Materials and Methods

Bacteria, chemicals, and growth mediumS. epidermidis and S. haemolyticus (wound isolates) were obtained from Leicester Public Health Laboratory, Leicester Royal Infirmary (Leicester, United Kingdom) and S. aureus strains Newman and 832–4 were obtained from the Department of Genetics, University of Leicester. All chemicals were purchased from Sigma with the exception of ⁵⁵FeCl₃ (specific activity 5 mCi/mg Fe), which was obtained from Amersham Life Science. SAPI medium, L-(−)-norepinephrine bitartrate and dopamine hydrochloride were prepared as described elsewhere [17, 26]. Rifampin was dissolved in DMSO and minocycline in distilled water; antibiotic stocks were stored at −20°C, and diluted into culture media immediately prior to use

Study designTwo experimental approaches were employed: (1) determination of the antibiotic MIC for staphylococci in the presence or absence of inotropes when inotropes and antibiotics were added simultaneously (2) addition of inotropes after staphylococci had been exposed to levels of antibiotic at or above the MIC. For the first experimental approach, initial determinations of antibiotic MICs were performed in serum-SAPI medium, to more closely approximate in vivo conditions [27]. After growing overnight in serum-SAPI medium, staphylococci were inoculated into fresh serum-SAPI medium at 1:50 dilution and cultured for 3 h until exponential growth was achieved (optical density at 600 nm [OD₆₀₀], 0.4; 10⁸ cfu/mL). Loss of viability in the antibiotic-treated cultures, compared with nontreated controls, was usually 3–4 logs. Replicate cultures were diluted to 10⁶ cfu per mL into fresh serum-SAPI medium supplemented simultaneously with increasing concentrations of rifampin or minocycline with or without the addition of 100 μmol/L of catecholamine (norepinephrine or dopamine) [15]. Cultures were then incubated for 24 hours at 37°C in a 5% CO₂ humidified incubator, and enumerated by use of pour-plate analysis on Luria broth agar [25]

To ensure maximal antibiotic sensitivity for the second experimental approach, overnight staphylococcal cultures grown in serum-SAPI medium were cultured twice in fresh serum-SAPI medium to mid-exponential phase (OD₆₀₀, 0.4; 10⁸ cfu/mL) before being supplemented with rifampin or minocycline at approximately 100 times the MIC (5 μg and 2 μg/mL, respectively); controls consisted of bacteria similarly prepared and cultured, but without the antibiotic, and diluted to give an equivalent OD₆₀₀ to that of the less well-grown, antibiotic-treated cultures. Antibiotic-treated cultures and control cultures were then incubated for an additional 4 h, and the degree of antibiotic damage was determined with viable counts of serially diluted cultures plated onto Luria agar (this method also produced a precise enumeration of the bacterial inoculum size for each analysis). Antibiotic-treated cultures and untreated controls were then serially diluted into fresh serum-SAPI medium that contained 100 μmol/L catecholamine. Unless otherwise stated, cultures were incubated at 37°C in a 5% CO₂ humidified incubator for up to 3 days, and viable growth levels were determined at the times indicated [25]

Catecholamine response and antagonism assaysCatecholamine antagonism assays were performed in serum-SAPI medium supplemented with concentrations of the compounds shown in the text. Controls comprised equivalent volumes of the solvent used to dissolve the catecholamine or antagonist. To determine whether an antagonist was directly inhibitory to bacterial growth, all antagonism of catecholamine-growth induction assays were also performed in the presence of a concentration of iron which overcomes the iron-limitation of serum-SAPI medium (100 μmol/L Fe(NO₃)₃) and allows maximal bacterial growth levels [14]. Unless stated otherwise, bacteria were inoculated into serum-SAPI medium at 50–100 cfu-mL. Cultures were incubated statically at 37°C in a 5% CO₂ humidified incubator for 24 h [26] and enumerated by pour-plate analysis [25]

Transferrin-iron uptake analysis⁵⁵Fe-labelled transferrin (⁵⁵Fe-Tf) was prepared as described elsewhere [25]. Exponentially growing staphylococci were inoculated at 10⁸ cfu/mL into serum-SAPI medium supplemented with 10⁵ cpm of ⁵⁵Fe-Tf with or without 100 μmol/L norepinephrine. Cultures were incubated at 37°C in a 5% CO₂ humidified incubator for 6 h, harvested by centrifugation at 5000 g for 5 min, washed in PBS, and assayed for cell numbers and ⁵⁵Fe incorporation by use of pour-plate analysis and scintillation counting as previously described [25]

Statistical analysisAll growth assays were performed in triplicate and performed on at least 3 separate occasions. Where appropriate, statistical analysis was performed by use of an unpaired t test in which a 2-tailed P value was calculated (Instat; GraphPad). Statistical significance was defined as P<.05

Results

Effect of catecholamine inotropes on sensitivity of S. epidermidisandS. aureusto rifampin and minocycline (first experimental approach) Figure 1A shows the growth of S. epidermidis in response to increasing concentrations of rifampin in the presence or absence of 100 μmol/L norepinephrine or dopamine. After incubation for 24 h, plate counts showed that the growth level of S. epidermidis in the presence of concentrations of rifampin near the MIC (0.05 μg/mL) was increased when catecholamines were present (P<.001), although the organisms’ overall antibiotic sensitivity was largely unchanged. Rifampin concentrations of 1 μg/mL and higher were markedly more bactericidal to S. epidermidis with the result that norepinephrine and dopamine were no longer as effective at increasing cell numbers, compared with their effect on nonsupplemented cultures (data not shown). When analogous experiments were performed for S. aureus Newman (figure 1B), a similar pattern of catecholamine-induced growth enhancement in the presence of rifampin was observed (P<.001). Analyses of changes in staphylococcal growth rates in response to a wide range minocycline concentrations also revealed that the simultaneous addition of inotropes did not significantly change the MIC, though, as with rifampin, the level of bacterial growth was increased at minocycline concentrations around the MIC (0.02 μg/mL; data not shown)

Effect of catecholamine inotropes on growth of staphylococci previously exposed to rifampin and minocycline (second experimental approach)The ability of bacteria to survive exposure to antimicrobial-coated CVCs in a damaged state and then enter the systemic circulation may be a relevant pathway by which staphylococci cause catheter-related BSI. To examine whether the presence of catecholamines could affect the survival of bacteria after the bacteria had been exposed to antibiotics, we analyzed the response of staphylococci that had been exposed for 4 h to rifampin or minocycline at 100 times the MIC (5 μg and 2 μg/mL, respectively), and then serially diluted directly into fresh serum-SAPI medium with or without 100 μmol/L catecholamine. All bacteria were then incubated for an additional 24 h and viable cells were enumerated as described above

RifampinFigure 2A shows that prior exposure of S. epidermidis and S. haemolyticus to rifampin resulted in ⩾3 log decrease in the number of viable cells. However, both norepinephrine and dopamine were able to significantly increase the growth level of S. epidermidis previously exposed to rifampin, even when the antibiotic carryover was near to the MIC (the 10⁻² dilution) (P<.001). Figure 2B shows a comparative growth profile for the untreated control cultures. Figure 2A shows growth levels after 24 h; the growth levels of the bacteria after a total of 72 h incubation are shown in figure 2C. Because the viable cell count of the S. epidermidis inoculating culture in figure 2A was 5.40×10⁴ cfu/mL, the number of viable cells in the 10⁻⁵ and 10⁻⁶ dilutions were nominally ⩽1 cell/mL. It can be seen that while there were only small increases in the numbers of bacteria in the cultures not supplemented with catecholamine, in the presence of norepinephrine or dopamine S. epidermidis reached the maximal level of growth supported by serum-SAPI medium, even though the carryover concentration of rifampin was still inhibitory (P<.001). Moreover, S. epidermidis isolates that had been previously exposed to 10 times the MIC of rifampin for as long as 6 days were still able to respond to norepinephrine and dopamine, as evidenced by the increased growth level when compared with cultures not supplemented with catecholamine (figure 2D) (P<.001). Similar results to those shown for S. epidermidis were also obtained for S. haemolyticus (figure 2E and 2F)

Previous work has shown that S. aureus and other coagulase-positive staphylococcal strains evidence little increase in growth in response to catecholamines in serum-SAPI medium [17]. This result was confirmed by the responses to norepinephrine and dopamine observed for S. aureus strains Newman and 832–4 cultures not treated with an antibiotic (figure 3B and 3D), which showed that the enhancement of growth was evident at only very low cell densities (⩽10 cfu/mL). However, antibiotic treatment of the S. aureus cultures (figure 3A and 3C) caused the bacteria to become more responsive to the catecholamine (P<.001). This being so, the growth profile observed over the S. aureus culture dilutions is different from that obtained for S. epidermidis and S. haemolyticus as shown in figure 2. Despite the inoculum for the rifampin-treated S. aureus cultures having had a similar viable cell count (2.21×10⁵ and 3.6×10⁵ cfu/mL, respectively) to that of the S. epidermidis and S. haemolyticus cultures (5.4×10⁴ and 1.14×10⁵ cfu/mL, respectively), S. aureus cells were detected at the end of the 24-h incubation period in all of the culture dilutions, and indeed the 10⁻⁴ dilution growth level values were similar to those observed in the presence of the catecholamines

MinocyclineThe growth profiles for staphylococci challenged with minocycline are shown in figures 4A–4D. The profiles for the response to catecholamine in control cultures not treated with antibiotics were similar to those seen in figures 2 and 3, and so have been omitted. It is apparent for all of the species tested that non–catecholamine dependent recovery from minocycline exposure was more extensive than that observed with rifampin, which suggests that exposure to minocycline was less stressful to the cells. However, it is also apparent that norepinephrine and dopamine significantly enhanced the recovery of staphylococci exposed to minocycline (P<.001)

Acquisition of transferrin-bound iron by staphylococciEarlier reports [14, 15, 17] have demonstrated that the growth of coagulase-negative staphylococci in inotrope-supplemented serum-SAPI medium is the result of inotrope-mediated provision of iron from the serum iron-binding protein transferrin. We therefore investigated whether the ability of S. aureus strains to grow effectively in the absence of inotrope support (figures 3B and 3D) was caused by possession of host-iron acquisition systems that were more effective than those possessed by coagulase-negative staphylococci. Iron-uptake assays were used to test the ability of each of the staphylococcal strains to acquire iron (in the form of ⁵⁵Fe) from ⁵⁵Fe-Tf in both the presence and absence of norepinephrine. As shown in table 1, significant incorporation of transferrin-derived ⁵⁵Fe in coagulase-negative staphylococci only occurred in the presence of norepinephrine, whereas in the absence of norepinephrine, both S. aureus cultures were able to acquire up to 10 times more iron, compared with the coagulase-negative organisms. However, norepinephrine supplementation still increased ⁵⁵Fe uptake from transferrin for S. aureus as described elsewhere [14]

Blocking responsiveness to catecholamine inS. epidermidisby use of catecholamine-receptor antagonistsRecent studies from our laboratories have demonstrated that catecholamine-induced growth in enteric bacteria can be blocked by use of drugs employed therapeutically as catecholamine receptor antagonists [23]. A range of α-adrenergic, β-adrenergic, and dopaminergic antagonists were therefore used to determine whether they could similarly block the responses of S. epidermidis to norepinephrine and dopamine. Table 2 shows that the nonselective β-adrenergic receptor antagonist propranolol had no effect on the ability of norepinephrine or dopamine to induce growth in S. epidermidis Other β-adrenergic antagonists, such as labetalol, atenolol, and yohimbine, were similarly without effect (data not shown). In marked contrast, the α-adrenergic antagonists phentolamine (table 2) and prazosin (data not shown) were able to inhibit norepinephrine-induced growth by ⩾3 log orders, in a concentration-dependent manner, compared with control cultures not supplemented with an antagonist (P<.001). None of the α- or β-antagonists induced growth in S. epidermidis when tested alone, even at concentrations of 500 μmol/L. Furthermore, addition of Fe(NO₃)₃ overcame antagonist blockage of growth induction (table 2), which indicated that growth inhibition by the α-adrenergic receptor antagonists did not result from any cellular toxicity due to the antagonist, but instead represented a specific antagonism of the bacterial response to the catecholamines

Interestingly, the results presented in table 2 also show that the α- and β-adrenergic antagonists caused little inhibition of the S. epidermidis response to dopamine. From the perspective of eukaryotic receptors this should not be surprising, given that dopamine does not operate through either α- or β-adrenergic receptors, but instead by interaction with specific dopamine receptors that are not targets for α- or β-adrenergic antagonists [28]. We therefore investigated whether dopaminergic antagonists had any effect on the response of S. epidermidis to dopamine. Inclusion of apomorphine and haloperidol (nonselective) and raclopride (D₁-specific) did not alter the ability of dopamine to induce growth in S. epidermidis (data not shown). However, the D₂ receptor antagonist chlorpromazine was able to block growth in response to dopamine by >3 log orders (P<.001) (table 2). Chlorpromazine had no significant effect on norepinephrine-induced growth and by itself did not induce growth. The addition of Fe(NO₃)₃ induced growth in dopamine-supplemented S. epidermidis cultures that contained chlorpromazine, similarly indicating that the inhibition observed had been the result of specific antagonism of the response to dopamine. Although earlier reports showed that catecholamines induce staphylococcal growth by facilitating acquisition of transferrin-complexed Fe [4, 7–13], analysis of catecholamine-mediated uptake of ⁵⁵Fe from ⁵⁵Fe-Tf in the presence of growth-inhibiting concentrations of the antagonists demonstrates that the α-adrenergic and dopaminergic antagonists do not exert their effects by blocking this process (data not shown)

Blocking catecholamine-induced rescue of bacteria exposed to antibioticsTo examine whether adrenergic or dopaminergic antagonists could directly block the ability of norepinephrine or dopamine to rescue antibiotic-damaged staphylococci, the catecholamine-resuscitation experiments illustrated by figures 2 and 3 were repeated with the inclusion of phentolamine and chlorpromazine. As seen in figure 5, both α-adrenergic and dopaminergic antagonists were able to specifically block norepinephrine and dopamine resuscitation of antibiotic-damaged S. epidermidis and S. haemolyticus. 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 (data not shown). For S. aureus (figure 5C), the antagonists were less potent, and at best phentolamine and chlorpromazine were able to reduce norepinephrine and dopamine resuscitation by a log in magnitude

Discussion

To our knowledge, the results presented in this report demonstrate for the first time that the catecholamine inotropes that are used in the clinical setting are capable of facilitating the recovery and growth of antibiotic-damaged staphylococci responsible for catheter-related BSIs. Of critical importance in the present study has been the use of very low bacterial inocula in an iron-restrictive, serum-based medium that more closely approximates the in vivo milieu. This extends previous work demonstrating that catecholamine inotropic drugs were capable of increasing both S. epidermidis planktonic growth levels [14] and biofilm growth on catheter materials [15] from very low initial bacterial inocula in an iron-restrictive, plasma- or serum-based medium. Taken together these reports echo the concerns recently expressed in a Lancet commentary by Singer [30] that addressed the use of inotropes in the treatment of sepsis [30]

Antimicrobial coatings for indwelling medical devices, such as those that incorporate rifampin (usually used in combination with minocycline, as for CVCs), are increasingly being used in the clinical setting to prevent colonization with opportunistic skin flora, of which S. epidermidis is one of the most pervasive [6–10]. However, controversy surrounds claims for the superiority of antimicrobial-impregnated CVCs, compared with CVCs not impregnated with antimicrobials, in preventing and reducing the prevalence of catheter-related BSIs [9–11]. Also, none of these clinical studies has examined efficacy specifically in the context of catecholamine inotrope usage. In demonstrating the ability of therapeutic levels of catecholamine inotropes to contribute to the survival and growth of staphylococci previously exposed to levels of rifampin or minocycline above the MIC, the present study may provide a clinically based mechanism to explain the inability of antimicrobial-impregnated CVCs to significantly alter the rates of catheter-related BSI, compared with nonimpregnated CVCs

Of considerable interest is our observation that inotropes were able to induce the growth of antibiotic-damaged S. aureus Previous work from our laboratories had shown that S. aureus strains showed little increase in growth in response to catecholamines in serum-based media [17]. This might be the result of possession of acquisition systems for host sequestered iron that enable them to independently overcome the iron-limitation of tissue fluids [29], and indeed the iron uptake analyses (table 1) confirmed that S. aureus was more efficient at accessing transferrin-complexed iron than either of the coagulase-negative strains. However, in the presence of a catecholamine inotrope, S. epidermidis and S. haemolyticus were able to assimilate levels of transferrin–derived iron similar to the levels achieved by both S. aureus strains. Additionally, the current work demonstrates that S. aureus cultures that had been stressed by prior exposure to antibiotics became more dependent on catecholamines and showed marked growth inhibition in their absence (P<.001), thereby suggesting that the antibiotic may have damaged the ability of the S. aureus strains to access host-sequestered iron

The data presented herein is also suggestive of potential new avenues for the prevention of staphylococcal responses to catecholamine inotropes (which can lead to biofilm formation [23]) based on the use of adrenergic and dopaminergic antagonists. As such, these observations unexpectedly extend the recent findings obtained from gram-negative enteric bacteria [23] to gram-positive species. They also provide an important perspective on the evolutionary development of responsiveness to catecholamine in staphylococcal species. Previous reports proposed that adrenergic and dopaminergic catecholamines induced staphylococcal growth via an apparently identical mechanism involving the provision of iron from the host iron-binding protein transferrin [14, 15]. Our latest work, however, has revealed this to be too simplistic an interpretation, given our demonstration that adrenergic and dopaminergic antagonists are selective in their ability to block staphylococcal responses to adrenergic and dopaminergic agonists, and that antagonist blocking of responsiveness to catecholamine is independent of catecholamine iron provision. These new findings indicate the presence of specific adrenergic- and dopaminergic-type receptors in the staphylococcal catecholamine-mediated growth pathways. The antagonist data shown in table 2 further suggests that these staphylococcal response systems resemble α-adrenergic receptors, but not β-adrenergic receptors, in the mechanism of norepinephrine growth induction and for a dopamine response system with D₂-dopaminergic–like specificity in dopamine-mediated growth induction, similar to that observed in enteric species [23]. This pharmacological similarity enables us to speculate that the evolution of bacterial catecholamine response pathways may have developed before the temporal separation of gram-positive and gram-negative species. Finally, from the clinical perspective, the present report may carry important implications related to diagnosis, and suggests that incorporation of catecholamine inotropes into clinical microbiological diagnostic media may provide a useful means to rescue otherwise damaged bacteria from clinical specimens that currently prove incapable of growth in standard diagnostic laboratory media