Exploring the applications of invertebrate host-pathogen models for in vivo biofilm infections

In the natural environment, microorganisms exist together in self-produced polymeric matrix biofilms. Often, several species, which can belong to both bacterial and fungal kingdoms, coexist and interact in ways which are not completely understood. Biofilm infections have become prevalent largely in medical settings because of the increasing use of indwelling medical devices such as catheters or prosthetics. These infections are resistant to common antimicrobial therapies because of the inherent nature of their structure. In terms of infectious biofilms, it is important to understand the microbe-microbe interactions and how the host immune system reacts in order to discover therapeutic targets. Currently, single infection immune response studies are thriving with the use of invertebrate models. This review highlights the advances in single microbial-host immune response as well as the promising aspects of polymicrobial biofilm study in five invertebrate models: Lemna minor (duckweed), Arabidopsis thaliana (thale cress), Dictyostelium discoideum (slime mold), Drosophila melanogaster (common fruit fly), and Caenorhabditis elegans (roundworm).     

A quorum sensing regulated small volatile molecule reduces acute virulence and promotes chronic infection phenotypes

A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections.     

Cystic fibrosis-niche adaptation of Pseudomonas aeruginosa reduces virulence in multiple infection hosts

The opportunistic pathogen Pseudomonas aeruginosa is able to thrive in diverse ecological niches and to cause serious human infection. P. aeruginosa environmental strains are producing various virulence factors that are required for establishing acute infections in several host organisms; however, the P. aeruginosa phenotypic variants favour long-term persistence in the cystic fibrosis (CF) airways. Whether P. aeruginosa strains, which have adapted to the CF-niche, have lost their competitive fitness in the other environment remains to be investigated. In this paper, three P. aeruginosa clonal lineages, including early strains isolated at the onset of infection, and late strains, isolated after several years of chronic lung infection from patients with CF, were analysed in multi-host model systems of acute infection. P. aeruginosa early isolates caused lethality in the three non-mammalian hosts, namely Caenorhabditis elegans, Galleria mellonella, and Drosophila melanogaster, while late adapted clonal isolates were attenuated in acute virulence. When two different mouse genetic background strains, namely C57Bl/6NCrl and Balb/cAnNCrl, were used as acute infection models, early P. aeruginosa CF isolates were lethal, while late isolates exhibited reduced or abolished acute virulence. Severe histopathological lesions, including high leukocytes recruitment and bacterial load, were detected in the lungs of mice infected with P. aeruginosa CF early isolates, while late isolates were progressively cleared. In addition, systemic bacterial spread and invasion of epithelial cells, which were detected for P. aeruginosa CF early strains, were not observed with late strains. Our findings indicate that niche-specific selection in P. aeruginosa reduced its ability to cause acute infections across a broad range of hosts while maintaining the capacity for chronic infection in the CF host.     

Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.

Respiratory infections with Pseudomonas aeruginosa and Burkholderia cepacia play a major role in the pathogenesis of cystic fibrosis (CF). This review summarizes the latest advances in understanding host-pathogen interactions in CF with an emphasis on the role and control of conversion to mucoidy in P. aeruginosa, a phenomenon epitomizing the adaptation of this opportunistic pathogen to the chronic chourse of infection in CF, and on the innate resistance to antibiotics of B. cepacia, person-to-person spread, and sometimes rapidly fatal disease caused by this organism. While understanding the mechanism of conversion to mucoidy in P. aeruginosa has progressed to the point where this phenomenon has evolved into a model system for studying bacterial stress response in microbial pathogenesis, the more recent challenge with B. cepacia, which has emerged as a potent bona fide CF pathogen, is discussed in the context of clinical issues, taxonomy, transmission, and potential modes of pathogenicity.     

Cross-kingdom interactions: Candida albicans and bacteria

Bacteria and fungi are found together in a myriad of environments and particularly in a biofilm, where adherent species interact through diverse signaling mechanisms. Yet, despite billions of years of coexistence, the area of research exploring fungal–bacterial interactions, particularly within the context of polymicrobial infections, is still in its infancy. However, reports describing a multitude of wide-ranging interactions between the fungal pathogen Candida albicans and various bacterial pathogens are on the rise. An example of a mutually beneficial interaction is coaggregation, a phenomenon that takes place in oral biofilms where the adhesion of C. albicans to oral bacteria is considered crucial for its colonization of the oral cavity. In contrast, the interaction between C. albicans and Pseudomonas aeruginosa is described as being competitive and antagonistic in nature. Another intriguing interaction is that occurring between Staphylococcus aureus and C. albicans , which although not yet fully characterized, appears to be initially synergistic. These complex interactions between such diverse and important pathogens would have significant clinical implications if they occurred in an immunocompromised host. Therefore, understanding the mechanisms of adhesion and signaling involved in fungal–bacterial interactions may lead to the development of novel therapeutic strategies for impeding microbial colonization and development of polymicrobial disease.     

Membrane-bound nitrate reductase is required for anaerobic growth in cystic fibrosis sputum

The autosomal recessive disorder cystic fibrosis (CF) affects approximately 70,000 people worldwide and is characterized by chronic bacterial lung infections with the opportunistic pathogen Pseudomonas aeruginosa. To form a chronic CF lung infection, P. aeruginosa must grow and proliferate within the CF lung, and the highly viscous sputum within the CF lung provides a likely growth substrate. Recent evidence indicates that anaerobic microenvironments may be present in the CF lung sputum layer. Since anaerobic growth significantly enhances P. aeruginosa biofilm formation and antibiotic resistance, it is important to examine P. aeruginosa physiology and metabolism in anaerobic environments. Measurement of nitrate levels revealed that CF sputum contains sufficient nitrate to support significant P. aeruginosa growth anaerobically, and mutational analysis revealed that the membrane-bound nitrate reductase is essential for P. aeruginosa anaerobic growth in an in vitro CF sputum medium. In addition, expression of genes coding for the membrane-bound nitrate reductase complex is responsive to CF sputum nitrate levels. These findings suggest that the membrane-bound nitrate reductase is critical for P. aeruginosa anaerobic growth with nitrate in the CF lung.     

Cystic fibrosis sputum supports growth and cues key aspects of Pseudomonas aeruginosa physiology

The opportunistic human pathogen Pseudomonas aeruginosa causes persistent airway infections in patients with cystic fibrosis (CF). To establish these chronic infections, P. aeruginosa must grow and proliferate within the highly viscous sputum in the lungs of CF patients. In this study, we used Affymetrix GeneChip microarrays to investigate the physiology of P. aeruginosa grown using CF sputum as the sole source of carbon and energy. Our results indicate that CF sputum readily supports high-density P. aeruginosa growth. Furthermore, multiple signals, which reduce swimming motility and prematurely activate the Pseudomonas quinolone signal cell-to-cell signaling cascade in P. aeruginosa, are present in CF sputum. P. aeruginosa factors critical for lysis of the common CF lung inhabitant Staphylococcus aureus were also induced in CF sputum and increased the competitiveness of P. aeruginosa during polymicrobial growth in CF sputum.     

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.     

Reliability of quantitative real-time PCR for bacterial detection in cystic fibrosis airway specimens

The cystic fibrosis (CF) airway microbiome is complex; polymicrobial infections are common, and the presence of fastidious bacteria including anaerobes make culture-based diagnosis challenging. Quantitative real-time PCR (qPCR) offers a culture-independent method for bacterial quantification that may improve diagnosis of CF airway infections; however, the reliability of qPCR applied to CF airway specimens is unknown. We sought to determine the reliability of nine specific bacterial qPCR assays (total bacteria, three typical CF pathogens, and five anaerobes) applied to CF airway specimens. Airway and salivary specimens from clinically stable pediatric CF subjects were collected. Quantitative PCR assay repeatability was determined using triplicate reactions. Split-sample measurements were performed to measure variability introduced by DNA extraction. Results from qPCR were compared to standard microbial culture for Pseudomonas aeruginosa, Staphylococcus aureus, and Haemophilus influenzae, common pathogens in CF. We obtained 84 sputa, 47 oropharyngeal and 27 salivary specimens from 16 pediatric subjects with CF. Quantitative PCR detected bacterial DNA in over 97% of specimens. All qPCR assays were highly reproducible at quantities≥10(2) rRNA gene copies/reaction with coefficient of variation less than 20% for over 99% of samples. There was also excellent agreement between samples processed in duplicate. Anaerobic bacteria were highly prevalent and were detected in mean quantities similar to that of typical CF pathogens. Compared to a composite gold standard, qPCR and culture had variable sensitivities for detection of P. aeruginosa, S. aureus and H. influenzae from CF airway samples. By reliably quantifying fastidious airway bacteria, qPCR may improve our understanding of polymicrobial CF lung infections, progression of lung disease and ultimately improve antimicrobial treatments.     

A bacterial cell to cell signal in the lungs of cystic fibrosis patients

Pseudomonas aeruginosa is an opportunistic pathogen that is a major cause of mortality in cystic fibrosis (CF) patients. This bacterium has numerous genes controlled by cell to cell signaling, which occurs through a complex circuitry of interconnected regulatory systems. One of the signals is the Pseudomonas Quinolone Signal (PQS), which was identified as 2-heptyl-3-hydroxy-4-quinolone. This intercellular signal controls the expression of multiple virulence factors and is required for virulence in an insect model of P. aeruginosa infection. Previous studies have implied that the intercellular signals of P. aeruginosa are important for human disease, and our goal was to determine whether PQS was produced during human infections. In this report, three types of samples from CF patients infected with P. aeruginosa were analyzed for the presence of PQS. Sputum, bronchoalveolar lavage fluid, and mucopurulent fluid from distal airways of end-stage lungs removed at transplant, all contained PQS, indicating that this cell to cell signal is produced in vivo by P. aeruginosa infecting the lungs of CF patients.     

Interspecies biofilms of Pseudomonas aeruginosa and Burkholderia cepacia.

The leading cause of morbidity and mortality in cystic fibrosis (CF) continues to be lung infections with Pseudomonas aeruginosa biofilms. Co-colonization of the lungs with P aeruginosa and Burkholderia cepacia can result in more severe pulmonary disease than P. aeruginosa alone. The interactions between P. aeruginosa biofilms and B. cepacia are not yet understood; one possible association being that mixed species biofilm formation may be part of the interspecies relationship. Using the Calgary Biofilm Device (CBD), members of all genomovars of the B. cepacia complex were shown to form biofilms, including those isolated from CF lungs. Mixed species biofilm formation between CF isolates of P. aeruginosa and B. cepacia was readily achieved using the CBD. Oxidation-fermentation lactose agar was adapted as a differential agar to monitor mixed biofilm composition. Scanning electron micrographs of the biofilms demonstrated that both species readily integrated in close association in the biofilm structure. Pseudomonas aeruginosa laboratory strain PAO1, however, inhibited mixed biofilm formation of both CF isolates and environmental strains of the B. cepacia complex. Characterization of the soluble inhibitor suggested pyocyanin as the active compound.     

Modelling Co-Infection of the Cystic Fibrosis Lung by Pseudomonas aeruginosa and Burkholderia cenocepacia Reveals Influences on Biofilm Formation and Host Response

The Gram-negative bacteria Pseudomonas aeruginosa and Burkholderia cenocepacia are opportunistic human pathogens that are responsible for severe nosocomial infections in immunocompromised patients and those suffering from cystic fibrosis (CF). These two bacteria have been shown to form biofilms in the airways of CF patients that make such infections more difficult to treat. Only recently have scientists begun to appreciate the complicated interplay between microorganisms during polymicrobial infection of the CF airway and the implications they may have for disease prognosis and response to therapy.To gain insight into the possible role that interaction between strains of P. aeruginosa and B. cenocepacia may play during infection, we characterised co-inoculations of in vivo and in vitro infection models. Co-inoculations were examined in an in vitro biofilm model and in a murine model of chronic infection. Assessment of biofilm formation showed that B. cenocepacia positively influenced P. aeruginosa biofilm development by increasing biomass. Interestingly, co-infection experiments in the mouse model revealed that P. aeruginosa did not change its ability to establish chronic infection in the presence of B. cenocepacia but co-infection did appear to increase host inflammatory response.Taken together, these results indicate that the co-infection of P. aeruginosa and B. cenocepacia leads to increased biofilm formation and increased host inflammatory response in the mouse model of chronic infection. These observations suggest that alteration of bacterial behavior due to interspecies interactions may be important for disease progression and persistent infection.     

Positive signature-tagged mutagenesis in Pseudomonas aeruginosa: tracking patho-adaptive mutations promoting airways chronic infection

The opportunistic pathogen Pseudomonas aeruginosa can establish life-long chronic infections in the airways of cystic fibrosis (CF) patients. Persistent lifestyle is established with P. aeruginosa patho-adaptive variants, which are clonal with the initially-acquired strains. Several reports indicated that P. aeruginosa adapts by loss-of-function mutations which enhance fitness in CF airways and sustain its clonal expansion during chronic infection. To validate this model of P. aeruginosa adaptation to CF airways and to identify novel genes involved in this microevolution, we designed a novel approach of positive-selection screening by PCR-based signature-tagged mutagenesis (Pos-STM) in a murine model of chronic airways infection. A systematic positive-selection scheme using sequential rounds of in vivo screenings for bacterial maintenance, as opposed to elimination, generated a list of genes whose inactivation increased the colonization and persistence in chronic airways infection. The phenotypes associated to these Pos-STM mutations reflect alterations in diverse aspects of P. aeruginosa biology which include lack of swimming and twitching motility, lack of production of the virulence factors such as pyocyanin, biofilm formation, and metabolic functions. In addition, Pos-STM mutants showed altered invasion and stimulation of immune response when tested in human respiratory epithelial cells, indicating that P. aeruginosa is prone to revise the interaction with its host during persistent lifestyle. Finally, sequence analysis of Pos-STM genes in longitudinally P. aeruginosa isolates from CF patients identified signs of patho-adaptive mutations within the genome. This novel Pos-STM approach identified bacterial functions that can have important clinical implications for the persistent lifestyle and disease progression of the airway chronic infection.     

IL-23 mediates inflammatory responses to mucoid Pseudomonas aeruginosa lung infection in mice

Patients with cystic fibrosis (CF) develop chronic Pseudomonas aeruginosa lung infection with mucoid strains of P. aeruginosa; these infections cause significant morbidity. The immunological response in these infections is characterized by an influx of neutrophils to the lung and subsequent lung damage over time; however, the underlying mediators to this response are not well understood. We recently reported that IL-23 and IL-17 were elevated in the sputum of patients with CF who were actively infected with P. aeruginosa; however, the importance of IL-23 and IL-17 in mediating this inflammation was unclear. To understand the role that IL-23 plays in initiating airway inflammation in response to P. aeruginosa, IL-23p19(-/-) (IL-23 deficient) and wild-type (WT) mice were challenged with agarose beads containing a clinical, mucoid isolate of P. aeruginosa. Levels of proinflammatory cytokines, chemokines, bacterial dissemination, and inflammatory infiltrates were measured. IL-23-deficient mice had significantly lower induction of IL-17, keratinocyte-derived chemokine (KC), and IL-6, decreased bronchoalveolar lavage (BAL) neutrophils, metalloproteinase-9 (MMP-9), and reduced airway inflammation than WT mice. Despite the reduced level of inflammation in IL-23p19(-/-) mice, there were no differences in the induction of TNF and interferon-gamma or in bacterial dissemination between the two groups. This study demonstrates that IL-23 plays a critical role in generating airway inflammation observed in mucoid P. aeruginosa infection and suggests that IL-23 could be a potential target for immunotherapy to treat airway inflammation in CF.     

Suppression of Drosophila cellular immunity by directed expression of the ExoS toxin GAP domain of Pseudomonas aeruginosa

We show here that transgenic Drosophila can be used to decipher the effect of a bacterial toxin on innate immunity and demonstrate the contribution of blood cells in fly resistance to bacterial infection. ExoS is a Pseudomonas aeruginosa exotoxin directly translocated into the host cell cytoplasm through the type III secretion system found in many Gram-negative bacteria. It contains a N-terminal GTPase activating (GAP) domain that prevents cytoskeleton reorganization by Rho family of GTPases in cell culture. Directed expression of the ExoS GAP domain (ExoSGAP) during fly eye morphogenesis inhibited Rac1-, Cdc42- and Rho-dependent signalling, demonstrating for the first time its activity on RhoGTPases in a whole organism. We further showed that fly resistance to P. aeruginosa infections was altered when ExoSGAP was expressed either ubiquitously or in haemocytes, but not when expressed into the fat body, the major source of NF-(kappa)B-dependent anti-microbial peptide synthesis. Fly sensitivity to infection was also observed with Gram-positive Staphylococcus aureus strain and was associated to a reduced phagocytosis capacity of ExoSGAP-expressing haemocytes. Our results highlight the major contribution of cellular immunity during the first hours after Drosophila infection by P. aeruginosa, an opportunist pathogen affecting patients with pathologies associated to a reduced leukocyte number.     

Metabolite cross-feeding enhances virulence in a model polymicrobial infection

Microbes within polymicrobial infections often display synergistic interactions resulting in enhanced pathogenesis; however, the molecular mechanisms governing these interactions are not well understood. Development of model systems that allow detailed mechanistic studies of polymicrobial synergy is a critical step towards a comprehensive understanding of these infections in vivo. In this study, we used a model polymicrobial infection including the opportunistic pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii to examine the importance of metabolite cross-feeding for establishing co-culture infections. Our results reveal that co-culture with S. gordonii enhances the pathogenesis of A. actinomycetemcomitans in a murine abscess model of infection. Interestingly, the ability of A. actinomycetemcomitans to utilize L-lactate as an energy source is essential for these co-culture benefits. Surprisingly, inactivation of L-lactate catabolism had no impact on mono-culture growth in vitro and in vivo suggesting that A. actinomycetemcomitans L-lactate catabolism is only critical for establishing co-culture infections. These results demonstrate that metabolite cross-feeding is critical for A. actinomycetemcomitans to persist in a polymicrobial infection with S. gordonii supporting the idea that the metabolic properties of commensal bacteria alter the course of pathogenesis in polymicrobial communities.     

Burkholderia cepacia complex bacteria: opportunistic pathogens with important natural biology

Interaction with plants around their roots and foliage forms the natural habitat for a wide range of gram-negative bacteria such as Burkholderia, Pseudomonas and Ralstonia. During these interactions many of these bacteria facilitate highly beneficial processes such as the breakdown of pollutants or enhancement of crop growth. All these bacterial species are also capable of causing opportunistic infections in vulnerable individuals, especially people with cystic fibrosis (CF). Here we will review the current understanding of the Burkholderia cepacia complex (Bcc) as a group of model opportunistic pathogens, contrasting their clinical epidemiology with their ecological importance. Currently, the B. cepacia complex is composed of nine formally named species groups which are all difficult to identify using phenotypic methods. Genetic methods such as 16S rRNA and recA gene sequence analysis have proven useful for Bcc species identification. Multilocus sequence typing (MLST) is also emerging as a very useful tool for both Bcc strain and species identification. Historically, Burkholderia cenocepacia was the most dominant Bcc pathogen in CF, however, probably as a result of strict infection control practices introduced to control the spread of this species, its prevalence has been reduced. Burkholderia multivorans is the now the most dominant Bcc infection encountered in the UK CF population, a changing epidemiology that also appears to be occurring in the US CF population. The distribution of Bcc species residing in the natural environment may vary considerably with the type of environment examined. Clonally identical Bcc strains have been found to occur in the natural environment and cause infection. The contamination of medical devices, disinfectants and pharmaceutical formulations has also been directly linked to several outbreaks of infection. In the last 10 years considerable progress has been made in understanding the natural biology and clinical infections caused by this fascinating group of bacteria.     

Multiple roles of TorD-like chaperones in the biogenesis of molybdoenzymes

Studies on cultured cells and in infection models have shown that cell density-dependent quorum-sensing (QS) controls many of the known virulence factors of Pseudomonas aeruginosa . However, it is less clear what role QS plays in chronic human lung infections associated with cystic fibrosis (CF). The involvement of QS in biofilm development, crucial to the establishment of long-term infections, suggests a role in the early stages of infection. However, the accumulation of QS mutants during chronic CF infections has been taken to indicate that any role diminishes thereafter. Here, we discuss the evidence for a continuing role for QS in P. aeruginosa CF infections, including QS activity in CF sputa and CF-relevant effects of QS-regulated products, such as pyocyanin. Bacterial population behaviour in CF is complex, and the exact roles of QS remains unclear. Therapeutic strategies directed against QS suggest that a greater understanding of bacterial populations during infection would be a valuable research goal from a clinical perspective.