A novel method for investigating Burkholderia cenocepacia infections in patients with cystic fibrosis and other chronic diseases of the airways

Wijers et al. BMC Microbiology (2016) 16:200 DOI 10.1186/s12866-016-0811-7 METHODOLOGY ARTICLE Open Access A novel method for investigating Burkholderia cenocepacia infections in patients with cystic fibrosis and other chronic diseases of the airways Christiaan D. M. Wijers, Ryan Vagedes and Christine Weingart* Abstract Background: Burkholderia cenocepacia is a Gram-negative, opportunistic pathogen that is a cause of morbidity and mortality in patients with cystic fibrosis (CF). Research efforts over the past few decades contributed to our understanding of these infections by identifying virulence factors. However, little is known about how this pathogen adapts to the harsh environment found inside the CF airways, which is characterized by a unique mucus containing high concentrations of inflammatory markers. The current study developed a novel model to further investigate this phenomenon. Results: Monolayers of human A549 lung carcinoma cells (HLCCs) were exposed to a mixture of artificial CF sputum medium (ASMDM) in tissue culture growth medium, and subsequently infected with B. cenocepacia K56-2 for 24 h. The data showed that this model supported B. cenocepacia growth. In addition, consistent with similar studies using current models such as CF airway tissue samples, HLCC viability was reduced by more than 70 % when grown in 60 % ASMDM and infected with B. cenocepacia compared to mock-infected controls and medium alone. Furthermore, the amount of B. cenocepacia cells associated with the HLCC monolayer was more than 10 times greater in 60 % ASMDM when compared to medium controls. Conclusions: These findings suggest that HLCC monolayers in 60 % ASMDM serve as a valid alternative to study B. cenocepacia infections in patients with CF, and possibly other chronic diseases of the airways. Furthermore, the results obtained in this study suggest an important role for CF sputum in B. cenocepacia pathogenesis. Keywords: Burkholderia, cenocepacia, Tissue culture, CF sputum, PCD sputum, Model Background Mucus plays an important protective role in our host defenses by trapping inhaled particles such as pathogens that are subsequently transported out of the airways by ciliated epithelial cells. For this mucociliary clearance to work effectively, the cilia must constantly be moving in the thin, moist layer of mucus. Some diseases such as cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) impair this clearance mechanism resulting in chronic lung infections. More specifically, mutations in the cystic fibrosis conductance regulator cause a disruption in the * Correspondence: Department of Biological Sciences, Denison University, 100 West College Street, Granville, OH 43023, USA transfer of Cl- across the cell membrane. Consequently, the mucus layer becomes thick and dehydrated preventing the successful removal of the particles. PCD patients have defective cilia structure causing the cilia to beat abnormally. Previous research indicated that there exists striking similarities in terms of mucus biophysical and chemical properties between PCD and CF sputum [1–3]. In addition to impaired mucociliary clearance, both diseases have a neutrophil-dominated inflammation in the airways. These conditions are challenging for many bacteria, however highly adaptable pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenza survive in the CF and PCD airways (see review [2, 4]) and B. cenocepacia thrives in the CF airways (see review [4–6]). © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wijers et al. BMC Microbiology (2016) 16:200 Burkholderia cenocepacia is a member of the Burkholderia cepacia Complex (BCC) that comprises 18 species that are Gram-negative opportunistic pathogens [7]. It is innately resistant to a wide array of antibiotics including aminoglycosides, quinolones, and β-lactams [8], (see review [4]). It possesses a variety of virulence factors such as cable pili, biofilm formation, degradative enzymes (see review [9]), (see review [10]), and it is transmitted from person-to-person [11] and from the environment [12]. While the majority of CF patients infected with B. cenocepacia experiences a gradual deterioration of lung function [6, 8], nearly 20 % of infected CF patients experience “cepacia syndrome”. This is a fatal case of necrotizing pneumonia sometimes associated with septicemia that may lead to death within one year [6, 13]. Because of these attributes, this pathogen poses a serious threat to CF patients. Over the past decade progress has led to a greater understanding of how B. cenocepacia causes disease in CF patients (see review [9]). However, because there is still no definite cure, further research is needed, on how this pathogen adapts to the harsh CF airways as such information could lead to new forms of treatment. Unfortunately, for investigative purposes, it can be very difficult to acquire sputum or tissue samples from CF patients, and there exists a need for an alternative, readily available method to investigate B. cenocepacia infections in CF patients and patients with similar chronic diseases of the airways. Fung et al. [14] developed an artificial sputum medium (ASMDM) that approximates the sputum found in CF patients in terms of components, concentrations of the components, and physical properties. In their study, they found that Pseudomonas aeruginosa, another opportunistic pathogen commonly found in CF airways (see review [4, 15]) grew normally and deeply invaded the ASMDM, suggesting that ASMDM mimicked the CF lower airway mucus well [14]. In addition, the fact that ASMDM seems to be a good substitute for CF mucus and a good growth medium for P. aeruginosa suggests that ASMDM would be a suitable growth medium for B. cenocepacia. Nevertheless, using ASMDM by itself is not sufficient to simulate the in vivo conditions in the lungs of CF patients, as this model lacks live host cells that can respond to a B. cenocepacia infection. Saijan et al. investigated B. cenocepacia infections in the presence of well-differentiated human CF cells and in non-CF human lung epithelial cells [16]. They found that infected CF cells contained significantly more bacteria in both the mucosal layer and inside the cell layer when compared to infected non-CF cells [16]. This study showed that B. cenocepacia penetrated the mucosal and cellular layers which is comparable to its activity in (...truncated)