Comparative Evaluation of the VITEK 2, Disk Diffusion, Etest, Broth Microdilution, and Agar Dilution Susceptibility Testing Methods for Colistin in Clinical Isolates, Including Heteroresistant Enterobacter cloacae and Acinetobacter baumannii Strains

Abstract

Increasing antibiotic resistance in gram-negative bacteria has recently renewed interest in colistin as a therapeutic option. The increasing use of colistin necessitates the availability of rapid and reliable methods for colistin susceptibility testing. We compared seven methods of colistin susceptibility testing (disk diffusion, agar dilution on Mueller-Hinton [MH] and Isosensitest agar, Etest on MH and Isosensitest agar, broth microdilution, and VITEK 2) on 102 clinical isolates collected from patient materials during a selective digestive decontamination or selective oral decontamination trial in an intensive-care unit. Disk diffusion is an unreliable method to measure susceptibility to colistin. High error rates and low levels of reproducibility were observed in the disk diffusion test. The colistin Etest, agar dilution, and the VITEK 2 showed a high level of agreement with the broth microdilution reference method. Heteroresistance for colistin was observed in six Enterobacter cloacae isolates and in one Acinetobacter baumannii isolate. This is the first report of heteroresistance to colistin in E. cloacae isolates. Resistance to colistin in these isolates seemed to be induced upon exposure to colistin rather than being caused by stable mutations. Heteroresistant isolates could be detected in the broth microdilution, agar dilution, Etest, or disk diffusion test. The VITEK 2 displayed low sensitivity in the detection of heteroresistant subpopulations of E. cloacae. The VITEK 2 colistin susceptibility test can therefore be considered to be a reliable tool to determine susceptibility to colistin in isolates of genera that are known not to exhibit resistant subpopulations. In isolates of genera known to (occasionally) exhibit heteroresistance, an alternative susceptibility testing method capable of detecting heteroresistance should be used.The polymyxins are a group of polypeptide antibiotics that were first isolated in 1947 from a spore-bearing soil bacillus (Bacillus polymyxa). Several chemically different polymyxins (A to E) could be isolated from different strains of this bacillus (19). Only polymyxin B and polymyxin E (colistin) have been used clinically. Systemic use of colistin was restricted, mainly because of reports of serious nephrotoxicity and the emergence of alternative, less toxic antibiotics. Polymyxin B use has continued in the topical treatment of skin, ear, and ocular diseases. Increasing antibiotic resistance in gram-negative bacteria has recently renewed interest in colistin as an intravenous therapeutic option. Colistin is now increasingly being used for life-threatening infections with multidrug-resistant gram-negative bacteria (6,7,13,14). The increasing use of colistin necessitates the availability of rapid and reliable methods for colistin susceptibility testing.Disk diffusion is a commonly used method for measuring colistin susceptibility. However, evaluation of in vitro susceptibility testing methods for colistin has shown testing errors with various disk diffusion methods compared t...

Comparison of methods for detection of plasmid-mediated and chromosomally encoded colistin resistance in Enterobacteriaceae

“…The low sensitivity to detect colistin heteroresistance has already been described for another automated system, i.e. the bioMérieux Vitek system [15].…”

<i>In Vitro</i> Activity of Colistin and Vancomycin or Azithromycin Combinations on Extensively Drug Resistant <i>Acinetobacter baumannii</i> Clinical Isolates

“…However, the optimal method for testing susceptibility of A. baumannii to colistin is still controversial. The drug diffusion through the agar is inconsistent and poor, making errors with disc diffusion common when compared to other methods [25]. The controversy comes from the absence of guidelines by the CLSI for disc diffusion.…”

Heteroresistance

“…Disc diffusion method or commercial automated systems may fail to detect most heteroresistant isolates (Lo-Ten-Foe et al, 2007;Tenover, 1999), and E-test results depend on the medium used (T.Y. Tan & Ng., 2007).…”

“…Heteroresistance to colistin was initially described among clinical A. baumannii (Li et al, 2006;C.H. Tan et al, 2007;Yau et al, 2009) and Enterobacter cloacae isolates (Lo-Ten-Foe et al, 2007). Intrestingly, in A. baumannii, this heteroresistance was documented to be related to previous colistin therapy (Hawley et al, 2008).…”