Heterogeneity of Pseudomonas aeruginosa in Brazilian Cystic Fibrosis Patients

The aim of this study was to assess the diversity and genomic variability of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients being treated at a university hospital in Brazil. Ninety-seven isolates of P. aeruginosa from 43 CF patients were characterized by macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE) and tested for susceptibility to 20 antimicrobial agents by broth microdilution. It was possible to evaluate single isolates from 20 patients and multiple isolates (two to seven) from 23 patients collected during a 22-month period. Among all of the unrelated patients, we detected only one pair of patients sharing a common strain. Among the 77 isolates from 23 patients who had multiple isolates analyzed, we identified 37 major types by PFGE, and five different colonization patterns were recognized. The isolates were susceptible to several antimicrobial agents, although consecutive isolates from the same patient may display differences in their susceptibilities. Mucoid isolates were more resistant (P < 0.001) than nonmucoid isolates to five antibiotics. Our results indicate that CF patients remain colonized by more than one strain of P. aeruginosa for long periods of time. In addition, the finding of several different genotypes in the same patient suggests that the colonizing strain may occasionally be replaced.     

Hypermutable Pseudomonas aeruginosa in Cystic Fibrosis Patients from Two Brazilian Cities

Hypermutable (HPM) strains of Pseudomonas aeruginosa have been found at high frequencies in cystic fibrosis (CF) patients in Europe. We report the results of testing for HPM frequencies, mutator genotype, and antimicrobial resistance of P. aeruginosa strains from Brazilian CF patients. A modified disk diffusion technique was used to quantify antibiotic-resistant subpopulations of an isolate, and estimations of the frequency of mutation to rifampin resistance were determined for 705 isolates from 149 patients attending clinics in two Brazilian cities. Mutations in the mutS gene were detected by sequencing assays. We found 194 (27.5%) HPM isolates in samples from 99 (66.4%) patients. Thirty-five HPM isolates (18.0%) from 31 (31.3%) patients exhibited a high increased spontaneous mutation rate compared with controls, and eight isolates from six patients displayed a defective mutS gene. The dominant HPM population was associated with very low antibiotic resistance levels, while HPM subpopulations were generally more resistant to antimicrobials. A relatively high prevalence of HPM P. aeruginosa in CF patients was associated with surprisingly low antibiotic resistance levels, in contrast to some earlier studies.     

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.     

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.     

Pseudomonas aeruginosa Exotoxin Pyocyanin Causes Cystic Fibrosis Airway Pathogenesis

The cystic fibrosis (CF) airway bacterial pathogen Pseudomonas aeruginosa secretes multiple virulence factors. Among these, the redox active exotoxin pyocyanin (PCN) is produced in concentrations up to 100 μmol/L during infection of CF and other bronchiectatic airways. However, the contributions of PCN during infection of bronchiectatic airways are not appreciated. In this study, we demonstrate that PCN is critical for chronic infection in mouse airways and orchestrates adaptive immune responses that mediate lung damage. Wild-type FVBN mice chronically exposed to PCN developed goblet cell hyperplasia and metaplasia, airway fibrosis, and alveolar airspace destruction. Furthermore, after 12 weeks of exposure to PCN, mouse lungs down-regulated the expression of T helper (Th) type 1 cytokines and polarized toward a Th2 response. Cellular analyses indicated that chronic exposure to PCN profoundly increased the lung population of recruited macrophages, CD4⁺ T cells, and neutrophils responsible for the secretion of these cytokines. PCN-mediated goblet cell hyperplasia and metaplasia required Th2 cytokine signaling through the Stat6 pathway. In summary, this study establishes that PCN is an important P. aeruginosa virulence factor capable of directly inducing pulmonary pathophysiology in mice, consistent with changes observed in CF and other bronchiectasis lungs.Cystic fibrosis (CF) is one of the most common fatal genetic disorders among the Caucasian population, affecting ∼30,000 individuals in the United States alone. CF is caused by mutations in the gene encoding the CF transmembrane regulator, which mediates anion (predominantly chloride, Cl⁻) conductance. Because of temperature-dependent misfolding and misprocessing in the cytoplasm, the most common CF transmembrane regulator mutation, ΔF508, exhibits reduced levels of CF transmembrane regulator localization to the apical membrane of lung epithelial cells, causing reduced levels of Cl⁻ secretion.^1,2 The main pathological feature of CF airways is the accumulation of thick, inspissated mucus, which has been attributed to mechanisms including excessive airway water and sodium absorption by airway epithelia leading to airway surface liquid volume depletion, increased mucus concentration, mucus adhesion to airway surfaces, and delayed mucus transport.^1,2 Defective mucociliary clearance has severe consequences in the lung as patients develop mucus obstruction of large and small airways, goblet cell hyperplasia, neutrophilic infiltration, and poor bacterial clearance, ultimately leading to scarring and airway fibrosis.^1,2 The major clinical problem for CF patients is a progressive loss of lung function caused by chronic lung infection with mucoid Pseudomonas aeruginosa, resulting in the death of >80% of patients.^1,2 The repeated cycles of pro- and anti- inflammatory responses triggered by P. aeruginosa-associated surface antigens, as well as secreted exoproducts, progressively compile the damage on CF lungs.^1,2,3,4Lung damage in P. aeruginosa-infected CF airways has been proposed to be partially because of imbalances between oxidants and antioxidants and between protease and antiprotease activities.^1,2,3,4 In normal airways, the antioxidant capacity exceeds the level of oxidant formation because of the presence of a variety of antioxidants including enzymes, vitamins, metal chelators and thiols. Collectively, these antioxidants protect cellular components from oxidative damage. In P. aeruginosa-infected CF airways, there is an abundant neutrophilic inflammatory response stimulated by both host and bacterial factors. Dysregulated inflammatory responses lead to high levels of cytotoxic phagocyte-derived reactive oxygen species (ROS). ROS are also produced by the redox-cycling activity of pyocyanin (PCN), a blue-colored tricyclic phenazine (Figure 1A) that is produced in concentrations up to 100 μmol/L by P. aeruginosa in CF airways.⁵ Notably, PCN-mediated ROS inhibit catalase activity, deplete cellular antioxidant reduced glutathione, and increase the oxidized reduced glutathione in the bronchiolar epithelial cells.^3,4 Excessive and continuous production of ROS and inhibition of antioxidant mechanisms overwhelm the antioxidant capacity, leading to tissue damage.Open in a separate windowFigure 1Biosynthesis and profiling of PCN in clinical isolates of P. aeruginosa from CF patients. A: PCN is synthesized from phenazine carboxylic acid via enzymatic modification by PhzM and PhzS. B: The majority of P. aeruginosa CF clinical isolates overproduced PCN.As an immunomodulator, PCN inhibits ciliary beating of airway epithelial cells,⁵ nitric oxide production by macrophages and endothelial cells,⁶ prostacyclin production by endothelial cells,⁷ oxidation of leukotriene B4 by neutrophils,⁸ and eicosanoid metabolism by platelets.⁹ PCN also enhances superoxide production,¹⁰ increases apoptosis in neutrophils,^11,12 and inactivates α1-protease inhibitor.¹³ In addition, PCN increases calcium signaling in human airway epithelial cells, stimulates interleukin (IL)-8 release, and inhibits regulated on activation normal T cell expressed and secreted and monocyte chemoattractant protein-1 release in human epithelial cells.^14,15,16,17 PCN furthermore inhibits the expression of IL-2 and its receptor.¹⁸ In animal models, PCN stimulates IL-8 release, neutrophil influx, and bronchoconstriction in sheep and decreases tracheal mucus velocity in sheep, guinea pigs, and baboons.^19,20,21,22Recently, we provided direct evidence that PCN participates in P. aeruginosa virulence using PCN-deficient mutants that were found to be attenuated in their ability to infect mouse lungs in an acute pneumonia model of infection when compared with isogenic wild-type bacteria.²³ These mutants also were less competitive than isogenic parental wild-type bacteria during competitive mixed infection using the agar bead model of chronic lung infections.²³ Thus, the production of PCN appears to confer a growth and/or survival advantage in mixed culture settings. These studies provide the most direct evidence for the importance of PCN in the P. aeruginosa-infected airway.PCN biosynthesis is regulated by the intercellular process of bacterial communication known as quorum sensing,^3,4 a process that is also critical for biofilm formation and full virulence.²⁴ PCN biosynthesis is increased during P. aeruginosa growth in biofilms in vitro.^25,26 As biofilms are the predominant mode of P. aeruginosa growth within CF airways,^1,2,4,27 not surprisingly, PCN concentrations of up to 100 μmol/L have been detected in pulmonary secretions of CF patients.⁵ Because the major CF pathogen is P. aeruginosa, lung epithelia and macrophages are constantly being exposed to PCN, which contribute to oxidative stress and immunomodulation. In this study, we examined whether continuous exposure of mouse lungs to PCN could result in some pathological features commonly found in CF airways of humans.     

Pulmonary Outcome in Cystic Fibrosis Is Influenced Primarily by Mucoid Pseudomonas aeruginosa Infection and Immune Status and Only Modestly by Genotype

Whether allelic variants of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) independently contribute to pulmonary outcome in CF patients has not been resolved. We used both cross-sectional and mixed-model longitudinal analyses of data from CF patients that were at least 12 years old to determine the influence on pulmonary function (percent predicted forced expiratory volume [FEV(1)]) of the CFTR gene genotype, gender, mucoid Pseudomonas aeruginosa (MPA) infection status, presence of total opsonic antibody to MPA, and, separately, the opsonic antibody activity specific to the mucoid exopolysaccharide (MEP) surface antigen. Two different factors were independently associated with the lack of MPA infection: a high level of MEP-specific opsonic activity (MSOA), implicating an immunologically based mechanism of resistance to infection, and a lack of any type of opsonic antibody to MPA, indicative of no significant exposure or infection. This latter phenotype was found in a subset of CF patients who carried at least one uncommon CFTR gene allele suggestive of a genetic basis for resistance to infection in this group of older CF patients. For CF patients in whom both CFTR gene alleles were identified by screening for the 12 most common variants (75% of alleles), cross-sectional analysis showed that MPA infection was best correlated with lower percent predicted FEV(1), while genotype (two versus one DeltaF508 CFTR gene allele) and a low level of MSOA were associated with increased risk of infection. A mixed-model analysis of longitudinal spirometric measurements that considered multiple risk factors to derive regression equations was used to determine which clinical parameters had the greatest effect on the annual rate of decline in percent predicted FEV(1). This analysis showed that the CFTR gene genotype only modestly modified the constant (y intercept) of the derived equations, while gender and MPA infection status had the largest effects on annual rates of decline in percent predicted FEV(1). These results indicate that the CFTR genotype is usually not a primary determinant of pulmonary function in most CF patients, but gender and MPA infection status are. Infection status is potentially influenced by both immunologic (a high level of MSOA) and genetic factors, such as carriage of a CFTR gene allele that leads to a diagnosis of CF but still confers resistance to infection that is comparable to that of the wild-type CFTR gene.     

Role of Adherence in the Pathogenesis of Pseudomonas aeruginosa Lung Infection in Cystic Fibrosis Patients

A correlation has been demonstrated between the in vitro adherence of Pseudomonas aeruginosa to upper respiratory tract epithelium and colonization of the respiratory tract by this organism. Twenty patients with cystic fibrosis (CF) and 20 age-matched controls were examined in this study. All of the CF patients but none of the controls were colonized with P. aeruginosa at the time of study. P. aeruginosa adherence to isolated epithelial cells, as determined by an in vitro assay, was 19.1 ± 1.1 bacteria per buccal epithelial cell in the CF patients and 2.3 ± 0.3 bacteria per cell in the controls (P < 0.01). P. aeruginosa strains of the mucoid colony type adhered in significantly lower numbers to buccal epithelial cells than did strains of the rough colony type (1.8 + 0.1 versus 24.8 ± 0.9, P < 0.001). This difference might explain the common observation that the initial pseudomonas colonization of the respiratory tract of CF patients is due to organisms of the rough colony type. We have further demonstrated that increased P. aeruginosa adherence in vitro varies directly with the loss of a protease-sensitive glycoprotein, fibronectin, from the cell surface, as well as increased levels of salivary proteases in CF patients. When examined by a direct radioimmune binding assay, buccal cells from CF patients possessed only 17% of the total cell surface fibronectin present on similar cells obtained from controls. Salivary protease levels, as measured by ¹²⁵I release from an ¹²⁵I-labeled insoluble fibrin matrix, were increased about threefold in CF patients versus controls. Thus, colonization of the respiratory tract by P. aeruginosa in CF patients correlates well with buccal cell adherence of this organism; increased adherence is associated with decreased amounts of fibronectin on respiratory epithelial cell surfaces and increased levels of salivary proteases.     

Epidemiological Analysis of Sequential Pseudomonas aeruginosa Isolates from Chronic Bronchiectasis Patients without Cystic Fibrosis

PCR fingerprinting was used for the epidemiological investigation of 64 Pseudomonas aeruginosa isolates collected from 16 chronic bronchiectasis patients without cystic fibrosis: 56% of the patients harbored one clone, 12.5% carried a single major type with minor variants, and 31.5% carried two clones. Only a minority of the acquisitions of antibiotic resistance was related to the acquisition of exogenous strains. Mucoid and nonmucoid sets of isolates did not display any consistent differences in their patterns. The genetic similarity among the clones ranged from 10 to 69%. Cross-infection or common-source exposure did not appear to have occurred.     

Serotypes and Antibiotic Susceptibilities of Pseudomonas aeruginosa Isolates from Single Sputa of Cystic Fibrosis Patients

A phenotypic characterization of Pseudomonas aeruginosa from single sputum samples of 21 typical cystic fibrosis patients indicated a high frequency of heterogeneity among isolates on the basis of differences in antibiotic resistance, colony morphology, pigmentation, and serotype. Two or more isolates with different but stable susceptibilities to carbenicillin, gentamycin, streptomycin, tetracycline, chloramphenicol, and sulfamethoxazole plus trimethoprim were detected in 38% of the sputa. Differences generally were independent of the mucoid state of the strain. O-antigen group determination with the Difco typing set showed that two or more serologically distinct strains were present in 10/21 sputum specimens. Nonmucoid derivatives of mucoid isolates almost always retained both the antibiotic susceptibilities and serotype of their parent strain. These data suggest that cystic fibrosis patients may be cocolonized/coinfected by different strains of P. aeruginosa more frequently than generally believed. Alternatively, phenotypically distinct strains from a single patient might arise as phenotypic dissociants from a single infecting strain. Because of the frequency and multiplicity of phenotypically distinct P. aeruginosa isolates which we obtained from our cystic fibrosis patients, it is important to select multiple isolates from sputum cultures for antimicrobial susceptibility testing so as to assess adequately the susceptibility of this organism to antibiotic therapy in cystic fibrosis. We recommend that several colonies of each distinguishable colony type of P. aeruginosa be pooled for the antibiogram.     

Platelet 3H-Serotonin Releasing Immune Complexes Induced by Pseudomonas aeruginosa in Cystic Fibrosis

In vitro formation of immune complexes was studied by ³H-serotonin release from human platelets by P. aeruginosa antigens in the presence of serum From 22 cystic fibrosis patients. chronically infected with mucoid P. aeruginosa (CF + P) and with a pronounced antibody response against these bacteria, and in 24 patients without P. aeruginosa (CF-P). All CF + P patients responded with ³H -serotonin release (16–34%), whereas CF-P patients released less than 15%. In the group of CF4-P patients the number of P. aeruginosa precipitins was correlated to the serotonin liter. Time courses indicated that SH-serotonin release was maximal between 2 and 5 min, and that no further release was observed up to 20 min. There was a gradual increase in ³H -serotonin release with higher platelet concentrations The response was not changed by complement inactivation. and fractionation of serum demonstrated that the serotonin release was dependent on the presence of the immuno-globulin fraction. These experiments support the suggestion of a type 111 reaction being invoked in the lung damage in CF + P patients and also suggest a possible involvement of serotonin in the inflammatory reaction during chronic P. aeruginosa lung infection.     

Specific Pseudomonas Immunoglobulin E Antibodies in Sera of Patients with Cystic Fibrosis

Immunoglobulin E antibodies to Psuedomonas aeruginosa were demonstrated in patients with cystic fibrosis colonized with the bacterium.     

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.     

Pseudomonas aeruginosa Genotype Prevalence in Dutch Cystic Fibrosis Patients and Age Dependency of Colonization by Various P. aeruginosa Sequence Types

The patient-to-patient transmission of highly prevalent Pseudomonas aeruginosa clones which are associated with enhanced disease progression has led to strict segregation policies for cystic fibrosis (CF) patients in many countries. However, little is known about the population structure of P. aeruginosa among CF patients. The aim of the present cross-sectional study was to determine the prevalence and genetic relatedness of P. aeruginosa isolates from CF patients who visited two major CF centers in The Netherlands in 2007 and 2008. These patients represented 45% of the Dutch CF population. P. aeruginosa carriage in the respiratory tract was determined by standard microbiological culture techniques, and all phenotypically different isolates in the first specimens recovered in 2007 and 2008 were genotyped by multilocus sequence typing. A total of 313 (57%) of 551 patients whose samples were cultured carried P. aeruginosa. Two sequence types (STs), ST406 and ST497, were found in 15% and 5% of the patients, respectively, and 60% of the patients harbored a strain that was also found in at least two other patients. The risk ratios for carrying ST406 and ST497 were 17.8 (95% confidence interval [CI], 7.2 to 43.6) for those aged between 15 and 24 years and 6 (95% CI, 1.4 to 26.1) for those aged >25 years. ST406 and ST497 were not genetically linked to previously described epidemic clones, which were also not found in this CF population. The population structure of P. aeruginosa in Dutch CF patients is characterized by the presence of two prevalent STs that are associated with certain age groups and that are not genetically linked to previously described epidemic clones.Pseudomonas aeruginosa is a ubiquitous, versatile bacterium that can infect humans as well as plants and animals. The species is infamous for causing nosocomial infections in immunocompromised patients and patients in intensive care units and is a major cause of morbidity and mortality in patients with cystic fibrosis (CF) (26).The widely held belief that CF patients acquire P. aeruginosa strains mainly from their inanimate environment, with most patients being colonized by unique strains, has been challenged by reports indicating that P. aeruginosa clones may frequently be transmitted between CF patients (3, 6, 18, 19, 23, 24). Some of these clones, such as the Liverpool epidemic strain and the Melbourne epidemic strain, have been associated with enhanced disease progression and higher rates of mortality, respectively (1, 13). In The Netherlands, the patient-to-patient transmission of P. aeruginosa was documented during a summer camp (4). These findings have led to strict segregation policies for CF patients in many countries, including The Netherlands. However, despite these studies, there is little information on the population structure of P. aeruginosa within populations of CF patients. We therefore investigated the prevalence and genetic relatedness of P. aeruginosa isolates compared to those of the international known genotypes in an unbiased cohort representing 45% of the CF population in The Netherlands in 2007 and 2008.     

Proteomic Identification of OprL as a Seromarker for Initial Diagnosis of Pseudomonas aeruginosa Infection of Patients with Cystic Fibrosis

Identification of new immunogenic antigens that diagnose initial Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF) alone or as an adjunct to microbiology is needed. In the present study, a proteomic analysis was performed to obtain a global assessment of the host immune response during the initial P. aeruginosa infection of patients with CF. Matrix-assisted laser desorption ionization-time of flight mass spectrometry was used to identify outer membrane protein L (OprL), a non-type III secretion system (TTSS) protein, as an early immunogenic protein during the initial P. aeruginosa infection of patients with CF. Longitudinal Western blot analysis of sera from 12 of 14 patients with CF detected antibodies to OprL during the initial P. aeruginosa infection. In addition, also detected were antibodies to ExoS, ExoU, or ExoS and ExoU, the latter indicating sequential P. aeruginosa infections during initial infections. Detection of serum reactivity to OprL, along with proteins of the TTSS, and in conjunction with microbiology may diagnose initial P. aeruginosa infections in patients with CF.Cystic fibrosis (CF) is an autosomal recessive multisystem disease which is caused by mutations in the CF transmembrane conductance receptor. Patients with CF have chronic respiratory infections which are the primary cause of morbidity and premature mortality (16). Patients with CF are infected with bacterial pathogens on an age-dependent timeline (16). Typically, Staphylococcus aureus and nonencapsulated Haemophilus influenzae are the first isolates from infants with CF (34, 35). However, Pseudomonas aeruginosa infections in children with CF are associated with progressive lung disease (30, 33). Microbiology is used for the diagnosis of P. aeruginosa, but successful P. aeruginosa isolation can be complicated in nonexpectorating populations of infants and young children with CF (4). The diagnosis and eradication of the initial P. aeruginosa infection with antibiotics to prevent chronic infection and mucoid transformation are important, since this diagnosis influences the quality of life and long-term patient survival (1, 2, 7, 11, 29, 30). Non-culture-based tests, like serology, should assist microbiology in the early diagnosis of P. aeruginosa infection.P. aeruginosa serology continues to be challenging without defined commercially available antigens licensed in the United States that reflect the molecular pathogenesis of P. aeruginosa upon adaptation to the host environment (13). Høiby (24) and Döring and Høiby (10) have detected an antibody response against a pool of antigens from common P. aeruginosa serotypes. Elevating antibody titers against this pool of antigens correlated with worsening P. aeruginosa infections and a poor clinical prognosis. The clinical progression of CF lung disease may be a reflection of the molecular pathogenesis of P. aeruginosa. Recent studies have correlated serological reactivity and known P. aeruginosa virulence factors. West et al. (42) showed that during the initial P. aeruginosa infection of children with CF, detection of serum antibodies to exotoxin A (ETA) and a P. aeruginosa lysate occurred earlier than detection of serum antibodies to elastase or alkaline phosphatase; subsequently, Corech et al. (6) detected antibodies to components of the type III secretion system (TTSS) at a time similar to that of P. aeruginosa Sup and earlier than ETA, showing the potential of measuring the antibody response to components of the TTSS as an indication of initial infection with P. aeruginosa in children with CF. This also indicated a role for TTSS in the initial P. aeruginosa pathogenesis of the CF lung.In the present study, a proteomic analysis was performed to obtain a global assessment of the host immune response during the initial P. aeruginosa infection of patients with CF. The goal was to identify a cellular component of P. aeruginosa that elicits an early immune response to P. aeruginosa infection to provide a stable immunogenic indication of P. aeruginosa infection relative to P. aeruginosa virulence factors that may fluctuate in expression during the course of P. aeruginosa infection, especially following transition from the acute to the chronic infection phase (41). Outer membrane protein L (OprL), a non-TTSS protein, was identified as an early immunogenic protein in the initial P. aeruginosa infection of patients with CF.     

Pseudomonas Aeruginosa Allergy in Cystic Fibrosis

Basophil histamine release by P. aeruginosa standard antigen was examined in cystic fibrosis patients chronically infected with mucoid P. aeruginosa (CF +P) and with pronounced antibody response against these bacteria, and in patients without P. aeruginosa infection (CF +P). All the patients showed eosinophil counts and total IgE, which did not differ significantly from that of normal persons. In the absence of patient's sera, histamine release was only found in two patients in the CF +P group, indicating that type I allergy to P. aeruginosa is not predominating in cystic fibrosis. In the presence of patients' sere significantly more of the CF+P patients responded to P. aeruginosa with histamine release compared with the CF-P patients. The response was lost by complement inactivation and regained by reconstitution of the complement activity. The involvement of a type III-mediated complement-dependent histamine release is therefore suggested in the pathogenesis of lung damage in cystic fibrosis.     

Evaluation of Reference Dilution Test Methods for Antimicrobial Susceptibility Testing of Pseudomonas aeruginosa Strains Isolated from Patients with Cystic Fibrosis

The development of multidrug-resistant Pseudomonas aeruginosa in patients with cystic fibrosis (CF) is most likely a consequence of increasing life expectancy and more prolonged exposure to antibiotics. The optimal method for antibiotic susceptibility testing of CF strains, particularly mucoid P. aeruginosa strains, is unknown. Antimicrobial susceptibilities of 48 CF strains (25 mucoid) and 50 non-CF strains to 12 anti-Pseudomonas agents were tested by both agar dilution and commercially custom-prepared broth microdilution plates (PML Microbiologicals, Portland, Oreg.) in three laboratories simultaneously to determine if broth microdilution could substitute for agar dilution as the reference method in subsequent studies. Comparison of MICs generated by agar dilution and broth microdilution demonstrated correlation coefficients (r) exceeding 0.85 for all agents tested; correlation was excellent for aminoglycosides (r >/= 0.92) and very good for beta-lactam agents including agents paired with a beta-lactamase inhibitor (r >/= 0.87) and for ciprofloxacin (r = 0.86). Correlation was not improved by 48-h readings, but correlation between 24- and 48-h readings ranged between 0.91 and 0.98 for both methods. Interlaboratory variations were minimal, as the percentage of acceptable variations was 94% for both methods, and serious discords were infrequent (<2% of comparisons). However, CF strains were more likely to have serious discords than were non-CF strains (P < 0. 0001), although mucoid strains were not more likely to have serious discords than were nonmucoid strains. In this study, MICs determined by custom-prepared broth microdilution compared favorably with MICs determined by agar dilution. Thus, this broth microdilution assay can serve as a reference method and facilitate future studies to determine the optimal method for antibiotic susceptibility testing of CF strains.