Geogenic particles induce bronchial susceptibility to non-typeable Haemophilus influenzae.

Exposure to geogenic (earth-derived) particulate matter (PM) is linked to an increased prevalence of bronchiectasis and other respiratory infections in Australian Indigenous communities. Experimental studies have shown that the concentration of iron in geogenic PM is associated with the magnitude of respiratory health effects, however, the mechanism is unclear. We investigated the effect of geogenic PM and iron oxide on the invasiveness of non-typeable Haemophilus influenzae (NTHi). Peripheral blood mononuclear cell-derived macrophages or epithelial cell lines (A549 & BEAS-2B) were exposed to whole geogenic PM, their primary constituents (haematite, magnetite or silica) or diesel exhaust particles (DEP). The uptake of bacteria was quantified by flow cytometry and whole genome sequencing (WGS) was performed on NTHi strains. Geogenic PM increased the invasiveness of NTHi in bronchial epithelial cells. Of the primary constituents, haematite also increased NTHi invasion with magnetite and silica having significantly less impact. Furthermore, we observed varying levels of invasiveness amongst NTHi isolates. WGS analysis suggested isolates with more genes associated with heme acquisition were more virulent in BEAS-2B cells. The present study suggests that geogenic particles can increase the susceptibility of bronchial epithelial cells to select bacterial pathogens in vitro, a response primarily driven by haematite content in the dust. This demonstrates a potential mechanism linking exposure to iron-laden geogenic PM and high rates of chronic respiratory infections in remote communities in arid environments.

Use of trans-complementation method to determine the effects of various ftsI mutations on ß-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae strains.

Haemophilus influenzae is a causative agent of serious infections, especially among children. ß-lactam antibiotics are commonly used for the treatment of these infections. Among H. influenzae isolates, ß-lactam resistance is due to the presence of ß-lactamase, or to mutations in the ftsI gene that generate altered PBP3 (penicillin-binding protein 3) with reduced affinity for ß-lactams (BLNAR-ß-lactamase-negative, ampicillin-resistant). Wild-type ftsI gene encoding for PBP3 was amplified in whole from ß-lactam susceptible H. influenzae Rd and cloned in pLS88 plasmid to obtain pADUTAS17, which was then used to transform known BLNAR strains, susceptible strains, and a strain (CF55) with wild-type ftsI but unexplained reduced ß-lactam susceptibility. Ampicillin and cefotaxime MICs (minimum inhibitory concentration) were determined after transformation with pLS88 and pADUTAS17 plasmids. The results showed that antibiotic susceptibilities were not affected by trans-complementation for isolates carrying wild-type ftsI gene. However, trans-complementation for all BLNAR strains showed decreases between - 0.957 and 0.5-fold for ampicillin and cefotaxime, confirming the role of the PBP3 substitutions in the BLNAR phenotype of these isolates. The first article showed that trans-complementation might be a useful tool in the investigation of decreased ß-lactam susceptibility in H. influenzae.

A heme-binding protein produced by Haemophilus haemolyticus inhibits non-typeable Haemophilus influenzae.

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.

New therapeutic targets for the prevention of infectious acute exacerbations of COPD: role of epithelial adhesion molecules and inflammatory pathways.

Chronic respiratory diseases are among the leading causes of mortality worldwide, with the major contributor, chronic obstructive pulmonary disease (COPD) accounting for approximately 3 million deaths annually. Frequent acute exacerbations (AEs) of COPD (AECOPD) drive clinical and functional decline in COPD and are associated with accelerated loss of lung function, increased mortality, decreased health-related quality of life and significant economic costs. Infections with a small subgroup of pathogens precipitate the majority of AEs and consequently constitute a significant comorbidity in COPD. However, current pharmacological interventions are ineffective in preventing infectious exacerbations and their treatment is compromised by the rapid development of antibiotic resistance. Thus, alternative preventative therapies need to be considered. Pathogen adherence to the pulmonary epithelium through host receptors is the prerequisite step for invasion and subsequent infection of surrounding structures. Thus, disruption of bacterial-host cell interactions with receptor antagonists or modulation of the ensuing inflammatory profile present attractive avenues for therapeutic development. This review explores key mediators of pathogen-host interactions that may offer new therapeutic targets with the potential to prevent viral/bacterial-mediated AECOPD. There are several conceptual and methodological hurdles hampering the development of new therapies that require further research and resolution.

A human origin strain Lactobacillus acidophilus DDS-1 exhibits superior in vitro probiotic efficacy in comparison to plant or dairy origin probiotics.

Background: The health benefits of probiotics are well established and known to be strain-specific. However, the role of probiotics obtained from different origins and their efficacy largely remains unexplored. The aim of this study is to investigate the in vitro efficacy of probiotics from different origins. Methods: Probiotic strains utilized in this study include Lactobacillus acidophilus DDS-1 (human origin), Bifidobacterium animalis ssp. lactis UABla-12 (human origin), L. plantarum UALp-05 (plant origin) and Streptococcus thermophilus UASt-09 (dairy origin). Screening assays such as in vitro digestion simulation, adhesion, cell viability and cytokine release were used to evaluate the probiotic potential. Results: All strains showed good resistance in the digestion simulation process, especially DDS-1 and UALp-05, which survived up to a range of 107 to 108 CFU/mL from an initial concentration of 109 CFU/mL. Two human colonic mucus-secreting cells, HT-29 and LS174T, were used to assess the adhesion capacity, cytotoxicity/viability, and cytokine quantification. All strains exhibited good adhesion capacity. No significant cellular cytotoxicity or loss in cell viability was observed. DDS-1 and UALp-05 significantly upregulated anti-inflammatory IL-10 and downregulated pro-inflammatory TNF-α cytokine production. All the strains were able to downregulate IL-8 cytokine levels. Conclusion: Of the 4 strains tested, DDS-1 demonstrated superior survival rates, good adhesion capacity and strong immunomodulatory effect under different experimental conditions.

Expression of acquired macrolide resistance genes in Haemophilus influenzae.

OBJECTIVES: To investigate the phenotypic effect of expression of selected acquired macrolide resistance genes (AMRGs) in non-typeable Haemophilus influenzae (NTHi). METHODS: The AMRGs erm(A), erm(B) and erm(C) were cloned into Escherichia coli JM109 using the shuttle vector pLS88; constructed plasmids extracted from suitable clones were used to transform H. influenzae Rd by electroporation. Erythromycin and azithromycin MICs for suitable transformants were determined by broth microdilution. AMRG expression was determined using quantitative PCR on transformant cDNA with locked nucleic acid dual-labelled hydrolysis probes. RESULTS: Expression of all AMRGs was observed in the transformants. Some variation in expression between the AMRGs was apparent, but expression of all genes was associated with a notable increase in erythromycin and azithromycin MICs compared with untransformed H. influenzae Rd. CONCLUSIONS: While the establishment of erm genes within WT populations of NTHi remains contentious, H. influenzae is capable of expression of erm. Expression may be associated with a subsequent decreased susceptibility to macrolides in isolates and future monitoring of these genes in NTHi isolates is warranted.

Reliability of Haemophilus influenzae biofilm measurement via static method, and determinants of in vitro biofilm production.

Information is lacking regarding the precision of microtitre plate (MTP) assays used to measure biofilm. This study investigated the precision of an MTP assay to measure biofilm production by nontypeable Haemophilus influenzae (NTHi) and the effects of frozen storage and inoculation technique on biofilm production. The density of bacterial final growth was determined by absorbance after 18-20 h incubation, and biofilm production was then measured by absorbance after crystal violet staining. Biofilm formation was categorised as high and low for each strain. For the high biofilm producing strains of NTHi, interday reproducibility of NTHi biofilm formation measured using the MTP assay was excellent and met the acceptance criteria, but higher variability was observed in low biofilm producers. Method of inoculum preparation was a determinant of biofilm formation with inoculum prepared directly from solid media showing increased biofilm production for at least one of the high producing strains. In general, storage of NTHi cultures at -80 °C for up to 48 weeks did not have any major effect on their ability to produce biofilm.

Interspecies transfer of the penicillin-binding protein 3-encoding gene ftsI between Haemophilus influenzae and Haemophilus haemolyticus can confer reduced susceptibility to ß-lactam antimicrobial agents.

Mutations in ftsI, encoding penicillin-binding protein 3, can cause decreased ß-lactam susceptibility in Haemophilus influenzae. Sequencing of ftsI from clinical strains has indicated interspecies recombination of ftsI between H. influenzae and Haemophilus haemolyticus. This study documented apparently unrestricted homologous recombination of ftsI between H. influenzae and H. haemolyticus in vitro. Transfer of ftsI from resistant isolates conferred similar but not identical increases in the MICs of susceptible strains of H. influenzae and H. haemolyticus.

Acquired macrolide resistance genes in Haemophilus influenzae?

OBJECTIVES: The objective of this study was to determine the prevalence of specific acquired macrolide resistance genes previously reported as present in clinical isolates of Haemophilus influenzae. METHODS: A collection of 172 clinical respiratory isolates of H. influenzae, including 59 isolates from cystic fibrosis patients and 27 from non-cystic fibrosis bronchiectasis patients with significant prior macrolide use, was established. This collection was tested for azithromycin susceptibility using Etest and screened for the presence of erm(A), erm(B), erm(C), erm(F), mef(A) and mef(E) using locked nucleic acid dual-labelled hydrolysis probes. RESULTS: The azithromycin MICs ranged from 0.09 to >256 mg/L, with 2 (1.2%) isolates susceptible, 163 (94.8%) intermediate and 7 (4%) resistant according to EUCAST breakpoints (susceptible, ≤0.12 mg/L; resistant, >4 mg/L). None of the acquired macrolide resistance genes erm(A), erm(B), erm(C), erm(F), mef(A) or mef(E) was detected in any of the isolates. CONCLUSIONS: The specific acquired macrolide resistance genes are not widespread in H. influenzae and the high prevalence of these genes previously reported might be unique to the specific circumstances of that study.

Relationship between clinical site of isolation and ability to form biofilms in vitro in nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen associated with a range of infections, including various lower respiratory infections, otitis media, and conjunctivitis. There is some debate as to whether or not NTHi produces biofilms and, if so, whether or not this is relevant to pathogenesis. Although many studies have examined the association between in vitro biofilm formation and isolates from a specific infection type, few have made comparisons from isolates from a broad range of isolates grouped by clinical source. In our study 50 NTHi from different clinical sources, otitis media, conjunctivitis, lower respiratory tract infections in both cystic fibrosis and non-cystic fibrosis patients, and nasopharyngeal carriage, plus 10 nasopharyngeal isolates of the commensal Haemophilus haemolyticus were tested for the ability to form biofilm by using a static microtitre plate crystal violet assay. A high degree of variation in biofilm forming ability was observed across all isolates, with no statistically significant differences observed between the groups, with the exception of the isolates from conjunctivitis. These isolates had uniformly lower biofilm forming ability compared with isolates from the other groups (p < 0.005).

Role of inter-species recombination of the ftsI gene in the dissemination of altered penicillin-binding-protein-3-mediated resistance in Haemophilus influenzae and Haemophilus haemolyticus.

OBJECTIVES: To screen the ftsI gene sequences obtained from clinical isolates of non-typeable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus for the presence of mosaic ftsI gene structures, and to evaluate the role of inter-species recombination of the ftsI gene in the formation and distribution of resistant ftsI genes. METHODS: The ftsI genes of 100 Haemophilus isolates comprising genetically defined ß-lactamase-negative ampicillin-susceptible (gBLNAS), ß-lactamase-positive ampicillin-resistant (gBLPAR), ß-lactamase-negative ampicillin-resistant (gBLNAR) and ß-lactamase-positive amoxicillin/clavulanate-resistant (gBLPACR) isolates of NTHi (n = 50) and H. haemolyticus (n = 50) were analysed in this study. Both the flanking regions and the full-length ftsI gene sequences of all study isolates were screened for mosaic structures using H. influenzae Rd and H. haemolyticus ATCC 33390 as reference parental sequences, and bioinformatics methods were used for recombination analysis using SimPlot. RESULTS: Of the 100 clinical isolates analysed 34% (34/100) harboured mosaic ftsI gene structures containing distinct ftsI gene fragments similar to both reference parental sequences. The inter-species recombination events were exclusively encountered in the ftsI gene of gBLNAR/gBLPACR isolates of both NTHi and H. haemolyticus, and were always associated with the formation of a mosaic fragment at the 3' end of the ftsI gene. There was no evidence supporting horizontal gene transfer (HGT) involving the entire ftsI gene among the clinical isolates in vivo. CONCLUSIONS: We provide evidence for the HGT and inter-species recombination of the ftsI gene among gBLNAR/gBLPACR isolates of NTHi and H. haemolyticus in a clinical setting, highlighting the importance of recombination of the ftsI gene in the emergence of altered penicillin-binding protein 3 and BLNAR-mediated resistance.

Heme sequestration by hemophilin from Haemophilus haemolyticus reduces respiratory tract colonization and infection with non-typeable Haemophilus influenzae.

Iron acquisition is a key feature dictating the success of pathogen colonization and infection. Pathogens scavenging iron from the host must contend with other members of the microbiome similarly competing for the limited pool of bioavailable iron, often in the form of heme. In this study, we identify a beneficial role for the heme-binding protein hemophilin (Hpl) produced by the non-pathogenic bacterium Haemophilus haemolyticus against its close relative, the opportunistic respiratory tract pathogen non-typeable Haemophilus influenzae (NTHi). Using a mouse model, we found that pre-exposure to H. haemolyticus significantly reduced NTHi colonization of the upper airway and impaired NTHi infection of the lungs in an Hpl-dependent manner. Further, treatment with recombinant Hpl was sufficient to decrease airway burdens of NTHi without exacerbating lung immunopathology or systemic inflammation. Instead, mucosal production of the neutrophil chemokine CXCL2, lung myeloperoxidase, and serum pro-inflammatory cytokines IL-6 and TNFα were lower in Hpl-treated mice. Mechanistically, H. haemolyticus suppressed NTHi growth and adherence to human respiratory tract epithelial cells through the expression of Hpl, and recombinant Hpl could recapitulate these effects. Together, these findings indicate that heme sequestration by non-pathogenic, Hpl-producing H. haemolyticus is protective against NTHi colonization and infection. IMPORTANCE: The microbiome provides a critical layer of protection against infection with bacterial pathogens. This protection is accomplished through a variety of mechanisms, including interference with pathogen growth and adherence to host cells. In terms of immune defense, another way to prevent pathogens from establishing infections is by limiting the availability of nutrients, referred to as nutritional immunity. Restricting pathogen access to iron is a central component of this approach. Here, we uncovered an example where these two strategies intersect to impede infection with the respiratory tract bacterial pathogen Haemophilus influenzae. Specifically, we find that a non-pathogenic (commensal) bacterium closely related to H. influenzae called Haemophilus haemolyticus improves protection against H. influenzae by limiting the ability of this pathogen to access iron. These findings suggest that beneficial members of the microbiome improve protection against pathogen infection by effectively contributing to host nutritional immunity.

Prevalence and mechanisms of ß-lactam resistance in Haemophilus haemolyticus.

OBJECTIVES: To compare the phenotypic and genotypic ß-lactam resistance profiles of non-typeable Haemophilus influenzae (NTHi) and the closely phylogenetically related Haemophilus haemolyticus. METHODS: XV-dependent Haemophilus species isolated as normal flora from nasopharyngeal and throat swabs (n = 312) were screened by PCR for markers to determine NTHi and H. haemolyticus identity. All NTHi and H. haemolyticus isolates were subsequently tested for susceptibilities to ampicillin and amoxicillin/clavulanate, and characterized with respect to the presence of blaTEM, blaROB and ftsI gene mutations. RESULTS: Of the 312 isolates, 236 (75%) were identified as NTHi, 61 (20%) as H. haemolyticus and 15 (5%) as equivocal. PCR for resistance genes showed 15.7% (37/236) of NTHi and 13.1% (8/61) of H. haemolyticus isolates were blaTEM positive and none was positive for blaROB. The blaTEM genes of both species were encoded on similar replicons and associated with the same promoter types. Altered penicillin-binding protein 3 due to the N526K substitution accounted for 31% of both NTHi (73/236) and H. haemolyticus (19/61) isolates, respectively. The presence of N526K in both NTHi and H. haemolyticus was associated with slightly raised ampicillin MICs compared with the H. influenzae Rd and H. haemolyticus ATCC 33390 control strains. In addition, some NTHi gBLNAR-associated substitutions were seen in H. haemolyticus with and without N526K, and appear to represent part of the baseline genotype of that species. CONCLUSIONS: The phenotypic and genotypic ß-lactam resistance in NTHi and H. haemolyticus is very similar, such that H. haemolyticus may represent a reservoir for ß-lactam resistance determinants for NTHi.

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

OBJECTIVES: 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: A total 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 ß-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 Hasegawa et 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.

Sequences of small blaTEM-encoding plasmids in Haemophilus influenzae and description of variants falsely negative for blaTEM by PCR.

OBJECTIVES: To characterize an unidentified ß-lactamase and associated genetic background in a bla(TEM) and bla(ROB) PCR-negative Haemophilus influenzae isolate, and characterize small bla(TEM)-encoding plasmids in a collection of H. influenzae. METHODS: The unidentified ß-lactamase gene was identified by cloning and sequencing the encoding plasmid. Strains with small bla(TEM) plasmids were identified using negative PCR for integrative conjugative elements, but positive bla(TEM) PCR; plasmids from selected isolates were sequenced. PCR for rep and divergent bla(TEM) were evaluated for detecting small plasmids on selected H. influenzae isolates. RESULTS: Small plasmids (4.8-5.5 kb) encoding bla(TEM) appear to be associated with remnants of Tn2 on a conserved plasmid core. The unidentified ß-lactamase was actually a TEM-1, with negative bla(TEM) PCR associated with a previously unrecognized deletion of bp 1-27 of bla(TEM) (Sutcliffe numbering) associated with a larger deletion within Tn2. This deletion was found in other isolates and may be more common than previously thought. PCR for the conserved rep gene appears useful for screening for small bla(TEM)-encoding plasmids or associated cryptic plasmids in H. influenzae. CONCLUSIONS: Small bla(TEM)-encoding plasmids in H. influenzae appear relatively conserved, but require further study to confirm this. PCR associated with the rep gene may be useful for studying these small plasmids. A deletion in part of bla(TEM) in some strains may interfere with some PCRs; therefore, care should be taken with primer selection or design and, preferably, regions within the open reading frame should be targeted.

An evaluation of SNP-based PCR methods for the detection of ß-lactamase-negative ampicillin-resistant Haemophilus influenzae.

Forty-four previously characterized strains of Haemophilus influenzae were used to evaluate the specificity of previously published SNP PCR primers for the detection of the N526K substitution in PBP3 of BLNAR isolates using real-time PCR. Hasegawa et al. primers that amplify strains without a substitution at 526 and fail to amplify strains with N526K were 100% sensitive and specific for detecting N526K. However, primer sets of Hasegawa et al. and Nakamura et al. designed to amplify strains with N526K, but not strains without a substitution, were unable to do this reliably because the primers were specific for N526K encoded by AAG and failed to amplify strains with N526K encoded by AAA. A review of N526K strains deposited on GenBank revealed an even distribution of AAG and AAA codons for N526K in European and Australian BLNAR strains, whereas only the AAG codon was seen in Japanese strains. The exclusive presence of the AAG codon in Japanese strains appears to be independent of the use of the SNP PCR primers evaluated here and remains unexplained.

Prevalence and genotypic characteristics of beta-lactamase-negative ampicillin-resistant Haemophilus influenzae in Australia.

OBJECTIVES: To determine the prevalence of ß-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae in Australia and characterize the associated amino acid substitutions in penicillin-binding protein 3. METHODS: Two hundred consecutive non-repeat clinical isolates of H. influenzae were collected and ß-lactamase-negative isolates were screened for reduced ampicillin susceptibility using an ampicillin 2 µg disc (breakpoint <17 mm) and Etest (breakpoint ≥0.25 mg/L). All screen-positive isolates had their ampicillin MICs determined by reference broth microdilution and their ftsI genes were sequenced. RESULTS: No BLNAR strains (MIC ≥4 mg/L) were found, but 5 (2.5%) low BLNAR (L-BLNAR) strains (MIC ≥2 mg/L) and 36 (18%) genetic BLNAR (gBLNAR) strains (R517H or N526K) were found. Of the gBLNAR strains, four had the R517H substitution and the remainder had N526K, while no strains had combined N526K and M377I/S385T/L389F substitutions. A number of strains with neither R517H nor N526K substitutions that did not meet the gBLNAR definition had other BLNAR-associated substitutions. CONCLUSIONS: BLNAR and L-BLNAR strains are uncommon in Australia, while gBLNAR strains are more common than previously recognized.

Haemophilin-Producing Strains of Haemophilus haemolyticus Protect Respiratory Epithelia from NTHi Colonisation and Internalisation.

Nontypeable Haemophilus influenzae (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, Haemophilus haemolyticus (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 hpl 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.

Oropharyngeal Carriage of hpl-Containing Haemophilus haemolyticus Predicts Lower Prevalence and Density of NTHi Colonisation in Healthy Adults.

Nontypeable Haemophilus influenzae (NTHi) is a major respiratory pathogen that initiates infection by colonising the upper airways. Strategies that interfere with this interaction may therefore have a clinically significant impact on the ability of NTHi to cause disease. We have previously shown that strains of the commensal bacterium Haemophilus haemolyticus (Hh) that produce a novel haem-binding protein, haemophilin, can prevent NTHi growth and interactions with host cells in vitro. We hypothesized that natural pharyngeal carriage of Hh strains with the hpl open reading frame (Hh-hpl+) would be associated with a lower prevalence and/or density of NTHi colonisation in healthy individuals. Oropharyngeal swabs were collected from 257 healthy adults in Australia between 2018 and 2019. Real-time PCR was used to quantitatively compare the oropharyngeal carriage load of NTHi and Hh populations with the Hh-hpl+ or Hh-hpl- genotype. The likelihood of acquiring/maintaining NTHi colonisation status over a two- to six-month period was assessed in individuals that carried either Hh-hpl- (n = 25) or Hh-hpl+ (n = 25). Compared to carriage of Hh-hpl- strains, adult (18-65 years) and elderly (>65 years) participants that were colonised with Hh-hpl+ were 2.43 or 2.67 times less likely to carry NTHi in their oropharynx, respectively. Colonisation with high densities of Hh-hpl+ correlated with a low NTHi carriage load and a 2.63 times lower likelihood of acquiring/maintaining NTHi colonisation status between visits. Together with supporting in vitro studies, these results encourage further investigation into the potential use of Hh-hpl+ as a respiratory probiotic candidate for the prevention of NTHi infection.