High efficacy treatment of murine Pseudomonas aeruginosa catheter-associated urinary tract infections using the c-di-GMP modulating anti-biofilm compound Disperazol in combination with ciprofloxacin.

Persistent urinary tract infections (UTIs) in hospitalized patients constitute an important medical problem. It is estimated that 75% of nosocomial UTIs are associated with urinary tract catheters with P. aeruginosa being a species that forms biofilms on these catheters. These infections are highly resistant to standard-of-care antibiotics, and the effects of the host immune defenses, which allows for development of persistent infections. With antibiotics losing their efficacy, new treatment options against resilient infections, such as catheter-associated urinary tract infections (CAUTIs), are critically needed. Central to our anti-biofilm approach is the manipulation of the c-di-GMP signaling pathway in P. aeruginosa to switch bacteria from the protective biofilm to the unprotected planktonic mode of life. We recently identified a compound (H6-335-P1), that stimulates the c-di-GMP degrading activity of the P. aeruginosa BifA protein which plummets the intracellular c-di-GMP content and induces dispersal of P. aeruginosa biofilm bacteria into the planktonic state. In the present study, we formulated H6-335-P1 as a hydrochloride salt (Disperazol), which is water-soluble and facilitates delivery via injection or oral administration. Disperazol can work as a monotherapy, but we observed a 100-fold improvement in efficacy when treating murine P. aeruginosa CAUTIs with a Disperazol/ciprofloxacin combination. Biologically active Disperazol reached the bladder 30 min after oral administration. Our study provides proof of concept that Disperazol can be used in combination with a relevant antibiotic for effective treatment of CAUTIs.

Inhibition of Pseudomonas aeruginosa quorum sensing by chemical induction of the MexEF-oprN efflux pump.

The cell-to-cell communication system quorum sensing (QS), used by various pathogenic bacteria to synchronize gene expression and increase host invasion potentials, is studied as a potential target for persistent infection control. To search for novel molecules targeting the QS system in the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, a chemical library consisting of 3,280 small compounds from LifeArc was screened. A series of 10 conjugated phenones that have not previously been reported to target bacteria were identified as inhibitors of QS in P. aeruginosa. Two lead compounds (ethylthio enynone and propylthio enynone) were re-synthesized for verification of activity and further elucidation of the mode of action. The isomeric pure Z-ethylthio enynone was used for RNA sequencing, revealing a strong inhibitor of QS-regulated genes, and the QS-regulated virulence factors rhamnolipid and pyocyanin were significantly decreased by treatment with the compounds. A transposon mutagenesis screen performed in a newly constructed lasB-gfp monitor strain identified the target of Z-ethylthio enynone in P. aeruginosa to be the MexEF-OprN efflux pump, which was further established using defined mex knockout mutants. Our data indicate that the QS inhibitory capabilities of Z-ethylthio enynone were caused by the drainage of intracellular signal molecules as a response to chemical-induced stimulation of the MexEF-oprN efflux pump, thereby inhibiting the autogenerated positive feedback and its enhanced signal-molecule synthesis.

Novel sampling technique maintaining the two-dimensional organization of microbes during cultivation from chronic wounds: The Imprint method.

This study aimed to develop and validate "the Imprint method,", a technique for sampling microbes from chronic wounds while preserving their two-dimensional spatial organization. We used nylon filters to sample bacteria and compared with sampling using Eswabs in 12 patients. The Imprint method identified a mean of 0.93 unique species more than Eswab (4.3 ± 2.2 and 3.4 ± 1.4 unique species, respectively; mean ± SD; n = 30). Accuracy between the Eswab and the Imprint method was 93.2% and in cases of disagreement between methods, Imprint had a higher sensitivity in 6/8 of the most prevalent species. In vitro validation confirmed that the Imprint method could transfer bacterial colonies while replicating their two-dimensional organization and the area covered by bacteria on the plate sampled. Clinical testing demonstrated that the imprint method is a rapid and feasible technique that identified more unique bacterial species than Eswab with a good agreement between methods but that Imprint was better at detecting important pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. The Imprint method is a novel technique that cultures and records the two-dimensional organization of microbes, providing an alternative or supplement to conventional surface culture using Eswab.

Single cells and bacterial biofilm populations in chronic wound infections.

Chronic wounds and chronic ulcers are an increasing problem associated with high health care burden and patient burden. The arrested healing of chronic wounds has, in part, been attributed to the presence of biofilms. Substantial research has documented the presence of biofilms in chronic wounds, and many mechanisms of host-pathogen interactions have been uncovered to explain the arrested healing. However, the paradigm of whether biofilms are only observed in chronic infections was recently challenged when biofilms were also observed in acute infections. Here, we characterize the distribution of bacteria in lower leg wounds with particular emphasis on Pseudomonas aeruginosa and Staphylococcus aureus by confocal laser scanning microscopy combined with PNA-FISH staining and routine culture of bacteria. We show that 40% of wounds contained either P. aeruginosa or S. aureus biofilms and demonstrate the presence of scattered single cells in tissues stained with a universal bacterial PNA-FISH probe. Thus, we demonstrate that chronic wounds do not only harbor bacteria organized in biofilms, but also carry populations of scattered single cells and small cell clusters of only a few bacteria. Our findings may influence diagnostic tools being developed to only target biofilms, where single-cell subpopulations thus may be overlooked and possibly lead to false-negative results.

What's in a name? Characteristics of clinical biofilms.

In vitro biofilms are communities of microbes with unique features compared to individual cells. Biofilms are commonly characterized by physical traits like size, adhesion, and a matrix made of extracellular substances. They display distinct phenotypic features, such as metabolic activity and antibiotic tolerance. However, the relative importance of these traits depends on the environment and bacterial species. Various mechanisms enable biofilm-associated bacteria to withstand antibiotics, including physical barriers, physiological adaptations, and changes in gene expression. Gene expression profiles in biofilms differ from individual cells but, there is little consensus among studies and so far, a 'biofilm signature transcriptome' has not been recognized. Additionally, the spatial and temporal variability within biofilms varies greatly depending on the system or environment. Despite all these variable conditions, which produce very diverse structures, they are all noted as biofilms. We discuss that clinical biofilms may differ from those grown in laboratories and found in the environment and discuss whether the characteristics that are commonly used to define and characterize biofilms have been shown in infectious biofilms. We emphasize that there is a need for a comprehensive understanding of the specific traits that are used to define bacteria in infections as clinical biofilms.

Inoculum Concentration Influences Pseudomonas aeruginosa Phenotype and Biofilm Architecture.

In infections, bacterial cells are often found as relatively small multicellular aggregates characterized by a heterogeneous distribution of phenotype, genotype, and growth rates depending on their surrounding microenvironment. Many laboratory models fail to mimic these characteristics, and experiments are often initiated from planktonic bacteria given optimal conditions for rapid growth without concerns about the microenvironmental characteristics during biofilm maturation. Therefore, we investigated how the initial bacterial concentration (henceforth termed the inoculum) influences the microenvironment during initial growth and how this affects the sizes and distribution of developed aggregates in an embedded biofilm model-the alginate bead biofilm model. Following 24 h of incubation, the viable biomass was independent of starting inoculum but with a radically different microenvironment which led to differences in metabolic activity depending on the inoculum. The inoculum also affected the number of cells surviving treatment with the antibiotic tobramycin, where the highest inoculum showed higher survival rates than the lowest inoculum. The change in antibiotic tolerance was correlated with cell-specific RNA content and O2 consumption rates, suggesting a direct role of metabolic activity. Thus, the starting number of bacteria results in different phenotypic trajectories governed by different microenvironmental characteristics, and we demonstrate some of the possible implications of such physiological gradients on the outcome of in vitro experiments. IMPORTANCE Biofilm aggregates grown in the alginate bead biofilm model bear resemblance to features of in vivo biofilms. Here, we show that changing the initial concentration of bacteria in the biofilm model leads to widely different behavior of the bacteria following an incubation period. This difference is influenced by the local conditions experienced by the bacteria during growth, which impact their response to antibiotic treatment. Our study provides a framework for manipulating aggregate sizes in in vitro biofilm models. It underlines the importance of how experiments are initiated, which can profoundly impact the outcomes and interpretation of microbiological experiments.

Biofilm Survival Strategies in Chronic Wounds.

Bacterial biofilms residing in chronic wounds are thought to have numerous survival strategies, making them extremely difficult to eradicate and resulting in long-term infections. However, much of our knowledge regarding biofilm persistence stems from in vitro models and experiments performed in vivo in animal models. While the knowledge obtained from such experiments is highly valuable, its direct translation to the human clinical setting should be undertaken with caution. In this review, we highlight knowledge obtained from human clinical samples in different aspects of biofilm survival strategies. These strategies have been divided into segments of the following attributes: altered transcriptomic profiles, spatial distribution, the production of extracellular polymeric substances, an altered microenvironment, inter-and intra-species interactions, and heterogeneity in the bacterial population. While all these attributes are speculated to contribute to the enhanced persistence of biofilms in chronic wounds, only some of them have been demonstrated to exist in human wounds. Some of the attributes have been observed in other clinical diseases while others have only been observed in vitro. Here, we have strived to clarify the limitations of the current knowledge in regard to this specific topic, without ignoring important in vitro and in vivo observations.

The structure-function relationship of Pseudomonas aeruginosa in infections and its influence on the microenvironment.

Pseudomonas aeruginosa is a human pathogen associated with both acute and chronic infections. While intensively studied, the basic mechanisms enabling the long-term survival of P. aeruginosa in the host, despite massive immune system attack and heavy antimicrobial treatment, remain to be identified. We argue that such infections may represent niche invasions by P. aeruginosa that influence the microenvironment by depleting host-derived substrate and activating the immune response. Bacteria embedded in cell aggregates establish a microenvironmental niche, where they endure the initial host response by slowing down their metabolism. This provides stable, lasting growth conditions with a constant, albeit slow supply of substrate and electron acceptors. Under such stable conditions, P. aeruginosa exhibits distinct adaptive traits, where its gene expression pattern reflects a life exposed to continuous attack by the host immune system and antimicrobials. Here, we review fundamental microenvironmental aspects of chronic P. aeruginosa infections and examine how their structural organization influences their in vivo microenvironment, which in turn affects the interaction of P. aeruginosa biofilm aggregates with the host immune system. We discuss how improving our knowledge about the microenvironmental ecology of P. aeruginosa in chronic infections can be used to combat persistent, hard-to-treat bacterial infections.

Nitric-oxide-driven oxygen release in anoxic Pseudomonas aeruginosa.

Denitrification supports anoxic growth of Pseudomonas aeruginosa in infections. Moreover, denitrification may provide oxygen (O2) resulting from dismutation of the denitrification intermediate nitric oxide (NO) as seen in Methylomirabilis oxyfera. To examine the prevalence of NO dismutation we studied O2 release by P. aeruginosa in airtight vials. P. aeruginosa rapidly depleted O2 but NO supplementation generated peaks of O2 at the onset of anoxia, and we demonstrate a direct role of NO in the O2 release. However, we were not able to detect genetic evidence for putative NO dismutases. The supply of endogenous O2 at the onset of anoxia could play an adaptive role when P. aeruginosa enters anaerobiosis. Furthermore, O2 generation by NO dismutation may be more widespread than indicated by the reports on the distribution of homologues genes. In general, NO dismutation may allow removal of nitrate by denitrification without release of the very potent greenhouse gas, nitrous oxide.

Identification of small molecules that interfere with c-di-GMP signaling and induce dispersal of Pseudomonas aeruginosa biofilms.

Microbial biofilms are involved in a number of infections that cannot be cured, as microbes in biofilms resist host immune defenses and antibiotic therapies. With no strict biofilm-antibiotic in the current pipelines, there is an unmet need for drug candidates that enable the current antibiotics to eradicate bacteria in biofilms. We used high-throughput screening to identify chemical compounds that reduce the intracellular c-di-GMP content in Pseudomonas aeruginosa. This led to the identification of a small molecule that efficiently depletes P. aeruginosa for c-di-GMP, inhibits biofilm formation, and disperses established biofilm. A combination of our lead compound with standard of care antibiotics showed improved eradication of an implant-associated infection established in mice. Genetic analyses provided evidence that the anti-biofilm compound stimulates the activity of the c-di-GMP phosphodiesterase BifA in P. aeruginosa. Our work constitutes a proof of concept for c-di-GMP phosphodiesterase-activating drugs administered in combination with antibiotics as a viable treatment strategy for otherwise recalcitrant infections.

Induction of Native c-di-GMP Phosphodiesterases Leads to Dispersal of Pseudomonas aeruginosa Biofilms.

A decade of research has shown that the molecule c-di-GMP functions as a central second messenger in many bacteria. A high level of c-di-GMP is associated with biofilm formation, whereas a low level of c-di-GMP is associated with a planktonic single-cell bacterial lifestyle. c-di-GMP is formed by diguanylate cyclases and is degraded by specific phosphodiesterases. We previously presented evidence that the ectopic expression of the Escherichia coli phosphodiesterase YhjH in Pseudomonas aeruginosa results in biofilm dispersal. More recently, however, evidence has been presented that the induction of native c-di-GMP phosphodiesterases does not lead to a dispersal of P. aeruginosa biofilms. The latter result may discourage attempts to use c-di-GMP signaling as a target for the development of antibiofilm drugs. However, here, we demonstrate that the induction of the P. aeruginosa c-di-GMP phosphodiesterases PA2133 and BifA indeed results in the dispersal of P. aeruginosa biofilms in both a microtiter tray biofilm assay and a flow cell biofilm system.

Sampling challenges in diagnosis of chronic bacterial infections.

In recent decades there has been an increase in knowledge of the distribution, species diversity and growth patterns of bacteria in human chronic infections. This has challenged standard diagnostic methods, which have undergone a development to both increase the accuracy of testing as well as to decrease the occurrence of contamination. In particular, the introduction of new technologies based on molecular techniques into the clinical diagnostic process has increased detection and identification of infectious pathogens. Sampling is the first step in the diagnostic process, making it crucial for obtaining a successful outcome. However, sampling methods have not developed at the same speed as molecular identification. The heterogeneous distribution and potentially small number of pathogenic bacterial cells in chronic infected tissue makes sampling a complicated task, and samples must be collected judiciously and handled with care. Clinical sampling is a step in the diagnostic process that may benefit from innovative methods based on current knowledge of bacteria present in chronic infections. In the present review, we describe and discuss different aspects that complicate sampling of chronic infections. The purpose is to survey representative scientific work investigating the presence and distribution of bacteria in chronic infections in relation to various clinical sampling methods.

Small Molecule Anti-biofilm Agents Developed on the Basis of Mechanistic Understanding of Biofilm Formation.

Microbial biofilms are the cause of persistent infections associated with various medical implants and distinct body sites such as the urinary tract, lungs, and wounds. Compared with their free living counterparts, bacteria in biofilms display a highly increased resistance to immune system activities and antibiotic treatment. Therefore, biofilm infections are difficult or impossible to treat with our current armory of antibiotics. The challenges associated with biofilm infections have urged researchers to pursue a better understanding of the molecular mechanisms that are involved in the formation and dispersal of biofilms, and this has led to the identification of several steps that could be targeted in order to eradicate these challenging infections. Here we describe mechanisms that are involved in the regulation of biofilm development in Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii, and provide examples of chemical compounds that have been developed to specifically inhibit these processes. These compounds include (i) pilicides and curlicides which inhibit the initial steps of biofilm formation by E. coli; (ii) compounds that interfere with c-di-GMP signaling in P. aeruginosa and E. coli; and (iii) compounds that inhibit quorum-sensing in P. aeruginosa and A. baumannii. In cases where compound series have a defined molecular target, we focus on elucidating structure activity relationship (SAR) trends within the particular compound series.

Oxidative stress response plays a role in antibiotic tolerance of Streptococcus mutans biofilms.

Knowledge about biofilm-associated antibiotic tolerance mechanisms is warranted in order to develop effective treatments against biofilm infections. We performed a screen of a Streptococcus mutans transposon mutant library for mutants with reduced biofilm-associated antimicrobial tolerance, and found that the spxA1 gene plays a role in tolerance towards gentamicin and other antibiotics such as vancomycin and linezolid. SpxA1 is a regulator of genes involved in the oxidative stress response in S. mutans. The oxidative stress response genes gor and ahpC were found to be up-regulated upon antibiotic treatment of S. mutans wild-type biofilms, but not spxA1 mutant biofilms. The gor gene product catalyses the formation of glutathione which functions as an important antioxidant during oxidative stress, and accordingly biofilm-associated antibiotic tolerance of the spxA1 mutant could be restored by exogenous addition of glutathione. Our results indicate that the oxidative stress response plays a role in biofilm-associated antibiotic tolerance of S. mutans, and add to the on-going debate on the role of reactive oxygen species in antibiotic mediated killing of bacteria.

Imaging N-Acyl Homoserine Lactone Quorum Sensing In Vivo.

In order to study N-acyl homoserine lactone (AHL)-based quorum sensing in vivo, we present a protocol using an Escherichia coli strain equipped with a luxR-based monitor system, which in the presence of exogenous AHL molecules expresses a green fluorescent protein (GFP). Lungs from mice challenged intratracheally with alginate beads containing both a Pseudomonas aeruginosa strain together with the E. coli monitor strain can be investigated at different time points postinfection. Epifluorescent or confocal scanning laser microscopy (CSLM) is used to detect the GFP-expressing E. coli monitor strain in the lung tissues, indicating production and excretion of AHLs in vivo by the infecting P. aeruginosa.

Qualitative and Quantitative Determination of Quorum Sensing Inhibition In Vitro.

The formation of biofilms in conjunction with quorum sensing (QS) regulated expression of virulence by opportunistic pathogens contributes significantly to immune evasion and tolerance to a variety of antimicrobial treatments. The present protocol describes methods to determine the in vitro efficacy of potential QS inhibitors (QSIs). Work on Pseudomonas aeruginosa has shown that chemical blockage of QS is a promising new antimicrobial strategy. Several live bacterial reporter systems have been developed to screen extracts and pure compounds for QSI activity. Here we describe the usage of reporter strains consisting of a lasB-gfp or rhlA-gfp fusion in P. aeruginosa for qualitative and quantitative evaluation of the inhibition of two of the major QS pathways, monitored as reduced expression of green fluorescence. By the use of an in vitro flow cell system it is possible to study the QSI activity by monitoring its ability to interfere with the protective functions of bacterial biofilm. For evaluation of the global effects of QSI compounds, we present a protocol for the DNA microarray based transcriptomics. Using these in vitro methods it is possible to evaluate the potential of various QSI compounds.

Metagenomic and metatranscriptomic analysis of saliva reveals disease-associated microbiota in patients with periodontitis and dental caries.

The taxonomic composition of the salivary microbiota has been reported to differentiate between oral health and disease. However, information on bacterial activity and gene expression of the salivary microbiota is limited. The purpose of this study was to perform metagenomic and metatranscriptomic characterization of the salivary microbiota and test the hypothesis that salivary microbial presence and activity could be an indicator of the oral health status. Stimulated saliva samples were collected from 30 individuals (periodontitis: n = 10, dental caries: n = 10, oral health: n = 10). Salivary microbiota was characterized using metagenomics and metatranscriptomics in order to compare community composition and the gene expression between the three groups. Streptococcus was the predominant bacterial genus constituting approx. 25 and 50% of all DNA and RNA reads, respectively. A significant disease-associated higher relative abundance of traditional periodontal pathogens such as Porphyromonas gingivalis and Filifactor alocis and salivary microbial activity of F. alocis was associated with periodontitis. Significantly higher relative abundance of caries-associated bacteria such as Streptococcus mutans and Lactobacillus fermentum was identified in saliva from patients with dental caries. Multiple genes involved in carbohydrate metabolism were significantly more expressed in healthy controls compared to periodontitis patients. Using metagenomics and metatranscriptomics we show that relative abundance of specific oral bacterial species and bacterial gene expression in saliva associates with periodontitis and dental caries. Further longitudinal studies are warranted to evaluate if screening of salivary microbial activity of specific oral bacterial species and metabolic gene expression can identify periodontitis and dental caries at preclinical stages.

Bacterial Biofilm Control by Perturbation of Bacterial Signaling Processes.

The development of effective strategies to combat biofilm infections by means of either mechanical or chemical approaches could dramatically change today's treatment procedures for the benefit of thousands of patients. Remarkably, considering the increased focus on biofilms in general, there has still not been invented and/or developed any simple, efficient and reliable methods with which to "chemically" eradicate biofilm infections. This underlines the resilience of infective agents present as biofilms and it further emphasizes the insufficiency of today's approaches used to combat chronic infections. A potential method for biofilm dismantling is chemical interception of regulatory processes that are specifically involved in the biofilm mode of life. In particular, bacterial cell to cell signaling called "Quorum Sensing" together with intracellular signaling by bis-(3'-5')-cyclic-dimeric guanosine monophosphate (cyclic-di-GMP) have gained a lot of attention over the last two decades. More recently, regulatory processes governed by two component regulatory systems and small non-coding RNAs have been increasingly investigated. Here, we review novel findings and potentials of using small molecules to target and modulate these regulatory processes in the bacterium Pseudomonas aeruginosa to decrease its pathogenic potential.

Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In Vivo-Like Characteristics.

Alginate beads represent a simple and highly reproducible in vitro model system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated with Pseudomonas aeruginosa and followed for up to 72 h. Confocal microscopy revealed that P. aeruginosa formed dense clusters similar in size to in vivo aggregates observed ex vivo in cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2 concentration decreased rapidly and reached anoxia ∼100 µm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3- as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulated P. aeruginosa confirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulated P. aeruginosa exhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth of P. aeruginosa in alginate beads represent a simple and flexible in vivo-like biofilm model system, wherein bacterial growth exhibits central features of in vivo biofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3- as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment.IMPORTANCEPseudomonas aeruginosa has been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygen-depleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation.

Disulfide Bond-Containing Ajoene Analogues As Novel Quorum Sensing Inhibitors of Pseudomonas aeruginosa.

Since its discovery 22 years ago, the bacterial cell-to-cell communication system, termed quorum sensing (QS), has shown potential as antipathogenic target. Previous studies reported that ajoene from garlic inhibits QS in opportunistic human pathogen Pseudomonas aeruginosa. In this study, screening of an in-house compound library revealed two sulfur-containing compounds which possess structural resemblance with ajoene and inhibit QS in bioreporter assay. Following a quantitative structure-activity relationship (SAR) study, 25 disulfide bond-containing analogues were synthesized and tested for QS inhibition activities. SAR study indicated that the allyl group could be replaced with other substituents, with the most active being benzothiazole derivative (IC50 = 0.56 µM). The compounds were able to reduce QS-regulated virulence factors (elastase, rhamnolipid, and pyocyanin) and successfully inhibit P. aeruginosa infection in murine model of implant-associated infection. Altogether, the QS inhibition activity of the synthesized compounds is encouraging for further exploration of novel analogues in antimicrobial drug development.