Rapid detection of urinary tract pathogens using microcalorimetry: principle, technique and first results.

UNLABELLED: What's known on the subject? and What does the study add? Microcalorimetry has been applied in several microbiological studies, but never in a clinical urological context. In addition, basic knowledge on the growth of urinary pathogens in urine is still scarce and data regarding the growth rate of many urinary pathogens in urine are still not available. The study demonstrates that this innovative application of microcalorimetry is useful in (i) investigating the growth of urinary pathogens in sterilized urine and (ii) as a rapid tool for diagnosis of urinary infection as well as for further identification of the causative infectious agent. OBJECTIVE: To investigate the value of isothermal microcalorimetry (IMC) in the detection and differentiation of common urinary tract pathogens in urine. IMC is a non-specific analytical tool for the measurement of heat in the microwatt range. PATIENTS AND METHODS: A microcalorimeter equipped with 48 channels was used. Detection was accomplished, and growth was monitored for four bacterial strains in sterilized urine at 37 °C by measuring metabolic heat flow (µW = µJ/s) as a function of time. The strains were Escherichia coli, Proteus mirabilis, Enterococcus faecalis and Staphylococcus aureus. RESULTS: Bacterial growth was detected after 3.1 to 17.1 h with decreasing inocula. The detection limit was 1 colony-forming unit (CFU)/mL for E. coli, 10 CFU/mL for P. mirabilis and E. faecalis and 10(3) CFU/mL for S. aureus. The total heat was highest in P. mirabilis ranging from 10 to 12 J, followed by E. coli (3-4 J), S. aureus (2-3 J) and E. faecalis (1.3-1.5 J). The shape of the heat flow curves was characteristic for each species independent of its initial concentration. CONCLUSIONS: IMC allows rapid detection of bacteriuria, much faster than conventional culture. Urinary tract pathogen detection after only 3.1 h is realistic. Clearly different heat flow patterns enable accurate pathogen differentiation. Due to expeditious identification of urine samples that contain only low colony counts (i.e. less than 10(3) CFU/mL), IMC may become a valuable screening tool for detecting the presence of significant bacteriuria.

Use of hydrodynamic forces to engineer cartilaginous tissues resembling the non-uniform structure and function of meniscus.

The aim of this study was to demonstrate that differences in the local composition of bi-zonal fibrocartilaginous tissues result in different local biomechanical properties in compression and tension. Bovine articular chondrocytes were loaded into hyaluronan-based meshes (HYAFF-11) and cultured for 4 weeks in mixed flask, a rotary Cell Culture System (RCCS), or statically. Resulting tissues were assessed histologically, immunohistochemically, by scanning electron microscopy and mechanically in different regions. Local mechanical analyses in compression and tension were performed by indentation-type scanning force microscopy and by tensile tests on punched out concentric rings, respectively. Tissues cultured in mixed flask or RCCS displayed an outer region positively stained for versican and type I collagen, and an inner region positively stained for glycosaminoglycans and types I and II collagen. The outer fibrocartilaginous capsule included bundles (up to 2 microm diameter) of collagen fibers and was stiffer in tension (up to 3.6-fold higher elastic modulus), whereas the inner region was stiffer in compression (up to 3.8-fold higher elastic modulus). Instead, molecule distribution and mechanical properties were similar in the outer and inner regions of statically grown tissues. In conclusion, exposure of articular chondrocyte-based constructs to hydrodynamic flow generated tissues with locally different composition and mechanical properties, resembling some aspects of the complex structure and function of the outer and inner zones of native meniscus.

Effects of scaffold composition and architecture on human nasal chondrocyte redifferentiation and cartilaginous matrix deposition.

We investigated whether the post-expansion redifferentiation and cartilage tissue formation capacity of adult human nasal chondrocytes can be regulated by controlled modifications of scaffold composition and architecture. As a model system, we used poly(ethylene glycol)-terephthalate-poly(butylene)-terephthalate block copolymer scaffolds from two compositions (low or high PEG content, resulting in different wettability) and two architectures (generated by compression molding or three-dimensional (3D) fiber deposition) with similar porosity and mechanical properties, but different interconnecting pore architectures. Scaffolds were seeded with expanded human chondrocytes and the resulting constructs assessed immunohistochemically, biochemically and at the mRNA expression level following up to 4 weeks of static culture. For a given 3D architecture, the more hydrophilic scaffold enhanced cell redifferentiation and cartilaginous tissue formation after 4 weeks culture, as assessed by higher mRNA expression of collagen type II, increased deposition of glycosaminoglycan (GAG) and predominance of type II over type I collagen immunostain. The fiber-deposited scaffolds, with a more accessible pore volume and larger interconnecting pores, supported increased GAG deposition, but only if a more hydrophilic composition was used. By applying controlled and selective modifications of chemico-physical scaffold parameters, we demonstrate that both scaffold composition and architecture are instructive for expanded human chondrocytes in the generation of 3D cartilaginous tissues. The observed effects of composition and architecture were likely to have been mediated, respectively, by differential serum protein adsorption and efficiency of nutrient/waste exchange.

Isothermal microcalorimetry provides new insights into biofilm variability and dynamics.

The purpose of this study was to investigate a three-species in vitro biofilm with peri-implantitis-related bacteria for its variability and metabolic activity. Streptococcus sanguinis, Fusobacterium nucleatum, and Porphyromonas gingivalis were suspended in simulated body fluid containing 0.2% glucose to form biofilms on polished, protein-coated implant-grade titanium disks over 72 h using a flow chamber system. Thereafter, biofilm-coated disks were characterized by scanning electron microscopy and fluorescence in situ hybridization/confocal laser scanning microscopy. To assess metabolic activity within the biofilms, their heat flow was recorded for 480 h at 37 °C by IMC. The microscopic methods revealed that the total number of bacteria in the biofilms varied slightly among specimens (2.59 × 10(4)  ± 0.67 × 10(4)  cells mm(-2) ), whereas all three species were found constantly with unchanged proportions (S. sanguinis 41.3 ± 4.8%, F. nucleatum 17.7 ± 2.1%, and P. gingivalis 41.0 ± 4.9%). IMC revealed minor differences in time-to-peak heat flow (20.6 ± 4.5 h), a trend consistent with the small variation in bacterial species proportions as shown by microscopy. Peak heat flow (35.8 ± 42.6 µW), mean heat flow (13.1 ± 22.0 µW), and total heat over 480 h (23.5 ± 37.2 J) showed very high variation. These IMC results may be attributed to differences in the initial cell counts and relative proportions of the three species, their distribution and embedment in exopolysaccharide matrix on the test specimens. The present results provide new insights into variability and dynamics of biofilms on titanium disks, aspects that should be explored in future studies of dental surfaces.

Closed ampoule isothermal microcalorimetry for continuous real-time detection and evaluation of cultured mammalian cell activity and responses.

Closed ampoule isothermal microcalorimetry (IMC) is a simple, powerful, nondestructive, and convenient technique that allows continuous, real-time detection and evaluation of cultured cell activity and responses. At a selected set temperature, IMC measures the heat flow between a sample and a heat sink and compares it to the heat-flow between a thermally inactive reference and the heat sink. Since heat flow rates are proportional to the rates of chemical reactions and changes of state, IMC provides a means for dynamically following these processes in any type of specimen - including ones containing cultured cells. The ability of IMC instruments to provide measurements in the microwatt (µJ/s) range allows one to detect and follow the activity (including replication) of low numbers of cells in culture (ca. 10(3)-10(5), depending on cell type). Closed ampoule IMC is increasingly being used in medical and environmental sciences. While a closed ampoule imposes limitations, it conversely provides simplicity and excellent control. Also, it is still usually possible with closed ampoules to follow mammalian cell activity and replication for several days. This chapter provides an overview of IMC measurement principles and provides examples of the use of IMC for evaluating cultured human and other mammalian cell activity and responses.

Use of an isothermal microcalorimetry assay to characterize microbial oxalotrophic activity.

Isothermal microcalorimetry (IMC) has been used in the past to monitor metabolic activities in living systems. A few studies have used it on ecological research. In this study, IMC was used to monitor oxalotrophic activity, a widespread bacterial metabolism found in the environment, and particularly in soils. Six model strains were inoculated in solid angle media with K-oxalate as the sole carbon source. Cupriavidus oxalaticus, Cupriavidus necator, and Streptomyces violaceoruber presented the highest activity (91, 40, and 55 µW, respectively) and a maximum growth rate (µmax h(-1) ) of 0.264, 0.185, and 0.199, respectively, among the strains tested. These three strains were selected to test the incidence of different oxalate sources (Ca, Cu, and Fe-oxalate salts) in the metabolic activity. The highest activity was obtained in Ca-oxalate for C. oxalaticus. Similar experiments were carried out with a model soil to test whether this approach can be used to measure oxalotrophic activity in field samples. Although measuring oxalotrophic activity in a soil was challenging, there was a clear effect of the amendment with oxalate on the metabolic activity measured in soil. The correlation between heat flow and growth suggests that IMC analysis is a powerful method to monitor bacterial oxalotrophic activity.

Acrylamide Polymer Double-Network Hydrogels: Candidate Cartilage Repair Materials with Cartilage-Like Dynamic Stiffness and Attractive Surgery-Related Attachment Mechanics.

BACKGROUND: In focal repair of joint cartilage and meniscus, initial stiffness and strength of repairs are generally much less than surrounding tissue. This increases early failure potential. Secure primary fixation of the repair material is also a problem. Acrylamide polymer double-network (DN) hydrogels are candidate-improved repair materials. DN gels have exceptional strength and toughness compared to ordinary gels. This stems from the double-network structure in which there is a high molar ratio of the second network to the first network, with the first network highly crosslinked and the second loosely crosslinked. Previous studies of acrylic PAMPS/PDMAAm and PAMPS/PAAm DN gels demonstrated physicochemical stability and tissue compatibility as well as the ability to foster cartilage formation. METHODS: Mechanical properties related to surgical use were tested in 2 types of DN gels. RESULTS: Remarkably, these >90%-water DN gels exhibited dynamic impact stiffness (E*) values (~1.1 and ~1.5 MPa) approaching swine meniscus (~2.9 MPa). Dynamic impact energy-absorbing capability was much lower (median loss angles of ~2°) than swine meniscus (>10°), but it is intriguing that >90%-water materials can efficiently store energy. Also, fine 4/0 suture tear-out strength approached cartilage (~2.1 and ~7.1 N v. ~13.5 N). Initial strength of attachment of DN gels to cartilage with acrylic tissue adhesive was also high (~0.20 and ~0.15 N/mm(2)). CONCLUSIONS: DN gel strength and toughness properties stem from optimized entanglement of the 2 network components. DN gels thus have obvious structural parallels with cartilaginous tissues, and their surgical handling properties make them ideal candidates for clinical use.

Use of isothermal microcalorimetry to monitor microbial activities.

Isothermal calorimetry measures the heat flow of biological processes, which is proportional to the rate at which a given chemical or physical process takes place. Modern isothermal microcalorimeters make measurements of less than a microwatt of heat flow possible. As a result, as few as 10 000-100 000 active bacterial cells in culture are sufficient to produce a real-time signal dynamically related to the number of cells present and their activity. Specimens containing bacteria need little preparation, and isothermal microcalorimetry (IMC) is a nondestructive method. After IMC measurements, the undisturbed samples can be evaluated by any other means desired. In this review, we present a basic description of microcalorimetry and examples of microbiological applications of IMC for medical and environmental microbiology. In both fields, IMC has been used to quantify microbial activity over periods of hours or even days. Finally, the recent development of highly parallel instruments (up to 48 channels) and the constantly decreasing costs of equipment have made IMC increasingly attractive for microbiology. Miniaturization of isothermal calorimeters provides an even wider range of possibilities.

Engineering human cell-based, functionally integrated osteochondral grafts by biological bonding of engineered cartilage tissues to bony scaffolds.

In this study, we aimed at developing and validating a technique for the engineering of osteochondral grafts based on the biological bonding of a chondral layer with a bony scaffold by cell-laid extracellular matrix. Osteochondral composites were generated by combining collagen-based matrices (Chondro-Gide) containing human chondrocytes with devitalized spongiosa cylinders (Tutobone) using a fibrin gel (Tisseel). We demonstrate that separate pre-culture of the chondral layer for 3 days prior to the generation of the composite allows for (i) more efficient cartilaginous matrix accumulation than no pre-culture, as assessed histologically and biochemically, and (ii) superior biological bonding to the bony scaffold than 14 days of pre-culture, as assessed using a peel-off mechanical test, developed to measure integration of bilayered materials. The presence of the bony scaffold induced an upregulation in the infiltrated cells of the osteoblast-related gene bone sialoprotein, indicative of the establishment of a gradient of cell phenotypes, but did not affect per se the quality of the cartilaginous matrix in the chondral layer. The described strategy to generate osteochondral plugs is simple to be implemented and--since it is based on clinically compliant cells and materials--is amenable to be readily tested in the clinic.

Microcalorimetry: a novel method for detection of microbial contamination in platelet products.

BACKGROUND: Measuring heat from replicating microorganisms in culture may be a rapid, accurate, and simple screening method for platelets (PLTs). Microcalorimetry for detection of microorganisms in in vitro contaminated PLT products was evaluated. STUDY DESIGN AND METHODS: Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus sanguinis, Escherichia coli, Propionibacterium acnes, and Candida albicans were inoculated in single-donor apheresis PLTs to achieve target concentrations of 10(5), 10(3), 10, or 1 colony-forming units (CFU) per mL of PLTs. Contaminated PLTs in growth medium were incubated at 37 degrees C for 5 days in a calorimeter. Positivity was defined as heat flow of at least 10 microW above the lowest value of the power-time curve. RESULTS: With microcalorimetry, inocula of 10 CFUs per mL PLTs could be detected with the following detection times: S. epidermidis (31.65 hr), S. aureus (24.24 hr), S. sanguinis (7.82 hr), E. coli (7.53 hr), P. acnes (73.57 hr), and C. albicans (43.77 hr). The detection time was less than 4 hr at 10(5) CFUs per mL PLTs for S. aureus, S. sanguinis, and E. coli. Noncontaminated PLTs remained negative. The total heat ranged from 2.8 (S. sanguinis) to 8.3 J (E. coli). The shape of the power-time curve was species-specific and independent from the initial concentration of microorganisms. CONCLUSION: The detection limit of microcalorimetry was 1 to 10 CFUs per mL PLTs. Microcalorimetry is a promising novel method for detection of contaminated PLTs. Applying this method to all PLT products could reduce the frequency of transfusion-related sepsis and prolong the shelf life of PLTs.