Colistin micro broth disk elution method in determining susceptibility to colistin in acinetobacter species

Abstract

Colistin use as a drug of last resort to treat multidrug-resistant organisms in humans has increased following its re-introduction. This has led to large patterns of resistance among various gram negative bacterial organisms hence the need to widely test for resistance or susceptibility of the various strains especially in the Uganda setting. The routine method that is currently being used by laboratories is the disc diffusion method where agar and colistin antimicrobial discs are used but is associated with false sensitive results. This is due to the fact that colistin is a drug of high molecular weight and hence doesn’t diffuse readily in the agar which makes the disc diffusion method unreliable. There is need therefore to evaluate other reliable methods that laboratories can adapt in the determination of colistin resistance. This study therefore aimed to use the colistin micro broth disk elution method (CMBDE), which is modified from the standard broth microdilution method to assess colistin susceptibility of acinetobacter species whose susceptibility and resistance was already established. 30 acinetobacter isolates were used together with four control gram negative strains i.e Klebsiella Pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa which were resistant and E.Coli which was susceptible to colistin. CMBDE was performed with four 10-ml cation-adjusted Mueller-Hinton broth tubes per isolate, to which 0, 1, 2, and 4 colistin 10µg disks were added, generating final concentrations in the tubes of 0 (growth control), 1, 2, and 4µg/ml respectively. Interpretation of results was done after an overnight incubation at 37˚c using a protocol from the Argentina National and Regional Reference Laboratory for Antimicrobial Resistance. There was an overall 57% agreement between results yielded by the CMBDE compared to those attained by the standard BMD. The sensitivity of the CMBDE method in comparison with the BMD was 71%, its specificity was 52%, its False Resistance Rate (FRR) was 48%, False Susceptibility Rate (FSR) was 29%, Resistance Predictive Value (RPV) rate was at 31%, Susceptibility Predictive Value (SPV) rate was at 86% and its overall accuracy was 57%. Very major errors occurred with most colistin susceptible strains of acinetobacter which tested resistant. This was probably due to low colistin concentrations than those intended which affected the sensitivity of the method. The specificity of the method was slightly high though not high enough to be adapted for use in routine laboratories. The first testing done with the CMBDE method didn’t give reliable results as compared to the standard BMD method. However, this was due to the very many errors that occurred during performance as this was the very first time of conducting the method. Repeat testing is therefore to be done in order to yield the final results that can be used to assess the reliability, precision, validity and accuracy of the method in determining the colistin susceptibility or resistance of the gram negative bacterial species.