Biology subjects

Bjarnsholt, T.

Publications and source records attributed to Bjarnsholt, T..

9 recordsLinked to original sources

bioRxiv2025

Harnessing machine learning to predict antibiotic susceptibility in Pseudomonas aeruginosa biofilms

Standard antibiotic susceptibility tests (ASTs) often fail to accurately predict treatment outcomes because they do not account for biofilm-specific mechanisms of reduced susceptibility. In the present study, we explored alternative approaches to predict tobramycin susceptibility of Pseudomonas aeruginosa biofilms that were experimentally evolved in physiologically relevant conditions. To this end, we used four analytical methods - whole-genome sequencing (WGS), matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS), isothermal microcalorimetry (IMC) and multi-excitation Raman spectroscopy (MX-Raman). Machine learning models were trained on data outputs from these methods to predict tobramycin susceptibility of our evolved strains and subsequently validated with a collection of clinical isolates. For minimal inhibitory concentration (MIC) predictions of the evolved strains, the highest accuracy{+/-}1 was achieved with MALDI-TOF MS (97.83 %), while for biofilm prevention concentration (BPC) predictions, Raman spectroscopy performed best with an accuracy{+/-}1 of 80.43 %. Overall, all analytical methods demonstrated comparable predictive performance, showing their potential for improving biofilm AST.

microbiology↗

bioRxiv2024

A method for separating bacterial cultures into single cells and defined size fractions of biofilm aggregates

In vitro microbiological experiments that aim to describe differences between planktonic and biofilm aggregate populations are challenging since liquid batch cultures contain a mix of both. Here, we present a simple method for fractioning a bacterial liquid batch culture into aggregates and single cells. Stackable cell strainers with mesh sizes of 30 m and 10 m were used to filtrate 6 day old batch cultures of Pseudomonas aeruginosa to produce size fractions of 0-10m and >30m. By confocal laser scanning microscopy measurements, we show that 95.5% of the total biomass was <10 m in the "0-10m size fraction" and that 92.5% of the total biomass was >30m in the ">30m size fraction". Furthermore, the adjustment of bacterial concentration using CFU/ml was validated by quantifying the total DNA of viable bacteria in the two size fractions after DNase treatment to deplete eDNA and DNA from dead bacteria. Surprisingly, this showed that adjusting the bacterial concentration using CFU/ml was a valid method with no significant differences in total DNA from viable bacteria.

microbiology↗

bioRxiv2023

What is Slough?A pilot study to define the proteomic and microbial composition of wound slough and its implications for wound healing.

Slough is a well-known feature of non-healing wounds. This study aims to determine the proteomic and microbiologic components of slough as well as interrogate the associations between wound slough components and wound healing. Twenty-three subjects with slow-to-heal wounds and visible slough were enrolled. Etiologies included venous stasis ulcers, post-surgical site infections, and pressure ulcers. Patient co-morbidities and wound healing outcome at 3-months post-sample collection was recorded. Debrided slough was analyzed microscopically, through untargeted proteomics, and high-throughput bacterial 16S-ribosomal gene sequencing. Microscopic imaging revealed wound slough to be amorphous in structure and highly variable. 16S-profiling found slough microbial communities to associate with wound etiology and location on the body. Across all subjects, slough largely consisted of proteins involved in skin structure and formation, blood-clot formation, and immune processes. To predict variables associated with wound healing, protein, microbial, and clinical datasets were integrated into a supervised discriminant analysis. This analysis revealed that healing wounds were enriched for proteins involved in skin barrier development and negative regulation of immune responses. While wounds that deteriorated over time started off with a higher baseline Bates-Jensen Wound Assessment Score and were enriched for anerobic bacterial taxa and chronic inflammatory proteins. To our knowledge, this is the first study to integrate clinical, microbiome, and proteomic data to systematically characterize wound slough and integrate it into a single assessment to predict wound healing outcome. Collectively, our findings underscore how slough components can help identify wounds at risk of continued impaired healing and serves as an underutilized biomarker.

microbiology↗

bioRxiv2023

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 3280 small compounds from LifeArc was screened. A series of ten 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 resynthesized 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 auto-generated positive feedback and its enhanced signal-molecule synthesis.

microbiology↗

bioRxiv2023

Biofilms and core pathogens shape the tumour microenvironment and immune phenotype in colorectal cancer

ObjectiveGrowing evidence links bacterial dysbiosis with colorectal cancer (CRC) carcinogenesis, characterized by an increased presence of core pathogens such as Bacteroides fragilis and Fusobacterium nucleatum. Here, we characterized the in situ biogeography and transcriptional interactions between bacteria and the host in mucosal colon biopsies. DesignThe influence of CRC core pathogens and biofilms on the tumour microenvironment (TME) was investigated in biopsies from patients with and without CRC (paired normal tissue and healthy tissue biopsies) using fluorescence in situ hybridization and dual-RNA sequencing. ResultsTissue-invasive, mixed-species biofilms enriched for B. fragilis and F. nucleatum were observed in CRC tissue, especially in right-sided tumours. Fusobacterium spp. was associated with increased bacterial biomass and inflammatory response in CRC samples. CRC samples with high bacterial activity demonstrated increased expression of pro-inflammatory cytokines, defensins, matrix-metalloproteases, and immunomodulatory factors. In contrast, the gene expression profiles of CRC samples with low bacterial activity resembled healthy tissue samples. Moreover, immune cell profiling showed that B. fragilis and F. nucleatum modulated the TME and correlated with increased infiltration of neutrophils and CD4+ T-cells. Overall, bacterial activity was critical for the immune phenotype and correlated with the infiltration of several immune cell subtypes, including M2 macrophages and regulatory T-cells. ConclusionBiofilms and core pathogens shape the TME and immune phenotype in CRC. Our results support that Fusobacterium spp. may provide a future therapeutic target to reduce biofilms and the inflammatory response in the TME while highlighting the importance of widening the scope of bacterial pathogenesis in CRC beyond core pathogens.

cancer biology↗

bioRxiv2020

Metabolic flux fingerprinting differentiates planktonic and biofilm states of Pseudomonas aeruginosa and Staphylococcus aureus

The challenges of defining the biofilm phenotype has been clear for decades. Many biomarkers for biofilm are known, but methods for identifying these are often invasive and/or complicated. These methods often rely on disrupting the biofilm matrix or examining virulence factors and compounds, which may only be expressed under certain conditions. We used microcalorimetric measurements of metabolic energy release to investigate whether unchallenged, planktonic Pseudomonas aeruginosa displayed differences in metabolism compared to surface-bound and non-attached biofilms. The pattern of energy release observed in the recorded microcalorimetric thermograms clearly depended on growth state, though the total energy expenditure was not different between growth states. To characterize these differences, we developed a classification pipeline utilizing machine learning algorithms to classify growth state, based on the observed patterns of energy release. With this approach, we could with high accuracy detect the growth form of blinded samples. To challenge the algorithm, we attempted to limit the amount of training data. By training the algorithm with only a single data point from each growth form, we obtained a mean accuracy of 90.5% using two principal components. Further validation of the classification pipeline showed that the approach was not limited to P. aeruginosa but could also be used for detection of gram-positive Staphylococcus aureus biofilm. We propose that microcalorimetric measurements, in combination with this new quantitative framework, can be used as a non-invasive biomarker to detect the presence of biofilm. These results could have a significant potential in clinical settings where the detection of biofilms in infections often means a different outcome and treatment regime for the patient.

microbiology↗

bioRxiv2019

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 aggregates 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.\n\nAuthor summaryThe role of extracellular DNA (eDNA) has been described in vitro to play a major role in biofilm formation and antibiotic tolerance, but never for biofilm infections in vivo.\n\nTwo important characteristics of human chronic bacterial infections are aggregated bacteria and white blood cells (WBC). Bacteria use eDNA to stabilize biofilms and WBC use eDNA to trap bacteria. Given the importance of eDNA for both bacteria and WBC we show here for the first time that bacterial biofilms do not co-localize with either bacterial or WBC-derived eDNA during chronic infections. Our in vivo findings show eDNA is located outside biofilms as opposed to incorporated within the biofilm as commonly observed in vitro.\n\nWe believe that understanding the interplay between biofilms and WBC during chronic infection is essential if we are to elucidate the mechanisms underlying persistent infection and ascertain why WBC fail to eradicate bacteria; this will enable us to develop new treatment strategies.

microbiology↗

bioRxiv2018

Oxygen restriction induces a viable but non-culturable population in bacteria

Induction of a non-culturable state has been demonstrated for many bacteria. In a clinical perspective, the lack of growth due to these non-culturable bacteria can have major consequences for the diagnosis and treatment of patients. Here we show how anoxic conditioning (restriction of molecular oxygen, O2) generates difficult-to-culture (DTC) bacteria during biofilm growth. A significant subpopulation of Pseudomonas aeruginosa entered a DTC state after anoxic conditioning, ranging from five to 90 % of the total culturable population, in both planktonic and biofilm models. Anoxic conditioning also generated DTC subpopulations of Staphylococcus aureus and Staphylococcus epidermidis. Growth of the DTC populations were achieved by substituting O2 with 10 mM NO3- as an alternative electron acceptor for anaerobic respiration or, in the case of P. aeruginosa, by adding sodium pyruvate or catalase as scavengers against reactive oxygen species (ROS) during aerobic respiration. An increase in normoxic plating due to addition of catalase suggests the molecule hydrogen peroxide as a possible mechanism for induction of DTC P. aeruginosa. Anoxic conditioning also generated a true viable but non-culturable (VBNC) population of P. aeruginosa that was not resurrected by substituting O2 with NO3- during anaerobic respiration. Moreover, bacterial detection in clinical samples was improved significantly by supplementing 10 mM NO3- to LB plates and incubating under anoxic conditions. These results demonstrate that habituation to an infectious anoxic micro-environment complicates diagnostic culturing of bacteria, especially in the case of chronic infections where oxygen is restricted due to the host immune response. ImportanceDiagnostics of bacteria from chronic infections by standard culture-based methods is challenging. Bacteria in a non-culturable state may contribute to the lack of culturing from these infections. Many stressors are known to induce a non-culturable state, among others the absence of molecular oxygen, which is evident in chronic infections due to high rates of oxygen consumption by the host response. In this study, we have shown that Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus epidermidis can enter a difficult-to-culture state after oxygen restriction. Regrowth was not possible using conventional normoxic plating where oxygen served as electron acceptor. Instead, regrowth was enabled during anoxic conditions with added nitrate as alternative electron acceptor. Our findings show that bacteria can habituate to their environment and that it has to be taken into consideration especially when culturing clinical samples e.g. from chronic infections.

microbiology↗

bioRxiv2017

Light assisted antibiotics

The overuse of antibiotics is accelerating the bacterial resistance, and therefore there is a need to reduce the amount of antibiotics used for treatment. Here, we demonstrate that specific wavelengths in a narrow range around 296 nm are able to eradicate bacteria in the biofilm state more effectively, than antibiotics and the combination of irradiation and antibiotics is even better, introducing a novel concept light assisted antibiotics. The investigated wavelength range was 249 nm to 338 nm with an approximate step of 5 nm. The novel concept can significantly reduce the amount of antibiotics needed for eradicating mature bacterial biofilms. The irradiation treatment was combined with tobramycin and its efficiency was compared to combinatory antibiotic treatment and highly concentrated antibiotic monotherapy. The eradication efficacies, on mature biofilms, achieved by light assisted antibiotic and by the antibiotic monotherapy at 10-fold higher concentration, were equivalent. The present achievement could motivate the development of light assisted antibiotic treatments for treating infections.

microbiology↗