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PCR screening for the N526K substitution in isolates of Haemophilus influenzae and Haemophilus haemolyticus


Witherden, EA and Kunde, D and Tristram, SG, PCR screening for the N526K substitution in isolates of Haemophilus influenzae and Haemophilus haemolyticus, Journal of Antimicrobial Chemotherapy, 68, (10) pp. 2255-2258. ISSN 0305-7453 (2013) [Refereed Article]

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Copyright 2013 the authors

DOI: doi:10.1093/jac/dkt189


Objectives: Firstly, to evaluate the current PBP3-S primers of Hasegawa et al. (Microb Drug Resist 2003; 9: 3946) 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 15761578 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.

Item Details

Item Type:Refereed Article
Keywords:PCR N526K haemophilus influenzae haemolyticus
Research Division:Biomedical and Clinical Sciences
Research Group:Medical microbiology
Research Field:Medical bacteriology
Objective Division:Health
Objective Group:Clinical health
Objective Field:Clinical health not elsewhere classified
UTAS Author:Witherden, EA (Dr Elizabeth Witherden)
UTAS Author:Kunde, D (Dr Dale Kunde)
UTAS Author:Tristram, SG (Dr Stephen Tristram)
ID Code:88598
Year Published:2013
Web of Science® Times Cited:8
Deposited By:Health Sciences A
Deposited On:2014-02-07
Last Modified:2014-11-25

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