-
Future Microbiology Jun 2017To design a highly specific and sensitive multiplex real-time PCR assay for the differentiation of the pathogen Haemophilus influenzae from its nonpathogenic...
AIM
To design a highly specific and sensitive multiplex real-time PCR assay for the differentiation of the pathogen Haemophilus influenzae from its nonpathogenic near-neighbor 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.
Topics: Bacterial Proteins; Genome, Bacterial; Genotype; Haemophilus; Haemophilus influenzae; Humans; Multiplex Polymerase Chain Reaction; Phylogeny; Proteins; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Sequence Analysis, DNA
PubMed: 28604066
DOI: 10.2217/fmb-2016-0215 -
European Journal of Microbiology &... Mar 2015Haemophilus influenzae is a key pathogen of upper respiratory tract infections. Its reliable discrimination from nonpathogenic Haemophilus spp. is necessary because... (Review)
Review
Haemophilus influenzae is a key pathogen of upper respiratory tract infections. Its reliable discrimination from nonpathogenic Haemophilus spp. is necessary because merely colonizing bacteria are frequent at primarily unsterile sites. Due to close phylogenetic relationship, it is not easy to discriminate H. influenzae from the colonizer Haemophilus haemolyticus. The frequency of H. haemolyticus isolations depends on factors like sampling site, patient condition, and geographic region. Biochemical discrimination has been shown to be nonreliable. Multiplex PCR including marker genes like sodC, fucK, and hpd or sequencing of the 16S rRNA gene, the P6 gene, or multilocus-sequence-typing is more promising. For the diagnostic routine, such techniques are too expensive and laborious. If available, matrix-assisted laser-desorption-ionization time-of-flight mass spectrometry is a routine-compatible option and should be used in the first line. However, the used database should contain well-defined reference spectra, and the spectral difference between H. influenzae and H. haemolyticus is small. Fluorescence in-situ hybridization is an option for less well-equipped laboratories, but the available protocol will not lead to conclusive results in all instances. It can be used as a second line approach. Occasional ambiguous results have to be resolved by alternative molecular methods like 16S rRNA gene sequencing.
PubMed: 25883794
DOI: 10.1556/EUJMI-D-14-00033 -
Clinical Microbiology Reviews Apr 2014The aim of this review is to provide a comprehensive update on the current classification and identification of Haemophilus and Aggregatibacter species with exclusive or... (Review)
Review
The aim of this review is to provide a comprehensive update on the current classification and identification of Haemophilus and Aggregatibacter species with exclusive or predominant host specificity for humans. Haemophilus influenzae and some of the other Haemophilus species are commonly encountered in the clinical microbiology laboratory and demonstrate a wide range of pathogenicity, from life-threatening invasive disease to respiratory infections to a nonpathogenic, commensal lifestyle. New species of Haemophilus have been described (Haemophilus pittmaniae and Haemophilus sputorum), and the new genus Aggregatibacter was created to accommodate some former Haemophilus and Actinobacillus species (Aggregatibacter aphrophilus, Aggregatibacter segnis, and Aggregatibacter actinomycetemcomitans). Aggregatibacter species are now a dominant etiology of infective endocarditis caused by fastidious organisms (HACEK endocarditis), and A. aphrophilus has emerged as an important cause of brain abscesses. Correct identification of Haemophilus and Aggregatibacter species based on phenotypic characterization can be challenging. It has become clear that 15 to 20% of presumptive H. influenzae isolates from the respiratory tracts of healthy individuals do not belong to this species but represent nonhemolytic variants of Haemophilus haemolyticus. Due to the limited pathogenicity of H. haemolyticus, the proportion of misidentified strains may be lower in clinical samples, but even among invasive strains, a misidentification rate of 0.5 to 2% can be found. Several methods have been investigated for differentiation of H. influenzae from its less pathogenic relatives, but a simple method for reliable discrimination is not available. With the implementation of identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry, the more rarely encountered species of Haemophilus and Aggregatibacter will increasingly be identified in clinical microbiology practice. However, identification of some strains will still be problematic, necessitating DNA sequencing of multiple housekeeping gene fragments or full-length 16S rRNA genes.
Topics: Aggregatibacter; Bacteriological Techniques; Haemophilus; Haemophilus Infections; Host Specificity; Humans; Molecular Diagnostic Techniques; Pasteurellaceae Infections; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 24696434
DOI: 10.1128/CMR.00103-13 -
Pathogens (Basel, Switzerland) Jan 2021Nontypeable (NTHi) is a significant respiratory tract pathogen responsible for infections that collectively pose a substantial health and socioeconomic burden. The...
Nontypeable (NTHi) is a significant respiratory tract pathogen responsible for infections that collectively pose a substantial health and socioeconomic burden. The clinical course of these infections is largely dictated by NTHi interactions with host respiratory epithelia, and thus, approaches that disrupt colonisation and invasion may have significant therapeutic potential. Survival, successful host-cell interactions, and pathogenesis are reliant on NTHi's ability to sequester host-derived haem. Previously, we demonstrated the therapeutic potential of exploiting this haem-dependence using a closely related competitor bacterium, (Hh). Hh strains capable of producing the novel haem-binding protein haemophilin (Hpl) possessed potent inhibitory activity by restricting NTHi access to haem in a broth co-culture environment. Here, we extend this work to cell culture models that more closely represent the human respiratory epithelium and show that Hh strains with high levels of expression protect epithelial cell line monolayers against adhesion and invasion by NTHi. Inhibitory activity was dependent on the level of Hpl production, which was stimulated by NTHi challenge and nasopharyngeal cell exposure. Provided these protective benefits translate to in vivo applications, Hpl-producing Hh may have probiotic utility against NTHi infections by inhibiting requisite nasopharyngeal colonisation.
PubMed: 33401487
DOI: 10.3390/pathogens10010029 -
Frontiers in Microbiology 2014Non-typeable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus are closely related bacteria that reside in the upper respiratory tract. NTHi is associated with... (Review)
Review
Non-typeable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus are closely related bacteria that reside in the upper respiratory tract. NTHi is associated with respiratory tract infections that frequently result in antibiotic prescription whilst H. haemolyticus is rarely associated with disease. NTHi and H. haemolyticus can be indistinguishable by traditional culture methods and molecular differentiation has proven difficult. This current review chronologically summarizes the molecular approaches that have been developed for differentiation of NTHi from H. haemolyticus, highlighting the advantages and disadvantages of each target and/or technique. We also provide suggestions for the development of new tools that would be suitable for clinical and research laboratories.
PubMed: 25520712
DOI: 10.3389/fmicb.2014.00664 -
Journal of Microbiology, Immunology,... Dec 2021This study aimed to investigate the clinical characteristics and outcomes of bacteremia caused by Haemophilus and Aggregatibacter species in patients who were treated at...
BACKGROUND/PURPOSE
This study aimed to investigate the clinical characteristics and outcomes of bacteremia caused by Haemophilus and Aggregatibacter species in patients who were treated at a medical center between 2006 and 2018.
METHODS
Haemophilus and Aggregatibacter isolates were identified up to the species level using Bruker Biotyper MALDI-TOF analysis and ancillary 16S rRNA gene sequencing analysis (in case of ambiguity). Clinical characteristics and outcomes of patients with bacteremia caused by these organisms were evaluated.
RESULTS
Sixty-five Haemophilus and Aggregatibacter species isolates causing bacteremia were identified from nonduplicated patients, including 51 (78.5%) Haemophilus influenzae, 6 (9.2%) Haemophilus parainfluenzae, 1 (1.5%) Haemophilus haemolyticus, 3 (4.6%) A. aphrophilus, and 4 (6.2%) A. segnis. Hospital mortality was observed in 18 (28.1%) of 64 patients with bacteremia caused by Haemophilus (n = 57) and Aggregatibacter species (n = 7). The majority of patients with bacteremia had community-acquired disease with low severity. The average Sequential Organ Failure Assessment (SOFA) score was low (4.4 ± 4.7). But, a higher SOFA score (adjusted odds ratio 2.5, 95% confidence interval 1.22-5.12; P = 0.01) was an independent factor predicting poor 7-day clinical outcomes in patients with community-acquired H. influenzae bacteremia (n = 39).
CONCLUSIONS
The overall hospital mortality of 28.1% was observed among patients with bacteremia due to Haemophilus and Aggregatibacter species. A higher SOFA score was and independent predictor of poor 7-day clinical outcomes in patients with community-acquired H. influenzae bacteremia.
Topics: Adult; Aged; Aggregatibacter; Anti-Bacterial Agents; Bacteremia; Female; Haemophilus; Hospital Mortality; Humans; Male; Microbial Sensitivity Tests; Middle Aged; Organ Dysfunction Scores; RNA, Ribosomal, 16S
PubMed: 33390332
DOI: 10.1016/j.jmii.2020.12.002 -
Microbiology Spectrum Apr 2024and are two of the most common bacterial genera in the human oral cavity, encompassing both commensals and pathogens of substantial ecological and medical...
UNLABELLED
and are two of the most common bacterial genera in the human oral cavity, encompassing both commensals and pathogens of substantial ecological and medical significance. In this study, we conducted a metapangenomic analysis of oral and species to uncover genomic diversity, phylogenetic relationships, and habitat specialization within the human oral cavity. Using three metrics-pangenomic gene content, phylogenomics, and average nucleotide identity (ANI)-we first identified distinct species and sub-species groups among these genera. Mapping of metagenomic reads then revealed clear patterns of habitat specialization, such as species predominantly in dental plaque, a distinctive sub-species group on the tongue dorsum, and . sp. HMT-036 predominantly in keratinized gingiva and buccal mucosa. In addition, we found that supragingival plaque samples contained predominantly only one out of the three taxa, , , and . sp. HMT-458, suggesting independent niches or a competitive relationship. Functional analyses revealed the presence of key metabolic genes, such as oxaloacetate decarboxylase, correlated with habitat specialization, suggesting metabolic versatility as a driving force. Additionally, heme synthesis distinguishes . sp. HMT-036 from closely related , suggesting that the availability of micronutrients, particularly iron, was important in the evolutionary ecology of these species. Overall, our study exemplifies the power of metapangenomics to identify factors that may affect ecological interactions within microbial communities, including genomic diversity, habitat specialization, and metabolic versatility.
IMPORTANCE
Understanding the microbial ecology of the mouth is essential for comprehending human physiology. This study employs metapangenomics to reveal that various and species exhibit distinct ecological preferences within the oral cavity of healthy individuals, thereby supporting the site-specialist hypothesis. Additionally, it was observed that the gene pool of different species correlates with their ecological niches. These findings shed light on the significance of key metabolic functions in shaping microbial distribution patterns and interspecies interactions in the oral ecosystem.
Topics: Humans; Aggregatibacter; Phylogeny; Ecosystem; Haemophilus; Mouth
PubMed: 38488280
DOI: 10.1128/spectrum.04017-23 -
Molecular Microbiology Feb 2020Commensal bacteria serve as an important line of defense against colonisation by opportunisitic pathogens, but the underlying molecular mechanisms remain poorly...
Commensal bacteria serve as an important line of defense against colonisation by opportunisitic pathogens, but the underlying molecular mechanisms remain poorly explored. Here, we show that strains of a commensal bacterium, Haemophilus haemolyticus, make hemophilin, a heme-binding protein that inhibits growth of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) in culture. We purified the NTHi-inhibitory protein from H. haemolyticus and identified the hemophilin gene using proteomics and a gene knockout. An x-ray crystal structure of recombinant hemophilin shows that the protein does not belong to any of the known heme-binding protein folds, suggesting that it evolved independently. Biochemical characterisation shows that heme can be captured in the ferrous or ferric state, and with a variety of small heme-ligands bound, suggesting that hemophilin could function under a range of physiological conditions. Hemophilin knockout bacteria show a limited capacity to utilise free heme for growth. Our data suggest that hemophilin is a hemophore and that inhibition of NTHi occurs by heme starvation, raising the possibility that competition from hemophilin-producing H. haemolyticus could antagonise NTHi colonisation in the respiratory tract.
Topics: Bacterial Proteins; Haemophilus; Haemophilus Infections; Haemophilus influenzae; Heme; Heme-Binding Proteins; Humans
PubMed: 31742788
DOI: 10.1111/mmi.14426 -
Journal of Clinical Microbiology Dec 2016Haemophilus haemolyticus has been recently discovered to have the potential to cause invasive disease. It is closely related to nontypeable Haemophilus influenzae (NT H....
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.
Topics: Base Sequence; Comparative Genomic Hybridization; DNA, Bacterial; Genome, Bacterial; Haemophilus Infections; Haemophilus influenzae; Humans; Limit of Detection; Lipoproteins; Phosphoribosylglycinamide Formyltransferase; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity; Sequence Analysis, DNA
PubMed: 27707939
DOI: 10.1128/JCM.01511-16