Summary Pseudomonas aeruginosa causes chronic lung infections in the airways of cystic fibrosis (CF) patients. Psl is an extracellular polysaccharide expressed by non-mucoid P. aeruginosa strains, which are believed to be initial colonizers. We hypothesized that Psl protects P. aeruginosa from host defences within the CF lung prior to their conversion to the mucoid phenotype. We discovered that serum opsonization significantly increased the production of reactive oxygen species (ROS) by neutrophils exposed to a psl-deficient mutant, compared with wild-type (WT) and Psl overexpressing strains (Psl++). Psl-deficient P. aeruginosa were internalized and killed by neutrophils and macrophages more efficiently than WT and Psl++ variants. Deposition of complement components C3, C5 and C7 was significantly higher on psl-deficient strains compared with WT and Psl++ bacteria. In an in vivo pulmonary competition assay, there was a 4.5-fold fitness advantage for WT over psl-deficient P. aeruginosa. Together, these data show that Psl inhibits efficient opsonization, resulting in reduced neutrophil ROS production, and decreased killing by phagocytes. This provides a survival advantage in vivo. Since phagocytes are critical in early recognition and control of infection, therapies aimed at Psl could improve the quality of life for patients colonized with P. aeruginosa.

Bordetellae are gram-negative bacteria that colonize the respiratory tracts of animals and humans. We and others have recently shown that these bacteria are capable of living as sessile communities known as biofilms on a number of abiotic surfaces. During the biofilm mode of existence, bacteria produce one or more extracellular polymeric substances that function, in part, to hold the cells together and to a surface. There is little information on either the constituents of the biofilm matrix or the genetic basis of biofilm development by Bordetella spp. By utilizing immunoblot assays and by enzymatic hydrolysis using dispersin B (DspB), a glycosyl hydrolase that specifically cleaves the polysaccharide poly-␤-1,6-N-acetyl-D-glucosamine (poly-␤-1,6-GlcNAc), we provide evidence for the production of poly-␤-1,6-GlcNAc by various Bordetella species (Bordetella bronchiseptica, B. pertussis, and B. parapertussis) and its role in their biofilm development. We have investigated the role of a Bordetella locus, here designated bpsABCD, in biofilm formation. The bps (Bordetella polysaccharide) locus is homologous to several bacterial loci that are required for the production of poly-␤-1,6-GlcNAc and have been implicated in bacterial biofilm formation. By utilizing multiple microscopic techniques to analyze biofilm formation under both static and hydrodynamic conditions, we demonstrate that the bps locus, although not essential at the initial stages of biofilm formation, contributes to the stability and the maintenance of the complex architecture of Bordetella biofilms.In contrast to the planktonic or free swimming mode of existence in laboratory settings, bacteria predominantly form surface-attached communities known as biofilms in their natural habitats. Biofilms are commonly defined as highly structured communities of cells that are encased in a self-produced polymeric organic matrix (7). Biofilms are increasingly being recognized as important contributors to chronic bacterial diseases (8,19,48). Biofilms provide protection from both the innate and the adaptive components of the immune system, and bacteria in biofilms are extremely recalcitrant to antibiotic therapy and other antimicrobial agents (15,20,31,40).We are studying the members of the bacterial genus Bordetella as a model system in order to understand how bacteria adapt to cope with the selective pressures inside mammalian hosts. Given the demonstrated importance of biofilms in contributing to bacterial persistence, we are interested in characterizing the role that biofilms play in Bordetella physiology and the infectious cycle within hosts. Bordetellae are small, aerobic, gram-negative coccobacilli that colonize the respiratory tracts of humans and animals (42). Bordetella pertussis, the human pathogen, results in the disease known as whooping cough, while B. bronchiseptica mainly infects animals and causes a variety of respiratory diseases (42, 64). B. parapertussis strains can be divided into two genetically distinct types: those that infect humans, causing a...

The majority of Bordetella sp. virulence determinants are regulated by the BvgAS signal transduction system. BvgAS mediates the control of multiple phenotypic phases and a spectrum of gene expression profiles specific to each phase in response to incremental changes in the concentrations of environmental signals. Studies highlighting the critical role of this signaling circuitry in the Bordetella infectious cycle have focused on planktonically growing bacterial cells. It is becoming increasingly clear that the major mode of bacterial existence in the environment and within the body is a surface-attached state known as a biofilm. Biofilms are defined as consortia of sessile microorganisms that are embedded in a matrix. During routine growth of Bordetella under agitating conditions, we noticed the formation of a bacterial ring at the air-liquid interface of the culture tubes. We show here that this surface adherence property reflects the ability of these organisms to form biofilms. Our data demonstrate that the BvgAS locus regulates biofilm development in Bordetella. The results reported in this study suggest that the Bvg-mediated control in biofilm development is exerted at later time points after the initial attachment of bacteria to the different surfaces. Additionally, we show that these biofilms are highly tolerant of a number of antimicrobials, including the ones that are currently recommended for treatment of veterinary and human infections caused by Bordetella spp. Finally, we discuss the significance of the biofilm lifestyle mode as a potential contributor to persistent infections.

Bordetellae are respiratory pathogens that infect both humans and animals. Bordetella bronchiseptica establishes asymptomatic and long-term to life-long infections of animal nasopharynges. While the human pathogen Bordetella pertussis is the etiological agent of the acute disease whooping cough in infants and young children, it is now being increasingly isolated from the nasopharynges of vaccinated adolescents and adults who sometimes show milder symptoms, such as prolonged cough illness. Although it has been shown that Bordetella can form biofilms in vitro, nothing is known about its biofilm mode of existence in mammalian hosts. Using indirect immunofluorescence and scanning electron microscopy, we examined nasal tissues from mice infected with B. bronchiseptica. Our results demonstrate that a wild-type strain formed robust biofilms that were adherent to the nasal epithelium and displayed architectural attributes characteristic of a number of bacterial biofilms formed on inert surfaces. We have previously shown that the Bordetella Bps polysaccharide encoded by the bpsABCD locus is critical for the stability and maintenance of three-dimensional structures of biofilms. We show here that Bps is essential for the formation of efficient nasal biofilms and is required for the colonization of the nose. Our results document a biofilm lifestyle for Bordetella in mammalian respiratory tracts and highlight the essential role of the Bps polysaccharide in this process and in persistence of the nares.Bacteria belonging to the genus Bordetella cause respiratory tract infections in both humans and animals (42). Bordetella pertussis is the etiological agent of pertussis, cases of which are steadily increasing in number, even in vaccinated populations (9). It has been proposed that the resurgence of pertussis is due in part to carriage within adolescent and adult populations because of waning immunity (3,4,9). Bordetella bronchiseptica has a broad host range and naturally infects a wide variety of nonhuman animals. It typically establishes asymptomatic infections but can cause atrophic rhinitis in pigs, kennel cough in dogs, snuffles in rabbits, and bronchopneumonia in guinea pigs (18,42).B. bronchiseptica is capable of establishing a chronic and asymptomatic infection and can be harvested from the nasal cavities of rats and mice for extended periods (1, 37). A convincing and frequently proposed hypothesis to explain long-term carriage is the ability of microorganisms to exist as biofilms. Bacterial biofilms are increasingly recognized as important contributors to chronic or persistent diseases. A biofilm is generally defined as a surface-attached population of one or more types of bacteria encased in a polymeric matrix, which can be composed of a number of different macromolecules, including nucleic acids, proteins, and polysaccharides (5). Numerous studies have documented the ability of biofilm bacteria to be recalcitrant to antibiotic treatments and to the host immune system (31,39,40,53).We and others have recently demonstra...