To the Editor—We read with great interest the recent publication by Gargis et al [1] regarding surveillance of Clostridioides difficile antimicrobial resistance in the United States between 2012 and 2017. The study utilized a convenience sample from the Centers for Disease Control and Prevention's Emerging Infections Program (EIP) to assess susceptibility changes to 6 antimicrobials, including metronidazole, and to test for genes and/or mutations associated with resistance to these agents using whole-genome sequencing. This effort reported a slight decrease in metronidazole minimum inhibitory concentrations (MICs) throughout the 5 years included, with 97.3% (577/593) and 100% (593/593) of the isolates considered susceptible based on European Committee on Antimicrobial Susceptibility Testing (EUCAST); ≤2 μg/mL) and Clinical and Laboratory Standards Institute (CLSI; ≤8 μg/mL) breakpoints, respectively.
Our group has conducted surveillance of C. difficile in Texas since 2011, and our epidemiology has largely resembled that from the EIP over the same time frame [2, 3]. In parallel, we used local clinical samples from this surveillance to investigate the mechanism and impact of metronidazole resistance [4, 5], during which we and others discovered that detection of metronidazole-resistant C. difficile requires molecularly intact heme in susceptibility testing agars [6, 7]. We also reported that extended exposure of agars to light produced more inaccurate MIC results, since heme is light sensitive [7]. Hence, detecting this resistance phenotype requires incorporating undegraded heme in the agar media and its subsequent protection from light during storage. Anaerobic susceptibility testing methods currently recommended by the CLSI, and thus used by Gargis et al, include incorporating 5 μg/mL of heme but without guidance regarding its integrity or storage [8]. These recommendations are of concern as their ability to accurately detect metronidazole resistance is dependent on local conditions, laboratory workflow, and storage. Indeed, our group MIC tested 29 currently available strains from the EIP Isolate Bank, which were among the strains reported by Gargis et al. When MIC tests were done with brain–heart infusion agars with and without heme and 2 CLSI-recommended media, we found that 20.7% (6/29) of the isolates were incorrectly classified as metronidazole susceptible (Table 1). Resistant strains were designated as showing heme-dependent metronidazole resistance.
Table 1.
A Comparison of Metronidazole Susceptibility Based on Heme Incorporation and Integrity
| Strain | RT | Gargis et al [1], 5 μg/mL Heme | Olaitan et al [5], BHI No Heme | Olaitan et al [5], BHI 5 μg/mL Heme | WCA, 5 μg/mL Heme | BRU, 5 μg/mL Heme, 5% v/v Defibrinated Sheep Blood | Phenotype |
|---|---|---|---|---|---|---|---|
| AR-1067 | 027 | 1 | 0.25 | 2 | 8 | 4 | HMR |
| AR-1068a | 056 | 0.25 | 0.25 | 1 | 1 | 0.25 | S |
| AR-1069 | 015 | 0.5 | 0.25 | 0.25 | 1 | 0.25 | S |
| AR-1070 | 002 | 0.25 | 0.25 | 0.25 | 1 | 0.25 | S |
| AR-1071 | 027 | 1 | 0.25 | 2 | 8 | 4 | HMR |
| AR-1072 | 027 | 1 | 0.25 | 2 | 4 | 4 | HMR |
| AR-1073 | 020 | 0.5 | 0.25 | 0.25 | 1 | 0.25–0.5 | S |
| AR-1074 | 002 | 0.5 | 0.25 | 0.25 | 1 | 0.5 | S |
| AR-1075 | 019 | 0.5 | 0.25 | 0.25 | 1 | 0.5 | S |
| AR-1076 | 027 | 0.25 | 0.25 | 2 | 4 | 4 | HMR |
| AR-1077 | 078 | 0.25 | 0.25 | 0.25 | 1 | 0.5 | S |
| AR-1078 | 106 | 0.5 | 0.25 | 0.25 | 1 | 0.25 | S |
| AR-1079 | 056 | 0.25 | 0.25 | 0.25 | 1 | 0.25 | S |
| AR-1081 | 014 | 0.25 | 0.25 | 0.25 | 1 | 0.25 | S |
| AR-1082 | 054 | 0.5 | 0.25 | 0.25 | 1 | 0.25 | S |
| AR-1083 | 078 | 0.25 | 0.25 | 0.25 | 1 | 0.25 | S |
| AR-1084 | 002 | 0.25 | 0.25 | 0.25 | 1 | 0.25 | S |
| AR-1085 | 106 | 0.25 | 0.25 | 0.25 | 1 | 0.25 | S |
| AR-1086 | 015 | 0.25 | 0.25 | 0.25 | 1 | 0.25–0.5 | S |
| AR-1087 | 106 | 0.5 | 0.25 | 0.25 | 1 | 0.25–0.5 | S |
| AR-1088 | 054 | 0.25 | 0.25 | 0.25 | 1 | 0.25–0.5 | S |
| AR-1089 | 106 | 0.5 | 0.25 | 0.25 | 1 | 0.25 | S |
| AR-1090 | 014 | 0.5 | 0.25 | 0.25 | 1 | 0.25 | S |
| AR-1091 | 014 | 0.5 | 0.25 | 0.25 | 1 | 0.25 | S |
| AR-1092 | 027 | 1 | 0.25 | 2 | 1 | 0.25 | HMR |
| AR-1093 | 106 | 0.5 | 0.25 | 0.25 | 1 | 0.25 | S |
| AR-1094 | 019 | 0.25 | 0.25 | 0.25 | 1 | 0.25 | S |
| AR-1095 | 027 | 0.5 | 0.25 | 2 | 4 | 4 | HMR |
| AR-1096 | 020 | 0.5 | 0.25 | 0.25 | 1 | 0.25 | S |
HMR is defined by an increase in MIC by at least 4-fold when compared with BHI media without heme and confirmed as resistant by MICs on WCA and BRU. MICs used 2 biological replicates and a range is reported where the 2 results differed. Controls were Clostridioides difficile CD196 (WCA MIC = 1 μg/mL; BRU MIC = 0.25 μg/mL) and C. difficile 17/27 (WCA MIC = 16 μg/mL; BRU MIC = 8 μg/mL). Agar plates were light protected by covering with foil. Abbreviations: BRU, Brucella agar (also contains vitamin K1 at 10 μg/mL); BHI, brain–heart infusion agar; HMR, heme-dependent metronidazole resistance; MIC, minimum inhibitory concentration; RT, ribotype; S, susceptible; v/v, volume/volume; WCA, Wilkins Chalgren agar.
AR-1068 is regarded as susceptible since resistance was not confirmed by WCA and BRU.
Accurate metronidazole susceptibility testing is necessary to quantify the clinical impact of resistance. This is indeed reflected in our study of 350 clinical isolates (MIC50: 0.25 μg/mL; MIC90: 2 μg/mL) [4], in which MICs were done with optimized methods of using fresh heme and its subsequent light protection in agars. Through classification and regression tree (CART) and multivariable analyses, a metronidazole MIC of 1 μg/mL or greater was found to correlate with increased odds of day-6 clinical failure in patients receiving metronidazole (odds ratio: 2.44; 95% confidence interval: 1.36 – 4.36; P = .003). As the first to associate metronidazole MICs with C. difficile infection clinical outcomes, we feel previous failures to do so may reflect inappropriate susceptibility testing practices. Furthermore, our clinical findings, in combination with pharmacokinetic data demonstrating that metronidazole fecal concentrations range from 0.25 to 9.5 μg/g stool [9], lead to question whether the current CLSI recommendation for metronidazole susceptibility is too high for C. difficile.
As another group interested in C. difficile surveillance, we commend the authors on this publication and similar efforts. As attention grows on evaluating C. difficile resistance to treatment antibiotics, we recommend those conducting surveillances to examine internal susceptibility testing methods to both more accurately appraise C. difficile epidemiology and the clinical impact of resistance. It is our opinion that C. difficile susceptibility guidance documents warrant updating to ensure the accurate and reproducible detection of metronidazole resistance.
Contributor Information
Anne J Gonzales-Luna, Department of Pharmacy Practice and Translational Research, University of Houston College of Pharmacy, University of Houston, Houston, Texas, USA.
Chetna Dureja, Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas, USA.
Taryn A Eubank, Department of Pharmacy Practice and Translational Research, University of Houston College of Pharmacy, University of Houston, Houston, Texas, USA.
Kevin W Garey, Department of Pharmacy Practice and Translational Research, University of Houston College of Pharmacy, University of Houston, Houston, Texas, USA.
Julian G Hurdle, Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, Texas, USA.
Notes
Disclaimer. The funders had no role in study design, data collection, interpretation of the findings, or in the writing and submission of the manuscript.
Financial support. This work was supported by the National Institute of Allergy and Infectious Diseases at the National Institutes of Health (grant number R01AI139261), as declared by K. W. G and J. G. H.
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