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.

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The present paper presents a hypothesis aimed at explaining why venous leg ulcers, pressure ulcers, and diabetic foot ulcers develop into a chronic state. We propose that the lack of proper wound healing is at least in part caused by inefficient eradication of infecting, opportunistic pathogens, a situation reminiscent of chronic Pseudomonas aeruginosa infections found in patients suffering from cystic fibrosis (CF). We have analyzed sections from chronic wounds by fluorescence in situ hybridization and found distinct microcolonies--the basal structures of bacterial biofilms. Several researchers have previously reported that another important hallmark of biofilm formation is development of increased tolerance to various antimicrobial measures and treatments. Furthermore, the immune response to infecting bacteria in the cystic fibrosis lung is dominated by polymorphonuclear neutrophils (PMNs), and we have recently shown that in vitro biofilms of P. aeruginosa produce a shielding mechanism that offers protection from the phagocytic activity of PMNs. We hypothesize that the presence of P. aeruginosa in biofilms, and the lack of concomitant elimination by attended PMNs, are the main causes of inefficient eradication by antibiotic treatment and antimicrobial activity of the innate immune system, respectively.

Bacteria survive in nature by forming biofilms on surfaces and probably most, if not all, bacteria (and fungi) are capable of forming biofilms. A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and extracellular DNA. Bacterial biofilms are resistant to antibiotics, disinfectant chemicals and to phagocytosis and other components of the innate and adaptive inflammatory defense system of the body. It is known, for example, that persistence of staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infections in cystic fibrosis patients are caused by biofilm growing mucoid strains. Gradients of nutrients and oxygen exist from the top to the bottom of biofilms and the bacterial cells located in nutrient poor areas have decreased metabolic activity and increased doubling times. These more or less dormant cells are therefore responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations. Bacteria in biofilms communicate by means of molecules, which activates certain genes responsible for production of virulence factors and, to some extent, biofilm structure. This phenomenon is called quorum sensing and depends upon the concentration of the quorum sensing molecules in a certain niche, which depends on the number of the bacteria. Biofilms can be prevented by antibiotic prophylaxis or early aggressive antibiotic therapy and they can be treated by chronic suppressive antibiotic therapy. Promising strategies may include the use of compounds which can dissolve the biofilm matrix and quorum sensing inhibitors, which increases biofilm susceptibility to antibiotics and phagocytosis.

Between 1 and 2% of the population in the developed world experiences a nonhealing or chronic wound characterized by an apparent arrest in a stage dominated by inflammatory processes. Lately, research groups have proposed that bacteria might be involved in and contribute to the lack of healing of these wounds. To investigate this, we collected and examined samples from chronic wounds obtained from 22 different patients, all selected because of suspicion of Pseudomonas aeruginosa colonization. These wound samples were investigated by standard culturing methods and peptide nucleic acid-based fluorescence in situ hybridization (PNA FISH) for direct identification of bacteria. By means of the culturing methods, Staphylococcus aureus was detected in the majority of the wounds, whereas P. aeruginosa was observed less frequently. In contrast, using PNA FISH, we found that a large fraction of the wounds contained P. aeruginosa. Furthermore, PNA FISH revealed the structural organization of bacteria in the samples. It appeared that P. aeruginosa aggregated as microcolonies imbedded in the matrix component alginate, which is a characteristic hallmark of the biofilm mode of growth. The present investigation suggests that bacteria present within these wounds tend to be aggregated in microcolonies imbedded in a self-produced matrix, characteristic of the biofilm mode of growth. Additionally, we must conclude that there exists no good correlation between bacteria detected by standard culturing methods and those detected by direct detection methods such as PNA FISH. This strongly supports the development of new diagnostic and treatment strategies for chronic wounds.