Screening of Periodontal Bacteria for Responses to Norepinephrine and Epinephrine

Once the model E. coli growth assay had been effectively adapted for use in assessing the growth of periodontal organisms, the purpose of subsequent experiments was to investigate the growth responses of 43 putative periodontal pathogens to norepinephrine and epinephrine under standard conditions of inoculum size, time course (24 or 48 h) and environmental growth conditions (anaerobic, 35°C) (Roberts et al. 2002)

This was the first study to demonstrate the direct role of stress hormones upon growth of those micro-organisms commonly found within sub-gingival plaque biofilms, although other studies have since demonstrated similar findings (Belay et al. 2003). A broad variation in the growth response of a large number of microorganisms to norepinephrine and epinephrine was demonstrated with both positive and negative effects being identified. Of the oral bacteria studied roughly, half the species showed significant catecholamine-induced growth enhancement or inhibition indicating that stress hormones might directly modulate the growth and composition of the sub-gingival biofilm. As periodontal disease is a consequence of host-parasite interactions, the effects of stress hormones seen within the sub-gingi-val biofilm may well provide one potential mechanism linking stress to periodontal disease progression.

The microbial complexes (Socransky et al. 1998) were used to group sub-gingi-val species within this study as they provide a model of the associations and interrelations between different organisms (Fig. 7.2). These complexes relate closely to clinical measures of inflammation and periodontal disease activity and were used to provide a biological framework against which the diversity of catecholamine growth responses could be investigated. Briefly, the organisms most strongly associated with severe chronic periodontitis are found on the right-side of Fig. 7.2. Other organisms are found within the sub-gingival plaque biofilm are likely to be opportunistic pathogens capitalising upon local environmental changes as the disease advances.

Interestingly, there was considerable variation in growth response to norepi-nephrine and epinephrine both within and between sub-gingival microbial complexes as well as between sub-species of micro-organisms depending on the nature of the supplementation. It was obvious from the Roberts et al. (2002) study that a broad spectrum of growth responses to catecholamines exists, ranging from growth stimulation to growth inhibition. This indicates that the relationship between the growth of periodontal organisms and catecholamine hormones is likely to be a complex one, with the relative proportions of the resultant flora generated by the differential effects of these varied responses and interactions. It should be emphasised

A.viscosus

A.viscosus

S. noxia

Fig. 7.2 Sub-gingival plaque microbial complexes

S. noxia

Fig. 7.2 Sub-gingival plaque microbial complexes that negative growth effects are as significant as positive growth effects in determining the resultant flora within a sub-gingival biofilm. Inhibitory growth effects were observed for the "Red Complex" organisms (P. gingivalis and T. forsythensis), and it is possible that these organisms (and others showing negative growth responses) utilise catecholamines for up-regulation of virulence expression rather than growth. For example, E. coli strains increase the production of Shiga-like toxins (Lyte et al. 1997a) and K99 pili (Lyte et al. 1997b) in response to norepinephrine. It is therefore possible that catecholamines may have a dual effect on both virulence expression and growth response of periodontal organisms, which are entirely separate responses. In addition to the direct effect of catecholamine hormones on periodon-tal microorganisms, those organisms demonstrating increased growth may indirectly influence those organisms demonstrating growth inhibition either by production of growth factors or virulence factors within the biofilm. The experiments within the (Roberts et al. 2002) study dealt with single strain cultures of planktonic organisms, and it would be of interest in the future to consider the growth responses of periodontal organisms to catecholamines when in polymicro-bial culture in a biofilm environment.

The study by (Roberts et al. 2002) investigated the growth effects of catecholamines on sub-gingival organisms. However, it is noticeable that many of the Actinomyces and Streptococci species demonstrated increases in growth to the catecholamines and are more commonly associated with the supra-gingival flora (Table 7.1). However, it has also been shown that the supra-gingival flora

Table 7.1 This table illustrates those oral organisms tested which responded with positive growth to norepinephine or epinephrine. + indicates a positive growth effect, - indicates a negative growth effect and those in within ( ) indicate a growth effect which was not statistically significant. No positive growth effects were observed from any organism found within the red complex

Table 7.1 This table illustrates those oral organisms tested which responded with positive growth to norepinephine or epinephrine. + indicates a positive growth effect, - indicates a negative growth effect and those in within ( ) indicate a growth effect which was not statistically significant. No positive growth effects were observed from any organism found within the red complex

Socransky's

ATCC

microbial complex

strain

Species

Norepinephrine

Epinephrine

Actinomyces

12104

A. naeslundii I

+

+

species

23860

A. gerencseriae

+

+

Purple complex

17929

A. odontolyticus I

+

+

Yellow complex

10558

S. gordonii

(+)

+

27335

S. intermedius

+

+

35037

S. oralis

(-)

+

49456

S. mitis

+

+

Green complex

23834

E. corrodens

+

+

33612

C. sputigena

+

+

33624

C. gingivalis

+

+

Orange complex

27823

S. constellatus

+

+

33236

C. gracilis

+

-

33270

P. micros

(+)

+

33693

F. periodonticum

(+)

+

49256

F nucleatum sp. vinc

-

+

Others

19696

N. mucosa

+

(+)

33271

E. saburreum

-

+

33397

S. anginosus

+

(-)

14201

L. buccalis

+

+

25175

S. mutans

+

+

27337

P. anaerobius

+

+

35308

P. denticola

+

+

plays a significant role in determining the composition of the sub-gingival flora (Dahlen et al. 1992); this may be another important influence on progression of periodontal disease.

Of considerable interest is the fact that a number of organisms associated with suppurative infections in humans responded positively to catecholamine supplementation (Roberts et al. 2002). Streptococcus constellatus, S. intermedius and S. anginosus are organisms associated with pus formation as well as Peptostreptococcus species (commonly isolated from dento-alveolar abscesses) and the Gram-negative anaerobic rods (including E. corrodens and Capnocytophagia species) that have been reported to cause lateral periodontal abscesses (Marsh and Martin 1999). Anecdotal evidence has existed for an association between stressful episodes and abscess formation for some time, and the positive growth changes in these organisms to stress hormones observed in this study, could provide one potential mechanism.

Other important periodontal diseases of considerable interest are the necrotising periodontal diseases. Two important organisms associated with these diseases are Fusobacterium nucleatum subsp. vincentii which showed positive growth effects with epinephrine and Prevotella intermedia which showed a tendency for increased growth with epinephrine (Loesche et al. 1982). The positive growth effects observed in these organisms with epinephrine could also provide a potential mechanism linking stress hormones and necrotising periodontal diseases. The spirochaetes are an important group of organisms also associated with these diseases, however the experimental medium could not support their growth.

In summary, the results suggest that changes in catecholamine levels may trigger a shift in the balance of sub-gingival species within that environment, with certain organisms able to utilise host-derived hormones to influence their growth and possible virulence expression. In this manner, biofilm composition could change in relation to a stressed host, with one or two organisms within a "complex" orchestrating change in response to their local environment. The growth effects of stress hormones could therefore have a major influence on periodontal disease pathogenesis.

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