Haemophilus influenzaepersists in biofilm communities in a smoke-exposed ferret model of COPD

Abstract

RationaleNon-typeable Haemophilus influenzae (NTHi) is a common inhabitant of the human nasopharynx and upper airways that can cause opportunistic infections of the airway mucosa including bronchopulmonary infections in patients with chronic obstructive pulmonary disease (COPD). It is clear that opportunistic infections contribute significantly to inflammatory exacerbations of COPD; however, there remains much to be learned regarding specific host and microbial determinants of persistence and/or clearance in this context.MethodsIn this study, we used a recently described ferret model for COPD, in which animals undergo chronic long-term exposure to cigarette smoke, to define host–pathogen interactions during COPD-related NTHi infections.ResultsNTHi bacteria colonised the lungs of smoke-exposed animals to a greater extent than controls, and elicited acute host inflammation and neutrophilic influx and activation, along with a significant increase in airway resistance and a decrease in inspiratory capacity consistent with inflammatory exacerbation; notably, these findings were not observed in air-exposed control animals. NTHi bacteria persisted within multicellular biofilm communities within the airway lumen, as evidenced by immunofluorescent detection of bacterial aggregates encased within a sialylated matrix as is typical of NTHi biofilms and differential bacterial gene expression consistent with the biofilm mode of growth.ConclusionsBased on these results, we conclude that acute infection with NTHi initiates inflammatory exacerbation of COPD disease. The data also support the widely held hypothesis that NTHi bacteria persist within multicellular biofilm communities in the lungs of patients with COPD.

The Role of Non-Typeable Haemophilus influenzae Biofilms in Chronic Obstructive Pulmonary Disease

“…Whilst these studies provide good evidence that NTHi has the capacity to form biofilms under nutrient-rich conditions, it does not confirm whether they actually form stable, persistent biofilms within the human COPD lung. Recently, however, the ferret COPD model, involving chronic long-term exposure to cigarette smoke, has provided immunofluorescent and gene expression evidence for NTHi biofilm formation and persistence ( Hunt et al., 2020 ). Here, smoke-exposed ferret lungs, which presented characteristic histological and immunological hallmarks of COPD, showed increased NTHi aggregation and the expression of four key biofilm-associated genes, pdgX, luxS, dps and hktE , that are together involved in growth, quorum sensing, environmental stress and oxidative stress tolerance ( Hunt et al., 2020 ).…”

NO donors and NO delivery methods for controlling biofilms in chronic lung infections

“…Nuclei were stained with DAPI (blue), and bacteria were stained with anti-NTHi polyclonal antibodies conjugated to Alexa 488 (green). Scale bar=50μm (Hunt et al 2020 ). Permissions to reuse images were obtained from journals and/or Copyright Clearance Centre.…”

“…H. influenzae is the most frequent microorganism isolated from a range of PCD patients (Alanin et al 2015 ), yet it was only confirmed in vitro that H. influenzae cells are more likely to form aggregates on the PCD epithelial cells than healthy control (Walker et al 2017 ). Biofilm growth mode has long been implicated in COPD despite the lack of direct evidence, and this hypothesis was only very recently supported by the detection of H. influenzae multicellular aggregates within the airway lumen of COPD ferret (Hunt et al 2020 ). Models of chronic lung infection including the implication of microbiota and biofilms have been set up, yet many more investigations are required to consolidate the biofilm growth mode in respiratory tracts of different patients.…”

Nontypeable Haemophilus influenzae redox recycling of protein thiols promotes resistance to oxidative killing and bacterial survival in biofilms in a smoke related infection model

“…RT-qPCR was performed using the Applied-Biosystems 7500 System, and oligonucleotide probes specific for pdgX, luxS, dps, hktE and omp26 (Table 2). The omp26 transcript was chosen as an endogenous control given that its expression does not vary between planktonic or biofilm mode of growth (44). The reaction mix used was the NEB Luna Universal following manufacturer's directions for cycling conditions (New England Biolabs, Ipswich, MA).…”

“…RT-qPCR was performed using the Applied-Biosystems 7500 System, and oligonucleotide probes specific for pdgX, luxS, dps, hktE and omp26 (Table 2). The omp26 transcript was chosen as an endogenous control given that its expression does not vary between planktonic or biofilm mode of growth (44) were normalized relative to omp26 levels from the same sample. Relative quantification of gene expression was determined using the comparative CT method (2 ΔΔCT ).…”