Christopher D. Sibley

A Novel Mechanism of Rapid Nuclear Neutrophil Extracellular Trap Formation in Response to Staphylococcus aureus

Neutrophil extracellular traps (NETs) are webs of DNA covered with antimicrobial molecules that constitute a newly described killing mechanism in innate immune defense. Previous publications reported that NETs take up to 3–4 h to form via an oxidant-dependent event that requires lytic death of neutrophils. In this study, we describe neutrophils responding uniquely to Staphylococcus aureus via a novel process of NET formation that did not require neutrophil lysis or even breach of the plasma membrane. The multilobular nucleus rapidly became rounded and condensed. During this process, we observed the separation of the inner and outer nuclear membranes and budding of vesicles, and the separated membranes and vesicles were filled with nuclear DNA. The vesicles were extruded intact into the extracellular space where they ruptured, and the chromatin was released. This entire process occurred via a unique, very rapid (5–60 min), oxidant-independent mechanism. Mitochondrial DNA constituted very little if any of these NETs. They did have a limited amount of proteolytic activity and were able to kill S. aureus. With time, the nuclear envelope ruptured, and DNA filled the cytoplasm presumably for later lytic NET production, but this was distinct from the vesicular release mechanism. Panton–Valentine leukocidin, autolysin, and a lipase were identified in supernatants with NET-inducing activity, but Panton–Valentine leukocidin was the dominant NET inducer. We describe a new mechanism of NET release that is very rapid and contributes to trapping and killing of S. aureus.

A polymicrobial perspective of pulmonary infections exposes an enigmatic pathogen in cystic fibrosis patients

Lung disease is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. A modest number of bacterial pathogens have been correlated with pulmonary function decline; however, microbiological and molecular evidence suggests that CF airway infection is polymicrobial. To obtain a more complete assessment of the microbial community composition and dynamics, we undertook a longitudinal study by using culture-independent and microbiological approaches. In the process, we demonstrated that within complex and dynamic communities, the Streptococcus milleri group (SMG) can establish chronic pulmonary infections and at the onset of 39% of acute pulmonary exacerbations, SMG is the numerically dominant pathogen. We report the comprehensive polymicrobial community dynamics of a CF lung infection in a clinically relevant context. If a given organism, such as Pseudomonas aeruginosa, becomes resistant to antibiotic therapy, an alternative treatment avenue may mediate the desired clinical response by effectively managing the composition of the microbial community.

Discerning the Complexity of Community Interactions Using a Drosophila Model of Polymicrobial Infections

A number of human infections are characterized by the presence of more than one bacterial species and are defined as polymicrobial diseases. Methods for the analysis of the complex biological interactions in mixed infections with a large number of microorganisms are limited and do not effectively determine the contribution of each bacterial species to the pathogenesis of the polymicrobial community. We have developed a novel Drosophila melanogaster infection model to study microbe–microbe interactions and polymicrobe–host interactions. Using this infection model, we examined the interaction of 40 oropharyngeal isolates with Pseudomonas aeruginosa. We observe three classes of microorganisms, one of which acts synergistically with the principal pathogen, while being avirulent or even beneficial on its own. This synergy involves microbe–microbe interactions that result in the modulation of P. aeruginosa virulence factor gene expression within infected Drosophila. The host innate immune response to these natural-route polymicrobial infections is complex and characterized by additive, suppressive, and synergistic transcriptional activation of antimicrobial peptide genes. The polymicrobial infection model was used to differentiate the bacterial flora in cystic fibrosis (CF) sputum, revealing that a large proportion of the organisms in CF airways has the ability to influence the outcome of an infection when in combination with the principal CF pathogen P. aeruginosa.

Culture Enriched Molecular Profiling of the Cystic Fibrosis Airway Microbiome

The microbiome of the respiratory tract, including the nasopharyngeal and oropharyngeal microbiota, is a dynamic community of microorganisms that is highly diverse. The cystic fibrosis (CF) airway microbiome refers to the polymicrobial communities present in the lower airways of CF patients. It is comprised of chronic opportunistic pathogens (such as Pseudomonas aeruginosa) and a variety of organisms derived mostly from the normal microbiota of the upper respiratory tract. The complexity of these communities has been inferred primarily from culture independent molecular profiling. As with most microbial communities it is generally assumed that most of the organisms present are not readily cultured. Our culture collection generated using more extensive cultivation approaches, reveals a more complex microbial community than that obtained by conventional CF culture methods. To directly evaluate the cultivability of the airway microbiome, we examined six samples in depth using culture-enriched molecular profiling which combines culture-based methods with the molecular profiling methods of terminal restriction fragment length polymorphisms and 16S rRNA gene sequencing. We demonstrate that combining culture-dependent and culture-independent approaches enhances the sensitivity of either approach alone. Our techniques were able to cultivate 43 of the 48 families detected by deep sequencing; the five families recovered solely by culture-independent approaches were all present at very low abundance (<0.002% total reads). 46% of the molecular signatures detected by culture from the six patients were only identified in an anaerobic environment, suggesting that a large proportion of the cultured airway community is composed of obligate anaerobes. Most significantly, using 20 growth conditions per specimen, half of which included anaerobic cultivation and extended incubation times we demonstrate that the majority of bacteria present can be cultured.

Phenotypic heterogeneity of Pseudomonas aeruginosa populations in a cystic fibrosis patient.

The opportunistic pathogen Pseudomonas aeruginosa chronically infects the lower airways of patients with cystic fibrosis. Throughout the course of infection this organism undergoes adaptations that contribute to its long-term persistence in the airways. While P. aeruginosa diversity has been documented, it is less clear to what extent within-patient diversity contributes to the overall population structure as most studies have been limited to the analysis of only a few isolates per patient per time point. To examine P. aeruginosa population structure in more detail we collected multiple isolates from individual sputum samples of a patient chronically colonized with P. aeruginosa. This strain collection, comprised of 169 clonal isolates and representing three pulmonary exacerbations as well as clinically stable periods, was assayed for a wide selection of phenotypes. These phenotypes included colony morphology, motility, quorum sensing, protease activity, auxotrophy, siderophore levels, antibiotic resistance, and growth profiles. Each phenotype displayed significant variation even within isolates of the same colony morphotype from the same sample. Isolates demonstrated a large degree of individuality across phenotypes, despite being part of a single clonal lineage, suggesting that the P. aeruginosa population in the cystic fibrosis airways is being significantly under-sampled.

Molecular methods for pathogen and microbial community detection and characterization: current and potential application in diagnostic microbiology.

Abstract Clinical microbiology laboratories worldwide have historically relied on phenotypic methods (i.e., culture and biochemical tests) for detection, identification and characterization of virulence traits (e.g., antibiotic resistance genes, toxins) of human pathogens. However, limitations to implementation of molecular methods for human infectious diseases testing are being rapidly overcome allowing for the clinical evaluation and implementation of diverse technologies with expanding diagnostic capabilities. The advantages and limitation of molecular techniques including real-time polymerase chain reaction, partial or whole genome sequencing, molecular typing, microarrays, broad-range PCR and multiplexing will be discussed. Finally, terminal restriction fragment length polymorphism (T-RFLP) and deep sequencing are introduced as technologies at the clinical interface with the potential to dramatically enhance our ability to diagnose infectious diseases and better define the epidemiology and microbial ecology of a wide range of complex infections.

Cystic fibrosis: a polymicrobial infectious disease.

Cystic fibrosis (CF) is the most common lethal genetic disease in the Caucasian population, and should be considered an infectious disease because of the basic pathophysiology. Chronic lower airway infections cause a progressive pathologic deterioration of lung tissue, a decline in pulmonary function and, ultimately, respiratory failure and death in 90% of CF patients. Historically, very few bacterial species have been implicated as principal CF pathogens. However, molecular evidence suggests the presence of a diverse mosaic of bacteria in CF lungs, and infections can be defined as polymicrobial. Here we review the work that supports this concept and we discuss the potential significance of the polymicrobial community in lung pathology. Understanding the dynamics of polymicrobial infections, the interplay between pathogen(s), normal oropharyngeal flora and the host immune system may lead to future advances in the therapeutic management of chronic lung infections.

The Streptococcus milleri group—An unrecognized cause of disease in cystic fibrosis: A case series and literature review

The “Streptococcus milleri” group (SMG) is increasingly recognized for their role in pyogenic infections including empyema and solid organ abscesses. However, SMG disease has rarely been identified in cystic fibrosis (CF). Inherent difficulties in both growing the organisms and distinguishing SMG from less virulent oropharyngeal viridans streptococci may have led to a decreased recognition of this as a CF pathogen. We report on six cases of SMG‐related infection over a 4‐year time‐frame occurring within an adult CF clinic in Canada, and a further four cases identified through a literature review. SMG manifested disease as bronchopulmonary exacerbations in 7 of 10 patients, and 4 of 10 patients had extra‐pulmonary dissemination of SMG infection. Noticeably, pulmonary exacerbations were frequently associated with atypically malodorous sputum. Furthermore, patients clinically responded to anti‐microbial therapies with no anti‐Pseudomonal activity. There was a consistent correlation of SMG disease and co‐colonization with P. aeruginosa leading to speculation of polymicrobial interactions resulting in enhanced virulence. SMG deserves considerable attention as a potential pathogen within the airways of patients with CF. Pediatr Pulmonol. 2008; 43:490–497.

Drosophila melanogaster as an animal model for the study of Pseudomonas aeruginosa biofilm infections in vivo.

Pseudomonas aeruginosa is an opportunistic pathogen capable of causing both acute and chronic infections in susceptible hosts. Chronic P. aeruginosa infections are thought to be caused by bacterial biofilms. Biofilms are highly structured, multicellular, microbial communities encased in an extracellular matrix that enable long-term survival in the host. The aim of this research was to develop an animal model that would allow an in vivo study of P. aeruginosa biofilm infections in a Drosophila melanogaster host. At 24 h post oral infection of Drosophila, P. aeruginosa biofilms localized to and were visualized in dissected Drosophila crops. These biofilms had a characteristic aggregate structure and an extracellular matrix composed of DNA and exopolysaccharide. P. aeruginosa cells recovered from in vivo grown biofilms had increased antibiotic resistance relative to planktonically grown cells. In vivo, biofilm formation was dependent on expression of the pel exopolysaccharide genes, as a pelB::lux mutant failed to form biofilms. The pelB::lux mutant was significantly more virulent than PAO1, while a hyperbiofilm strain (PAZHI3) demonstrated significantly less virulence than PAO1, as indicated by survival of infected flies at day 14 postinfection. Biofilm formation, by strains PAO1 and PAZHI3, in the crop was associated with induction of diptericin, cecropin A1 and drosomycin antimicrobial peptide gene expression 24 h postinfection. In contrast, infection with the non-biofilm forming strain pelB::lux resulted in decreased AMP gene expression in the fly. In summary, these results provide novel insights into host-pathogen interactions during P. aeruginosa oral infection of Drosophila and highlight the use of Drosophila as an infection model that permits the study of P. aeruginosa biofilms in vivo.

The polymicrobial nature of airway infections in cystic fibrosis: Cangene Gold Medal Lecture.

Microbial communities characterize the airways of cystic fibrosis (CF) patients. Members of these diverse and dynamic communities can be thought of as pathogens, benign commensals, or synergens--organisms not considered pathogens in the traditional sense but with the capacity to alter the pathogenesis of the community through microbe-microbe or polymicrobe-host interactions. Very few bacterial pathogens have been implicated as clinically relevant in CF; however, the CF airway microbiome can be a reservoir of previously unrecognized but clinically relevant organisms. A combination of culture-dependent and culture-independent approaches provides a more comprehensive perspective of CF microbiology than either approach alone. Here we review these concepts, highlight the future challenges for CF microbiology, and discuss the implications for the management of CF airway infections. We suggest that the success of treatment interventions for chronic CF lung disease will rely on the context of the microbes within microbial communities. The microbiology of CF airways may serve as a model to investigate the emergent properties of other clinically relevant microbial communities in the human body.