Niels Høiby

Antibiotic resistance of bacterial biofilms

A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and DNA. Bacterial biofilms cause chronic infections because they show increased tolerance to antibiotics and disinfectant chemicals as well as resisting phagocytosis and other components of the bodys defence system. The persistence of, for example, staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infection in cystic fibrosis patients is caused by biofilm-growing mucoid strains. Characteristically, gradients of nutrients and oxygen exist from the top to the bottom of biofilms and these gradients are associated with decreased bacterial metabolic activity and increased doubling times of the bacterial cells; it is these more or less dormant cells that are responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations as well as with quorum-sensing-regulated mechanisms. Conventional resistance mechanisms such as chromosomal beta-lactamase, upregulated efflux pumps and mutations in antibiotic target molecules in bacteria also contribute to the survival of biofilms. Biofilms can be prevented by early aggressive antibiotic prophylaxis or therapy and they can be treated by chronic suppressive therapy. A promising strategy may be the use of enzymes that can dissolve the biofilm matrix (e.g. DNase and alginate lyase) as well as quorum-sensing inhibitors that increase biofilm susceptibility to antibiotics.

Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors

Traditional treatment of infectious diseases is based on compounds that kill or inhibit growth of bacteria. A major concern with this approach is the frequent development of resistance to antibiotics. The discovery of communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria without interfering with growth. Compounds that can override communication signals have been found in the marine environment. Using Pseudomonas aeruginosa PAO1 as an example of an opportunistic human pathogen, we show that a synthetic derivate of natural furanone compounds can act as a potent antagonist of bacterial quorum sensing. We employed GeneChip® microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug specifically targeted quorum sensing systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa biofilms increased bacterial susceptibility to tobramycin and SDS. In a mouse pulmonary infection model, the drug inhibited quorum sensing of the infecting bacteria and promoted their clearance by the mouse immune response.

Inhibition of quorum sensing in Pseudomonas aeruginosa biofilm bacteria by a halogenated furanone compound

Novel molecular tools have been constructed which allow for in situ detection of N-acyl homoserine lactone (AHL)-mediated quorum sensing in Pseudomonas aeruginosa biofilms. The reporter responds to AHL activation of LasR by expression of an unstable version of the green-fluorescent protein (Gfp). Gfp-based reporter technology has been applied for non-destructive, single-cell-level detection of quorum sensing in laboratory-based P. aeruginosa biofilms. It is reported that a synthetic halogenated furanone compound, which is a derivative of the secondary metabolites produced by the Australian macroalga Delisea pulchra, is capable of interfering with AHL-mediated quorum sensing in P. aeruginosa. It is demonstrated that the furanone compound specifically represses expression of a PlasB-gfp reporter fusion without affecting growth or protein synthesis. In addition, it reduces the production of important virulence factors, indicating a general effect on target genes of the las quorum sensing circuit. The furanone was applied to P. aeruginosa biofilms established in biofilm flow chambers. The Gfp-based analysis reveals that the compound penetrates microcolonies and blocks cell signalling and quorum sensing in most biofilm cells. The compound did not affect initial attachment to the abiotic substratum. It does, however, affect the architecture of the biofilm and enhances the process of bacterial detachment, leading to a loss of bacterial biomass from the substratum.

Why chronic wounds will not heal: a novel hypothesis

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. 1,2 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.

Antibiotic treatment of initial colonization with Pseudomonas aeruginosa postpones chronic infection and prevents deterioration of pulmonary function in cystic fibrosis

Chronic pulmonary infection with Pseudomonas aeruginosa (PA) develops in most patients with cystic fibrosis (CF) and is associated with a poor prognosis. Much effort has been directed toward treating the chronic infection, but it is almost impossible to eradicate it once established; therefore, prevention is preferable. Since 1989 CF patients at the Danish CF Center in Copenhagen have been treated with an intensive three‐step‐protocol consisting of colistin inhalations and oral ciprofloxacin at the time of initial PA colonization. This study compares 48 patients treated according to this intensive protocol with 43 historic controls. The study was carried out over 44 months and included 218 patient‐years. Only 16% of the treated patients developed chronic PA infection after 3 1/2 years compared with 72% of the control patients (Kaplan Meier estimate, P < 0.005, log rank test). This indicates that aggressive treatment prevented or delayed chronic PA infection in 78% of the patients for 3 1/2 years. Furthermore, aggressive treatment maintained or increased pulmonary function (forced vital capacity and forced expiratory volume in 1 second in percent of predicted values) during the year after inclusion compared with the control group, in which pulmonary function declined (P < 0.01, Mann‐Whitney test). Although some of the treated patients eventually developed chronic PA infection, these patients had significantly better pulmonary function at the onset of chronic PA infection compared with control patients (P < 0.001, Mann‐Whitney test). When the different steps in the intensive three‐step‐protocol were analyzed, there was a trend suggesting that 3 months of high‐dose treatment with colistin inhalation and oral ciprofloxacin produced the best results in terms of postponement or prevention of chronic PA infection (P < 0.05). Pediatr. Pulmonol. 1997; 23:330–335.

Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients

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.

Association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis

Mannose-binding lectin (MBL) is a key factor in innate immunity, and lung infections are the leading cause of morbidity and mortality in cystic fibrosis (CF). Accordingly, we investigated whether MBL variant alleles, which are associated with recurrent infections, might be risk factors for CF patients. In 149 CF patients, different MBL genotypes were compared with respect to lung function, microbiology, and survival to end-stage CF (death or lung transplantation). The lung function was significantly reduced in carriers of MBL variant alleles when compared with normal homozygotes. The negative impact of variant alleles on lung function was especially confined to patients with chronic Pseudomonas aeruginosa infection. Burkholderia cepacia infection was significantly more frequent in carriers of variant alleles than in homozygotes. The risk of end-stage CF among carriers of variant alleles increased 3-fold, and the survival time decreased over a 10-year follow-up period. Moreover, by using a modified life table analysis, we estimated that the predicted age of survival was reduced by 8 years in variant allele carriers when compared with normal homozygotes. Presence of MBL variant alleles is therefore associated with poor prognosis and early death in patients with CF.

Prevention of chronic Pseudomonas aeruginosa colonisation in cystic fibrosis by early treatment

To assess whether chronic pulmonary colonisation with Pseudomonas aeruginosa in cystic fibrosis is preventable, 26 patients who had never received anti-pseudomonas chemotherapy were randomly allocated to groups receiving either no anti-pseudomonas chemotherapy or oral ciprofloxacin and aerosol inhalations of colistin twice daily for 3 weeks, whenever Ps aeruginosa was isolated from routine sputum cultures. During the 27 months of the trial, infection with Ps aeruginosa became chronic in significantly fewer treated than untreated subjects (2 [14%] vs 7 [58%]; p less than 0.05) and there were significantly fewer Ps aeruginosa isolates in routine sputum cultures in the treated group (49/214 [23%] vs 64/158 [41%]; p = 0.0006). Thus, chronic colonisation with Ps aeruginosa can be prevented in cystic fibrosis by early institution of anti-pseudomonas chemotherapy.

N-Acylhomoserine-lactone-mediated communication between pseudomonas aeruginosa and Burkholderia cepacia in mixed biofilms

Pseudomonas aeruginosa and Burkholderia cepacia are capable of forming mixed biofilms in the lungs of cystic fibrosis patients. Both bacteria employ quorum-sensing systems, which rely on N-acylhomoserine lactone (AHL) signal molecules, to co-ordinate expression of virulence factors with the formation of biofilms. As both bacteria utilize the same class of signal molecules the authors investigated whether communication between the species occurs. To address this issue, novel Gfp-based biosensors for non-destructive, in situ detection of AHLs were constructed and characterized. These sensors were used to visualize AHL-mediated communication in mixed biofilms, which were cultivated either in artificial flow chambers or in alginate beads in mouse lung tissue. In both model systems B. cepacia was capable of perceiving the AHL signals produced by P. aeruginosa, while the latter strain did not respond to the molecules produced by B. cepacia. Measurements of extracellular proteolytic activities of defined quorum-sensing mutants grown in media complemented with AHL extracts prepared from culture supernatants of various wild-type and mutant strains supported the view of unidirectional signalling between the two strains.

The clinical impact of bacterial biofilms.

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.