Antimicrobial resistance is an increasing concern for the treatment of many human pathogens, including the respiratory tract pathobiont, Haemophilus influenzae (NTHi), which causes upper and lower respiratory tract infections. NTHi isolates are increasingly resistant to common antibiotics, with reported ampicillin resistance rates ranging from 7.4% to 69.4% [1], and resistance to trimethoprim-sulfamethoxazole also increasing [2].
Here we have determined the genome sequences of 49 recent NTHi clinical isolates from Australia. The isolates had an average genome size of 1.85Mb ± 0.05Mb with ~1763 protein encoding genes. Comparison of gene contents revealed a small core genome of 1315 protein coding genes, with 1342 genes classified as cloud genes in keeping with previously reported values [3] and the ability of NTHi to acquire genetic material through natural competence.
No prevalent MLST classes were detected in the group of isolates, but 19 strains contained genes encoding AMR determinants. As expected, genes encoding β-lactamases were most common, but determinants for resistances to aminoglycosides, chloramphenicol, sulfonamides and tetracyclines were also present. Interestingly several strains encoded multiple resistance determinants, with 2 being potential multi-drug resistant. Analysis of the PBP3 genes for mutations conferring intrinsic resistant to β-lactam antibiotics identified 28 strains carrying relevant mutations, but only 11 strains contained the combinations of mutations associated with β-lactamase negative ampicillin resistant NTHi.
Phenotypic verification of expected AMR phenotypes using disk diffusion assays for 15 strains confirmed the expected phenotypes for 8 out of 15 strains, but also uncovered additional, unexpected resistances. Overall, 60% of these strains were ampicillin resistant, and 3 strains were classified as multi-drug resistant after showing resistance to ≥3 antibiotics.
These results indicate a clear increase in antibiotic resistance in Australian NTHi isolates since the last published survey in 2006 [4], highlighting the need for closer monitoring of these isolates across Australia.