George A. O’Toole

The developmental model of microbial biofilms: ten years of a paradigm up for review

For the past ten years, the developmental model of microbial biofilm formation has served as the major conceptual framework for biofilm research; however, the paradigmatic value of this model has begun to be challenged by the research community. Here, we critically evaluate recent data to determine whether biofilm formation satisfies the criteria requisite of a developmental system. We contend that the developmental model of biofilm formation must be approached as a model in need of further validation, rather than utilized as a platform on which to base empirical research and scientific inference. With this in mind, we explore the experimental approaches required to further our understanding of the biofilm phenotype, highlighting evolutionary and ecological approaches as a natural complement to rigorous mechanistic studies into the causal basis of biofilm formation. Finally, we discuss a second model of biofilm formation that serves as a counterpoint to our discussion of the developmental model. Our hope is that this article will provide a platform for discussion about the conceptual underpinnings of biofilm formation and the impact of such frameworks on shaping the questions we ask, and the answers we uncover, during our research into these microbial communities.

Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway.

The cystic fibrosis (CF) lung is chronically inflamed and infected by Pseudomonas aeruginosa, which is a major cause of morbidity and mortality in this genetic disease. Although aerosolization of Tobramycin into the airway of CF patients improves outcomes, the lungs of CF patients, even those receiving antibiotic therapy, are persistently colonized by P. aeruginosa. Recent studies suggest that the antibiotic resistance of P. aeruginosa in the CF lung is due to the formation of drug resistant biofilms, which are defined as communities of microbes associated with surfaces or interfaces, and whose growth is facilitated by thick and dehydrated mucus in the CF lung. In this review, we discuss some of the current models used to study biofilm formation in the context of biotic surfaces, such as airway cells, as well as the contribution of host-derived factors, including DNA, actin and mucus, to the formation of these microbial communities. We suggest that better in vitro models are required, both to understand the interaction of P. aeruginosa with the host airway, and as models to validate new therapeutics, whether targeted at bacteria or host.

Unique microbial communities persist in individual cystic fibrosis patients throughout a clinical exacerbation

BackgroundCystic fibrosis (CF) is caused by inherited mutations in the cystic fibrosis transmembrane conductance regulator gene and results in a lung environment that is highly conducive to polymicrobial infection. Over a lifetime, decreasing bacterial diversity and the presence of Pseudomonas aeruginosa in the lung are correlated with worsening lung disease. However, to date, no change in community diversity, overall microbial load or individual microbes has been shown to correlate with the onset of an acute exacerbation in CF patients. We followed 17 adult CF patients throughout the course of clinical exacerbation, treatment and recovery, using deep sequencing and quantitative PCR to characterize spontaneously expectorated sputum samplesResultsWe identified approximately 170 bacterial genera, 12 of which accounted for over 90% of the total bacterial load across all patient samples. Genera abundant in any single patient sample tended to be detectable in most samples. We found that clinical stages could not be distinguished by absolute Pseudomonas aeruginosa load, absolute total bacterial load or the relative abundance of any individual genus detected, or community diversity. Instead, we found that the microbial structure of each patient’s sputum microbiome was distinct and resilient to exacerbation and antibiotic treatment.ConclusionConsistent with previously reported sputum microbiome studies we found that total and relative abundance of genera at the population level were remarkably stable for individual patients regardless of clinical status. Patient-by-patient analysis of diversity and relative abundance of each individual genus revealed a complex microbial landscape and highlighted the difficulty of identifying a universal microbial signature of exacerbation. Overall, at the genus level, we find no evidence of a microbial signature of clinical stage.

New yeast recombineering tools for bacteria

Recombineering with Saccharomyces cerevisiae is a powerful methodology that can be used to clone multiple unmarked pieces of DNA to generate complex constructs with high efficiency. Here, we introduce two new tools that utilize the native recombination enzymes of S. cerevisiae to facilitate the manipulation of DNA. First, yeast recombineering was used to make directed nested deletions in a bacteria-yeast shuttle plasmid using only one or two single stranded oligomers, thus obviating the need for a PCR step. Second, we have generated several new shuttle vectors for yeast recombineering capable of replication in a wide variety of bacterial genera. As a demonstration of utility, some of the approaches and vectors generated in this study were used to make a pigP deletion mutation in the opportunistic pathogen Serratia marcescens.

Characterization and quantification of the fungal microbiome in serial samples from individuals with cystic fibrosis

BackgroundHuman-associated microbial communities include fungi, but we understand little about which fungal species are present, their relative and absolute abundances, and how antimicrobial therapy impacts fungal communities. The disease cystic fibrosis (CF) often involves chronic airway colonization by bacteria and fungi, and these infections cause irreversible lung damage. Fungi are detected more frequently in CF sputum samples upon initiation of antimicrobial therapy, and several studies have implicated the detection of fungi in sputum with worse outcomes. Thus, a more complete understanding of fungi in CF is required.ResultsWe characterized the fungi and bacteria in expectorated sputa from six CF subjects. Samples were collected upon admission for systemic antibacterial therapy and upon the completion of treatment and analyzed using a pyrosequencing-based analysis of fungal internal transcribed spacer 1 (ITS1) and bacterial 16S rDNA sequences. A mixture of Candida species and Malassezia dominated the mycobiome in all samples (74%–99% of fungal reads). There was not a striking trend correlating fungal and bacterial richness, and richness showed a decline after antibiotic therapy particularly for the bacteria. The fungal communities within a sputum sample resembled other samples from that subject despite the aggressive antibacterial therapy. Quantitative PCR analysis of fungal 18S rDNA sequences to assess fungal burden showed variation in fungal density in sputum before and after antibacterial therapy but no consistent directional trend. Analysis of Candida ITS1 sequences amplified from sputum or pure culture-derived genomic DNA from individual Candida species found little (<0.5%) or no variation in ITS1 sequences within or between strains, thereby validating this locus for the purpose of Candida species identification. We also report the enhancement of the publically available Visualization and Analysis of Microbial Population Structures (VAMPS) tool for the analysis of fungal communities in clinical samples.ConclusionsFungi are present in CF respiratory sputum. In CF, the use of intravenous antibiotic therapy often does not profoundly impact bacterial community structure, and we observed a similar stability in fungal species composition. Further studies are required to predict the effects of antibacterials on fungal burden in CF and fungal community stability in non-CF populations.

Surface attachment induces Pseudomonas aeruginosa virulence.

Significance Pseudomonas aeruginosa is a pathogen that kills a remarkably wide range of hosts. The environmental cues that regulate P. aeruginosa virulence have remained unclear. Here, we develop a rapid imaging-based virulence assay to quantify virulence. We find that association with rigid surfaces induces virulence toward multiple hosts. Virulence induction depends on the mechanical, but not chemical, properties of the surfaces and requires the surface-exposed protein PilY1, which has homology to the mechanosensitive von Willebrand factor A domain. Specific mutation of this mechanosensitive domain is sufficient to constitutively activate virulence independent of surface attachment. Mechanosensitive virulence induction can explain how P. aeruginosa infects a broad range of hosts while tightly regulating virulence. Consistently, association with one host induces virulence toward other hosts. Pseudomonas aeruginosa infects every type of host that has been examined by deploying multiple virulence factors. Previous studies of virulence regulation have largely focused on chemical cues, but P. aeruginosa may also respond to mechanical cues. Using a rapid imaging-based virulence assay, we demonstrate that P. aeruginosa activates virulence in response to attachment to a range of chemically distinct surfaces, suggesting that this bacterial species responds to mechanical properties of its substrates. Surface-activated virulence requires quorum sensing, but activating quorum sensing does not induce virulence without surface attachment. The activation of virulence by surfaces also requires the surface-exposed protein PilY1, which has a domain homologous to a eukaryotic mechanosensor. Specific mutation of the putative PilY1 mechanosensory domain is sufficient to induce virulence in non–surface-attached cells, suggesting that PilY1 mediates surface mechanotransduction. Triggering virulence only when cells are both at high density and attached to a surface—two host-nonspecific cues—explains how P. aeruginosa precisely regulates virulence while maintaining broad host specificity.

A Hierarchical Cascade of Second Messengers Regulates Pseudomonas aeruginosa Surface Behaviors

ABSTRACT  Biofilms are surface-attached multicellular communities. Using single-cell tracking microscopy, we showed that a pilY1 mutant of Pseudomonas aeruginosa is defective in early biofilm formation. We leveraged the observation that PilY1 protein levels increase on a surface to perform a genetic screen to identify mutants altered in surface-grown expression of this protein. Based on our genetic studies, we found that soon after initiating surface growth, cyclic AMP (cAMP) levels increase, dependent on PilJ, a chemoreceptor-like protein of the Pil-Chp complex, and the type IV pilus (TFP). cAMP and its receptor protein Vfr, together with the FimS-AlgR two-component system (TCS), upregulate the expression of PilY1 upon surface growth. FimS and PilJ interact, suggesting a mechanism by which Pil-Chp can regulate FimS function. The subsequent secretion of PilY1 is dependent on the TFP assembly system; thus, PilY1 is not deployed until the pilus is assembled, allowing an ordered signaling cascade. Cell surface-associated PilY1 in turn signals through the TFP alignment complex PilMNOP and the diguanylate cyclase SadC to activate downstream cyclic di-GMP (c-di-GMP) production, thereby repressing swarming motility. Overall, our data support a model whereby P. aeruginosa senses the surface through the Pil-Chp chemotaxis-like complex, TFP, and PilY1 to regulate cAMP and c-di-GMP production, thereby employing a hierarchical regulatory cascade of second messengers to coordinate its program of surface behaviors. IMPORTANCE Biofilms are surface-attached multicellular communities. Here, we show that a stepwise regulatory circuit, involving ordered signaling via two different second messengers, is required for Pseudomonas aeruginosa to control early events in cell-surface interactions. We propose that our studies have uncovered a multilayered “surface-sensing” system that allows P. aeruginosa to effectively coordinate its surface-associated behaviors. Understanding how cells transition into the biofilm state on a surface may provide new approaches to prevent formation of these communities. Biofilms are surface-attached multicellular communities. Here, we show that a stepwise regulatory circuit, involving ordered signaling via two different second messengers, is required for Pseudomonas aeruginosa to control early events in cell-surface interactions. We propose that our studies have uncovered a multilayered “surface-sensing” system that allows P. aeruginosa to effectively coordinate its surface-associated behaviors. Understanding how cells transition into the biofilm state on a surface may provide new approaches to prevent formation of these communities.

Nanoscale adhesion forces of Pseudomonas aeruginosa type IV Pili.

A variety of bacterial pathogens use nanoscale protein fibers called type IV pili to mediate cell adhesion, a primary step leading to infection. Currently, how these nanofibers respond to mechanical stimuli and how this response is used to control adhesion is poorly understood. Here, we use atomic force microscopy techniques to quantify the forces guiding the adhesion of Pseudomonas aeruginosa type IV pili to surfaces. Using chemical force microscopy and single-cell force spectroscopy, we show that pili strongly bind to hydrophobic surfaces in a time-dependent manner, while they weakly bind to hydrophilic surfaces. Individual nanofibers are capable of withstanding forces up to 250 pN, thereby explaining how they can resist mechanical stress. Pulling on individual pili yields constant force plateaus, presumably reflecting conformational changes, as well as nanospring properties that may help bacteria to withstand physiological shear forces. Analysis of mutant strains demonstrates that these mechanical responses originate solely from type IV pili, while flagella and the cell surface localized and proposed pili-associated adhesin PilY1 play no direct role. We also demonstrate that bacterial–host interactions involve constant force plateaus, the extension of bacterial pili, and the formation of membrane tethers from host cells. We postulate that the unique mechanical responses of type IV pili unravelled here enable the bacteria to firmly attach to biotic and abiotic surfaces and thus maintain attachment when subjected to high shear forces under physiological conditions, helping to explain why pili play a critical role in colonization of the host.

Cystic Fibrosis Lung Infections: Polymicrobial, Complex, and Hard to Treat.

Historically, H. influenzae and S. aureus were considered to be the primary organisms infecting the airways of infants and children with CF, followed by P. aeruginosa or Burkholderia cepacia complex during adulthood. These bacteria continue to play important roles in CF respiratory infections and clinical outcome; however, with the advent of improved culture methods and culture-independent approaches, including deep sequencing technology, it is clear that the airways of patients with CF are chronically colonized with complex, polymicrobial infections. In 2003, Rogers and colleagues revolutionized our understanding of CF lung infections through their identification of complex bacterial communities in sputum and bronchoscopy samples using a culture-independent, molecular-based approach, terminal restriction fragment length polymorphism profiling. This study was the first to recognize the role of highly prevalent and diverse bacterial species previously unrecognized in CF lung infections, including obligate anaerobes [1]. Since then, several additional studies utilizing culture-independent approaches have confirmed that the airways of patients with CF are chronically colonized with diverse bacterial, fungal, and viral taxa [2–5]. These polymicrobial communities are highly individualized to each patient and promote intricate inter-microbial and host—pathogen interactions, which alter the lung environment, impact response to treatment, and direct the course of disease (summarized in Fig 1). Core genera, including Streptococcus, Pseudomonas, Prevotella, Veillonella, Neisseria, Porphorymonas, and Catonella are detected in abundance in the majority of adult sputum samples [7]. In addition to this core, deep sequencing typically identifies 50–200 unique operational taxonomic units in a single CF respiratory sample. Furthermore, the advancement of molecular identification approaches has greatly enhanced our recognition of the diverse fungi (including Candida spp.,Malassezia spp., and Aspergillus spp.) and viruses (including influenza and respiratory syncytial virus) that co-inhabit the lungs of patients with CF [2,4,8]. Accompanying the explosion of organisms recognized in these multi-domain communities comes the task of determining the role of such microbes in CF lung disease. In the recently accepted context of highly individualized, complex, polymicrobial communities, answering the question of “who’s the pathogen?” has become a non-trivial challenge of both clinical diagnostic and academic research focus. Emerging pathogens (microbes that directly contribute to disease progression and poor patient outcome) of interest are bacterial (Streptococcusmilleri group spp., nontuberculous mycobacterium), fungal (Trichosporon spp.), and viral (rhinovirus). Meanwhile, other organisms previously considered to be pathogenic in the context of CF lung infections are now more widely thought of as normal microbiota and unlikely pathogens,

The microbiome in pediatric cystic fibrosis patients: the role of shared environment suggests a window of intervention

BackgroundCystic fibrosis (CF) is caused by mutations in the CFTR gene that predispose the airway to infection. Chronic infection by pathogens such as Pseudomonas aeruginosa leads to inflammation that gradually degrades lung function, resulting in morbidity and early mortality. In a previous study of CF monozygotic twins, we demonstrate that genetic modifiers significantly affect the establishment of persistent P. aeruginosa colonization in CF. Recognizing that bacteria other than P. aeruginosa contribute to the CF microbiome and associated pathology, we used deep sequencing of sputum from pediatric monozygotic twins and nontwin siblings with CF to characterize pediatric bacterial communities and the role that genetics plays in their evolution.FindingsWe found that the microbial communities in sputum from pediatric patients living together were much more alike than those from pediatric individuals living apart, regardless of whether samples were taken from monozygous twins or from nontwin CF siblings living together, which we used as a proxy for dizygous twins. In contrast, adult communities were comparatively monolithic and much less diverse than the microbiome of pediatric patients.ConclusionTaken together, these data and other recent studies suggest that as patients age, the CF microbiome becomes less diverse, more refractory to treatment and dominated by mucoid P. aeruginosa, as well as being associated with accelerated pulmonary decline. Our studies show that the microbiome of pediatric patients is susceptible to environmental influences, suggesting that interventions to preserve the community structure found in young CF patients might be possible, perhaps slowing disease progression.