Cefotaxime resistance in invasive Haemophilus influenzae isolates in Germany 2016-19: prevalence, epidemiology and relevance of PBP3 substitutions.

BACKGROUND: Haemophilus influenzae can cause invasive infections, in which cefotaxime is among the first-line antibiotics for treatment. The prevalence of cefotaxime-resistant H. influenzae in Europe is reported to be on a low level. Nevertheless, systematic studies with a large set of invasive isolates are scarce. OBJECTIVES: To provide prevalence data for cefotaxime resistance in invasive H. influenzae isolates in Germany 2016-19 and investigate the epidemiological relevance of PBP3 mutations known to elevate the cefotaxime MIC. METHODS: Cefotaxime susceptibility of invasive H. influenzae isolates, collected in the national laboratory surveillance programme, was examined by gradient agar diffusion (GAD) testing. Cefotaxime resistance was determined according to EUCAST guidelines (resistance breakpoint MIC >0.125 mg/L). Therefore, the MIC for all resistant isolates was verified by broth microdilution method (BMD). WGS was performed to investigate the genetic relationship of cefotaxime-resistant isolates and to analyse alterations in the PBP3. An analysis of the geographic distribution of the resistant isolates was performed. RESULTS: From 2016 to 2019, the German National Reference Laboratory for Meningococci and H. influenzae received 2432 invasive H. influenzae isolates from blood and CSF. According to GAD results, 27 strains were resistant to cefotaxime. BMD confirmed the resistance in 22 of these isolates, which equals a prevalence of cefotaxime resistance of 0.90% in invasive H. influenzae in Germany. Among cefotaxime-resistant isolates cgMLST revealed three clusters. PBP3 analysis showed previously described mutations in our strains. In comparison with cefotaxime-susceptible strains, the alterations L389F and Y557H were significantly associated with cefotaxime resistance, but were not present in all resistant strains. Geographic analysis showed that the disease cases with cefotaxime-resistant H. influenzae were evenly spread throughout the population in Germany. CONCLUSIONS: Cefotaxime is still well suited for the treatment of invasive H. influenzae infections. Rarely occurring cefotaxime resistance is caused by sporadic mutations. The role of PBP3 mutations needs further investigation.

Molecular epidemiology of imipenem resistance in invasive Haemophilus influenzae infections in Germany in 2016.

BACKGROUND: The carbapenems imipenem and meropenem play an important role in the empirical anti-infective treatment of critically ill patients. Carbapenem resistance in Haemophilus influenzae (Hi) has rarely been reported. OBJECTIVES: We provide prevalence data for resistance to carbapenems from laboratory surveillance of invasive Hi infections in Germany in 2016. METHODS: Phenotypic susceptibility testing against ampicillin, amoxicillin/clavulanate, cefotaxime and imipenem was carried out on 474 isolates from blood and CSF. The isolates were collected as part of the national laboratory surveillance programme. Imipenem-resistant strains were further tested for meropenem susceptibility. Molecular analysis was done by ftsI sequencing to detect mutations in PBP3, by acrR sequencing to detect alterations in the regulatory protein of the AcrAB-TolC efflux pump and by MLST. RESULTS: No resistance to meropenem was detected. Cefotaxime resistance was rare (n = 3; 0.6%). Imipenem resistance was found in 64 strains (13.5%) using gradient agar diffusion and was confirmed in 26 isolates by broth microdilution (5.5%). Imipenem resistance occurred predominantly in Hi that were ß-lactamase negative but ampicillin resistant and in those that were ß-lactamase positive but nevertheless amoxicillin/clavulanate resistant. This finding suggested a ß-lactamase-independent mechanism. Accordingly, sequence analysis of PBP3 identified previously described mutations. MLST of the imipenem-resistant strains, which were all non-typeable Hi, revealed a high diversity. CONCLUSIONS: We conclude that imipenem, but not meropenem, resistance is frequent in Hi. It is likely to be supported by PBP3 mutations.