2.1.1 Defined mixed culture in the isothermal microcalorimeter
As reviewed in the previous chapter, microcalorimetry has had vast microbiological applications in various fields (section 1.4.5 and 1.4.6). Despite all the applications, only few studies have been conducted on defined mixed culture with the aim of investigating the relationship between two or more bacteria (Schaffer et al., 2004, Kong et al., 2012, Vazquez et al., 2014).
In this chapter, the growth and behaviour of Pseudomonas aeruginosa, Staphyloccocus
aureus and Escherichia coli were studied as mixed culture to determine if the
microcalorimeter could detect their relative growth. These 3 non-fastiduous species, previously characterised in the microcalorimeter (O'Neill et al., 2003, Gaisford et al., 2009, Said et al., 2014) were to be used in subsequent susceptibility test with some commercial probiotic strains.
P. aeruginosa is an opportunistic human pathogen that can cause various infections, for
example infections of the ear, eyes, skin, urethra and respiratory tract in cystic fibrosis and patients with severe burns, as well as other immunocompromised and chronically debilitated individuals (Garrity et al., 2005). It is a very adaptable bacterium, known to colonize several environmental niches; one of the most common species in nature (Garrity et al., 2005, Khan et al., 2010). Its numbers are very low in the faeces but numbers can increase significantly after antibiotic treatment (Levison, 1977).
S. aureus forms part of the normal human microbiome, and colonizes the skin, skin
glands, mucous membranes, especially anterior nares (Schleifer and Bell, 2009). It is an important cause of nosocomial infections and has caused considerable mobidity and mortality in hospitalised patients (Wisplinghoff et al., 2004, de Kraker et al., 2011). It is associated with surgical infections, lung infections, urogenital tract infections, skin
E. coli is an essential part of the gut microbiota. Most strains are non-pathogenic but
certain serotypes can cause diseases of the intestine and other parts of the body. They possess a variety of virulence factors responsible for their pathogenicity in healthy patients. For example, enteropathogenic and enterotoxigenic E. coli cause diarrhoea. Uropathogenic E. coli, neonetal meningitis E. coli and other types are described (Scheutz and Strockbine, 2005).
2.1.2 Standardization of inocula: cryopreservation
As a prerequisite for using bacteria as “standard reagents”, ensuring that any difference between experiments is solely due to deliberate changes made, an objective was set to develop a cryopreservation protocol for the microorganisms, which, while maintaining uniformity of inocula, would give viable cell recoveries of at least 95%.
It must be noted that microbiological assays could be subject to inconsistencies between experiments because of variations in the metabolic performance of inocula used (Beezer et al., 1976). While this variation is tolerated in some assays because of other standardization protocol in place by a particular method, for quantitative characterization or identification by microcalorimetry (for example, antibiotic susceptibility testing) this may not be the case (Beezer et al., 1976). The source of variation of an inoculum can be credited to the volume or size, the phase of growth and particularly the history of the inoculum being used (Beezer et al., 1976). To avoid batch-to-batch variability, it is important that inocula are rigorously standardized. Standardization of inocula can be achieved through the development of standardized freezing and storage protocols for large volumes of inocula (Beezer et al., 1976, Gaisford et al., 2009). This will ensure that a consistent inoculum is used for each assay since the microorganisms would be sourced from the same batch per assay or would have gone through a similar cultivation process.
In the frozen state, the microorganisms are expected to remain viable, without any alterations in genotypic or phenotypic characteristics (Tedeschi and De Paoli, 2011). They should also be easily restored to the same condition they were before freezing or cryopreservation (Beezer et al., 1976). Conventionally, microbiological stocks are preserved by storing at -4oC to -80oC. Storage at these temperatures has however been shown to be less effective than at -160oC in liquid nitrogen vapour (McDaniel and Bailey, 1968) or liquid nitrogen storage at -196oC (Stapert et al., 1964, Sokolski et al.,
1964, Hwang, 1966, Cowman and Speck, 1965, Beezer et al., 1976, Gaisford et al., 2009). Freezing in liquid nitrogen at -196oC can store living microorganisms for several years while maintaining viability and reproducibility (Beezer et al., 1976, Gaisford et al., 2009, Tedeschi and De Paoli, 2011). For instance, some bacteria have been shown to store for up to 30 years without loss in viability (Gherna, 1981, Reimer and Carroll, 2004).
A number of factors are known to affect the effectiveness of cryopreservation of microorganisms, for example, the composition of the freezing medium, the storage temperature, the cooling rate and warming rate (Meryman, 1971, Beezer et al., 1976, Hubalek, 2003, Morgan et al., 2006, Tedeschi and De Paoli, 2011). The cooling rate during the freezing process is a critical factor for cell viability and recovery (Beezer et al., 1976). The optimal cooling rate for most bacteria has been suggested to be 1oC per min (Mazur et al., 1972) even though some bacteria will withstand less than ideal cooling rates (Tedeschi and De Paoli, 2011). Very slow cooling rate results in death of cells caused by changes in osmotic pressure whereas very fast freezing rate can cause cell death from mechanical damage of cell membranes caused by recrystallization of intracellular ice (Meryman, 1971). Another critical aspect in freezing is the efficiency of a cryoprotective agent in protecting cells from injury and death (Meryman, 1971). As already pointed out, during freezing and defrosting processes, cells could suffer severe osmotic stress and ice crystal damage. A way of circumventing these possible lethal effects is to include a cryoprotectant prior to freezing. Addition of cryoprotectants has been shown to improve survivability of cells (Harrison, 1956, Postgate and Hunter, 1961, Nash et al., 1963, Baumann and Reinbold, 1966, Gibson et al., 1966, Green and Woodford, 1992, Vekeman et al., 2013).
There are different types of cryoprotectants and classifications: extensively reviewed by Meryman, (1971) and Hubalek, (2003). Briefly, there are those that penetrate the cells: methanol, ethanol, ethylene glycol, propylene glycol, dimethylformamide, dimethyl sulfoxide (DMSO), methylacetamide and glycerol. There are others that do not penetrate the cells: mannitol, sorbitol, dextran, methyl cellulose, albumin, gelatin, other proteins, polyvinylpyrrolidone (PVP) etc., (Meryman, 1971). The non-penetrating ones provide only extracellular protection whilst the penetrating ones can also delay intracellular freezing and provide both intracellular and extracellular protection (Meryman, 1971,
For the cryopreservation of the organisms, the effectiveness of different cryoprotective agents was tested in order to select the best cryoprotectant for the organisms to give recoveries of 95% or more. Storage of the stock was to be in liquid nitrogen because of the highlighted advantages from previous experiences (Beezer et al., 1976, Gaisford et al., 2009).