Compromised Host Defense on Pseudomonas aeruginosa Biofilms: Characterization of Neutrophil and Biofilm Interactions

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms on tissues and other surfaces. We characterized the interaction of purified human neutrophils with P. aeruginosa, growing in biofilms, with regard to morphology, oxygen consumption, phagocytosis, and degranulation. Scanning electron and confocal laser microscopy indicated that the neutrophils retained a round, unpolarized, unstimulated morphology when exposed to P. aeruginosa PAO1 biofilms. However, transmission electron microscopy demonstrated that neutrophils, although rounded on their dorsal side, were phagocytically active with moderate membrane rearrangement on their bacteria-adjacent surfaces. The settled neutrophils lacked pseudopodia, were impaired in motility, and were enveloped by a cloud of planktonic bacteria released from the biofilms. The oxygen consumption of the biofilm/neutrophil system increased 6- and 8-fold over that of the biofilm alone or unstimulated neutrophils in suspension, respectively. H2O2 accumulation was transient, reaching a maximal measured value of 1 μM. Following contact, stimulated degranulation was 20–40% (myeloperoxidase, β-glucuronidase) and 40–80% (lactoferrin) of maximal when compared with formylmethionylleucylphenylalanine plus cytochalasin B stimulation. In summary, after neutrophils settle on P. aeruginosa biofilms, they become phagocytically engorged, partially degranulated, immobilized, and rounded. The settling also causes an increase in oxygen consumption of the system, apparently resulting from a combination of a bacterial respiration and escape response and the neutrophil respiratory burst but with little increase in the soluble concentration of H2O2. Thus, host defense becomes compromised as biofilm bacteria escape while neutrophils remain immobilized with a diminished oxidative potential.

The Limitations of In Vitro Experimentation in Understanding Biofilms and Chronic Infection

“…In the biofilm state, the enclosing matrix of extracellular substances is capable of protecting underlying cells from the immune system, such as PMN phagocytosis [22,23]. In addition to this, biofilms are capable of suppressing the antimicrobial action of PMNs through the production of various virulence factors [19,24]. P. aeruginosa growing in biofilms has been shown to actively kill PMNs through the secretion of rhamnolipids which reduce the host's ability to clear infection [25].…”

Yersinia pestis Biofilm in the Flea Vector and Its Role in the Transmission of Plague

“…The ECM that surrounds bacteria in a biofilm has been shown in some cases to protect against phagocytosis and/or intracellular killing, cationic antimicrobial peptides, and antibiotics (Costerton et al 1999;Jesaitis et al 2003;Vuong et al 2004b). It is likely that the infectious inoculum delivered by a flea consists of small clumps of Y. pestis that are associated with a complex ECM (Fig.…”

Calcium-Induced Virulence Factors Associated with the Extracellular Matrix of MucoidPseudomonas aeruginosaBiofilms

“…P. aeruginosa pulmonary infections are considered biofilm-associated diseases, where the bacteria colonize the surface of pulmonary tissue (36), are often encapsulated in extracellular polysaccharide matrix material (25,36,48), secrete quorumsensing signaling compounds (58), and have increased resistance to antibiotics (reviewed in reference 59) and to host defenses (2,30,45). Mutations in the cystic fibrosis transmembrane regulator of CF patients lead to physiological alteration of pulmonary fluid, with the most characteristic manifestation being the increased concentrations of thick pulmonary mucus that includes DNA and actin from lysed neutrophils (38).…”