Comparative genome analysis identifies novel nucleic acid diagnostic targets for use in the specific detection of Haemophilus influenzae

Coughlan, Helena; Reddington, Kate; Tuite, Nina; Boo, Teck Wee; Cormican, Martin; Barrett, Louise M.; Smith, Terry J.; Clancy, Eoin; Barry, Thomas

doi:10.1016/j.diagmicrobio.2015.06.013

Cited by 8 publications

(8 citation statements)

References 44 publications

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“…influenzae was the first bacterial species to have its genome sequenced, in 1995 (54). WGS has been used to describe the population structure of H. influenzae (55), provide insight into genomic diversity (56), identify targets for strain differentiation and diagnostics (57), and characterize virulence properties (58)(59)(60)(61). Our study is unique in that we examined a large collection of prospectively collected H. influenzae, including strains that persisted for months to years in the human respiratory tract, to answer questions related to in vivo adaptation of this exclusively human pathogen in the airways of its human host.…”

Section: Discussionmentioning

confidence: 99%

Haemophilus influenzae genome evolution during persistence in the human airways in chronic obstructive pulmonary disease

Pettigrew

Ahearn

Gent³

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Nontypeable (NTHi) exclusively colonize and infect humans and are critical to the pathogenesis of chronic obstructive pulmonary disease (COPD). In vitro and animal models do not accurately capture the complex environments encountered by NTHi during human infection. We conducted whole-genome sequencing of 269 longitudinally collected cleared and persistent NTHi from a 15-y prospective study of adults with COPD. Genome sequences were used to elucidate the phylogeny of NTHi isolates, identify genomic changes that occur with persistence in the human airways, and evaluate the effect of selective pressure on 12 candidate vaccine antigens. Strains persisted in individuals with COPD for as long as 1,422 d. Slipped-strand mispairing, mediated by changes in simple sequence repeats in multiple genes during persistence, regulates expression of critical virulence functions, including adherence, nutrient uptake, and modification of surface molecules, and is a major mechanism for survival in the hostile environment of the human airways. A subset of strains underwent a large 400-kb inversion during persistence. NTHi does not undergo significant gene gain or loss during persistence, in contrast to other persistent respiratory tract pathogens. Amino acid sequence changes occurred in 8 of 12 candidate vaccine antigens during persistence, an observation with important implications for vaccine development. These results indicate that NTHi alters its genome during persistence by regulation of critical virulence functions primarily by slipped-strand mispairing, advancing our understanding of how a bacterial pathogen that plays a critical role in COPD adapts to survival in the human respiratory tract.

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Section: Discussionmentioning

confidence: 99%

Haemophilus influenzae genome evolution during persistence in the human airways in chronic obstructive pulmonary disease

Pettigrew

Ahearn

Gent³

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…Other approaches for genome comparison to identify potential diagnostic targets have been described. In a study on Haemophilus influenzae three genome sequences were compared for selection of putative diagnostic PCR targets, and subsequent sequence alignment and development of primers and probes [ 29 ]. Although this was a very successful approach, it required a lot of manual work and laboratory resources.…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, the approach for comparison of a few genomes, described by Coughlan et al . [ 29 ], is still very suitable for organisms for which only a few genomes are available in public databases. M-GCAT [ 30 ], and MAUVE [ 31 ] are also computer programs that are able to compare large numbers of whole genome sequences in a short time, but they can only show sequence similarities and dissimilarities.…”

Section: Discussionmentioning

confidence: 99%

Development of a real-time PCR for detection of Staphylococcus pseudintermedius using a novel automated comparison of whole-genome sequences

et al. 2017

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Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and cats and occasionally causes infections in humans. S. pseudintermedius is often resistant to multiple classes of antimicrobials. It requires a reliable detection so that it is not misidentified as S. aureus. Phenotypic and currently-used molecular-based diagnostic assays lack specificity or are labour-intensive using multiplex PCR or nucleic acid sequencing. The aim of this study was to identify a specific target for real-time PCR by comparing whole genome sequences of S. pseudintermedius and non-pseudintermedius.Genome sequences were downloaded from public repositories and supplemented by isolates that were sequenced in this study. A Perl-script was written that analysed 300-nt fragments from a reference genome sequence of S. pseudintermedius and checked if this sequence was present in other S. pseudintermedius genomes (n = 74) and non-pseudintermedius genomes (n = 138). Six sequences specific for S. pseudintermedius were identified (sequence length between 300–500 nt). One sequence, which was located in the spsJ gene, was used to develop primers and a probe. The real-time PCR showed 100% specificity when testing for S. pseudintermedius isolates (n = 54), and eight other staphylococcal species (n = 43). In conclusion, a novel approach by comparing whole genome sequences identified a sequence that is specific for S. pseudintermedius and provided a real-time PCR target for rapid and reliable detection of S. pseudintermedius.

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“…The phoB gene has been previously been described in the literature as a suitable diagnostics assay target for the species specific identification of H. influenzae using real-time PCR (Coughlan et al, 2015). In this study we have further validated this target and have successfully demonstrated the suitability of the phoB mRNA transcript for use in a duplex real-time NASBA diagnostics assay for the specific detection of H. influenzae.…”

Section: Discussionmentioning

confidence: 74%

“…PhoB has recently been described in the literature as species specific target for H. influenzae using real-time PCR (Coughlan et al, 2015). Based on our in silico evaluation, this diagnostics target demonstrated suitable characteristics for evaluation as a H. influenzae species specific mRNA diagnostics target for use in a real-time NASBA assay.…”

Section: Diagnostics Target Identificationmentioning

confidence: 91%

Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis

Clancy

Coughlan

Higgins

et al. 2016

Journal of Microbiological Methods

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Comparative genome analysis identifies novel nucleic acid diagnostic targets for use in the specific detection of Haemophilus influenzae

Cited by 8 publications

References 44 publications

Haemophilus influenzae genome evolution during persistence in the human airways in chronic obstructive pulmonary disease

Haemophilus influenzae genome evolution during persistence in the human airways in chronic obstructive pulmonary disease

Development of a real-time PCR for detection of Staphylococcus pseudintermedius using a novel automated comparison of whole-genome sequences

Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis

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