Detection of Colistin Resistance in Carbapenem Resistant Enterobacteriaceae by Reference Broth Microdilution and Comparative Evaluation of Three Other Methods

Abstract

Objective Challenges in susceptibility testing of colistin along with increase in the prevalence of colistin-resistant carbapenemase-producing Enterobacteriaceae (CRE) pathogens needs addressal. Evaluation of user-friendly methods is necessary as an alternative to broth microdilution (BMD), the reference susceptibility testing method, for routine implementation in diagnostic clinical microbiology laboratories. Genotypic detection of the plasmid-mediated colistin resistance is also needed for infection control purposes. Materials and Methods Colistin susceptibility of 200 nonduplicate clinical CRE isolates from December 2017 to June 2019 was determined by BMD, agar dilution (AD), E test, and rapid polymyxin NP test and interpreted as per the European Committee on Antimicrobial Susceptibility Testing. The results of AD, E test, and NP test were compared with that of BMD, considering minimal inhibitory concentration (MIC) ≤ 2 µg/mL as susceptible and > 2 µg/mL as resistant. Presence of any plasmid-mediated colistin resistance (mcr-1 and 2) was evaluated in 27 colistin-resistant CRE isolates by polymerase chain reaction. Statistical Analysis Performance of different phenotypic methods was analyzed by comparing MIC results of AD and E test with that of reference BMD method. Agreement between BMD and the other two methods was expressed in terms of categorical agreement and essential agreement. Errors were expressed as very major error (VME: false-susceptible) and major error (ME: false-resistance) by AD/E test. VME and ME of 3% disagreement were considered unacceptable. Results Colistin resistance was found in 27 (13.5%) isolates by BMD method. The VME rates of both AD (11%) and E test (37%) could not meet the Clinical and Laboratory Standards Institute recommendation (< 3% VME rate is acceptable) as alternative tests to the reference BMD. Colistin NP test showed sensitivity and specificity of 85% and 98%, respectively. The percentage discordant result in NP test was highest in Enterobacter spp. (17%). None of the 27 colistin resistant isolates showed presence of mcr-1 and mcr-2 genes. Conclusion High VME rate in AD and E tests precludes their use as alternatives to BMD for colistin susceptibility testing. NP test with moderate sensitivity but excellent specificity can be a good alternative for testing colistin susceptibility in CRE isolates, except in Enterobacter spp. Absence of mcr-1 and mcr-2 gene necessitates the exploration of other mechanisms of colistin resistance.

Assessment of Colistin Susceptibility among Carbapenem-Resistant Clinical Isolates

“…Klebsiella spp were the most frequent CRGNB isolates (36.1%) followed by Escherichia coli (32.9%), Pseudomonas aeruginosa (12.9%), Acinetobacter spp (8.4%), Enterobacter spp (5.8%) and Citrobacter spp (3.9%). Matching with carbapenem resistance rates (54.5% and 55.8%) in previous studies 17,4 . On the contrary, lower rates (18.5% and 23.1%) were reported in other studies 18-19 .…”

“…Intensive Care Units were the main source of these samples (50.32%), followed by surgical departments (18.71%), nursery (14.84%), medical departments (9.03 %) and burn unit (7.1%). Kra et al 17 reported the same observations. While, Saed et al mentioned that tracheal aspirate was the main source of CRGNB isolates 20 .…”

Colistin Susceptibility among Carbapenem Resistant Klebsiella pneumoniae Isolated from Menoufia University Hospitals

“…Our results demonstrated that, fosfomycin was the most effective antibiotic (93.5%) against CPKp followed by tigecycline (92.5%), gentamicin (90.3%), amikacin (86.0%), trimethoprim-sulfamethoxazole (81.7%), colistin (81.7%) and then β-lactam/βlactamase inhibitors (76.3%). That is higher than the susceptibility in Kar et al 26 study who found that, netilmicin, amikacin, gentamicin, trimethoprimsulfamethoxazole, piperacillin-tazobactam and fosfomycin were the most effective against CRE by only 23%, 21%, 20.5%, 15.5%, 8.5% and 5.5% respectively. And around the susceptibility pattern of Ajlan et al 19 study, who detected that the most effective drug against CRGNB isolates was colistin (72.3%), followed by ceftazidime-avibactam (67.7%), then tigecycline (63.2%).…”

Evaluating the drop test method in measuring colistin susceptibility of Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa

“…In our study, we evaluated a larger number of strains (n=309) than previous studies, where the number of strains analysed ranged from 75 to 271 isolates [9,11,14,15,20]. Overall, the drop test met the FDA-established criteria for assessing antimicrobial susceptibility [18].…”