Table 4.

Summary of Clinical Evidence for NTHi Biofilms (ex-vivo).

Evidence	Model/Experiment	Experimental Effects	References
Biofilm promoting adhesin genes	PCR analysis of 108 clinical NTHi strains for adhesin genes correlated with biofilm formatting in-vitro	Variability in the presence of key adhesin genes including hifA (22%), hmw (48%), hia (57%), hap (22%) and siaB (38%), of which hemagglutinating pili and Hia were significantly associated with increased biofilm forming capacity. Biofilm forming strains of NTHi were significantly more likely to be identified in patients with chronic (90%) rather than acute (63%) respiratory infections.	(Nishi et al., 2006)
TEM showing biofilm adhesion to cell surfaces	Ex-vivo BALF imaged using TEM	Analysis of BAL from CF patients has provided good evidence of NTHi biofilm attached to cell surfaces, using transition election microscopy techniques These clinical NTHi isolates also formed mature biofilms on cultured airway epithelial cells, showing microcolony formation and EPS production.	(Starner et al., 2006)
Biofilm NTHi isolates	61 patients hospitalised with lower respiratory tract infection patients. NTHi isolated and grown in-vitro	Claimed to have an association with NTHi biofilm formation and increased hospitalisation duration. However, the evidence of biofilm production was poor, with only 10% actually forming biofilms, the majority of which (80%) were weak biofilm producers with no strong biofilms produced. Additionally, biofilms were no more significantly resistant to antimicrobials than non-biofilms so did not display a major hallmark of biofilm formation.	(Martinez-Resendez et al., 2016)
Biofilm NTHi isolates	Sarcoidosis patient sputum samples	Haemophilus spp. was identified in both 31/37 healthy and 30/31 sarcoidosis patients, 67% of H. influenzae isolates formed biofilm, all of which were weak using the standard crystal violet assay and classification.	(Kosikowska et al., 2016)
Immune response to biofilm strategies	6 COPD patient derived NTHi isolates and 18 COPD patients for ELISA analysis	COPD patient derived NTHi strains express greater pdgx under biofilm than planktonic growth conditions. ELISA detects pdgx antibodies in 44.4% COPD patients’ respiratory tract by ELISA showing host immune response to biofilm-survival strategies.	(Murphy et al., 2005)