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Pseudomonas aeruginosa-Catecholamine Inotrope Interactions
CHEST
Original Research CRITICAL CARE
Pseudomonas aeruginosa-Catecholamine Inotrope Interactions A Contributory Factor in the Development of Ventilator-Associated Pneumonia? Primrose P. Freestone, PhD; Robert A. Hirst, PhD; Sara M. Sandrini, PhD; Fathima Sharaff, MSc; Helen Fry, MD; Stefan Hyman; and Chris O’Callaghan, DM, PhD
Background: Ventilated patients receiving intensive care are at significant risk of acquiring a ventilator-associated pneumonia that is associated with significant morbidity and mortality. Despite intensive research, it is still unclear why Pseudomonas aeruginosa, a microbe that rarely causes pneumonia outside of intensive care, is responsible for so many of these infections. Methods: We investigated whether medications frequently prescribed to patients in the ICU, the catecholamine inotropes, were affecting the growth and virulence of P aeruginosa. Effects of clinically attainable concentrations of inotropes on P aeruginosa pathogenicity were explored using in vitro growth and virulence assays and an ex vivo model of infection using ciliated human respiratory epithelium. Results: We found that inotropes were potent stimulators of P aeruginosa growth, producing up to 50-fold increases in bacterial numbers via a mechanism involving inotrope delivery of transferriniron, internalization of the inotrope, and upregulation of the key pseudomonal siderophore pyoverdine. Inotropes also markedly increased biofilm formation on endotracheal tubing and enhanced the biofilm production and toxicity of P aeruginosa in its interaction with respiratory epithelium. Importantly, catecholamine inotropes also facilitated the rapid recovery of P aeruginosa from tobramycin antibiotic challenge. We also tested out the effect of the inotropes vasopressin and phenylephrine on the growth and virulence of P aeruginosa and found that, in contrast to the catecholamines, these drugs had no stimulatory effect. Conclusions: Collectively, our results suggest that catecholamine inotrope-bacterial interactions may be an unexpected contributory factor to the development of P aeruginosa-ventilator-associated pneumonia. CHEST 2012; 142(5):1200–1210 Abbreviations: ALI 5 air-liquid interface; CFU 5 colony-forming unit; ET 5 endotracheal tube
who require ventilation while receiving Patients intensive care are susceptible to infection by opportunistic pathogens1-6 that rarely cause infections in healthy individuals. Up to 20% develop a severe
Manuscript received October 18, 2011; revision accepted April 1, 2012. Affiliations: From the Department of Infection, Immunity and Inflammation (Drs Freestone, Hirst, Sandrini, Fry, and O’Callaghan and Ms Sharaff), University of Leicester School of Medicine; the Division of Child Health (Drs Hirst and O’Callaghan); and the Electron Microscopy Laboratory (Mr Hyman), University of Leicester; and the Institute of Lung Health (Drs Hirst and O’Callaghan), Leicester, England. Funding/Support: Drs Freestone, Sandrini, and O’Callaghan are funded by the SPARKS Children’s Charity (www.sparks.org.uk), England [Grant 09 LCS 01].
ventilator-related pneumonia that is associated with significant mortality and prolonged stays in the ICU.5 Gram-negative bacilli are responsible for approximately 60% of ventilator-associated infections, with Pseudomonas aeruginosa responsible for up to onehalf of all cases.3 The reasons for the high rate of infection by these organisms remain unclear. Our Correspondence to: Chris O’Callaghan, DM, PhD, Department of Respiratory Medicine, Portex Unit, Institute of Child Health, University College London (UCL), 30 Guildford St, London, WC1N 1EH, England; e-mail: [email protected] © 2012 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.11-2614
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laboratory and others have shown that catecholamine stress hormones and certain of their metabolites can stimulate the growth and infectivity of a range of infectious bacteria.7-13 This is of concern because catecholamines as inotropic drugs, particularly norepinephrine and dopamine, are prescribed for up to 50% of patients in the ICU,14 which led us to hypothesize that they may also increase the growth and virulence of bacteria such as P aeruginosa. Infused inotropes affect lung liquid clearance,15 and it is well known that the lungs are an active site of inotrope metabolism.16 Studies using injected 14C-labeled l-DOPA and l-dopamine showed that inotrope accumulation occurs in the lung within an hour of systemic administration.17 The respiratory tissues, via the endotracheal tube (ET), may also be used as a direct site for systemic administration of inotropes.15 Because dopamine and norepinephrine are present within respiratory mucus,18 it is likely that bacteria colonizing the respiratory tract and catecholamines will at some point occupy the same spatial location within the intensive care patient. Therefore, the aim of this study was to determine if exposure of P aeruginosa to catecholamine inotropes commonly prescribed to patients, at levels that may be encountered in patients during intensive care management, affected aspects of its virulence relevant to its ability to cause pneumonia. Materials and Methods Strains, Growth Induction, and Antibiotic Sensitivity Assays The P aeruginosa strains used in this study were a clinical isolate from a patient with ventilator-associated pneumonia (Public Health Laboratory, Leicester Royal Infirmary, England) and P aeruginosa reference strain PA14. The serum-SAPI medium used for growth assays was prepared as described previously.8,11 L-(-)-norepinephrine bitartrate, epinephrine bitartrate, and dopamine hydrochloride were obtained from Sigma. Vasopressin (20 units/mL, 600 units/mg) and phenylephrine (10 mg/mL) were obtained from the Department of Pharmacy, Leicester Royal Infirmary, University Hospitals of Leicester. Horseradish peroxidase conjugated transferrin (Cat. No. 009-030-050) was obtained from Jackson ImmunoResearch Laboratories. Tobramycin (80 mg/2 mL) was obtained from King Pharmaceuticals. 55FeCl3 (IES, specific activity 5 mCi/mg iron) and 3H-norepinephrine (TRK584,l-[7,8-3H] norepinephrine) were obtained from Amersham Life Sciences. P aeruginosa was inoculated at low cell density (50-100 colonyforming units [CFU]/mL) into serum-SAPI (a serum-supplemented minimal medium8,11) to more closely approximate in vivo conditions.7 Bacterial inoculum sizes were determined by pour-plate analysis, and cultures were incubated statically at 37°C in a 5% CO2 incubator. All analyses used inotrope concentrations within the range reported in the plasma of dopamine-medicated patients.19,20 Norepinephrine and epinephrine were used at 5 mM and dopamine at 5 mM plus 1 mM norepinephrine, to reflect the enzymatic conversions that occur in vivo.21 After incubation, bacterial numbers were enumerated using Luria agar.11-13
To assess the role of transferrin in the mechanism of inotrope growth induction, cultures were incubated for 1 h at 37°C in a 5% CO2 incubator with 0.1 mg/mL of horseradish peroxidase conjugated transferrin, harvested by centrifugation at 5,000 3 g for 5 min, and washed twice in phosphate-buffered saline. Washed cell suspensions were resuspended in phosphate-buffered saline, serially diluted in twofold steps, and 10-mL aliquots spotted onto nitrocellulose; horseradish peroxidase conjugated transferrin binding was visualized using enhanced chemiluminescence. To investigate inotrope effects on transferrin-iron uptake, and inotrope internalization, P aeruginosa cultures were inoculated into serum-SAPI supplemented with 1 3 105 counts per min of 55Fe-transferrin or 0.5 3 105 counts per min/mL 3H-norepinephrine, with and without the addition of norepinephrine. 55Fe-transferrin was prepared as described previously.12 Cultures were incubated as described for the growth assays and were assayed for cell numbers and radiolabel incorporation using scintillation counting.12 P aeruginosa intracellular iron levels were measured from total protein using inductively coupled plasma optical emission spectroscopy. P aeruginosa tobramycin sensitivity assays were performed in serum-SAPI using a minimum inhibitory concentration methodology.22 Exponential P aeruginosa cultures (about 105 CFU/mL) were added to serum-SAPI supplemented with increasing tobramycin concentrations in the presence/absence of norepinephrine or dopamine.23 Cultures were incubated for 24 h and enumerated for bacterial numbers as described previously. Production of the P aeruginosa siderophore pyoverdine was measured using a fluorimetric assay24 and quantified in terms of relative fluorescent units per normalized optical density/mL of bacterial culture. Analysis of Inotrope Effects on P aeruginosa Biofilm Formation Inotrope effects on P aeruginosa attachment, swimming, and twitching motility were measured as previously described.25,26 Briefly, twitching motility assays were performed in Luria broth medium solidified with 1.0% agar supplemented with various concentrations of catecholamines. Plates were dried overnight at room temperature, and P aeruginosa strains were point inoculated at the bottom of the Petri plate. After 3 days, the twitching distance along the plastic-agar interface (at the bottom of the agar plate) was measured. Initial attachment on polyvinylchloride was visualized by staining with the FilmTracer LIVE/DEAD biofilm viability kit (Invitrogen; Life Technologies Corp) using a Nikon Ti inverted fluorescence microscope (Nikon Corp) set at 480/500 nm emission and excitation maxima. Sterile polyvinylchloride ET sections were used for ET-biofilm analyses. To ensure similarity in growth rates between control and inotrope-treated cultures, P aeruginosa was added at 104 CFU/mL to serum-SAPI and incubated at 37°C in a 5% CO2 incubator for 48 h. ET sections were washed twice with phosphate-buffered saline, once with 0.1 M sodium phosphate buffer pH 7.2 to remove residual protein, and then processed for scanning electron microscopy as described previously,13 using a Hitachi S3000H scanning electron microscope (Hitachi Ltd). Inotrope effects on P aeruginosa interaction with respiratory cells were studied using a human-ciliated respiratory epithelium air-liquid interface (ALI) cell culture prepared from nasal biopsy cells.27,28 Ciliary beat frequency and ciliary beat amplitude measurements were analyzed as described previously21,22 (e-Appendix 1). To visualize host-pathogen interactions in real time, P aeruginosa at an inoculum of 106 CFU/mL was used to infect the ALI cultures; the inotrope alone was added to the uninfected control culture. Ciliary function, ultrastructure analyses of the bacterial-epithelial cell cultures, and bacterial growth levels were obtained at hourly intervals for 5 h.
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Statistics All experiments were performed in at least triplicate; unless stated otherwise, numerical data shown are expressed as mean ⫾ SD. Where appropriate, statistical analysis was first performed using one-way analysis of variance and, if significant, an unpaired t test. Statistical significance was indicated by a P value of ,. 05.
Results Inotropes Increase P aeruginosa Growth and Pyoverdine Synthesis Previous analyses of P aeruginosa-catecholamine interactions used norepinephrine levels of 50 mM and higher.7,8,11 However, in the current study, we found that incubation of an inoculum of , 102 CFU/mL of P aeruginosa strains clinical isolate and PA14 in serum-SAPI for 18 h with 5 mM concentrations of the inotropes norepinephrine and dopamine (drug levels that have been reported in the blood of patients medicated with inotrope19,20) resulted in up to 50-fold increases in bacterial numbers over unsupplemented control cultures (P , .01 for both strains) (Fig 1A). Dose response analyses using other inotrope concentrations (e-Fig 1) revealed that enhancement of P aeruginosa growth also occurred with lower concentrations of norepinephrine and dopamine (1 and 3 mM), although this was to a lower level than seen with 5 mM. Iron limitation by transferrin is primarily responsible for the innate bacteriostatic nature of serum and blood, so we investigated if the mechanism by which inotropes induced P aeruginosa growth involved the inotrope enabling the bacteria to access transferrincomplexed iron. Figure 1B shows that transferrin was directly bound by both P aeruginosa strains during growth. The addition of 55Fe-transferrin (Fig 1C) to culture media showed that the inotrope increased bacterial acquisition of transferrin-55Fe. Inclusion of 3H-norepinephrine also revealed that the inotrope was directly internalized by the P aeruginosa during the catecholamine-induced growth process (Fig 1C) (P , .001). Following longer incubation (48 h), the control and inotrope cultures reached a similar cell density (Fig 1D); inductively coupled plasma optical emission spectroscopy measurement of the intracellular iron content of the cultures confirmed that the inotropes were increasing internal iron levels of the bacteria. The photo insets in Figure 1D show that inotropesupplemented cultures were greener in color and displayed more biofilm than did control subjects. Pyoverdine is a green ferric-iron-sequestering siderophore often seen in P aeruginosa cultures.24,29 Fluorimetric assays of the P aeruginosa culture supernatants (Fig 1E) confirmed the presence of pyoverdine and showed (n 5 4) that the inotropes induced both strains to synthesize significantly more siderophore than did control subjects (P , .001).
Inotropes Enhance Recovery of Tobramycin-Treated P aeruginosa Figure 2 shows that during incubation with increasing concentrations of the antipseudomonal antibiotic tobramycin, the P aeruginosa strains clinical isolate (Fig 2A ) and PA14 (Fig 2B) underwent a dosedependent reduction in bacterial numbers. However, viable counts indicated that compared with control subjects, the numbers of P aeruginosa in the presence of concentrations of tobramycin near the minimum inhibitory concentration (approximately 0.1 mg/mL for strain clinical isolate and 1.0 mg/mL for PA14) increased up to 100-fold when an inotrope was present (P , .0001). Inotropes Enhance P aeruginosa Biofilm Formation on ETs The ET is an area with a recognized of risk of development of Pseudomonas-associated pneumonia.1-6 Attachment is the first stage in Pseudomonas biofilm formation, and Figure 3A reveals that for both strains, clinically attainable concentrations of the catecholamines significantly enhanced attachment (P . .01) to polyvinylchloride (an ET polymer). The epifluorescence micrographs in Figure 3A show that the inotropes also enhanced P aeruginosa cell-cell attachment and microcolony formation (n 5 3). As well as production of factors for attachment, motility within a Pseudomonas biofilm is important for its establishment.25 Swimming motility (Fig 3B) with the exception of norepinephrine was not significantly affected by the addition of 5 mM inotrope concentrations, although higher drug levels (50 mM) were stimulatory (data not shown). Twitching motility was enhanced by the inotropes, although only for strain clinical isolate (Fig 3C), because PA14 had generally low motility. Figure 3D shows scanning electron microscopy images of P aeruginosa clinical isolate directly seeded onto ET sections and incubated in the presence of norepinephrine, dopamine, and epinephrine. Relative to control cultures, inotrope exposure consistently induced marked increases in biofilm formation on the ET (n 5 4). A very similar inotropeinduced enhancement of ET biofilm production was also obtained with P aeruginosa strain PA14 (data not shown). Figure 3D shows the biofilm that remains attached to the ET after scanning electron microscopy processing. A fuller picture of the architecture of inotropeinduced P aeruginosa biofilms is revealed in Figure 4, which shows fragments of norepinephrine-induced biofilms that have become detached from the ET. Figure 4A shows a section of biofilm before scanning electron microscopy processing, viewed using a light microscope. The characteristic mushroom structures
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Figure 1. Inotropes increase Pseudomonas aeruginosa growth and pyoverdine production. A, NE and dopamine effects on growth of P aeruginosa strains CI and PA14 after 18-h incubation (n 5 4). B, Transferrin binding by the CI and PA14 P aeruginosa strains occurs during growth; the upper two panels show transferrin binding blots of a twofold dilution of the two cultures (initial cell density around 2 3 108 CFU/mL); the control consists of similar numbers of bacteria to the 1:2 dilution, but incubated without H-Tf; the activity of 1.0 and 0.1 mg of H-Tf is shown in the lower panel. C, After normalizing cell densities between control and inotrope-treated cultures, representative bacterial internalizations of iron (n 5 4) (in the form 55Fe from 55Fe-transferrin) (right panel) are increased in the presence of the inotrope; tritiated norepinephrine internalization is shown in the left panel. D, P aeruginosa CI and PA14 growth levels after 48-h incubation in serum-SAPI medium; the intracellular iron levels of control, NE, and dopamine-treated cultures (n 5 4) are shown for each culture; SDs for each set of data are shown in brackets. Representative images of the cultures are shown in the inserts. E, Typical measurements (n 5 5) of the pyoverdine levels of the 48-h CI and PA14 cultures. The initial inoculum for strains CI and PA14 in experiments A and C to E were 78, 99, 88, and 93 CFU/mL, respectively. *Statistical significance (P , .01). CFU 5 colony-forming units; CI 5 clinical isolate; H-Tf 5 horseradish peroxidase transferrin; NE 5 norepinephrine; NS 5 not significant.
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Figure 2. Inotropes enhance P aeruginosa recovery from antibiotic challenge. The data show how inotropes assist P aeruginosa in resisting a tobramycin challenge. Incubation of P aeruginosa strains CI and PA14 with tobramycin for 24 h decreased viable bacterial counts at the higher tobramycin concentrations (control bar [clear]). The presence of either norepinephrine (black) or dopamine (gray) during this time resulted in a marked increase in viable bacteria at 24 h. Data shown are the mean⫾ SD of three separate analyses of triplicate assays. A, CI. B, PA14. *Statistical significance (P , .01). See Figure 1 legend for expansion of abbreviations.
indicative of a mature biofilm are strikingly visible. Figure 4B shows more-detailed scanning electron microscopy images of a similar P aeruginosa biofilm fragment. The underside differs markedly from the upper side and is flatter and more uniform in appearance. In contrast, the upper side contains the mushroom-like projections shown in Figure 4A. The full fragment image reveals that the thickness of the biofilm is variable; however, the side edge view gives an indication of how dense the biofilm may have been prior to the scanning electron microscopy processing. Effect of Inotropes on Interaction of P aeruginosa With Respiratory Epithelium We used an ex vivo ALI model of cultured human ciliated epithelium27,28 to investigate the inotrope effects on the interaction of P aeruginosa with human respiratory epithelia. Additional supporting data for Figure 5 can be found in Videos 1-6, which show the P aeruginosa-ALI infection time course. Figures 5B and 5C show that throughout the 5-h study, the uninfected control maintained a normal ciliary beat frequency and beat pattern, indicating that the inotrope had not affected ciliary function (P . .05). P aeruginosa attachment to host cells was evident within 1 h of infection in both inotrope and noninotrope bacterial
cultures. Inotrope-mediated biofilm enhancement was evident by 1 h and was clearly present by 2 h, becoming progressively more extensive over time. Figures 5A and 5D show that by 5 h, the inotrope had induced the formation of extensive sheets of bacteria and exopolysaccharide covering most of the epithelial cell surface (P 5 .02). Figures 5B and 5C show that, although the cilia managed to continue beating under the burden of the biofilm with an unchanged frequency, the viscous drag of bacteria and exopolysaccharide attached to the cilia tips significantly reduced the amplitude of the cilia tip distance traveled (P , .01).27 By the end of the 5-h time course, signs of cytotoxicity in the epithelial cells exposed to the inotrope-treated P aeruginosa were evident, with reductions occurring in cilia function such as beat frequency, and extrusion of cells from the epithelial culture surface (data not shown). The non-inotrope-treated P aeruginosa culture also showed production of biofilm during the course of the incubation (P 5 .03) (Figs 5A, 5D) but this was always significantly less extensive than the inotrope-treated bacteria (P 5 .02) and, by 5 h, considerable areas of the noninotrope-treated epithelial cell cultures were still free of biofilm. Very similar results to those shown for strain clinical isolate were also obtained for P aeruginosa strain PA14 (data not shown). P aeruginosa Interaction With Noncatecholamine Inotropes We additionally tested whether the noncatecholamine inotropic agents phenylephrine (neosynephrine) and vasopressin had any stimulatory effects on our P aeruginosa strains. Figure 6 examines the response of P aeruginosa to a wide range of vasopressin and phenylephrine concentrations, from therapeutic serum levels to at least two log orders above.30-33 It is clear that neither drug had any effect on growth (Figs 6A, 6B) or initiation of biofilm formation (Figs 6C, 6D) at any of the concentrations tested. Other aspects of P aeruginosa virulence, such as pyoverdine production, or swimming and twitching motility, were similarly unaffected by the levels of phenylephrine and vasopressin shown in Figure 6 (data not shown). Discussion Ventilator-associated pneumonia, a hospital-acquired infection, remains a major cause of morbidity and death, despite intensive research.1-6 P aeruginosa, a pathogen that rarely causes pneumonia outside of intensive care, is responsible for a high proportion of these infections.1-6 In this study, we presented evidence that levels of catecholamine inotropes that are within the ranges reported in the plasma of
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Figure 3. P aeruginosa biofilm formation on endotracheal tubing (ET) is enhanced in the presence of catecholamine inotropes. A, Results of a crystal violet attachment assay demonstrating that, relative to control subjects, 5 mM concentrations of the inotropes significantly increased P aeruginosa attachment to polyvinylchloride. **P . .001. The panels underneath the histograms show that the inotropes are also enhancing other aspects of pseudomonal biofilm formation, including exopolysaccharide production and cell-cell attachment. B, Only NE has an effect on P aeruginosa swimming motility. *P . .01; **P . .001. C, Twitching motility of clinical strain CI is enhanced by the inotropes; the twitching motility values of the CI image are shown in the accompanying histogram; in contrast, the reference strain PA14 is largely nonmotile. **P . .001. D, Scanning electron micrographs of inotrope enhancement of P aeruginosa attachment and biofilm formation on ET. P aeruginosa strain CI was inoculated at 8 3 105 CFU/mL onto sterile ET sections in the absence or presence of NE (5 mM), Epi (5 mM), and Dop (5 mM) in serum-SAPI medium for 48 h (n 5 3). The four sets of panels show representative scanning electron micrographs of the ET biofilms at increasing magnification; the scales for each image are shown in the bottom right corner of the micrograph. Note that the growth levels of the control and inotrope-supplemented cultures were not significantly different (P 5 .16) (data not shown), indicating that the effects seen are not due to growth differences and that the inotropes are directly enhancing biofilm-responsive genes. Dop 5 dopamine; Epi 5 epinephrine. See Figure 1 legend for expansion of other abbreviations. journal.publications.chestnet.org
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Figure 4. Architecture of inotrope-induced P aeruginosa biofilms. A, A 48-hour-old NE-induced biofilm fragment (from P aeruginosa strain clinical isolate) that has been detached from an endotracheal tube section; the image shown was obtained using a 3 40 light microscope objective. B, Scanning electron micrographs of the upper, lower, and side architectural views of a similarly prepared biofilm fragment. The scales shown are for the bars on the scanning electron micrographs. Similar types of biofilm structure were also seen with Dop and Epi-treated P aeruginosa (data not shown). See Figure 1 and 3 legends for expansion of abbreviations.
inotrope-supplemented patients19,20 can greatly increase aspects of P aeruginosa virulence relevant to its capacity to cause ventilator-associated pneumonia. We also analyzed the effects of the noncatecholamine inotropes vasopressin and phenylephrine and found that, in contrast to the catecholamines, neither compound at any of the concentrations tested was stimulatory to the growth and virulence of our P aeruginosa strains. All infectious bacteria require iron for growth in vivo, and iron limitation of host fluids by proteins such as transferrin is a key primary host defense. We found that the mechanism by which inotropes increased P aeruginosa growth involved the inotrope enabling the bacteria to access the iron within transferrin coupled with the direct internalization of the catecholamine. Mechanistically, by binding the transferrin, the closer proximity probably allows more efficient iron removal by P aeruginosa siderophores and better internalization of the sequestered iron. Further, despite inotropes increasing the intracellular iron levels of P aeruginosa, which might be expected to inhibit expression of iron-regulated genes,34 upregulation of synthesis of pyoverdine, a key pseudomonal siderophore that is normally under tight iron repression, was observed. Pyoverdine can remove iron from transferrin, which has been shown to be important in P aeruginosa growth in lung secretions.35 In addition to its role in scavenging host iron, pyoverdine is unusual in having signaling functions and influencing P aeruginosa host colonization, biofilm formation,36 and virulence factor synthesis.29 This suggests that inotrope exposure could be even more wide reaching in its effects on promoting the ability of P aeruginosa to cause infection. Resistance to antibiotics is an increasing problem in the control of hospital-acquired infections,6,23,37
making of particular interest our discovery that inotropes can catalyze the recovery of tobramycin-treated P aeruginosa. As well as being associated with increased resistance to antibiotics and immune attack, biofilm formation by P aeruginosa is an aspect of its virulence vitally important in its capacity to colonize the ET, lung, and other tissues.1-6,37,38 We showed that those inotropes most frequently administered to ventilated patients (norepinephrine, epinephrine, and dopamine) all markedly increased P aeruginosa biofilm formation on ETs. This is an important discovery because bacterial colonization of the ET is a factor thought to predispose ventilated patients to development of pneumonia.1-6,39 We additionally used an ex vivo human model of respiratory infection to show that inotropes also enhanced the ability of P aeruginosa to directly colonize ciliated respiratory epithelia via increased host cell attachment and biofilm formation. Note that this is the first time that a human model of infection has been used in the examination of catecholaminebacteria interactions. Previous in vitro bacteria-catecholamine studies, including those employing animal infection models, have routinely used catecholamine concentrations of 50 to 2,000 mM (these studies are reviewed in Freestone et al7). An important finding of this report is that, to the best of our knowledge, we are the first to demonstrate the stimulatory effects of inotropes on P aeruginosa pathogenicity using much lower clinically attainable drug concentrations. In its usage as the most widely employed inotrope, dopamine is typically infused at 1 to 15 mg/kg/min, and steadystate levels in plasma vary according to infusion levels and general metabolic fitness, with acutely ill patients showing slower elimination rates.39 Girbes et al20
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Figure 5. Catecholamine inotropes enhance P aeruginosa biofilm formation on human airway epithelial cells. Inotrope NE influences P aeruginosa biofilm formation on human ciliated respiratory epithelia. PA was inoculated at 2 3 106 CFU/mL onto a differentiated airway epithelium as described in the “Materials and Methods” section; NE was used at 5 mM. A, Time course of P aeruginosa biofilm coverage; 䊊 5 uninfected control; ⵧ 5 P aeruginosa: no inotrope; 䊏 5 P aeruginosa + inotrope. B, Tip distance traveled of free cilia and of cilia covered by the P aeruginosa biofilm. 䊊 5 uninfected control; ⵧ 5 P aeruginosa with no inotrope: cilia beat amplitude outside the biofilm; 䊏 5 P aeruginosa with no inotrope: cilia beat amplitude inside the biofilm. 䉭 5 P aeruginosa 1 inotrope: cilia beat amplitude outside the biofilm; 䉱 5 P aeruginosa 1 inotrope: cilia beat amplitude inside the biofilm. C, Analysis of the cilia beat frequency (CBF) of free cilia, and cilia covered by biofilm. 䊊 5 CBF of uninfected control. ⵧ 5 P aeruginosa no inotrope: CBF outside the biofilm; 䊏 5 P aeruginosa no inotrope: CBF inside the biofilm; 䉭 5 P aeruginosa 1 inotrope: CBF outside the biofilm; 䉱 5 P aeruginosa 1 inotrope: CBF inside the biofilm. *Significance of P . .01; **P . .001. D, Representative light microscopy photographs (n 5 3) of the appearance of noninfected respiratory epithelia (control), and respiratory epithelia infected with only PA or PA plus NE (5 mM). The arrows are indicative of the presence of P aeruginosa biofilm. PA 5 P aeruginosa strain clinical isolate. See Figure 1 legend for expansion of other abbreviations.
showed that in septic shock patients medicated with 6 mg/kg/min dopamine, catecholamine plasma levels in some patients rose to . 5 mM, the concentration used in our assays. Within the circulation, dopamine
undergoes enzymatic conversion to norepinephrine and epinephrine,21 which has relevance to the current study, because Thompson et al19 showed that in some cardiac surgery patients treated with 3 mg/kg/min
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Figure 6. Effects of the noncatecholamine inotropes vasopressin and phenylephrine on P aeruginosa growth and virulence. A, Effects of a range of vasopressin concentrations on the growth of P aeruginosa strains CI and PA14 after 18-h incubation (n 5 4). B, Result of a crystal violet attachment assay demonstrating that none of the vasopressin concentrations tested had any effect on attachment of either P aeruginosa strain (n 5 4). C, Similarly, none of the phenylephrine concentrations tested increased the growth of P aeruginosa. D, Phenylephrine also had no effect on the surface attachment of either strain (n 5 4). See Figure 1 legend for expansion of abbreviations.
dopamine, norepinephrine plasma levels rose to 9.24 mM. The lungs are pharmacologically active and are responsible for extraction of around 20% of infused inotropes, increasing to 33% in the critically ill.31,32 Lung inotrope uptake systems are so effective that they have been proposed to be alternate sites
for inotrope administration. Endotracheal administration of epinephrine has been recommended if IV access is contraindicated, and up to 2 mg of epinephrine may be applied directly via the ET.15 Epinephrine (as a 300-mM solution) is also occasionally nebulized directly through the ET to reduce airway
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inflammation40; our study showed that epinephrine doses of a fraction of this value increased P aeruginosa colonization of the ET. It is probable that within the ventilated patient, the outer surface of the ET is in direct contact with airway secretions, which is significant because mucus itself contains catecholamines.18 In addition to being given inotropes, ventilated patients are both chronically and acutely stressed, and significant endogenous increases in plasma norepinephrine and epinephrine have been associated with procedures such as ET suctioning.41 Thus, there is considerable opportunity for bacteria colonizing the ET to come into direct contact with both endogenous and administered catecholamines. Conclusions In conclusion, this study suggests that administration of inotropes to patients in the ICU, particularly if high doses are given systemically or via direct local application, may be an unappreciated risk factor in the development of ventilator-associated pneumonia by P aeruginosa. Further investigations are therefore needed to determine whether the higher circulating catecholamines of the ventilated patient,19,20 combined with the presence of administered inotropes and naturally occurring catecholamines in mucosal secretions, are acting to promote the growth and virulence of a bacterium that rarely cause pneumonia outside of intensive care. Acknowledgments Author contributions: Dr O’Callaghan is the guarantor of the manuscript and takes responsibility for the integrity of the data and accuracy of the data analysis. Dr Freestone: conceived and designed the study, the carrying out of many of the experiments, analysis of the data, and writing and revision of the manuscript. Dr Hirst: contributed to parts of the study design, carrying out of some of the experiments, analysis of the data, and writing of some of the manuscript. Dr Sandrini: contributed to the carrying out of some of the experiments, analysis of the data, and writing of some of the manuscript. Ms Sharaff: contributed to the carrying out of some of the experiments, analysis of the data, and writing of some of the manuscript. Dr Fry: contributed to the carrying out of some of the experiments, analysis of the data, and writing of some of the manuscript. Mr Hyman: contributed to the carrying out of some of the experiments, analysis of the data, and writing of some of the manuscript. Dr O’Callaghan: conceived and designed the study and contributed to the writing and revision of the manuscript. Role of sponsors: The sponsor had no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript. Additional information: The e-Appendix, e-Figure, and Videos can be found in the “Supplemental Materials” area of the online article.
References 1. Koerner RJ. Contribution of endotracheal tubes to the pathogenesis of ventilator-associated pneumonia. J Hosp Infect. 1997;35(2):83-89.
2. Morehead RS, Pinto SJ. Ventilator-associated pneumonia. Arch Intern Med. 2000;160(13):1926-1936. 3. Garau J, Gomez L. Pseudomonas aeruginosa pneumonia. Curr Opin Infect Dis. 2003;16(2):135-143. 4. Ramirez P, Ferrer M, Torres A. Prevention measures for ventilator-associated pneumonia: a new focus on the endotracheal tube. Curr Opin Infect Dis. 2007;20(2):190-197. 5. Shorr AF, Kollef MH. Ventilator-associated pneumonia: insights from recent clinical trials. Chest. 2005;128(5)(suppl 2): 583S-591S. 6. Fagon JY, Rello J. Targeted antibiotic management of ventilator-associated pneumonia. Clin Microbiol Infect. 2006; 12(s9):17-22. 7. Freestone PP, Sandrini SM, Haigh RD, Lyte M. Microbial endocrinology: how stress influences susceptibility to infection. Trends Microbiol. 2008;16(2):55-64. 8. Lyte M, Ernst S. Catecholamine induced growth of gram negative bacteria. Life Sci. 1992;50(3):203-212. 9. Alverdy J, Holbrook C, Rocha F, et al. Gut-derived sepsis occurs when the right pathogen with the right virulence genes meets the right host: evidence for in vivo virulence expression in Pseudomonas aeruginosa. Ann Surg. 2000;232(4): 480-489. 10. Bailey MT, Engler H, Sheridan JF. Stress induces the translocation of cutaneous and gastrointestinal microflora to secondary lymphoid organs of C57BL/6 mice. J Neuroimmunol. 2006;171(1-2):29-37. 11. Freestone PP, Haigh RD, Williams PH, Lyte M. Stimulation of bacterial growth by heat-stable, norepinephrineinduced autoinducers. FEMS Microbiol Lett. 1999;172(1): 53-60. 12. Freestone PP, Williams PH, Haigh RD, Maggs AF, Neal CP, Lyte M. Growth stimulation of intestinal commensal Escherichia coli by catecholamines: a possible contributory factor in trauma-induced sepsis. Shock. 2002;18(5):465-470. 13. Lyte M, Freestone PP, Neal CP, et al. Stimulation of Staphylococcus epidermidis growth and biofilm formation by catecholamine inotropes. Lancet. 2003;361(9352): 130-135. 14. Smythe MA, Melendy S, Jahns B, Dmuchowski C. An exploratory analysis of medication utilization in a medical intensive care unit. Crit Care Med. 1993;21(9):1319-1323. 15. Raymondos K, Panning B, Leuwer M, et al. Absorption and hemodynamic effects of airway administration of adrenaline in patients with severe cardiac disease. Ann Intern Med. 2000;132(10):800-803. 16. Adir Y, Azzam ZS, Lecuona E, et al. Augmentation of endogenous dopamine production increases lung liquid clearance. Am J Respir Crit Care Med. 2004;169(6):757-763. 17. Shindo H, Nakajima E, Kawai K, Miyakoshi N, Tanaka K. Studies on the metabolism of D- and L-isomers of 3,4-dihydroxyphenylalanine (DOPA). 3. Absorption, distribution and excretion of D- and L-DOPA-14C in rats following intravenous and oral administration. Chem Pharm Bull (Tokyo). 1973;21(4):817-825. 18. Lucero MT, Squires A. Catecholamine concentrations in rat nasal mucus are modulated by trigeminal stimulation of the nasal cavity. Brain Res. 1998;807(1-2):234-236. 19. Thompson JP, Boyle JR, Thompson MM, Strupish J, Bell PR, Smith G. Cardiovascular and catecholamine responses during endovascular and conventional abdominal aortic aneurysm repair. Eur J Vasc Endovasc Surg. 1999;17(4): 326-333. 20. Girbes AR, Patten MT, McCloskey BV, Groeneveld AB, Hoogenberg K. The renal and neurohumoral effects of the addition of low-dose dopamine in septic critically ill patients. Intensive Care Med. 2000;26(11):1685-1689.
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21. Goldstein DS, Eisenhofer G, Kopin IJ. Sources and significance of plasma levels of catechols and their metabolites in humans. J Pharmacol Exp Ther. 2003;305(3):800-811. 22. Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother. 2001;48(suppl 1):5-16. 23. Freestone PP, Haigh RD, Lyte M. Catecholamine inotrope resuscitation of antibiotic-damaged staphylococci and its blockade by specific receptor antagonists. J Infect Dis. 2008; 197(7):1044-1052. 24. Adonizio A, Kong KF, Mathee K. Inhibition of quorum sensing-controlled virulence factor production in Pseudomonas aeruginosa by South Florida plant extracts. Antimicrob Agents Chemother. 2008;52(1):198-203. 25. O’Toole GA, Kolter R. Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol. 1998;30(2):295-304. 26. O’Toole GA, Kolter R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol. 1998;28(3):449-461. 27. O’Callaghan CL, Sikand K, Rutman A, Hirst RA. The effect of viscous loading on brain ependymal cilia. Neurosci Lett. 2008;439(1):56-60. 28. Hirst RA, Rutman A, Williams G, O’Callaghan C. Ciliated air-liquid cultures as an aid to diagnostic testing of primary ciliary dyskinesia. Chest. 2010;138(6):1441-1447. 29. Lamont IL, Beare PA, Ochsner U, Vasil AI, Vasil ML. Siderophore-mediated signaling regulates virulence factor production in Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 2002;99(10):7072-7077. 30. Martinsson A, Bevegård S, Hjemdahl P. Analysis of phenylephrine in plasma: initial data about the concentration-effect relationship. Eur J Clin Pharmacol. 1986;30(4):427-431.
31. Landry DW, Levin HR, Gallant EM, et al. Vasopressin deficiency contributes to the vasodilation of septic shock. Circulation. 1997;95(5):1122-1125. 32. Russell JA, Walley KR, Singer J, et al; VASST Investigators. Vasopressin versus norepinephrine infusion in patients with septic shock. N Engl J Med. 2008;358(9):877-887. 33. Tsuneyoshi I, Yamada H, Kakihana Y, Nakamura M, Nakano Y, Boyle WA III. Hemodynamic and metabolic effects of lowdose vasopressin infusions in vasodilatory septic shock. Crit Care Med. 2001;29(3):487-493. 34. Ratledge C, Dover LG. Iron metabolism in pathogenic bacteria. Annu Rev Microbiol. 2000;54:881-941. 35. Lamont IL, Konings AF, Reid DW. Iron acquisition by Pseudomonas aeruginosa in the lungs of patients with cystic fibrosis. Biometals. 2009;22(1):53-60. 36. Yang L, Nilsson M, Gjermansen M, Givskov M, TolkerNielsen T. Pyoverdine and PQS mediated subpopulation interactions involved in Pseudomonas aeruginosa biofilm formation. Mol Microbiol. 2009;74(6):1380-1392. 37. Moreau-Marquis S, Stanton BA, O’Toole GA. Pseudomonas aeruginosa biofilm formation in the cystic fibrosis airway. Pulm Pharmacol Ther. 2008;21(4):595-599. 38. Mesaros N, Nordmann P, Plésiat P, et al. Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium. Clin Microbiol Infect. 2007;13(6):560-578. 39. Juste RN, Moran L, Hooper J, Soni N. Dopamine clearance in critically ill patients. Intensive Care Med. 1998;24(11): 1217-1220. 40. Stannard W, O’Callaghan C. Management of croup. Paediatr Drugs. 2002;4(4):231-240. 41. Schmidt C, Kraft K. Beta-endorphin and catechomaline concentrations during chronic and acute stress in intensive care patients. Eur J Med Res. 1996;1(1):528-532.
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Original Research CRITICAL CARE
Pseudomonas aeruginosa-Catecholamine Inotrope Interactions A Contributory Factor in the Development of Ventilator-Associated Pneumonia? Primrose P. Freestone, PhD; Robert A. Hirst, PhD; Sara M. Sandrini, PhD; Fathima Sharaff, MSc; Helen Fry, MD; Stefan Hyman; and Chris O’Callaghan, DM, PhD
Background: Ventilated patients receiving intensive care are at significant risk of acquiring a ventilator-associated pneumonia that is associated with significant morbidity and mortality. Despite intensive research, it is still unclear why Pseudomonas aeruginosa, a microbe that rarely causes pneumonia outside of intensive care, is responsible for so many of these infections. Methods: We investigated whether medications frequently prescribed to patients in the ICU, the catecholamine inotropes, were affecting the growth and virulence of P aeruginosa. Effects of clinically attainable concentrations of inotropes on P aeruginosa pathogenicity were explored using in vitro growth and virulence assays and an ex vivo model of infection using ciliated human respiratory epithelium. Results: We found that inotropes were potent stimulators of P aeruginosa growth, producing up to 50-fold increases in bacterial numbers via a mechanism involving inotrope delivery of transferriniron, internalization of the inotrope, and upregulation of the key pseudomonal siderophore pyoverdine. Inotropes also markedly increased biofilm formation on endotracheal tubing and enhanced the biofilm production and toxicity of P aeruginosa in its interaction with respiratory epithelium. Importantly, catecholamine inotropes also facilitated the rapid recovery of P aeruginosa from tobramycin antibiotic challenge. We also tested out the effect of the inotropes vasopressin and phenylephrine on the growth and virulence of P aeruginosa and found that, in contrast to the catecholamines, these drugs had no stimulatory effect. Conclusions: Collectively, our results suggest that catecholamine inotrope-bacterial interactions may be an unexpected contributory factor to the development of P aeruginosa-ventilator-associated pneumonia. CHEST 2012; 142(5):1200–1210 Abbreviations: ALI 5 air-liquid interface; CFU 5 colony-forming unit; ET 5 endotracheal tube
who require ventilation while receiving Patients intensive care are susceptible to infection by opportunistic pathogens1-6 that rarely cause infections in healthy individuals. Up to 20% develop a severe
Manuscript received October 18, 2011; revision accepted April 1, 2012. Affiliations: From the Department of Infection, Immunity and Inflammation (Drs Freestone, Hirst, Sandrini, Fry, and O’Callaghan and Ms Sharaff), University of Leicester School of Medicine; the Division of Child Health (Drs Hirst and O’Callaghan); and the Electron Microscopy Laboratory (Mr Hyman), University of Leicester; and the Institute of Lung Health (Drs Hirst and O’Callaghan), Leicester, England. Funding/Support: Drs Freestone, Sandrini, and O’Callaghan are funded by the SPARKS Children’s Charity (www.sparks.org.uk), England [Grant 09 LCS 01].
ventilator-related pneumonia that is associated with significant mortality and prolonged stays in the ICU.5 Gram-negative bacilli are responsible for approximately 60% of ventilator-associated infections, with Pseudomonas aeruginosa responsible for up to onehalf of all cases.3 The reasons for the high rate of infection by these organisms remain unclear. Our Correspondence to: Chris O’Callaghan, DM, PhD, Department of Respiratory Medicine, Portex Unit, Institute of Child Health, University College London (UCL), 30 Guildford St, London, WC1N 1EH, England; e-mail: [email protected] © 2012 American College of Chest Physicians. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians. See online for more details. DOI: 10.1378/chest.11-2614
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laboratory and others have shown that catecholamine stress hormones and certain of their metabolites can stimulate the growth and infectivity of a range of infectious bacteria.7-13 This is of concern because catecholamines as inotropic drugs, particularly norepinephrine and dopamine, are prescribed for up to 50% of patients in the ICU,14 which led us to hypothesize that they may also increase the growth and virulence of bacteria such as P aeruginosa. Infused inotropes affect lung liquid clearance,15 and it is well known that the lungs are an active site of inotrope metabolism.16 Studies using injected 14C-labeled l-DOPA and l-dopamine showed that inotrope accumulation occurs in the lung within an hour of systemic administration.17 The respiratory tissues, via the endotracheal tube (ET), may also be used as a direct site for systemic administration of inotropes.15 Because dopamine and norepinephrine are present within respiratory mucus,18 it is likely that bacteria colonizing the respiratory tract and catecholamines will at some point occupy the same spatial location within the intensive care patient. Therefore, the aim of this study was to determine if exposure of P aeruginosa to catecholamine inotropes commonly prescribed to patients, at levels that may be encountered in patients during intensive care management, affected aspects of its virulence relevant to its ability to cause pneumonia. Materials and Methods Strains, Growth Induction, and Antibiotic Sensitivity Assays The P aeruginosa strains used in this study were a clinical isolate from a patient with ventilator-associated pneumonia (Public Health Laboratory, Leicester Royal Infirmary, England) and P aeruginosa reference strain PA14. The serum-SAPI medium used for growth assays was prepared as described previously.8,11 L-(-)-norepinephrine bitartrate, epinephrine bitartrate, and dopamine hydrochloride were obtained from Sigma. Vasopressin (20 units/mL, 600 units/mg) and phenylephrine (10 mg/mL) were obtained from the Department of Pharmacy, Leicester Royal Infirmary, University Hospitals of Leicester. Horseradish peroxidase conjugated transferrin (Cat. No. 009-030-050) was obtained from Jackson ImmunoResearch Laboratories. Tobramycin (80 mg/2 mL) was obtained from King Pharmaceuticals. 55FeCl3 (IES, specific activity 5 mCi/mg iron) and 3H-norepinephrine (TRK584,l-[7,8-3H] norepinephrine) were obtained from Amersham Life Sciences. P aeruginosa was inoculated at low cell density (50-100 colonyforming units [CFU]/mL) into serum-SAPI (a serum-supplemented minimal medium8,11) to more closely approximate in vivo conditions.7 Bacterial inoculum sizes were determined by pour-plate analysis, and cultures were incubated statically at 37°C in a 5% CO2 incubator. All analyses used inotrope concentrations within the range reported in the plasma of dopamine-medicated patients.19,20 Norepinephrine and epinephrine were used at 5 mM and dopamine at 5 mM plus 1 mM norepinephrine, to reflect the enzymatic conversions that occur in vivo.21 After incubation, bacterial numbers were enumerated using Luria agar.11-13
To assess the role of transferrin in the mechanism of inotrope growth induction, cultures were incubated for 1 h at 37°C in a 5% CO2 incubator with 0.1 mg/mL of horseradish peroxidase conjugated transferrin, harvested by centrifugation at 5,000 3 g for 5 min, and washed twice in phosphate-buffered saline. Washed cell suspensions were resuspended in phosphate-buffered saline, serially diluted in twofold steps, and 10-mL aliquots spotted onto nitrocellulose; horseradish peroxidase conjugated transferrin binding was visualized using enhanced chemiluminescence. To investigate inotrope effects on transferrin-iron uptake, and inotrope internalization, P aeruginosa cultures were inoculated into serum-SAPI supplemented with 1 3 105 counts per min of 55Fe-transferrin or 0.5 3 105 counts per min/mL 3H-norepinephrine, with and without the addition of norepinephrine. 55Fe-transferrin was prepared as described previously.12 Cultures were incubated as described for the growth assays and were assayed for cell numbers and radiolabel incorporation using scintillation counting.12 P aeruginosa intracellular iron levels were measured from total protein using inductively coupled plasma optical emission spectroscopy. P aeruginosa tobramycin sensitivity assays were performed in serum-SAPI using a minimum inhibitory concentration methodology.22 Exponential P aeruginosa cultures (about 105 CFU/mL) were added to serum-SAPI supplemented with increasing tobramycin concentrations in the presence/absence of norepinephrine or dopamine.23 Cultures were incubated for 24 h and enumerated for bacterial numbers as described previously. Production of the P aeruginosa siderophore pyoverdine was measured using a fluorimetric assay24 and quantified in terms of relative fluorescent units per normalized optical density/mL of bacterial culture. Analysis of Inotrope Effects on P aeruginosa Biofilm Formation Inotrope effects on P aeruginosa attachment, swimming, and twitching motility were measured as previously described.25,26 Briefly, twitching motility assays were performed in Luria broth medium solidified with 1.0% agar supplemented with various concentrations of catecholamines. Plates were dried overnight at room temperature, and P aeruginosa strains were point inoculated at the bottom of the Petri plate. After 3 days, the twitching distance along the plastic-agar interface (at the bottom of the agar plate) was measured. Initial attachment on polyvinylchloride was visualized by staining with the FilmTracer LIVE/DEAD biofilm viability kit (Invitrogen; Life Technologies Corp) using a Nikon Ti inverted fluorescence microscope (Nikon Corp) set at 480/500 nm emission and excitation maxima. Sterile polyvinylchloride ET sections were used for ET-biofilm analyses. To ensure similarity in growth rates between control and inotrope-treated cultures, P aeruginosa was added at 104 CFU/mL to serum-SAPI and incubated at 37°C in a 5% CO2 incubator for 48 h. ET sections were washed twice with phosphate-buffered saline, once with 0.1 M sodium phosphate buffer pH 7.2 to remove residual protein, and then processed for scanning electron microscopy as described previously,13 using a Hitachi S3000H scanning electron microscope (Hitachi Ltd). Inotrope effects on P aeruginosa interaction with respiratory cells were studied using a human-ciliated respiratory epithelium air-liquid interface (ALI) cell culture prepared from nasal biopsy cells.27,28 Ciliary beat frequency and ciliary beat amplitude measurements were analyzed as described previously21,22 (e-Appendix 1). To visualize host-pathogen interactions in real time, P aeruginosa at an inoculum of 106 CFU/mL was used to infect the ALI cultures; the inotrope alone was added to the uninfected control culture. Ciliary function, ultrastructure analyses of the bacterial-epithelial cell cultures, and bacterial growth levels were obtained at hourly intervals for 5 h.
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Statistics All experiments were performed in at least triplicate; unless stated otherwise, numerical data shown are expressed as mean ⫾ SD. Where appropriate, statistical analysis was first performed using one-way analysis of variance and, if significant, an unpaired t test. Statistical significance was indicated by a P value of ,. 05.
Results Inotropes Increase P aeruginosa Growth and Pyoverdine Synthesis Previous analyses of P aeruginosa-catecholamine interactions used norepinephrine levels of 50 mM and higher.7,8,11 However, in the current study, we found that incubation of an inoculum of , 102 CFU/mL of P aeruginosa strains clinical isolate and PA14 in serum-SAPI for 18 h with 5 mM concentrations of the inotropes norepinephrine and dopamine (drug levels that have been reported in the blood of patients medicated with inotrope19,20) resulted in up to 50-fold increases in bacterial numbers over unsupplemented control cultures (P , .01 for both strains) (Fig 1A). Dose response analyses using other inotrope concentrations (e-Fig 1) revealed that enhancement of P aeruginosa growth also occurred with lower concentrations of norepinephrine and dopamine (1 and 3 mM), although this was to a lower level than seen with 5 mM. Iron limitation by transferrin is primarily responsible for the innate bacteriostatic nature of serum and blood, so we investigated if the mechanism by which inotropes induced P aeruginosa growth involved the inotrope enabling the bacteria to access transferrincomplexed iron. Figure 1B shows that transferrin was directly bound by both P aeruginosa strains during growth. The addition of 55Fe-transferrin (Fig 1C) to culture media showed that the inotrope increased bacterial acquisition of transferrin-55Fe. Inclusion of 3H-norepinephrine also revealed that the inotrope was directly internalized by the P aeruginosa during the catecholamine-induced growth process (Fig 1C) (P , .001). Following longer incubation (48 h), the control and inotrope cultures reached a similar cell density (Fig 1D); inductively coupled plasma optical emission spectroscopy measurement of the intracellular iron content of the cultures confirmed that the inotropes were increasing internal iron levels of the bacteria. The photo insets in Figure 1D show that inotropesupplemented cultures were greener in color and displayed more biofilm than did control subjects. Pyoverdine is a green ferric-iron-sequestering siderophore often seen in P aeruginosa cultures.24,29 Fluorimetric assays of the P aeruginosa culture supernatants (Fig 1E) confirmed the presence of pyoverdine and showed (n 5 4) that the inotropes induced both strains to synthesize significantly more siderophore than did control subjects (P , .001).
Inotropes Enhance Recovery of Tobramycin-Treated P aeruginosa Figure 2 shows that during incubation with increasing concentrations of the antipseudomonal antibiotic tobramycin, the P aeruginosa strains clinical isolate (Fig 2A ) and PA14 (Fig 2B) underwent a dosedependent reduction in bacterial numbers. However, viable counts indicated that compared with control subjects, the numbers of P aeruginosa in the presence of concentrations of tobramycin near the minimum inhibitory concentration (approximately 0.1 mg/mL for strain clinical isolate and 1.0 mg/mL for PA14) increased up to 100-fold when an inotrope was present (P , .0001). Inotropes Enhance P aeruginosa Biofilm Formation on ETs The ET is an area with a recognized of risk of development of Pseudomonas-associated pneumonia.1-6 Attachment is the first stage in Pseudomonas biofilm formation, and Figure 3A reveals that for both strains, clinically attainable concentrations of the catecholamines significantly enhanced attachment (P . .01) to polyvinylchloride (an ET polymer). The epifluorescence micrographs in Figure 3A show that the inotropes also enhanced P aeruginosa cell-cell attachment and microcolony formation (n 5 3). As well as production of factors for attachment, motility within a Pseudomonas biofilm is important for its establishment.25 Swimming motility (Fig 3B) with the exception of norepinephrine was not significantly affected by the addition of 5 mM inotrope concentrations, although higher drug levels (50 mM) were stimulatory (data not shown). Twitching motility was enhanced by the inotropes, although only for strain clinical isolate (Fig 3C), because PA14 had generally low motility. Figure 3D shows scanning electron microscopy images of P aeruginosa clinical isolate directly seeded onto ET sections and incubated in the presence of norepinephrine, dopamine, and epinephrine. Relative to control cultures, inotrope exposure consistently induced marked increases in biofilm formation on the ET (n 5 4). A very similar inotropeinduced enhancement of ET biofilm production was also obtained with P aeruginosa strain PA14 (data not shown). Figure 3D shows the biofilm that remains attached to the ET after scanning electron microscopy processing. A fuller picture of the architecture of inotropeinduced P aeruginosa biofilms is revealed in Figure 4, which shows fragments of norepinephrine-induced biofilms that have become detached from the ET. Figure 4A shows a section of biofilm before scanning electron microscopy processing, viewed using a light microscope. The characteristic mushroom structures
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Figure 1. Inotropes increase Pseudomonas aeruginosa growth and pyoverdine production. A, NE and dopamine effects on growth of P aeruginosa strains CI and PA14 after 18-h incubation (n 5 4). B, Transferrin binding by the CI and PA14 P aeruginosa strains occurs during growth; the upper two panels show transferrin binding blots of a twofold dilution of the two cultures (initial cell density around 2 3 108 CFU/mL); the control consists of similar numbers of bacteria to the 1:2 dilution, but incubated without H-Tf; the activity of 1.0 and 0.1 mg of H-Tf is shown in the lower panel. C, After normalizing cell densities between control and inotrope-treated cultures, representative bacterial internalizations of iron (n 5 4) (in the form 55Fe from 55Fe-transferrin) (right panel) are increased in the presence of the inotrope; tritiated norepinephrine internalization is shown in the left panel. D, P aeruginosa CI and PA14 growth levels after 48-h incubation in serum-SAPI medium; the intracellular iron levels of control, NE, and dopamine-treated cultures (n 5 4) are shown for each culture; SDs for each set of data are shown in brackets. Representative images of the cultures are shown in the inserts. E, Typical measurements (n 5 5) of the pyoverdine levels of the 48-h CI and PA14 cultures. The initial inoculum for strains CI and PA14 in experiments A and C to E were 78, 99, 88, and 93 CFU/mL, respectively. *Statistical significance (P , .01). CFU 5 colony-forming units; CI 5 clinical isolate; H-Tf 5 horseradish peroxidase transferrin; NE 5 norepinephrine; NS 5 not significant.
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Figure 2. Inotropes enhance P aeruginosa recovery from antibiotic challenge. The data show how inotropes assist P aeruginosa in resisting a tobramycin challenge. Incubation of P aeruginosa strains CI and PA14 with tobramycin for 24 h decreased viable bacterial counts at the higher tobramycin concentrations (control bar [clear]). The presence of either norepinephrine (black) or dopamine (gray) during this time resulted in a marked increase in viable bacteria at 24 h. Data shown are the mean⫾ SD of three separate analyses of triplicate assays. A, CI. B, PA14. *Statistical significance (P , .01). See Figure 1 legend for expansion of abbreviations.
indicative of a mature biofilm are strikingly visible. Figure 4B shows more-detailed scanning electron microscopy images of a similar P aeruginosa biofilm fragment. The underside differs markedly from the upper side and is flatter and more uniform in appearance. In contrast, the upper side contains the mushroom-like projections shown in Figure 4A. The full fragment image reveals that the thickness of the biofilm is variable; however, the side edge view gives an indication of how dense the biofilm may have been prior to the scanning electron microscopy processing. Effect of Inotropes on Interaction of P aeruginosa With Respiratory Epithelium We used an ex vivo ALI model of cultured human ciliated epithelium27,28 to investigate the inotrope effects on the interaction of P aeruginosa with human respiratory epithelia. Additional supporting data for Figure 5 can be found in Videos 1-6, which show the P aeruginosa-ALI infection time course. Figures 5B and 5C show that throughout the 5-h study, the uninfected control maintained a normal ciliary beat frequency and beat pattern, indicating that the inotrope had not affected ciliary function (P . .05). P aeruginosa attachment to host cells was evident within 1 h of infection in both inotrope and noninotrope bacterial
cultures. Inotrope-mediated biofilm enhancement was evident by 1 h and was clearly present by 2 h, becoming progressively more extensive over time. Figures 5A and 5D show that by 5 h, the inotrope had induced the formation of extensive sheets of bacteria and exopolysaccharide covering most of the epithelial cell surface (P 5 .02). Figures 5B and 5C show that, although the cilia managed to continue beating under the burden of the biofilm with an unchanged frequency, the viscous drag of bacteria and exopolysaccharide attached to the cilia tips significantly reduced the amplitude of the cilia tip distance traveled (P , .01).27 By the end of the 5-h time course, signs of cytotoxicity in the epithelial cells exposed to the inotrope-treated P aeruginosa were evident, with reductions occurring in cilia function such as beat frequency, and extrusion of cells from the epithelial culture surface (data not shown). The non-inotrope-treated P aeruginosa culture also showed production of biofilm during the course of the incubation (P 5 .03) (Figs 5A, 5D) but this was always significantly less extensive than the inotrope-treated bacteria (P 5 .02) and, by 5 h, considerable areas of the noninotrope-treated epithelial cell cultures were still free of biofilm. Very similar results to those shown for strain clinical isolate were also obtained for P aeruginosa strain PA14 (data not shown). P aeruginosa Interaction With Noncatecholamine Inotropes We additionally tested whether the noncatecholamine inotropic agents phenylephrine (neosynephrine) and vasopressin had any stimulatory effects on our P aeruginosa strains. Figure 6 examines the response of P aeruginosa to a wide range of vasopressin and phenylephrine concentrations, from therapeutic serum levels to at least two log orders above.30-33 It is clear that neither drug had any effect on growth (Figs 6A, 6B) or initiation of biofilm formation (Figs 6C, 6D) at any of the concentrations tested. Other aspects of P aeruginosa virulence, such as pyoverdine production, or swimming and twitching motility, were similarly unaffected by the levels of phenylephrine and vasopressin shown in Figure 6 (data not shown). Discussion Ventilator-associated pneumonia, a hospital-acquired infection, remains a major cause of morbidity and death, despite intensive research.1-6 P aeruginosa, a pathogen that rarely causes pneumonia outside of intensive care, is responsible for a high proportion of these infections.1-6 In this study, we presented evidence that levels of catecholamine inotropes that are within the ranges reported in the plasma of
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Figure 3. P aeruginosa biofilm formation on endotracheal tubing (ET) is enhanced in the presence of catecholamine inotropes. A, Results of a crystal violet attachment assay demonstrating that, relative to control subjects, 5 mM concentrations of the inotropes significantly increased P aeruginosa attachment to polyvinylchloride. **P . .001. The panels underneath the histograms show that the inotropes are also enhancing other aspects of pseudomonal biofilm formation, including exopolysaccharide production and cell-cell attachment. B, Only NE has an effect on P aeruginosa swimming motility. *P . .01; **P . .001. C, Twitching motility of clinical strain CI is enhanced by the inotropes; the twitching motility values of the CI image are shown in the accompanying histogram; in contrast, the reference strain PA14 is largely nonmotile. **P . .001. D, Scanning electron micrographs of inotrope enhancement of P aeruginosa attachment and biofilm formation on ET. P aeruginosa strain CI was inoculated at 8 3 105 CFU/mL onto sterile ET sections in the absence or presence of NE (5 mM), Epi (5 mM), and Dop (5 mM) in serum-SAPI medium for 48 h (n 5 3). The four sets of panels show representative scanning electron micrographs of the ET biofilms at increasing magnification; the scales for each image are shown in the bottom right corner of the micrograph. Note that the growth levels of the control and inotrope-supplemented cultures were not significantly different (P 5 .16) (data not shown), indicating that the effects seen are not due to growth differences and that the inotropes are directly enhancing biofilm-responsive genes. Dop 5 dopamine; Epi 5 epinephrine. See Figure 1 legend for expansion of other abbreviations. journal.publications.chestnet.org
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Figure 4. Architecture of inotrope-induced P aeruginosa biofilms. A, A 48-hour-old NE-induced biofilm fragment (from P aeruginosa strain clinical isolate) that has been detached from an endotracheal tube section; the image shown was obtained using a 3 40 light microscope objective. B, Scanning electron micrographs of the upper, lower, and side architectural views of a similarly prepared biofilm fragment. The scales shown are for the bars on the scanning electron micrographs. Similar types of biofilm structure were also seen with Dop and Epi-treated P aeruginosa (data not shown). See Figure 1 and 3 legends for expansion of abbreviations.
inotrope-supplemented patients19,20 can greatly increase aspects of P aeruginosa virulence relevant to its capacity to cause ventilator-associated pneumonia. We also analyzed the effects of the noncatecholamine inotropes vasopressin and phenylephrine and found that, in contrast to the catecholamines, neither compound at any of the concentrations tested was stimulatory to the growth and virulence of our P aeruginosa strains. All infectious bacteria require iron for growth in vivo, and iron limitation of host fluids by proteins such as transferrin is a key primary host defense. We found that the mechanism by which inotropes increased P aeruginosa growth involved the inotrope enabling the bacteria to access the iron within transferrin coupled with the direct internalization of the catecholamine. Mechanistically, by binding the transferrin, the closer proximity probably allows more efficient iron removal by P aeruginosa siderophores and better internalization of the sequestered iron. Further, despite inotropes increasing the intracellular iron levels of P aeruginosa, which might be expected to inhibit expression of iron-regulated genes,34 upregulation of synthesis of pyoverdine, a key pseudomonal siderophore that is normally under tight iron repression, was observed. Pyoverdine can remove iron from transferrin, which has been shown to be important in P aeruginosa growth in lung secretions.35 In addition to its role in scavenging host iron, pyoverdine is unusual in having signaling functions and influencing P aeruginosa host colonization, biofilm formation,36 and virulence factor synthesis.29 This suggests that inotrope exposure could be even more wide reaching in its effects on promoting the ability of P aeruginosa to cause infection. Resistance to antibiotics is an increasing problem in the control of hospital-acquired infections,6,23,37
making of particular interest our discovery that inotropes can catalyze the recovery of tobramycin-treated P aeruginosa. As well as being associated with increased resistance to antibiotics and immune attack, biofilm formation by P aeruginosa is an aspect of its virulence vitally important in its capacity to colonize the ET, lung, and other tissues.1-6,37,38 We showed that those inotropes most frequently administered to ventilated patients (norepinephrine, epinephrine, and dopamine) all markedly increased P aeruginosa biofilm formation on ETs. This is an important discovery because bacterial colonization of the ET is a factor thought to predispose ventilated patients to development of pneumonia.1-6,39 We additionally used an ex vivo human model of respiratory infection to show that inotropes also enhanced the ability of P aeruginosa to directly colonize ciliated respiratory epithelia via increased host cell attachment and biofilm formation. Note that this is the first time that a human model of infection has been used in the examination of catecholaminebacteria interactions. Previous in vitro bacteria-catecholamine studies, including those employing animal infection models, have routinely used catecholamine concentrations of 50 to 2,000 mM (these studies are reviewed in Freestone et al7). An important finding of this report is that, to the best of our knowledge, we are the first to demonstrate the stimulatory effects of inotropes on P aeruginosa pathogenicity using much lower clinically attainable drug concentrations. In its usage as the most widely employed inotrope, dopamine is typically infused at 1 to 15 mg/kg/min, and steadystate levels in plasma vary according to infusion levels and general metabolic fitness, with acutely ill patients showing slower elimination rates.39 Girbes et al20
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Figure 5. Catecholamine inotropes enhance P aeruginosa biofilm formation on human airway epithelial cells. Inotrope NE influences P aeruginosa biofilm formation on human ciliated respiratory epithelia. PA was inoculated at 2 3 106 CFU/mL onto a differentiated airway epithelium as described in the “Materials and Methods” section; NE was used at 5 mM. A, Time course of P aeruginosa biofilm coverage; 䊊 5 uninfected control; ⵧ 5 P aeruginosa: no inotrope; 䊏 5 P aeruginosa + inotrope. B, Tip distance traveled of free cilia and of cilia covered by the P aeruginosa biofilm. 䊊 5 uninfected control; ⵧ 5 P aeruginosa with no inotrope: cilia beat amplitude outside the biofilm; 䊏 5 P aeruginosa with no inotrope: cilia beat amplitude inside the biofilm. 䉭 5 P aeruginosa 1 inotrope: cilia beat amplitude outside the biofilm; 䉱 5 P aeruginosa 1 inotrope: cilia beat amplitude inside the biofilm. C, Analysis of the cilia beat frequency (CBF) of free cilia, and cilia covered by biofilm. 䊊 5 CBF of uninfected control. ⵧ 5 P aeruginosa no inotrope: CBF outside the biofilm; 䊏 5 P aeruginosa no inotrope: CBF inside the biofilm; 䉭 5 P aeruginosa 1 inotrope: CBF outside the biofilm; 䉱 5 P aeruginosa 1 inotrope: CBF inside the biofilm. *Significance of P . .01; **P . .001. D, Representative light microscopy photographs (n 5 3) of the appearance of noninfected respiratory epithelia (control), and respiratory epithelia infected with only PA or PA plus NE (5 mM). The arrows are indicative of the presence of P aeruginosa biofilm. PA 5 P aeruginosa strain clinical isolate. See Figure 1 legend for expansion of other abbreviations.
showed that in septic shock patients medicated with 6 mg/kg/min dopamine, catecholamine plasma levels in some patients rose to . 5 mM, the concentration used in our assays. Within the circulation, dopamine
undergoes enzymatic conversion to norepinephrine and epinephrine,21 which has relevance to the current study, because Thompson et al19 showed that in some cardiac surgery patients treated with 3 mg/kg/min
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Figure 6. Effects of the noncatecholamine inotropes vasopressin and phenylephrine on P aeruginosa growth and virulence. A, Effects of a range of vasopressin concentrations on the growth of P aeruginosa strains CI and PA14 after 18-h incubation (n 5 4). B, Result of a crystal violet attachment assay demonstrating that none of the vasopressin concentrations tested had any effect on attachment of either P aeruginosa strain (n 5 4). C, Similarly, none of the phenylephrine concentrations tested increased the growth of P aeruginosa. D, Phenylephrine also had no effect on the surface attachment of either strain (n 5 4). See Figure 1 legend for expansion of abbreviations.
dopamine, norepinephrine plasma levels rose to 9.24 mM. The lungs are pharmacologically active and are responsible for extraction of around 20% of infused inotropes, increasing to 33% in the critically ill.31,32 Lung inotrope uptake systems are so effective that they have been proposed to be alternate sites
for inotrope administration. Endotracheal administration of epinephrine has been recommended if IV access is contraindicated, and up to 2 mg of epinephrine may be applied directly via the ET.15 Epinephrine (as a 300-mM solution) is also occasionally nebulized directly through the ET to reduce airway
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inflammation40; our study showed that epinephrine doses of a fraction of this value increased P aeruginosa colonization of the ET. It is probable that within the ventilated patient, the outer surface of the ET is in direct contact with airway secretions, which is significant because mucus itself contains catecholamines.18 In addition to being given inotropes, ventilated patients are both chronically and acutely stressed, and significant endogenous increases in plasma norepinephrine and epinephrine have been associated with procedures such as ET suctioning.41 Thus, there is considerable opportunity for bacteria colonizing the ET to come into direct contact with both endogenous and administered catecholamines. Conclusions In conclusion, this study suggests that administration of inotropes to patients in the ICU, particularly if high doses are given systemically or via direct local application, may be an unappreciated risk factor in the development of ventilator-associated pneumonia by P aeruginosa. Further investigations are therefore needed to determine whether the higher circulating catecholamines of the ventilated patient,19,20 combined with the presence of administered inotropes and naturally occurring catecholamines in mucosal secretions, are acting to promote the growth and virulence of a bacterium that rarely cause pneumonia outside of intensive care. Acknowledgments Author contributions: Dr O’Callaghan is the guarantor of the manuscript and takes responsibility for the integrity of the data and accuracy of the data analysis. Dr Freestone: conceived and designed the study, the carrying out of many of the experiments, analysis of the data, and writing and revision of the manuscript. Dr Hirst: contributed to parts of the study design, carrying out of some of the experiments, analysis of the data, and writing of some of the manuscript. Dr Sandrini: contributed to the carrying out of some of the experiments, analysis of the data, and writing of some of the manuscript. Ms Sharaff: contributed to the carrying out of some of the experiments, analysis of the data, and writing of some of the manuscript. Dr Fry: contributed to the carrying out of some of the experiments, analysis of the data, and writing of some of the manuscript. Mr Hyman: contributed to the carrying out of some of the experiments, analysis of the data, and writing of some of the manuscript. Dr O’Callaghan: conceived and designed the study and contributed to the writing and revision of the manuscript. Role of sponsors: The sponsor had no role in the design of the study, the collection and analysis of the data, or in the preparation of the manuscript. Additional information: The e-Appendix, e-Figure, and Videos can be found in the “Supplemental Materials” area of the online article.
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