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Ciprofloxacin plus erythromycin or ambroxol ameliorates endotracheal tube-associated Pseudomonas aeruginosa biofilms in a rat model
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Original Article
Ciprofloxacin plus erythromycin or ambroxol ameliorates endotracheal tube-associated Pseudomonas aeruginosa biofilms in a rat model Chen Cheng a , Lizhong Du b , Jialin Yu a,∗ , Qi Lu a , Yu He a , Tao Ran a a b
Department of Neonatology, Children’s Hospital, Chongqing Medical University, Chongqing, China The Children’s Hospital, Zhejiang University School of Medicine, China
a r t i c l e
i n f o
Article history: Received 3 March 2015 Received in revised form 27 September 2015 Accepted 2 October 2015 Keywords: Pseudomonas aeruginosa Biofilm Ambroxol Erythromycin Ciprofloxacin Combination
a b s t r a c t Background and objective: Pseudomonas aeruginosa is a multi-drug resistant bacterium, with its biofilm-growing mucoid (alginate-producing) strains being particularly resistant. As atomized drug administration is a common practice in pediatric patients, we compared the effect of inhalational therapy with erythromycin plus ciprofloxacin, with that of ambroxol plus ciprofloxacin, against biofilm producing strains of P. aeruginosa. Results: Both combined treatment regimens were associated with a significant reduction in bacterial counts in endotracheal (ET) tubes and lungs, as compared to that observed with ambroxol and erythromycin monotherapies (P < 0.05). Ciprofloxacin plus ambroxol appeared to have a higher efficacy than ciprofloxacin plus erythromycin, both in lowering bacterial counts (P < 0.05) and in disrupting the structural integrity of biofilm. Histopathological changes in the lungs were milder in the two combined treatment groups, as compared to that in groups treated with single drugs. Conclusion: Erythromycin or ambroxol in combination with ciprofloxacin could eliminate P. aeruginosa biofilms. When combined with ciprofloxacin, ambroxol outperformed erythromycin in eradicating P. aeruginosa biofilm. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction With the widespread use of mechanical ventilation in newborns, especially in the preemies, the incidence of respiratory tract infection with biofilm-producing micro-organisms has increased in recent years. Pseudomonas aeruginosa (P. aeruginosa), which belongs to Gram-negative opportunistic pathogen [1], is one of the commonest causes of nosocomial respiratory infections. P. aeruginosa is also one of the most common causes of ventilatorassociated pneumonia in neonates [2]. These infections are difficult
Abbreviations: CFU, colony-forming units; CLSI, Clinical Laboratory Standard Institute standard method; EPS, extracellular polymeric substances; ETT, endotracheal tube; DPB, diffuse panbronchiolitis; GMD, GDP-mannose dehydrogenase; LB, Luria broth; IL, interleukin; LPS, lipopolysaccharide; MIC, minimum inhibitory concentration; PA, Pseudomonas aeruginosa; PS, pulmonary surfactant; SD, SpragueDawley; SEM, scanning electron microscopy; TSB, trypticase soy broth; TNF, tumor necrosis factor; VAP, ventilator associated pneumonia. ∗ Corresponding author at: Department of Neonatology, Children’s Hospital, Chongqing Medical University, 136, Zhongshan Road, Yuzhong District, Chongqing 400014, China. E-mail address: [email protected] (J. Yu).
to treat due to the development of a bacterial biofilm, a term that refers to the matrix-enclosed bacterial populations adherent to each other and/or to surfaces or interfaces with a tenacious extracellular matrix mostly made up of polysaccharides and proteins [2,3]. The biofilms serve as a protective sanctuary for bacteria, and allows for diffusion of nutrients and communication between distant layers within the biofilm community [4]. Several studies have demonstrated that biofilms are important for the persistence of chronic rhinosinusitis, pulmonary infections in cystic fibrosis, chronic otitis media, and device-related infections [4]. Ciprofloxacin, a broad-spectrum antibiotic, has a higher efficacy against Gram-negative bacteria as compared to that against grampositive bacteria. The susceptibility of P. aeruginosa to ciprofloxacin has been demonstrated in vitro as well as in vivo, especially when it is amongst its planktonic brethren [5]. However, the drug is known to have serious side effects in children and its safety in neonates is not yet established. In our previous study (as yet unpublished), we found that inhalational mode of administration of ciprofloxacin was found to be safer in neonates owing to the relatively low concentration achieved in blood.
http://dx.doi.org/10.1016/j.prp.2015.10.003 0344-0338/© 2015 Elsevier GmbH. All rights reserved.
Please cite this article in press as: C. Cheng, et al., Ciprofloxacin plus erythromycin or ambroxol ameliorates endotracheal tube-associated Pseudomonas aeruginosa biofilms in a rat model, Pathol. – Res. Pract (2015), http://dx.doi.org/10.1016/j.prp.2015.10.003
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Ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl] benzylamine) is a mucolytic agent that is known to have antioxidant and anti-inflammatory effect in patients with pulmonary infection [6]. Recent reports suggest that ambroxol also promotes the permeability of ciprofloxacin across P. aeruginosa biofilms and reduces the volume of extracellular polymeric substances (EPS) [5]. Giovanna et al. found that ambroxol can help alleviate voriconazole resistance of Candida parapsilosis biofilm [7]. According to Li et al., ambroxol treatment appeared to disrupt the integrity of the biofilm on the intubation equipment and decrease the bacterial load [8]. Erythromycin, a macrolide, has proved effective in patients with infected cystic fibrosis and Pseudomonas biofilm disease [9]. Towako et al. also demonstrated that the clinical efficacy of erythromycin in diffuse panbronchiolitis (DPB) may be due, at least in part, to the reduction in P. aeruginosa biofilm formation [10]. Moreover, erythromycin is bactericidal for P. aeruginosa, especially on prolonged exposure [11]. Since both ambroxol and erythromycin appear to counter the effect of biofilms, and since ciprofloxacin administered by inhalational route does not influence its concentration in the blood, we hypothesized that nebulized ciprofloxacin combined with ambroxol or erythromycin will have a superior efficacy in treating P. aeruginosa infection. In the present study, we examined the effect of combined treatment on acute lung infections caused by P. aeruginosa with biofilm formation in an endotracheal intubation rat model, and compared the efficacy of the two combination treatments.
2. Materials and methods 2.1. Bacterial strains and their culture A mucoid strain of P. aeruginosa O1 (PAO1), maintained at −70 ◦ C, was obtained from the West China School of Medicine, Sichuan University. This bacterial strain was precultivated in 20% Luria broth (LB), diluted 1:5, and grown overnight at 37 ◦ C in a neutral pH. The bacteria was suspended in saline, centrifugated (3000 g, 4 ◦ C, 10 min) and harvested, resuspended in sterile saline, and fixed to 109 colony-forming units (CFU)/mL, as estimated by turbidimetry. Stock cultures were maintained at −70 ◦ C in 30% glycerol. 2.2. Animals Male and female Sprague-Dawley (SD) (approximately 7-weekold, weighing 200–220 g), rats were purchased from Chongqing Tengxin Biological Technology Limited Company. All rats were housed in a pathogen-free environment and received unlimited sterile food and water in the Laboratory Animal Center at the Children’s Hospital of Chongqing Medical University (Chongqing, China). The experimental protocol was approved by the Animal Care and Use Committee at the Chongqing Medical University. 2.3. Erythromycin and ciprofloxacin susceptibility and MIC culture Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method according to the Clinical Laboratory Standard Institute standard method (CLSI, 2005) [12]. Strains were grown either in trypticase soy broth (TSB; Difco) containing MIC of the antibiotics or in antibiotic-free medium for 48 h at 37 ◦ C.
2.4. Preparation of tubes covered with bacteria biofilms PAO1 was cultured on a Luria-Bertani medium. Intubation tubes with 3.0 mm diameter, were cut to 1.0 cm lengths, and immersed in 1 mL of 0.5 McIntosh turbidimetric concentration (approximately 1.5 × 108 mL−1 ), bacterium-saline suspensions for 3 days at 37 ◦ C. Biofilm was formed on the inner surface of these inoculation tubes. 2.5. Experimental model of endotracheal tube precoated with biofilms and drug administration Before the rats were intubated, the tubes were thoroughly rinsed with 3 mL sterile saline to remove any planktonic bacteria, and the inner fluid was sucked to keep the airway unobstructed. Rats were weighed and anesthetized with a subcutaneous injection of 0.3 mL/g of chloral hydrate. Intratracheal placement and fixation of tubes was done by tracheotomy. After intubation, rats were allowed to recover from anesthesia, eat, and drink spontaneously. Ambroxol was obtained from Boehringer Ingelheim (Shanghai, China), and erythromycin was purchased from Dalian Meiluo (Dalian, China). Rats were housed in a closed chamber (length × width × height: 40 × 30 × 34 cm). An ultrasonic nebulizer (BOY SX; PARI, Starnberg, Germany) was used to deliver an aerosol output at a rate of 6 mL/min [13]. Saline, ambroxol, erythromycin were nebulized respectively. When used in combination, ciprofloxacin was nebulized first and immediately followed by ambroxol or erythromycin. Biofilm-covered intubation model was established in 40 SD rats and these were randomly divided into 5 groups and were administered sterile saline (control); 1 MIC erythromycin; ambroxol (1.07 mg/mL); and 1 MIC erythromycin plus 8 MIC ciprofloxacin; and ambroxol (1.07 mg/mL) plus 8 MIC ciprofloxacin, respectively. Each of the rats was administered the assigned treatment once a day for 7 days. 2.6. Bacteriological and histopathological examination After one-week treatment, all rats were sacrificed by injecting 0.3 mL/kg of 10% chloral hydrate, and the left and right lungs were excised separately. For bacteriological examination, the right lungs were homogenized, including the implanted tube, and cultured quantitatively. Bacterial enumeration was performed by serially diluting samples on Mueller-Hinton II agar plates, incubating the Table 1 The bacteria counts in infected lungs. Groups Saline Ambroxol Erythromycin Ery + Cipro Amb + Cipro
Bacteria counts (CFU/ml, X¯ ± S) 139.250 101.625 109.625 57.750 22.250
± ± ± ± ±
42.0162 40.4190 33.4747 37.8295 17.3184
t
P a
1.825 −0.431b 1.560c
0.089 0.673 0.142
2.431d
0.037
Ery, erythromycin, Amb, ambroxol, Cipro, ciprofloxacin. a Comparison between saline group and ambroxol group. b Comparison between ambroxol group and erythromycin group. c Comparison between saline group and erythromycin group. d Erythromycin plus ciprofloxacin group compared with ambroxol plus ciprofloxacin group. The t value of saline group compared with erythromycin combined group and ambroxol combined group respectively were 4.077, and 7.282; P value for saline group compared with erythromycin combined group and ambroxol combined group, respectively, were 0.001, 0.000; the t value of ambroxol group compared with erythromycin combined group and ambroxol combined group respectively were 2.242, 5.106, the P value of ambroxol group compared with erythromycin combined group and ambroxol combined group respectively were 0.042, 0.001; the t value of erythromycin group compared with erythromycin combined group and ambroxol combined group, respectively, were 2.905, 6.557, the P value of erythromycin group compared with erythromycin combined group and ambroxol combined group, respectively, were 0.012 and 0.000.
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plates at 37 ◦ C in air for 12 h, followed by counting of the colonies to estimate the conoly-forming unit (CFU) in the lungs. For histopathological analysis, the left lungs were fixed in 10% formalin buffer, stained with hematoxylin and eosin for light microscopic examination. For scanning electron microscopy, the intubation tubes were retrieved aseptically, longitudinally sectioned, rinsed with 3 mL sterile saline to remove the planktonic bacteria and secretion. The biofilm were kept on the inner surface of the inoculation tube. These specimens were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer for 2 h at 4 ◦ C, 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%), freeze-dried, and finally coated with platinum palladium by using an ion sputter and examined under a scanning electron microscope [14]. 2.7. Statistical analysis All data are expressed as mean and standard deviation (SD). Inter-group differences were assessed using the approximate Student’s t test. A P value of <0.05 was considered as statistically significant. Statistical analyses were performed using the SPSS software, version 10.0. 3. Results 3.1. MICs of erythromycin and ciprofloxacin All PAO1 strains were susceptible to both erythromycin and ciprofloxacin. The MICs of erythromycin and ciprofloxacin were 31.25 mg/L and 0.5 mg/L, respectively.
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3.2. Bacterial counts of the lungs and tubes At the completion of the one-week long inhalational treatment, there was a considerable inter-group variability in the bacterial counts in lungs (Table 1). Ambroxol or erythromycin plus ciprofloxacin treatment was associated with significantly lower bacterial counts as compared to those associated with treatment with a single drug (P < 0.05; Fig. 1A). Further, the combination of ambroxol plus ciprofloxacin appeared to have a better synergistic inhibitory effect as compared to that of erythromycin plus ciprofloxacin treatment (P < 0.05; Fig. 1A). The results of bacterial counts on the biofilm-covered tubes were consistent with that observed in the respective lungs (Fig. 1B; Table 2). The results indicate that the combined treatment was associated with the highest efficacy in restraining the growth of PAO1, and that the ambroxol plus ciprofloxacin outperformed the erythromycin plus ciprofloxacin combination. 3.3. Scanning electron microscopy of the intubated tube The biofilm structure on the inner surface of the endotracheal tubes was examined in situ by scanning electron microscopy. The differences in the biofilm structure after the 7-day treatment were discernible in each group. In the saline-treated control group, the inner surface of the ET tube was covered with a thick membrane, which appeared as a dense, tightly formed structure contains innumerable bacteria and fibrous structures. The agglomerated bacteria were surrounded by thick mucosal fluid (Fig. 2a). The biofilms were observed to be thinner and less fibrous structure after treatment with ambroxol plus erythromycin (Fig. 2b
Fig. 1. Bacterial counts in the lungs (A) and the inner surface of the intubated tubes (B). Inter-group differences were assessed by approximate Student’s t test. 䊏 indicates a statistically significant difference from the saline controlled-treated group (P < 0.05); • indicates a statistically significant difference from the ambroxol group (P < 0.05); indicates a statistically significant difference from the erythromycin group (P < 0.05); indicates a statistically significant difference from the erythromycin plus ciprofloxacin group.
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Table 2 Bacteria counts of the inner surface of the intubated tubes. Groups Saline Ambroxol Erythromycin Ery + Cipro Amb + Cipro
Bacteria counts (104 CFU/ml, X¯ ± S) 170.000 127.625 133.500 70.375 38.125
± ± ± ± ±
48.3263 39.0163 33.6876 35.7768 19.1045
3.4. Histopathological changes in the lungs t
P a
1.930 −0.322b 1.752c
0.075 0.752 0.104
2.249d
0.047
Ery, erythromycin, Amb, ambroxol, Cipro, ciprofloxacin. a Comparison between saline group and ambroxol group. b Comparison between ambroxol group and erythromycin group. c Comparison between saline group and erythromycin group. d Erythromycin combined ciprofloxacin group compared with ambroxol combined ciprofloxacin group. The t value of saline group compared with erythromycin combined group and ambroxol combined group, respectively, were 4.686 and 7.718, the P value of saline group compared with erythromycin combined group and ambroxol combined group respectively were 0.000, 0.000; the t value of ambroxol group compared with erythromycin combined group and ambroxol combined group, respectively, were 3.059 and 5.872, the P value of ambroxol group compared with erythromycin combined group and ambroxol combined group, respectively, were 0.009, 0.000; the t value of erythromycin group compared with erythromycin combined group and ambroxol combined group, respectively, were 3.633 and 6.966, the P value of erythromycin group compared with erythromycin combined group and ambroxol combined group, respectively, were 0.003 and 0.000.
and c). Moreover, the biofilm appeared to be more diffusing in the groups that received combination treatment as compared to that in groups that received single drug treatment (Fig. 2d and e).
The rats in the control group appeared to have the most serious infection with the lungs showing severe congestion and significant inflammatory exudates in the pulmonary alveoli (Fig. 3a). Rats treated with ambroxol or erythromycin alone showed milder inflammation than that observed in the control group; however, lymphocytic infiltration and inflammatory exudates were distinctly observable (Fig. 3b and c). As expected, rats treated with combination treatment showed a markedly lesser degree of lung inflammation with only a mild lymphocytic infiltration (Fig. 3d and e). 4. Discussion With the continued advances in medical science in recent years, chronic infections associated with bacterial biofilms have increasingly garnered attention. P. aeruginosa, the most common pathogen in neonate intensive care units, is ranked among the top five organisms causing pulmonary, bloodstream, urinary tract, surgical site, and soft tissue infections [15]. Given their critical role in the pathogenesis of P. aeruginosa infection, biofilms formed by P. aeruginosa have been an obvious target for therapeutic interventions. However, biofilms are antibiotic resistance, which increases the challenge of treating and preventing mortality due to P. aeruginosa infection in intubated patients on mechanical ventilator. The search for newer adjuvant drugs that are effective against
Fig. 2. SEM of the inner surface of the intubated tube: saline treatment (a); ambroxol treatment (b); erythromycin treatment (c); erythromycin combined ciprofloxacin treatment (d); ambroxol combined ciprofloxacin treatment (e).
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Fig. 3. Histopathological changes in the lungs: saline treatment (a); ambroxol treatment (b); erythromycin treatment (c); erythromycin plus ciprofloxacin treatment (d); ambroxol plus ciprofloxacin treatment (e). H&E stain ×400.
biofilms is an ongoing imperative. Ambroxol has been shown to disrupt the structural integrity of biofilms on endotracheal tubes [16], in addition to curbing the growth of bacteria and the alginate content within biofilms. Moreover, it is also known to have a synergistic effect when administered in combination with ciprofloxacin [17]. Inhalational administration of ciprofloxacin for pulmonary infection confers the added advantage of attaining adequate local concentration of the drug with minimal systemic effects owing to poor absorption into blood circulation. Nebulization is a key mode of drug administration in children with respiratory diseases such as cystic fibrosis, asthma, acute bronchiolitis, and neonatal hypoxemic respiratory failure, and has a beneficial effect in mitigating airway inflammation and hyper responsiveness [18–21]. Owing to the inherent advantages of nebulization, in terms of safety, convenience and its non-invasive nature, it is a method-of-choice for drug administration in pediatric patients. We conducted this experiment in order to search for an effective and suitable way to counter infections associated with formation of P. aeruginosa biofilms on endotracheal tubes, a major contributor to VAP in neonates. In this study, we did not seek to assess the relative efficacy of nebulization vis-a-vis intravenous or intraperitoneal route of drug administration. Rather, the objective was to find a safe and practical way to treat neonates with VAP; therefore, we only used nebulization as the mode of drug administration in this study. In the ETT rat model, we observed that use of nebulized erythromycin plus ciprofloxacin, or ambroxol plus ciprofloxacin combinations was associated with reduced bacterial counts in both lungs and endotracheal tubes, disruption of the structural integrity of biofilms and mitigation of the inflammatory response.
The primary content of mucoid biofilms is alginate, which serves to protect the bacteria in a relatively harsh environment where the bacteria are continually exposed to oxidative stress and immunological response [22]. O-Acetylation or overproduction of alginate influences biofilm architecture and sensitivity to the antibiotic [23]. With the help of alginate, bacteria embedded into the biofilm are less susceptible to antibiotics. -Lactam antibiotic, aminoglycosides and fluoroquinolone antibiotics are effective against rapidly dividing P. aeruginosa. However, most of them have a poor effect on bacteria in the stationary phase. Ciprofloxacin inhibits bacterial DNA helicase, which prevents bacteria from replicating. Relatively, it has an excellent germicidal efficacy in nongrowing P. aeruginosa cells. Ciprofloxacin belongs to a positively charged group of antibiotics which can attach to the surface of catheter associated P. aeruginosa biofilms for more than 48 h. The alginate barrier of P. aeruginosa biofilms tends to attract positively charged antibiotics; therefore, a high concentration of ciprofloxacin is achieved on the surface of biofilms improving the bactericidal effect [5]. Moreover, ciprofloxacin can ruin the structure of aggregated bacteria. In an earlier study, we observed a comparable effect of ciprofloxacin and ambroxol, when used alone, in eliminating biofilms and bacterial aggregates in lungs and ETT; however, the effect was milder than that observed with the combination treatment [5]. This is attributable to the improved permeability of ciprofloxacin through P. aeruginosa biofilms caused by ambroxol. Erythromycin is a macrolide antibiotic used to treat respiratory infections. Tamaoki et al. found that lipopolysaccharide (LPS) could cause acute lung injury, microvascular leakage, and neutrophil recruitment in the trachea, erythromycin protects against these changes [24]. In addition to its antimicrobial actions,
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erythromycin may have immunomodulating effects, such as attenuation of neutrophil chemotaxis, inhibition of proinflammatory cytokines. In vitro studies have shown that macrolide sub-MIC reduces expression of P. aeruginosa exotoxins, exoenzymes and lectins, which are known to mediate P. aeruginosa biofilm formation [25]. Schultz et al. demonstrated that local production of cytokines plays an important role in the pathogenesis of pneumonia, and that in an in vitro study erythromycin inhibited tumor necrosis factor (TNF)-␣ production in whole blood stimulated with P. aeruginosa. However, erythromycin only reduced the production of interleukin (IL)-10, IL-6, IFN-␥ and IL-8 at the highest concentration (10−3 M), which is relatively difficult to attain in the airways [26]. In our study, we found that erythromycin had a mild effect in decreasing viability of P. aeruginosa, which is consistent with the findings reported by Takesh’s team [27]. In addition to being a mucolytic agent, ambroxol is known to have an antioxidant and anti-inflammatory effect in the treatment of pulmonary infectious diseases [6]. In a recent study, ambroxol appeared to reduce the production of alginate by P. aeruginosa by increasing the expression of mucA gene and decreasing expression of algD, algU, and algR genes. In addition, ambroxol treatment was also associated with decreased activity of diphospho-dmannose dehydrogenase (GDP-mannose dehydrogenase; GMD) [28]. The genes and GMD mentioned above are critical to the formation of alginate. Thus, ambroxol appears to reduce the production of alginate at two levels: genetic expression and enzyme activity. Ambroxol is also known to stimulate the synthesis and secretion of exogenous pulmonary surfactant (PS) in lungs of P. aeruginosainduced infection in rats [29], and appeared to exert a protective effect on cellular lipids against oxidative stress related to endotoxemia or inflammatory responses [29]. The mucolytic, antioxidant and anti-inflammatory properties of ambroxol, in addition to its inhibitory effect on alginate production, explain why the viable cell counts of P. aeruginosa from ETT and infected lungs in the ambroxol group were lower in comparison to that in the control group, but without any significant difference. The SEM of the bacterial biofilm and histopathological changes in lungs had no obvious differences from that in the control group, which is different from the result of Fang Li’s team [27]. In our experiment, we administered ambroxol at the dose of 1.07 mg/mL, whereas Fang Li’s team adopted a dose of 3.75 mg/mL. However, when ambroxol or erythromycin was used in combination with ciprofloxacin synergistically and respectively, viable bacterial counts or SEM findings and histopathological changes showed a much greater effect than that observed in the control group or in ambroxol/erythromycin alone groups. Moreover, ambroxol combined group was better than erythromycin combined group in eradicating biofilms. We speculate that the difference is attributable to the direct effect of ambroxol on biofilm-alginate, by modulating the relevant genes and enzyme activity. In conclusion, we demonstrate that erythromycin or ambroxol combined with ciprofloxacin could eliminate P. aeruginosa biofilms. Ambroxol appeared to outperform erythromycin with a higher efficacy in this respect. Ambroxol plus ciprofloxacin appears to be a good candidate therapy for treatment of P. aeruginosa infections associated with endotracheal intubation. This study serves to provide a theoretical basis for the use of a combination of ambroxol with other sensitive antibiotics, as a strategy to counter biofilms.
5. Conclusion Erythromycin or ambroxol in combination with ciprofloxacin could reduce or even eliminate P. aeruginosa biofilms. Ciprofloxacin
plus ambroxol appears to have a higher efficacy in eradicating P. aeruginosa biofilms as compared to that of ambroxol plus erythromycin. Authors’ contributions Jialin Yu, Lizhong Du and Chen Cheng conceived of the study, and participated in its design and coordination and helped to draft the manuscript. Qi Lu and Yu He participated in the modified the manuscript. Tao Ran helped performed the statistical analysis. All authors read and approved the final manuscript. Funding All phases of this study were supported by the National Natural Science Foundation of China (No. 81370744), Doctoral Degree Funding from Chinese Ministry of Education (No. 20135503110009), the subproject of National Science & Technology Pillar Program during the 12th Five-year Plan Period in China (No. 2012BAI04B05). Competing interest The authors declare that they have no competing interests. Acknowledgments We would like to thank Qing Ai for her technical assistance. In addition, we are grateful to Guanxin Liu and Hongdong Li for helpful discussions. References [1] H.A. Abbas, F.M. Serry, E.M. EL-Masry, Combating Pseudomonas aeruginosa biofilms by potential biofilm inhibitors, Asian J. Res. Pharm. Sci. 2 (2012) 66–72. [2] T. Bauer, A. Torres, R. Ferrer, C. Heyer, C. Schultze-Werninghaus, K. Rasche, Biofilm formation in endotracheal tubes. Association between pneumonia and the persistence of pathogens, Monaldi Arch. Chest Dis. 57 (2002) 84–87. [3] J. Pintucci, S. Corno, M. Garotta, Biofilms and infections of the upper respiratory tract, Eur. Rev. Med. Pharmacol. Sci. 14 (2010) 683–690. [4] Liu Y-CC, J.C. Post, Biofilms in pediatric respiratory and related infections, Curr. Allergy Asthma Rep. 9 (2009) 449–455. [5] Q. Lu, J. Yu, L. Bao, T. Ran, H. Zhong, Effects of combined treatment with ambroxol and ciprofloxacin on catheter-associated Pseudomonas aeruginosa biofilms in a rat model, Chemotherapy 59 (2013) 51–56. [6] K.M. Beeh, J. Beier, A. Esperester, L.D. Paul, Anti-inflammatory properties of ambroxol, Eur. J. Med. Res. 13 (12) (2008) 557–562. [7] G. Pulcrano, D. Panellis, G. Domenico, F. Rossano, M.R. Catania, Ambroxol influences voriconazole resistance of Candida parapsilosis biofilm, FEMS Yeast Res. 12 (2012) 430–438. [8] F. Li, W. Wang, L. Hu, L. Li, J. Yu, Effect of ambroxol on pneumonia caused by Pseudomonas aeruginosa with biofilm formation in an endotracheal intubation rat model, Chemotherapy 57 (2011) 173–180. [9] H. Kobayashi, Biofilm disease: its clinical manifestation and therapeutic possibilities of macrolides, Am. J. Med. 99 (1995) 26–30. [10] T. Nagata, H. Mukae, J. Kadota, T. Hayashi, T. Fujii, M. Kuroki, et al., Effect of erythromycin on chronic respiratory infection caused by Pseudomonas aeruginosa with biofilm formation in an experimental murine model, Antimicrob. Agents Chemother. 48 (2004) 2251–2259. [11] K. Tateda, Y. Ishii, T. Matsumoto, N. Furuya, M. Nagashima, T. Matsunaga, et al., Direct evidence for antipseudomonal activity of macrolides: exposure-dependent bactericidal activity and inhibition of protein synthesis by erythromycin, clarithromycin, and azithromycin, Antimicrob. Agents Chemother. 40 (1996) 2271–2275. [12] D. Amsterdam, Susceptibility testing of antimicrobials in liquid media, Antibiot. Lab. Med. 4 (1996) 52–111. [13] N. Kirschvink, G. Vincke, L. Fiévez, C. Onclinx, D. Wirth, M. Belleflamme, et al., Repeated cadmium nebulizations induce pulmonary MMP-2 and MMP-9 production and enphysema in rats, Toxicology 211 (2005) 36–48. [14] B. Chen, J. Yu, G. Liu, L. Hu, L. Li, F. Li, et al., Electron microscopic analysis of biofilm on tracheal tubes removed from intubated neonates and the relationship between bilofilm and lower respiratory infection, Chin. J. Pediatr. 45 (2007) 655–660.
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Original Article
Ciprofloxacin plus erythromycin or ambroxol ameliorates endotracheal tube-associated Pseudomonas aeruginosa biofilms in a rat model Chen Cheng a , Lizhong Du b , Jialin Yu a,∗ , Qi Lu a , Yu He a , Tao Ran a a b
Department of Neonatology, Children’s Hospital, Chongqing Medical University, Chongqing, China The Children’s Hospital, Zhejiang University School of Medicine, China
a r t i c l e
i n f o
Article history: Received 3 March 2015 Received in revised form 27 September 2015 Accepted 2 October 2015 Keywords: Pseudomonas aeruginosa Biofilm Ambroxol Erythromycin Ciprofloxacin Combination
a b s t r a c t Background and objective: Pseudomonas aeruginosa is a multi-drug resistant bacterium, with its biofilm-growing mucoid (alginate-producing) strains being particularly resistant. As atomized drug administration is a common practice in pediatric patients, we compared the effect of inhalational therapy with erythromycin plus ciprofloxacin, with that of ambroxol plus ciprofloxacin, against biofilm producing strains of P. aeruginosa. Results: Both combined treatment regimens were associated with a significant reduction in bacterial counts in endotracheal (ET) tubes and lungs, as compared to that observed with ambroxol and erythromycin monotherapies (P < 0.05). Ciprofloxacin plus ambroxol appeared to have a higher efficacy than ciprofloxacin plus erythromycin, both in lowering bacterial counts (P < 0.05) and in disrupting the structural integrity of biofilm. Histopathological changes in the lungs were milder in the two combined treatment groups, as compared to that in groups treated with single drugs. Conclusion: Erythromycin or ambroxol in combination with ciprofloxacin could eliminate P. aeruginosa biofilms. When combined with ciprofloxacin, ambroxol outperformed erythromycin in eradicating P. aeruginosa biofilm. © 2015 Elsevier GmbH. All rights reserved.
1. Introduction With the widespread use of mechanical ventilation in newborns, especially in the preemies, the incidence of respiratory tract infection with biofilm-producing micro-organisms has increased in recent years. Pseudomonas aeruginosa (P. aeruginosa), which belongs to Gram-negative opportunistic pathogen [1], is one of the commonest causes of nosocomial respiratory infections. P. aeruginosa is also one of the most common causes of ventilatorassociated pneumonia in neonates [2]. These infections are difficult
Abbreviations: CFU, colony-forming units; CLSI, Clinical Laboratory Standard Institute standard method; EPS, extracellular polymeric substances; ETT, endotracheal tube; DPB, diffuse panbronchiolitis; GMD, GDP-mannose dehydrogenase; LB, Luria broth; IL, interleukin; LPS, lipopolysaccharide; MIC, minimum inhibitory concentration; PA, Pseudomonas aeruginosa; PS, pulmonary surfactant; SD, SpragueDawley; SEM, scanning electron microscopy; TSB, trypticase soy broth; TNF, tumor necrosis factor; VAP, ventilator associated pneumonia. ∗ Corresponding author at: Department of Neonatology, Children’s Hospital, Chongqing Medical University, 136, Zhongshan Road, Yuzhong District, Chongqing 400014, China. E-mail address: [email protected] (J. Yu).
to treat due to the development of a bacterial biofilm, a term that refers to the matrix-enclosed bacterial populations adherent to each other and/or to surfaces or interfaces with a tenacious extracellular matrix mostly made up of polysaccharides and proteins [2,3]. The biofilms serve as a protective sanctuary for bacteria, and allows for diffusion of nutrients and communication between distant layers within the biofilm community [4]. Several studies have demonstrated that biofilms are important for the persistence of chronic rhinosinusitis, pulmonary infections in cystic fibrosis, chronic otitis media, and device-related infections [4]. Ciprofloxacin, a broad-spectrum antibiotic, has a higher efficacy against Gram-negative bacteria as compared to that against grampositive bacteria. The susceptibility of P. aeruginosa to ciprofloxacin has been demonstrated in vitro as well as in vivo, especially when it is amongst its planktonic brethren [5]. However, the drug is known to have serious side effects in children and its safety in neonates is not yet established. In our previous study (as yet unpublished), we found that inhalational mode of administration of ciprofloxacin was found to be safer in neonates owing to the relatively low concentration achieved in blood.
http://dx.doi.org/10.1016/j.prp.2015.10.003 0344-0338/© 2015 Elsevier GmbH. All rights reserved.
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Ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl] benzylamine) is a mucolytic agent that is known to have antioxidant and anti-inflammatory effect in patients with pulmonary infection [6]. Recent reports suggest that ambroxol also promotes the permeability of ciprofloxacin across P. aeruginosa biofilms and reduces the volume of extracellular polymeric substances (EPS) [5]. Giovanna et al. found that ambroxol can help alleviate voriconazole resistance of Candida parapsilosis biofilm [7]. According to Li et al., ambroxol treatment appeared to disrupt the integrity of the biofilm on the intubation equipment and decrease the bacterial load [8]. Erythromycin, a macrolide, has proved effective in patients with infected cystic fibrosis and Pseudomonas biofilm disease [9]. Towako et al. also demonstrated that the clinical efficacy of erythromycin in diffuse panbronchiolitis (DPB) may be due, at least in part, to the reduction in P. aeruginosa biofilm formation [10]. Moreover, erythromycin is bactericidal for P. aeruginosa, especially on prolonged exposure [11]. Since both ambroxol and erythromycin appear to counter the effect of biofilms, and since ciprofloxacin administered by inhalational route does not influence its concentration in the blood, we hypothesized that nebulized ciprofloxacin combined with ambroxol or erythromycin will have a superior efficacy in treating P. aeruginosa infection. In the present study, we examined the effect of combined treatment on acute lung infections caused by P. aeruginosa with biofilm formation in an endotracheal intubation rat model, and compared the efficacy of the two combination treatments.
2. Materials and methods 2.1. Bacterial strains and their culture A mucoid strain of P. aeruginosa O1 (PAO1), maintained at −70 ◦ C, was obtained from the West China School of Medicine, Sichuan University. This bacterial strain was precultivated in 20% Luria broth (LB), diluted 1:5, and grown overnight at 37 ◦ C in a neutral pH. The bacteria was suspended in saline, centrifugated (3000 g, 4 ◦ C, 10 min) and harvested, resuspended in sterile saline, and fixed to 109 colony-forming units (CFU)/mL, as estimated by turbidimetry. Stock cultures were maintained at −70 ◦ C in 30% glycerol. 2.2. Animals Male and female Sprague-Dawley (SD) (approximately 7-weekold, weighing 200–220 g), rats were purchased from Chongqing Tengxin Biological Technology Limited Company. All rats were housed in a pathogen-free environment and received unlimited sterile food and water in the Laboratory Animal Center at the Children’s Hospital of Chongqing Medical University (Chongqing, China). The experimental protocol was approved by the Animal Care and Use Committee at the Chongqing Medical University. 2.3. Erythromycin and ciprofloxacin susceptibility and MIC culture Minimum inhibitory concentrations (MICs) were determined using the broth microdilution method according to the Clinical Laboratory Standard Institute standard method (CLSI, 2005) [12]. Strains were grown either in trypticase soy broth (TSB; Difco) containing MIC of the antibiotics or in antibiotic-free medium for 48 h at 37 ◦ C.
2.4. Preparation of tubes covered with bacteria biofilms PAO1 was cultured on a Luria-Bertani medium. Intubation tubes with 3.0 mm diameter, were cut to 1.0 cm lengths, and immersed in 1 mL of 0.5 McIntosh turbidimetric concentration (approximately 1.5 × 108 mL−1 ), bacterium-saline suspensions for 3 days at 37 ◦ C. Biofilm was formed on the inner surface of these inoculation tubes. 2.5. Experimental model of endotracheal tube precoated with biofilms and drug administration Before the rats were intubated, the tubes were thoroughly rinsed with 3 mL sterile saline to remove any planktonic bacteria, and the inner fluid was sucked to keep the airway unobstructed. Rats were weighed and anesthetized with a subcutaneous injection of 0.3 mL/g of chloral hydrate. Intratracheal placement and fixation of tubes was done by tracheotomy. After intubation, rats were allowed to recover from anesthesia, eat, and drink spontaneously. Ambroxol was obtained from Boehringer Ingelheim (Shanghai, China), and erythromycin was purchased from Dalian Meiluo (Dalian, China). Rats were housed in a closed chamber (length × width × height: 40 × 30 × 34 cm). An ultrasonic nebulizer (BOY SX; PARI, Starnberg, Germany) was used to deliver an aerosol output at a rate of 6 mL/min [13]. Saline, ambroxol, erythromycin were nebulized respectively. When used in combination, ciprofloxacin was nebulized first and immediately followed by ambroxol or erythromycin. Biofilm-covered intubation model was established in 40 SD rats and these were randomly divided into 5 groups and were administered sterile saline (control); 1 MIC erythromycin; ambroxol (1.07 mg/mL); and 1 MIC erythromycin plus 8 MIC ciprofloxacin; and ambroxol (1.07 mg/mL) plus 8 MIC ciprofloxacin, respectively. Each of the rats was administered the assigned treatment once a day for 7 days. 2.6. Bacteriological and histopathological examination After one-week treatment, all rats were sacrificed by injecting 0.3 mL/kg of 10% chloral hydrate, and the left and right lungs were excised separately. For bacteriological examination, the right lungs were homogenized, including the implanted tube, and cultured quantitatively. Bacterial enumeration was performed by serially diluting samples on Mueller-Hinton II agar plates, incubating the Table 1 The bacteria counts in infected lungs. Groups Saline Ambroxol Erythromycin Ery + Cipro Amb + Cipro
Bacteria counts (CFU/ml, X¯ ± S) 139.250 101.625 109.625 57.750 22.250
± ± ± ± ±
42.0162 40.4190 33.4747 37.8295 17.3184
t
P a
1.825 −0.431b 1.560c
0.089 0.673 0.142
2.431d
0.037
Ery, erythromycin, Amb, ambroxol, Cipro, ciprofloxacin. a Comparison between saline group and ambroxol group. b Comparison between ambroxol group and erythromycin group. c Comparison between saline group and erythromycin group. d Erythromycin plus ciprofloxacin group compared with ambroxol plus ciprofloxacin group. The t value of saline group compared with erythromycin combined group and ambroxol combined group respectively were 4.077, and 7.282; P value for saline group compared with erythromycin combined group and ambroxol combined group, respectively, were 0.001, 0.000; the t value of ambroxol group compared with erythromycin combined group and ambroxol combined group respectively were 2.242, 5.106, the P value of ambroxol group compared with erythromycin combined group and ambroxol combined group respectively were 0.042, 0.001; the t value of erythromycin group compared with erythromycin combined group and ambroxol combined group, respectively, were 2.905, 6.557, the P value of erythromycin group compared with erythromycin combined group and ambroxol combined group, respectively, were 0.012 and 0.000.
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plates at 37 ◦ C in air for 12 h, followed by counting of the colonies to estimate the conoly-forming unit (CFU) in the lungs. For histopathological analysis, the left lungs were fixed in 10% formalin buffer, stained with hematoxylin and eosin for light microscopic examination. For scanning electron microscopy, the intubation tubes were retrieved aseptically, longitudinally sectioned, rinsed with 3 mL sterile saline to remove the planktonic bacteria and secretion. The biofilm were kept on the inner surface of the inoculation tube. These specimens were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer for 2 h at 4 ◦ C, 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%), freeze-dried, and finally coated with platinum palladium by using an ion sputter and examined under a scanning electron microscope [14]. 2.7. Statistical analysis All data are expressed as mean and standard deviation (SD). Inter-group differences were assessed using the approximate Student’s t test. A P value of <0.05 was considered as statistically significant. Statistical analyses were performed using the SPSS software, version 10.0. 3. Results 3.1. MICs of erythromycin and ciprofloxacin All PAO1 strains were susceptible to both erythromycin and ciprofloxacin. The MICs of erythromycin and ciprofloxacin were 31.25 mg/L and 0.5 mg/L, respectively.
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3.2. Bacterial counts of the lungs and tubes At the completion of the one-week long inhalational treatment, there was a considerable inter-group variability in the bacterial counts in lungs (Table 1). Ambroxol or erythromycin plus ciprofloxacin treatment was associated with significantly lower bacterial counts as compared to those associated with treatment with a single drug (P < 0.05; Fig. 1A). Further, the combination of ambroxol plus ciprofloxacin appeared to have a better synergistic inhibitory effect as compared to that of erythromycin plus ciprofloxacin treatment (P < 0.05; Fig. 1A). The results of bacterial counts on the biofilm-covered tubes were consistent with that observed in the respective lungs (Fig. 1B; Table 2). The results indicate that the combined treatment was associated with the highest efficacy in restraining the growth of PAO1, and that the ambroxol plus ciprofloxacin outperformed the erythromycin plus ciprofloxacin combination. 3.3. Scanning electron microscopy of the intubated tube The biofilm structure on the inner surface of the endotracheal tubes was examined in situ by scanning electron microscopy. The differences in the biofilm structure after the 7-day treatment were discernible in each group. In the saline-treated control group, the inner surface of the ET tube was covered with a thick membrane, which appeared as a dense, tightly formed structure contains innumerable bacteria and fibrous structures. The agglomerated bacteria were surrounded by thick mucosal fluid (Fig. 2a). The biofilms were observed to be thinner and less fibrous structure after treatment with ambroxol plus erythromycin (Fig. 2b
Fig. 1. Bacterial counts in the lungs (A) and the inner surface of the intubated tubes (B). Inter-group differences were assessed by approximate Student’s t test. 䊏 indicates a statistically significant difference from the saline controlled-treated group (P < 0.05); • indicates a statistically significant difference from the ambroxol group (P < 0.05); indicates a statistically significant difference from the erythromycin group (P < 0.05); indicates a statistically significant difference from the erythromycin plus ciprofloxacin group.
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Table 2 Bacteria counts of the inner surface of the intubated tubes. Groups Saline Ambroxol Erythromycin Ery + Cipro Amb + Cipro
Bacteria counts (104 CFU/ml, X¯ ± S) 170.000 127.625 133.500 70.375 38.125
± ± ± ± ±
48.3263 39.0163 33.6876 35.7768 19.1045
3.4. Histopathological changes in the lungs t
P a
1.930 −0.322b 1.752c
0.075 0.752 0.104
2.249d
0.047
Ery, erythromycin, Amb, ambroxol, Cipro, ciprofloxacin. a Comparison between saline group and ambroxol group. b Comparison between ambroxol group and erythromycin group. c Comparison between saline group and erythromycin group. d Erythromycin combined ciprofloxacin group compared with ambroxol combined ciprofloxacin group. The t value of saline group compared with erythromycin combined group and ambroxol combined group, respectively, were 4.686 and 7.718, the P value of saline group compared with erythromycin combined group and ambroxol combined group respectively were 0.000, 0.000; the t value of ambroxol group compared with erythromycin combined group and ambroxol combined group, respectively, were 3.059 and 5.872, the P value of ambroxol group compared with erythromycin combined group and ambroxol combined group, respectively, were 0.009, 0.000; the t value of erythromycin group compared with erythromycin combined group and ambroxol combined group, respectively, were 3.633 and 6.966, the P value of erythromycin group compared with erythromycin combined group and ambroxol combined group, respectively, were 0.003 and 0.000.
and c). Moreover, the biofilm appeared to be more diffusing in the groups that received combination treatment as compared to that in groups that received single drug treatment (Fig. 2d and e).
The rats in the control group appeared to have the most serious infection with the lungs showing severe congestion and significant inflammatory exudates in the pulmonary alveoli (Fig. 3a). Rats treated with ambroxol or erythromycin alone showed milder inflammation than that observed in the control group; however, lymphocytic infiltration and inflammatory exudates were distinctly observable (Fig. 3b and c). As expected, rats treated with combination treatment showed a markedly lesser degree of lung inflammation with only a mild lymphocytic infiltration (Fig. 3d and e). 4. Discussion With the continued advances in medical science in recent years, chronic infections associated with bacterial biofilms have increasingly garnered attention. P. aeruginosa, the most common pathogen in neonate intensive care units, is ranked among the top five organisms causing pulmonary, bloodstream, urinary tract, surgical site, and soft tissue infections [15]. Given their critical role in the pathogenesis of P. aeruginosa infection, biofilms formed by P. aeruginosa have been an obvious target for therapeutic interventions. However, biofilms are antibiotic resistance, which increases the challenge of treating and preventing mortality due to P. aeruginosa infection in intubated patients on mechanical ventilator. The search for newer adjuvant drugs that are effective against
Fig. 2. SEM of the inner surface of the intubated tube: saline treatment (a); ambroxol treatment (b); erythromycin treatment (c); erythromycin combined ciprofloxacin treatment (d); ambroxol combined ciprofloxacin treatment (e).
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Fig. 3. Histopathological changes in the lungs: saline treatment (a); ambroxol treatment (b); erythromycin treatment (c); erythromycin plus ciprofloxacin treatment (d); ambroxol plus ciprofloxacin treatment (e). H&E stain ×400.
biofilms is an ongoing imperative. Ambroxol has been shown to disrupt the structural integrity of biofilms on endotracheal tubes [16], in addition to curbing the growth of bacteria and the alginate content within biofilms. Moreover, it is also known to have a synergistic effect when administered in combination with ciprofloxacin [17]. Inhalational administration of ciprofloxacin for pulmonary infection confers the added advantage of attaining adequate local concentration of the drug with minimal systemic effects owing to poor absorption into blood circulation. Nebulization is a key mode of drug administration in children with respiratory diseases such as cystic fibrosis, asthma, acute bronchiolitis, and neonatal hypoxemic respiratory failure, and has a beneficial effect in mitigating airway inflammation and hyper responsiveness [18–21]. Owing to the inherent advantages of nebulization, in terms of safety, convenience and its non-invasive nature, it is a method-of-choice for drug administration in pediatric patients. We conducted this experiment in order to search for an effective and suitable way to counter infections associated with formation of P. aeruginosa biofilms on endotracheal tubes, a major contributor to VAP in neonates. In this study, we did not seek to assess the relative efficacy of nebulization vis-a-vis intravenous or intraperitoneal route of drug administration. Rather, the objective was to find a safe and practical way to treat neonates with VAP; therefore, we only used nebulization as the mode of drug administration in this study. In the ETT rat model, we observed that use of nebulized erythromycin plus ciprofloxacin, or ambroxol plus ciprofloxacin combinations was associated with reduced bacterial counts in both lungs and endotracheal tubes, disruption of the structural integrity of biofilms and mitigation of the inflammatory response.
The primary content of mucoid biofilms is alginate, which serves to protect the bacteria in a relatively harsh environment where the bacteria are continually exposed to oxidative stress and immunological response [22]. O-Acetylation or overproduction of alginate influences biofilm architecture and sensitivity to the antibiotic [23]. With the help of alginate, bacteria embedded into the biofilm are less susceptible to antibiotics. -Lactam antibiotic, aminoglycosides and fluoroquinolone antibiotics are effective against rapidly dividing P. aeruginosa. However, most of them have a poor effect on bacteria in the stationary phase. Ciprofloxacin inhibits bacterial DNA helicase, which prevents bacteria from replicating. Relatively, it has an excellent germicidal efficacy in nongrowing P. aeruginosa cells. Ciprofloxacin belongs to a positively charged group of antibiotics which can attach to the surface of catheter associated P. aeruginosa biofilms for more than 48 h. The alginate barrier of P. aeruginosa biofilms tends to attract positively charged antibiotics; therefore, a high concentration of ciprofloxacin is achieved on the surface of biofilms improving the bactericidal effect [5]. Moreover, ciprofloxacin can ruin the structure of aggregated bacteria. In an earlier study, we observed a comparable effect of ciprofloxacin and ambroxol, when used alone, in eliminating biofilms and bacterial aggregates in lungs and ETT; however, the effect was milder than that observed with the combination treatment [5]. This is attributable to the improved permeability of ciprofloxacin through P. aeruginosa biofilms caused by ambroxol. Erythromycin is a macrolide antibiotic used to treat respiratory infections. Tamaoki et al. found that lipopolysaccharide (LPS) could cause acute lung injury, microvascular leakage, and neutrophil recruitment in the trachea, erythromycin protects against these changes [24]. In addition to its antimicrobial actions,
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erythromycin may have immunomodulating effects, such as attenuation of neutrophil chemotaxis, inhibition of proinflammatory cytokines. In vitro studies have shown that macrolide sub-MIC reduces expression of P. aeruginosa exotoxins, exoenzymes and lectins, which are known to mediate P. aeruginosa biofilm formation [25]. Schultz et al. demonstrated that local production of cytokines plays an important role in the pathogenesis of pneumonia, and that in an in vitro study erythromycin inhibited tumor necrosis factor (TNF)-␣ production in whole blood stimulated with P. aeruginosa. However, erythromycin only reduced the production of interleukin (IL)-10, IL-6, IFN-␥ and IL-8 at the highest concentration (10−3 M), which is relatively difficult to attain in the airways [26]. In our study, we found that erythromycin had a mild effect in decreasing viability of P. aeruginosa, which is consistent with the findings reported by Takesh’s team [27]. In addition to being a mucolytic agent, ambroxol is known to have an antioxidant and anti-inflammatory effect in the treatment of pulmonary infectious diseases [6]. In a recent study, ambroxol appeared to reduce the production of alginate by P. aeruginosa by increasing the expression of mucA gene and decreasing expression of algD, algU, and algR genes. In addition, ambroxol treatment was also associated with decreased activity of diphospho-dmannose dehydrogenase (GDP-mannose dehydrogenase; GMD) [28]. The genes and GMD mentioned above are critical to the formation of alginate. Thus, ambroxol appears to reduce the production of alginate at two levels: genetic expression and enzyme activity. Ambroxol is also known to stimulate the synthesis and secretion of exogenous pulmonary surfactant (PS) in lungs of P. aeruginosainduced infection in rats [29], and appeared to exert a protective effect on cellular lipids against oxidative stress related to endotoxemia or inflammatory responses [29]. The mucolytic, antioxidant and anti-inflammatory properties of ambroxol, in addition to its inhibitory effect on alginate production, explain why the viable cell counts of P. aeruginosa from ETT and infected lungs in the ambroxol group were lower in comparison to that in the control group, but without any significant difference. The SEM of the bacterial biofilm and histopathological changes in lungs had no obvious differences from that in the control group, which is different from the result of Fang Li’s team [27]. In our experiment, we administered ambroxol at the dose of 1.07 mg/mL, whereas Fang Li’s team adopted a dose of 3.75 mg/mL. However, when ambroxol or erythromycin was used in combination with ciprofloxacin synergistically and respectively, viable bacterial counts or SEM findings and histopathological changes showed a much greater effect than that observed in the control group or in ambroxol/erythromycin alone groups. Moreover, ambroxol combined group was better than erythromycin combined group in eradicating biofilms. We speculate that the difference is attributable to the direct effect of ambroxol on biofilm-alginate, by modulating the relevant genes and enzyme activity. In conclusion, we demonstrate that erythromycin or ambroxol combined with ciprofloxacin could eliminate P. aeruginosa biofilms. Ambroxol appeared to outperform erythromycin with a higher efficacy in this respect. Ambroxol plus ciprofloxacin appears to be a good candidate therapy for treatment of P. aeruginosa infections associated with endotracheal intubation. This study serves to provide a theoretical basis for the use of a combination of ambroxol with other sensitive antibiotics, as a strategy to counter biofilms.
5. Conclusion Erythromycin or ambroxol in combination with ciprofloxacin could reduce or even eliminate P. aeruginosa biofilms. Ciprofloxacin
plus ambroxol appears to have a higher efficacy in eradicating P. aeruginosa biofilms as compared to that of ambroxol plus erythromycin. Authors’ contributions Jialin Yu, Lizhong Du and Chen Cheng conceived of the study, and participated in its design and coordination and helped to draft the manuscript. Qi Lu and Yu He participated in the modified the manuscript. Tao Ran helped performed the statistical analysis. All authors read and approved the final manuscript. Funding All phases of this study were supported by the National Natural Science Foundation of China (No. 81370744), Doctoral Degree Funding from Chinese Ministry of Education (No. 20135503110009), the subproject of National Science & Technology Pillar Program during the 12th Five-year Plan Period in China (No. 2012BAI04B05). Competing interest The authors declare that they have no competing interests. Acknowledgments We would like to thank Qing Ai for her technical assistance. In addition, we are grateful to Guanxin Liu and Hongdong Li for helpful discussions. References [1] H.A. Abbas, F.M. Serry, E.M. EL-Masry, Combating Pseudomonas aeruginosa biofilms by potential biofilm inhibitors, Asian J. Res. Pharm. Sci. 2 (2012) 66–72. [2] T. Bauer, A. Torres, R. Ferrer, C. Heyer, C. Schultze-Werninghaus, K. Rasche, Biofilm formation in endotracheal tubes. Association between pneumonia and the persistence of pathogens, Monaldi Arch. Chest Dis. 57 (2002) 84–87. [3] J. Pintucci, S. Corno, M. Garotta, Biofilms and infections of the upper respiratory tract, Eur. Rev. Med. Pharmacol. Sci. 14 (2010) 683–690. [4] Liu Y-CC, J.C. Post, Biofilms in pediatric respiratory and related infections, Curr. Allergy Asthma Rep. 9 (2009) 449–455. [5] Q. Lu, J. Yu, L. Bao, T. Ran, H. Zhong, Effects of combined treatment with ambroxol and ciprofloxacin on catheter-associated Pseudomonas aeruginosa biofilms in a rat model, Chemotherapy 59 (2013) 51–56. [6] K.M. Beeh, J. Beier, A. Esperester, L.D. Paul, Anti-inflammatory properties of ambroxol, Eur. J. Med. Res. 13 (12) (2008) 557–562. [7] G. Pulcrano, D. Panellis, G. Domenico, F. Rossano, M.R. Catania, Ambroxol influences voriconazole resistance of Candida parapsilosis biofilm, FEMS Yeast Res. 12 (2012) 430–438. [8] F. Li, W. Wang, L. Hu, L. Li, J. Yu, Effect of ambroxol on pneumonia caused by Pseudomonas aeruginosa with biofilm formation in an endotracheal intubation rat model, Chemotherapy 57 (2011) 173–180. [9] H. Kobayashi, Biofilm disease: its clinical manifestation and therapeutic possibilities of macrolides, Am. J. Med. 99 (1995) 26–30. [10] T. Nagata, H. Mukae, J. Kadota, T. Hayashi, T. Fujii, M. Kuroki, et al., Effect of erythromycin on chronic respiratory infection caused by Pseudomonas aeruginosa with biofilm formation in an experimental murine model, Antimicrob. Agents Chemother. 48 (2004) 2251–2259. [11] K. Tateda, Y. Ishii, T. Matsumoto, N. Furuya, M. Nagashima, T. Matsunaga, et al., Direct evidence for antipseudomonal activity of macrolides: exposure-dependent bactericidal activity and inhibition of protein synthesis by erythromycin, clarithromycin, and azithromycin, Antimicrob. Agents Chemother. 40 (1996) 2271–2275. [12] D. Amsterdam, Susceptibility testing of antimicrobials in liquid media, Antibiot. Lab. Med. 4 (1996) 52–111. [13] N. Kirschvink, G. Vincke, L. Fiévez, C. Onclinx, D. Wirth, M. Belleflamme, et al., Repeated cadmium nebulizations induce pulmonary MMP-2 and MMP-9 production and enphysema in rats, Toxicology 211 (2005) 36–48. [14] B. Chen, J. Yu, G. Liu, L. Hu, L. Li, F. Li, et al., Electron microscopic analysis of biofilm on tracheal tubes removed from intubated neonates and the relationship between bilofilm and lower respiratory infection, Chin. J. Pediatr. 45 (2007) 655–660.
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Please cite this article in press as: C. Cheng, et al., Ciprofloxacin plus erythromycin or ambroxol ameliorates endotracheal tube-associated Pseudomonas aeruginosa biofilms in a rat model, Pathol. – Res. Pract (2015), http://dx.doi.org/10.1016/j.prp.2015.10.003