Biofilm aggregates
NTHi can form biofilms (Figure-2), which are aggregated bacterial communities encased in an ECM of polysaccharides, proteins and extracellular DNA (eDNA) that adhere to a surface.92As biofilms mature, the resident bacteria undergo a series of behavioral changes, including differential gene expression and increased ECM production, in response to increasing cell density and environmental stresses from decreasing oxygen tension and nutrient availability.93 Crucial bacterial factors for biofilm formation include LOS, pili, eDNA, and a functioning quorum sensing system (QSS). Bacteria utilize QSS for inter-cellular communication where cells collectively regulate their gene expression in response to cell density as sensed by the concentration of small soluble autoinducer signal molecules produced and secreted by bacteria.94In NTHi , biofilm development and dispersal are mediated by autoinducer-2 (AI-2), which is controlled by the LuxS/RbsB system regulating LOS and pili expression, including PilA, P2 and P6. DNA-NET-like structures captured by DNA-binding proteins from the DNABII family form a mesh network within biofilms where they help make up important structural components.95
The ECM of biofilms helps protect NTHi from environmental stresses, including acting as a physiochemical barrier to cellular and innate host defenses and to antimicrobial agents. For example, in addition to stimulating biofilm formation, eDNA binds the AMP, human beta-defensin-3, while peroxiredoxin-glutaredoxin and catalase protect against neutrophil-induced oxidative stress, and IgA proteases cleaving IgA are found on the ECM.96 There are several potential mechanisms within biofilms capable of mediating antimicrobial resistance. These include the (i) ECM limiting antibiotic access to bacteria within the biofilm, (ii) negatively charged eDNA sequestering positively charged antibiotics, (iii) hypoxic and nutrient restricted environment deep within biofilms transforming bacteria into a semi-dormant, metabolically inert state rendering antibiotics relying upon active cell growth and division ineffective, and (iv) horizontal gene transfer of antibiotic resistance genes that result in deactivating enzymes, altered membrane permeability and cellular targets, and upregulated multidrug efflux pumps.97 Nevertheless, despite much in-vitro and experimental model work on biofilms, evidence for their presence in patients with CSLD is relatively limited. They have however, been detected in the sputum of adults with CF and COPD,98,99 and in bronchoalveolar lavage (BAL) fluid from children with PBB and bronchiectasis.100Importantly, by using fluorescent in-situ hybridization (FISH) staining in a small subset of BAL samples, researchers were able to demonstrate the polymicrobial nature of the biofilms, which included in addition toNTHi other recognised respiratory pathogens (Streptococcus pneumoniae, M. catarrhalis ) and upper airway commensals, such asPrevotella species.100