Haemophilus influenzae may be untypable by the multilocus sequence typing scheme due to a complete deletion of the fucose operon

Ridderberg, Winnie; Fenger, Mette G.; Nørskov‐Lauritsen, Niels

doi:10.1099/jmm.0.018424-0

Cited by 27 publications

(33 citation statements)

References 15 publications

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“…Six (M19067, M19102, M19104, M19171, M19365, and M19444) of the 46 isolates negative for both biomarker genes grouped with H. influenzae by the 16S assay. The fuculose operon, including the fucK gene, may be deleted in some H. influenzae strains (17,21). The results seen with the hpd PCR-negative isolates may have been due to deletions at that locus or to sequence variation at any of the primer or probe binding sites.…”

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confidence: 96%

Evaluation of New Biomarker Genes for Differentiating Haemophilus influenzae from Haemophilus haemolyticus

et al. 2012

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dPCR detecting the protein D (hpd) and fuculose kinase (fucK) genes showed high sensitivity and specificity for identifying Haemophilus influenzae and differentiating it from H. haemolyticus. Phylogenetic analysis using the 16S rRNA gene demonstrated two distinct groups for H. influenzae and H. haemolyticus. Haemophilus species account for approximately 10% of the bacterial flora in the human respiratory tract (9). At least 8 different Haemophilus species are found to colonize the pharyngeal cavity of humans (13) Discrimination between the two organisms can be challenging due to similarities in colony and cell morphology, biochemical characteristics, and genetic background. Hemolytic H. haemolyticus can be differentiated from H. influenzae by production of clear (beta) hemolysis on horse blood agar. The hemolysis activity may be lost after subculture, however, and nonhemolytic H. haemolyticus strains are often misidentified as H. influenzae (10,13,15,16,18).DNA-DNA hybridization is the gold standard for identifying bacterial species (2-4). Because of the complexity of DNA-DNA hybridization, 16S rRNA gene sequence similarity is widely used as a tool to identify bacteria at the species level and assist with differentiating between closely related bacterial species (8). Several molecular tools have been assessed for differentiation of H. haemolyticus from H. influenzae, such as the fuculose kinase gene fucK, the H. influenzae adherence and penetration protein gene hap, the [Cu, Zn]-superoxide dismutase gene sodC, the immunoglobulin A protease gene iga, the lipopolysaccharide (LPS) gene lgtC, and the outer membrane protein P6 (1, 16-18, 24, 26). fucK has been proposed as a reliable marker gene for discrimination of H. influenzae from other Haemophilus species (17). A real-time PCR assay targeting the Haemophilus protein D gene (hpd) has been developed to identify H. influenzae (28). The hpd assay is highly specific for H. influenzae and provides a potential tool to differentiate H. influenzae from H. haemolyticus, including nonhemolytic H. haemolyticus. In this study, both hpd and fucK PCR assays were evaluated against 16S rRNA gene phylogeny for distinguishing H. influenzae from H. haemolyticus by the use of Haemophilus isolates identified during a carriage study in Minnesota (12,22 All isolates were further characterized using the hpd and fucK PCR assays with crude DNA as the template (28). All of the 31 hemolytic H. haemolyticus and 128 other Haemophilus species isolates were negative by both hpd and fucK PCR assays. Of the 245 presumed H. influenzae isolates, 121 (49.4%) were positive for both the hpd and fucK genes, which is the expected genotype for H. influenzae, and 98 (40.0%) were negative for both hpd and fucK,

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confidence: 96%

Evaluation of New Biomarker Genes for Differentiating Haemophilus influenzae from Haemophilus haemolyticus

et al. 2012

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“…Two H. haemolyticus and seven H. influenzae strains were negative for both purT and hpd genes. The results seen with purT and hpd PCR-negative isolates may have been due to deletions at that locus or to sequence variation at any of the primer or probe binding sites (30,32).…”

Section: Discussionmentioning

confidence: 99%

Comparative Genomic Analysis of Haemophilus haemolyticus and Nontypeable Haemophilus influenzae and a New Testing Scheme for Their Discrimination

Rishishwar

Sivadas

et al. 2016

J Clin Microbiol

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g Haemophilus haemolyticus has been recently discovered to have the potential to cause invasive disease. It is closely related to nontypeable Haemophilus influenzae (NT H. influenzae). NT H. influenzae and H. haemolyticus are often misidentified because none of the existing tests targeting the known phenotypes of H. haemolyticus are able to specifically identify H. haemolyticus. Through comparative genomic analysis of H. haemolyticus and NT H. influenzae, we identified genes unique to H. haemolyticus that can be used as targets for the identification of H. haemolyticus. A real-time PCR targeting purT (encoding phosphoribosylglycinamide formyltransferase 2 in the purine synthesis pathway) was developed and evaluated. The lower limit of detection was 40 genomes/PCR; the sensitivity and specificity in detecting H. haemolyticus were 98.9% and 97%, respectively. To improve the discrimination of H. haemolyticus and NT H. influenzae, a testing scheme combining two targets (H. haemolyticus purT and H. influenzae hpd, encoding protein D lipoprotein) was also evaluated and showed 96.7% sensitivity and 98.2% specificity for the identification of H. haemolyticus and 92.8% sensitivity and 100% specificity for the identification of H. influenzae, respectively. The dual-target testing scheme can be used for the diagnosis and surveillance of infection and disease caused by H. haemolyticus and NT H. influenzae. Haemophilus haemolyticus is a human commensal that colonizes the respiratory tract. It shares high similarities in morphology, biochemistry, and genetics with nontypeable Haemophilus influenzae (NT H. influenzae) (1-3). NT H. influenzae has emerged as the major cause of invasive H. influenzae disease since the implementation of H. influenzae serotype b (H. influenzae b) vaccine (4). NT H. influenzae infections such as childhood otitis media and respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD) result in an enormous social and economic burden to societies (5). H. haemolyticus was recently reported to cause invasive disease (1). These cases were previously misidentified as NT H. influenzae due to the lack of proper identification methods to discriminate between the two species (6). Reevaluation of NT H. influenzae cases from previous years revealed that about 2% (7/374) of the NT H. influenzae invasive cases reported from Active Bacterial Core surveillance (ABCs) were in fact caused by H. haemolyticus (1).Misidentification of H. haemolyticus as NT H. influenzae has been repeatedly reported by multiple research groups, with up to 40% of isolates being misidentified as NT H. influenzae using classical phenotypic methods in clinical microbiology laboratories (1, 7-11). The misidentification of H. haemolyticus has a potential impact on accurate assessment of the prevalence of antibioticresistant NT H. influenzae (12). Therefore, a rapid and accurate method is needed to distinguish H. haemolyticus from NT H. influenzae and to better understand the epidemiology of H. haemolyticus infections....

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“…The species misclassification of these strains by the original authors [28] exemplifies the difficulties associated with accurate speciation of Haemophilus spp. Other researchers [13,[15][16] have reported that certain H. influenzae isolates may lack fucK, one of the seven genes used in the H. influenzae MLST scheme [14]. The absence of this gene has historically confounded H. influenzae speciation attempts such as the probable incorrect assignment of fucK-negative H. influenzae strains as H. haemolyticus [42].…”

Section: • Phylogenomic Analysis Of Haemophilus Spp For Species Detementioning

confidence: 99%

“…The fucP gene resides within the fucose operon [13], which also encodes the multilocus sequencing typing (MLST) 'housekeeping' gene, fucK [14]. However, there is a growing body of evidence that certain H. influenzae strains lack some, or all, genes within the fucose operon [13,[15][16][17]. For example, a recent study using multilocus sequence analysis across 60 clinical Haemophilus spp.…”

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confidence: 99%

“…This assay targets fucP, a highly conserved gene that encodes for L-fucose permease, a major facilitator superfamily transporter involved in L-fucose uptake [12]. The fucP gene resides within the fucose operon [13], which also encodes the multilocus sequencing typing (MLST) 'housekeeping' gene, fucK [14]. However, there is a growing body of evidence that certain H. influenzae strains lack some, or all, genes within the fucose operon [13,[15][16][17].…”

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Simultaneous Identification of Haemophilus Influenzae and Haemophilus Haemolyticus using Real-Time PCR

et al. 2017

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Aim: To design a highly specific and sensitive multiplex real-time PCR assay for the differentiation of the pathogen Haemophilus influenzae from its nonpathogenic nearneighbor Haemophilus haemolyticus. Materials & methods: A comparison of 380 Haemophilus spp. genomes was used to identify loci specific for each species. Novel PCR assays targeting H. haemolyticus (hypD) and H. influenzae (siaT) were designed. Results & discussion: PCR screening across 143 isolates demonstrated 100% specificity for hypD and siaT. These two assays were multiplexed with the recently described fucP assay for further differentiation among H. influenzae. Conclusion: The triplex assay provides rapid, unambiguous, sensitive and highly specific genotyping results for the simultaneous detection of hypD and siaT, including fucose-positive H. influenzae (fucP), in a single PCR.

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Haemophilus influenzae may be untypable by the multilocus sequence typing scheme due to a complete deletion of the fucose operon

Abstract: Haemophilus influenzae may be untypable by the multilocus sequence typing scheme due to a complete deletion of the fucose operon

Cited by 27 publications

References 15 publications

Evaluation of New Biomarker Genes for Differentiating Haemophilus influenzae from Haemophilus haemolyticus

Evaluation of New Biomarker Genes for Differentiating Haemophilus influenzae from Haemophilus haemolyticus

Comparative Genomic Analysis of Haemophilus haemolyticus and Nontypeable Haemophilus influenzae and a New Testing Scheme for Their Discrimination

Simultaneous Identification of Haemophilus Influenzae and Haemophilus Haemolyticus using Real-Time PCR

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