Comparing Quantitative and Qualitative Methods for Detecting the In Vitro Activity of Colistin against Different Gram-Negative Bacilli

Research Article

J Bacteriol Mycol. 2021; 8(5): 1181.

Comparing Quantitative and Qualitative Methods for Detecting the In Vitro Activity of Colistin against Different Gram-Negative Bacilli

Shams N1#, AlHiraky H1#, Moulana N1, Riahi M1, Alsowaidi K1, Albukhati K1, Zughaie SM2,3, Eltai NO4*

1Department of Health Sciences, Biomedical Sciences, QU Health, Qatar University, Doha, Qatar

2College of Medicine, QU Health, Qatar University, Doha, Qatar

3Biomedical and Pharmaceutical Research Unit, QU Health, Qatar University, Doha, Qatar

4Biomedical Research Centre, Microbiology Unit Qatar University, Doha, Qatar

#These authors contributed equally to work

*Corresponding author: Nahla O. Eltai, Biomedical Research Centre, Microbiology Unit Qatar University, Doha, Qatar

Received: June 24, 2021; Accepted: July 15, 2021; Published: July 22, 2021


The surge in the prevalence of Multidrug-Resistant (MDR) Gram-negative bacterial infections with limited treatment led to colistin reusing to treat MDR infections. This study aimed to determine economical, simple, and reliable colistin susceptibility testing methods as an alternative to the microdilution technique. We compared seven colistin susceptibility testing methods, including quantitative and qualitative, namely: Disk diffusion, E-test, ComASPTM SensiTest Colistin, Colistin broth disk elution, and colistin agar test CHROMagarTM COL-APSE, and BD Phoenix ID/AST automated identification and susceptibility testing system to the gold standard Broth Microdilution (BMD). Whole-genome sequencing was performed on all isolates to determine if the genetic resistant factors affect the phenotypic profile of the colistin resistance. Our results revealed that disk diffusion is still an ineffective method for measuring colistin susceptibility in Gram-negative Bacilli with the highest major error (31.75%), the lowest Kappa 0 (0%), and categorical agreement (68.25%) values. Phoenix, and CompASPTM SensiTest colistin methods have remained superior in reproducibility, sturdiness, and simplicity of use, similar to the currently recommended broth microdilution procedure; with high sensitivity of 95.56%, and 97.73%, specificity of 95.24, and 100%, and Kappa values of 0.89 and 0.95, respectively. This study revealed that Phoenix, and ComASPTM SensiTest colistin methods are recommended for routine microbiology laboratories with a large workload.

Keywords: Colistin; Gram-negative bacilli; Resistance; Microdilution; MIC


Colistin is a cationic polypeptide antibiotic that belongs to the family polymyxin, including polymyxin B and polymyxin E (colistin). It is the last resort for treating multidrug resistance Gramnegative Bacilli (MDR-GNB), mainly against Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), and Pseudomonas aeruginosa (P. aeruginosa) [1,2]. It is synthesized naturally by Paenibacillus polymyxa and is rapidly bactericidal to Gram-negative bacteria. Colistin targets lipid A of Lipopolysaccharide (LPS) in the Gram-Negative Bacilli (GNB) outer layer cell membrane. Its central role is to inhibit cell membrane function by increasing its permeability to absorb polymyxins substances. This increase in permeability leads to the destruction of inner macromolecules and cell ions, essential for cell survival [2,3]. However, several MDR-GNB bacteria developed their defense mechanisms against colistin. This resistance development could be either chromosomal or plasmid-mediated. The chromosomal resistance can occur due to mutation/insertion in LPS biosynthesis genes (lpx, pmrA/B, mgrB, and phoP/Q). On the other hand, the plasmid-mediated resistance is acquired by the horizontal gene transmission, Encoding Phosphoethanolamine Transferase (pEtN) enzymes [4]. As a result, the cell membrane permeability will be reduced, and polymyxin binding will be suppressed [1,5-7].

Natural resistance to colistin has been reported on several bacteria, mainly chromosomal mutations in some Gram-negative species. However, recently plasmid-mediated colistin resistance emerged in clinically significant Gram-negative bacilli. The emergent resistance was first reported in China in November 2015 through the mcr-1 gene [8, 9]. Since then, strains carrying the mcr-1 gene, especially E. coli, have been isolated worldwide from humans [10], food-producing animals [11], and the environment [12]. Another plasmid-carried colistin-resistant gene (mcr-2) was detected in E. coli isolates from pigs in Belgium [13]. To date, ten mobile mcr genes have been described [14,15]. Colistin was first introduced as an antibiotic in 1952 and was used until the early 1980s to treat infections caused by GNB. Its use was discontinued because of problems with toxicity, such as nephrotoxicity and neurotoxicity [16]. The increase of multidrug resistance among clinically significant GNB renewed interest in colistin as a last resort therapeutic option for treating MDR-GNB bacteria. In clinical practice, there are some situations where clinician needs to use colistin; therefore, it is critical for clinical laboratories to determine colistin resistance to prevent prolonged administration to the patient for whom it would not be effective.

The poor diffusion and binding of colistin to plastics lead to its complicated phenotypic resistance testing [17]. In addition, some studies noted discrepancies in phenotypic colistin resistance results obtained by different methods. Besides, no reference method has been demarcated to compare colistin susceptibility testing results [4]. As such, both the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommend only Broth Microdilution (BMD) [17]. However, the method is impractical for clinical laboratories due to the large sample load in routine microbiology laboratories [18]. Thus, an alternative, more practical method is needed. Several commercial methods are available for colistin Minimum Inhibitory Concentration (MIC) measurement, but only a few studies describe their efficacy compared to each other. In this context, we conducted this study to evaluate and compare the different available quantitative and qualitative colistin susceptibility testing methods on some GNB to find the feasible colistin test. These methods include disk diffusion (Kirby- Bauer test), gradient diffusion method (E- test), CHROMagarTM COL-APSE, ComASPTM colistin broth microdilution, colistin broth disk elution, agar dilution, BD Phoenix ID/ AST automated identification, and susceptibility testing system.

Additionally, we performed Whole-Genome Sequencing (WGS) to identify the genetic determinants of colistin resistance and find if it affects the phenotypic testing method. The study outcomes will provide a framework for clinical laboratories to select the appropriate method to test colistin resistance.

Materials and Methods

Bacterial strains

Two sets of previously collected bacterial isolates (n=63) were employed in this study. The first set comprises isolated human pathogens collected from Hamad Medical Corporation (HMC), Doha, Qatar, during routine diagnostic testing (n=37). The second set was obtained from poultry fecal samples (n=26). Non-duplicate bacterial strains were characterized and identified as GNB via Biomic V 3 (Giles Scientific, USA) or Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MAL-DI-TOF MS) (Bruker Daltonik GmbH, Leipzig, Germany). These isolates include 43 strains of E. coli, 18 strains of K. Pneumoniae, and two strains of P. aeruginosa. Clinical Isolates were selected because of their resistance to third-generation cephalosporins and/or colistin. E. coli isolates from poultry fecal samples were obtained from farms using colistin as a prophylaxis and growth promotor. The reference strains E. coli ATCC® 25922, K. pneumoniae ATCC® 700603, and P. aeruginosa ATCC® 27853 were used as quality control organisms in all experiments. This study was approved by Qatar University Institutional Biosafety Committee (QU-IBC) number QU-IBC-2020/030.

Phenotypic methods to detect colistin resistance

The subsequent techniques were applied to all 63 isolates to determine their susceptibility to colistin, and readings were interpreted according to the CLSI [19], NCCLS [20], and Gales et al. [21] (Supplementary Table S1).

Disk diffusion method (Kirby-Bauer test)

Bacterial isolates were cultured on nutrient agar plates for 18- 24hrs at 37°C. After incubation, 2 to 3 pure colonies were suspended in a phosphate buffer solution (Atom Scientific, UK) to achieve an inoculum equivalent to 0.5 McFarland Standard as measured by DensiCHEK PLUS (bioMérieux, France). The suspension was then swabbed onto a Mueller-Hinton (MH) agar plate (Himedia, Mumbai, India) and allowed to dry completely. Next, antibiotic disks impregnated with 10μg colistin (Liofilchem®, Roseto Degli Abruzzi, Italy) were applied to the agar surface and incubated at 37°C for 24hrs. The zone of inhibition was measured in mm (Figure 1). These values were interpreted according to the CLSI [19].