Background: Concern about Haemophilus influenzae infection has been increasing over recent decades. Given the emergence of H. influenzae with severe drug resistance, we assessed the prevalence of as well as risk factors and potential therapies for extensively drug-resistant (XDR) H. influenzae infection in Taiwan. Results: In total, 2091 H. influenzae isolates with disk diffusion-based antibiotic susceptibility testing from 2007 to 2018 were enrolled. H. influenzae strains resistant to ampicillin, chloramphenicol, levofloxacin, and trimethoprimsulfamethoxazole tended to be isolated from patient wards (≧41%), whereas those resistant to amoxicillinclavulanate, cefotaxime, and cefuroxime were more likely to be isolated from intensive care units (approximately 50%). XDR H. influenzae was first identified in 2007, and its incidence did not significantly change thereafter. Overall prevalence of single, multiple, and extensively drug-resistant H. influenzae over 2007-2018 was 21.5% (n = 450), 26.6% (n = 557), and 2.5% (n = 52), respectively. A stepwise logistic regression analysis revealed that blood culture (odds ratio: 4.069, 95% confidence intervals: 1.339-12.365, P = 0.013) was an independent risk factor for XDR H. influenzae infection. No nosocomial transmission of XDR H. influenzae observed. Antibiotic susceptibility testing results demonstrated that cefotaxime was effective against 78.8% (n = 41) of the XDR strains. Conclusions: The presence of XDR H. influenzae strains was identified in Taiwan, and cefotaxime was efficacious against most of these strains.

Background Cefepime and aztreonam are highly efficacious against H. influenzae, and resistant strains are rare. In this study, we isolated cefepime- and aztreonam-nonsusceptible H. influenzae strains and addressed the molecular basis of their resistance to cefepime and aztreonam. Methods Two hundred and 28 specimens containing H. influenzae were screened, of which 32 isolates were enrolled and applied to antimicrobial susceptibility testing and whole-genome sequencing. Genetic variations that were detected in all nonsusceptible isolates with statistical significance by Fisher’s exact tests were identified as cefepime or aztreonam nonsusceptibility related. Functional complementation assays were conducted to assess the in vitro effects of proteins with sequence substitutions on drug susceptibility. Results Three H. influenzae isolates were nonsusceptible to cefepime, one of which was also nonsusceptible to aztreonam. Genes encoding TEM, SHV and CTX-M extended-spectrum β-lactamases were not detected in the cefepime- and aztreonam-nonsusceptible isolates. Five genetic variations in four genes and 10 genetic variations in five genes were associated with cefepime and aztreonam nonsusceptibility, respectively. Phylogenetic analyses revealed that changes in FtsI were correlated strongly with the MIC of cefepime and moderately with aztreonam. FtsI Thr532Ser-Tyr557His cosubstitution linked to cefepime nonsusceptibility and Asn305Lys-Ser385Asn-Glu416Asp cosubstitution to aztreonam nonsusceptibility. Functional complementation assays revealed that these cosubstitutions increased MICs of cefepime and aztreonam in susceptible H. influenzae isolates, respectively. Conclusions Genetic variations relevant to resistant phenotypes of cefepime and aztreonam nonsusceptibility in H. influenzae were identified. Moreover, the effects of FtsI cosubstitutions on increasing MICs of cefepime and aztreonam in H. influenzae were demonstrated.

Background Non-typeable Haemophilus influenzae (NTHi) has become the major cause of invasive H. influenzae diseases in the post-H. influenzae type b vaccine era. The emergence of multidrug-resistant (MDR) NTHi is a growing public health problem. Herein, we investigated the molecular basis of MDR in NTHi. The isolated NTHi were subjected to antimicrobial susceptibility testing for 12 agents. Whole genome and plasmid sequencing were conducted and analyzed to identify significant genetic variations and plasmid-encoded genes conferred antibiotic resistance. Results Thirteen (50%) MDR NTHi isolates were obtained; of these, 92.3% were non-susceptible to ampicillin, 30.8% to amoxicillin-clavulanate, 61.5% to cefuroxime, 61.5% to ciprofloxacin/levofloxacin, 92.3% to trimethoprim-sulfamethoxazole, 30.8% to tetracycline, and 7.7% to azithromycin. Eight ampicillin-resistant isolates were β-lactamase positive; of these, 6 carried blaTEM-1 and 2 carried blaROB-1, whereas 4 were β-lactamase negative. Genetic variations in mrdA, mepA, and pbpG were correlated with amoxicillin-clavulanate non-susceptibility, whereas variations in ftsI and lpoA conferred cefuroxime resistance. Five variations in gyrA, 2 in gyrB, 3 in parC, 1 in parE, and 1 in the parC-parE intergenic region were associated with levofloxacin/ciprofloxacin non-susceptibility. Among these genes, 8 variations were linked to high-level levofloxacin resistance. Six variations in folA were associated with trimethoprim-sulfamethoxazole resistance. Plasmid-bearing tet(B) and mef(A) genes were responsible for tetracycline and azithromycin resistance in 4 and 1 MDR isolates, respectively. Conclusions This study clarified the molecular epidemiology of MDR in NTHi. This can benefit the monitoring of drug resistance trends in NTHi and the adequate medical management of patients with NTHi infection.

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