Adaptation of Pseudomonas aeruginosa biofilms to tobramycin and the quorum sensing inhibitor C-30 during experimental evolution requires multiple genotypic and phenotypic changes.

In the present study we evaluated the fitness, antimicrobial susceptibility, metabolic activity, gene expression, in vitro production of virulence factors and in vivo virulence of experimentally evolved Pseudomonas aeruginosa PAO1. These strains were previously evolved in the presence of tobramycin and the quorum sensing inhibitor furanone C-30 (C-30) and carried mutations in mexT and fusA1. Compared to the wild-type (WT), the evolved strains show a different growth rate and different metabolic activity, suggesting they have an altered fitness. mexT mutants were less susceptible to C-30 than WT strains; they also show reduced susceptibility to chloramphenicol and ciprofloxacin, two substrates of the MexEF-OprN efflux pump. fusA1 mutants had a decreased susceptibility to aminoglycoside antibiotics, and an increased susceptibility to chloramphenicol. The decreased antimicrobial susceptibility and decreased susceptibility to C-30 was accompanied by a changed metabolic activity profile during treatment. The expression of mexE was significantly increased in mexT mutants and induced by C-30, suggesting that MexEF-OprN exports C-30 out of the bacterial cell. The in vitro production of virulence factors as well as virulence in two in vivo models of the strains evolved in the presence of C-30 was unchanged compared to the virulence of the WT. Finally, the evolved strains were less susceptible towards tobramycin (alone and combined with C-30) in an in vivo mouse model. In conclusion, this study shows that mutations acquired during experimental evolution of P. aeruginosa biofilms in the presence of tobramycin and C-30, are accompanied by an altered fitness, metabolism, mexE expression and in vitro and in vivo antimicrobial susceptibility.

A collagen-based layered chronic wound biofilm model for testing antimicrobial wound products.

A new in vitro chronic wound biofilm model was recently published, which provided a layered scaffold simulating mammalian tissue composition on which topical wound care products could be tested. In this paper, we updated the model even further to mimic the dynamic influx of nutrients from below as is the case in a chronic wound. The modified in vitro model was created using collagen instead of agar as the main matrix component and contained both Staphylococcus aureus and Pseudomonas aeruginosa. The model was cast in transwell inserts and then placed in wound simulating media, which allowed for an exchange of nutrients and waste products across a filter. Three potential wound care products and chlorhexidine digluconate 2% solution as a positive control were used to evaluate the model. The tested products were composed of hydrogels made from completely biodegradable starch microspheres carrying different active compounds. The compounds were applied topically and left for 2-4 days. Profiles of oxygen concentration and pH were measured to assess the effect of treatments on bacterial activity. Confocal microscope images were obtained of the models to visualise the existence of microcolonies. Results showed that the modified in vitro model maintained a stable number of the two bacterial species over 6 days. In untreated models, steep oxygen gradients developed and pH increased to >8.0. Hydrogels containing active compounds alleviated the high oxygen consumption and decreased pH drastically. Moreover, all three hydrogels reduced the colony forming units significantly and to a larger extent than the chlorhexidine control treatment. Overall, the modified model expressed several characteristics similar to in vivo chronic wounds.

Optical Properties of Corals Distort Variable Chlorophyll Fluorescence Measurements.

Pulse-amplitude-modulated (PAM) fluorimetry is widely used in photobiological studies of corals, as it rapidly provides numerous photosynthetic parameters to assess coral ecophysiology. Coral optics studies have revealed the presence of light gradients in corals, which are strongly affected by light scattering in coral tissue and skeleton. We investigated whether coral optics affects variable chlorophyll (Chl) fluorescence measurements and derived photosynthetic parameters by developing planar hydrogel slabs with immobilized microalgae and with bulk optical properties similar to those of different types of corals. Our results show that PAM-based measurements of photosynthetic parameters differed substantially between hydrogels with different degrees of light scattering but identical microalgal density, yielding deviations in apparent maximal electron transport rates by a factor of 2. Furthermore, system settings such as the measuring light intensity affected F 0, Fm , and Fv /Fm in hydrogels with identical light absorption but different degrees of light scattering. Likewise, differences in microalgal density affected variable Chl fluorescence parameters, where higher algal densities led to greater Fv /Fm values and relative electron transport rates. These results have important implications for the use of variable Chl fluorimetry in ecophysiological studies of coral stress and photosynthesis, as well as other optically dense systems such as plant tissue and biofilms.

Light Sheet Microscopy Imaging of Light Absorption and Photosynthesis Distribution in Plant Tissue.

In vivo variable chlorophyll fluorescence measurements of photosystem II (PSII) quantum yields in optically dense systems are complicated by steep tissue light gradients due to scattering and absorption. Consequently, externally measured effective PSII quantum yields may be composed of signals derived from cells differentially exposed to actinic light, where cells located deeper inside tissues receive lower irradiance than cells closer to the surface and can display distinct photophysiological status. We demonstrate how measured distributions of PSII quantum yields in plant tissue change under natural tissue light gradients as compared with conventionally measured quantum yields with even exposure to actinic light. This was achieved by applying actinic irradiance perpendicular to one side of thallus cross sections of the aquatic macrophyte Fucus vesiculosus with laser light sheets of defined spectral composition, while imaging variable chlorophyll fluorescence from cross sections with a microscope-mounted pulse amplitude-modulated imaging system. We show that quantum yields are highly affected by light gradients and that traditional surface-based variable chlorophyll fluorescence measurements result in substantial underestimations and/or overestimations, depending on incident actinic irradiance. We present a method for using chlorophyll fluorescence profiles in combination with integrating sphere measurements of reflectance and transmittance to calculate depth-resolved photon absorption profiles, which can be used to correct apparent PSII electron transport rates to photons absorbed by PSII. Absorption profiles of the investigated aquatic macrophyte were different in shape from what is typically observed in terrestrial leaves, and based on this finding, we discuss strategies for optimizing photon absorption via modulation of the structural organization of phytoelements according to in situ light environments.

In Situ Hydrogen Dynamics in a Hot Spring Microbial Mat during a Diel Cycle.

UNLABELLED: Microbes can produce molecular hydrogen (H2) via fermentation, dinitrogen fixation, or direct photolysis, yet the H2 dynamics in cyanobacterial communities has only been explored in a few natural systems and mostly in the laboratory. In this study, we investigated the diel in situ H2 dynamics in a hot spring microbial mat, where various ecotypes of unicellular cyanobacteria (Synechococcus sp.) are the only oxygenic phototrophs. In the evening, H2 accumulated rapidly after the onset of darkness, reaching peak values of up to 30 µmol H2 liter(-1) at about 1-mm depth below the mat surface, slowly decreasing to about 11 µmol H2 liter(-1) just before sunrise. Another pulse of H2 production, reaching a peak concentration of 46 µmol H2 liter(-1), was found in the early morning under dim light conditions too low to induce accumulation of O2 in the mat. The light stimulation of H2 accumulation indicated that nitrogenase activity was an important source of H2 during the morning. This is in accordance with earlier findings of a distinct early morning peak in N2 fixation and expression of Synechococcus nitrogenase genes in mat samples from the same location. Fermentation might have contributed to the formation of H2 during the night, where accumulation of other fermentation products lowered the pH in the mat to less than pH 6 compared to a spring source pH of 8.3. IMPORTANCE: Hydrogen is a key intermediate in anaerobic metabolism, and with the development of a sulfide-insensitive microsensor for H2, it is now possible to study the microdistribution of H2 in stratified microbial communities such as the photosynthetic microbial mat investigated here. The ability to measure H2 profiles within the mat compared to previous measurements of H2 emission gives much more detailed information about the sources and sinks of H2 in such communities, and it was demonstrated that the high rates of H2 formation in the early morning when the mat was exposed to low light intensities might be explained by nitrogen fixation, where H2 is formed as a by-product.

Nanoparticle-based measurements of pH and O2 dynamics in the rhizosphere of Zostera marina L.: effects of temperature elevation and light-dark transitions.

Seagrasses can modulate the geochemical conditions in their immediate rhizosphere through the release of chemical compounds from their below-ground tissue. This is a vital chemical defence mechanism, whereby the plants detoxify the surrounding sediment. Using novel nanoparticle-based optical O2 and pH sensors incorporated in reduced and transparent artificial sediment, we investigated the spatio-temporal dynamics of pH and O2 within the entire rhizosphere of Zostera marina L. during experimental manipulations of light and temperature. We combined such measurements with O2 microsensor measurements of the photosynthetic productivity and respiration of seagrass leaves. We found pronounced pH and O2 microheterogeneity within the immediate rhizosphere of Z. marina, with higher below-ground tissue oxidation capability and rhizoplane pH levels during both light exposure of the leaf canopy and elevated temperature, where the temperature-mediated stimuli of biogeochemical processes seemed to predominate. Low rhizosphere pH microenvironments appeared to correlate with plant-derived oxic microzones stimulating local sulphide oxidation and thus driving local proton generation, although the rhizoplane pH levels generally where much higher than the bulk sediment pH. Our data show that Z. marina can actively alter its rhizosphere pH microenvironment alleviating the local H2 S toxicity and enhancing nutrient availability in the adjacent sediment via geochemical speciation shift.

Pronounced gradients of light, photosynthesis and O2 consumption in the tissue of the brown alga Fucus serratus.

Macroalgae live in an ever-changing light environment affected by wave motion, self-shading and light-scattering effects, and on the thallus scale, gradients of light and chemical parameters influence algal photosynthesis. However, the thallus microenvironment and internal gradients remain underexplored. In this study, microsensors were used to quantify gradients of light, O2 concentration, variable chlorophyll fluorescence, photosynthesis and O2 consumption as a function of irradiance in the cortex and medulla layers of Fucus serratus. The two cortex layers showed more efficient light utilization compared to the medulla, calculated both from electron transport rates through photosystem II and from photosynthesis-irradiance curves. At moderate irradiance, the upper cortex exhibited onset of photosynthetic saturation, whereas lower thallus layers exhibited net O2 consumption. O2 consumption rates in light varied with depth and irradiance and were more than two-fold higher than dark respiration. We show that the thallus microenvironment of F. serratus exhibits a highly stratified balance of production and consumption of O2 , and when the frond was held in a fixed position, high incident irradiance levels on the upper cortex did not saturate photosynthesis in the lower thallus layers. We discuss possible photoadaptive responses and consequences for optimizing photosynthetic activity on the basis of vertical differences in light attenuation coefficients.

Antimicrobial susceptibility testing of bone and joint pathogens using isothermal microcalorimetry.

The rise in osteomyelitis and periprosthetic joint infections, in combination with increasing life expectancy and the prevalence of diabetes, underscores the urgent need for rapid and accurate diagnostic tools. Conventional culture-based methods are often time-consuming and prone to false-negatives, leading to prolonged and inappropriate antibiotic treatments. This study aims to improve osteomyelitis diagnostics by decreasing the time to detection and the time to an antibiotic susceptibility result to enable a targeted treatment using isothermal microcalorimetry (IMC). IMC measures heat flow in real-time, providing insights into bacterial metabolism without the need for labeling. Using clinical isolates from bone infections, assessing their response to antibiotics through IMC, we demonstrated that IMC could detect bacteria within 4 h and determine antimicrobial susceptibility profiles within 2-22 h (median 4.85, range 1.28-21.78). This is significantly faster than traditional methods. A decision tree, based on antibiotic susceptibility, accurately categorized pathogens, achieving high accuracy (74-100%), sensitivity (100%), and specificity (65-100%). These findings suggest that IMC could redefine diagnostics of bone and joint infections and potentially infections in general, offering timely and precise treatment guidance, thereby improving patient outcomes and reducing health care burdens. Further optimization and clinical validation are needed to fully integrate IMC into routine diagnostics.

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.

Bacterial micro-aggregates as inoculum in animal models of implant-associated infections.

Is it time to rethink the inoculum of animal models of implant-associated infections (IAI)? Traditionally, animal models of IAI are based on inoculation with metabolically active overnight cultures of planktonic bacteria or pre-grown surface-attached biofilms. However, such inoculums do not mimic the clinical initiation of IAI. Therefore, the present study aimed to develop a clinically relevant inoculum of low metabolic micro-aggregated bacteria. The porcine Staphylococcus aureus strain S54F9 was cultured in Tryptone Soya Broth (TSB) for seven days to facilitate the formation of low metabolic micro-aggregates. Subsequently, the aggregated culture underwent filtration using cell strainers of different pore sizes to separate micro-aggregates. Light microscopy was used to evaluate the aggregate formation and size in the different fractions, while isothermal microcalorimetry was used to disclose a low metabolic activity. The micro-aggregate fraction obtained with filter size 5-15 µm (actual measured mean size 32 µm) was used as inoculum in a porcine implant-associated osteomyelitis (IAO) model and compared to a standard overnight planktonic inoculum and a sham inoculum of 0.9 % saline. The micro-aggregate and planktonic inoculums caused IAO with the re-isolation of S. aureus from soft tissues, bones, and implants. However, compared to their planktonic counterpart, neither of the micro-aggregate inoculated animals showed signs of osteomyelitis, i.e., sequester, osteolysis, and pus at gross inspection. Furthermore, inoculation with low metabolic micro-aggregates resulted in a strong healing response with pronounced osteoid formation, comparable to sham animals. In conclusion, the formation and separation of low metabolic bacterial micro-aggregates into various size fractions is possible, however, planktonic bacteria were still seen in all size fractions. Inoculation with micro-aggregates caused a less-aggressive osteomyelitis i.e. combination of infected tissue and strong healing response. Therefore, the use of low metabolic micro-aggregates could be a relevant inoculum for animal models of less-aggressive and thereby slower developing IAI and add in to our understanding of the host-implant-bacteria interactions in slow-onset low-grade infections.

The chronic wound characterisation study and biobank: a study protocol for a prospective observational cohort investigation of bacterial community composition, inflammatory responses and wound-healing trajectories in non-healing wounds.

INTRODUCTION: Chronic wounds affect 1%-2% of the global population, with rising incidence due to ageing and lifestyle-related diseases. Bacterial biofilms, found in 80% of chronic wounds, and scattered single-cell bacteria may hinder healing. Microbes are believed to negatively impact healing by exacerbating inflammation and host immune response. METHODS AND ANALYSIS: The primary objective of the chronic wound characterisation (CWC) study is to investigate chronic wounds through a prospective observational cohort study exploring bacterial community composition, inflammatory responses and the influence of bacteria on wound-healing trajectories. The CWC study will be investigated through two cohorts: the predictive and in-depth.The predictive cohort includes patients with a chronic wound scheduled for mechanical debridement. The debrided material will be collected for dual RNA sequencing and 16s ribosomal RNA gene sequencing, as well as samples for microbial culturing and a photo to assess the wound. Clinical data is recorded, and healing and/or other clinical endpoints are established through medical records.The in-depth cohort includes and follows patients undergoing split-thickness skin grafting. Extensive sampling (ESwabs, biopsies, tape strips, debrided material and a sample of the skin graft) will be performed on surgery and patients will be seen at two follow-up visits. Samples will be analysed through culturing and next-generation sequencing methods. A biobank will be established comprising longitudinal clinical samples and clinical data. ETHICS AND DISSEMINATION: The study has been approved by the board of health ethics, Capital Region of Denmark, under protocol number H-20032214. The study findings will be disseminated through peer-reviewed publications and showcased at both national and international conferences and meetings within the domains of microbiology, wound healing and infection.

The non-attached biofilm aggregate.

Biofilms have conventionally been perceived as dense bacterial masses on surfaces, following the five-step model of development. Initial biofilm research focused on surface-attached formations, but detached aggregates have received increasing attention in the past decade due to their pivotal role in chronic infections. Understanding their nature sparked fervent discussions in biofilm conferences and scientific literature. This review consolidates current insights on non-attached aggregates, offering examples of their occurrence in nature and diseases. We discuss their formation and dispersion mechanisms, resilience to antibiotics and immune-responses, drawing parallels to surface-attached biofilms. Moreover, we outline available in vitro models for studying non-attached aggregates.

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.

Protocol to assess metabolic activity of Pseudomonas aeruginosa by measuring heat flow using isothermal calorimetry.

Here, we present a protocol for assessing metabolic activity of bacterial populations by measuring heat flow using isothermal calorimetry. We outline the steps for preparing the different growth models of Pseudomonas aeruginosa and performing continuous metabolic activity measurements in the calScreener. We detail simple principal component analysis to differentiate between metabolic states of different populations and probabilistic logistic classification to assess resemblance to wild-type bacteria. This protocol for fine-scale metabolic measurement can aid in understanding microbial physiology. For complete details on the use and execution of this protocol, please refer to Lichtenberg et al. (2022).1.

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.

Pharmacokinetics of Locally Applied Antibiotic Prophylaxis for Implant-Based Breast Reconstruction.

Importance: Antibiotic irrigation of breast implants is widely used internationally, but no clinical study has investigated the pharmacokinetics of antibiotic prophylaxis in the breast implant pocket. Objectives: To evaluate how long locally applied gentamicin, cefazolin, and vancomycin concentrations in the implant pocket remain above the minimum inhibitory concentration (MIC) for the most common bacterial infections and to measure systemic uptake. Design, Setting, and Participants: This prospective cohort study was performed at the Department of Plastic Surgery and Burns Treatment, Rigshospitalet, Copenhagen, Denmark, between October 25, 2021, and September 22, 2022, among 40 patients undergoing implant-based breast reconstruction who were part of the ongoing BREAST-AB trial (Prophylactic Treatment of Breast Implants With a Solution of Gentamicin, Vancomycin and Cefazolin Antibiotics for Women Undergoing Breast Reconstructive Surgery: a Randomized Controlled Trial). Patients were randomized to receive locally applied gentamicin, cefazolin, and vancomycin or placebo. Samples were obtained from the surgical breast drain and blood up to 10 days postoperatively. Exposures: The breast implant and the implant pocket were irrigated with 160 µg/mL of gentamicin, 2000 µg/mL of cefazolin, and 2000 µg/mL of vancomycin in a 200-mL saline solution. Main Outcomes and Measures: The primary outcome was the duration of antibiotic concentrations above the MIC breakpoint for Staphylococcus aureus according to the Clinical and Laboratory Standards Institute: gentamicin, 4 µg/mL; cefazolin, 2 µg/mL; and vancomycin, 2 µg/mL. Secondary outcomes included the time above the MIC for Pseudomonas aeruginosa and other relevant bacteria, as well as systemic uptake. Results: The study included 40 patients (median age, 44.6 years [IQR, 38.3-51.4 years]; median body mass index, 23.9 [IQR, 21.7-25.9]) with a median number of 3 drain samples (range, 1-10 drain samples) and 2 blood samples (range, 0-6 blood samples). Vancomycin and cefazolin remained above the MIC for S aureus significantly longer than gentamicin (gentamicin, 0.9 days [95% CI, 0.5-1.2 days] for blood samples vs 6.9 days [95% CI, 2.9 to 10.9 days] for vancomycin [P = .02] vs 3.7 days [95% CI, 2.2-5.2 days] for cefazolin [P = .002]). The gentamicin level remained above the MIC for P aeruginosa for 1.3 days (95% CI, 1.0-1.5 days). Only cefazolin was detectable in blood samples, albeit in very low concentrations (median concentration, 0.04 µg/mL [range, 0.007-0.1 µg/mL]). Conclusions and Relevance: This study suggests that patients treated with triple-antibiotic implant irrigation during breast reconstruction receive adequate prophylaxis for S aureus and other common implant-associated, gram-positive bacteria. However, the protection against P aeruginosa may be inadequate.

Cyclic-di-GMP signaling controls metabolic activity in Pseudomonas aeruginosa.

Bacteria in biofilms are embedded in extracellular matrix and display low metabolic activity, partly due to insufficient diffusive exchange of metabolic substrate. The extracellular matrix and low metabolic activity both contribute to the high antibiotic tolerance-the hallmark of biofilm bacteria. The second messenger molecule, c-di-GMP, regulates biofilm development in Pseudomonas aeruginosa, where high internal levels lead to biofilm formation and low levels are associated with planktonic bacteria. Using a microcalorimetric approach, we show that c-di-GMP signaling is a major determinant of the metabolic activity of P. aeruginosa, both in planktonic culture and in two biofilm models. The high c-di-GMP content of biofilm bacteria forces them to rapidly spend a large amount of energy on the production of exopolysaccharides, resulting in a subsequent low metabolic state. This suggests that the low metabolic state of bacteria in mature biofilms, to some extent, is a consequence of a c-di-GMP-regulated survival strategy.

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.