Bacterial Growth May be Stimulated Experimentally by a Range of Catechols

It is clear that catechols ingested in the diet, or their derivatives, may be found in plasma and tissues at submicromolar and micromolar levels. These are comparable with, or higher than, normal plasma concentrations of norepinephrine and epinephrine, which are in the nanomolar range (Benedict and Grahame-Smith 1978); and, against this background, we shall shortly examine the evidence from two studies (Coulanges et al. 1998; Freestone et al. 2007c) that have shown that dietary catechols can stimulate bacterial growth in a manner similar to that observed with the neuroendocrine catecholamines, norepineph-rine and epinephrine.

However, it is important to keep in mind that a substantial proportion, possibly approaching 50% in some cases (see Prior et al. 2006), of dietary catechol derivatives in the plasma and tissues may be conjugates, in which one of the catecholic hydroxyl groups is methylated, glucuronidated or sulphated. Furthermore, these compounds may not be free in solution. Quercetin incubated with human plasma becomes almost entirely bound to plasma proteins, chiefly albumin (Boulton et al. 1998) and other flavonoids behave similarly, although sulphation and glycosidation may substantially reduce the binding affinity (Dufour and Dangles 2005). The potential effect of protein binding is important because the stimulation of bacterial growth in iron-restricted medium that occurs in response to norepinephrine appears to occur concomitantly with uptake of the catecholamine into the bacterial cell (Freestone et al. 2000). However, the role of norepinephrine uptake in relation to both iron uptake and growth stimulation still remains unclear (Freestone et al. 2003, 2007b). It is not yet known whether the iron uptake and growth stimulation that occur in response to dietary catechols, and which are discussed below, are associated with the bacterial uptake of these compounds. In any event, it remains to be established how far the reported effects of catechols on bacterial growth and behaviour are affected by protein-binding of the catechol. However, the iron-restricted, serum-SAPI medium employed by Freestone et al. (2000, 2007c) contains, by definition, the proteins present in adult bovine serum, suggesting that the binding of catechols to proteins is not an issue, at least in this experimental set-up.

In the first of the two studies examining the effects of catechols on bacterial growth, Coulanges et al. (1998) examined the growth-promoting effects of a range of catechols upon a number of Listeria species, which (as far as is known) are unable to biosynthesise siderophores. Their ability to overcome growth inhibition induced by the iron-chelator, tropolone, was measured in disk diffusion assays. A number of compounds possessing a catechol grouping were effective in relieving growth inhibition. These included dopamine, epinephrine and norepi-nephrine and DL-DOPA, the siderophores pyoverdine and rhodotorulic acid, and plant-derived catechols including caffeic acid, esculetin, quercetin and rutin. Salicylic acid (o-hydroxybenzoic acid) was ineffective, as was dihydroxybenzoic acid (the isomer was not specified). This study therefore demonstrated that the relief of tropolone-induced growth inhibition in Listeria monocytogenes that had previously been observed to occur with catecholamines (Coulanges et al. 1997) was not restricted to these compounds but could occur also with a range of nonamine catechols.

In the second study, and following work summarised by Freestone et al. (2002) and Lyte (2004a), Freestone et al. (2007c) examined the growth of E. coli O157:H7 and Salmonella enterica SV Enteriditis in response to a number of catechols commonly consumed in the diet, and to fruit and vegetable extracts known to contain catechols. In the case of the individual catechols, they determined growth responses both in iron-restricted medium and in iron-replete medium and followed up these experiments with measurements of the uptake of 55Fe from 55Fe-labelled transferrin and lactoferrin. Marked differences in growth were observed, depending upon whether a rich medium (Luria broth) or an iron-restricted medium (serum-SAPI)

was employed (Figs. 4.2 and 4.3). In the rich medium, none of the compounds tested - catechin, caffeic acid, chlorogenic (5-0-caffeoylquinic) acid and tannic acid - had any significant effect upon growth, with the exception of tannic acid, which became progressively more inhibitory to growth with increasing concentration (up to 200 |xg ml-1). In contrast, in the iron-restricted medium (and hence broadly consistent with the findings in Listeria of Coulanges et al. 1998), all four catechol compounds tested promoted growth, by a factor of around 4 log-orders, with saturation occurring at 50-100 |M (50 |g ml-1 for tannic acid). Similarly, all the fruit and vegetable extracts tested (apple, carrot, grape, pear, plum, orange and strawberry), and infusions of tea and coffee, promoted growth in the iron-restricted medium. Study of the effects of tea flavanols (see Fig. 4.4) revealed that the ability to promote growth in the iron-restricted medium was restricted to those compounds possessing a 3,4-dihydroxyphenyl (i.e. catecholic) B-ring ((+)-catechin, (-)-cate-chin gallate and (-)-epicatechin gallate); trihydroxy-compounds with an additional hydroxyl group in the 5-position of the B-ring ((-)-epigallocatechin, (-)-epigallo-catechin gallate and (-)-gallocatechin gallate) failed to promote growth.

The work of Freestone et al. (2007c) revealed important mechanistic similarities between the responses to dietary catechols and the responses to neuroendocrine catecholamines. All of the catechols and plant extracts that promoted growth in the iron-restricted medium were also able to stimulate the uptake of 55Fe from 55Fe-transferrin or 55Fe-lactoferrin in both organisms studied (Fig. 4.5). Their activity in this respect was therefore in general comparable with that of the neuroendocrine catecholamines, typified by norepinephrine. Further similarity with the behaviour of the catecholamines was shown by the absence of any growth promotion by dietary catechols, or by fruit or vegetable extracts, in E. coli siderophore biosynthesis (entA) and transport (tonB) mutants. Mutations in these genes were previously shown to prevent norepinephrine-stimulated growth of E. coli (Burton et al. 2002; Freestone et al. 2003).

The evidence from these studies by Freestone et al. (2007c) and Coulanges et al. (1998) therefore show conclusively that the behaviour of the catecholamines in promoting growth in iron-restricted medium is not unique but instead is common to a diverse range of compounds all possessing the catechol structure. Thus, any consideration of the role of catecholamines in the promotion of bacterial growth (and potentially of virulence) needs to be broadened to take account of the occurrence and effects of dietary catechols, but subject to the caveats outlined at the beginning of this section.

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