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

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

Exploring the resilience and stability of a defined human gut microbiota consortium: An isothermal microcalorimetric study.

The gut microbiota significantly contributes to human health and well-being. The aim of this study was to evaluate the stability and resilience of a consortium composed of three next-generation probiotics (NGPs) candidates originally found in the human gut. The growth patterns of Akkermansia muciniphila, Bacteroides thetaiotaomicron, and Faecalibacterium prausnitzii were studied both individually and consortium. The growth kinetics of Akkermansia muciniphila (A. muciniphila), Bacteroides thetaiotaomicron (B. thetaiotaomicron), and Faecalibacterium prausnitzii (F. prausnitzii) were characterized both individually and in consortium using isothermal microcalorimetry and 16S ribosomal RNA next-generation sequencing. The consortium reached stability after three passages and demonstrated resilience to changes in its initial composition. The concentration of butyrate produced was nearly twice as high in the consortium compared to the monoculture of F. prausnitzii. The experimental conditions and methodologies used in this article are a solid foundation for developing further complex consortia.

Proof of concept: real-time viability and metabolic profiling of probiotics with isothermal microcalorimetry.

Isothermal microcalorimetry (IMC) is a potent analytical method for the real-time assessment of microbial metabolic activity, which serves as an indicator of microbial viability. This approach is highly relevant to the fields of probiotics and Live Biotherapeutic Products (LBPs), offering insights into microbial viability and growth kinetics. One important characteristic of IMC is its ability to measure microbial metabolic activity separately from cellular enumeration. This is particularly useful in situations where continuous tracking of bacterial activity is challenging. The focus on metabolic activity significantly benefits both probiotic research and industrial microbiology applications. IMC's versatility in handling different media matrices allows for the implementation of viability assessments under conditions that mirror those found in various industrial environments or biological models. In our study, we provide a proof of concept for the application of IMC in determining viability and growth dynamics and their correlation with bacterial count in probiotic organisms. Our findings reinforce the potential of IMC as a key method for process enhancement and accurate strain characterization within the probiotic sector. This supports the broader objective of refining the systematic approach and methods used during the development process, thereby providing detailed insights into probiotics and LBPs.

Efficacy of different bioactive glass S53P4 formulations in biofilm eradication and the impact of pH and osmotic pressure.

AIM: The challenging properties of biofilm-associated infections and the rise of multidrug-resistant bacteria are prompting the exploration of alternative treatment options. This study investigates the efficacy of different bioactive glass (BAG) formulations - alone or combined with vancomycin - to eradicate biofilm. Further, we study the influence of BAG on pH and osmotic pressure as important factors limiting bacterial growth. METHOD: Different BAG S53P4 formulations were used for this study, including (a) powder (<45 µm), (b) granules (500-800 µm), (c) a cone-shaped scaffold and (d) two putty formulations containing granules with no powder (putty A) or with additional powder (putty B) bound together by a synthetic binder. Inert glass beads (1.0-1.3 mm) were included as control. All formulations were tested in a concentration of 1750 mg/ml in Müller-Hinton-Broth against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE). Vancomycin was tested at the minimum-inhibitory concentration for each strain. Changes in pH and osmolality over time were assessed at 0 h, 24 h, 72 h and 168 h. RESULTS: All tested BAG formulations showed antibiofilm activity against MRSA and MRSE. Powder and putty B were the most effective formulations suppressing biofilm leading to its complete eradication after up to 168 h of co-incubation, followed by granules, scaffold and putty A. In general, MRSE appeared to be more susceptible to bioactive glass compared to MRSA. The addition of vancomycin had no substantial impact on biofilm eradication. We observed a positive correlation between a higher pH and higher antibiofilm activity. CONCLUSIONS: BAG S53P4 has demonstrated efficient biofilm antibiofilm activity against MRSA and MRSE, especially in powder-containing formulations, resulting in complete eradication of biofilm. Our data indicate neither remarkable increase nor decrease in antimicrobial efficacy with addition of vancomycin. Moreover, high pH appears to have a direct antimicrobial impact; the role of high osmolality needs further investigation.

Bacterial isolates from positive paired venous catheter and peripheral blood cultures taken during parenteral nutrition were the same species but different strains: A case report.

OBJECTIVE: The same microbial species isolated from blood simultaneously drawn from a central venous catheter hub and a peripheral vein (paired blood cultures) during parenteral nutrition may be assumed to represent the same strain. This case report provides an example of this assumption being incorrect along with a comparator example of it being correct. This has implications for interpretation of differential time to positivity and differential quantitative blood cultures during investigation of suspected intraluminal intravascular catheter or cannula bloodstream infection. CASE DESCRIPTION: Two patients ages ≥18 y prescribed parenteral nutrition each had positive paired blood cultures that had been taken for suspected catheter bloodstream infection because of temperature spikes ≥38°C. The paired Staphylococcus epidermidis isolates from the first patient and the paired Enterococcus faecium isolates from the second patient were each tested beyond routine clinical care to establish if they could be different strains. The central and peripheral isolates of Staphylococcus epidermidis from the first patient were different strains based on hospital-reported antibiograms, genomic DNA profiles, thermograms, and weaker growth and different sizes of colonies of the central strain compared with the peripheral strain. There were no such differences for the isolates of Enterococcus faecium from the second patient. RESULTS: The central and peripheral isolates of Staphylococcus epidermidis from the first patient were different strains based on hospital-reported antibiograms, genomic DNA profiles, thermograms, and weaker growth and different sizes of colonies of the central strain compared with the peripheral strain. There were no such differences for the isolates of Enterococcus faecium from the second patient. CONCLUSION: This case report indicates consideration should be given to reporting whether bacteria have been identified at either species or strain level if differential time to positivity or differential quantitative blood cultures are used to define catheter or cannula bloodstream infection.

The potential for isothermal microcalorimetry to detect venous catheter infection isolates and establish antibiograms.

OBJECTIVES: Because bloodstream infection and venous catheter (or cannula) bloodstream infection are associated with high morbidity and cost, early identification and treatment are important. Isothermal microcalorimetry can detect microbial growth using thermal power (heat flow), essentially in real time. The aim of this study was to examine the potential of this technique in clinical practice. METHODS: Thermal power of wild-type bacteria (Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae, and Enterococcus faecium) isolated from blood cultures of adult inpatients receiving parenteral nutrition in routine clinical practice was measured at 37°C every 10s using a Thermometric 2277 instrument. Temporal patterns of heat flow were used to detect the presence of bacteria, differentiate between them, and test their antibiotic sensitivity. Within and between batch reproducibility (% coefficient of variation [%CV]) was also established. RESULTS: Isothermal microcalorimetry always correctly detected the absence or presence of wild-type bacteria. Thermograms differed distinctly between species. Key thermographic features, such as peak heights, timing of peak heights, and interval between peak heights, were highly reproducible within each species (within-batch %CV usually about ≤1%, although between-batch %CV was usually higher). The antibiotic sensitivities (tested only for S. epidermidis and K. pneumoniae) confirmed the results obtained from the hospital laboratory. CONCLUSIONS: Isothermal microcalorimetry is a promising and highly reproducible real-time measurement technique with potential application to the investigation, species identification, and targeted antibiotic treatment of bloodstream infection and venous catheter (or cannula) bloodstream infection.

Isothermal microcalorimetry improves accuracy and time to bacterial detection of periprosthetic joint infection after total joint arthroplasty.

Isothermal microcalorimetry (IMC) was evaluated compared to conventional cultures to determine the clinical performance for diagnosing periprosthetic joint infection (PJI) of hip/knee replacements. We prospectively collected three to five deep tissue samples per patient from 152 patients undergoing conversion or revision hip/knee arthroplasty from July 2020 to November 2022. Cultures and IMC for each sample were compared for concordance, median time to detection (TTD), and diagnostic performance based on 2013 Musculoskeletal Infection Society criteria. Secondary analyses involved patients on antibiotics at sampling. The 152 total patients had 592 tissue samples (mean 3.9 ± 0.3) with sample concordance between cultures and IMC of 90%. IMC demonstrated a sensitivity of 83%, specificity of 100%, negative predictive value (NPV) of 89%, and positive predictive value (PPV) of 100% for PJI. Cultures resulted in 69% sensitivity, 100% specificity, 81% NPV, and 100% PPV. The accuracy of IMC was 93% compared to 87% for cultures (P < 0.001). The median TTD of PJI by cultures was 51 (21-410) hours compared to 10 (0.5-148) hours for IMC (P < 0.001). For 39 patients on chronic antibiotics, sensitivity in PJI detection was 93%, specificity 100%, NPV 85%, and PPV 100% by IMC compared to 79% sensitivity, 100% specificity, 65% NPV, and 100% PPV for cultures. The accuracy was 95% for IMC compared to 85% for cultures (P < 0.001) with median TTD of 12 (0.5-127) hours compared to 52 (21-174) hours (P < 0.001). Utilizing IMC for PJI detection improves TTD by nearly 2 days while improving diagnostic accuracy compared to cultures, particularly in patients on chronic antibiotics.

Possibilities and limitations of α-relaxation data of amorphous freeze-dried cakes to predict long term IgG1 antibody stability.

The value of correlating global α-relaxations with long term protein stability after freeze-drying is inconsistently reported. This study aims to clarify whether and to what extend the long term stability of a freeze-dried protein formulation can be predicted with this method. For this purpose, the α-relaxation parameter τß [h] of freshly prepared freeze-dried products is obtained by isothermal microcalorimetry. The concept is, that molecular movements in the amorphous matrix are strongly reduced in cakes with longer relaxation time and the product should therefore be more resistant against aggregation. To increase τß in comparison to a conventional freeze-drying cycle, aggressive drying cycles including structural collapse of the product as well as tempering protocols after freeze-drying are applied. The τß values are correlated with the aggregation rate of a freeze-dried IgG1 monoclonal antibody measured with high performance size exclusion chromatography. The antibody was used in its market formulation and 6 further compositions. A weak correlation between α-relaxation times and IgG1 aggregation was found. A higher mobility level through increased residual moisture helped to improve the correlation.

Distinct Effects of Chemical Toxicity and Radioactivity on Metabolic Heat of Cultured Cells Revealed by "Isotope-Editing".

Studying the toxicity of chemical compounds using isothermal microcalorimetry (IMC), which monitors the metabolic heat from living microorganisms, is a rapidly expanding field. The unprecedented sensitivity of IMC is particularly attractive for studies at low levels of stressors, where lethality-based data are inadequate. We have revealed via IMC the effect of low dose rates from radioactive ß--decay on bacterial metabolism. The low dose rate regime (<400 µGyh-1) is typical of radioactively contaminated environmental sites, where chemical toxicity and radioactivity-mediated effects coexist without a predominance or specific characteristic of either of them. We found that IMC allows distinguishing the two sources of metabolic interference on the basis of "isotope-editing" and advanced thermogram analyses. The stable and radioactive europium isotopes 153Eu and 152Eu, respectively, were employed in monitoring Lactococcus lactis cultures via IMC. ß--emission (electrons) was found to increase initial culture growth by increased nutrient uptake efficiency, which compensates for a reduced maximal cell division rate. Direct adsorption of the radionuclide to the biomass, revealed by mass spectrometry, is critical for both the initial stress response and the "dilution" of radioactivity-mediated damage at later culture stages, which are dominated by the chemical toxicity of Eu.