Choice of Growth Medium

It has generally been observed that propagation of bacteria in traditional rich microbiological media, which typically allow good growth, frequently does not promote the strong expression of virulence determinants. Indeed, many bacteria will only express their adhesins, invasins or toxins when grown under the same temperature, pH, nutritional or atmospheric conditions that they would face on entry into the host. Consequently, when undertaking microbial endocrinology experiments, where the effects of neuroendocrine stress hormones on the growth and virulence of bacteria are being investigated, it would seem obvious that it is essential to try to mimic, as far as possible, the stressful environments in which the bacteria actually encounters those hormones in the host. Indeed, a failure to provide such an in vivo like environment may likely result in erroneous conclusions as to the influence those neurohormones may have on bacterial growth and/or virulence. A possible example of this is the work of Straub et al. (2006) who used Luria broth as a test medium for analysing the effect of norepinephrine on bacteria, and thereby its potential role in a model of bacteremia. Based upon the lack of any obvious effect of the catecholamine in this media, they concluded that norepinephrine was not directly interacting with the bacterial species investigated; however, considering the ever increasing number of reports of catecholamine-bacterial interactions (Freestone et al. 2008) it seems very premature to rule out such a role without further studies using culture media, which might more accurately reflect the in vivo conditions.

For their initial microbial endocrinology work Lyte and Ernst (1992, 1993) developed serum-SAPI, a minimal salts medium supplemented with 30% adult bovine serum that would provide host-like conditions for their experiments. Due to its limited nutrient availability, iron restriction and the presence of host defence proteins, such as antibodies and complement, serum-SAPI presents a highly stressful bacteriostatic environment. Indeed most bacteria grow very poorly in serum-SAPI without supplementation (Lyte and Ernst 1992, 1993; Lyte 2004). Many subsequent analyses of neuroendocrine catecholamine hormone interactions with bacteria have used serum-SAPI as their medium of choice; this primarily involves those studies concerning growth induction (Lyte and Ernst 1992, 1993; Freestone et al. 1999, 2000, 2002, 2003, 2007a, b; Kinney et al. 1999, 2000; Neal et al. 2001; Belay et al. 2003; O'Donnell et al. 2006; Nakano et al. 2007b) but also includes some studies on virulence factor expression (Lyte et al. 1996, 1997a, b; Nakano et al. 2007a) or global transcriptional changes (Dowd 2007; Bearson et al. 2008). The growth inhibition inherent to serum-SAPI is primarily due to the iron restriction imposed by the serum iron-binding protein transferrin as evidenced by the fact that addition of excess of ferric iron will alleviate the growth repression; however, addition of cat-echolamine hormones can also promote growth by allowing the bacteria access to the iron bound up in transferrin (Freestone et al. 2000, 2003; Anderson and Armstrong 2008). Indeed, this stimulation in growth with catecholamines can be very large with up to 5 log orders difference in the numbers of bacteria with some species when compared with unsupplemented control cultures (Lyte and Ernst 1992, 1993, Lyte et al. 1996; Freestone et al. 1999, 2002; Neal et al. 2001). Some researchers have used modifications of serum-SAPI for work with fastidious oral pathogens (Roberts et al. 2002) and other research groups, working with bacteria with complex nutritional requirements, have used media composed of rich microbiological medias (e.g. Tryptic Soy Broth, Mueller Hinton Broth, Stainer-Scholte medium), which were then treated with chelators to remove iron, prior to supplementation with serum to render them bacteriostatic (Coulanges et al. 1997; Anderson and Armstrong 2006; Cogan et al. 2007).

In contrast with those researchers investigating catecholamine induced growth, many studies looking at catecholamine modulation of virulence determinant expression, particularly those using enteropathogenic and enterhemorrhagic E. coli (Sperandio et al. 2003; Walters and Sperandio 2006; Kendall et al. 2007), have used non-restrictive growth media which do not contain serum. In these studies, the researchers have typically grown their bacteria in cell culture media, such as Dulbecco's Modified Eagle medium (DMEM), which have been previously demonstrated to induce high-level expression of E. coli virulence determinants. Whilst the use of such growth media may make analysis of changes in virulence determinant expression much easier, it must be kept in mind that these media are not representative of the conditions which the bacteria will encounter within the host.

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