Search

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.

Aim: To make an inoculum for induction of Implant-Associated Osteomyelitis (IAO) in pigs based on bacterial aggregates resembling those found on the human skin, i.e. aggregates of 5-15 µm with low metabolic activity. The aggregates were evaluated and compared to a standard planktonic bacterial inoculum. Method: The porcine Staphylococcus aureus strain S54F9 was cultured in Tryptone Soya Broth for seven days. Subsequently, the culture was filtered through cell strainers with pore sizes of 15 µm and 5 µm, respectively. The fraction of 5-15 µm aggregates in the top of the 5 µm filter was collected as the aggregate-inoculum. The separation of aggregates into different size fractions was evaluated by light microscopy. The metabolism of the aggregate-inoculum and a standard overnight planktonic inoculum was evaluated with isothermal microcalorimetry.In total, six female minipigs were allocated into three groups (n=2), receiving different inoculums. Group A: overnight planktonic inoculum; 104 CFU S. aureus (S54F9), Group B: seven days old 5-15 µm aggregate-inoculum; 104 CFU S. aureus (S54F9), Group C: saline. All inoculums were placed in a pre-drilled implant cavity in the right tibia of the pig and a sterile stainless-steel implant was inserted. The pigs were euthanized seven days after surgery. Postmortem macroscopic pathology, microbiology, computed tomography and histopathology were performed. Results: The separation of aggregates into different size fractions was done successfully by the filtering method. Isothermal microcalorimetry showed, a delayed Time-to-peak metabolic activity of the aggregate-inoculum compared to the planktonic inoculum. S. aureus was isolated from subcutis, bone and implants from all animals in groups A and B. Both group A animals showed osteomyelitis at gross inspection with suppuration and sequestration, while groups B and C animals had no macroscopic lesions. From CT scans, both group A animals also showed positive signs of osteomy

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

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, metabo-lism, mexE expression and in vitro and in vivo antimicrobial susceptibility.

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.

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.

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.

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.

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.

Bacterial biofilms residing in chronic wounds are thought to have numerous survival strate-gies, 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 sub-stances, 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.

Daptomycin is recommended for the treatment of Staphylococcus aureus infections due to its bactericidal activity. However, its mechanism of action is poorly understood. The involvement of reactive oxygen species (ROS) in the bactericidal activity of daptomycin has been proved against planktonic S. aureus, but not against the biofilm of S. aureus. Therefore, we evaluated if ROS contributes to the effect of daptomycin against biofilm of S. aureus. Biofilms of wild type, catalase deficient and daptomycin-resistant S. aureus strains were grown in microtiter-plates. After three days, the biofilms were exposed to daptomycin with or without thiourea in the presence of a ROS indicator. After overnight incubation, the amount of ROS and the percentage of surviving bacteria were determined. The bacterial survival was higher and the amount of ROS was lower in the wild type than in the catalase deficient biofilm, demonstrating a protective effect of catalase against daptomycin. The induction of cytotoxic ROS formation by daptomycin was verified by the addition of thiourea, which reduced the amount of ROS and protected the wild type biofilm against high concentrations of daptomycin. Accordingly, only the highest concentration of daptomycin reduced the bacterial survival and increased the ROS formation in the resistant biofilm. In conclusion, daptomycin induced the production of cytotoxic levels of endogenous ROS in S. aureus biofilm and the presence of catalase protected the biofilm against the lethality of the induced ROS.

Chloroflexus aggregans is a metabolically versatile, thermophilic, anoxygenic phototrophic member of the phylum Chloroflexota (formerly Chloroflexi), which can grow photoheterotrophically, photoautotrophically, chemoheterotrophically, and chemoautotrophically. In hot spring-associated microbial mats, C. aggregans co-exists with oxygenic cyanobacteria under dynamic micro-environmental conditions. To elucidate the predominant growth modes of C. aggregans, relative transcription levels of energy metabolism-and CO2 fixation-related genes were studied in Nakabusa Hot Springs microbial mats over a diel cycle and correlated with microscale in situ measurements of O2 and light. Metatranscriptomic analyses indicated two periods with different modes of energy metabolism of C. aggregans: (1) phototrophy around midday and (2) chemotrophy in the early morning hours. During midday, C. aggregans mainly employed photoheterotrophy when the microbial mats were hyperoxic (400–800 µmol L−1 O2 ). In the early morning hours, relative transcription peaks of genes encoding uptake hydrogenase, key enzymes for carbon fixation, respiratory complexes as well as enzymes for TCA cycle and acetate uptake suggest an aerobic chemomixotrophic lifestyle. This is the first in situ study of the versatile energy metabolism of C. aggregans based on gene transcription patterns. The results provide novel insights into the metabolic flexibility of these filamentous anoxygenic phototrophs that thrive under dynamic environmental conditions.

Background: Intake of probiotic bacteria may prevent oral Candida infection. Objective: To screen the antifungal activity of 14 Lactobacillus candidate strains of human origin, against six opportunistic C. albicans and non-albicans species. A second aim was to study the acid production of the four strains showing the strongest antifungal activity. Methods: We used an agar overlay growth inhibition assay to the assess the antifungal activity of the lactobacilli. The acid-producing capacity was measured with pH micro-sensors. Results: All 14 Lactobacillus candidates inhibited the growth of the Candida spp. The four best-performing strains were L. rhamnosus DSM 32992 (oral origin), L. rhamnosus DSM 32991 (oral), L. jensenii 22B42 (vaginal), and L. rhamnosus PB01 (vaginal). The difference between L. rhamnosus DSM 32992 and the other three strains was statistically significant (p < 0.001). The Candida spp. differed in susceptibility; C. parapsilosis was highly inhibited, while C. krusei was not or slightly inhibited. The oral L. rhamnosus DSM 32992 and DSM 32991 strains showed the lowest pH-values. Conclusion: Screening of probiotic lactobacilli showed significant strain-dependent variations in their antifungal capacity in a pH-dependent mode. Two strains of oral origin were most effective. A further characterization seems justified to elaborate on their probiotic properties.

Chronic infections present a serious economic burden to health-care systems. The severity and prevalence of chronic infections are continuously increasing due to an aging population and an elevated number of lifestyle related diseases such as diabetes. Treatment of chronic infections has proven difficult, mainly due to the presence of biofilms that render bacteria more tolerant toward antimicrobials and the host immune response. Chronic infections have been described to harbor several different bacterial species and it has been hypothesized that microscale interactions and mixed-species consortia are present as described for most natural occurring biofilms i.e., aquatic systems and industrial settings, but also for some commensal human biofilms i.e., the mouth microbiota. However, the presence of mixed-species biofilms in chronic infections is most often an assumption based on culture-based methods and/or by means of molecular approaches, such as PCR and sequencing performed from homogenized bulk tissue samples. These methods disregard the spatial organization of the bacterial community and thus valuable information on biofilm aggregate composition, spatial organization, and possible interactions between different species is lost. Hitherto, only few studies have made visual in situ presentations of mixed-species biofilms in chronic infections, which is pivotal for the description of bacterial composition, spatial distribution, and interspecies interaction on the microscale. In order for bacteria to interact (synergism, commensalism, mutualism, competition, etc.) they need to be in close proximity to each other on the scale where they can affect e.g., solute concentrations. We argue that visual proof of mixed species biofilms in chronic infections is scarce compared to what is seen in e.g., environmental biofilms and call for a debate on the importance of mixed-species biofilm in chronic infections.

The antibacterial efficacy of a copper-silver alloy coating under conditions resembling build up of dry surface bacterial biofilms is successfully demonstrated according to US EPA test methods with a ≥99.9% reduction of test organisms over a 24 h period. A tailor-made confocal imaging protocol is designed to visualize in situ the killing of bacterial biofilms at the copper-silver alloy surface and monitor the kinetics for 100 min. The copper-silver alloy coating eradicates a biofilm of Gram-positive bacteria within 5 min while a biofilm of Gram-negative bacteria are killed more slowly. In situ pH monitoring indicates a 2-log units increase at the interface between the metallic surface and bacterial biofilm; however, the viability of the bacteria is not directly affected by this raise (pH 8.0-9.5) when tested in buffer. The OH- production, as a result of the interaction between the electrochemically active surface and the bacterial biofilm under environmental conditions, is thus one aspect of the contact-mediated killing of the copper-silver alloy coating and not the direct cause of the observed antibacterial efficacy. The combination of oxidation of bacterial cells, release of copper ions, and local pH raise characterizes the antibacterial activity of the copper-silver alloy-coated dry surface.