Beilharz, K., et al., 2023. Protocol to assess metabolic activity of Pseudomonas aeruginosa by measuring heat flow using isothermal calorimetry. STAR Protocols.
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.
Bokhari, M.H., et al., 2023. Use of Isothermal Microcalorimetry to Measure Cellular Heat Production in Thermogenic Adipocytes. Lodhi, I.J. (eds) Thermogenic Fat. Methods in Molecular Biology, vol 2662. Humana, New York, NY
Induction of thermogenesis in brown and brite adipocytes has recently emerged as a therapeutic target for novel anti obesogenic therapies necessitating the development of methods that can accurately measure heat production in these cells. Modern isothermal microcalorimetric techniques allow for the high throughput quantitative measurement of cellular heat production with limited sample material. Here, we describe the application of this technique for the measurement of thermogenesis in both floating and adherent adipocytes from various murine depots and human cell lines.
Level II, diagnostic study.
Maybin, JM. et al., 2023. Cold atmospheric pressure plasma-antibiotic synergy in Pseudomonas aeruginosa biofilms is mediated via oxidative stress response. Biofilm
Cold atmospheric-pressure plasma (CAP) has emerged as a potential alternative or adjuvant to conventional antibiotics for the treatment of bacterial infections, including those caused by antibiotic-resistant pathogens. The potential of sub-lethal CAP exposures to synergise conventional antimicrobials for the eradication of Pseudomonas aeruginosa biofilms is investigated in this study. The efficacy of antimicrobials following or in the absence of sub-lethal CAP pre-treatment in P. aeruginosa biofilms was assessed. CAP pre-treatment resulted in an increase in both planktonic and biofilm antimicrobial sensitivity for all three strains tested (PAO1, PA14, and PA10548), with both minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs) of individual antimicrobials, being significantly reduced following CAP pre-treatment of the biofilm (512-fold reduction with ciprofloxacin/gentamicin; and a 256-fold reduction with tobramycin). At all concentrations of antimicrobial used, the combination of sub-lethal CAP exposure and antimicrobials was effective at increasing time-to-peak metabolism, as measured by isothermal microcalorimetry, again indicating enhanced susceptibility. CAP is known to damage bacterial cell membranes and DNA by causing oxidative stress through the in situ generation of reactive oxygen and nitrogen species (RONS). While the exact mechanism is not clear, oxidative stress on outer membrane proteins is thought to damage/perturb cell membranes, confirmed by ATP and LDH leakage, allowing antimicrobials to penetrate the bacterial cell more effectively, thus increasing bacterial susceptibility. Transcriptomic analysis, reveals that cold-plasma mediated oxidative stress caused upregulation of P. aeruginosa superoxide dismutase, cbb3 oxidases, catalases, and peroxidases, and upregulation in denitrification genes, suggesting that P. aeruginosa uses these enzymes to degrade RONS and mitigate the effects of cold plasma mediated oxidative stress. CAP treatment also led to an increased production of the signalling molecule ppGpp in P. aeruginosa, indicative of a stringent response being established. Although we did not directly measure persister cell formation, this stringent response may potentially be associated with the formation of persister cells in biofilm cultures. The production of ppGpp and polyphosphate may be associated with protein synthesis inhibition and increase efflux pump activity, factors which can result in antimicrobial tolerance. The transcriptomic analysis also showed that by 6 h post-treatment, there was downregulation in ribosome modulation factor, which is involved in the formation of persister cells, suggesting that the cells had begun to resuscitate/recover. In addition, CAP treatment at 4 h post-exposure caused downregulation of the virulence factors pyoverdine and pyocyanin; by 6 h post-exposure, virulence factor production was increasing. Transcriptomic analysis provides valuable insights into the mechanisms by which P. aeruginosa biofilms exhibits enhanced susceptibility to antimicrobials. Overall, these findings suggest, for the first time, that short CAP sub-lethal pre-treatment can be an effective strategy for enhancing the susceptibility of P. aeruginosa biofilms to antimicrobials and provides important mechanistic insights into cold plasma-antimicrobial synergy. Transcriptomic analysis of the response to, and recovery from, sub-lethal cold plasma exposures in P. aeruginosa biofilms improves our current understanding of cold plasma biofilm interactions.
Doucette, C.C., et al., 2023. Optogenetic Activation of UCP1-Dependent Thermogenesis in Brown Adipocytes. ISCIENCE
Brown adipocytes are unique in that they expend energy and produce heat to maintain euthermia through expression of uncoupling protein-1 (UCP1). Given their propensity to stimulate weight loss and promote resistance to obesity, they are a compelling interventional target for obesity-related disorders. Here, we tested whether an optogenetic approach could be used to directly activate UCP1-dependent thermogenesis in brown adipocytes. We generated brown adipocytes expressing a bacterial-derived photoactivatable adenylyl cyclase (bPAC) that, upon blue light stimulation, increases UCP1 expression, fuel uptake and thermogenesis. This unique system allows for precise, chemical free, temporal control of UCP1-dependent thermogenesis, which can aid in our understanding of brown adipocyte biology and development of therapies that target obesity-related disorders.
Im H, et al., 2023. Targeting NAD+ regeneration enhances antibiotic susceptibility of Streptococcus pneumoniae during invasive disease. PLoS Biol.
Anaerobic bacteria are responsible for half of all pulmonary infections. One such pathogen is Streptococcus pneumoniae (Spn), a leading cause of community-acquired pneumonia, bacteremia/sepsis, and meningitis. Using a panel of isogenic mutants deficient in lactate, acetyl-CoA, and ethanol fermentation, as well as pharmacological inhibition, we observed that NAD(H) redox balance during fermentation was vital for Spn energy generation, capsule production, and in vivo fitness. Redox balance disruption in fermentation pathway-specific fashion substantially enhanced susceptibility to killing in antimicrobial class-specific manner. Blocking of alcohol dehydrogenase activity with 4-methylpyrazole (fomepizole), an FDA-approved drug used as an antidote for toxic alcohol ingestion, enhanced susceptibility of multidrug-resistant Spn to erythromycin and reduced bacterial burden in the lungs of mice with pneumonia and prevented the development of invasive disease. Our results indicate fermentation enzymes are de novo targets for antibiotic development and a novel strategy to combat multidrug-resistant pathogens.
Bové M., et al., 2023. Adaptation of Pseudomonas aeruginosa biofilms to tobramycin and the quorum sensing inhibitor C-30 during experimental evolution requires multiple genotypic and phenotypic changes. Microbiology.
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.
Xiang, Z., et al., 2023. Human Tooth as a Fungal Niche: Candida albicans Traits in Dental Plaque Isolates. mBio.
Candida albicans, a fungus typically found in the mucosal niche, is frequently detected in biofilms formed on teeth (dental plaque) of toddlers with severe childhood caries, a global public health problem that causes rampant tooth decay. However, knowledge about fungal traits on the tooth surface remains limited. Here, we assess the phylogeny, phenotype, and interkingdom interactions of C. albicans isolated from plaque of diseased toddlers and compare their properties to reference strains, including 529L (mucosal isolate). C. albicans isolates exhibit broad phenotypic variations, but all display cariogenic traits, including high proteinase activity, acidogenicity, and acid tolerance. Unexpectedly, we find distinctive variations in filamentous growth, ranging from hyphal defective to hyperfilamentous. We then investigate the ability of tooth isolates to form interkingdom biofilms with Streptococcus mutans (cariogenic partner) and Streptococcus gordonii (mucosal partner). The hyphal-defective isolate lacks cobinding with S. gordonii, but all C. albicans isolates develop robust biofilms with S. mutans irrespective of their filamentation state. Moreover, either type of C. albicans (hyphae defective or hyperfilamentous) enhances sucrose metabolism and biofilm acidogenicity, creating highly acidic environmental pH (<5.5). Notably, C. albicans isolates show altered transcriptomes associated with pH, adhesion, and cell wall composition (versus reference strains), further supporting niche-associated traits. Our data reveal that C. albicans displays distinctive adaptive mechanisms on the tooth surface and develops interactions with pathogenic bacteria while creating an acidogenic state regardless of fungal morphology, contrasting with interkingdom partnerships in mucosal infections. Human tooth may provide new insights into fungal colonization/adaptation, interkingdom biofilms, and contributions to disease pathogenesis.
Molendijk, M. M., et al., 2023. Microcalorimetry: A Novel Application to Measure In Vitro Phage Susceptibility of Staphylococcus aureus in Human Serum. Viruses
Infections involving antibiotic resistant Staphylococcus aureus (S. aureus) represent a major challenge to successful treatment. Further, although bacteriophages (phages) could be an alternative to antibiotics, there exists a lack of correlation in phage susceptibility results between conventional in vitro and in vivo assays. This discrepancy may hinder the potential implementation of bacteriophage therapy. In this study, the susceptibility of twelve S. aureus strains to three commercial phage cocktails and two single phages was assessed. These S. aureus strains (including ten clinical isolates, five of which were methicillin-resistant) were compared using four assays: the spot test, efficiency of plating (EOP), the optical density assay (all in culture media) and microcalorimetry in human serum. In the spot test, EOP and optical density assay, all cocktails and single phages lysed both methicillin susceptible and methicillin resistant S. aureus strains. However, there was an absence of phage-mediated lysis in high concentrations of human serum as measured using microcalorimetry. As this microcalorimetry-based assay more closely resembles in vivo conditions, we propose that microcalorimetry could be included as a useful addition to conventional assays, thereby facilitating more accurate predictions of the in vivo susceptibility of S. aureus to phages during phage selection for therapeutic purposes.
Coenye. T., et al., 2022. Biofilm antimicrobial susceptibility through an experimental evolutionary lens. NPJ Biofilm Microbiomes
Experimental evolution experiments in which bacterial populations are repeatedly exposed to an antimicrobial treatment, and examination of the genotype and phenotype of the resulting evolved bacteria, can help shed light on mechanisms behind reduced susceptibility. In this review we present an overview of why it is important to include biofilms in experimental evolution, which approaches are available to study experimental evolution in biofilms and what experimental evolution has taught us about tolerance and resistance in biofilms. Finally, we present an emerging consensus view on biofilm antimicrobial susceptibility supported by data obtained during experimental evolution studies.
Lichtenberg, M., et al., 2022. Inoculum Concentration Influences Pseudomonas aeruginosa Phenotype and Biofilm Architecture. Microbiology Spectrum.
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.
Lichtenberg, M., et al., 2022. 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.
Keywords: CP: Microbiology; Pseudomonas aeruginosa; biofilm; c-di-GMP; exopolysaccharides; matrix; metabolism; microcalorimetry.
Antonelli, A., et al., 2022. Isothermal microcalorimetry vs checkerboard assay to evaluate in-vitro synergism of meropenem–amikacin and meropenem–colistin combinations against multi-drug-resistant Gram-negative pathogens. I. J. Ant. Mic. Ag.
To evaluate the activity of meropenem–amikacin and meropenem–colistin combinations with checkerboard broth microdilution (CKBM) compared with isothermal microcalorimetry (ITMC) assays against a multi-centric collection of multi-drug-resistant Gram-negative clinical isolates; and to compare the fractional inhibitory concentration (FIC) index and time to results of CKBM and ITMC.
A collection of 333 multi-drug-resistant Gram-negative clinical isolates showing reduced susceptibility to meropenem (121 Klebsiella pneumoniae, 14 Escherichia coli, 130 Pseudomonas aeruginosa and 68 Acinetobacter baumannii) isolated from different centres (Florence, Madrid, Rotterdam and Stockholm) was included in the study. The antimicrobial activity of meropenem–amikacin and meropenem–colistin combinations was evaluated with CKBM and ITMC. FIC index results were interpreted as synergistic/additive and indifferent for values ≤0.5/0.5<x≤1 and >1, respectively. Whole-genome sequencing data of a subset of strains were used to evaluate their clonality.
In total, 254 and 286 strains were tested with meropenem–colistin and meropenem–amikacin combinations with ITMC and CKBM, respectively. Synergistic/additive effects were observed for 46 strains (20 K. pneumoniae, four E. coli, 22 P. aeruginosa) and 20 strains (three K. pneumoniae, 11 P. aeruginosa and six A. baumannii) with meropenem–amikacin and meropenem–colistin combinations, respectively, with CKBM. ITMC showed good concordance with CKBM, with 89.5% and 92.2% of cases interpreted within the same FIC index category for meropenem–amikacin and meropenem–colistin combinations, respectively. Most of the synergistic/additive effects were detected within 6 h by ITMC.
ITMC showed very good concordance with CKBM against a large collection of multi-drug-resistant Gram-negative clinical isolates, and could be implemented for the rapid evaluation of in-vitro activity of antimicrobial combinations.
Baede , V. O., et al., 2022. Dehydration Tolerance in Epidemic versus Nonepidemic MRSA Demonstrated by Isothermal Microcalorimetry. Micro. Spectrum
Zalavras, Charalampos G., 2022. CORR Insights®: Isothermal Microcalorimetry Improves the Time to Diagnosis of Fracture-related Infection Compared With Conventional Tissue Cultures. Clin. Orthop. Relat. Res.
Cho, H., et al., 2022. Pathobiont-mediated spatial structuring enhances biofilm virulence in childhood oral disease. Res. Square.
Microbiome studies are revealing complex microbiota in biofilm-mediated human diseases commonly linked with specific bacterial pathogens. Streptococcus mutans has been implicated as the primary pathogen in childhood dental caries (tooth decay). While the role of polymicrobial communities is appreciated, it remains unclear whether other microorganisms are active contributors, inactive cohabitants, or interact with known pathogens such as S. mutans . Here, we integrate multi-omics of human dental plaque (biofilm) from two community-based samples of preschool-age children in a discovery-validation pipeline involving bioinformatics, laboratory, and in vivo experimental approaches to identify disease-relevant inter-species interactions. In metagenomics and metatranscriptomics analyses among 416 preschool-age children, we identify 16 taxa strongly associated with childhood caries. Using multiscale imaging, virulence assays, microcalorimetry, and computational analyses, we investigate biofilm formation dynamics, microscale spatial arrangement, and metabolic activity by Selenomonas sputigena, Prevotella salivae and Leptotrichia wadei either individually or with S. mutans . Notably, we discover that the flagellated S. sputigena , a subgingival anaerobe with previously unknown role in supragingival biofilm virulence, becomes trapped in foreign streptococcal exoglucans, loses its motility but actively proliferates to build a honeycomb-like multicellular superstructure encapsulating S. mutans and enhances acidogenesis. Rodent model experiments reveal a previously unrecognized ability of S. sputigena to colonize supragingival tooth surfaces. While incapable of causing caries on its own, S. sputigena exacerbates the disease severity in vivo when co-infected with S. mutans , causing extensive lesions on tooth enamel. Our data reveal a pathobiont from a disparate habitat in a prevalent disease that cooperates with a known pathogen to build a unique 3D spatial structure and heighten biofilm virulence—which may be relevant to other polymicrobial diseases.
Cichos, K. H., et al., 2022. Isothermal Microcalorimetry Improves the Time to Diagnosis of Fracture-related Infection Compared with Conventional Tissue Cultures. Clin. Orthop. Relat. Res.
A consensus definition recently was formulated for fracture-related infection, which centered on confirmatory criteria including conventional cultures that take time to finalize and have a 10% to 20% false-negative rate. During this time, patients are often on broad-spectrum antibiotics and may remain hospitalized until cultures are finalized to adjust antibiotic regimens.
(1) What is the diagnostic accuracy of isothermal microcalorimetry, and how does its accuracy compare with that of conventional cultures? (2) Does isothermal microcalorimetry decrease time to detection (or diagnosis) of fracture-related infection compared with conventional cultures? (3) Does isothermal microcalorimetry have a diagnostic accuracy or time advantage over conventional cultures in patients on chronic suppressive antibiotics?
Between July 2020 and August 2021, we treated 310 patients with concerns for infection after prior fracture repair surgery. Of those, we considered all patients older than 18 years of age with fixation hardware in place at the time of presentation as potentially eligible. All included patients returned to the operating room with cultures obtained and assessed by both isothermal microcalorimetry and conventional cultures, and all were diagnosed using the consensus criteria for fracture-related infection. Based on that, 81% (250 of 310) of patients were eligible; a further 51% (157 of 310) were excluded because of the following reasons: the capacity of the isothermal microcalorimetry instrument limited the throughput on that day (34% [106 of 310]), they had only swab cultures obtained in surgery (15% [46 of 310]), or they had less than 3 months follow-up after surgery for infectious concerns (2% [5 of 310]), leaving 30% (93 of 310) of the originally identified patients for analysis. We obtained two to five cultures from each patient during surgery, which were sent to our clinical microbiology laboratory for standard processing (conventional cultures). This included homogenization of each tissue sample individually and culturing for aerobic, anaerobic, acid-fast bacilli, and fungal culturing. The remaining homogenate from each sample was then taken to our orthopaedic research laboratory, resuspended in growth media, and analyzed by isothermal microcalorimetry for a minimum of 24 hours. Aerobic and anaerobic cultures were maintained for 5 days and 14 days, respectively. Overall, there were 93 patients (59 males), with a mean age of 43 ± 14 years and a mean BMI of 28 ± 8 kg/m2, and 305 tissue samples (mean 3 ± 1 samples per patient) were obtained and assessed by conventional culturing and isothermal microcalorimetry. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy of isothermal microcalorimetry to diagnose fracture-related infection were compared with conventional cultures using a McNemar test based on the consensus definition of fracture-related infection. This consensus criteria is comprised of two levels of certainty for the diagnostic variables. The first is confirmatory criteria, where infection is considered definitely present and includes the presence of fistula/sinus tract/wound breakdown, purulent drainage or the presence of pus, presence of microorganisms in deep tissue specimens on histopathologic examination, presence of more than five neutrophils/high-powered field by histopathologic examination (only for chronic/late onset cases), and identification of phenotypically indistinguishable pathogens by conventional culture from at least two separate deep tissue/implant specimens. The second is suggestive criteria in which further investigation is required to achieve confirmatory status. Fracture-related infection was diagnosed for this study to minimize subjectivity based on the presence of at least one of the confirmatory criteria as documented by the managing surgeon. When suggestive criteria were present without confirmatory criteria, patients were considered negative for fracture-related infection and followed further in clinic after surgical exploration (n = 25 patients). All 25 patients deemed not to have fracture-related infection were considered infection-free at latest follow-up (range 3 to 12 months). The time to detection or diagnosis was recorded and compared via the Mann-Whitney U test.
Using the consensus criteria for fracture-related infection, there were no differences with the numbers available between isothermal microcalorimetry and conventional cultures in terms of sensitivity (87% [95% confidence interval 77% to 94%] versus 81% [95% CI 69% to 89%]), specificity (100% [95% CI 87% to 100%] versus 96% [95% CI 79% to 99%]), PPV (100% [95% CI 90% to 100%] versus 98% [95% CI 89% to 99%]), NPV (74% [95% CI 60% to 84%] versus 65% [95% CI 52% to 75%]), or accuracy (90% [95% CI 83% to 96%] versus 85% [95% CI 76% to 91%]; p = 0.13). The concordance by sample between conventional cultures and isothermal microcalorimetry was 85%. Isothermal microcalorimetry had a shorter median (range) time to detection or diagnosis compared with conventional cultures (2 hours [0.5 to 66] versus 51 hours [18 to 147], difference of medians 49 hours; p < 0.001). Additionally, 32 patients used antibiotics for a median (range) duration of 28 days (7 to 1095) before presentation. In these unique patients, there were no differences with the numbers available between isothermal microcalorimetry and conventional cultures in terms of sensitivity (89% [95% CI 71% to 98%] versus 74% [95% CI 53% to 88%]), specificity (100% [95% CI 48% to 100%] versus 83% [95% CI 36% to 99%]), PPV (100% [95% CI 85% to 100%] versus 95% [95% CI 77% to 99%]), NPV (63% [95% CI 37% to 83%] versus 42% [95% CI 26% to 60%]), or accuracy (91% [95% CI 75% to 98%] versus 78% [95% CI 57% to 89%]; p = 0.17). Isothermal microcalorimetry again had a shorter median (range) time to detection or diagnosis compared with conventional cultures (1.5 hours [0.5 to 48] versus 51.5 hours [18 to 125], difference of medians 50 hours; p < 0.001).
Given that isothermal microcalorimetry considerably decreases the time to the diagnosis of a fracture-related infection without compromising the accuracy of the diagnosis, managing teams may eventually use isothermal microcalorimetry—pending developmental improvements and regulatory approval—to rapidly detect infection and begin antibiotic management while awaiting speciation and susceptibility testing to modify the antibiotic regimen. Given the unique thermograms generated, further studies are already underway focusing on speciation based on heat curves alone. Additionally, increased study sizes are necessary for both overall fracture-related infection diagnostic accuracy and test performance on patients using long-term antibiotics given the promising results with regard to time to detection for this groups as well.
Level of Evidence
Level II, diagnostic study.
Huo, Z et al., 2022. Perfusion-Based Bioreactor Culture and Isothermal Microcalorimetry for Preclinical Drug Testing with the Carbonic Anhydrase Inhibitor SLC-0111 in Patient-Derived Neuroblastoma. Int. J. Mol. Sci.
Neuroblastoma is a rare disease. Rare are also the possibilities to test new therapeutic options for neuroblastoma in clinical trials. Despite the constant need to improve therapy and outcomes for patients with advanced neuroblastoma, clinical trials currently only allow for testing few substances in even fewer patients. This increases the need to improve and advance preclinical models for neuroblastoma to preselect favorable candidates for novel therapeutics. Here we propose the use of a new patient-derived 3D slice-culture perfusion-based 3D model in combination with rapid treatment evaluation using isothermal microcalorimetry exemplified with treatment with the novel carbonic anhydrase IX and XII (CAIX/CAXII) inhibitor SLC-0111. Patient samples showed a CAIX expression of 18% and a CAXII expression of 30%. Corresponding with their respective CAIX expression patterns, the viability of SH-EP cells was significantly reduced upon treatment with SLC-0111, while LAN1 cells were not affected. The inhibitory effect on SH-SY5Y cells was dependent on the induction of CAIX expression under hypoxia. These findings corresponded to thermogenesis of the cells. Patient-derived organotypic slice cultures were treated with SLC-0111, which was highly effective despite heterogeneity of CAIX/CAXII expression. Thermogenesis, in congruence with the findings of the histological observations, was significantly reduced in SLC-0111-treated samples. In order to extend the evaluation time, we established a perfusion-based approach for neuroblastoma tissue in a 3D perfusion-based bioreactor system. Using this system, excellent tissue quality with intact tumor cells and stromal structure in neuroblastoma tumors can be maintained for 7 days. The system was successfully used for consecutive drug response monitoring with isothermal microcalorimetry. The described approach for drug testing, relying on an advanced 3D culture system combined with a rapid and highly sensitive metabolic assessment, can facilitate development of personalized treatment strategies for neuroblastoma.
Sultan A. R. et al., 2022. Real time monitoring of Staphylococcus aureus biofilm sensitivity towards antibiotics with isothermal microcalorimetry. PLoS ONE
Biofilm-associated infections with Staphylococcus aureus are difficult to treat even after administration of antibiotics that according to the standard susceptibility assays are effective. Currently, the assays used in the clinical laboratories to determine the sensitivity of S. aureus towards antibiotics are not representing the behaviour of biofilm-associated S. aureus, since these assays are performed on planktonic bacteria. In research settings, microcalorimetry has been used for antibiotic susceptibility studies. Therefore, in this study we investigated if we can use isothermal microcalorimetry to monitor the response of biofilm towards antibiotic treatment in real-time. We developed a reproducible method to generate biofilm in an isothermal microcalorimeter setup. Using this system, the sensitivity of 5 methicillin-sensitive S. aureus (MSSA) and 5 methicillin-resistant S. aureus (MRSA) strains from different genetic lineages were determined towards: flucloxacillin, cefuroxime, cefotaxime, gentamicin, rifampicin, vancomycin, levofloxacin, clindamycin, erythromycin, linezolid, fusidic acid, co-trimoxazole, and doxycycline. In contrast to conventional assays, our calorimetry-based biofilm susceptibility assay showed that S. aureus biofilms, regardless MSSA or MRSA, can survive the exposure to the maximum serum concentration of all tested antibiotics. The only treatment with a single antibiotic showing a significant reduction in biofilm survival was rifampicin, yet in 20% of the strains, emerging antibiotic resistance was observed. Furthermore, the combination of rifampicin with flucloxacillin, vancomycin or levofloxacin was able to prevent S. aureus biofilm from becoming resistant to rifampicin. Isothermal microcalorimetry allows real-time monitoring of the sensitivity of S. aureus biofilms towards antibiotics in a fast and reliable way.
Bao X. et al., 2021. Organic acids and their salts potentiate the activity of selected antibiotics against Pseudomonas aeruginosa biofilms grown in a synthetic cystic fibrosis sputum medium. Antimicrobial Agents and Chemotherapy.
The failure of antibiotic therapy in respiratory tract infections in cystic fibrosis is partly due to the high tolerance observed in Pseudomonas aeruginosa biofilms. This tolerance is mediated by changes in bacterial metabolism linked to growth in biofilms, opening up potential avenues for novel treatment approaches based on modulating metabolism. The goal of the present study was to identify carbon sources that increase the inhibiting and/or eradicating activity of tobramycin, ciprofloxacin and ceftazidime against P. aeruginosa PAO1 biofilms grown in a synthetic cystic fibrosis sputum medium (SCFM2) and to elucidate their mode of action. After screening 69 carbon sources, several combinations of antibiotics + carbon sources that showed markedly higher anti-biofilm activity than antibiotics alone were identified. D,L-malic acid and sodium acetate could potentiate both biofilm inhibiting and eradicating activity of ciprofloxacin and ceftazidime, respectively, while citric acid could only potentiate biofilm inhibitory activity of tobramycin. The mechanisms underlying the increased biofilm eradicating activity of combinations ciprofloxacin/D,L-malic acid and ceftazidime/sodium acetate are similar but not identical. Potentiation of ceftazidime activity by sodium acetate was linked to increased metabolic activity, a functional TCA cycle, increased ROS production and high intracellular pH, whereas the latter was not required for D,L-malic acid potentiation of ciprofloxacin. Finally, our results indicate that the potentiation of antibiotic activity by carbon sources is strain dependent.
Bokhari, M.H. et al., 2021. Isothermal microcalorimetry measures UCP1-mediated thermogenesis in mature brite adipocytes. Commun Biol.
The activation of thermogenesis in adipose tissue has emerged as an important target for the development of novel anti-obesity therapies. Using multi-well isothermal microcalorimetry, we have demonstrated that mature murine brown and brite adipocytes produce quantifiable heat upon β3-AR stimulation, independently of any anaerobic mechanisms. Additionally, in brite adipocytes lacking UCP1 protein, β3-AR stimulation still induces heat production, albeit to a much lower extent than in their wildtype counterparts, suggesting that UCP1 is an essential component of adrenergic induced thermogenesis in murine brite adipocytes exvivo. Similarly, we could observe an increase in heat production in human-derived adipocytes (hMADS) upon β-AR stimulation. Collectively, these results establish the use of isothermal microcalorimetry as a sensitive and accurate technique for measuring thermogenic responses in intact mature brite adipocytes from murine and human origin.
Pini, N., et al., 2021. Increased Proliferation of Neuroblastoma Cells under Fructose Metabolism Can Be Measured by Isothermal Microcalorimetry. Children
Neuroblastoma, like other cancer types, has an increased need for energy. This results in an increased thermogenic profile of the cells. How tumor cells optimize their energy efficiency has been discussed since Warburg described the fact that tumor cells prefer an anaerobic to an aerobic metabolism in the 1920s. An important question is how far the energy efficiency is influenced by the substrate. The aim of this study was to investigate how the metabolic activity of neuroblastoma cells is stimulated by addition of glucose or fructose to the medium and if this can be measured accurately by using isothermal microcalorimetry. Proliferation of Kelly and SH-EP Tet-21/N cells was determined in normal medium, in fructose-enriched, in glucose-enriched and in a fructose/glucose-enriched environment. Heat development of cells was measured by isothermal microcalorimetry. The addition of fructose, glucose or both to the medium led to increases in the metabolic activity of the cells, resulting in increased proliferation under the influence of fructose. These changes were reflected in an enhanced thermogenic profile, mirroring the results of the proliferation assay. The tested neuroblastoma cells prefer fructose metabolism over glucose metabolism, a quality that provides them with a survival benefit under unfavorable low oxygen and low nutrient supply when fructose is available. This can be quantified by measuring thermogenesis.
Bergsten, H. et al., 2021. Adjunctive rifampicin increases antibiotic efficacy in group A streptococcal tissue infection models. Antimicrob Agents Chemother
7 September 2021
Gros, S. et al., 2021. Metastatic Esophageal Carcinoma Cells Exhibit Reduced Adhesion Strength and Enhanced Thermogenesis. Cells
Despite continuous improvements in multimodal therapeutic strategies, esophageal carcinoma maintains a high mortality rate. Metastases are a major life-limiting component; however, very little is known about why some tumors have high metastatic potential and others not. In this study, we investigated thermogenic activity and adhesion strength of primary tumor cells and corresponding metastatic cell lines derived from two patients with metastatic adenocarcinoma of the esophagus. We hypothesized that the increased metastatic potential of the metastatic cell lines correlates with higher thermogenic activity and decreased adhesion strength. Our data show that patient-derived metastatic esophageal tumor cells have a higher thermogenic profile as well as a decreased adhesion strength compared to their corresponding primary tumor cells. Using two paired esophageal carcinoma cell lines of primary tumor and lymph nodes makes the data unique. Both higher specific thermogenesis profile and decreased adhesion strength are associated with a higher metastatic potential. They are in congruence with the clinical patient presentation. Understanding these functional, biophysical properties of patient derived esophageal carcinoma cell lines will enable us to gain further insight into the mechanisms of metastatic potential of primary tumors and metastases. Microcalorimetric evaluation will furthermore allow for rapid assessment of new treatment options for primary tumor and metastases aimed at decreasing the metastatic potential.
Bové, M. et al., 2021. The quorum sensing inhibitor furanone C-30 rapidly loses its tobramycin potentiating activity against Pseudomonas aeruginosa biofilms during experimental evolution. Antimicrobial Agents and Chemotherapy
The use of quorum sensing inhibitors (QSI) has been proposed as an alternative strategy to combat antibiotic resistance. QSI reduce the virulence of a pathogen without killing it and it is claimed that resistance to such compounds is less likely to develop although there is a lack of experimental data supporting this hypothesis. Additionally, such studies are often carried out in conditions that do not mimic the in vivo situation. In the present study, we evaluated whether a combination of the QSI furanone C-30 and the aminoglycoside antibiotic tobramycin is ‘evolution-proof’ when it is used to eradicate Pseudomonas aeruginosa biofilms grown in a synthetic cystic fibrosis sputum medium. We found that the biofilm eradicating activity of the tobramycin/furanone C-30 combination decreased already after 5 treatment cycles. The antimicrobial susceptibility of P. aeruginosa to tobramycin decreased 8-fold after 16 cycles of treatment with the tobramycin/furanone C-30 combination. Furthermore, microcalorimetry revealed changes in the metabolic activity of P. aeruginosa exposed to furanone C-30, tobramycin, and the combination. Whole-genome sequencing analysis of the evolved strains exposed to the combination identified mutations in mexT, fusA1 and parS, genes known to be involved in antibiotic resistance. In P. aeruginosa treated with furanone C-30 alone, a deletion in mexT was also observed. Our data indicate that furanone C-30 is not ‘evolution-proof’ and quickly becomes ineffective as a tobramycin potentiator.
Kragh, K. et al., 2021. Effective antimicrobial combination in vivo treatment predicted with microcalorimetry screening. Journal of Antimicrobial Chemotherapy
The worldwide emergence of antibiotic resistance calls for effective exploitation of existing antibiotics. Antibiotic combinations with different modes of action can synergize for successful treatment. In the present study, we used microcalorimetry screening to identify synergistic combination treatments against clinical MDR isolates. The synergistic effects were validated in a murine infection model.
Cirnski, K. et al., 2020. Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry. JoVE
The elucidation of the mode-of-action of a novel antibiotic is a challenging task in the drug discovery process. The goal of the method described here is the application of isothermal microcalorimetry using calScreener in antibacterial profiling to provide additional insight into drug-microbe interactions.
Lichtenberg, M. et al., 2020. Metabolic flux fingerprinting differentiates planktonic and biofilm states of Pseudomonas aeruginosa and Staphylococcus aureus. bioRxiv
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.
Tellapragda, C. et al., 2020. Isothermal microcalorimetry minimal inhibitory concentration testing in extensively drug resistant Gram-negative bacilli – A multicenter study. Clinical Microbiology and Infection
We evaluated performance of an isothermal microcalorimetry (IMC) method for determining the minimal inhibitory concentrations (MICs) among extensively drug resistant (XDR) Gram-negative bacilli.
Gros, S. et al., 2019. Personalized Treatment Response Assessment for Rare Childhood Tumors Using Microcalorimetry–Exemplified by Use of Carbonic Anhydrase IX and Aquaporin 1 Inhibitors. International Journal of Molecular Sciences, 20(20), 4984
We present a novel approach to a personalized therapeutic concept for solid tumors. We illustrate this on a rare childhood tumor for which only a generalized treatment concept exists using carbonic anhydrase IX and aquaporin 1 inhibitors. The use of microcalorimetry as a refined in vitro method for evaluation of drug susceptibility in organotypic slice culture has not previously been established. Rapid microcalorimetric drug response assessment can refine a general treatment concept when it is applied in cases in which tumors do not respond to conventional chemo-radiation treatment. For solid tumors, which do not respond to classical treatment, and especially for rare tumors without an established protocol rapid microcalorimetric drug response testing presents an elegant novel approach to test alternative therapeutic approaches. While improved treatment concepts have led to improved outcome over the past decades, the prognosis of high risk disease is still poor and rethinking of clinical trial design is necessary. A small patient population combined with the necessity to assess experimental therapies for rare solid tumors rather at the time of diagnosis than in relapsed or refractory patients provides great challenges. The possibility to rapidly compare established protocols with innovative therapeutics presents an elegant novel approach to refine and personalize treatment.
Abdillahi, S. et al., 2018. Collagen VI Contains Multiple Host Defense Peptides with Potent In Vivo Activity. The Journal of Immunology, 201(3), pp.1007–1020
Collagen VI is a ubiquitous extracellular matrix component that forms extensive microfibrillar networks in most connective tissues. In this study, we describe for the first time, to our knowledge, that the collagen VI von Willebrand factor type A–like domains exhibit a broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria in human skin infections in vivo. In silico sequence and structural analysis of VWA domains revealed that they contain cationic and amphipathic peptide sequence motifs, which might explain the antimicrobial nature of collagen VI. In vitro and in vivo studies show that these peptides exhibited significant antibacterial activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa through membrane disruption. Our findings shed new light on the role of collagen VI–derived peptides in innate host defense and provide templates for development of peptide-based antibacterial therapies.
Kriszt, R. et al., 2017. Optical visualisation of thermogenesis in stimulated single-cell brown adipocytes. Scientific Reports, 7(1), pp.1–14.
The identification of brown adipose deposits in adults has led to significant interest in targeting this metabolically active tissue for treatment of obesity and diabetes.
Improved methods for the direct measurement of heat production as the signature function of brown adipocytes (BAs), particularly at the single cell level, would be of substantial benefit to these ongoing efforts. Here, we report the first application of a small molecule-type thermosensitive fluorescent dye, ERthermAC, to monitor thermogenesis in BAs derived from murine brown fat precursors and in human brown fat cells differentiated from human neck brown preadipocytes.
ERthermAC accumulated in the endoplasmic reticulum of BAs and displayed a marked change in fluorescence intensity in response to adrenergic stimulation of cells, which corresponded to temperature change. ERthermAC fluorescence intensity profiles were congruent with mitochondrial depolarisation events visualised by the JC-1 probe. Moreover, the averaged fluorescence intensity changes across a population of cells correlated well with dynamic changes such as thermal power, oxygen consumption, and extracellular acidification rates.
These findings suggest ERthermAC as a promising new tool for studying thermogenic function in brown adipocytes of both murine and human origins.
Wadsö, I. et al., 2017. A well-plate format isothermal multi-channel microcalorimeter for monitoring the activity of living cells and tissues. Thermochimica Acta, 652, pp.141–149.
Design and properties are reported for a novel type of multi-channel isothermal microcalorimeter. It is equipped with 48 calorimetric units (channels) and is primarily intended for use as a monitor of the activity of living cells, tissues and small animals. Calorimetric vessels are positioned in a holder with the format of a 48-well microtiter plate.
At most, 47 samples can be measured simultaneously; one vessel is then used as reference. The standard configuration is 32 sample positions using 16 channels as references in a twin calorimeter setup. The detection limit is then 0.1 μW. Sample volumes are usually 100 μl–300 μl. The 24 h baseline stability is typically 0.2 μW (room temperature variation ≤1 °C). The instrument was designed considering feasible uses in applied biology, especially in pharmaceutical and clinical laboratories and in environmental work.
However, it can be employed as a general monitor of slow processes in different fields of biology and for non-biological systems, including accurate determination of their thermal powers (heat production rates). In the present report, properties of the instrument are characterized by chemical calibration experiments and in measurements of growth of bacteria and mammalian cells.
Astasov-Frauenhoffer, M. et al., 2017. Exopolysaccharides regulate calcium flow in cariogenic biofilms. PLoS ONE, 12(10), pp.1–14.
Caries-associated biofilms induce loss of calcium from tooth surfaces in the presence of dietary carbohydrates. Exopolysaccharides (EPS) provide a matrix scaffold and an abundance of primary binding sites within biofilms. The role of EPS in binding calcium in cariogenic biofilms is only partially understood. Thus, the aim of the present study is to investigate the relationship between the calcium dissolution rates and calcium tolerance of caries-associated bacteria and yeast as well as to examine the properties of EPS to quantify its binding affinity for dissolved calcium. Calcium dissolution was measured by dissolution zones on Pikovskaya’s agar.
Calcium tolerance was assessed by isothermal microcalorimetry (IMC) by adding CaCl2 to the bacterial cultures. Acid-base titration and Fourier transform infrared (FTIR) spectroscopy were used to identify possible functional groups responsible for calcium binding, which was assessed by isothermal titration calorimetry (ITC). Lactobacillus spp. and mutans streptococci demonstrated calcium dissolution in the presence of different carbohydrates.
All strains that demonstrated high dissolution rates also revealed higher rates of calcium tolerance by IMC. In addition, acidic functional groups were predominantly identified as possible binding sites for calcium ions by acid-base titration and FTIR. Finally, ITC revealed EPS to have a higher binding affinity for calcium compared, for example, to lactic acid. In conclusion, this study illustrates the role of EPS in terms of the calcium tolerance of cariogenic microbiota by determining the ability of EPS to control free calcium concentrations within the biofilms as a self-regulating mode of action in the pathogenesis of dental caries.
Flores, D. et al., 2016. A novel isothermal microcalorimetry tool to assess drug effects on Ancylostoma ceylanicum and Necator americanus. Applied Microbiology and Biotechnology, 100(2), pp.837–846.
Soil-transmitted helminths, which affect the poorest communities, worldwide cause a range of symptoms and morbidity, yet few treatment options are available and drug resistance is a concern.
To improve and accelerate anthelminthic drug discovery, novel drug screening tools such as isothermal microcalorimetry (IMC) have been tested with great potential. In this study, we used a novel microcalorimeter, the calScreener™, to study the viability on the hookworms Necator americanus and Ancylostoma ceylanicum as well as the whipworm Trichuris muris. Significant heat flow signals could be obtained with already one adult worm per channel for all three species. High-amplitude oscillations were observed for the hookworms; however, adult T. muris showed a twofold heat flow decrease during the first 24 h.
Antinematodal effects of ivermectin and levamisole at 1, 10, and 100 μg/ml were evaluated on adult N. americanus and A. ceylanicum. Levamisole-treated hookworms showed a decline in heat flow and oscillation amplitude in a dose-response manner. Heat flow for ivermectin-treated hookworms increased proportionally with increased concentrations of ivermectin, though the wavelet analysis showed an opposite trend as observed by flatter wavelets. In conclusion, the calScreener™ is an excellent tool to study drug effects on intestinal hookworms at the adult worm stage as it offers a lower detection limit than other IMC devices and the possibility to monitor worm viability online.
Braissant, O. et al., 2015. Isothermal microcalorimetry accurately detects bacteria, tumorous microtissues, and parasitic worms in a label-free well-plate assay. Biotechnology Journal, 10(3), pp.460–468.
Isothermal microcalorimetry is a label-free assay that allows monitoring of enzymatic and metabolic activities. The technique has strengths, but most instruments have a low throughput, which has limited their use for bioassays. Here, an isothermal microcalorimeter, equipped with a vessel holder similar to a 48-well plate, was used. The increased throughput of this microcalorimeter makes it valuable for biomedical and pharmaceutical applications.
Our results show that the sensitivity of the instrument allows the detection of 3 × 10(4) bacteria per vial. Growth of P. mirabilis in Luria Broth medium was detected between 2 and 9 h with decreasing inoculum. The culture released 2.1J with a maximum thermal power of 76 μW. The growth rate calculated using calorimetric and spectrophotometric data were 0.60 and 0.57 h(-1) , respectively. Additional insight on protease activities of P. mirabilis matching the last peak in heat production could be gathered as well. Growth of tumor microtissues releasing a maximum thermal power of 2.1 μW was also monitored and corresponds to a diameter increase of the microtissues from ca. 100 to 428 μm. This opens new research avenues in cancer research, diagnostics, and development of new antitumor drugs.
For parasitic worms, the technique allows assessment of parasite survival using motor and metabolic activities even with a single worm.
Jansson M, 2015. Fighting Resistance With Calorimetry: New Tools for Antimicrobial Drug Development. American Laboratory Nov/Dec2015
Antibiotic-resistant bacteria are a growing concern, and with good reason—an estimated 23,000 deaths and two million illnesses occur annually in the U.S.1 Major contributing factors are overuse, lack of regulation and scarcity of novel antibiotics.
Jansson M, 2014. Label-Free Cell-Based Assays and the Holistic Calorimetry Approach. American Laboratory Nov/Dec2014
The cell-based assay arena is slowly entering center stage due to the major and growing medical problems facing the world—both financial (i.e., drug development) and curative (i.e., antibiotic resistance). In a modern 3-D holistic format, the mature technique of calorimetry is the answer to some of these problems.
This article describes how and why measurement of the total metabolic response of a biological system is a valuable proposition and a true time- and cost-efficient complement to existing assays.