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