Pathogen In Cystic Fibrosis Patients Research Articles

Structural Study of the Lipopolysaccharide O‐Antigen Produced by the Emerging Cystic Fibrosis Pathogen Pandoraea pulmonicola

AbstractThis is the first report on the molecular structure of the O‐chain of the lipopolysaccharide produced by the emerging and highly virulent clinical isolate Pandoraea pulmonicola strain LMG 18108, an opportunistic human pathogen in cystic fibrosis patients. Monosaccharide analysis and 2D NMR spectroscopy revealed a novel polysaccharide, the structure of which consists of the trisaccharide repeating unit [→2‐β‐D‐Quip3NAcyl‐(1→4)‐α‐D‐GalpNAc‐(1→3)‐α‐D‐GlcpNAc‐1→]n. The quinovosamine residue was found to be linked by a very unusual acyl substituent, a five‐membered‐ring acyl residue, the 3‐hydroxy‐2,3‐dimethyl‐5‐oxoprolyl group.

First Description of an RND-Type Multidrug Efflux Pump in Achromobacter xylosoxidans, AxyABM

Achromobacter xylosoxidans is an emerging pathogen in cystic fibrosis patients. The multidrug resistance of these bacteria remains poorly understood. We have characterized in a clinical strain the first resistance-nodulation-cell division (RND)-type multidrug efflux pump in this species: AxyABM. The inactivation of the transporter component axyB gene led to decreased MICs of cephalosporins (except cefepime), aztreonam, nalidixic acid, fluoroquinolones, and chloramphenicol.

Studying the Effect of Alginate Overproduction on <I>Pseudomonas aeruginosa</I> Biofilm by Atomic Force Microscopy

Pseudomonas aeruginosa is the most important pathogen in cystic fibrosis patients and forms biofilms in the lung. P. aeruginosa strains isolated from the lungs of the patients have a mucoid phenotype overproducing alginate. The phenotype forms highly structured biofilms which are more resistant to antibiotics than biofilms formed by its nonmucoid phenotype. Conversion to the alginate-overproducing phenotype occurs through a mutation in rpoN gene in the strains. The biofilms formed by the alginate-overproducing phenotype are highly sticky, but their stickiness has not been measured. Herein, the stickiness of biofilms formed by the rpoN mutant was measured by atomic force microscopy (AFM). Confocal laser scanning microscopy showed that the biofilms formed by the slowly-growing rpoN mutant were more structured than those formed by the wild-type strain. AFM analysis indicated that the biofilms formed by the rpoN mutant were stickier than those formed by the wild type strain during the attachment and establishment stages, but the difference in stickiness was greatly reduced during the maturation stage possibly due to the cytosolic contents released from dead cells in the biofilms formed by the wild type. These results suggest that the alginate overproduction greatly affects the physical properties (topography and stickiness) of P. aeruginosa biofilms as well as the physiological properties (cell death and growth) of the bacterial cells inside the biofilms.

Microbial ecology and adaptation in cystic fibrosis airways

Chronic infections in the respiratory tracts of cystic fibrosis (CF) patients are important to investigate, both from medical and from fundamental ecological points of view. Cystic fibrosis respiratory tracts can be described as natural environments harbouring persisting microbial communities with Pseudomonas aeruginosa as a dominant pathogen. Various factors contribute to the complexity of this ecosystem, including community composition, dynamics and interactions, as well as heterogeneous distribution and fluctuation of components of the immune system, antibiotics and nutrients. All these elements constitute the selective forces that drive the evolution of the microbes after they migrate from the outer environment to human airways. Pseudomonas aeruginosa adapts to the new environment through genetic changes and exhibits a special lifestyle in chronic CF airways. Understanding the persistent colonization of microbial pathogens in CF patients in the context of ecology and evolution will expand our knowledge of the pathogenesis of chronic infections and improve therapeutic strategies.

Requirement for asparagine in the aquaporin NPA sequence signature motifs for cation exclusion

Two highly conserved NPA motifs are a hallmark of the aquaporin (AQP) family. The NPA triplets form N-terminal helix capping structures with the Asn side chains located in the centre of the water or solute-conducting channel, and are considered to play an important role in AQP selectivity. Although another AQP selectivity filter site, the aromatic/Arg (ar/R) constriction, has been well characterized by mutational analysis, experimental data concerning the NPA region--in particular, the Asn position--is missing. Here, we report on the cloning and mutational analysis of a novel aquaglyceroporin carrying one SPA motif instead of the NPA motif from Burkholderia cenocepacia, an epidemic pathogen of cystic fibrosis patients. Of 1357 AQP sequences deposited in RefSeq, we identified only 15 with an Asn exchange. Using direct and phenotypic permeability assays, we found that Asn and Ser are freely interchangeable at both NPA sites without affecting protein expression or water, glycerol and methylamine permeability. However, other mutations in the NPA region led to reduced permeability (S186C and S186D), to nonfunctional channels (N64D), or even to lack of protein expression (S186A and S186T). Using electrophysiology, we found that an analogous mammalian AQP1 N76S mutant excluded protons and potassium ions, but leaked sodium ions, providing an argument for the overwhelming prevalence of Asn over other amino acids. We conclude that, at the first position in the NPA motifs, only Asn provides efficient helix cap stabilization and cation exclusion, whereas other small residues compromise structural stability or cation exclusion but not necessarily water and solute permeability.

The Pseudomonas aeruginosa generalized transducing phage φPA3 is a new member of the φKZ-like group of ‘jumbo’ phages, and infects model laboratory strains and clinical isolates from cystic fibrosis patients

Pseudomonas aeruginosa is an important pathogen in cystic fibrosis patients, and a model organism for the study of nosocomially acquired infections, biofilms and intrinsic multidrug resistance. In this study we characterize ϕPA3, a new generalized transducing bacteriophage for P. aeruginosa. ϕPA3 transduced chromosomal mutations between PAO1 strains, and infected multiple P. aeruginosa clinical isolates as well as the P. aeruginosa model laboratory strains PAK and PA14. Electron microscopy imaging was used to classify ϕPA3 in the order Caudovirales and the family Myoviridae. The genome of ϕPA3 was sequenced and found to contain 309,208 bp, the second-largest bacteriophage currently deposited in GenBank. The genome contains 378 ORFs and five tRNAs. Many ORF products in the ϕPA3 genome are similar to proteins encoded by P. aeruginosa phage ϕKZ and Pseudomonas chlororaphis phage 201ϕ2-1, and so ϕPA3 was classified genetically as a member of the ϕKZ-like group of phages. This is the first report of a member of this group of phages acting as a generalized transducer. Given its wide host range, high transduction efficiency and large genome size, the 'jumbo' phage ϕPA3 could be a powerful tool in functional genomic analysis of diverse P. aeruginosa strains of fundamental and clinical importance.

Mutations that permit residual CFTR function delay acquisition of multiple respiratory pathogens in CF patients

BackgroundLung infection by various organisms is a characteristic feature of cystic fibrosis (CF). CFTR genotype effects acquisition of Pseudomonas aeruginosa (Pa), however the effect on acquisition of other infectious organisms that frequently precede Pa is relatively unknown. Understanding the role of CFTR in the acquisition of organisms first detected in patients may help guide symptomatic and molecular-based treatment for CF.MethodsLung infection, defined as a single positive respiratory tract culture, was assessed for 13 organisms in 1,381 individuals with CF. Subjects were divided by predicted CFTR function: 'Residual': carrying at least one partial function CFTR mutation (class IV or V) and 'Minimal' those who do not carry a partial function mutation. Kaplan-Meier estimates were created to assess CFTR effect on age of acquisition for each organism. Cox proportional hazard models were performed to control for possible cofactors. A separate Cox regression was used to determine whether defining infection with Pa, mucoid Pa or Aspergillus (Asp) using alternative criteria affected the results. The influence of severity of lung disease at the time of acquisition was evaluated using stratified Cox regression methods by lung disease categories.ResultsSubjects with 'Minimal' CFTR function had a higher hazard than patients with 'Residual' function for acquisition of 9 of 13 organisms studied (HR ranging from 1.7 to 3.78 based on the organism studied). Subjects with minimal CFTR function acquired infection at a younger age than those with residual function for 12 of 13 organisms (p-values ranging: < 0.001 to 0.017). Minimal CFTR function also associated with younger age of infection when 3 alternative definitions of infection with Pa, mucoid Pa or Asp were employed. Risk of infection is correlated with CFTR function for 8 of 9 organisms in patients with good lung function (>90%ile) but only 1 of 9 organisms in those with poorer lung function (<50%ile).ConclusionsResidual CFTR function correlates with later onset of respiratory tract infection by a wide spectrum of organisms frequently cultured from CF patients. The protective effect conferred by residual CFTR function is diminished in CF patients with more advanced lung disease.

Adhesion to and biofilm formation on IB3-1 bronchial cells by Stenotrophomonas maltophilia isolates from cystic fibrosis patients

BackgroundStenotrophomonas maltophilia has recently gained considerable attention as an important emerging pathogen in cystic fibrosis (CF) patients. However, the role of this microorganism in the pathophysiology of CF lung disease remains largely unexplored. In the present study for the first time we assessed the ability of S. maltophilia CF isolates to adhere to and form biofilm in experimental infection experiments using the CF-derived bronchial epithelial IB3-1cell line. The role of flagella on the adhesiveness of S. maltophilia to IB3-1 cell monolayers was also assessed by using fliI mutant derivative strains.ResultsAll S. maltophilia CF isolates tested in the present study were able, although at different levels, to adhere to and form biofilm on IB3-1 cell monolayers. Scanning electron and confocal microscopy revealed S. maltophilia structures typical of biofilm formation on bronchial IB3-1 cells. The loss of flagella significantly (P < 0.001) decreased bacterial adhesiveness, if compared to that of their parental flagellated strains. S. maltophilia CF isolates were also able to invade IB3-1 cells, albeit at a very low level (internalization rate ranged from 0.01 to 4.94%). Pre-exposure of IB3-1 cells to P. aeruginosa PAO1 significantly increased S. maltophilia adhesiveness. Further, the presence of S. maltophilia negatively influenced P. aeruginosa PAO1 adhesiveness.ConclusionsThe main contribution of the present study is the finding that S. maltophilia is able to form biofilm on and invade CF-derived IB3-1 bronchial epithelial cells, thus posing a rationale for the persistence and the systemic spread of this opportunistic pathogen in CF patients. Experiments using in vivo models which more closely mimic CF pulmonary tissues will certainly be needed to validate the relevance of our results.

Jean‐Marc Reyrat (29/04/1967–28/10/2009)

Jean‐Marc Reyrat (29/04/1967–28/10/2009)

Pseudomonas aeruginosa uses a cyclic‐di‐GMP‐regulated adhesin to reinforce the biofilm extracellular matrix

Pseudomonas aeruginosa, the principal pathogen of cystic fibrosis patients, forms antibiotic-resistant biofilms promoting chronic colonization of the airways. The extracellular (EPS) matrix is a crucial component of biofilms that provides the community multiple benefits. Recent work suggests that the secondary messenger, cyclic-di-GMP, promotes biofilm formation. An analysis of factors specifically expressed in P. aeruginosa under conditions of elevated c-di-GMP, revealed functions involved in the production and maintenance of the biofilm extracellular matrix. We have characterized one of these components, encoded by the PA4625 gene, as a putative adhesin and designated it cdrA. CdrA shares structural similarities to extracellular adhesins that belong to two-partner secretion systems. The cdrA gene is in a two gene operon that also encodes a putative outer membrane transporter, CdrB. The cdrA gene encodes a 220 KDa protein that is predicted to be rod-shaped protein harbouring a β-helix structural motif. Western analysis indicates that the CdrA is produced as a 220 kDa proprotein and processed to 150 kDa before secretion into the extracellular medium. We demonstrated that cdrAB expression is minimal in liquid culture, but is elevated in biofilm cultures. CdrAB expression was found to promote biofilm formation and auto-aggregation in liquid culture. Aggregation mediated by CdrA is dependent on the Psl polysaccharide and can be disrupted by adding mannose, a key structural component of Psl. Immunoprecipitation of Psl present in culture supernatants resulted in co-immunoprecipitation of CdrA, providing additional evidence that CdrA directly binds to Psl. A mutation in cdrA caused a decrease in biofilm biomass and resulted in the formation of biofilms exhibiting decreased structural integrity. Psl-specific lectin staining suggests that CdrA either cross-links Psl polysaccharide polymers and/or tethers Psl to the cells, resulting in increased biofilm structural stability. Thus, this study identifies a key protein structural component of the P. aeruginosa EPS matrix.

Ochrobactrum and Agrobacterium spp.: emerging opportunistic pathogens in cystic fibrosis patients?

Ochrobactrum and Agrobacterium spp.: emerging opportunistic pathogens in cystic fibrosis patients?

The structure of the O-specific polysaccharide from the lipopolysaccharide of Burkholderia anthina

The pathogenic mechanisms of Gram-negative infection in cystic fibrosis are only just beginning to be explored at molecular level. Several virulence factors have been defined, one of the most important is the lipopolysaccharide molecule. In order to fully understand the mechanisms of bacterial infection and host recognition a full structure/activity study of lipopolysaccharide is needed. In the present paper, we define the complete structure of the O-specific polysaccharide from the lipopolysaccharide from Burkholderia anthina, an uncommon pathogen of cystic fibrosis patients.

Burkholderia cepacia complex: epithelial cell–pathogen confrontations and potential for therapeutic intervention

Burkholderia cepacia complex (Bcc) is an important and virulent pathogen in cystic fibrosis patients. The interactions between this pathogen and the host lung epithelium are being widely investigated but remain to be elucidated. The complex is very versatile and its interactions with the lung epithelial cells are many and varied. The first steps in the interaction are penetration of the mucosal blanket and subsequent adherence to the epithelial cell surface. A range of epithelial receptors have been reported to bind to Bcc. The next step in pathogenesis is the invasion of the lung epithelial cell and also translocation across the epithelium to the serosal side. Furthermore, pathogenesis is mediated by a range of virulence factors that elicit their effects on the epithelial cells. This review outlines these interactions and examines the therapeutic implications of understanding the mechanisms of pathogenesis of this difficult, antibiotic-resistant, opportunistic pathogen.

A polymicrobial perspective of pulmonary infections exposes an enigmatic pathogen in cystic fibrosis patients

Lung disease is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients. A modest number of bacterial pathogens have been correlated with pulmonary function decline; however, microbiological and molecular evidence suggests that CF airway infection is polymicrobial. To obtain a more complete assessment of the microbial community composition and dynamics, we undertook a longitudinal study by using culture-independent and microbiological approaches. In the process, we demonstrated that within complex and dynamic communities, the Streptococcus milleri group (SMG) can establish chronic pulmonary infections and at the onset of 39% of acute pulmonary exacerbations, SMG is the numerically dominant pathogen. We report the comprehensive polymicrobial community dynamics of a CF lung infection in a clinically relevant context. If a given organism, such as Pseudomonas aeruginosa, becomes resistant to antibiotic therapy, an alternative treatment avenue may mediate the desired clinical response by effectively managing the composition of the microbial community.

Aerosolized antibiotics in cystic fibrosis: current and future trends

Chronic bacterial infection is one of the key challenges in caring for patients with cystic fibrosis (CF). Effective infection control requires appropriate administration of antibiotics. Inhaled antibiotics play a pivotal role in CF management. Pathogens in CF patients target the lumen of the lung, a cavity that is difficult to access via the oral or intravenous route. Aerosolized antibiotics deliver effective local drug concentrations to the infection site and decrease the risk of systemic side effects associated with oral or intravenous antibiotics. However, effective aerosolized antibiotic therapy must ensure reliable drug delivery to the lower respiratory tract with a beneficial pharmacodynamic profile and high potency.

Acetobacter indonesiensisPneumonia after Lung Transplant

To the Editor: Unusual and multiresistant bacterial infections are increasingly reported in cystic fibrosis (CF) patients (1). On January 25, 2007, a 31-year-old man with CF (mutation ΔF 508 and I 507) was admitted to our institution in Marseille, France, for lung transplantation. His immunosuppressive regimen included IV cyclosporin A (for the first 6 days with conversion to oral tacrolimus thereafter), azathioprine, and corticosteroids. Induction therapy that used antithymocyte globulin was administered for the first 3 days (Thymoglobuline, Genzyme Corporation, Naarden, the Netherlands). Since 2003, the patient was chronically colonized by methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (susceptible only to colistin sulfomethate), and Candida albicans. Preemptive treatment with antimicrobial agents including colistin sulfomethate, tobramycin sulfate, ceftazidime, and linezolid was administered, starting on posttransplant day 1; prophylactic caspofungin, followed by inhaled amphotericin B, was given for the first month. Six and 9 days, respectively, after surgery, sputa from the patient showed P. aeruginosa and MRSA. On postoperative day 11, the patient’s clinical condition worsened. Leukocytes increased to 13.84 × 109/L. In addition to P. aeruginosa (104 CFU/mL) and MRSA (103 CFU/mL), culture of later sputum samples yielded the growth of 104 CFU/mL of gram-negative, catalase-positive, and oxidase-negative bacillus (isolate 7120034) on CEPACIA agar (AES, Combourg, France) after 72 hours of incubation at 30°C. API 20NE, API 20E, and VITEK 2 Auto system (bioMerieux, Marcy l’Etoile, France) did not identify the bacillus. This bacterium was multiresistant to antimicrobial agents, including colistin, and was susceptible only to imipenem, rifampin, and aminoglycosides. The final identification of this isolate as Acetobacter indonesiensis was achieved after partial sequencing of 16S rRNA gene, as previously described (2) (GenBank accession no. AJ199841, 99% similarity). The sequence of our isolate has been deposited in GenBank under the accession no. {type:entrez-nucleotide,attrs:{text:EF681860,term_id:157381183,term_text:EF681860}}EF681860. The phylogenetic position of isolate 7120034 among other gram-negative bacteria is shown in the Figure. Figure Phylogenetic tree showing the position of Acetobacter indonesiensis (isolate 7120034, GenBank accession no. {type:entrez-nucleotide,attrs:{text:EF681860,term_id:157381183,term_text:EF681860}}EF681860), in boldface, within acetic acid ... Tobramycin was stopped at day 11, colistin and ceftazidime were stopped at postoperative day 14, linezolid was maintained for 10 additional days, and gentamicin (for 13 days) was added to the patient’s drug therapy on February 10. Despite this treatment, A. indonesiensis was cultured from sputa obtained on February 16 (104 CFU/mL) and February 20 (102 CFU/mL), respectively. Six days later, leukocytes decreased to 8.62 × 109/L, and the patient’s condition improved. He was discharged at the beginning of March. During 7 months of follow-up, the A. indonesiensis strain was not found again. Acetic acid bacteria are gram-negative bacilli classified into the genera Acetobacter, Gluconobacter, Gluconacetobacter, Acidomonas, and the recently described genus Asaia; these bacteria belong to the α subgroup of Proteobacteria (3). Isolates of this family are recognized as food-associated bacteria and are able to grow at acidic pH (4). Three species have been reported as emerging pathogens in humans: Asaia bogorensis (in a case of peritonitis in a patient with a peritoneal dialysis catheter [5]); Granulibacter bethesdensis (in 3 cases of lymphadenitis associated with chronic granulomatous disease [6]); and Acetobacter cibinongensis (a recent case of bacteremia in a patient receiving chronic hemodialysis for end-stage renal failure [7]). A. indonesiensis has been isolated from fruits and flowers in Indonesia (8); only 3 reports on it have been published (3,8,9). In our patient, we believe that this multidrug-resistant bacterium was the primary cause of the infection because the patient was eventually cured after an adapted antimicrobial drug therapy. Because this bacterium grows easily at acidic pH (4), a classic condition in the CF airway surface liquid, acidity might contribute to bacterial adhesion and colonization (10). Because acetic acid bacteria have never been isolated from human flora, the source of the contamination for our patient remains unknown. We also report the antimicrobial drug susceptibility of this bacterium. It was multiresistant, especially to colistin. Antimicrobial drug susceptibility results were obtained by using Vitek 2 Auto system because of the absence of growth on Mueller-Hinton agar. This pattern was also the case for G. bethesdensis (6). The antimicrobial susceptibility of A. cibinongensis could not be validated because of the lack of interassay reproducibility (7). Initial antimicrobial drug therapy for this patient with amoxicillin, pristinamycin, and cefazolin did not cure the patient; he was eventually cured after the therapy was switched to tobramycin (7). Our findings reemphasize the emergence of new colistin-resistant pathogens in CF patients, as recently reported for Inquilinus limosus (1). The increased clinical use of nebulized colistin for P. aeruginosa infection in CF patients may select specific colistin-resistant bacteria in such populations. In summary, this report of a respiratory tract infection caused by A. indonesiensis after lung transplantation in a French CF patient supports that this multidrug-resistant bacterium may be an emerging opportunistic pathogen in immunocompromized patients, including CF patients with a lung transplant.

Inquilinus limosus: an easily missed emerging resistant respiratory pathogen in cystic fibrosis patients

Inquilinus limosus: an easily missed emerging resistant respiratory pathogen in cystic fibrosis patients

Pulmonary bacterial pathogens in cystic fibrosis patients and antibiotic therapy: a tool for the health workers.

Cystic fibrosis is the most common and best known genetic disease involving a defect in transepithelial Cl- transport by mutations in the CF gene on chromosome 7, which codes for the cystic fibrosis transmembrane conductance regulator protein (CFTR). The most serious symptoms are observed in the lungs, augmenting the risk of bacterial infection. The objective of this review was to describe the bacterial pathogens colonizing patients with cystic fibrosis. A systematic search was conducted using the international bibliographic databanks SCIELO, HIGHWIRE, PUBMED, SCIRUS and LILACS to provide a useful and practical review for healthcare workers to make them aware of these microorganisms. Today, B. cepacia, P. aeruginosa and S. aureus are the most important infectious agents in cystic fibrosis patients. However, healthcare professionals must pay attention to emerging infectious agents in these patients, because they represent a potentially serious future problem. Therefore, these pathogens should be pointed out as a risk to these patients, and hospitals all over the world must be prepared to detect and combat these bacteria.

Exopolysaccharides produced by Inquilinus limosus, a new pathogen of cystic fibrosis patients: novel structures with usual components

The major cause of morbidity and mortality in patients with cystic fibrosis, an autosomal recessive disorder, is chronic microbial colonisation of the major airways that leads to exacerbation of pulmonary infection. Several different microbes colonise cystic fibrosis lungs, and Pseudomonas aeruginosa is one of the most threatening, since the establishment of mucoid (alginate producing) strains is ultimately associated with the patient’s death. Very recently a new bacterium, named Inquilinus limosus, was repeatedly found infecting the respiratory tract of cystic fibrosis patients. Its multi-resistance characteristic to antibiotics might result in the spreading of I. limosus infection among the cystic fibrosis community, as recently happened with strains of the Burkholderia cepacia complex. Since exopolysaccharides are recognised as important virulence factors in lung infections, the primary structure of the polysaccharide produced by I. limosus strain LMG 20952 T was investigated as the first step in understanding its role in pathogenesis. The structure was determined by means of methylation analysis, acid degradations, mass spectrometry and NMR spectroscopy. The results showed that the bacterium produced a mixture constituted of the following polymers: [3)-[4,6- O-(1-carboxyethylidene)]-β- d-Glc p(1→] n [2)-[4,6- O-(1-carboxyethylidene)]-α- d-Man p(1→] n Both polymers were completely substituted with pyruvyl ketal groups, a novel structural characteristic not previously found in bacterial polysaccharides. The absolute configuration of all pyruvyl groups was S. Inspection of possible local conformations assumed by the two polysaccharide chains showed features, which might provide interesting clues for understanding structure–function relationships.

Quorum-Sensing Mutations Affect Attachment and Stability of Burkholderia cenocepacia Biofilms

Biofilm formation in Burkholderia cenocepacia has been shown to rely in part on acylhomoserine lactone-based quorum sensing. For many other bacterial species, it appears that both the initial adherence and the later stages of biofilm maturation are affected when quorum sensing pathways are inhibited. In this study, we examined the effects of mutations in the cepIR and cciIR quorum-sensing systems of Burkholderia cenocepacia K56-2 with respect to biofilm attachment and antibiotic resistance. We also examined the role of the cepIR system in biofilm stability and structural development. Using the high-throughput MBEC assay system to produce multiple equivalent biofilms, the biomasses of both the cepI and cepR mutant biofilms, measured by crystal violet staining, were less than half of the value observed for the wild-type strain. Attachment was partially restored upon providing functional gene copies via multicopy expression vectors. Surprisingly, neither the cciI mutant nor the double cciI cepI mutant was deficient in attachment, and restoration of the cciI gene resulted in less attachment than for the mutants. Meanwhile, the cciR mutant did show a significant reduction in attachment, as did the cciR cepIR mutant. While there was no change in antibiotic susceptibility with the individual cepIR and cciIR mutants, the cepI cciI mutant biofilms were more sensitive to ciprofloxacin. A significant increase in sensitivity to removal by sodium dodecyl sulfate was seen for the cepI and cepR mutants. Flow cell analysis of the individual cepIR mutant biofilms indicated that they were both structurally and temporally impaired in attachment and development. These results suggest that biofilm structural defects might be present in quorum-sensing mutants of B. cenocepacia that affect the stability and resistance of the adherent cell mass, providing a basis for future studies to design preventative measures against biofilm formation in this species, an important lung pathogen of cystic fibrosis patients.