Standardization of isothermal microcalorimetry in urinary tract infection detection by using artificial urine.

PURPOSE: Isothermal microcalorimetry (IMC) has recently been reported as a new method to rapidly detect urinary tract pathogens (UTP). However, further application of microcalorimetry in the clinical setting requires a standardized procedure. An important step toward such standardization is to use a reproducible growth medium. In this study, we investigated the potential of artificial urine in combination with microcalorimetry for detection of common UTP. METHODS: A microcalorimeter equipped with 48 channels was used. Detection was accomplished, and growth was monitored for four bacterial strains in artificial 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. RESULT: Bacterial growth was detected after 3-32 h with decreasing inoculums down to 1 CFU. The gram-negative strains grew and were detected faster than their gram-positive counterparts. The growth rates the different strains were 0.75 ± 0.11 for E. coli, 0.74 ± 0.10 for E. faecalis, 1.31 ± 0.04 for P. mirabilis, and 0.56 ± 0.20 for S. aureus. The shape of individual heat flow curves was characteristic for each species independent of its initial concentration. CONCLUSIONS: IMC allows rapid detection of UTP in artificial urine. Clearly, different heat flow patterns enable accurate pathogen differentiation. UTP detection after only 4 h is realistic. The rapid detection of UTP tested in standardized artificial urine proves the diagnostic potential of IMC and warrants further microcalorimetric studies in the clinical setting of urinary tract infections.

Measuring the Metabolic Activity of Mature Mycobacterial Biofilms Using Isothermal Microcalorimetry.

Measuring metabolic activity and response of biofilm to different conditions or compounds is of general interest but is also expected to help in developing new antibiofilm compounds and potentially new treatments. Current culture-based and microscopic methods although of much use have several drawbacks. Isothermal calorimetry can be useful in this context by allowing measurements of the metabolic activity of biofilm grown and maintained on solid medium. Biofilms prepared on membranes were placed in calorimetry vials containing solid medium. Sealed vials were introduced in an isothermal calorimeter, and the rate of metabolic heat production was monitored over time. We chose mycobacteria as an example for this paper as working with mycobacterial biofilms is notoriously difficult.

Drug susceptibility testing of mature Mycobacterium tuberculosis H37Ra and Mycobacterium smegmatis biofilms with calorimetry and laser spectroscopy.

Biofilms are more resistant to antibiotics and antimicrobial stressors than planktonic bacteria; however, only a limited number of standardized assays enable investigation of this phenomenon. Here, we utilized non-invasive and independent techniques, including isothermal microcalorimetry (IMC) and tunable diode laser absorption spectroscopy (TDLAS), to measure the effect of isoniazid on metabolic activity and respiratory capability of mature Mycobacterium tuberculosis H37Ra (an avirulent strain) and Mycobacterium smegmatis biofilms. We detected only minor changes in metabolic heat production and respiratory rates (O2 and CO2) for mature M. smegmatis biofilms after antibiotic exposure. However, mature M. tuberculosis biofilms showed greater sensitivity to antibiotic treatment, with isoniazid exhibiting dose-dependent effects on metabolic activity and respiration. Specifically, treatment of M. tuberculosis biofilms with 250 µg/ml and 1 mg/ml isoniazid decreased the rate of heat production by 33% and 40%, respectively, oxygen consumption by 18% and 55%, respectively, and carbon dioxide production by 27% and 64%, respectively. These effects were prominent even after regrowth of antibiotic-treated M. tuberculosis H37Ra biofilms on fresh medium. Our data therefore suggest that IMC and TDLAS are appropriate for drug susceptibility testing of mature biofilms, and these techniques may facilitate study of microbial resistance to antimicrobial compounds from a bioenergetic perspective.

Metabolic activity of mature biofilms of Mycobacterium tuberculosis and other non-tuberculous mycobacteria.

Mycobacteria are classified into two groups, fast- and slow-growing. Often, fast-growing mycobacteria are assumed to have a higher metabolic activity than their slower counterparts, but in mature biofilms this assumption might not be correct. Indeed, when measuring the metabolic activity of mycobacterial biofilms with two independent non-invasive techniques (isothermal microcalorimetry and tunable diode laser absorption spectrometry), mature biofilms of slow- and fast-growing species appeared more alike than expected. Metabolic heat production rate was 2298 ± 181 µW for M. smegmatis and 792 ± 81 µW for M. phlei, while M. tuberculosis and M. bovis metabolic heat production rates were between these values. These small differences were further confirmed by similar oxygen consumption rates (3.3 ± 0.2 nMole/s and 1.7 ± 0.3 nMole/s for M. smegmatis and M. tuberculosis, respectively). These data suggest that the metabolic potential of slow-growing mycobacterial biofilms has been underestimated, particularly for pathogenic species.

A combined application of tunable diode laser absorption spectroscopy and isothermal micro-calorimetry for calorespirometric analysis.

Calorespirometry is the simultaneous analysis of the rate of heat emission (Rq), O2 consumption (RO2) and CO2 production (RCO2) by living systems such as tissues or organism cultures. The analysis provides useful knowledge about thermodynamic parameters relevant for e.g. biotechnology where parameter based yield maximization (fermentation) is relevant. The determination of metabolism related heat emission is easy and normally done by a calorimeter. However, measuring the amount of consumed O2 and produced CO2 can be more challenging, as additional preparation or instrumentation might be needed. Therefore, tunable diode laser absorption spectroscopy (TDLAS) was investigated as an alternative approach for respirometric analysis in order to facilitate the data collection procedure. The method determines by a spectroscopic laser non-invasively CO2 and O2 gas concentration changes in the respective vial headspaces. The gathered growth data from Pseudomonas aeruginosa cultured in two different scarce media was used to compute respiratory quotient (RQ) and calorespirometric ratios (CRCO2 [Rq/RCO2], CRO2 [Rq/RO2]). A comparison of the computed (experimental) values (for RQ, CRCO2 and CRO2) with values reported in the literature confirmed the appropriateness of TDLAS in calorespirometric studies. Thus, it could be demonstrated that TDLAS is a well-performing and convenient way to evaluate non-invasively respiratory rates during calorespirometric studies. Therefore, the technique is definitively worth to be investigated further for its potential use in research and in diverse productive environments.

Growth of mycobacteria in urine determined by isothermal microcalorimetry: implications for urogenital tuberculosis and other mycobacterial infections.

OBJECTIVE: To overcome the limitations of current urine-based diagnostic assays of urogenital tuberculosis, we used isothermal microcalorimetry to detect the metabolic activity of Mycobacterium tuberculosis and other commonly neglected pathogenic mycobacteria in urine and accurately determine their growth parameters. METHODS: A microcalorimeter equipped with 48 channels was used. Detection was accomplished, and growth was monitored for 4 different Mycobacterium species in sterilized and modified urine at 37 °C by measuring metabolic heat flow (µW = µJ/s) as a function of time. These strains were M. smegmatis, M. phlei, M. kansasii, and M. tuberculosis. The data were integrated to perform curve fitting and extract the growth parameter from the raw data. RESULTS: In sterilized urine, M. smegmatis showed the fastest growth rate (0.089 ± 0.017 [h(-1)]), followed by M. phlei (0.072 ± 0.016 [h(-1)]) and M. kansasii (0.007 ± 0.001 [h(-1)]). No growth of M. tuberculosis was detected in sterilized urine. However, in serum-supplemented urine, growth of M. tuberculosis was observed within 3 weeks at a growth rate of 0.008 ± 0.001 [h(-1)]. Biofilm formation was enhanced in the serum supplemented urine. CONCLUSION: Isothermal microcalorimetry allows rapid and accurate detection of mycobacterial growth in urine. Given the absence of data on the mycobacterial growth in urine, isothermal microcalorimetry could be used to unravel key aspects of Mycobacterium physiology in the urinary tract and potentially contribute to improvement in the diagnosis and treatment of urogenital tuberculosis.

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.