Molecular characterisation of the clonal emergence of high-level ciprofloxacin-monoresistant Haemophilus influenzae in the Region of Southern Denmark

• We report the appearance of high-level ciprofloxacin-resistant Haemophilus influenzae in the Region of Southern Denmark.
• The strains were non-encapsulated, biotype III and were demonstrated by whole-genome sequencing to be clonal belonging to a single clade with an unknown MLST type (double-locus variant of ST196).
• The strains were monoresistant to ciprofloxacin with an MIC >32 mg/L.
• The resistome revealed both previously characterised and novel putative resistance-related mutations in gyrA, parC and parE.
• No epidemiological link between the patients could be demonstrated, but isolates (although multiresistant) with an identical MLST profile and mutations in the QRDR have recently been reported in Spain.

Haemophilus influenzae is an important human pathogen usually susceptible to quinolones. Here we report the emergence of high-level ciprofloxacin-monoresistant H. influenzae in the Region of Southern Denmark. Four isolates were collected for phenotypic and molecular characterisation using whole-genome sequencing (WGS). During an 18-month period, the occurrence of high-level ciprofloxacin-monoresistant H. influenzae in patients aged 1–77 years from sputum, ear and eye samples was detected. An epidemiological link between the patients could not be identified. The isolates were non-encapsulated, biotype III and were demonstrated by WGS to be clonal belonging to a single clade with an unknown multilocus sequence type (double-locus variant of ST196). The antibiogram demonstrated that they were all monoresistant to ciprofloxacin with a minimum inhibitory concentration (MIC) >32 mg/L. In silico resistome analysis revealed identical, both previously characterised and novel, putative resistance-related mutations in gyrA (S84L and D88 N), parC (K20R, S84I, D356A or T356A, and M481I) and parE (E151 K, I159A, D420 N and S599A) in all isolates. The isolates were otherwise negative for any resistance genes. This is the first description of the clonal emergence of high-level monoresistant H. influenzae due to amino acid substitutions in gyrA, parC and parE.