotic which has been used as a therapeutic option for carbapenem-and multidrug-resistant Gram-negative bacteria, like Acinetobacter baumannii. This pathogen produces life-threatening infections, mainly in patients admitted to intensive care units. Rapid detection of resistance to colistin may improve patient outcomes and prevent the spread of resistance. For this purpose, Micromax technology was evaluated in four isogenic A. baumannii strains with known mechanisms of resistance to colistin and in 66 isolates (50 susceptible and 16 resistant). Two parameters were determined, DNA fragmentation and cell wall damage. To assess DNA fragmentation, cells trapped in a microgel were incubated with a lysing solution to remove the cell wall, and the released nucleoids were visualized under fluorescence microscopy. Fragmented DNA was observed as spots that diffuse from the nucleoid. To assess cell wall integrity, cells were incubated with a lysis solution which removes only weakened cell walls, resulting in nucleoid release exclusively in affected cells. A dose-response relationship was demonstrated between colistin concentrations and the percentages of bacteria with DNA fragmentation and cell wall damage, antibiotic effects that were delayed and less frequent in resistant strains. Receiver operating characteristic (ROC) curves demonstrated that both DNA fragmentation and cell wall damage were excellent parameters for identifying resistant strains. Obtaining <11% of bacteria with cell wall damage after incubation with 0.5 g/ml colistin identified resistant strains of A. baumannii with 100% sensitivity and 96% specificity. Results were obtained in 3 h 30 min. This is a simple, rapid, and accurate assay for detecting colistin resistance in A. baumannii, with strong potential value in critical clinical situations.

Development of New Tools to Detect Colistin-Resistance among Enterobacteriaceae Strains

“…The Micromax assay (Halotech DNA SL, Madrid, Spain) is based on the detection of DNA fragmentation and cell wall damage in the presence of colistin [86]. Bacteria are incubated for 60 min with 0.5 µ g/ml of colistin, trapped in a microgel, and then incubated with a lysis solution to remove weakened cell walls.…”

Polymyxins: Antibacterial Activity, Susceptibility Testing, and Resistance Mechanisms Encoded by Plasmids or Chromosomes

“…The Micromax assay is based on the detection of released nucleotides, indicating cell wall damage, in the presence of colistin (104). After incubation with 0.5 g/ml of colistin, strains are considered resistant to colistin if Յ11% of bacteria present cell wall damage.…”

Global Dissemination of Carbapenemase-Producing Klebsiella pneumoniae: Epidemiology, Genetic Context, Treatment Options, and Detection Methods

“…This situation demonstrates the need for the development of accurate and reliable methods for detecting colistin resistance. Recently, several methods for the identification of colistin resistance were reported, including various routine colistin MIC testing methods, such as BMD, BMD-P80, AD, Etest, MTS, and Vitek2 ( Dafopoulou et al, 2015 ; Humphries, 2015 ); capillary electrophoresis method according to characteristic surface properties of bacteria ( Sautrey et al, 2015 ); and the micromax assay based on evaluation of the efficacy of antibiotics that affect cell wall integrity ( Tamayo et al, 2013 ). Because a recent report showed that the mcr-1 gene, involved in the modification of lipid A, is a major contributor to colistin resistance in Gram-negative bacteria ( Liu et al, 2016 ), detection of this gene may be important in the detection of colistin resistance.…”