Biology subjects

Coenye, T.

Publications and source records attributed to Coenye, T..

9 recordsLinked to original sources

bioRxiv2026

Evaluation of a novel isothermal microcalorimetry-based sterility test

Parenteral drugs must meet strict release criteria to ensure patient safety upon administration. Sterility is a critical requirement, and is typically assessed using the compendial U.S. Pharmacopoeia (USP) <71> sterility test. However, with the growing demand to reduce batch release times, the tests 14-day incubation period is becoming a concern, emphasizing the need for more rapid alternatives. Here, we compared the performance of the calScreener+ isothermal microcalorimetry (IMC) device (Symcel) to that of the compendial USP <71> sterility test, using a panel of sixteen microorganisms (six USP <71> reference strains and ten field isolates) in two inoculum sizes (100 and 5 CFU). The IMC-based method detected a higher number of positive samples compared to the compendial method (95.8% vs 87.5%; p < 0.05). Furthermore, IMC was consistently faster, reducing mean detection times from 43 hours to 19 hours at 100 CFU and from 46 hours to 28 hours at 5 CFU (p < 0.001). In conclusion, the calScreener+ IMC device shows promise as a rapid and sensitive alternative to the compendial sterility test, with the potential to speed up batch releases without compromising patient safety.

microbiology↗

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

Identification of echinacoside as a tobramycin potentiator against Pseudomonas aeruginosa aggregates

Cyclic diguanylate (c-di-GMP) is a central biofilm regulator, where increased intracellular levels promote biofilm formation and antibiotic tolerance. Targeting the c-di-GMP network is a promising anti-biofilm approach. Most agents reported previously decreased c-di-GMP to eliminate surface-attached biofilms, which did not recapitulate in vivo biofilms well and may thus impede their clinical impact. Here, the expression profile of genes encoding proteins associated with c-di-GMP metabolism was analysed among 32 Pseudomonas aeruginosa strains grown as suspended aggregates in synthetic sputum or planktonic cells. A diguanylate cyclase, SiaD, proved essential for auto-aggregation under in vivo-like conditions. Virtual screening against SiaD identified echinacoside as an inhibitor, which reduced intracellular c-di-GMP levels and aggregate sizes and potentiated the efficacy of tobramycin against aggregates established by >80% of tested strains. This synergistic effect was also observed for in vivo-like 3-D alveolar cells infected by cytotoxic P. aeruginosa, demonstrating its high potential as an adjunctive therapy for recalcitrant P. aeruginosa infections.

microbiology↗

bioRxiv2023

Genome-Scale Modeling of Rothia mucilaginosa Reveals Insights into Metabolic Capabilities and Therapeutic Strategies for Cystic Fibrosis

BackgroundCystic fibrosis (CF) is an inherited genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in the production of sticky and thick mucosal fluids. This leads to an environment that facilitates the colonization of various microorganisms, some of which can cause acute and chronic lung infections, while others may have a positive influence on the disease process. Rothia mucilaginosa, an oral commensal, is relatively abundant in the lungs of CF patients. Recent studies have unveiled the anti-inflammatory properties of R. mucilaginosa using in vitro three-dimensional (3-D) lung epithelial cell cultures and in vivo mouse models relevant to chronic lung diseases. Apart from a potentially beneficial anti-inflammatory role in chronic lung diseases, R. mucilaginosa has been associated with severe infections. This dual nature highlights the bacteriums complexity and diverse impact on health and disease. However, its metabolic capabilities and genotype-phenotype relationships remain largely unknown. ResultsTo gain insights into the cellular metabolism and genetic content of R. mucilaginosa, we developed the first manually curated genome-scale metabolic model, iRM23NL. Through growth kinetic experiments and high-throughput phenotypic microarray testings, we defined its complete catabolic phenome. Subsequently, we assessed the models effectiveness in accurately predicting growth behaviors and utilizing multiple substrates. We used constraint-based modeling techniques to formulate novel hypotheses that could expedite the development of antimicrobial strategies. More specifically, we detected putative essential genes and assessed their effect on metabolism under varying nutritional conditions. These predictions could offer novel potential antimicrobial targets without laborious large-scale screening of knock-outs and mutant transposon libraries. ConclusionOverall, iRM23NL demonstrates a solid capability to predict cellular phenotypes and holds immense potential as a valuable resource for accurate predictions in advancing antimicrobial therapies. Moreover, it can guide metabolic engineering to tailor R. mucilaginosas metabolism for desired performance.

systems biology↗

bioRxiv2022

What are the reference strains of Acinetobacter baumannii referring to?

We assembled the whole genome sequence (WGS) of a collection of 43 non-redundant modern clinical isolates and four broadly used reference strains of Acinetobacter baumannii. Comparison of these isolates and their WGS confirmed the high heterogeneity in capsule loci, sequence types, the presence of virulence and antibiotic resistance genes. However, a significant portion of clinical isolates strongly differ when compared to several reference strains in the light of colony morphology, cellular density, capsule production, natural transformability and in vivo virulence. These genetic and phenotypic differences between current circulating strains of A. baumannii and established reference strains could hamper the study of A. baumannii as an entity. The broadly used reference strains led to the current state of the art of the A. baumannii field, however, we propose that established reference strains in the A. baumannii field should be carefully used, because of the high genetic and phenotypic heterogeneities. In this study, we generated a collection of high-quality nucleotide sequences of 43 modern clinical isolates with the corresponding multi-level phenotypic characterizations. Beside the contribution of novel fundamental observations generated in this study, the phenotypic and genetic data, along with the bacterial strains themselves, will be further accessible using the first open access online platform called "Acinetobase". Therefore, a rational choice of modern strains will be possible to select the ones that suit the needs of specific biological questions.

microbiology↗

bioRxiv2020

DNA methylation epigenetically regulates gene expression in Burkholderia cenocepacia

Respiratory tract infections by the opportunistic pathogen Burkholderia cenocepacia often lead to severe lung damage in cystic fibrosis (CF) patients. New insights in how to tackle these infections might emerge from the field of epigenetics, as DNA methylation has shown to be an important regulator of gene expression. The present study focused on two DNA methyltransferases (MTases) in B. cenocepacia strains J2315 and K56-2, and their role in regulating gene expression. In silico predicted DNA MTase genes BCAL3494 and BCAM0992 were deleted in both strains, and the phenotypes of the resulting deletion mutants were studied: deletion mutant {Delta}BCAL3494 showed changes in biofilm structure and cell aggregation, {Delta}BCAM0992 was less motile. B. cenocepacia wild type cultures treated with sinefungin, a known DNA MTase inhibitor, exhibited the same phenotype as DNA MTase deletion mutants. Single-Molecule Real-Time sequencing was used to characterize the methylome of B. cenocepacia, including methylation at the origin of replication, and motifs CACAG and GTWWAC were identified as targets of BCAL3494 and BCAM0992, respectively. All genes with methylated motifs in their putative promoter region were identified and qPCR experiments showed an upregulation of several genes, including biofilm and motility related genes, in MTase deletion mutants with unmethylated motifs, explaining the observed phenotypes in these mutants. In summary, our data confirm that DNA methylation plays an important role in regulating the expression of B. cenocepacia genes involved in biofilm formation and motility. ImportanceCF patients diagnosed with B. cenocepacia infections often experience rapid deterioration of lung function, known as cepacia syndrome. B. cenocepacia has a large multi-replicon genome and a lot remains to be learned about regulation of gene expression in this organism. From studies in other (model) organisms, it is known that epigenetic changes through DNA methylation play an important role in this regulation. The identification of B. cenocepacia genes of which the expression is regulated by DNA methylation and identification of the regulatory systems involved in this methylation are likely to lead to new insights in how to tackle B. cenocepacia infections in CF patients.

microbiology↗

bioRxiv2019

Low iron-induced small RNA BrrF regulates central metabolism and oxidative stress responses in Burkholderia cenocepacia

BrrF is a Fur-regulated small RNA highly upregulated in Burkholderia cenocepacia under conditions of iron depletion. Its computationally predicted targets include iron-containing enzymes of the tricarboxylic acid (TCA) cycle such as aconitase and succinate dehydrogenase, as well as iron-containing enzymes responsible for the oxidative stress response, such as superoxide dismutase and catalase. Phenotypic and gene expression analysis of BrrF deletion and overexpression mutants show that the regulation of these genes is BrrF-dependent. Expression of acnA, fumA, sdhA and sdhC was downregulated during iron depletion in the wild type strain, but not in a BrrF deletion mutant. TCA cycle genes not predicted as target for BrrF were not affected in the same manner by iron depletion. Likewise, expression of sodB and katB was dowregulated during iron depletion in the wild type strain, but not in a BrrF deletion mutant. BrrF overexpression reduced aconitase and superoxide dismutase activities and increased sensitivity to hydrogen peroxide. All phenotypes and gene expression changes of the BrrF deletion mutant could be complemented by overexpressing BrrF in trans. Overall, BrrF acts as a regulator of central metabolism and oxidative stress response, possibly as an iron-sparing measure to maintain iron homeostasis under conditions of iron starvation. ImportanceRegulatory small RNAs play an essential role in maintaining cell homeostasis in bacteria in response to environmental stresses such as iron starvation. Prokaryotes generally encode a large number of RNA regulators, yet their identification and characterisation is still in its infancy for most bacterial species. Burkholderia cenocepacia is an opportunistic pathogen with high innate antimicrobial resistance, which can cause the often fatal cepacia syndrome in individuals with cystic fibrosis. In this study we characterise a small RNA which is involved in the response to iron starvation, a condition that pathogenic bacteria are likely to encounter in the host.

microbiology↗

bioRxiv2019

Non-hierarchical, RhlR-regulated acyl-homoserine lactone quorum sensing in a cystic fibrosis isolate of Pseudomonas aeruginosa

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of airway infection in cystic fibrosis (CF) patients. P. aeruginosa employs several hierarchically arranged and interconnected quorum sensing (QS) regulatory circuits to produce a battery of virulence factors such as elastase, phenazines, and rhamnolipids. The QS transcription factor LasR sits atop this hierarchy, and activates the transcription of dozens of genes, including that encoding the QS regulator RhlR. Paradoxically, inactivating lasR mutations are frequently observed in isolates from CF patients with chronic P. aeruginosa infections. In contrast, mutations in rhlR are rare. We have recently shown that in CF isolates, the QS circuitry is often \"rewired\" such that RhlR acts in a LasR-independent manner. To begin understanding how QS activity differs in this \"rewired\" background, we characterized QS activation and RhlR-regulated gene expression in P. aeruginosa E90, a LasR-null, RhlR-active chronic infection isolate. In this isolate, RhlR activates the expression of 53 genes in response to increasing cell density. The genes regulated by RhlR include several that encode virulence factors. Some, but not all, of these genes are present in the QS regulon described in the well-studied laboratory strain PAO1. We also demonstrate that E90 produces virulence factors at similar concentrations to that of PAO1. Unlike PAO1, cytotoxicity by E90 in a three-dimensional lung epithelium cell model is also RhlR-regulated. These data illuminate a \"rewired\" LasR-independent RhlR regulon in chronic infection isolates and suggest that RhlR may be a target for therapeutic development in chronic infections.\n\nAUTHOR SUMMARYPseudomonas aeruginosa is a prominent cystic fibrosis (CF) pathogen that uses quorum sensing (QS) to regulate virulence. In laboratory strains, the key QS regulator is LasR. Some isolates from patients with chronic CF infections appear to use an alternate QS circuitry in which another transcriptional regulator, RhlR, mediates QS. We show that a LasR-null CF clinical isolate engages in QS through RhlR and remains capable of inducing cell death in an in vivo-like lung epithelium cell model. Our findings support the notion that LasR-null clinical isolates can engage in RhlR QS and highlight the centrality of RhlR gene regulation in chronic P. aeruginosa infections.

microbiology↗

bioRxiv2018

Various evolutionary trajectories lead to loss of the tobramycin-potentiating activity of the quorum sensing inhibitor baicalin hydrate in Burkholderia cenocepacia biofilms

Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant and -tolerant bacteria. As these potentiators typically do not interfere with essential processes of bacteria, it has been hypothesized that they are less likely to induce resistance than conventional antibiotics. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop resistance towards one such adjuvant, baicalin hydrate (BH), a quorum sensing inhibitor known to increase antibiotic-induced oxidative stress. Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After each cycle of treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to treatments with TOB and TOB+BH, compared to the start population, and the potentiating effect of BH towards TOB was lost. Whole genome sequencing was performed to probe which changes were involved in the reduced effect of BH and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter), as well as a partial deletion of two larger regions. No changes in the minimal inhibitory or minimal bactericidal concentration of TOB or changes in the number of persister cells were observed in the evolved populations. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that resistance towards antibiotic-potentiating activity can develop rapidly in B. cenocepacia J2315 biofilms and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as potential mechanisms.\n\nImportanceBacteria show a markedly reduced susceptibility to antibiotics when growing in a biofilm, which hampers effective treatment of biofilm-related infections. The use of potentiators that increase the activity of antibiotics against biofilms has been proposed as a solution to this problem, but it is unclear whether resistance to these potentiators could develop. Using an experimental evolution approach, we convincingly demonstrate that Burkholderia cenocepacia biofilms rapidly develop resistance towards the tobramycin-potentiating activity of baicalin hydrate. Whole genome sequencing revealed that there are different mechanisms that lead to this resistance, including mutations resulting in metabolic changes, changes in production of intracellular levels of reactive oxygen species, and differences in transporter-mediated tobramycin uptake. Our study suggests that this form of combination therapy is not evolution-proof and highlights the usefulness of experimental evolution to identify mechanisms of resistance and tolerance in biofilm-grown bacteria.

microbiology↗