My study focuses on Non-typeable Haemophilus Influenzae (NTHi) and Corynebacterium spp, both of which reside in the human upper respiratory tract (URT).

NTHi is a pathobiont that naturally inhabits our respiratory tract, however, upon perturbation it has been shown to cause a variety of lung diseases such as Chronic Obstructive Pulmonary Disease (COPD) and Cystic Fibrosis. It has also been identified by WHO as a priority pathogen for the development of new treatments due to increasing evidence of antimicrobial resistance. Corynebacterium spp, a commensal species, has previously been shown to have antimicrobial activity against two other key pathobionts that also reside in the URT; Staphylococcus aureus and Streptococcus pneumoniae. However, it’s interactions with NTHi is still yet to be investigated.

The primary objective of my PhD project is to investigate the interplay between Corynebacterium spp and NTHi, and determine if Corynebacterium spp has potential to be used as a future bacteriotherapy for NTHi infection.

Using the duet system, different strains belonging to both species will first be assessed for planktonic growth through absorbance measurements (OD600) over 24 hours. Following this, samples will be taken from the wells for analysis with H1-NMR to determine changes in metabolic profiles and identify key metabolites or by-products that may play an important role in regulating NTHi growth. Biofilm formation will then be assessed through crystal violet staining to evaluate changes in biofilm biomass.

Co-culture experiments are typically performed for 24 – 48 hours.

Three 24-well co-culture plates are typically used to screen the effect of a single C. accolens strain against six different NTHi strains, including controls.

Using the Duet system I’m aiming to evaluate the impact of Corynebacterium spp on NTHi planktonic growth rate over 24 hours (OD600 readings), metabolic activity (sampling of media from the duet wells at 24 hours for NMR analysis), and biofilm formation (crystal violet staining of biofilm formed on the surface of the duet wells). These assays will complement other conventional competition assays to establish full metabolic reference spectra for NTHi andCorynebacterium spp during co-culture.

This will determine whether there is mutualism or competition in metabolite consumption between NTHi and Corynebacterium spp, and also whether there are any by-products that impact growth. In addition to this, I will also assess the effect of C. accolens on NTHi colonisation of respiratory epithelial cells using an advanced co-culture model.

Conventional co-culture assays that are being performed in this project are focusing on evaluating the effect of spent media from Corynebacterium spp on NTHi (absent Corynebacterium spp cells), and also the effect of direct interaction of both species during co-culture. The Duet co-culture system provides us with an exciting opportunity to complement these experiments by being able to grow both species at the same time with physical separation, thereby removing the temporal and spatial limitations imposed by the other systems, potentially identifying time-dependent effects relating to their metabolism.