PMC9199433_01
Time scale annotated with exposure to antibiotics (including dosages), along with results from phenotypic antimicrobial susceptibility testing (AST) to relevant antibiotics for five H. influenzae isolates sampled between November 2013 and January 2018. The Hi-Alpha and Hi-Beta isolates were not available for genomic characterization, and their potential phylogenetic relationship to the Hi-117, Hi226 and Hi-197 isolates could not be explored. Routine AST results for the five isolates represent primary testing with disk diffusion and/or gradient diffusion, whereas reference AST results were produced retrospectively by determination of broth microdilution (BMD) MIC using custom panels. Disk diffusion and BMD were done according to the standards of the EUCAST, while gradient diffusion was performed according to the manufacturer's recommendations. AST results were interpreted using EUCAST clinical breakpoints (v. 12.0), except azithromycin (no clinical breakpoints), for which susceptibility categorization was based on the epidemiological cut-off value (4 mg/L). bid, twice daily; qd, once daily; Trim-sulfa, trimethoprim-sulfamethoxazole; S, susceptible; I, intermediately susceptible (changed to "susceptible, increased exposure" from 2019); R, resistant; ND, no data.
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The first isolation of H. influenzae from a sputum sample was in November 2013, susceptible to beta-lactam antibiotics and trimethoprim-sulfamethoxazole (Hi-Alpha;  Figure 1 ). Microbiological samples were procured by bronchoscopy and H. influenzae was identified as sole pathogen (Hi-Beta;  Figure 1 ). H. influenzae was again isolated from sputum in April 2016 (Hi-117;  Figure 1 ). Cultivation of sputum in February 2017 revealed H. influenzae (Hi-226) with a resistance pattern similar to Hi-117 from 2016 ( Figure 1 ). H. influenzae cultivated from sputum (Hi-197,  Figure 1 ), was resistant to trimethoprim-sulfamethoxazole.   Figure 1  shows a time scale with antibiotic exposure, along with results from phenotypic antimicrobial susceptibility testing (AST) to relevant antibiotics for five H. influenzae isolates, identified as the sole pathogen from samples representative for lower respiratory tract, between November 2013 and January 2018.