Effect of Ambroxol on Pneumonia Caused by ...

Experimental Chemotherapy Received: March 23, 2010 Accepted after revision: August 11, 2010 Published online: April 1, 2011

Chemotherapy 2011;57:173–180 DOI: 10.1159/000323622

Effect of Ambroxol on Pneumonia Caused by Pseudomonas aeruginosa with Biofilm Formation in an Endotracheal Intubation Rat Model Fang Li a Wenlei Wang b Linyan Hu a Luquan Li a Jialin Yu a

Departments of a Neonatology and b PICU, Children’s Hospital of Chongqing Medical University, Key Laboratory of Developmental Diseases in Childhood, Chongqing Medical University, Ministry of Education, Chongqing, China

Key Words Ambroxol ⴢ Pneumonia ⴢ Pseudomonas aeruginosa ⴢ Biofilm ⴢ Endotracheal intubation

Abstract Background: Pseudomonas aeruginosa, especially the mucoid phenotype, is responsible for most of the morbidity and mortality in ventilator-associated pneumonia. Although ambroxol is widely used in neonatal lung problems as a mucolytic as well as an antioxidant agent, its anti-infective role is not well demonstrated by studies in vivo. Objective: To explore the effect of ambroxol on the biofilms of mucoid P. aeruginosa and on the associated lung infection using a rat model. Methods: We developed a rat model of acute lung infection by endotracheal intubation with a tube covered with mucoid P. aeruginosa biofilm. Then, we studied the effect of ambroxol on the biofilm using saline treatment as a control. Subsequently, we studied the microstructure of the biofilm, bacterial count in the tubes and lungs, pathological changes that occurred in the lungs, and the cytokine response. Results: Alteration of the microstructure of the biofilm with ambroxol treatment was demonstrated by scanning electron microscopy. The bacterial counts on the biofilm-covered tube in the ambroxol-treated group were significantly lower than those in the saline-treated group on both post-bacterial challenge days 4 and 7 (p ! 0.05). The

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bacterial counts in lungs of the ambroxol-treated group and of the saline-treated group on post-bacterial challenge day 7 were not significantly different (p 1 0.05). The pathological changes in lungs were milder with the effect of ambroxol. The cytokine responses, namely the level of IFN- ␥ and the ratio of IFN- ␥ and IL-10, were also reduced with the effect of ambroxol. Conclusion: We demonstrated that the ambroxol treatment could destroy the structure of the biofilm on the tube used for intubation and decrease the bacterial load. Further, the reduced cytokine response and milder pathological changes in lungs in an endotracheal intubation rat model indicate that ambroxol can attenuate the damage caused by biofilm-associated infection in the lung. Copyright © 2011 S. Karger AG, Basel

Introduction

Ventilator-associated pneumonia (VAP) is the second most common health care-associated infection among pediatric and neonatal intensive care unit patients [1, 2]. Recent literature suggests that pediatric VAP is associated with increased morbidity, antibiotic use, cost, and length of hospital stay among the patients admitted to a pediatric intensive care unit and neonatal intensive care unit (NICU) [3]. One of the most commonly isolated organisms in VAP patients is Pseudomonas aeruginosa [4, Jialin Yu 136, Zhongshan Road, Yuzhong District Chongqing 400014 (China) Tel. +86 023 6363 5567 E-Mail rematalili @ yahoo.com.cn

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Chemotherapy 2011;57:173–180

Materials and Methods Bacterial Strains and Their Culture A mucoid strain of P. aeruginosa was isolated from the catheter of a mechanically ventilated newborn at the Children’s Hospital of Chongqing Medical University. This bacterial strain was precultivated in 20% Luria broth (LB), diluted 1:5, and grown at a neutral pH at 37 ° C overnight. The bacteria were then suspended in saline, harvested by centrifugation (3,000 g, 4 ° C, 10 min), resuspended in sterile saline, and adjusted to 109 colony-forming units (CFU)/ml, as estimated by turbidimetry. Stock cultures were maintained at –70 ° C in 30% glycerol.

Preparation of Endotracheal Tubes Precoated with Bacteria The tubes for endotracheal intubation, disposable sterile plastic scalp acupuncture tubes of 3.0 mm in diameter, were cut to 1 cm in length, and they were immersed in the bacterial suspensions for 7 days at 37 ° C. The biofilm was formed on the inner surface of these inoculation tubes. To estimate the bacterial count on these biofilms, the bacteria were detached from the tubes by using a concussion machine (Shanghai Facility Factory; UR 513) for 5 min. Only viable bacteria were counted. The number of bacteria at 7 days after incubation and before endotracheal intubation was 6.32 8 0.57 log10 CFU/tube (mean and standard deviation; n = 10).

Animals Used for the Study Eighty 8-week-old specific pathogen-free female SpragueDawley rats, weighing 300–350 g, were purchased from Chongqing Medical University Laboratory Animals Center (Chongqing, China). All rats were housed in a pathogen-free environment and received sterile food and water in the Laboratory Animal Center at the Children’s Hospital of Chongqing Medical University. The experimental protocol was approved by the Animal Care and Use Committee, Chongqing Medical University. Rat Infection Model and Drug Administration Eighty-eight Sprague-Dawley rats were randomly allocated to 4 groups: 22 rats with biofilm-covered tube intubation were given ambroxol (because we add the data of macroscopic description of the lungs in each group at two time points, respectively); 22 rats with biofilm-covered tube intubation were given saline as control; another 22 rats with sterile tube intubation were given saline as control, and the remaining 22 rats, without any intervention, were used as normal controls. Infection was induced in rats by using the method described previously by Yanagihara et al. [14], with modifications. Briefly, rats were weighed and anesthetized with a subcutaneous injection of 0.3 ml/g of chloral hydrate before challenge. The tube was intratracheally placed with tracheotomy and fixed within the trachea. After intubation, rats were allowed to recover from anesthesia, eat, and drink spontaneously. Ambroxol was purchased from Boehringer Ingelheim as an intravenous solution (7.5 mg/ml). On the 2nd day of intervention, ambroxol solution (3 ml/kg) or saline (3 ml/kg) was injected intravenously once a day. Ten rats in each group were sacrificed on the 4th and 7th day, respectively, by injecting 2 ml of 20% pentobarbital kg–1. Specimen Collection and Examination Using Scanning Electron Microscopy The biofilm on the inner surface of the inoculation tube was formed as mentioned before. Then, the tubes of each group were

Li /Wang /Hu /Li /Yu


5]. P. aeruginosa, a Gram-negative opportunistic human pathogen, is an important causative agent of a variety of acute and chronic infections, including burn wound infections, as well as infections of the urinary tract, eye, ear, and respiratory tract [6]. P. aeruginosa is well adapted to the hospital environment because of its nutritional versatility, minimal growth requirements, and extensive acquisition of antibiotic resistance [7] and therefore causes nosocomial infection on indwelling devices, such as catheters used in mechanical ventilation and in the urinary tract. The indwelling materials enable the formation of biofilm. Biofilm consists of cells and their secreted insoluble extracellular polymers, which are largely alginate in mucoid isolates. They are recalcitrant to chemical biocides and to antibiotics. The development of biofilm infections on indwelling devices may lead to prolonged hospitalization, device malfunction, or even mortality. The principal strategy for managing biofilm infections relies on antibiotics to kill the bacteria embedded in the biofilm. However, the organisms on biofilm cultures are found to be much more difficult to eradicate than those on suspended cultures [8]. The former needs a much higher minimal inhibitory concentration of antibiotics, and it is difficult to reach that level in the circulation. One alternative approach to control biofilm formation could be inhibition of the production of the biofilm matrix material. In recent years, chemical agents, besides antibiotics, possessing potent antimicrobial properties have attracted considerable attention [9]. Ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl] benzylamine), a mucolytic agent, seems to have additional antioxidant and anti-inflammatory properties in the treatment of pulmonary infectious diseases characterized by impaired mucociliary clearance [10]. Furthermore, ambroxol is known to stimulate the formation and release of surfactant by type II pneumocytes, and it protects cellular lipids from oxidative stress related to endotoxemia or inflammatory responses [11]. Recently, it was observed that ambroxol affects the structure of biofilm formed by wild-type P. aeruginosa (PAO1) and facilitates the permeability of ciprofloxacin through P. aeruginosa biofilm and the contents of exopolysaccharide [12]. We previously found that ambroxol can reduce the production of alginate in mucoid P. aeruginosa biofilm [13]. However, the effects of ambroxol on P. aeruginosa lung infections are still unknown. In the present study, we aimed to examine the effect of ambroxol on acute lung infections caused by P. aeruginosa with biofilm formation in an endotracheal intubation rat model.

removed from the culture. These specimens were fixed for 2 h at 4 ° C with 2% glutaraldehyde in 0.1 M phosphate buffer, followed by refixation for 2 h at 4 ° C in 1% osmium acid in the same buffer, dehydration in a series of aqueous ethanol solutions (50–100%), and freeze-drying. The specimens were coated with platinumpalladium by using an ion sputter and observed by using a JSM35C scanning electron microscope (JEOL, Tokyo, Japan).

Bacteriology of the Endotracheal Tube Randomly selected rats in each of the intubated groups were prepared for quantitative bacteriology. The intubation tubes were taken out aseptically and cut open longitudinally. One section was agitated in 1 ml of aseptic saline for 3 h with an ultrasonotor (1 MHz), and 100 ␮l of appropriately serial diluted bacterial fluid was plated on an LB plate, incubated at 37 ° C, and inspected for colonies of P. aeruginosa overnight.

Macroscopic Description of the Lungs and Histopathological Changes in the Lungs After treatment, 11 animals in each group were sacrificed on the 4th and 7th day, respectively. The lungs were excised under aseptic conditions. One lung was dried naturally for macroscopic observation. Macroscopic lung pathology was expressed as the lung index of macroscopic pathology (LIMP), which was calculated by dividing the area of the left lung showing pathological changes by the total area of the whole lung. In addition, the gross pathological changes in the lungs were also assigned the following scores according to the severity of the inflammation: I = normal lungs; II = swollen lungs, hyperemia, and small atelectasis (!10 mm2); III = pleural adhesions and atelectasis (!40 mm2), and IV = abscesses, large atelectasis, and hemorrhages. As for the other 10 lungs, most parts of lungs were homogenized and prepared for bacterial count and determination of cytokines. Bacterial enumeration in the lungs was performed by serially diluting samples on LB agar plates, incubating the plates overnight at 37 ° C in air, and counting colonies on the plates to estimate the number of CFUs. The remaining parts of lungs from all rats in each group were fixed in 10% formalin buffer and subjected to histopathological examination. Lung histological examination was performed as described previously [15]. Cellular alterations in the lungs were classified as acute or chronic inflammation by a scoring system based on the proportion of polymorphonuclear neutrophils (PMNs) and mononuclear leukocytes (MNs) in the inflammatory foci [8]. Acute inflammation was defined as cellular infiltration in which PMNs were predominant (90% PMNs and 10% MNs), and chronic inflammation was considered when MNs were predominant (10% PMNs and 90% MNs), which included lymphocytes and plasma cells and the presence of granulomas. Both macroscopic and histopathological evaluations were performed as a double-blind study to avoid bias.

Statistical Analysis Data were expressed as the mean and standard deviation. Differences between groups were examined for statistical significance by using the unpaired Student t test. p ! 0.05 denoted the presence of a statistically significant difference. Statistical analysis was performed by using the SPSS software, version 11.5.

Results

Examination of Intubated Tube Using a Scanning Electron Microscope We examined the microstructure of biofilm on the inner wall of the tube in situ with a scanning electron microscope. In the saline-treated group, the inner wall of the tube was covered with a relatively smooth, integrated, thick layer with many granular particles on its surface on day 4 (fig. 1a). The layer became thicker with mucosal mass and mixed inflammatory cells on the 7th postoperative day (fig. 1d). On the other hand, the biofilm in rats treated with ambroxol formed irregularly cracked skin-like pieces with a rough surface on day 4 (fig. 1b). After treatment with ambroxol for 7 days, the biofilm on the tube became thinner with a cracked tilelike pattern with few inflammatory cells (fig. 1e). On the tubes without biofilm, there were few bacterial colonies of mixed nature adhering to the inner surface of the tube on the 4th postoperative day (fig. 1c). The number of bacteria was found to be increased on the 7th postoperative day (fig. 1f).

Cytokines and Anti-Alginate IgG Determination Two milliliters of fresh blood was obtained, and serum was extracted by centrifugation (13,200 g for 7 min). Interferon-␥ (IFN-␥) and interleukin-10 (IL-10) in the serum were determined using enzyme-linked immunosorbent assay kits (Nordic BioSite). Standard curves were constructed for IFN-␥ (range: 31.25–2,000 pg/ml; sensitivity: !10 pg/ml) and IL-10 (range: 31.25–2,000 pg/ ml; sensitivity: ! 20 pg/ml).

Effect of Ambroxol on Pneumonia

Bacterial Counts of the Tube and Lungs Bacterial counts of the tube and lung were determined by the plate-dilution method. The bacterial counts on the biofilm-covered tube in the ambroxol-treated group were significantly lower than those in the saline-treated group on both postbacterial challenge days 4 and 7 (p ! 0.05). The bacterial counts on the tube in the saline control group were significantly lower (p ! 0.05) than on the biofilm-covered tube in the ambroxol-treated group on both days 4 and 7 (fig. 2a). The bacterial counts in lungs of the ambroxol-treated group were higher than those in the saline-treated group on postbacterial challenge day 4, but they were not significantly different on day 7 (p 1 0.05). The bacterial counts in the lungs of the saline control group were significantly lower (p ! 0.05) than in the lungs of the ambroxol-treated group with a biofilm-covered tube on both days 4 and 7 (fig. 2b).


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Fig. 2. a Bacterial count on the inner surface of the intubated tube on days 4 and 7 after operation. b Bacterial count in lung tissue. Differences were statistically significant compared with the bio-

film saline-treated group (* p ! 0.05). Differences were not statistically significant compared with the biofilm saline-treated group (+ p 1 0.05). BF = Biofilm.

Macroscopic Description and Histopathological Changes in the Lungs Lungs in both groups with biofilm-covered tubes mainly showed signs of abscess and emphysema (fig. 3); in the biofilm saline-treated group it was mainly a large area of lung consolidation, lung adhesion, or abscess, whereas in the biofilm ambroxol-treated group, it was dominated

by lung atelectasis; the area of abscess is smaller than in the biofilm saline-treated group. As for LIMP score (fig. 4), the level in the biofilm ambroxol-treated group is slightly lower than that in the biofilm saline-treated group on day 4 after the bacterial challenge, and on day 7, the level in the biofilm ambroxol-treated group is much lower compared with that in the biofilm saline-treated group.

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Saline control BF ambroxol-treated BF saline-treated Bacterial count in lungs (log CFU)

Counts of bacterial colony in BF on the tube (log CFU)

Fig. 1. Scanning electron microscopy of the inner surface of the tube: on the 4th day of administration of saline (a) and ambroxol (b) and on the 7th day of administration of saline (d) and ambroxol (e). Scanning electron microscopy of the control group without biofilm formation on the inner surface of the tube on the 4th day (c) and 7th day (f).

Fig. 3. Macroscopic changes in the lung of

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rats. On day 4 of infection with biofilmcovered tube intubation: ambroxol-treated (a) and saline-treated (b), and with sterile tube intubation (c). On day 7 of infection with biofilm-covered tube intubation: ambroxol-treated (d) and saline-treated (e), and with sterile tube intubation (f).

ambroxol treatment (fig. 5e). The inflammatory responses in the lung of rats on days 4 and 7 were milder in the group with intubation of a sterile tube rather than a biofilm-covered tube (fig. 5c, f).

Saline control BF ambroxol-treated BF saline-treated 0.30

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Fig. 4. Lung LIMP score. Differences were statistically significant

compared with the biofilm saline-treated group (* p ! 0.05). Differences were not statistically significant compared with the biofilm saline-treated group (+ p 1 0.05). BF = Biofilm.

Cytokine Response The response of IL-10 and IFN-␥ after infection was determined by ELISA. There was no significant difference (p 1 0.05) in IL-10 levels between the biofilm-covered groups on day 4 and day 7 (fig. 6a) after infection. However, the level of IFN-␥ of the biofilm-covered group with ambroxol treatment was significantly lower (p ! 0.05) than that of the biofilm-covered group with saline treatment at both time points (fig. 6b). The ratio of IFN-␥ and IL-10 was significantly (p ! 0.05) different between the biofilm-covered groups on day 4 and day 7 (fig. 6c) after infection.

On day 4 after the bacterial challenge, significant inflammation was found in the lung tissues with signs of neutrophil infiltration in the lungs of rats intubated with biofilm-covered tubes. The changes in the lungs of rats with saline treatment (fig. 5b) were not different from those treated with ambroxol (fig. 5a). On day 7 after the infection, the histopathological changes in both groups intubated with biofilm-covered tubes were predominated by significant inflammation. However, in the lungs of the ambroxol-treated group, less neutrophil infiltration (fig. 5d) was found compared with lungs of rats without

VAP accounts for approximately 90% of infections in patients requiring assisted ventilation [16]. Bacteria adhere to tubes and proliferate, causing clinical infection and disease. Biofilm formation on the intubated tube may lead to the development of antibiotic-resistant VAP. One important agent that causes these infections is P. aeruginosa, and it has characteristic pathological effects, including fimbriation, interaction with host defense, and, most importantly, their adhesive and biofilm-formation abilities [17]. The increased resistance of P. aeruginosa to antibiotics is attributed to mechanisms, including the increasing mutations [18], the protection from the biofilm,



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Discussion

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Fig. 5. Histopathological changes in the lung of rats. Hematoxylin and eosin staining. Magnification !200. On day 4 of infection with biofilm-covered tube intubation: ambroxol-treated (a) and salinetreated (b), and with sterile tube intubation (c). On day 7 of infection with biofilm-covered tube intubation: ambroxol-treated (d) and saline-treated (e), and with sterile tube intubation (f).

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significant compared with the biofilm saline-treated group (* p ! 0.05). Differences were not statistically significant compared with the biofilm saline-treated group (+ p 1 0.05). BF = Biofilm.

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Fig. 6. Serum cytokine response at different time points: the level of IL-10 on the 4th and on the 7th day (a); the level of IFN-␥ on the 4th and on the 7th day (b), and the ratio of IFN-␥ and IL-10 on the 4th and on the 7th day (c). Differences were statistically

Ratio of IFN-␥ and IL-10 in serum (OD 450)

1.2

ed tube was observed in situ by scanning electron microscopy, and we found that the structure of biofilm was destroyed in the biofilm ambroxol-treated group. The increased release of bacteria in the biofilm can cause higher bacterial loads in the lung, as shown in our study. However, a higher bacterial load did not cause more severe histopathological changes in the lung. The damage to the lung by P. aeruginosa is likely to be determined by the balance of bacterial load and the immune response caused by bacteria. The immune response to bacterial infections is modulated by T helper cells via changes in cytokine levels. Severity of infection is correlated with the imbalance between pro- and anti-inflammatory cytokine levels. Constitutive production of IL-10 may help to prevent local tissue destruction by inhibiting the proinflammatory cytokine IFN-␥ production and higher mortality. Yu et al. [24] observed higher mortality and more severe lung pathology in the IL-10-deficient mice compared to controls. It has been shown that bronchoalveolar lavage fluid of cystic fibrosis patients with chronic infection by mucoid strains has significantly lower levels of IL-10 [25]. Song et al. [8] demonstrated that the severity of lung pathology and PMN infiltration with the alginate overproduction strain may be due to an increase in the proinflammatory cytokines IFN-␥ and the decrease in the anti-inflammatory cytokine IL-10. In this study, we found no difference in the IL-10 level between the biofilm ambroxol-treated group and the biofilm saline-treated group, but a lower level of IFN-␥ and a lower ratio of IFN␥ and IL-10 in the biofilm of the ambroxol-treated group compared with the biofilm of the saline-treated group. It seemed that ambroxol had no significant effect on the IL-10 level. However, with the effect of ambroxol, the level of IFN-␥ decreased and the balance between pro- and anti-inflammatory cytokine levels was destroyed, which might have contributed to the milder pathological changes in the lungs of the biofilm ambroxol-treated group. In vitro studies demonstrated that alginate protected the bacteria from phagocytosis, opsonization, antibodies, complement, and PMN infiltration [26]. Earlier research with analyses of both micro- and macroscopic pathology revealed that alginate-negative PAOalgD infection resulted in milder pathological changes compared with alginate-positive PAOmucA22 strains [8]. Given the effect of ambroxol on alginate in vitro as our previous study demonstrated and the role of alginate in immune response, we assume that the protective effect of ambroxol on lung infection is associated with its effect on alginate. So this might be another interesting study.



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and exposure of the organisms to some special environment, such as anaerobic, acidic, and nutrient-depleted [19]. Biofilm formation is very important among them. Transport limitation, neutralization effect, and physiological adaption are often quoted to explain the enhanced antibiotic resistance within the biofilm. The formation of P. aeruginosa biofilm poses a physical barrier to antibiotics [20]. The adsorption of positively charged antibiotics (such as the aminoglycosides) to the negatively charged alginate polymers can retard penetration. The low metabolic status of the bacteria inside the biofilm reduces the activity of antibiotics. In mucoid isolates, alginate is the main primary structural matrix. Alginate is a virulence factor that contributes to bacterial adherence and persistence, formation of microcolonies and biofilm growth, reduction of polymorphonuclear leukocyte (PMN) chemotaxis, suppression of lymphocyte and PMN function, formation of a physical barrier to the immune system and antibiotics, and resistance to opsonic killing by PMNs and macrophages [21]. Hence, alginate has increasingly become the target of anti-biofilm research. Ambroxol, a known mucolytic agent, seems to have additional antioxidant and anti-inflammatory properties. Furthermore, ambroxol is known to stimulate the formation and release of surfactant by type II pneumocytes, and it protects cellular lipids from oxidative stress related to endotoxemia or inflammatory responses [10]. In our previous study, we reported the anti-biofilm properties of ambroxol against mucoid P. aeruginosa biofilm and its influence on the production of alginate in vitro [13]. In the present study, we further studied the effect of ambroxol on mucoid P. aeruginosa biofilm in vivo. Animal models with lung infection were recently established. Yanagihara et al. [14] put a biofilm-covered tube with an inner needle through the oral cavity, via the vocal cords, into the trachea. The inner needle was pulled out, and the outer sheath was gently pushed to place the precoated tube into the main bronchus. Pedersen et al. [22] established a chronic lung infection model by direct intratracheal challenge with bacterial suspension. Intratracheal intubation through the oral cavity, as described by Yanagihara et al. [14], is particularly complicated in a rat, and the inner wall of the tube covered with biofilm may be destroyed with the inner needle. The method described by Moser et al. [23], though appearing to be simple, does not adequately represent catheter-associated infections. In our study, the tube was intratracheally placed with tracheotomy and fixed in the trachea in order to prevent the rupture of biofilm. It could be easily carried out with careful sterilization and manipulation. The intubat-

Most studies in the literature on the biofilm-forming mucoid P. aeruginosa were performed in vitro, whereas few studies were performed in vivo. Our study has focused on the effect of ambroxol on mucoid P. aeruginosa biofilm in vivo. Ambroxol seems to attenuate the severity of lung infection caused by mucoid P. aeruginosa biofilm, although the bacteria cannot be eliminated. The destructive effect of ambroxol on the biofilm structure would release more bacteria, which is likely to increase the bac-

terial load in lung during a certain period. Hence, combining ambroxol and antibiotics seems to be an effective strategy against biofilm-associated infections. Acknowledgments We would like to thank Bing Deng and Xiaoping Zhang for their technical assistance. This work was supported by the National Natural Science Foundation of China (No. 30772363 and 81070513).

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