Assessment of trimethoprim-sulfamethoxazole susceptibility testing methods for fastidious Haemophilus spp

• Published in: Clinical microbiology and infection Vol. 26; no. 7; pp. 944.e1 - 944.e7
• Main Authors: Sierra, Y., Tubau, F., González-Díaz, A., Carrera-Salinas, A., Moleres, J., Bajanca-Lavado, P., Garmendia, J., Domínguez, M. Ángeles, Ardanuy, C., Martí, S.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.07.2020
• Subjects: Anti-Bacterial Agents - pharmacology; Antimicrobial susceptibility testing methods; Bacterial Proteins - genetics; clinical resistance breakpoint; Culture Media - chemistry; Drug Resistance, Multiple, Bacterial; EUCAST; Haemophilus Infections - microbiology; Haemophilus influenzae; Haemophilus influenzae - drug effects; Haemophilus influenzae - genetics; Haemophilus parainfluenzae; Haemophilus parainfluenzae - drug effects; Haemophilus parainfluenzae - genetics; High-Throughput Nucleotide Sequencing; Humans; Microbial Sensitivity Tests; Mutation; Promoter Regions, Genetic; Resistance-related determinants; Trimethoprim, Sulfamethoxazole Drug Combination - pharmacology; Trimethoprim-sulfamethoxazole; Whole Genome Sequencing; Trimethoprim-sulfamethoxazole; EUCAST; Antimicrobial susceptibility testing methods; clinical resistance breakpoint; Haemophilus parainfluenzae; Resistance-related determinants; Haemophilus influenzae
• ISSN: 1198-743X; 1469-0691
• DOI: 10.1016/j.cmi.2019.11.022

To compare the determinants of trimethoprim-sulfamethoxazole resistance with established susceptibility values for fastidious Haemophilus spp., to provide recommendations for optimal trimethoprim-sulfamethoxazole measurement. We collected 50 strains each of Haemophilus influenzae and Haemophilus parainfluenzae at Bellvitge University Hospital. Trimethoprim-sulfamethoxazole susceptibility was tested by microdilution, E-test and disc diffusion using both Mueller–Hinton fastidious (MH-F) medium and Haemophilus test medium (HTM) following EUCAST and CLSI criteria, respectively. Mutations in folA, folP and additional determinants of resistance were identified in whole-genome-sequenced isolates. Strains presented generally higher rates of trimethoprim-sulfamethoxazole resistance when grown on HTM than on MH-F, independent of the methodology used (average MIC 2.6-fold higher in H. influenzae and 1.2-fold higher in H. parainfluenzae). The main resistance-related determinants were as follows: I95L and F154S/V in folA; 3- and 15-bp insertions and substitutions in folP; acquisition of sul genes; and FolA overproduction potentially linked to mutations in -35 and -10 promoter motifs. Of note, 2 of 19 H. influenzae strains (10.5%) and 9 of 33 H. parainfluenzae strains (27.3%) with mutations and assigned as resistant by microdilution were inaccurately considered susceptible by disc diffusion. This misinterpretation was resolved by raising the clinical resistance breakpoint of the EUCAST guidelines to ≤30 mm. Given the routine use of disc diffusion, a significant number of strains could potentially be miscategorized as susceptible to trimethoprim-sulfamethoxazole despite having resistance-related mutations. A simple modification to the current clinical resistance breakpoint given by the EUCAST guideline for MH-F ensures correct interpretation and correlation with the reference standard method of microdilution.