ABSTRACTThe proportions ofHaemophilus influenzaeresistant to ampicillin and other β-lactam antibiotics have been low in Sweden compared to other countries in the Western world. However, a near-doubled proportion of nasopharyngeal SwedishH. influenzaeisolates with resistance to β-lactams has been observed in the last decade. In the present study, the epidemiology and mechanisms of antimicrobial resistance ofH. influenzaeisolates from blood and cerebrospinal fluid in southern Sweden from 1997 to 2010 (n= 465) were studied. Antimicrobial susceptibility testing was performed using disk diffusion, and isolates with resistance to any tested β-lactam were further analyzed in detail. We identified a significantly increased (P= 0.03) proportion of β-lactam-resistant invasiveH. influenzaeduring the study period, which was mainly attributed to a significant recent increase of β-lactamase-negative β-lactam-resistant isolates (P= 0.04). Furthermore, invasive β-lactamase-negative β-lactam-resistantH. influenzaeisolates from 2007 and onwards were found in higher proportions than the corresponding proportions of nasopharyngeal isolates in a national survey. Multiple-locus sequence typing (MLST) of this group of isolates did not completely separate isolates with different resistance phenotypes. However, one cluster of β-lactamase-negative ampicillin-resistant (BLNAR) isolates was identified, and it included isolates from all geographical areas. A truncated variant of a β-lactamase gene with a promoter deletion,blaTEM-1-PΔ dominated among the β-lactamase-positiveH. influenzaeisolates. Our results show that the proportions of β-lactam-resistant invasiveH. influenzaehave increased in Sweden in the last decade.