Improved Accuracy of Cefepime Susceptibility Testing for Extended-Spectrum-Beta-Lactamase-Producing Enterobacteriaceae with an On-Demand Digital Dispensing Method

Abstract

Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae generally cannot be treated with penicillins and cephalosporins. However, some later-generation cephalosporins, including cefepime, are poorly hydrolyzed by specific ESBL enzymes, and certain strains demonstrate in vitro susceptibility to these agents, potentially affording additional treatment opportunities. Moreover, the ability to adjust both the dose and dosing interval of beta-lactam agents allows the treatment of strains with elevated MICs that were formerly classified in the intermediate range. The ability to treat strains with elevated cefepime MICs is codified in new susceptible dose-dependent (SDD) breakpoints promulgated by the Clinical and Laboratory Standards Institute. In the interest of validating and implementing new cefepime SDD criteria, we evaluated the performances of Vitek 2, disk diffusion, and a MicroScan panel compared to that of reference broth microdilution (BMD) during the testing of 64 strains enriched for presumptive ESBL phenotype (based on nonsusceptibility to ceftriaxone). Surprisingly, categorical agreement with BMD was only 47.6%, 57.1%, and 44.6% for the three methods, respectively. Given these findings, we tested the performance of the HP D300 inkjet-assisted broth microdilution digital dispensing method (DDM), which was previously described by our group as an atwill testing alternative. In contrast to commercial methods, DDM results correlated well with the reference method, with 86% categorical agreement, 91.1% evaluable essential agreement, and no major or very major errors. The reproducibility and accuracy of MIC determinations were statistically equivalent to BMD. Our results provide support for the use of the DDM as a BMD equivalent methodology that will enable hospital-based clinical laboratories to support cefepime MIC-based dosing strategies.KEYWORDS ESBL, antimicrobial susceptibility testing, broth microdilution, cefepime, digital dispensing, extended-spectrum beta-lactamase producer, inkjet, susceptibility testing, susceptible dose dependent, verification F ew treatment options remain for extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) (1). Therefore, the rapidly increasing worldwide prevalence of these strains is of great concern (2-8). Notably, in China, ESBL-E comprise over half of all Escherichia coli bloodstream infections (9), and colonization rates of intensive care unit patients in India have been reported to reach 90% (10). In the United States, prevalence has been rising, especially in certain vulnerable populations, such as patients in adult and pediatric intensive care units, patients with prolonged hospital stays, and patients with exposure to multiple antibiotics (11-15).

Synergistic Activity of Colistin-Containing Combinations against Colistin-Resistant Enterobacteriaceae

“…Checkerboard array testing. To create checkerboard arrays, serial 2-fold dilutions of 2 antibiotics were dispensed in orthogonal titrations by the D300 instrument using the DDM previously developed by our group (17,47,52). Titrations consisted of 11 2-fold dilutions of colistin (0.016 to 16 g/ml) and 13 2-fold dilutions of all other antibiotics.…”

Development of MAST: A Microscopy-Based Antimicrobial Susceptibility Testing Platform

“…9 We previously determined through extensive analysis that polysorbate-20 at the concentrations used in assay wells has no effect on MIC determinations for all antibiotics examined. 5, 10 …”

“…1 BMD experiments were performed in triplicate for each antibiotic/organism combination with the modal result recorded as the reference MIC. 5 …”

“…3 However, these tests are limited in that they incorporate only predetermined panels of relatively common antimicrobials; do not include recently approved agents developed for multidrug-resistant pathogens; 4 require a long (>8–20 hour) incubation; and for MIC surrogate methods, may not always correlate with reference MIC results, a problem particularly noted for multidrug-resistant organisms. 5–7 …”

The Poisoned Well: Enhancing the Predictive Value of Antimicrobial Susceptibility Testing in the Era of Multidrug Resistance

“…The availability of automated liquid handlers and other programmable antibiotic dispensing systems means that the previously time-consuming and error-prone process of preparing subdoubling dilutions is no longer a true impediment. Finer dilutions could be discontinuous and concentrate around critical decision points, such as cutoffs bordering safety margins and breakpoints, and could include finer gradations bracketing quality control strain ranges to allow greater sensitivity to detect subtle drift in panel performance (25,26). While decreasing dilution intervals will not reduce the biological variability that may play a role in inconsistent MIC results, it will provide increased data about the true location of the MIC within the dilution series.…”