Defining antimicrobial susceptibility testing methods and breakpoints among species.

species can cause opportunistic infections which are difficult to treat due to a variety of antimicrobial resistance (AMR) mechanisms. As such, antimicrobial susceptibility testing (AST) is a cornerstone to successful treatment for these organisms. Currently, there are no specific Clinical and Laboratory Standards Institute (CLSI) AST guidelines for species. The purpose of this study was to (i) establish tentative CLSI M45 MIC and disk diffusion (DD) breakpoints (BPs), (ii) evaluate the modified carbapenem inactivation method (mCIM), and (iii) understand the mechanisms mediating AMR among species. Contemporary MIC data from multiple sources were collated to define the tentative epidemiological cutoff values (tECVs), and the MIC distributions were used to inform the establishment of MIC BPs for various agents. A disk-to-MIC correlate study with 91 isolates from across the United States was completed by testing reference broth microdilution and DD from the same inoculum and applying the dBETS software to establish DD BPs. Lastly, the mCIM and whole-genome sequencing (WGS) were pursued. The tECVs for piperacillin-tazobactam, imipenem, and meropenem were 1, 2, and 0.5 µg/mL, respectively. Disk correlates met CLSI M23 acceptance criteria with a few exceptions related to a small number of isolates, resulting in high minor errors. WGS revealed that 82 (90.1%) isolates harbored a variant with predominating (90.2%). Nineteen isolates harbored acquired beta-lactamase genes, including 16 , 2 , and 1 The mCIM had a sensitivity of 100% and specificity of 87%. Upon review, the CLSI M45 committee set tentative MIC and DD BPs for piperacillin-tazobactam, imipenem, meropenem, and trimethoprim-sulfamethoxazole.IMPORTANCE species can cause opportunistic infections which may be difficult to treat due to a variety of antimicrobial resistance mechanisms. Antimicrobial susceptibility testing is a critical component of patient treatment for these infections. Currently, the Clinical and Laboratory Standards Institute M100 non-Enterobacterales susceptibility test interpretive criteria and methodology are utilized for by clinical laboratories and likely do not accurately predict susceptibility results for species. This study was designed to establish tentative MIC and disk diffusion breakpoints specific to species.