Investigation of the Mechanism and Chemistry Underlying Staphylococcus aureus' Ability to Inhibit Pseudomonas aeruginosa Growth In Vitro.

Pseudomonas aeruginosa inhibits or eradicates Staphylococcus aureus in most in vitro settings. Nonetheless, P. aeruginosa and S. aureus are commonly isolated from chronically infected, nonhealing wounds and lungs of people with cystic fibrosis (CF). Therefore, we hypothesized that S. aureus could protect itself from P. aeruginosa through glucose-derived metabolites, such as small organic acids, preventing it from being eradicated. This in vitro study demonstrated that S. aureus populations, in the presence of glucose, secrete one or more substances that efficiently eradicate P. aeruginosa in a concentration-dependent manner. These substances had a molecular mass lower than three kDa, were hydrophilic, heat- and proteinase-resistant, and demonstrated a pH-dependent effect. Nuclear magnetic resonance analysis identified acetoin, acetic acid, and oligopeptides or cyclic peptides in glucose-grown S. aureus supernatants. All the tested wild-type and clinical S. aureus strain inhibited P. aeruginosa growth. Thus, we proposed a model in which a cocktail of these compounds, produced by established S. aureus populations in glucose presence, facilitated these two species' coexistence in chronic infections. IMPORTANCE Chronic infections affect a growing part of the population and are associated with high societal and personal costs. Multiple bacterial species are often present in these infections, and multispecies infections are considered more severe than single-species infections. Staphylococcus aureus and Pseudomonas aeruginosa often coexist in chronic infections. However, the interactions between these two species and their coexistence in chronic infections are not fully understood. By exploring in vitro interactions, we found a novel S. aureus-mediated inhibition of P. aeruginosa, and we suggested a model of the coexistence of the two species in chronic infections. With this study, we enhanced our understanding of the pathogenesis of chronic multispecies infections, which is crucial to paving the way for developing improved treatment strategies.

The origin of extracellular DNA in bacterial biofilm infections in vivo.

Extracellular DNA (eDNA) plays an important role in both the aggregation of bacteria and in the interaction of the resulting biofilms with polymorphonuclear leukocytes (PMNs) during an inflammatory response. Here, transmission electron and confocal scanning laser microscopy were used to examine the interaction between biofilms of Pseudomonas aeruginosa and PMNs in a murine implant model and in lung tissue from chronically infected cystic fibrosis patients. PNA FISH, DNA staining, labeling of PMN DNA with a thymidine analogue and immunohistochemistry were applied to localize bacteria, eDNA, PMN-derived eDNA, PMN-derived histone H3 (H3), neutrophil elastase (NE) and citrullinated H3 (citH3). Host-derived eDNA was observed surrounding bacterial biofilms but not within the biofilms. H3 localized to the lining of biofilms while NE was found throughout biofilms. CitH3, a marker for neutrophil extracellular traps (NETs) was detected only sporadically indicating that most host-derived eDNA in vivo was not a result of NETosis. Together these observations show that, in these in vivo biofilm infections with P. aeruginosa, the majority of eDNA is found external to the biofilm and derives from the host.

Interleukin-26 (IL-26) is a novel anti-microbial peptide produced by T cells in response to staphylococcal enterotoxin.

Anti-microbial peptides are produced at outer and inner surfaces by epithelia and innate immune cells in response to bacterial infection. Staphylococcus aureus is an enterotoxin producing, Gram-positive pathogen, which is a major cause of soft tissue infections and life-threatening bacteremia and sepsis. Here we show that (i) skin T cells in chronic wounds infected with S. aureus express interleukin-26 (IL-26) in situ, (ii) staphylococcal enterotoxins (SE) trigger IL-26 expression in T cell lines and primary skin T cells, and (iii) IL-26 triggers death and inhibits biofilm formation and growth of S. aureus. Thus, we provide novel evidence that IL-26 is an anti-microbial peptide produced by T cells in response to SE. Accordingly, we propose that IL-26 producing T cells take part in the innate immune response to SE producing S. aureus and thus play a novel role in the primary innate immune defense in addition to their classical role in adaptive immunity.

Activation of pulmonary and lymph node dendritic cells during chronic Pseudomonas aeruginosa lung infection in mice.

The majority of cystic fibrosis (CF) patients acquire chronic Pseudomonas aeruginosa lung infection, resulting in increased mortality and morbidity. The chronic P. aeruginosa lung infection is characterized by bacteria growing in biofilm surrounded by polymorphonuclear neutrophils (PMNs). However, the infection is not eradicated and the inflammatory response leads to gradual degradation of the lung tissue. In CF patients, a Th2-dominated adaptive immune response with a pronounced antibody response is correlated with poorer outcome. Dendritic cells (DCs) are crucial in bridging the innate immune system with the adaptive immune response. Once activated, the DCs deliver a set of signals to uncommitted T cells that induce development, such as expansion of regulatory T cells and polarization of Th1, Th2 or Th17 subsets. In this study, we characterized DCs in lungs and regional lymph nodes in BALB/c mice infected using intratracheal installation of P. aeruginosa embedded in seaweed alginate in the lungs. A significantly elevated concentration of DCs was detected earlier in the lungs than in the regional lymph nodes. To evaluate whether the chronic P. aeruginosa lung infection leads to activation of DCs, costimulatory molecules CD80 and CD86 were analyzed. During infection, the DCs showed significant elevation of CD80 and CD86 expression in both the lungs and the regional lymph nodes. Interestingly, the percentage of CD86-positive cells was significantly higher than the percentage of CD80-positive cells in the lymph nodes. In addition, cytokine production from Lipopolysaccharides (LPS)-stimulated DCs was analyzed demonstrating elevated production of IL-6, IL-10 and IL-12. However, production of IL-12 was suppressed earlier than IL-6 and IL-10. These results support that DCs are involved in skewing of the Th1/Th2 balance in CF and may be a possible treatment target.

In silico analyses of metagenomes from human atherosclerotic plaque samples.

BACKGROUND: Through several observational and mechanistic studies, microbial infection is known to promote cardiovascular disease. Direct infection of the vessel wall, along with the cardiovascular risk factors, is hypothesized to play a key role in the atherogenesis by promoting an inflammatory response leading to endothelial dysfunction and generating a proatherogenic and prothrombotic environment ultimately leading to clinical manifestations of cardiovascular disease, e.g., acute myocardial infarction or stroke. There are many reports of microbial DNA isolation and even a few studies of viable microbes isolated from human atherosclerotic vessels. However, high-resolution investigation of microbial infectious agents from human vessels that may contribute to atherosclerosis is very limited. In spite of the progress in recent sequencing technologies, analyzing host-associated metagenomes remain a challenge. RESULTS: To investigate microbiome diversity within human atherosclerotic tissue samples, we employed high-throughput metagenomic analysis on: (1) atherosclerotic plaques obtained from a group of patients who underwent endarterectomy due to recent transient cerebral ischemia or stroke. (2) Presumed stabile atherosclerotic plaques obtained from autopsy from a control group of patients who all died from causes not related to cardiovascular disease. Our data provides evidence that suggest a wide range of microbial agents in atherosclerotic plaques, and an intriguing new observation that shows these microbiota displayed differences between symptomatic and asymptomatic plaques as judged from the taxonomic profiles in these two groups of patients. Additionally, functional annotations reveal significant differences in basic metabolic and disease pathway signatures between these groups. CONCLUSIONS: We demonstrate the feasibility of novel high-resolution techniques aimed at identification and characterization of microbial genomes in human atherosclerotic tissue samples. Our analysis suggests that distinct groups of microbial agents might play different roles during the development of atherosclerotic plaques. These findings may serve as a reference point for future studies in this area of research.

Antibiofilm Properties of Acetic Acid.

Bacterial biofilms are known to be extremely tolerant toward antibiotics and other antimicrobial agents. These biofilms cause the persistence of chronic infections. Since antibiotics rarely resolve these infections, the only effective treatment of chronic infections is surgical removal of the infected implant, tissue, or organ and thereby the biofilm. Acetic acid is known for its antimicrobial effect on bacteria in general, but has never been thoroughly tested for its efficacy against bacterial biofilms. In this article, we describe complete eradication of both Gram-positive and Gram-negative biofilms using acetic acid both as a liquid and as a dry salt. In addition, we present our clinical experience of acetic acid treatment of chronic wounds. In conclusion, we here present the first comprehensive in vitro and in vivo testing of acetic acid against bacterial biofilms.

Autofluorescence in samples obtained from chronic biofilm infections--"all that glitters is not gold".

When looking at tissue sections of ex vivo samples, autofluorescence can be a major cause of artifacts and misinterpretations. We here reiterate evidence that autofluorescing granules, often hemosiderin but also ceroid or mucinogen granules, are severe obstacles when imaging and diagnosing biofilm infections through fluorescent imaging techniques. We used confocal laser scanning microscopy with spectral analysis for autofluorescence detection as well as standard histological stains in order to identify the culprit and show that these granules might very well be mistaken for bacterial biofilms. Furthermore, we hypothesize that the increased amount of autofluorescing granules may be a consequence of prolonged inflammation as a consequence of chronic biofilm infections.

Physiological levels of nitrate support anoxic growth by denitrification of Pseudomonas aeruginosa at growth rates reported in cystic fibrosis lungs and sputum.

Chronic Pseudomonas aeruginosa lung infection is the most severe complication in patients with cystic fibrosis (CF). The infection is characterized by the formation of biofilm surrounded by numerous polymorphonuclear leukocytes (PMNs) and strong O2 depletion in the endobronchial mucus. We have reported that O2 is mainly consumed by the activated PMNs, while O2 consumption by aerobic respiration is diminutive and nitrous oxide (N2O) is produced in infected CF sputum. This suggests that the reported growth rates of P. aeruginosa in lungs and sputum may result from anaerobic respiration using denitrification. The growth rate of P. aeruginosa achieved by denitrification at physiological levels (~400 µM) of nitrate (NO(-) 3) is however, not known. Therefore, we have measured growth rates of anoxic cultures of PAO1 and clinical isolates (n = 12) in LB media supplemented with NO(-) 3 and found a significant increase of growth when supplementing PAO1 and clinical isolates with ≥150 µM NO(-) 3 and 100 µM NO(-) 3, respectively. An essential contribution to growth by denitrification was demonstrated by the inability to establish a significantly increased growth rate by a denitrification deficient ΔnirS-N mutant at <1 mM of NO(-) 3. Activation of denitrification could be achieved by supplementation with as little as 62.5 µM of NO(-) 3 according to the significant production of N2O by the nitrous oxide reductase deficient ΔnosZ mutant. Studies of the promoter activity, gene transcripts, and enzyme activity of the four N-oxide reductases in PAO1 (Nar, Nir, Nor, Nos) further verified the engagement of denitrification, showing a transient increase in activation and expression and rapid consumption of NO(-) 3 followed by a transient increase of NO(-) 2. Growth rates obtained by denitrification in this study were comparable to our reported growth rates in the majority of P. aeruginosa cells in CF lungs and sputum. Thus, we have demonstrated that denitrification is required for P. aeruginosa growth in infected endobronchial CF mucus.

Polymorphonuclear leukocytes restrict growth of Pseudomonas aeruginosa in the lungs of cystic fibrosis patients.

Cystic fibrosis (CF) patients have increased susceptibility to chronic lung infections by Pseudomonas aeruginosa, but the ecophysiology within the CF lung during infections is poorly understood. The aim of this study was to elucidate the in vivo growth physiology of P. aeruginosa within lungs of chronically infected CF patients. A novel, quantitative peptide nucleic acid (PNA) fluorescence in situ hybridization (PNA-FISH)-based method was used to estimate the in vivo growth rates of P. aeruginosa directly in lung tissue samples from CF patients and the growth rates of P. aeruginosa in infected lungs in a mouse model. The growth rate of P. aeruginosa within CF lungs did not correlate with the dimensions of bacterial aggregates but showed an inverse correlation to the concentration of polymorphonuclear leukocytes (PMNs) surrounding the bacteria. A growth-limiting effect on P. aeruginosa by PMNs was also observed in vitro, where this limitation was alleviated in the presence of the alternative electron acceptor nitrate. The finding that P. aeruginosa growth patterns correlate with the number of surrounding PMNs points to a bacteriostatic effect by PMNs via their strong O2 consumption, which slows the growth of P. aeruginosa in infected CF lungs. In support of this, the growth of P. aeruginosa was significantly higher in the respiratory airways than in the conducting airways of mice. These results indicate a complex host-pathogen interaction in chronic P. aeruginosa infection of the CF lung whereby PMNs slow the growth of the bacteria and render them less susceptible to antibiotic treatment while enabling them to persist by anaerobic respiration.

Bacterial biofilm formation and treatment in soft tissue fillers.

Injection of soft tissue fillers plays an important role in facial reconstruction and esthetic treatments such as cosmetic surgery for lip augmentation and wrinkle smoothening. Adverse events are an increasing problem, and recently, it has been suggested that bacteria are the cause of a vast fraction these. We developed a novel mouse model and evaluated hyaluronic acid gel, calcium hydroxyl apatite microspheres, and polyacrylamide hydrogel for their potential for sustaining bacterial infections and their possible treatments. We were able to culture Pseudomonas aeruginosa, Staphylococcus epidermidis, and Probionibacterium acnes in all three gels. When contaminated gels were left for 7 days in a mouse model, we found sustainment of bacterial infection with the permanent gel, less with the semi-permanent gel, and no growth within the temporary gel. Evaluation of treatment strategies showed that once the bacteria had settled (into biofilms) within the gels, even successive treatments with high concentrations of relevant antibiotics were not effective. Our data substantiate bacteria as a cause of adverse reactions reported when using tissue fillers, and the sustainability of these infections appears to depend on longevity of the gel. Most importantly, the infections are resistant to antibiotics once established but can be prevented using prophylactic antibiotics.

Pseudomonas aeruginosa biofilms: mechanisms of immune evasion.

The opportunistic gram-negative bacterium Pseudomonas aeruginosa is implicated in many chronic infections and is readily isolated from chronic wounds, medical devices, and the lungs of cystic fibrosis patients. P. aeruginosa is believed to persist in the host organism due to its capacity to form biofilms, which protect the aggregated, biopolymer-embedded bacteria from the detrimental actions of antibiotic treatments and host immunity. A key component in the protection against innate immunity is rhamnolipid, which is a quorum sensing (QS)-regulated virulence factor. QS is a cell-to-cell signaling mechanism used to coordinate expression of virulence and protection of aggregated biofilm cells. Rhamnolipids are known for their ability to cause hemolysis and have been shown to cause lysis of several cellular components of the human immune system, for example, macrophages and polymorphonuclear leukocytes (PMNs). In this chapter, the interplay between P. aeruginosa and the PMNs in chronic infections is discussed with focus on the role of rhamnolipids and extracellular DNA.

The in vivo biofilm.

Bacteria can grow and proliferate either as single, independent cells or organized in aggregates commonly referred to as biofilms. When bacteria succeed in forming a biofilm within the human host, the infection often becomes very resistant to treatment and can develop into a chronic state. Biofilms have been studied for decades using various in vitro models, but it remains debatable whether such in vitro biofilms actually resemble in vivo biofilms in chronic infections. In vivo biofilms share several structural characteristics that differ from most in vitro biofilms. Additionally, the in vivo experimental time span and presence of host defenses differ from chronic infections and the chemical microenvironment of both in vivo and in vitro biofilms is seldom taken into account. In this review, we discuss why the current in vitro models of biofilms might be limited for describing infectious biofilms, and we suggest new strategies for improving this discrepancy.

Complete genome sequence of the cystic fibrosis pathogen Achromobacter xylosoxidans NH44784-1996 complies with important pathogenic phenotypes.

Achromobacter xylosoxidans is an environmental opportunistic pathogen, which infects an increasing number of immunocompromised patients. In this study we combined genomic analysis of a clinical isolated A. xylosoxidans strain with phenotypic investigations of its important pathogenic features. We present a complete assembly of the genome of A. xylosoxidans NH44784-1996, an isolate from a cystic fibrosis patient obtained in 1996. The genome of A. xylosoxidans NH44784-1996 contains approximately 7 million base pairs with 6390 potential protein-coding sequences. We identified several features that render it an opportunistic human pathogen, We found genes involved in anaerobic growth and the pgaABCD operon encoding the biofilm adhesin poly-ß-1,6-N-acetyl-D-glucosamin. Furthermore, the genome contains a range of antibiotic resistance genes coding efflux pump systems and antibiotic modifying enzymes. In vitro studies of A. xylosoxidans NH44784-1996 confirmed the genomic evidence for its ability to form biofilms, anaerobic growth via denitrification, and resistance to a broad range of antibiotics. Our investigation enables further studies of the functionality of important identified genes contributing to the pathogenicity of A. xylosoxidans and thereby improves our understanding and ability to treat this emerging pathogen.

Clearance of Pseudomonas aeruginosa foreign-body biofilm infections through reduction of the cyclic Di-GMP level in the bacteria.

Opportunistic pathogenic bacteria can engage in biofilm-based infections that evade immune responses and develop into chronic conditions. Because conventional antimicrobials cannot efficiently eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. It has recently been established that the secondary messenger cyclic diguanosine monophosphate (c-di-GMP) functions as a positive regulator of biofilm formation in several different bacteria. In the present study we investigated whether manipulation of the c-di-GMP level in bacteria potentially can be used for biofilm control in vivo. We constructed a Pseudomonas aeruginosa strain in which a reduction in the c-di-GMP level can be achieved via induction of the Escherichia coli YhjH c-di-GMP phosphodiesterase. Initial experiments showed that induction of yhjH expression led to dispersal of the majority of the bacteria in in vitro-grown P. aeruginosa biofilms. Subsequently, we demonstrated that P. aeruginosa biofilms growing on silicone implants, located in the peritoneal cavity of mice, dispersed after induction of the YhjH protein. Bacteria accumulated temporarily in the spleen after induction of biofilm dispersal, but the mice tolerated the dispersed bacteria well. The present work provides proof of the concept that modulation of the c-di-GMP level in bacteria is a viable strategy for biofilm control.

P. aeruginosa in the paranasal sinuses and transplanted lungs have similar adaptive mutations as isolates from chronically infected CF lungs.

BACKGROUND: Pseudomonas aeruginosa cells are present as biofilms in the paranasal sinuses and the lungs of chronically infected cystic fibrosis (CF) patients. Since different inflammatory responses and selective antibiotic pressures are acting in the sinuses compared with the lungs, we compared the adaptive profiles of mucoid and non-mucoid isolates from the two locations. METHODS: We studied the genetic basis of phenotypic diversification and gene expression profiles in sequential lung and sinus P. aeruginosa isolates from four chronically infected CF patients, including pre- and post-lung transplantation isolates. RESULTS: The same phenotypes caused by similar mutations and similar gene expression profiles were found in mucoid and non-mucoid isolates from the paranasal sinuses and from the lungs before and after transplantation. CONCLUSION: Bilateral exchange of P. aeruginosa isolates between the paranasal sinuses and the lungs occurs in chronically infected patients and extensive sinus surgery before the lung transplantation might prevent infection of the new lung.

A non-fatal case of invasive zygomycete (Lichtheimia corymbifera) infection in an allogeneic haematopoietic cell transplant recipient.

Post-transplant infections in allogeneic haematopoietic cell transplant (allo-HCT) recipients often have severe consequences. This is especially the case when dealing with zygomycete infections where the result is often fatal. A major problem when dealing with zygomycete infections is the need for an accurate and fast diagnosis as the phylum is highly resistant towards the conventional antifungals. We herein describe a non-fatal case of Lichtheimia corymbifera infection in an allo-HCT recipient.

Interactions between polymorphonuclear leukocytes and Pseudomonas aeruginosa biofilms on silicone implants in vivo.

Chronic infections with Pseudomonas aeruginosa persist because the bacterium forms biofilms that are tolerant to antibiotic treatment and the host immune response. Scanning electron microscopy and confocal laser scanning microscopy were used to visualize biofilm development in vivo following intraperitoneal inoculation of mice with bacteria growing on hollow silicone tubes, as well as to examine the interaction between these bacteria and the host innate immune response. Wild-type P. aeruginosa developed biofilms within 1 day that trapped and caused visible cavities in polymorphonuclear leukocytes (PMNs). In contrast, the number of cells of a P. aeruginosa rhlA mutant that cannot produce rhamnolipids was significantly reduced on the implants by day 1, and the bacteria were actively phagocytosed by infiltrating PMNs. In addition, we identified extracellular wire-like structures around the bacteria and PMNs, which we found to consist of DNA and other polymers. Here we present a novel method to study a pathogen-host interaction in detail. The data presented provide the first direct, high-resolution visualization of the failure of PMNs to protect against bacterial biofilms.

Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition.

Bacterial biofilms are imaged by various kinds of microscopy including confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). One limitation of CLSM is its restricted magnification, which is resolved by the use of SEM that provides high-magnification spatial images of how the single bacteria are located and interact within the biofilm. However, conventional SEM is limited by the requirement of dehydration of the samples during preparation. As biofilms consist mainly of water, the specimen dehydration might alter its morphology. High magnification yet authentic images are important to understand the physiology of biofilms. We compared conventional SEM, Focused Ion Beam (FIB)-SEM and CLSM with SEM techniques [cryo-SEM and environmental-SEM (ESEM)] that do not require dehydration. In the case of cryo-SEM, the biofilm is not dehydrated but kept frozen to obtain high-magnification images closer to the native state of the sample. Using the ESEM technique, no preparation is needed. Applying these methods to biofilms of Pseudomonas aeruginosa showed us that the dehydration of biofilms substantially influences its appearance and that a more authentic biofilm image emerges when combining all methods.