Abstract
Nontypeable Haemophilus influenzae (NTHi) is a common airway commensal and opportunistic pathogen that persists within surface-attached biofilm communities. In this study, we tested the hypothesis that bacterial stress-responses are activated within biofilms. Transcripts for several factors associated with bacterial resistance to environmental stress were increased in biofilm cultures as compared to planktonic cultures. Among these, a homolog of the DNA-binding protein from starved cells (dps) was chosen for further study. An isogenic NTHi 86-028NP dps mutant was generated and tested for resistance to environmental stress, revealing a significant survival defects in high-iron conditions, which was mediated by oxidative stress and was restored by genetic complementation. As expected, NTHi 86-028NP dps had a general stress-response defect, exhibiting decreased resistance to many types of environmental stress. While no differences were observed in density and structure of NTHi 86-028NP and NTHi 86-028NP dps biofilms, bacterial survival was decreased in NTHi 86-028NP dps biofilms as compared to the parental strain. The role of dps persistence in vivo was tested in animal infection studies. NTHi 86-028NP dps had decreased resistance to clearance after pulmonary infection of elastase-treated mice as compared to NTHi 86-028NP, whereas minimal differences were observed in clearance from mock-treated mice. Similarly, lower numbers of NTHi 86-028NP dps were recovered from middle-ear effusions and bullar homogenates in the chinchilla model for otitis media (OM). Therefore, we conclude that Dps promotes bacterial survival within NTHi biofilm communities both in vitro and in chronic infections in vivo.
Highlights
Nontypeable Haemophilus influenzae (NTHi) is a nearly universal nasopharyngeal commensal that can cause opportunistic airway infections, including bronchopulmonary infections in patients with chronic obstructive pulmonary disease (COPD) and otitis media (OM) and sinusitis infections in children
It is known that biofilms can resist killing by some microbicidal factors due to decreased permeation of the biofilm due to the inherent diffusion barrier provided by the biofilm matrix (Anderl et al, 2000)
Additional evidence supports the conclusion that anaerobicity, nutrient limitation, or other environmental conditions within biofilms lead to metabolic resistance of bacteria within a biofilm (Lewis, 2001; Anderl et al, 2003; Kim et al, 2009)
Summary
Nontypeable Haemophilus influenzae (NTHi) is a nearly universal nasopharyngeal commensal that can cause opportunistic airway infections, including bronchopulmonary infections in patients with chronic obstructive pulmonary disease (COPD) and otitis media (OM) and sinusitis infections in children. Persistent infections caused by NTHi and other mucosal opportunists involve the formation of biofilm communities that enhance bacterial resistance to clearance (Swords et al, 2004; Hong et al, 2007a,b, 2009). An NTHi 86-028NP dps null mutant was generated, and shown to have increased susceptibility to environmental stress, consistent with prior work with other bacterial species This mutant was compared with the parental strain using in vitro biofilm assays and in animal infection models for COPD-related infections and OM. The results clearly show that Dps promotes survival of NTHi 86-028NP within biofilm communities, as well as resistance to host clearance in vivo These data indicate that the NTHi stress-response may promote survival during chronic infection, and may be an important target for antimicrobial therapy during chronic infections
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