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PCR screening for the N526K substitution in isolates of Haemophilus influenzae and Haemophilus haemolyticus
journal contribution
posted on 2023-05-17, 21:50 authored by Witherden, EA, Kunde, D, Stephen TristramStephen TristramObjectives: Firstly, to evaluate the current PBP3-S primers of Hasegawa et al. (Microb Drug Resist 2003; 9: 39–46) and develop new primers for the amplification of N526 in isolates of Haemophilus haemolyticus. Secondly, to develop a new PCR assay for the detection (by amplification) of the N526K substitution, encoded by either the AAA or AAG single nucleotide polymorphism (SNP) at position 1576–1578 of the ftsI gene, in isolates of both Haemophilus influenzae and H. haemolyticus. Methods: Atotal of 50 H. influenzae and 50 H. haemolyticus isolates, comprising N526 and N526K genotypes,were used to evaluate the performance of SNP-based PCR primers for the detection of the b-lactamase-negative ampicillin resistance (BLNAR)-defining N526K substitution in H. influenzae and H. haemolyticus, using a real-time PCR platform. Results: The PBP3-S primers of Hasegawaet al. failed to amplify H. haemolyticus isolates, irrespective of their N526/N526K status, owing to an inability of the forward primer to bind the H. haemolyticus ftsI sequence, giving an overall sensitivity of 100% and a specificity of 40% when using all of the isolates. However, the PBP3-N526 and PBP3-N526K PCR primers designed in this study were 100% sensitive and specific, and 84% sensitive and 100% specific, respectively, for the detection of N526K-positive isolates. Conclusions: Although antibiotic resistance surveillance studies on H. influenzae should include a definitive test for H. influenzae/H. haemolyticus identification, the new primers from this study will not only allow for PCR characterization of both H. influenzae and H. haemolyticus with respect to the N526K BLNAR substitutions, they will also stop incorrect characterization of susceptible H. haemolyticus isolates as low-BLNAR H. influenzae.
Funding
Clifford Craig Foundation
History
Publication title
Journal of Antimicrobial ChemotherapyVolume
68Issue
10Pagination
2255-2258ISSN
0305-7453Department/School
School of Health SciencesPublisher
Oxford University PressPlace of publication
Great Clarendon St, Oxford, England, Ox2 6DpRights statement
Copyright 2013 the authorsRepository Status
- Restricted