Fighting Back Blockade of Staphylococcal Catecholamine Responsiveness

Previously we had shown that blockade of catecholamine growth responsiveness in enteric bacteria was possible using drugs employed therapeutically as catecholamine receptor antagonists (Freestone et al. 2007). To determine if we could similarly inhibit staphylococcal responses to the inotropes, we examined the ability of a range of a and b-adrenergic and dopaminergic antagonists to block S. epidermidis responses to norepinephrine and dopamine. As can be seen in Table 8.1, the b-adrenergic receptor antagonist propranolol had no effect on the ability of either catecholamine to stimulate growth of S. epidermidis. Other b-adrenergic antagonists such as labetalol, atenolol, and yohimbine also had no effect (data not shown). However, the a-adrenergic antagonists phentolamine and prazosin (data not shown) were able to inhibit growth induction by norepinephrine by over three log-orders.

1.0E+08

1.0E+08

Dilution Factor b 1.0E+09

1.0E+08

1.0E+07

1.0E+05

1.0E+04

1.0E+08

1.0E+07

1.0E+05

1.0E+04

Dilution Factor

Fig. 8.4 Effect of catecholamine inotropes on coagulase-positive staphylococci pre-exposed to rifampin. Replicates of an exponentially growing S. aureus strain Newman cultures grown in serum-SAPI medium were incubated for 4 h at 37°C with 5 mg per ml rifampin (100 times MIC). The antibiotic-treated (inoculum size 2.21 x 105 CFU per ml) (a) and control cultures (inoculum size 2.22 x 108 CFU per ml) (b) were each then serially diluted in tenfold dilution steps into fresh serum-SAPI with no additions (white bar) or supplemented with norepinephrine (black bar) or dopamine (grey bar) each at a concentration of 100 mM. Test and control were incubated at 37°C for 24 h and enumerated for viable cell levels using pour-plate analysis. The asterisk, *, indicates a statistically significant increase in growth level over the corresponding non-catecholamine ino-trope supplemented control culture (P < 0.0001). This figure was taken with permission from Freestone et al. (2008)

None of the a- or b-antagonists when tested alone induced growth of S. epidermidis, even at 500 |M. Furthermore, addition of Fe overcame the antagonist blockade of growth induction (Table 8.1), indicating that growth inhibition by the a-adrenergic receptor antagonists was not due to any cellular toxicity of the antagonist, but instead represents a specific antagonism of the staphylococcal response to the catecholamines.

a 1.0E+09 1.0E+08 _ 1.0E+07 E 1.0E+06 o. 1.0E+05 g 1.0E+04 ° 1.0E+03 1.0E+02 1.0E+01

**

*

1*

*

1*

1

1

1

1

1

r

1.0E+03

Fig. 8.5 Effect of catecholamine inotropes on staphylococci pre-exposed to minocycline. Replicates of an exponentially growing S. epidermidis and S. aureus strain Newman cultures grown in serum-SAPI medium were incubated for 4 h at 37°C with 2 mg per ml minocycline (100 times MIC). The antibiotic treated S. epidermidis culture (inoculum size 5.40 x 104 CFU per ml) (a) and S. aureus Newman culture (inoculum size 8.90 x 104 CFU per ml) (b) were each then serially diluted in tenfold dilution steps into fresh serum-SAPI with no additions (white bar) or supplemented with norepinephrine (black bar) or dopamine (grey bar) each at a concentration of 100 mM. The two sets of cultures were then incubated at 37°C for 24 h and enumerated for viable cell levels using pour-plate analysis. The asterisk, *, indicates a statistically significant increase in growth level over the corresponding non-catecholamine inotrope supplemented control culture (P < 0.0001). This figure was taken with permission from Freestone et al. (2008)

The next question we asked was whether we could use phentolamine and chlo-rpromazine to prevent the inotrope from rescuing the growth of antibiotic-treated staphylococci. Figure 8.6 shows the effect of including the antagonists in the cate-cholamine resuscitation experiments described in Figs. 8.3 and 8.4. As seen in Fig. 8.6, the a-adrenergic and dopaminergic antagonists were able to specifically block norepinephrine and dopamine resuscitation of rifampin-damaged S. epider-midis and S. aureus. Further work showed that the inotrope blocking activity of a single addition of the antagonists was retained for at least 3 days for both strains

Table 8.1 Blockade of catecholamine-induced staphylococcal growth responsiveness

Antagonist concentration (|j,M)

Table 8.1 Blockade of catecholamine-induced staphylococcal growth responsiveness

Antagonist concentration (|j,M)

Antagonist

CA

0

0.1

1

10

20

50

75

100

200

300

N/A»

Phentolamine

NE

8.01a

7.94

7.86

6.90

6.08

5.15

4.90

4.60

3.75

3.75

3.90

NE + Fec

8.21

8.13

8.13

8.13

8.06

8.06

8.04

8.15

8.09

7.99

Dop

8.01

7.96

Propranolol

NE

8.02

8.02

7.99

8.03

7.68

7.76

7.91

7.90

7.81

7.60

4.00

Dop

8.01

7.95

Chloipromazine

Dop

8.00

7.98

7.98

7.81

7.34

6.92

6.66

6.35

5.27

4.81

4.03

Dop + Fec

8.06

8.08

8.08

8.16

8.11

8.08

8.08

8.08

8.11

8.06

NE

8.03

7.81

S. epidennidis was inoculated at 102CFU per ml into serum-SAPI medium containing the catecholamine (CA) plus the concentrations of antagonists shown in the table, incubated statically for 24 h 37°C in a 5% CO, humidified incubator and measured for growth ("expressed as log~10CFU per ml) using pour plate counts. Growth levels of non-catecholamine supplemented cultures (bN/A) are shown for comparison purposes. Norepinephrine (NE) and dopamine (Dop)

were both used at 50(.iM and Fe (Fe(NO ) ) at 100(.iM. Results shown are representative data from at least three separate experiments; all data points showed variation of no more than 5%Data table was taken with permission from Freestone et al. (2008) cNote that Fe was included to show that the effects of the antagonists were not due to toxicity of the compounds.

1.0E+09

1.0E+08

Dilution Factor

1.0E+09

1.0E+08

Dilution Factor

Fig. 8.6 Effects of adrenergic and dopaminergic antagonists on the ability of catecholamines to rescue of growth of antibiotic-stressed staphylococci. Replicates of exponentially growing cultures of the bacteria shown were cultured in serum-SAPI medium and then pre-incubated with 5 mg per ml rifampin as described in the legends to Fig. 8.3, and similarly further processed except that additional catecholamine assays containing norepinephrine were supplemented with 200 mM phentolamine and dopamine supplemented with 200 mM chlorpromazine. Test and non-catecholamine inotrope supplemented controls were incubated at 37°C for 24 h, and enumerated for viable cell levels using pour-plate analysis. Viable counts of the inoculating antibiotic treated S. epidermidis, and S. aureus Newman cultures were 2.21 x 105 and 7.8 x 105 CFU per ml respectively. The values shown represent means of triplicate plate counts; standard error of the mean was not greater than 7% for all cultures shown. (a) S. epidermidis, (b) S. aureus White bar no additions (control), black bar 100 mM norepinephrine, right diagonal hatch 100 mM norepinephrine plus 200 mM phentolamine, gray bar 100 mM dopamine, left diagonal hatch 100 mM dopamine plus 200 mM chlorpromazine. This figure was taken with permission from Freestone et al. (2008)

(data not shown). For S. aureus (Fig. 8.6b), the antagonists were less potent, and at best phentolamine and chlorpromazine were able to reduce norepinephrine and dopamine resuscitation by at most a log order in magnitude.

0 0

Post a comment