Comparative Evaluation of Colistin-Susceptibility Testing in Carbapenem-Resistant Klebsiella pneumoniae Using VITEK, Colistin Broth Disc Elution, and Colistin Broth Microdilution.

PURPOSE: The study aimed to compare the results of colistin-susceptibility testing performed using the automated VITEK system, colistin broth microdilution (BMD), and colistin broth disk elution (CBDE) methods. MATERIALS AND METHODS: This exploratory study was conducted in a tertiary care center in South India. Carbapenem-resistant Klebsiella pneumoniae (n = 49) isolates collected from a clinical microbiology laboratory over six months (March-September 2023) were used for the study. RESULTS: Among the 49 carbapenem-resistant Klebsiella pneumoniae isolates, 42 were found to be susceptible to carbapenem by all three methods. Seven isolates were found to be resistant to colistin using BMD and CBDE methods. Two isolates were incorrectly detected as colistin-susceptible, and one isolate was wrongly categorized as colistin-resistant using the automated VITEK system. CONCLUSION:  CBDE is a reliable and cost-effective method that can be adopted in the routine microbiology laboratory for colistin-susceptibility testing, as it does not require any specialized equipment or techniques and is 100% consistent with the gold standard BMD method. Although the automated VITEK system is used in most routine microbiological laboratories for antibiotic-susceptibility testing, it cannot be reliably used for colistin-susceptibility testing due to its high error rates (very major error rate of 28.5%; major error rate of 2.4%).

Colistin Susceptibility Testing by Colistin Broth Disk Elution MIC Method among Carbapenem-resistant Gram-negative Blood Culture Clinical Isolates in a Tertiary Care Setting, East Delhi, India.

Background: Antibiotic resistance is a growing concern for bloodstream infections (BSIs), especially with the emergence of multidrug-resistant (MDR) gram-negative bacteria. In this study, we aimed to assess the pattern of colistin susceptibility using the colistin broth disc elution (CBDE) method among carbapenem-resistant gram-negative clinical isolates from blood cultures in a high burden tertiary healthcare setting in East Delhi. Methods: A total of 106 carbapenem-resistant gram-negative clinical isolates were tested. The most common isolates were Klebsiella pneumoniae, Escherichia coli, Enterobacter species, and Klebsiella oxytoca by CBDE method. Result: All the carbapenem resistant gram-negative bacterial blood culture isolates showed intermediate colistin susceptibility. This was statistically significant by chi-square test (p<0.5). Conclusion: This study highlights the need to monitor colistin resistance trends in the face of increasing antimicrobial resistance. Accurate surveillance of emerging colistin resistance is crucial for effective management of BSIs caused by carbapenem-resistant gram-negative bacteria.

Comparative Evaluation of Colistin Broth Disk Elution Method With Two Commercially Available Systems for Colistin Susceptibility Testing Against Carbapenem-Resistant Klebsiella pneumoniae: A Single-Center Exploratory Study.

PURPOSE: The purpose is to explore the diagnostic utility of colistin broth disk elution (CBDE) as a simple and reliable method of colistin susceptibility testing. MATERIALS AND METHODS: An exploratory study was undertaken in a tertiary care teaching hospital in Uttarakhand, from September 2021 to March 2022, after obtaining approval from the Institute Ethics Committee. Twenty-five non-repetitive carbapenem-resistant Klebsiella pneumoniae clinical isolates were included in the study. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and BD Phoenix M50 system were used to perform species-level identification and antibiotic susceptibility testing (AST), respectively, as per the manufacturer's instructions. AST results (including those of colistin) were interpreted as per the CLSI guidelines 2022. The test isolates were further subjected to additional in vitro colistin susceptibility testing using a commercially available Mikrolatest colistin susceptibility testing kit and CBDE, respectively. RESULTS: The in vitro colistin resistance rates varied from 8% by BD Phoenix system to 20% by Mikrolatest kit and 32% by CBDE, respectively. For colistin susceptibility, a higher CA was observed between the BD Phoenix system and CBDE (64.71%) than between the Mikrolatest kit and CBDE (31.60%). Overall, a statistically significant fair agreement was observed between the BD Phoenix system and CBDE (Kappa: 0.312; 95% CI: 0.036 to 0.660) and Mikrolatest MIC colistin kit and CBDE (Kappa: 0.286; 95% CI: 0.111 to 0.683), respectively. CONCLUSIONS: In vitro colistin testing remains a significant challenge globally. Although the present study results are inconclusive due to the small sample size, we should conduct multi-centric studies globally, taking a considerable sample size representing different Gram-negative bacilli to generate conclusive evidence on the utility of CBDE as a reliable method of colistin susceptibility testing.

Colistin Susceptibility Testing by Rapid Colistin Disk Elution Test Among Enterobacteriaceae in Low-Resource Setting.

We modified rapid polymyxin Nordmann-Poirel (RPNP) test, called rapid colistin disk elution (RCDE) test, for detecting colistin resistance in Gram-negative bacilli and evaluated its performance compared with colistin broth disk elution (CBDE) test recommended by Clinical and Laboratory Standards Institute (CLSI). The RCDE test was performed by using a 10-µg colistin disk in 2.7 mL volume (final colistin concentration of 3.7 µg/mL) of either cation-adjusted Mueller-Hinton broth or phenol red broth base media with bacterial inoculum of 1-µL loop, and 1-4 and 16-20 hr incubation for Enterobacteriaceae and Acinetobacter baumannii isolates, respectively. Both tests were evaluated in 236 Enterobacteriaceae and 49 A. baumannii isolates using broth microdilution as reference method. Among the Enterobacteriaceae isolates, categorical agreement and very major error (VME or false intermediate susceptibility) rate were 98.3% and 5.4%, respectively, for the RCDE test, compared with 97.9% and 7.1%, respectively, for the CBDE test. Both tests had major error (ME or false resistance) rate of 0.6%. For the A. baumannii isolates, the RCDE and CBDE tests gave high VME rates of 8.3% and 16.7%, respectively. The RCDE test showed good performance comparable with the CBDE test but is cheaper and more rapid (3 hr) and convenient, thus suggesting as an alternative for detecting colistin resistance among Enterobacteriaceae in low-income countries.

Multicenter Evaluation of Colistin Broth Disk Elution and Colistin Agar Test: a Report from the Clinical and Laboratory Standards Institute.

Susceptibility testing of the polymyxins (colistin and polymyxin B) is challenging for clinical laboratories. The Clinical and Laboratory Standards Institute (CLSI) Antimicrobial Susceptibility Testing Subcommittee evaluated two methods to enable accurate testing of these agents. These methods were a colistin broth disk elution (CBDE) and a colistin agar test (CAT), the latter of which was evaluated using two inoculum volumes, 1 µl (CAT-1) and 10 µl (CAT-10). The methods were evaluated using a collection of 270 isolates of Enterobacterales, 122 Pseudomonas aeruginosa isolates, and 106 Acinetobacter spp. isolates. Overall, 94.4% of CBDE results were in essential agreement and 97.9% in categorical agreement (CA) with reference broth microdilution MICs. Nine very major errors (VME; 3.2%) and 3 major errors (ME; 0.9%) were observed. With the CBDE, 98.6% CA was observed for Enterobacterales (2.5% VME, 0% ME), 99.3% CA was observed for P. aeruginosa (0% VME, 0.7% ME), and 93.1% CA was observed for Acinetobacter spp. (5.6% VME, 3.3% ME). Overall, CA was 94.9% with 6.8% VME using CAT-1 and improved to 98.3% with 3.9% VME using CAT-10. No ME were observed using either CAT-1 or CAT-10. Using the CAT-1/CAT-10, the CA observed was 99.4%/99.7% for Enterobacterales (1%/0.5% VME), 98.7%/100% for P. aeruginosa (8.3%/0% VME), and 88.5%/92.3% for Acinetobacter spp. (21.4%/14.3% VME). Based on these data, the CLSI antimicrobial susceptibility testing (AST) subcommittee endorsed the CBDE and CAT-10 methods for colistin testing of Enterobacterales and P. aeruginosa.

Two-Site Evaluation of the Colistin Broth Disk Elution Test To Determine Colistin In Vitro Activity against Gram-Negative Bacilli.

Limited methods for colistin MIC determination are available to clinical microbiology laboratories. The purpose of this study was to evaluate the accuracy of the colistin broth disk elution (CBDE) test compared to that of broth microdilution (BMD) for identifying colistin MICs. CBDE was compared to colistin BMD using a collection of Gram-negative bacilli tested at two U.S. microbiology laboratories. The isolates tested included 121 retrospective clinical isolates, 45 prospective clinical isolates, and 6 mcr-1-positive Escherichia coli isolates. CBDE was performed with four 10-ml cation-adjusted Mueller-Hinton broth tubes per isolate, to which 0, 1, 2, and 4 colistin 10-µg disks were added, generating final concentrations in the tubes of 0 (growth control), 1, 2, and 4 µg/ml, respectively. MICs were evaluated visually and interpreted using Clinical and Laboratory Standards Institute breakpoints. Site 2 also compared CBDE to the reference broth macrodilution (BMAD) method (n = 110 isolates). Overall, CBDE yielded a categorical agreement (CA) and essential agreement (EA) of 98% and 99%, respectively, compared to the results of colistin BMD. Very major errors occurred for mcr-1-producing strains, with MICs fluctuating from 2 to 4 µg/ml on repeat testing. The results for all other isolates were in CA with those of BMD. CBDE versus BMAD had an EA of 100% and a CA of 100%. Compared to currently used techniques, CBDE is an easy and practical method to perform colistin MIC testing. Some mcr-1-producing isolates yielded MICs of 2 µg/ml by CBDE and 4 µg/ml by BMD. As such, the results for isolates with colistin MICs of 2 µg/ml by CBDE should be confirmed by the reference BMD method, and isolates with MICs of ≥2 µg/ml should be evaluated for the presence of mcr genes.