BACKGROUNDHemophilus influenzae (Hi) is one of the major pediatric bacterial pneumonia pathogens that heavily threatens children's lives and global health. With widespread usage as first-line treatment, the prevalence of β-lactam-resistant strains is increasing sharply. In order to treat Hi more effectively, a systematic study on the antibiotic resistance profiles, β-lactamase-negative ampicillin-resistant (BLNAR) strains isolation rate, and potential BLNAR resistance mechanism in our region is needed.METHODSThis study analyzed antimicrobial susceptibility of Hi, and clinical data of Hi-infected patients retrospectively. BLNAR and β-lactamase-positive ampicillin-clavulanate resistant strains (BLPACR) were confirmed by the Kirby-Bauer method and β-lactamase test. ftsI gene in BLNAR was sequenced to find out whether resistance was induced by penicillin-binding protein mutation. Ampicillin susceptibility test with or without efflux pump inhibitors were done to assess efflux pump contribution in BLNAR. RT-PCR was performed to evaluate the efflux pump genes' transcription levels.RESULTSA total of 2,561 Hi strains were isolated in our hospital from January 2016 to December 2019. Male to female ratio was 1.52:1. Median age was 10 months. Infant (< 3 years old) infection accounted for 83.72%. Hi resistance rates to sulfamethoxazole-trimethoprim, ampicillin, cefathiamidine, cefaclor, cefuroxime, cephalothin, amoxicillin-clavulanate, tetracycline, chloramphenicol, ofloxacin, cefotaxime, and rifampin were 84.28%, 78.01%, 49.80%, 41.98%, 36.58%, 33.64%, 4.55%, 4.1%, 3.37%, 1.77%, 0.99%, and 0.12%, respectively, while 1.33% were BLNAR. BLNARs were classified into four groups by mutation patterns in ftsI gene and most strains were divided to Group Ⅲ/Ⅲ-like. EmrB, ydeA and norM transcription levels in some ampicillin-resistant strains were higher than their sensitive counterparts.CONCLUSIONSAmpicillin is not sufficiently effective as a first-line Hi infection treatment. However, ampicillin-clavulanate and cefotaxime may be a better choice. Efflux pumps, emrB, ydeA and norM play roles in the high resistance to ampicillin.