Kinetic analysis of microcalorimetric data derived from microbial growth: Basic theoretical, practical and industrial considerations.

We report here a mathematical framework for the quantitative interpretation of exponential bacterial growth measured with isothermal microcalorimetry. The method allows determination of many parameters that define the exponential growth phase. To automate the analysis, we also wrote a coding program, so that the approach could be embedded in a commercial setting. As an exemplar, we apply the method to a commercial probiotic product. The outcome was that we could identify characteristic parameters of growth (including rate constant and doubling time), and hence authenticate product quality, within 15 h. This compares favourably with the current 7-10 days required for conventional microbiological assessment (to allow release of product for bottling and marketing) via plating methods. The method would lend itself to growth analysis of single and mixed bacterial cultures.

Influence of probiotic bacteria on gut microbiota composition and gut wall function in an in-vitro model in patients with Parkinson's disease.

We report here the potential role of a 4-strain probiotic suspension for use with patients with Parkinson's disease (PD). Stool samples from a group of three patients with diagnosed PD were used to create microbiotas in an in-vitro gut model. The effects of dosing with an oral probiotic suspension (Symprove) on bacterial composition and metabolic activity in the microbiotas was evaluated over 48 h and compared with healthy controls. Additionally, the effect of probiotic dosing on epithelial tight-junction integrity, production of inflammatory markers and wound healing were evaluated in cell culture models. In general, the relative proportions of the main bacterial phyla in the microbiotas of PD patients differed from those of healthy subjects, with levels of Firmicutes raised and levels of Bacteroidetes reduced. Dosing with probiotic resulted in a change in bacterial composition in the microbiotas over a 48 h period. Several other indicators of gut health changed upon dosing with the probiotic; production of short chain fatty acids (SCFAs) and lactate was stimulated, levels of anti-inflammatory cytokines (IL-6, IL-10) increased and levels of pro-inflammatory cytokines and chemokines (MCP-1 and IL-8) decreased. Tight junction integrity was seen to improve with probiotic dosing and wound healing was seen to occur faster than a control. The data suggest that if development and/or progression of PD is influenced by gut microbiota dysbiosis then supplementation of the diet with a properly formulated probiotic may be a useful adjunct to standard treatment in clinic.

A 4-strain probiotic supplement influences gut microbiota composition and gut wall function in patients with ulcerative colitis.

Symprove, a multi-strain probiotic, has been shown to exert a mild anti-inflammatory effect in patients with ulcerative colitis (UC). We examined stool samples from 3 patients with UC in order to create microbiotas in an in-vitro gut model. The effects of Symprove on bacterial diversity and metabolic activity in the microbiotas was evaluated over 48 h. In addition, the influence of probiotic dosing on epithelial tight-junction integrity, production of inflammatory markers and wound healing were evaluated in cell culture models. The relative proportions of the main bacterial phyla in UC patients differed from those of healthy subjects studied previously; levels of Firmicutes were lowered and levels of Bacteroidetes were raised. Addition of Symprove changed the bacterial composition in the microbiotas over a 48 h period. Several other factors generally implicated in good gut health changed after dosing with probiotic; production of short chain fatty acids (SCFAs) and lactate was stimulated, levels of anti-inflammatory cytokines (IL-6, IL-10) increased, levels of pro-inflammatory cytokines and chemokines (MCP-1 and IL-8) decreased, epithelial tight junction integrity improved and wound healing occurred faster than a control. The results imply it is not the simple addition of probiotic bacteria that improves gut health. Rather, the probiotic bacteria generate lactate, which then stimulates growth of commensal gut bacteria, raising SCFA levels (particularly butyrate). The increased butyrate concentration positively influences inflammation response and time of wound healing.

Development of a flow system for studying biofilm formation on medical devices with microcalorimetry.

Isothermal microcalorimetry (IMC) is particularly suited to the study of microbiological samples in complex or heterogeneous environments because it does not require optical clarity of the sample and can detect metabolic activity from as few as 10(4) CFU/mL cells. While the use of IMC for studying planktonic cultures is well established, in the clinical environment bacteria are most likely to be present as biofilms. Biofilm prevention and eradication present a number of challenges to designers and users of medical devices and implants, since bacteria in biofilm colonies are usually more resistant to antimicrobial agents. Analytical tools that facilitate investigation of biofilm formation are therefore extremely useful. While it is possible to study pre-prepared biofilms in closed ampoules, better correlation with in vivo behaviour can be achieved using a system in which the bacterial suspension is flowing. Here, we discuss the potential of flow microcalorimetry for studying biofilms and report the development of a simple flow system that can be housed in a microcalorimeter. The use of the flow system is demonstrated with biofilms of Staphylococcus aureus.

An in vitro test of the efficacy of an anti-biofilm wound dressing.

Broad-spectrum antimicrobial agents, such as silver, are increasingly being formulated into medicated wound dressings in order to control colonization of wounds by opportunistic pathogens. Medicated wound dressings have been shown in-vitro to be effective against planktonic cultures, but in-vivo bacteria are likely to be present in biofilms, which makes their control and eradication more challenging. Recently, a functional wound dressing (AQUACEL(®) Ag+ Extra™ (AAg + E)) has been developed that in addition to silver contains two agents (ethylenediaminetetraacetic acid (EDTA) and benzethonium chloride (BC)) designed to disrupt biofilms. Here, the efficacy of AAg + E is demonstrated using a biofilm model developed in an isothermal microcalorimeter. The biofilm was seen to remain viable in the presence of unmedicated dressing, silver-containing dressing or silver nitrate solution. In the presence of AAg + E, however, the biofilm was eradicated. Control experiments showed that neither EDTA nor BC alone had a bactericidal effect, which means it is the synergistic action of EDTA and BC disrupting the biofilm with silver being bactericidal that leads to the product's efficacy.

An in vitro test of the efficacy of silver-containing wound dressings against Staphylococcus aureus and Pseudomonas aeruginosa in simulated wound fluid.

An isothermal microcalorimetric assay was used to quantify the efficacy of a silver-containing wound dressing against two common wound pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. The growth patterns of the two species were unique and varied depending on the environment in which the organisms were grown. Addition of non-silver-containing dressing altered the growth kinetics while addition of silver (contained either in a dressing or as AgNO3 solution) was seen to elicit inhibition and/or kill depending on concentration. Tests were conducted in nutrient broth and simulated wound fluid. It was found that minimum inhibitory and minimum bactericidal concentration values were higher in simulated wound fluid and under anaerobic conditions. Bioavailability of silver from the wound dressing was 35% against S. aureus in nutrient broth and 68% against both species in simulated wound fluid. The data highlight the importance of developing and conducting in vitro assays in biorelevant media.