Role of pili in adherence of Pseudomonas aeruginosa to mammalian buccal epithelial cells.

Adherence of Pseudomonas aeruginosa organisms to the upper respiratory epithelium of seriously ill patients in vitro is correlated with subsequent colonization of the respiratory tract by this opportunistic pathogen. The role of pili in the attachment to epithelial cells of P. aeruginosa was studied in an in vitro system employing human buccal epithelial cells and P. aeruginosa pretreated by various means. Pretreatment of the bacteria with proteases, heat, or Formalin caused a significant decrease in adherence. A decrease when compared with controls was also noted in the adherence of P. aeruginosa organisms to buccal epithelial cells preincubated with purified pili prepared from the strain used for adherence testing; however, pili prepared from a heterologous strain failed to block adherence. Similar results were obtained in serological studies when antisera to purified pili prepared from the strain used for adherence testing decreased adherence, whereas heterologous antiserum to pili did not decrease adherence. From these results it appears that pili mediate the adherence of P. aeruginosa organisms to human buccal epithelial cells.     

Asialo GM1 is a receptor for Pseudomonas aeruginosa adherence to regenerating respiratory epithelial cells.

We investigated the implication of asialo GM1 as an epithelial receptor in the increased Pseudomonas aeruginosa affinity for regenerating respiratory epithelial cells from cystic fibrosis (CF) and non-CF patients. Human respiratory epithelial cells were obtained from nasal polyps of non-CF subjects and of CF patients homozygous for the delta F 508 transmembrane conductance regulator protein (CFTR) mutation and cultured according to the explant-outgrowth model. At the periphery of the outgrowth, regenerating respiratory epithelial cells spreading over the collagen I matrix with lamellipodia were observed, characteristic of respiratory epithelial wound repair after injury. P aeruginosa adherence to regenerating respiratory epithelial cells was found to be significantly greater in the delta F 508 homozygous CF group than in the non-CF group (P < 0.001). In vitro competitive binding inhibition assays performed with rabbit polyclonal antibody against asialo GM1 demonstrated that blocking asialo GM1 reduces P. aeruginosa adherence to regenerating respiratory epithelial cells in delta F 508 homozygous cultures (P < 0.001) as well as in non-CF cultures (P < 0.001). Blocking of asialo GM1 was significantly more efficient in CF patients than in non-CF subjects (P < 0.05). Distribution of asialo GM1 as determined by preembedding labelling and immunoelectron microscopy clearly demonstrated the specific apical membrane expression of asialo GM1 by regenerating respiratory epithelial cells, whereas other cell phenotypes did not apically express asialo GM1. These results demonstrate that (i) asialo GM1 is an apical membrane receptor for P. aeruginosa expressed at the surface of CF and non-CF regenerating respiratory epithelial cells and (ii) asialo GM1 is specifically recovered in regenerating respiratory epithelium. These results suggest that in CF, epithelial repair represents the major event which exposes asialo GM1 for P. aeruginosa adherence.     

Unveiling the early events of Pseudomonas aeruginosa adaptation in cystic fibrosis airway environment using a long-term in vitro maintenance

Pseudomonas aeruginosa chronic infections are the major cause of high morbidity and mortality in cystic fibrosis (CF) patients due to the use of sophisticated mechanisms of adaptation, including clonal diversification into specialized CF-adapted phenotypes. In contrast to chronic infections, very little is known about what occurs after CF lungs colonization and at early infection stages.This study aims to investigate the early events of P. aeruginosa adaptation to CF environment, in particular, to inspect the occurrence of clonal diversification at early stages of infection development and its impact on antibiotherapy effectiveness.To mimic CF early infections, three P. aeruginosa strains were long-term grown in artificial sputum (ASM) over 10 days and phenotypic diversity verified through colony morphology characterization. Biofilm sub- and inhibitory concentrations of ciprofloxacin were applied to non- and diversified populations to evaluate antibiotic effectiveness on P. aeruginosa eradication.Our results demonstrated that clonal diversification might occur after ASM colonization and growth. However, this phenotypic diversification did not compromise ciprofloxacin efficacy in P. aeruginosa eradication since a biofilm minimal inhibitory dosage would be applied. The expected absence of mutators in P. aeruginosa populations led us to speculate that clonal diversification in the absence of ciprofloxacin treatments could be driven by niche specialization. Yet, biofilm sub-inhibitory concentrations of ciprofloxacin seemed to overlap niche specialization as “fitter” variants emerged, such as mucoid, small colony and pinpoint variants, known to be highly resistant to antibiotics. The pathogenic potential of all emergent colony morphotypes-associated bacteria, distinct from the wild-morphotypes, revealed that P. aeruginosa evolved to a non-swimming phenotype. Impaired swimming motility seemed to be one of the first evolutionary steps of P. aeruginosa in CF lungs that could pave the way for further adaptation steps including biofilm formation and progress to chronic infection. Based on our findings, impaired swimming motility seemed to be a candidate to disease marker of P. aeruginosa infection development. Despite our in vitro CF model represents a step forward towards in vivo scenario simulation and provided valuable insights about the early events, more and distinct P. aeruginosa strains should be studied to strengthen our results.     

Stationary biofilm growth normalizes mutation frequencies and mutant prevention concentrations in Pseudomonas aeruginosa from cystic fibrosis patients

Bacterial biofilms play an important role in the persistent colonization of the respiratory tract in cystic fibrosis (CF) patients. The trade‐offs among planktonic or sessile modes of growth, mutation frequency, antibiotic susceptibility and mutant prevention concentrations (MPCs) were studied in a well‐defined collection of 42 CF Pseudomonas aeruginosa isolates. MICs of ciprofloxacin, tobramycin, imipenem and ceftazidime increased in the biofilm mode of growth, but not the MPCs of the same drugs. The mutation frequency median was significantly higher in planktonic conditions (1.1 × 10^?8) than in biofilm (9.9 × 10^?9) (p 0.015). Isolates categorized as hypomutable increased their mutation frequency from 3.6 × 10^?9 in the planktonic mode to 6 × 10^?8 in biofilm, whereas normomutators (from 9.4 × 10^?8 to 5.3 × 10^?8) and hypermutators (from 1.6 × 10^?6 to 7.7 × 10^?7) decreased their mutation frequencies in biofilm. High and low mutation frequencies in planktonic growth converge into the normomutable category in the biofilm mode of growth of CF P. aeruginosa, leading to stabilization of MPCs. This result suggests that once the biofilm mode of growth has been established, the propensity of CF P. aeruginosa populations to evolve towards resistance is not necessarily increased.     

Interferon-gamma (IFN-γ) treatment decreases the inflammatory response in chronic Pseudomonas aeruginosa pneumonia in rats

In a rat model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis (CF), we studied whether the inflammatory response could be altered by intraperitoneal treatment with recombinant rat interferon-gamma (rrIFN-γ). Rats were treated either before or after intratracheal challenge with P. aeruginosa embedded in alginate beads. Rats treated after challenge had a significant reduction in the severity of macroscopic lung inflammation compared with rats treated before challenge (P =0·004) and controls (P =0·003). The histopathology in controls was dominated by numerous polymorphonuclear leucocytes (PMN) (≥90%) surrounding the alginate beads like in CF. This could be caused by a Th2-like response. In contrast, a complete shift to a chronic-type inflammation dominated by mononuclear leucocytes (≥90% lymphocytes and plasma cells) and granulomas was observed in both rrIFN-γ-treated groups of rats. This could be caused by a Th1-like response. There was no significant difference in lethality between the groups, and the antibody titres against P. aeruginosa sonicate and alginate were similar in the treated rats and controls. Since the ongoing lung tissue damage in CF patients has been shown to be caused by elastase secreted by PMN, which dominate the P. aeruginosa lung infection, our findings offer a possible new strategy of modifying the inflammatory response in CF patients.     

Pseudomonas aeruginosa: Immune Status in Patients with Cystic Fibrosis

In order to have a better understanding of the clinical significance of Pseudomonas aeruginosa, circulating and secretory antibodies were measured. Of 100 patients diagnosed as having cystic fibrosis (CF) and an atypical mucoid P. aeruginosa cultured from their sputum, each possessed serum precipitins. These immunoprecipitates, however, were not detected in the sera of 40 CF patients, some of whom were chronically ill with pulmonary colonization by typically rough-smooth strains of P. aeruginosa. The sera of 46 CF patients and 27 CF patient parents not colonized by P. aeruginosa were negative for the precipitins. The sera from 15 of 45 chronically ill patients not having CF, however, but harboring P. aeruginosa, also possessed serum precipitins. The sera from 85 subjects not having CF and not clinically infected with P. aeruginosa were negative for precipitins. Serum hemagglutination titers as high as 1:4096 were measured in older CF patients having advanced pulmonary disease and who were infected with mucoid P. aeruginosa. Salivary titers ranged from 1:8 to 1:64. Increased levels of both circulating and secretory antibodies of the immunoglobulin A and G classes were demonstrated in patients with CF. Once a patient with CF becomes colonized with P. aeruginosa a process of conversion from the rough and smooth forms to the mucoid form is almost inevitable. Although the mucoid form predominates in the sputum, intermediates of the various colony types are often present. Serum precipitins were demonstrable only after the appearance of mucoid strains in the sputum of patients with CF. Although antibiotics tend to reduce the number of mucoid microorganisms, they are rarely, if ever, eradicated from these patients'' lungs. Recurrent episodes of servere pulmonary infection and the evidence of increasing antibody formation to mucoid strains indicates the invasiveness of these particular strains.     

Pseudomonas aeruginosa and Periodontal Pathogens in the Oral Cavity and Lungs of Cystic Fibrosis Patients: a Case-Control Study

Cystic fibrosis (CF) is the most frequent lethal genetic disease in the Caucasian population. Lung destruction is the principal cause of death by chronic Pseudomonas aeruginosa colonization. There is a high prevalence of oropharyngeal anaerobic bacteria in sputum of CF patients. This study was carried out due to the lack of results comparing subgingival periodontal pathogenic bacteria between the oral cavity and lungs in patients with CF in relation with P. aeruginosa presence. Our first goal was to detect P. aeruginosa in oral and sputum samples by culture and molecular methods and to determine clonality of isolates. In addition, subgingival periodontal anaerobic bacteria were searched for in sputum. A cross-sectional pilot case-control study was conducted in the CF Reference Center in Roscoff, France. Ten CF patients with a ΔF508 homozygous mutation (5 chronically colonized [CC] and 5 not colonized [NC]) were enrolled. P. aeruginosa was detected in saliva, sputum, and subgingival plaque samples by real-time quantitative PCR (qPCR). Subsequently, periodontal bacteria were also detected and quantified in subgingival plaque and sputum samples by qPCR. In CC patients, P. aeruginosa was recovered in saliva and subgingival plaque samples. Sixteen P. aeruginosa strains were isolated in saliva and sputum from this group and compared by pulsed-field gel electrophoresis (PFGE). Subgingival periodontal anaerobic bacteria were found in sputum samples. A lower diversity of these species was recovered in the CC patients than in the NC patients. The presence of the same P. aeruginosa clonal types in saliva and sputum samples underlines that the oral cavity is a possible reservoir for lung infection.     

Effectiveness of bacteriophages in the sputum of cystic fibrosis patients

Bacteriophages have been shown to be effective for treating acute infections of the respiratory tract caused by antibiotic-resistant bacteria in animal models, but no evidence has yet been presented of their activity against pathogens in complex biological samples from chronically infected patients. We assessed the efficacy of a cocktail of ten bacteriophages infecting Pseudomonas aeruginosa following its addition to 58 sputum samples from cystic fibrosis (CF) patients collected at three different hospitals. Ten samples that did not contain P. aeruginosa were not analysed further. In the remaining 48 samples, the addition of bacteriophages led to a significant decrease in the levels of P. aeruginosa strains, as shown by comparison with controls, taking two variables (time and bacteriophages) into account (p = 0.024). In 45.8% of these samples, this decrease was accompanied by an increase in the number of bacteriophages. We also tested each of the ten bacteriophages individually against 20 colonies from each of these 48 samples and detected bacteriophage-susceptible bacteria in 64.6% of the samples. An analysis of the clinical data revealed no correlation between patient age, sex, duration of P. aeruginosa colonization, antibiotic treatment, FEV1 (forced expiratory volume in the first second) and the efficacy of bacteriophages. The demonstration that bacteriophages infect their bacterial hosts in the sputum environment, regardless of the clinical characteristics of the patients, represents a major step towards the development of bacteriophage therapy to treat chronic lung infections.     

Pseudomonas aeruginosa Alginate Promotes Burkholderia cenocepacia Persistence in Cystic Fibrosis Transmembrane Conductance Regulator Knockout Mice

Pseudomonas aeruginosa, a major respiratory pathogen in cystic fibrosis (CF) patients, facilitates infection by other opportunistic pathogens. Burkholderia cenocepacia, which normally infects adolescent patients, encounters alginate elaborated by mucoid P. aeruginosa. To determine whether P. aeruginosa alginate facilitates B. cenocepacia infection in mice, cystic fibrosis transmembrane conductance regulator knockout mice were infected with B. cenocepacia strain BC7 suspended in either phosphate-buffered saline (BC7/PBS) or P. aeruginosa alginate (BC7/alginate), and the pulmonary bacterial load and inflammation were monitored. Mice infected with BC7/PBS cleared all of the bacteria within 3 days, and inflammation was resolved by day 5. In contrast, mice infected with BC7/alginate showed persistence of bacteria and increased cytokine levels for up to 7 days. Histological examination of the lungs indicated that there was moderate to severe inflammation and pneumonic consolidation in isolated areas at 5 and 7 days postinfection in the BC7/alginate group. Further, alginate decreased phagocytosis of B. cenocepacia by professional phagocytes both in vivo and in vitro. P. aeruginosa alginate also reduced the proinflammatory responses of CF airway epithelial cells and alveolar macrophages to B. cenocepacia infection. The observed effects are specific to P. aeruginosa alginate, because enzymatically degraded alginate or other polyuronic acids did not facilitate bacterial persistence. These observations suggest that P. aeruginosa alginate may facilitate B. cenocepacia infection by interfering with host innate defense mechanisms.Respiratory failure due to lung infection is the major cause of mortality in cystic fibrosis (CF) patients. CF airways are colonized by more than one opportunistic bacterial pathogen, and Pseudomonas aeruginosa is a major pathogen. The other opportunistic bacterial pathogens that are frequently isolated from CF airways include Haemophilus influenzae, Staphylococcus aureus, the Burkholderia cepacia complex (BCC), Stenotrophomonas maltophilia, and methicillin-resistant S. aureus (7). Most individuals with CF experience a characteristic age-related pattern of pulmonary colonization and intermittent exacerbations involving H. influenzae and S. aureus, followed by P. aeruginosa (4, 5). Similarly, accumulating evidence suggests that P. aeruginosa can promote colonization by less commonly observed bacteria, such as S. maltophilia, Achromobacter xylosoxidans, and Mycobacterium abscessus (43). P. aeruginosa has also been implicated in promoting BCC pathogenesis by increasing the adherence of BCC to respiratory epithelial cells and upregulating the expression of BCC virulence factors (17, 31, 32).Chronic P. aeruginosa infections are often associated with a mucoid phenotype due to the production of large quantities of the acidic exopolysaccharide alginate (5). Alginate is an important extracellular virulence factor and has been shown to impair host innate defenses related to phagocytes (1, 13, 15, 18, 26, 30). In CF airways, P. aeruginosa is found in the airway lumen, and hence one may expect large amounts of alginate in airways along with host products. Sputum samples from CF patients have been shown to contain 50 to 200 μg/ml alginate (23, 30). In fact, it is likely that there are much higher concentrations of alginate in CF airways, as sputum samples are mixed with host secretions and hence the concentration of alginate may be underestimated. Since BCC infection generally occurs in patients who have been chronically colonized with mucoid P. aeruginosa, we hypothesized that alginate in the airways may prevent detection of BCC by phagocytes and facilitate colonization of CF lungs by BCC. To test this hypothesis, we infected gut-corrected CF mice with Burkholderia cenocepacia strain BC7 suspended in either phosphate-buffered saline (PBS) (BC7/PBS), P. aeruginosa alginate (BC7/alginate), or enzymatically degraded alginate (BC7/ED-alginate) and examined the persistence of bacteria and the associated lung inflammation. We also examined the effects of alginate on phagocytosis of B. cenocepacia by macrophages and neutrophils and the proinflammatory responses of airway epithelial cells to B. cenocepacia infection.     

Oral bacteria modulate invasion and induction of apoptosis in HEp-2 cells by Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important opportunistic bacterial pathogen, causing infections of the respiratory and other organ systems in susceptible hosts. P. aeruginosa infection is initiated by adhesion to and invasion of mucosal epithelial cells. The failure of host defenses to eliminate P. aeruginosa from mucosal surfaces results in P. aeruginosa proliferation, sometimes followed by overt infection and tissue destruction. There is growing evidence that associates poor oral health and respiratory infection. An in vitro model system for bacterial invasion of respiratory epithelial cells was used to investigate the influence of oral bacteria on P. aeruginosa epithelial cell invasion. Oral pathogens including Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter (Actinobacillus) actinomycetemcomitans increased invasion of P. aeruginosa into HEp-2 cells from one- to threefold. In contrast, non-pathogenic oral bacteria such as Actinomyces naeslundii and Streptococcus gordonii showed no significant influence on P. aeruginosa invasion. P. aeruginosa together with oral bacteria stimulated greater cytokine production from HEp-2 cells than did P. aeruginosa alone. P. aeruginosa in combination with periodontal pathogens also increased apoptosis of HEp-2 cells and induced elevated caspase-3 activity. These results suggest that oral bacteria, especially periodontal pathogens, may foster P. aeruginosa invasion into respiratory epithelial cells to enhance host cell cytokine release and apoptosis.     

Genotypic diversity of Pseudomonas aeruginosa in cystic fibrosis siblings in Qatar using AFLP fingerprinting

Pseudomonas aeruginosa is one of the primary pathogens in patients with cystic fibrosis (CF) and a major cause of morbidity and mortality. Reports of the spread of epidemic or transmissible strains of P. aeruginosa within and across CF centers raised the possibility of clonal spread among siblings with CF. This work reports the genotypic relatedness of P. aeruginosa in CF patients with the CFTR I1234V mutation, and to determine whether the genotypes are identical among CF siblings and among different families with the same CFTR mutation. Sixty-six P. aeruginosa isolates were obtained from sputa/deep-pharyngeal swabs from 27 CF patients belonging to 17 families. Genotypic relatedness was assessed using amplified fragment-length polymorphism (AFLP) fingerprinting. Twenty-three distinct genotypes of P. aeruginosa were identified. Eleven families each had one distinct genotype. In the other 6 families more than one genotype was observed; 3 families each showed two genotypes, 2 families each had three genotypes and 1 family had four genotypes of P. aeruginosa. In several cases, siblings with CF from the same family harbored the same strain of P. aeruginosa, which were different from the genotypes in other families. On the other hand, there was an overlap in P. aeruginosa between closely related families. Some patients show persistent colonization with the same genotype of P. aeruginosa over the longitudinal period. The presence of the same genotypes in siblings of the same family and closely related families suggests cross-transmission of P. aeruginosa or acquisition from common environmental exposure.     

Localization of Burkholderia cepacia Complex Bacteria in Cystic Fibrosis Lungs and Interactions with Pseudomonas aeruginosa in Hypoxic Mucus

The localization of Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) lungs, alone or during coinfection with Pseudomonas aeruginosa, is poorly understood. We performed immunohistochemistry for Bcc and P. aeruginosa bacteria on 21 coinfected or singly infected CF lungs obtained at transplantation or autopsy. Parallel in vitro experiments examined the growth of two Bcc species, Burkholderia cenocepacia and Burkholderia multivorans, in environments similar to those occupied by P. aeruginosa in the CF lung. Bcc bacteria were predominantly identified in the CF lung as single cells or small clusters within phagocytes and mucus but not as “biofilm-like structures.” In contrast, P. aeruginosa was identified in biofilm-like masses, but densities appeared to be reduced during coinfection with Bcc bacteria. Based on chemical analyses of CF and non-CF respiratory secretions, a test medium was defined to study Bcc growth and interactions with P. aeruginosa in an environment mimicking the CF lung. When test medium was supplemented with alternative electron acceptors under anaerobic conditions, B. cenocepacia and B. multivorans used fermentation rather than anaerobic respiration to gain energy, consistent with the identification of fermentation products by high-performance liquid chromatography (HPLC). Both Bcc species also expressed mucinases that produced carbon sources from mucins for growth. In the presence of P. aeruginosa in vitro, both Bcc species grew anaerobically but not aerobically. We propose that Bcc bacteria (i) invade a P. aeruginosa-infected CF lung when the airway lumen is anaerobic, (ii) inhibit P. aeruginosa biofilm-like growth, and (iii) expand the host bacterial niche from mucus to also include macrophages.     

Non-Tuberculous Mycobacteria multispecies biofilms in cystic fibrosis: development of an in vitro Mycobacterium abscessus and Pseudomonas aeruginosa dual species biofilm model

Lung disease in cystic fibrosis (CF) is characterized by the progressive colonization of the respiratory tract by different bacteria, which develop polymicrobial biofilms. In the past decades, there has been an increase in the number of CF patients infected with Non-Tuberculous Mycobacteria (NTM). Although Mycobacterium abscessus is the main NTM isolated globally, little is known about M. abscessus multispecies biofilm formation. In the present study we developed an in vitro model to study the phenotypic characteristics of biofilms formed by M. abscessus and Pseudomonas aeruginosa, a major pathogen in CF. For that purpose, dual species biofilms were grown on polycarbonate membranes with a fixed concentration of P. aeruginosa and different inoculums of M. abscessus. The biofilms were sampled at 24, 48, and 72 h and bacteria were quantified in specific media. The results revealed that the increasing initial concentration of M. abscessus in dual species biofilms had an effect on its population only at 24 and 48 h, whereas P. aeruginosa was not affected by the different concentrations used of M. abscessus. Time elapsed increased biofilm formation of both species, specially between 24 and 48 h. According to the results, the conditions to produce a mature dual species biofilm in which the relative species distribution remained stable were 72 h growth of the mixed microbial culture at a 1:1 ratio. A significant decrease in mycobacterial population in dual compared to single species biofilms was found, suggesting that P. aeruginosa has a negative influence on M. abscessus. Finally, in a proof of concept experiment, young and mature dual species biofilms were exposed to clarithromycin.     

Polymorphonuclear Leukocytes Restrict Growth of Pseudomonas aeruginosa in the Lungs of Cystic Fibrosis Patients

Cystic fibrosis (CF) patients have increased susceptibility to chronic lung infections by Pseudomonas aeruginosa, but the ecophysiology within the CF lung during infections is poorly understood. The aim of this study was to elucidate the in vivo growth physiology of P. aeruginosa within lungs of chronically infected CF patients. A novel, quantitative peptide nucleic acid (PNA) fluorescence in situ hybridization (PNA-FISH)-based method was used to estimate the in vivo growth rates of P. aeruginosa directly in lung tissue samples from CF patients and the growth rates of P. aeruginosa in infected lungs in a mouse model. The growth rate of P. aeruginosa within CF lungs did not correlate with the dimensions of bacterial aggregates but showed an inverse correlation to the concentration of polymorphonuclear leukocytes (PMNs) surrounding the bacteria. A growth-limiting effect on P. aeruginosa by PMNs was also observed in vitro, where this limitation was alleviated in the presence of the alternative electron acceptor nitrate. The finding that P. aeruginosa growth patterns correlate with the number of surrounding PMNs points to a bacteriostatic effect by PMNs via their strong O₂ consumption, which slows the growth of P. aeruginosa in infected CF lungs. In support of this, the growth of P. aeruginosa was significantly higher in the respiratory airways than in the conducting airways of mice. These results indicate a complex host-pathogen interaction in chronic P. aeruginosa infection of the CF lung whereby PMNs slow the growth of the bacteria and render them less susceptible to antibiotic treatment while enabling them to persist by anaerobic respiration.     

Season is associated with Pseudomonas aeruginosa acquisition in young children with cystic fibrosis

Pseudomonas aeruginosa, the principal respiratory pathogen in cystic fibrosis (CF) patients, is ubiquitous in the environment. Initial P. aeruginosa isolates in CF patients are generally environmental in nature. However, little information regarding seasonality of P. aeruginosa acquisition is available. We conducted a retrospective study to evaluate the seasonality of initial P. aeruginosa acquisition in young children with CF in the USA using the Cystic Fibrosis Foundation National Patient Registry from 2003 to 2009. Additionally, we assessed whether seasonal acquisition varied by climate zone. A total of 4123 children met inclusion criteria and 45% (n = 1866) acquired P. aeruginosa during a mean 2.0 years (SD 0.2 years) of follow up. Compared with winter, increased P. aeruginosa acquisition was observed in summer (incidence rate ratio (IRR): 1.22; 95% CI: 1.07–1.40) and autumn (IRR: 1.34; 95% CI: 1.18–1.52), with lower acquisition observed in spring (IRR: 0.81; 95% CI: 0.70–0.94). Seasonal variations in P. aeruginosa acquisition rates in the temperate and continental climate zones were similar to those in the overall cohort. In contrast, no significant seasonal effect was observed in the dry climate zone. In a corresponding analysis, no seasonal difference was observed in the rate of acquisition of Staphylococcus aureus, another common CF respiratory pathogen. These results provide preliminary support that climatic factors may be associated with initial P. aeruginosa acquisition in CF patients. Investigation and identification of specific risk factors, as well as awareness of seasonal variation, could potentially inform clinical recommendations including increased awareness of infection control and prevention strategies.     

Low Rates of Pseudomonas aeruginosa Misidentification in Isolates from Cystic Fibrosis Patients

Pseudomonas aeruginosa is an important cause of pulmonary infection in cystic fibrosis (CF). Its correct identification ensures effective patient management and infection control strategies. However, little is known about how often CF sputum isolates are falsely identified as P. aeruginosa. We used P. aeruginosa-specific duplex real-time PCR assays to determine if 2,267 P. aeruginosa sputum isolates from 561 CF patients were correctly identified by 17 Australian clinical microbiology laboratories. Misidentified isolates underwent further phenotypic tests, amplified rRNA gene restriction analysis, and partial 16S rRNA gene sequence analysis. Participating laboratories were surveyed on how they identified P. aeruginosa from CF sputum. Overall, 2,214 (97.7%) isolates from 531 (94.7%) CF patients were correctly identified as P. aeruginosa. Further testing with the API 20NE kit correctly identified only 34 (59%) of the misidentified isolates. Twelve (40%) patients had previously grown the misidentified species in their sputum. Achromobacter xylosoxidans (n = 21), Stenotrophomonas maltophilia (n = 15), and Inquilinus limosus (n = 4) were the species most commonly misidentified as P. aeruginosa. Overall, there were very low rates of P. aeruginosa misidentification among isolates from a broad cross section of Australian CF patients. Additional improvements are possible by undertaking a culture history review, noting colonial morphology, and performing stringent oxidase, DNase, and colistin susceptibility testing for all presumptive P. aeruginosa isolates. Isolates exhibiting atypical phenotypic features should be evaluated further by additional phenotypic or genotypic identification techniques.The accurate identification of Pseudomonas aeruginosa is a critical component of cystic fibrosis (CF) patient management. Once established within CF lungs, P. aeruginosa is rarely eradicated, leading to increased treatment requirements and an accelerated decline in pulmonary function, quality of life, and life expectancy (10, 13, 27). Emerging evidence indicates that aggressive antipseudomonal therapy at the time of initial acquisition may eliminate P. aeruginosa, preventing the development of chronic infection for months or even years (37). Similarly, separating patients with P. aeruginosa from other CF patients may reduce the spread of multiple-antibiotic-resistant strains capable of person-to-person transmission (16). Such strategies are contingent upon the early and correct identification of these organisms (30).While there is much emphasis on misidentifying P. aeruginosa as another species (39), less attention is paid to falsely identifying other species as P. aeruginosa. Nevertheless, accurate identification of P. aeruginosa is important, as this may avoid prolonged and sometimes unnecessary antibiotic treatments, which could select for other antibiotic-resistant pathogens (6). Similarly, in CF clinics where cohort isolation is practiced as an infection control measure, false identification could mean exposure of the CF patient to potentially transmissible bacteria (2, 17, 28, 33).While most clinical strains of P. aeruginosa are easily identified, respiratory isolates from patients with CF can present a taxonomic challenge (15, 24). Phenotypic identification of P. aeruginosa from patients with CF is often complicated by slow growth, auxotrophic metabolic activity, loss of pigment production, multiple antibiotic resistance, atypical colonial morphology, and development of mucoid exopolysaccharide (14, 25). Commercial identification platforms are also considered unreliable (18, 21, 39). Moreover, CF respiratory secretions may contain other nonfermenting gram-negative bacilli, such as Achromobacter, Stenotrophomonas, and Burkholderia species, which can further impede the identification of P. aeruginosa (29, 32, 35, 39).Although several molecular strategies have been developed recently (1, 35, 39), most clinical microbiology laboratories still identify P. aeruginosa by traditional phenotypic techniques. However, there are few published data describing the frequency at which bacterial species in CF sputum are falsely identified as P. aeruginosa by phenotypic methods. In this study, we used P. aeruginosa-specific duplex real-time (PAduplex) PCR assays, phenotypic analysis, amplified rRNA gene restriction analysis (ARDRA), and partial 16S rRNA gene sequence analysis to assess the rate and extent of misidentification of P. aeruginosa isolates in CF sputum by Australian clinical microbiology laboratories.     

Interleukin-8 Production by Human Airway Epithelial Cells in Response to Pseudomonas aeruginosa Clinical Isolates Expressing Type a or Type b Flagellins

Pseudomonas aeruginosa lung infection is a major cause of morbidity and mortality worldwide. P. aeruginosa flagellin, the main structural protein of the flagellar filament, is a virulence factor with proinflammatory activity on respiratory epithelial cells. P. aeruginosa bacteria express one of two isoforms of flagellin (type a or b) that differ in their primary amino acid sequences as well as in posttranslational glycosylation. In this study, the distribution of type a and b flagellins among 3 P. aeruginosa laboratory strains and 14 clinical isolates (1 ulcerative keratitis, 3 cystic fibrosis, and 10 acute pneumonia isolates) was determined, and their abilities to stimulate interleukin-8 (IL-8) production by human airway epithelial cells was compared. By comparison with the PAK (type a) and PAO1 (type b) prototype laboratory strains, 10/14 (71.4%) of clinical isolates expressed type a and 4/14 (28.6%) expressed type b flagellins. Among four cell lines surveyed, BEAS-2B cells were found to give the greatest difference between constitutive and flagellin-stimulated IL-8 production. All 17 flagellins stimulated IL-8 production by BEAS-2B cells (range, 700 to 4,000 pg/ml). However, no discernible differences in IL-8 production were evident when comparing type a versus type b flagellins or flagellins from laboratory versus clinical strains or among the clinical strains.Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium with a unipolar flagellum. P. aeruginosa is a clinically important opportunistic human pathogen, and its respiratory tract infections are a leading cause of morbidity and mortality in patients with cystic fibrosis, ventilator-associated pneumonia, and compromised immune systems (6). Hospital-acquired pneumonia constitutes the second leading type of nosocomial infection, and P. aeruginosa is the most commonly isolated bacterium from these cases (36). P. aeruginosa lung colonization in cystic fibrosis patients induces a neutrophil-dominated airway inflammatory response that, if untreated, ultimately leads to lung failure and death (41). P. aeruginosa also causes severe eye and urinary tract infections in immunocompromised patients, particularly those with HIV, and in individuals with severe burn wounds (42). Despite antibiotic treatment, mortality rates as high as 40% may occur in acute infections, and multidrug-resistant isolates are increasingly reported (11).Respiratory epithelial cells play a crucial role in the inflammatory response during P. aeruginosa infection (33). Airway epithelial cells produce cytokines and chemokines that initiate and amplify host innate and adaptive immune responses following bacterial colonization. For example, epithelial cells exposed to P. aeruginosa produce interleukin-8 (IL-8), the major chemokine associated with neutrophil extravasation from the vasculature into the lumen of the airways (17). IL-8 and neutrophils are present in increased amounts in the lungs of patients with P. aeruginosa infections (8). A diverse array of P. aeruginosa gene products stimulate IL-8 production by respiratory epithelial cells, including flagellin and pilin, the primary structural proteins of bacterial flagella and pili respectively (9).In addition to its ability to stimulate a proinflammatory host response, P. aeruginosa flagellin also constitutes a bacterial virulence factor. Multiple studies have demonstrated a role for P. aeruginosa flagella in the pathogenesis of experimental and clinical diseases (16, 22, 25). Using a burned-mouse model, nonflagellated P. aeruginosa strains expressing a mutant flagellin gene showed a significant decrease in virulence that was restored when flagellin expression was reinstated (29). Pulmonary infection of mice with P. aeruginosa devoid of flagella also resulted in reduced airway colonization and decreased mortality compared with those in mice infected with flagellated bacteria (12). Because flagella are one of the most immunostimulatory products of P. aeruginosa, it should be possible to modulate airway inflammation and reduce mortality using flagellin-based therapeutics without predisposing the host to invasive bacterial infection. However, to develop such therapies, the immunostimulatory bacterial component, as well as the epithelial cell responses that are activated, requires thorough characterization.Flagellins isolated from laboratory reference strains of P. aeruginosa have been classified as type a or b based upon molecular mass and reactivity with specific antisera (28). The type a flagellins have more variable molecular masses (45 to 52 kDa), whereas the type b proteins show an invariant size of about 53 kDa (1, 4). The discrepancy in sizes between type a and b flagellins results from differences in their primary amino acid sequences as well as in posttranslational glycosylation (4, 39, 43-45). The P. aeruginosa flagellar typing system was developed based upon the analysis of defined laboratory strains, and to our knowledge, clinical isolates, particularly from acute bacterial pneumonia patients, have not been extensively characterized in this manner. Therefore, the present study was undertaken to assess the distribution of type a and b flagellins among a panel of P. aeruginosa clinical isolates and to compare the abilities of the two protein isoforms to stimulate a proinflammatory response by respiratory epithelial cells.     

Role of Flagella in Pathogenesis of Pseudomonas aeruginosa Pulmonary Infection

Pseudomonas aeruginosa strains are opportunistic pathogens associated with infections in immunocompromised hosts and patients with cystic fibrosis. Like many other mucosal pathogens, P. aeruginosa cells express flagella which provide motility and chemotaxis toward preferred substrates but also provide a ligand for clearance by phagocytic cells. We tested the role of flagella in the initial stages of respiratory tract infection by comparing the virulence of fliC mutants in a neonatal mouse model of pneumonia. In the absence of fliC, there was no mortality, compared with 30% mortality attributed to the parental strain PAK or 15% mortality associated with infection due to a pilA mutant PAK/NP (P < 0.0001). The fliC mutants caused pneumonia in only 25% of the mice inoculated, regardless of whether there was expression of the pilus, whereas the parental strain was associated with an 80% rate of pneumonia. Histopathological studies demonstrated that the fliC mutants caused very focal inflammation and that the organisms did not spread through the lungs as seen in infection due to either PAK or PAK/NP. Purified flagellin elicited an intense inflammatory response in the mouse lung. ¹²⁵I-labeled flagellin bound to the glycolipids GM1 and GD_1a and to asialoGM1 in an in vitro binding assay. However, flagellin-mediated binding to epithelial gangliosides was a relatively unusual event, as quantified by binding assays of wild-type or fliC mutant organisms to CHO Lec-2 cells with membrane-incorporated GM1. Fla⁺ organisms but not fliC mutants were efficiently taken up by murine macrophages. P. aeruginosa flagella are important in the establishment of respiratory tract infection and may act as a tether in initial interactions with epithelial membranes. This function is offset by the contribution of flagella to host clearance mechanisms facilitating phagocytic clearance and the role of flagellar genes in mucin binding and clearance.

Flagella are highly complex bacterial organelles which are unusually well conserved among diverse bacterial species. Over 50 genes are involved in the synthesis and function of flagella, suggesting that their preservation and role in chemotaxis and motility are important in the survival of many organisms (24). Flagella facilitate the acquisition of essential nutrients; thus, it seems likely that these organelles have a role in the virulence of pathogenic organisms. However, flagella are known to be highly immunogenic (16), and in certain settings Fla⁺ bacteria may be more readily cleared than Fla⁻ organisms (1).The contribution of flagella to the virulence of the opportunistic pathogen Pseudomonas aeruginosa has been examined in several animal models of infection (11, 19). Fla⁻ mutants were less invasive than motile strains in a mouse burn infection, and the administration of antiflagellum antibody had a significant protective effect (8). In a model of mucosal colonization, globally defective RpoN mutants (Pil⁻ Fla⁻) of P. aeruginosa were more deficient in their ability to colonize than to persist within the murine gastrointestinal tract (21). A rat model of Pseudomonas pneumonia was used to demonstrate the efficacy of human antiflagellum monoclonal antibody in attenuating pulmonary infection and reducing the spread of the organism within the rat lung (13). In these instrumented animals, large inocula of bacteria were instilled directly into the trachea. These studies do not directly address how flagella function in either the initial colonization of the upper respiratory tract or the subsequent infection of the lung by aspiration. Several flagellar genes, although not the flagellin structural gene fliC, have been shown to contribute to binding to respiratory mucin (2, 25). Attachment to mucin facilitates clearance from the respiratory tract via the normal mucociliary escalator. Flagella can function as ligands for macrophages and polymorphonuclear leukocytes (PMNs) which clear organisms from mucosal surfaces. In vivo selection of Fla⁻ mutants of P. aeruginosa has been demonstrated in human pulmonary infection in cystic fibrosis (CF) (14, 15). Although the first of sequential Pseudomonas isolates from a patient were motile and piliated, such as environmental strains of P. aeruginosa, genotypically identical isolates over the course of a chronic infection had an RpoN-like phenotype and failed to express functional flagella (15). There may be significant selective pressure for the emergence of Fla⁻ mutants which are less efficiently cleared by phagocytic cells (16, 29). Some respiratory pathogens such as Bordetella pertussis are nonmotile, and recent studies suggest that a bovine pathogen, Bordetella bronchoseptica, which can be motile, specifically turns off flagellar expression during the initial stages of infection (1).Since P. aeruginosa flagella are antigenically relatively homogeneous, falling into two large groups based on structural analysis and reactivity to antisera (28), they may provide a useful target for vaccine development. The ability of antiflagellum antibody to ameliorate infection in the rat lung suggests that flagella may provide a useful target for immune intervention and may be most effective as a strategy to block the initial infection of the respiratory tract by motile strains. However, the importance of normal flagellar function in the earliest stages of P. aeruginosa respiratory infection has not been established for genetically characterized strains. The purpose of this study was to examine (i) the virulence of wild-type and fliC mutant strains in the development of murine pneumonia and (ii) participation of flagella in stimulation of the subsequent immune response to the organism.     

Antibody response of rabbits and cystic fibrosis patients to an alginate-specific outer membrane protein of a mucoid strain of Pseudomonas aeruginosa

The aim of this work was to study the immunogenicity of an outer membrane (OM) protein (AIgE; 54 kDa) which is produced solely by mucoid, i.e. alginate-producing strains of P. aeruginosa. The source of AIgE used for our study was the mucoid strain CF3/M1 originally isolated from sputum of a cystic fibrosis (CF) patient. The purified non-denatured protein served as antigen to raise polyclonal monospecific anti-AIgE antibodies in rabbits and to assay sera from 41 cystic fibrosis (CF) patients for anti-AIgE antibodies. According to clinical protocols the sputa of 22 CF patients were positive for P. aeruginosa , 18 were negative and one case was unknown. Our ELISA studies showed that high titers of anti-AIgE antibodies (IgG) corresponded well with the infection status of the CF patients. None of 23 control sera derived from healthy volunteers contained significant levels of anti-AIgE antibodies. Thus the ELISA should be considered as a sensitive diagnostic tool for the early detection of mucoid P. aeruginosa infections in CF patients. Furthermore, we suggest to include AIgE in a multicomponent experimental vaccine for potential protection of non-colonized CF patients from colonization with mucoid P. aeruginosa.