Heteroresistance is a cause of discrepant antibiotic susceptibility testing results
Authors: Tugba Ozturk, David S Weiss
Journal: The Lancet Microbe (2024)

Antibiotic susceptibility testing (AST) can be an essential step in antibiotic therapy and guides the use of drugs considered to be effective in vitro against a given isolate. Conflicting results obtained from susceptibility tests can lead to confusion about the selection of antibiotics for treatment, confound the work of standards bodies such as the Clinical & Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing, and negatively affect patient care. The causes of discrepancies in AST results are not generally clear. Broth microdilution (BMD) is considered the gold standard AST; when results of different tests contradict those of BMD, the other tests are considered erroneous. Discrepant susceptibility test results have been widely reported for the novel beta-lactam cefiderocol, particularly in cases of carbapenem-resistant Acinetobacter baumannii (CRAB).1Albano M Karau MJ Schuetz AN Patel R Comparison of agar dilution to broth microdilution for testing in vitro activity of cefiderocol against Gram-negative bacilli.J Clin Microbiol. 2020; 59e00966-20Crossref PubMed Scopus (26) Google Scholar CRAB has a high rate of cefiderocol heteroresistance, a phenomenon in which an isolate harbours a minor subpopulation of cells that are resistant to a given antibiotic,2Band VI Hufnagel DA Jaggavarapu S et al.Antibiotic combinations that exploit heteroresistance to multiple drugs effectively control the infections.Nat Microbiol. 2019; 4: 1627-1635Crossref PubMed Scopus (0) Google Scholar and can be detected using a population analysis profile (PAP) test (appendix pp 2, 5). In the present study, we investigated cefiderocol heteroresistance as a potential cause of discrepant AST results in 211 clinical samples (appendix p 1). The cefiderocol susceptibility status of CRAB (n=108) and carbapenem-resistant Enterobacterales (n=103) isolates was compared using standard AST methods (BMD, disc diffusion [DD], agar dilution) and PAP (appendix pp 1–3). We found that discrepant AST results can be caused by a subset of heteroresistant isolates with a resistant cell frequency that is too low to be consistently detected as resistant but too high to be classified as susceptible. PAP classified 95 of 211 (45%) strains as heteroresistant and 116 (55%) of 211 strains as susceptible to cefiderocol (appendix p 4). Among the strains classified susceptible by PAP, the three standard tests were in agreement in classifying 115 (99·1%) of 116 susceptible strains; discrepant findings were noted only for one isolate (one [0·9%] of 116). In contrast, the standard tests had a higher rate of discrepant results (appendix p 6) for strains classified heteroresistant on the basis of PAP (24 [25·3%] of 95 strains [p<0·0001]) than the discrepancy rate for those classified as susceptible by PAP (appendix p 4). All standard tests were in agreement in classifying nine (9·5%) of 95 heteroresistant isolates as non-susceptible, and the average number of resistant cells in these strains was one in 1230 cells (appendix pp 4, 7). The standard tests were also in agreement in classifying 62 (65%) of 95 heteroresistant isolates susceptible, and the average resistant cell frequency of these isolates was one in 42 267 cells. In contrast, the standard tests had discrepancies in results for 24 (25·3%) of 95 heteroresistant isolates, with an average resistant cell frequency of one in 7178 cells (appendix p 4). Collectively, these data indicate that a subset of heteroresistant isolates with an intermediate frequency of resistant cells accounted for almost all discrepancies from standard AST. Notably, the CLSI observed a lack of reproducibility for cefiderocol BMD, especially for CRAB isolates with a minimum inhibitory concentration (MIC) higher than 2 μg/mL (CLSI breakpoint is 16 μg/mL) and for isolates with colonies within the zone of clearing in DD, complicating the interpretation of the results.3Simner PJ Palavecino EL Satlin MJ et al.Potential for inaccurate cefiderocol susceptibility results: a CLSI AST subcommittee advisory.J Clin Microbiol. 2023; 61e0160022Crossref Scopus (7) Google Scholar,4Simner PJ Patel R Cefiderocol antimicrobial susceptibility testing considerations: the Achilles' heel of the Trojan horse?.J Clin Microbiol. 2020; 59e00951-20Crossref Scopus (72) Google Scholar All CRAB isolates with an MIC of 2 μg/mL or higher (appendix p 8) and those with colonies in the zone of clearing in DD were classified as cefiderocol heteroresistant by PAP. Taken together, these data indicate that heteroresistance might explain the discrepancy in AST results and suggest that higher MICs in the susceptible range, colonies within the zone of clearing, or both should not be ignored and could be used as markers of heteroresistance. The results show that a biological phenomenon brings about at least some testing discrepancies and provide greater clarity in understanding AST, which might help to guide future recommendations and selection of breakpoints. DSW has pending patents related to heteroresistance. TO declares no competing interests. The authors thank Jacob E Choby for critically reading the manuscript. This work was funded by an NIH grant (AI158080) to DSW. DSW was also funded by the Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Disease Award. Download .pdf (1.03 MB) Help with pdf files Supplementary appendix