Feel the heat of antimicrobial resistance

Used by Antoine Lavoisier in the 18th Century, calorimetry is one of the oldest methods of chemical analysis. But sometimes the oldies are the best says Dr Magnus Jansson. Here he tells us why this 200 year-old method could help us solve one of the biggest problems of our generation

Antibiotic-resistant bacteria present laboratory researchers with a major R&D challenge – for which there is a highly viable solution at hand. The development of antibiotic-resistant compounds has in the main followed the traditional Pasteur-style microbiology approach – predominantly involving culturing, plating and manual operation. The critical problem with molecular-based, non-culture methods – which invariably rely on DNA typing or proteomics – is that they are often costly, have low specificity and are unable to differentiate between living, dead, and highly persistent dormant bacteria. What has been lacking until recently is a sensitive, label-free cell-based assay that has the capability to measure bacterial activity in real time with minimum effort. Scientists, committed to innovative solutions, went beyond Pasteur to find an alternative that provides the much needed solution the industry has been searching for. The answer is calorimetry and advanced technology that brings the application of this effective approach to life.

At its core, calorimetry measures the power produced in a cell culture at any given time as Joules/second (W)

Up until recently, the calorimetry-based monitoring of living systems had fallen out of use due to an industry perception that the approach is too complex. But the pressing need to tackle antibiotic resistance and technological advancement is fuelling demand for this approach. Calorimetry-based cell monitoring, and the accuracy of the data produced, are uniquely suited to the development of novel antibiotics. The calorimetry approach, brought into operation through the advanced technologies now available, provides researchers and clinicians dealing with bacterial infections with tests that are both accurate and fast. This means that the technique can serve as a reliable scientific tool to determine whether an antibiotic should be used in the patient, the type of antibiotic to utilise and the choice of therapy to apply.

[caption id="attachment_56494" align="alignnone" width="500"] Calorimetry was first used in the late 1700s to measure the heat released by chemical reactions.[/caption]

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 tested. Calorimetry provides label-free, non-destructive measurement, and therefore makes post experimental analysis possible, while being independent of sample morphology. This means assays can be performed on bacteria in solution as well as on solid media, including three-dimensional matrices such as bone biopsies and surgical and dental implant materials.

With the calorimetry approach, the bacterial growth assay can be performed in both liquid and solid media

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, reflected in their 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 importantly, antimicrobial sensitivity. By integrating the metabolic power over time to accumulated heat over time (in Joules), a growth curve is established that is 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 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 compound’s bioavailability is a highly important consideration. Since the calorimetric measurement is a measure of the total metabolism, bioavailability 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 because the metabolic status and the treatment efficacy are clearly different. Colonisation in complex matrices like bone can be difficult to assay. ‘Normal’ assays cannot provide a representative sample of bacteria colonising three-dimensional surfaces. Consequently, large deviations may be found when using microscopy, fluorescence and molecular methods. The heat produced by bacterial metabolism in 3D 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 colonisation of, for example dental and surgical implant materials. A wide range of possible growth conditions allow for the study of both aerobic and difficult-to-grow anaerobic systems, as well as for monitoring tuberculosis and other slow-growing mycobacteria. Correct quantification of the number of cells, as well as the number of living cells, can be a challenge in antibiotic development. Since 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 efficacy numbers. 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 it can be difficult to distinguish 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 needed. This permits 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 biofilms² 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. Potentiating treatments are being used more frequently to increase antibiotic efficacy. Multiple modes of action of combined therapies and the use of potentiating compounds with no inherent antibiotic properties can be monitored using a calorimetric assay. Since there is no need to know the mechanism of action prior to the experiment, unbiased phenotype screening is achieved. Technologies, like for example the 32-channel calScreener technology from SymCel, enable efficient screening of lead compounds and dose response measurements, performing calorimetric assays in a microtiter plate-based format. Small sample volumes and multiple parallel channels increase throughput, with pre-sterilised single-use consumables suited for bacterial growth. Calorimetry-based monitoring of the spread of resistant strains is rapid, sensitive and cost-effective. Combining detection with an indication-based panel of antibiotics will make it possible to identify the presence of an infecting agent and to determine the correct antibiotic treatment in a matter of hours as opposed to days.

References 1. Antibiotic resistance threats in the United States, Centers for Disease Control and Prevention, 2013;http://www.cdc.gov/ drugresistance/pdf/ar-threats-2013-508.pdf. 2. Hay, M.; Thomas, D.W. et al. Clinical development success rates for investigational drugs. Nature Biotechnol.2014, 32(1), 40–51; doi:10.1038/nbt.2786.)

Author

Magnus Jansson is chief scientific officer at SymCel Sverige AB.

Contact magnus.jansson@symcel.se www.symcel.se