Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients

Abstract

The present study was undertaken to investigate the appearance and location of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung and in sputum. Samples include preserved tissues of CF patients who died due to chronic P. aeruginosa lung infection prior to the advent of intensive antibiotic therapy, explanted lungs from 3 intensively treated chronically P. aeruginosa infected CF patients and routine sputum from 77 chronically P. aeruginosa infected CF patients. All samples were investigated microscopically using hematoxylin-eosin (HE), Gram and alcian-blue stain, PNA FISH and immunofluorescence for alginate.Investigation of the preserved tissues revealed that prior to aggressive antibiotic therapy, P. aeruginosa infection and destruction of the CF lung correlated with the occurrence of mucoid (alginate) bacteria present in aggregating structures surrounded by pronounced polymorphonuclear-leukocyte (PMN) inflammation in the respiratory zone (9/9). Non-mucoid bacteria were not observed here, and rarely in the conductive zone (1/9). However, in the explanted lungs, the P. aeruginosa aggregates were also mucoid but in contrast to the autopsies, they were very rare in the respiratory zone but abundant in the sputum of the conductive zone (3/3), which also contained abundances of PMNs (3/3). Non-mucoid and planktonic P. aeruginosa were also observed here (3/3).In conclusion, the present intensive antibiotic therapy of chronic P. aeruginosa infections, at the Copenhagen CF Centre, seems to restrain but not eradicate the bacteria from the conductive zone, whereas the remaining healthy respiratory zone appears to be protected, for a long period, from massive biofilm infection. This strongly suggests that the conductive zone serves as a bacterial reservoir where the bacteria are organized in mucoid biofilms within the mucus, protected against antibiotics and host defenses.

A Winogradsky-based culture system shows an association between microbial fermentation and cystic fibrosis exacerbation

“…This treatment strategy is inherently biased towards aerobic microbes that grow on media selected by clinical microbiologists (Burns and Rolain, 2014). There is growing evidence that CF microbes survive in a low oxygen environment and many grow anaerobically deep in the mucus plugs of CF lungs (Worlitzsch et al, 2002(Worlitzsch et al, , 2009Bjarnsholt et al, 2009;Lipuma, 2010). Thus conventional culture on microbiological media misses important components of the CF microbial community that may contribute to exacerbations and provides antibiotic resistance profiles from microbes not grown in a state in which they cause disease.…”

The clinical impact of bacterial biofilms

“…P. aeruginosa is able to survive by switching to the biofilm mode of growth (Figures 3, 4), which provides tolerance to the inflammatory defense mechanism and antibiotic therapy. Biofilm adaptation also allows survival in the aerobic respiratory zone, the conductive zone of the lungs which contain anaerobic sputum, and the paranasal sinuses where the mucus also has a decreased oxygen concentration [2][3][35][36][37][38][39][40]. During the adaptation, mucoid (biofilm mode of growth) and non-mucoid phenotypes are split off due to mutations ( Figure 5, Table 3).…”

“…Biofilm growth occurs on natural surfaces such as teeth [4], heart valves (endocarditis) [5], in the lungs of cystic fibrosis (CF) patients causing chronic bronchopneumonia [2], in the middle ear in patients with chronic and secretory otitis media [6][7], in chronic rhinosinusitis [8], in chronic The bold fonts indicate biofilm specific features [3].…”

“…The recommended method for treatment of chronic P. aeruginosa biofilm infection is chronic suppressive antibiotic therapy [2,48] that is started when the chronic infection is diagnosed (continuous colonization at the monthly bacteriological examinations for 6 months and/ or increased level of antibodies against P. aeruginosa) [49]. The chronic suppressive therapy consists of daily nebulized colistin or tobramycin for the rest of the patient's life combined with either regular 2-week courses every 3 months of intravenous anti-pseudomonas antibiotics (combination therapy of 2 antibiotics: tobramycin or colistin + ceftazidime, or piperacillin/tazobactam, or carbapenem, or aztreonam, or ciprofloxacin) or ad hoc intravenous therapy when clinical manifestations occur [48].…”

“…Biofilm growing bacteria cause chronic infections [1] which are characterized by persisting inflammation and tissue damage [2]. Chronic infections, including foreign body infections, are infections that 1) persist in spite of antibiotic therapy, the innate and adaptive immune system and inflammatory response of the host, and 2) in contrast to colonization, are characterized by immune response and persisting pathology [3] (Table 1).…”