Catecholamine Inotropes Induce Staphylococcal Growth and Biofilm Formation on Intravascular Catheters

The requirement for iron in growth is recognized for the vast majority of pathogenic and commensal bacteria including the staphylococci (Ratledge and Dover 2000). Under normal conditions, the human body seeks to severely limit the availability of free iron to approximately 10-18 M through the production of the iron sequestering transferrin (blood) and lactoferrin (mucosal secretions and the gastrointestinal tract), to a concentration of iron that is below the level required to support the growth of bacteria. In an effort to obtain iron from host iron-binding proteins such as transferrin, bacteria have developed a variety of mechanisms including ferric reductase, transferrin and lactoferrin binding proteins, and ferric iron sequestering siderophores (Ratledge and Dover 2000). The importance of iron as a determinant of S. epidermidis biofilm formation has been demonstrated in studies that have employed iron restriction. For example, staphylococcal strains of S. epidermidis which were initially biofilm negative, were induced to form biofilms when grown in iron-starved medium (Deighton and Borland 1993), and iron limitation generally induces slime production by the staphylococci (Baldassarri et al. 2001).

Work from our laboratories has shown that catecholamine inotropic drugs may have additional side effects to those recognized pharmacologically. We have shown that they can serve as an etiological factor in the staphylococcal colonization of indwelling medical devices due to their ability to stimulate S. epidermidis growth and biofilm formation (Freestone et al. 1999; Neal et al. 2001; Lyte et al. 2003). The exposure of less than 100 CFU per ml of S. epidermidis to pharmacologically relevant concentrations of dopamine, dobutamine, and norepinephrine resulted in only 24 h of a greater than 10,000-fold increased growth in serum and blood-based media. The mechanism of this growth enhancement involved the drugs enabling the staphylococci access to normally inaccessible transferrin-sequestered iron (Freestone et al. 2000; Neal et al. 2001; Lyte et al. 2003; Freestone et al. 2008) (see also Chap. 3). Importantly, as well as inducing growth in normally bacteriostatic host tissue fluids, inotropes can also enhance other aspects of C-NS physiology highly relevant to their presentation in the ICU, specifically biofilm formation. This is demonstrated in Fig. 8.1, which shows a series of scanning electron micrographs of the biofilm formation that occurs when less than 100 S. epidermidis cells were seeded onto intravenous catheter grade polystyrene in the presence of tissue fluids they will encounter in vivo (plasma) and incubated without (a) and with (b-f) concentrations of norepinephrine that would be administered via the intravenous catheter. As is

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Fig. 8.1 Catecholamine-induced biofilm formation in Staphylococcus epidermidis. The image panels show scanning electron micrographs of biofilms of S. epidermidis adhering to polystyrene after overnight growth in freshly prepared plasma-SAPI medium in the absence (a) or presence (b-f) of 0.1 mM norepinephrine as described in Lyte et al. (2003). Initial inocula for both S. epidermidis cultures were approximately 102 CFU per ml. Higher magnification scanning electron micrographs showing details of the bacteria-exopolysaccharide clusters are shown in panels (b)-(f). This figure was taken with permission from Freestone and Lyte (2008)

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Fig. 8.1 Catecholamine-induced biofilm formation in Staphylococcus epidermidis. The image panels show scanning electron micrographs of biofilms of S. epidermidis adhering to polystyrene after overnight growth in freshly prepared plasma-SAPI medium in the absence (a) or presence (b-f) of 0.1 mM norepinephrine as described in Lyte et al. (2003). Initial inocula for both S. epidermidis cultures were approximately 102 CFU per ml. Higher magnification scanning electron micrographs showing details of the bacteria-exopolysaccharide clusters are shown in panels (b)-(f). This figure was taken with permission from Freestone and Lyte (2008)

clearly visible, exposure of S. epidermidis to the inotrope led to massive increases in biofilm formation and production of exopolysaccharide (Lyte et al. 2003). The increasing magnification shows the mushroom like structures of bacteria and exopolysaccharide, all of which became evident in less than 48 h.

The findings demonstrated in Fig. 8.1 are clinically significant, as bacterial colonization of intravenous catheters, predominantly by the C-NS, is recognized as the most common infection encountered in the intensive care setting. There are also a number of methodological issues in the data shown in Fig. 8.1 that are worth noting. In demonstrating catecholamine inotrope induction of S. epidermidis biofilm, we attempted to make the analytical conditions close to those likely to be occurring in the clinical scenario. We therefore used two methodological aspects that were markedly different from the previous studies of bacterial biofilm formation. Only 10-100 S. epidermidis cells were used to seed the polystyrene plastic section on which the biofilm was to be established, a low inoculum chosen to reflect the number of bacteria likely to be encountered at the beginning of the catheter colonisation. This is in contrast to many prior biofilm studies which have used several log orders higher levels of bacteria to establish biofilms. The second important difference was the use of culture conditions that employed a plasma supplemented minimal media. This again differs from studies which have used rich microbiological media to study biofilm formation, and in so doing, it may be argued, do not realistically reflect in vivo conditions in which host factors present in plasma, which are recognized to play a role in initial bacterial adhesion, are not present.

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