The rapid polymyxin NP test is easy-to-perform, rapid, sensitive, and specific. It detects polymyxin-resistant and -susceptible isolates from any enterobacterial species, regardless of the molecular mechanism of polymyxin resistance. This test offers the possibility of detecting polymyxin resistance from bacterial cultures from infected samples or from selective media before any antimicrobial drug susceptibility testing results are obtained. Results are obtained at least 16 h sooner with this test than with the reference broth microdilution method. This test is as reliable as the reference dilution technique but much less cumbersome and is not based on diffusion of large polymyxin molecules in agar (as are the Etest and the disk-diffusion techniques), which therefore prevents false susceptibility results. A commercial test for research-use only is available for determining MICs of polymyxins (TREK Diagnostic Systems, Inc., Cleveland, OH, USA); however, this test is adapted to testing series of isolates rather than single isolates, and results are available in 16–20 h. Sensitivity and specificity of the rapid polymyxin NP test were high (99.3% and 95.4%, respectively), making it a potential useful clinical technique.
The rapid polymyxin NP test can be performed on cultured bacteria grown on media such as Luria Bertani, Mueller-Hinton, Uriselect-4, eosin-methylene blue, blood agar, and chocolate agar. Interference may be observed with colonies grown on acidifying culture media such as Drigalski, Mac Conkey, and bromocresol purple agars. The test has been optimized in its present form; testing under other conditions (e.g., changes in pH indicator, inoculum size, glucose and pH indicator concentrations; preparation of the solution with non-cation–adjusted culture medium; or use of other polystyrene-containing trays) gave less optimal results.
The rapid polymyxin NP test uses commercially available polymyxin B and colistin sulfate powders, which are unspecified mixtures of chemically related compounds that differ by single amino acid changes and fatty acyl moieties. The changes of the relative proportion of the polymyxin components of these mixtures is poorly defined and a potential source of variability.
We believe that the rapid polymyxin NP test may useful for first-step screening of polymyxin resistance because use of molecular-based techniques for identification of all polymyxin-resistance mechanisms cannot be foreseen. Indeed, polymyxin resistance in Enterobacteriaceae may be associated with many nonrelated mechanisms, some of which are known (defects in outer-membrane proteins, structural modification of lipopolysaccharide, efflux overexpression;) and others still unknown.
This study was subject to at least 4 limitations. First, we did not assess the ability of the rapid polymyxin NP test to detect heteroresistant isolates with low MICs for polymyxin by broth microdilution; such isolates are problematic to detect. Second, the rapid polymyxin NP test involves an orange-to-yellow color change, which is readily apparent for resistant organisms, but interpretation may require more vigilance from laboratory technicians testing organisms with low-level resistance; thus, larger scale studies in different laboratories are needed to fully evaluate the reliability of rapid polymyxin NP test performance. Third, we could not determine the mechanism for colistin resistance in 43 isolates for which several mechanisms of colistin resistance may exist and may be expressed in various ways. Last, we did not evaluate the rapid polymyxin NP test in species of bacteria with metabolic pathways that differed from those of Enterobacteriaceae; further work is needed to adapt the rapid polymyxin NP test to detection of polymyxin-resistant Pseudomonas aeruginosa and Acinetobacter baumannii, which have different metabolic pathways.
The rapid polymyxin NP test can be used to determine susceptibility or resistance to polymyxins in countries facing endemic spread of carbapenemase producers and for which polymyxins are last-resort drugs. The test can also rapidly identify carriers of polymyxin-resistant isolates, leading to rapid implementation of adequate hygiene measures to control their spread. This test can also support the development of novel polymyxin-like molecules, facilitating patient enrollment in pivotal clinical trials.
We thank the following colleagues, who provided polymyxin-resistant isolates: Adrian Brink, Jean-Winoc Decousser, Javier Fernandez-Dominguez, Jean-Yves Madec, Maria-Virginia Villegas, and Mesut Yilmaz.
This work was financed by the University of Fribourg, Switzerland. An international patent form, corresponding to this test, has been filed on behalf of the University of Fribourg.
Emerging Infectious Diseases. 2016;22(6):1038-1043. © 2016 Centers for Disease Control and Prevention (CDC)