Microenvironmental characteristics and physiology of biofilms in chronic infections of CF patients are strongly affected by the host immune response

Abstract

Jensen PØ, Kolpen M, Kragh KN, K€ uhl M. Microenvironmental characteristics and physiology of biofilms in chronic infections of CF patients are strongly affected by the host immune response. APMIS 2017; 125: 276-288.In vitro studies of Pseudomonas aeruginosa and other pathogenic bacteria in biofilm aggregates have yielded detailed insight into their potential growth modes and metabolic flexibility under exposure to gradients of substrate and electron acceptor. However, the growth pattern of P. aeruginosa in chronic lung infections of cystic fibrosis (CF) patients is very different from what is observed in vitro, for example, in biofilms grown in flow chambers. Dense in vitro biofilms of P. aeruginosa exhibit rapid O 2 depletion within <50-100 lm due to their own aerobic metabolism. In contrast, in vivo investigations show that P. aeruginosa persists in the chronically infected CF lung as relatively small cell aggregates that are surrounded by numerous PMNs, where the activity of PMNs is the major cause of O 2 depletion rendering the P. aeruginosa aggregates anoxic. High levels of nitrate and nitrite enable P. aeruginosa to persist fueled by denitrification in the PMN-surrounded biofilm aggregates. This configuration creates a potentially long-term stable ecological niche for P. aeruginosa in the CF lung, which is largely governed by slow growth and anaerobic metabolism and enables persistence and resilience of this pathogen even under the recurring aggressive antimicrobial treatments of CF patients. As similar slow growth of other CF pathogens has recently been observed in endobronchial secretions, there is now a clear need for better in vitro models that simulate such in vivo growth patterns and anoxic microenvironments in order to help unravel the efficiency of existing or new antimicrobials targeting anaerobic metabolism in P. aeruginosa and other CF pathogens. We also advocate that host immune responses such as PMN-driven O 2 depletion play a central role in the formation of anoxic microniches governing bacterial persistence in other chronic infections such as chronic wounds.

Metabolic flux fingerprinting differentiates planktonic and biofilm states ofPseudomonas aeruginosaandStaphylococcus aureus

“…P. aeruginosa is able to grow anaerobically with high biomass yield on NO3 - (Strohm et al, 2007;Line et al, 2014) and further on arginine and pyruvate fermentation (Eschbach et al, 2004;Schreiber et al, 2006) and reduction-oxidation reactions of self-produced phenazines (Price-Whelan et al, 2007). This supports the current setup for studying clinical biofilm where multiple studies have showed that bacterial metabolism is halted by electron acceptor availability (Kragh et al, 2014;Jensen et al, 2017) rather than carbon source.…”

The O2-independent pathway of ubiquinone biosynthesis is essential for denitrification inPseudomonas aeruginosa

“…Although its contribution is still poorly understood in the host, anaerobic respiration of P. aeruginosa is likely significant to promote virulence mechanisms in chronic lung infections (13). Indeed, according to O2 assays directly in the lungs of CF patients, the infected endobronchial mucus is subject to severe hypoxia or even anoxia (31).…”

“…First, it may be due to the biofilm structure per se may establish an oxygen gradient across. However, this hypothesis is currently questioned due to the strong morphological differences between in vivo and in vitro biofilm production, the last being much more thicker than the first, for example (13). The second hypothesis is that accelerated O2 consumption in the biofilm may result from activated polymorphonuclear leukocytes that produce superoxide (32) and to a lesser extend nitric oxide (33).…”

“…This process called denitrification allows the reduction of soluble nitrate (NO3 -) and nitrite (NO2 -) to gaseous nitrous oxide (N2O) or molecular nitrogen (N2) (12). Because P. aeruginosa-infected mucus in CF airway is depleted of oxygen and enriched in nitrate/nitrite, the anaerobic metabolism of P. aeruginosa via the denitrification pathway is believed to be important for its pathogenicity (13). Four sequential reactions involving metalloenzymes are needed to reduce nitrate to N2, i.e.…”

Oxygen restriction generates difficult-to-culture pathogens

“…In addition, other cells of the host may also consume O 2 , increasing the likelihood of bacteria experiencing anoxia (Worlitzsch et al, 2002). Many chronic infections are thought to contain bacteria in the biofilm mode of growth (Costerton et al, 2003) and endogenous O 2 depletion inside the biofilm (Sønderholm et al, 2018), along with intense O 2 consumption by the host immune response (Worlitzsch et al, 2002;Kolpen et al, 2010;Trunk et al, 2010;Kragh et al, 2014;James et al, 2016;Jensen et al, 2017), may therefore increase the number of VBNC bacteria.…”