Bacterial isolates were stored at −80 °C in brain heart infusion broth +20% glycerol. Isolates were cultured on chocolate agar plates with incubation at 35 °C +5% CO2 for 18–24 h and all tests were performed on a subculture of a single isolated colony. Identities of all isolates were confirmed by standard biochemical tests (Kilian, 2003) and 16S rRNA gene sequencing (Lau et al., 2004). HCS assay Biotypes were assigned according to Kilian’s biotyping scheme based on three biochemical reactions: urease, indole and ornithine decarboxylase (Kilian, 1976). The nontypeable nature

of all 125 isolates was confirmed by slide agglutination test using antisera against all six serotypes purchased from commercial sources (Difco, Oakville, ON, Canada; Denka Seiken, Tokyo, Japan). The absence of both the serotype-specific and the capsule transport, GS 1101 bexA, genes was confirmed by PCR using primers described by Falla et al. (1994). β-Lactamase production was detected using Dryslide Nitrocefin (BBL, Becton Dickinson, Oakville, ON, Canada). Disc diffusion test was carried out as described by the Clinical Laboratory Standards Institute (CLSI, 2008). The following antibiotics (Oxoid, Nepean,

ON, Canada) were tested: ampicillin (2 and 10 μg), amoxicillin–clavulanic acid (30 μg), cefaclor (30 μg), ceftriaxone (30 μg), chloramphenicol (30 μg), ciprofloxacin Amine dehydrogenase (5 μg), clarithromycin (15 μg), moxifloxacin (5 μg), sulfamethoxazole–trimethoprim (25 μg), azithromycin (15 μg), imipenem (10 μg), levofloxacin (5 μg) and tetracycline (30 μg). Detection of β-lactamase-negative ampicillin-resistant (BLNAR) strains was accomplished using two concentrations of ampicillin (Karpanoja et al., 2004). Hi BLNAR strain ATCC 49247 was used as a control in

each experiment. MLST was carried out by PCR amplification of seven housekeeping genes according to the previously described method (Meats et al., 2003), and the assignment of STs was conducted using the Hi MLST website (http://haemophilus.mlst.net/). Genetic relationships between isolates based on MLST data were also analysed by eburst (Feil et al., 2004) and concatenated sequences of the seven housekeeping gene loci using software available from the Hi MLST website cited above. Seventy isolates (56%) were from invasive disease cases and were recovered from normally sterile body sites (blood, 61 isolates; CSF, eight isolates; liver abscess, one isolate). The other 55 isolates (44%) were from the respiratory tract. The breakdown of the invasive isolates by year is as follows: eight isolates from 2000, eight from 2001, four from 2002, three from 2003, 13 from 2004, 20 from 2005 and 14 from 2006. Invasive isolates were from patients whose ages ranged from 1 day to 94 years.