A new highly innovative approach for tackling antimicrobial drug resistance – calorimetry has arrive

Laboratory researchers have been presented with a major R&D challenge with antibiotic-resistant bacteria. Fortunately, a solution to this problem is readily available now.

Going beyond Pasteur – the benefits and growing application of the calorimetry approach in the field

The development of antibiotic-resistant compounds has mainly adopted the traditional Pasteur-style microbiology approach – predominantly involving culturing, plating and manual operation. The fundamental problems with molecular-based, non-culture methods, which invariably rely on DNA typing or proteomics, are threefold - they are often costly, have low specificity and are unable to differentiate between living, dead, and highly persistent dormant bacteria. What the R&D community has lacked until recently is a sensitive, label-free cell-based assay possessing the capability to measure bacterial activity in real time with the minimal effort. As a consequence, scientists, committed to the development of innovative solutions, went beyond Pasteur to find an alternative that provides the effective solution that the industry has been after. The answer is calorimetry and advanced technology that brings the application of this effective approach to life.

Calorimetry-based microbial measurements

The calorimetry-based monitoring of living systems had fallen out of use, until recently, due to an industry perception that the approach is too complex. However, demand has been fuelled for calorimetry from a combination of the pressing and high priority need to tackle antibiotic resistance, together with technological advancement. Calorimetry-based cell monitoring, with the accuracy of the data it generates, are uniquely suited to the development of novel antibiotics.

The calorimetry approach, brought into operation through advanced technologies that are now available, provides researchers and clinicians dealing with bacterial infections with tests that are both accurate and at the same time fast – serving as a highly reliable scientific tool for determining whether an antibiotic should be used in the patient, the type of antibiotic to utilize and the choice of therapy to apply.

At its core, calorimetry measures the power produced in a cell culture at any given time as Joules/second (W). The heat generated is a measure of the metabolic processes in the cells and, as a consequence, gives a true phenotype fingerprint of the organism measured. Different bacteria and treatments create unique heat profiles that reveal significant information about the system being tested. Calorimetry provides a label-free, non-destructive measurement - making post experimental analysis possible, whilst being independent of sample morphology. This means that assays can be performed both on bacteria in solution as well as on solid media, including three-dimensional matrices such as surgical and dental implant materials and bone biopsies

One of the unique properties of calorimetry-based metabolic monitoring of bacterial growth is that the pattern of energy expenditure is species, as well as strain, specific - over time, each bacterium gives rise to a specific growth pattern as heat production. This can be used to quantify the number of bacteria and to determine the species. The bacterial load determination is similar to a quantitative PCR measurement, in which the curves are identical in shape, but different numbers of cycles are needed to reach the detection limit. Furthermore, different loads of bacteria require a varying number of cell divisions to reach the detection limit concerned. The metabolic output assay therefore becomes quantitative as well as qualitative. Minor changes in growth behaviour, such as metabolic pathway mutations, are detected, as are biofilm formation and, most significantly, antimicrobial sensitivity.

Through integrating the metabolic power over time to accumulated heat over time (in Joules), a growth curve is established equivalent to a traditional growth curve (as measured by optical density of the cell culture). From this data, it is possible to calculate both the lag time and the maximum growth rate of the culture concerned. This forms the basis for determining the effect of antibiotic treatment.

A prolonged lag phase is indicative of a bactericidal action, since the starting number of live bacteria will be less, and a decrease in growth rate will suggest a bacteriostatic effect. The starting number of bacteria will not change, but the cell division time will increase. This data can be used to qualify the mechanism of action, based on the inhibiting properties and the curve shape, compared to substances with known mechanisms of action. Dose-response curves, plotting dose against lag time and dose against maximal growth rate, are easy to derive from the data. Furthermore, the total energy release for a given time frame, plotted against concentration, is a measure of the total biomass formation and can be used as a measurement of antibacterial efficiency.

A highly important consideration is a compound’s bioavailability. Since the calorimetric measurement is a measure of the total metabolism, bioavailability to the bacterium p is a non-issue since it is accounted for in the measurement. Compare this to culturing assays on solid media, where the diffusion of compound in the media can lead to measurement errors. The formation of biofilms can also be a concern in the potency measurements of antibiotics, since the efficacy of antibiotics differs between planktonic and biofilm growth. Biofilm formation can be monitored by calorimetry due to the fact that the metabolic status and the treatment efficacy are clearly different.

Colonization in complex matrices like bone can be difficult to assay. "Normal" assays cannot provide a representative sample of bacteria colonizing three-dimensional surfaces. Consequently, large deviations may be found when using microscopy, and molecular, fluorescence and microscopy methods. The heat produced by bacterial metabolism in 3-D matrices can be measured regardless of the sample properties, thereby enabling new areas of investigation.

With the calorimetry approach, the bacterial growth assay can be performed in both liquid and solid media. This enables different properties to be studied during the colonization of, for example, surgical and dental implant materials. A wide range of possible growth conditions enable the study of both aerobic and difficult-to-grow anaerobic systems, as well as the monitoring tuberculosis and other slow-growing mycobacteria.

Correct quantification of the number of cells, as well as the number of living cells, can be challenging in antibiotic development. As a consequence of the fact that many bacteria give rise to clustered cells, biofilms, etc., there will likely be a misrepresentation when using standard plating/growth analysis. A single colony may originate from a cluster of living bacteria, thus giving false numbers for efficacy. Calorimeter-based assays account only for the actual number of metabolic active, live cells. This also has implications for the comparison to DNA or protein-based assays, where there can be difficulty distinguishing between the number of live active cells and DNA/protein remaining in inactive/dead cells protected by biofilm. It is easy to monitor the metabolic activity for prolonged times using calorimetry - a typical assay will run from just a few hours up to days or weeks if required. This allows for the monitoring of persister cells or cells derived with antibiotic resistance from biofilm formation. These cells produce metabolic activity at a lower but constant rate during a prolonged time and can be distinguished in the assay. Up to 80% of all infections are complicated by bacteria forming biofilms,2 and antibiotics typically developed using bacteria in planktonic growth may be largely ineffective for treating biofilm-derived infections. The possible degradation and instability of tested compounds can allow regrowth of persister cells; this can be easily monitored by following the total metabolic state of the culture for a prolonged period of time.

Increasing efficacy

Potentiating treatments are being used more regularly so that antibiotic efficacy can be increased. The use of potentiating compounds with no inherent antibiotic properties and multiple modes of action of combined therapies can be monitored using a calorimetric assay. Unbiased phenotype screening is achieved since there is no need to know the mechanism of action prior to the experiment,

Conclusion

Efficient screening of lead compounds and dose response measurements, performing calorimetric assays in a microtiter plate-based format is being made possible by the advent of advanced technologies – a prime example of which is the 32-channel calScreener technology. Small sample volumes and multiple parallel channels increase throughput, with presterilized single-use consumables ideally suited for bacterial growth.

Calorimetry is a ground-breaking approach to effectively monitor the spread of resistant strains – the method is cost-effective, rapid and sensitive. The capability to identify the presence of an infecting agent and to determine the correct antibiotic treatment in just hours instead of days will be made possible by combining detection with an indication-based panel of antibiotics.