Cable Pili Research Articles

Regulation and production of Tcf, a cable-like fimbriae from Salmonella enterica serovar Typhi.

tcf (Typhi colonization factor) is one of the 12 putative chaperone/usher fimbrial clusters present in the Salmonella enterica serovar Typhi genome. We investigated the production, expression and regulation of tcf as well as its role during interaction with human cells. The tcf gene cluster was cloned and induced in Escherichia coli and S. Typhi, and the production of intertwined fibres similar to the Cbl (cable) pili of Burkholderia cepacia was observed on the bacterial surface by electron microscopy. In S. Typhi, tcf was expressed more after growth in M63 minimal medium than in standard Luria-Bertani medium. Analysis of the promoter region identified putative binding sites for the global regulators RcsB, ArgR and Fur. The expression of tcf was measured in isogenic strains lacking these global regulators. Under the conditions tested, the results showed that tcf expression was higher in the fur mutant and was regulated by iron concentration. Fur may regulate these fimbriae indirectly via the small RNAs RyhB1 and RyhB2. An isogenic mutant harbouring a deletion of the tcf cluster did not demonstrate any defect in adhesion or invasion of human epithelial cells, or in phagocytosis or survival in macrophages, when compared to the WT serovar Typhi strain. However, the tcf cluster contributed to adherence to human epithelial cells when introduced into E. coli. Thus, tcf genes encode functional fimbriae that can act as an adhesin and may contribute to colonization during typhoid fever.

Identification, molecular characterisation and antimicrobial susceptibility of genomovars of the Burkholderia cepacia complex in Spain

Burkholderia spp. strains collected in Spain over a 13-year period from patients with cystic fibrosis (CF) (n = 148), non-CF patients (n = 103) and from environmental sources (n = 64) were characterised. One hundred and forty-one of the examined strains were involved in seven suspected nosocomial disease outbreaks. Strains were identified by their 16s rRNA and recA genes. Their genetic relatedness, the possession of cable pili and the B. cepacia epidemic strain marker (BCESM), and their susceptibility to antimicrobial agents were studied using pulsed-field gel electrophoresis (PFGE), cblA and esmR genes analysis, and by the E-test, respectively. The genomovar distribution for the 315 strains was as follows: B. stabilis 29.5 %, B. cepacia 14.9 %, B. multivorans 11.1 %, B. cenocepacia IIIA 9.5 %, B. vietnamiensis 3.8 %, B. cenocepacia IIIB 3.5 %, and B. ambifaria and B. pyrrocinia 0.3 % each. The genetic diversity of the B. cepacia complex (Bcc) was ample, with 57 different SpeI types, showing a genetic similarity of 36.4-96.6 %. No strain carried cblA, whereas 25 B. cenocepacia genotypes harboured BCESM (23 from patients with CF). Antimicrobial resistance rates to tobramycin (TOB; 86 %) and imipenem (IPM; 67 %) were high. The strains from patients with CF showed significantly greater resistance to piperacillin (PIP), levofloxacin (LVX) and co-trimoxazole (SXT) than those isolated from non-CF patients (p < 0.05). In conclusion, B. cenocepacia was the most prevalent genomovar found in patients with CF (19.1 %), whereas B. cepacia was the most common among non-CF patients (20.7 %). B. stabilis (47.6 %) was the most common environmental genomovar. Susceptibility to antimicrobial agents depended on genomovar status and strain origin.

Host Evasion by Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic respiratory pathogen of individuals with cystic fibrosis (CF). Some strains of B. cenocepacia are highly transmissible and resistant to almost all antibiotics. Approximately one-third of B. cenocepacia infected CF patients go on to develop fatal “cepacia syndrome.” During the last two decades, substantial progress has been made with regards to evasion of host innate defense mechanisms by B. cenocepacia. Almost all strains of B. cenocepacia have the capacity to survive and replicate intracellularly in both airway epithelial cells and macrophages, which are primary sentinels of the lung and play a pivotal role in clearance of infecting bacteria. Those strains of B. cenocepacia, which express both cable pili and the associated 22 kDa adhesin are also capable of transmigrating across airway epithelium and persist in mouse models of infection. In this review, we will discuss how this type of interaction between B. cenocepacia and host may lead to persistence of bacteria as well as lung inflammation in CF patients.

Cable Pili and the Associated 22 Kda Adhesin Contribute to Burkholderia Cenocepacia Persistence In Vivo

BackgroundInfection by Burkholderia cenocepacia in cystic fibrosis (CF) patients is associated with poor clinical prognosis. Previously, we demonstrated that one of the highly transmissible strains, BC7, expresses cable pili and the associated 22 kDa adhesin, both of which contribute to BC7 binding to airway epithelial cells. However, the contribution of these factors to induce inflammation and bacterial persistence in vivo is not known.Methodology/Principal FindingsWild-type BC7 stimulated higher IL-8 responses than the BC7 cbl and BC7 adhA mutants in both CF and normal bronchial epithelial cells. To determine the role of cable pili and the associated adhesin, we characterized a mouse model of B. cenocepacia, where BC7 are suspended in Pseudomonas aeruginosa alginate. C57BL/6 mice were infected intratracheally with wild-type BC7 suspended in either alginate or PBS and were monitored for lung bacterial load and inflammation. Mice infected with BC7 suspended in PBS completely cleared the bacteria by 3 days and resolved the inflammation. In contrast, mice infected with BC7 suspended in alginate showed persistence of bacteria and moderate lung inflammation up to 5 days post-infection. Using this model, mice infected with the BC7 cbl and BC7 adhA mutants showed lower bacterial loads and mild inflammation compared to mice infected with wild-type BC7. Complementation of the BC7 cblS mutation in trans restored the capacity of this strain to persist in vivo. Immunolocalization of bacteria revealed wild-type BC7 in both airway lumen and alveoli, while the BC7 cbl and BC7 adhA mutants were found mainly in airway lumen and peribronchiolar region.Conclusions and Significance B. cenocepacia suspended in alginate can be used to determine the capacity of bacteria to persist and cause lung inflammation in normal mice. Both cable pili and adhesin contribute to BC7-stimulated IL-8 response in vitro, and BC7 persistence and resultant inflammation in vivo.

Bovine lactoferrin interacts with cable pili of Burkholderia cenocepacia

In this study we evaluated the ability of lactoferrin, the most abundant antimicrobial protein in airway secretions, to bind the surface structures of a Burkholderia strain cystic fibrosis-isolated. Burkholderia cenocepacia is a gram-negative bacterium involved as respiratory pathogen in cystic fibrosis patient infections. This bacterium possesses filamentous structures, named cable pili that have been proposed as virulence factors because of their ability to bind to respiratory epithelia and mucin. Previously, we demonstrated that bovine lactoferrin was able to influence the efficiency of invasion of different iron-regulated morphological forms of B. cenocepacia. Bovine lactoferrin showed to efficiently inhibit invasion of alveolar epithelial cells by free-living bacteria or iron-induced aggregates or biofilm. Results of the present study demonstrate that bovine lactoferrin is also able to specifically bind to B. cenocepacia cells and show that cable pili are involved in this interaction. The attachment of bovine lactoferrin to pili led to a reduced binding of bacterial cells to mucin. Since cable pili are implicated in mediating the bacterial interactions with mucin and epithelial cells, lactoferrin binding to these structures could play an important role in neutralizing bacterial infection in cystic fibrosis patients.

Strains of Burkholderia cenocepacia genomovar IIIA possessing the cblA gene that are distinct from ET12

Three strains of Burkholderia cenocepacia genomovar IIIA that were polymerase chain reaction positive for cblA, bcrA, and the epidemic strain marker, but were distinct from representatives of ET12 by pulsed-field gel electrophoresis, are described. One of these strains was shown to express cable pili by electron microscopy.

Gene expression changes linked to antimicrobial resistance, oxidative stress, iron depletion and retained motility are observed when Burkholderia cenocepacia grows in cystic fibrosis sputum.

BackgroundBacteria from the Burkholderia cepacia complex (Bcc) are the only group of cystic fibrosis (CF) respiratory pathogens that may cause death by an invasive infection known as cepacia syndrome. Their large genome (> 7000 genes) and multiple pathways encoding the same putative functions make virulence factor identification difficult in these bacteria.MethodsA novel microarray was designed to the genome of Burkholderia cenocepacia J2315 and transcriptomics used to identify genes that were differentially regulated when the pathogen was grown in a CF sputum-based infection model. Sputum samples from CF individuals infected with the same B. cenocepacia strain as genome isolate were used, hence, other than a dilution into a minimal growth medium (used as the control condition), no further treatment of the sputum was carried out.ResultsA total of 723 coding sequences were significantly altered, with 287 upregulated and 436 downregulated; the microarray-observed expression was validated by quantitative PCR on five selected genes. B. cenocepacia genes with putative functions in antimicrobial resistance, iron uptake, protection against reactive oxygen and nitrogen species, secretion and motility were among the most altered in sputum. Novel upregulated genes included: a transmembrane ferric reductase (BCAL0270) implicated in iron metabolism, a novel protease (BCAL0849) that may play a role in host tissue destruction, an organic hydroperoxide resistance gene (BCAM2753), an oxidoreductase (BCAL1107) and a nitrite/sulfite reductase (BCAM1676) that may play roles in resistance to the host defenses. The assumptions of growth under iron-depletion and oxidative stress formulated from the microarray data were tested and confirmed by independent growth of B. cenocepacia under each respective environmental condition.ConclusionOverall, our first full transcriptomic analysis of B. cenocepacia demonstrated the pathogen alters expression of over 10% of the 7176 genes within its genome when it grows in CF sputum. Novel genetic pathways involved in responses to antimicrobial resistance, oxidative stress, and iron metabolism were revealed by the microarray analysis. Virulence factors such as the cable pilus and Cenocepacia Pathogenicity Island were unaltered in expression. However, B. cenocepacia sustained or increased expression of motility-associated genes in sputum, maintaining a potentially invasive phenotype associated with cepacia syndrome.

Identification of Sodium Chloride-Regulated Genes in Burkholderia cenocepacia

Previous studies have suggested that the airways of cystic fibrosis (CF) patients have elevated sodium chloride (NaCl) levels due to the malfunctioning of the CF transmembrane conductance regulator protein. For bacteria to survive in this high-salt environment, they must adjust by altering the regulation of gene expression. Among the different bacteria inhabiting the airways of CF patients is the opportunistic pathogen Burkholderia cenocepacia. Previous studies have indicated that B. cenocepacia produces a toxin and cable pili under high osmolar conditions. We used transposon mutagenesis to identify NaCl-regulated genes in the clinical strain B. cenocepacia K56-2. Six transconjugants were induced with increasing NaCl concentration. The DNA flanking the transposon was sequenced and five distinct open reading frames were identified encoding the following putative proteins: an integrase, an NAD-dependent deacetylase, TolB, an oxidoreductase, and a novel hypothetical protein. The collective results of this study provide important information about the physiology of B. cenocepacia when faced with osmotic stress and suggest the identity of significant virulence mechanisms in this opportunistic pathogen.

Differential interaction of bacterial species from the Burkholderia cepacia complex with human airway epithelial cells

To increase knowledge of the pathogenic potential of the Burkholderia cepacia complex (BCC), we investigated the effects of reference strains of the nine BCC species on human bronchial epithelial cells in vitro. B. multivorans exhibited the highest rates of adherence to and internalization by host cells. Two out of three clinical isolates recovered from cystic fibrosis patients confirmed the B. multivorans high adhesiveness. All four B. multivorans isolates exhibited an aggregated pattern of adherence but any of them expressed cable pili. When bacteria were centrifuged onto cell cultures to circumvent their poor adhesiveness, B. pyrrocinia exhibited the highest internalization rate, followed by B. multivorans. The percentages of apoptotic cells in cultures infected with B. cepacia, B. multivorans, B. cenocepacia (subgroups IIIA and IIIB), B. stabilis and B. vietnamiensis were significantly higher than in control non-infected cultures. All nine BCC species triggered a similar release of the inflammatory cytokine IL-8, that was not reduced by cell treatment with cytochalasin D. Hence, our data demonstrate, for the first time, that all BCC species exhibit a similar ability to induce the expression of host immune mediators whereas they differ on their ability to adhere to, invade and kill airway epithelial cells.

Molecular Typing and Presence of Genetic Markers Among Strains of Banana Finger-Tip Rot Pathogen, Burkholderia cenocepacia, in Taiwan

ABSTRACT Burkholderia cenocepacia (genomovar III of B. cepacia complex), the causal agent of banana finger-tip rot, is a common plant-associated bacterium but also an important opportunistic pathogen of humans. To better understand the nature of B. cenocepacia from banana, the genetic variation among B. cenocepacia isolates from various banana-growing regions in southern Taiwan was examined. Forty-four serial isolates recovered from diseased banana stigmata from three banana-growing regions during the periods ranging from 2002 to 2004 were investigated. All B. cenocepacia isolates picked from quinate-yeast extract tetracycline-polymyxin semiselective medium could cause onion maceration and were polymerase chain reaction (PCR) positive for bcscV, which is a type III secretion gene present in all members of the B. cepacia complex except B. cepacia (formerly genomovar I). Genetic diversity was assessed using recA PCR restriction fragment length polymorphism, recA nucleotide sequence analysis, and pulsed-field gel electrophoresis assays. The assays revealed the genetic variability among the isolates and also allowed us to trace the relationship among isolates. The isolates all were assigned to genomovar III and consisted of two groups, A and B, which corresponded to recA lineage IIIA and IIIB. The group B strains were separated into B1 and B2 subgroups and the B1 strains were further divided into distinct lineages. The B1 strains were the most frequently detected and occurred in all regions tested. There was no significant difference between strains from each subgroup in the virulence on banana fingers of cv. Cavendish. PCR assays were further used to determine whether B. cenocepacia from banana contained the cable pilus subunit gene (cblA), IS1356, and B. cepacia epidemic strain marker (BCESM), which are DNA markers associated with epidemic B. cepacia clinic strains. The results indicated that cblA and IS1356 were absent but the BCESM was found in all isolates. The present study revealed that banana is a natural reservoir of genetically diversified B. cenocepacia strains.

Cable Pili and the 22-Kilodalton Adhesin Are Required for Burkholderia cenocepacia Binding to and Transmigration across the Squamous Epithelium

Burkholderia cenocepacia strains expressing both cable (Cbl) pili and the 22-kDa adhesin bind to cytokeratin 13 (CK13) strongly and invade squamous epithelium efficiently. It has not been established, however, whether the gene encoding the adhesin is located in the cbl operon or what specific contribution the adhesin and Cbl pili lend to binding and transmigration or invasion capacity of B. cenocepacia. By immunoscreening an expression library of B. cenocepacia isolate BC7, we identified a large gene (adhA) that encodes the 22-kDa adhesin. Isogenic mutants lacking expression of either Cbl pili (cblA or cblS mutants) or the adhesin (adhA mutant) were constructed to assess the individual role of Cbl pili and the adhesin in mediating B. cenocepacia binding to and transmigration across squamous epithelium. Relative to the parent strain, mutants of Cbl pili showed reduced binding (50%) to isolated CK13, while the adhesin mutant showed almost no binding (0 to 8%). Mutants lacking either cable pili or the adhesin were compromised in their ability to bind to and transmigrate across the squamous epithelium compared to the wild-type strain, although this deficiency was most pronounced in the adhA mutant. These results indicate that both Cbl pili and the 22-kDa adhesin are necessary for the optimal binding to CK13 and transmigration properties of B. cenocepacia.

Genetic characterization of a multicomponent signal transduction system controlling the expression of cable pili in Burkholderia cenocepacia.

Cable pili are peritrichous organelles expressed by certain strains of Burkholderia cenocepacia, believed to facilitate colonization of the lower respiratory tract in cystic fibrosis patients. The B. cenocepacia cblBACDS operon encodes the structural and accessory proteins required for the assembly of cable pili, as well as a gene designated cblS, predicted to encode a hybrid sensor kinase protein of bacterial two-component signal transduction systems. In this study we report the identification of two additional genes, designated cblT and cblR, predicted to encode a second hybrid sensor kinase and a response regulator, respectively. Analyses of the deduced amino acid sequences of the cblS and cblT gene products revealed that both putative sensor kinases have transmitter and receiver domains and that the cblT gene product has an additional C-terminal HPt domain. Mutagenesis of the cblS, cblT, or cblR gene led to a block in expression of CblA, the major pilin subunit, and a severe decrease in cblA transcript abundance. Using transcriptional fusion analyses, the decrease in the abundance of the cblA transcript in the cblS, cblT, and cblR mutants was shown to be due to a block in transcription from the cblB-proximal promoter, located upstream of the cblBACDS operon. Furthermore, ectopic expression of either cblS or cblR in wild-type B. cenocepacia strain BC7 led to a significant increase, while ectopic expression of cblT resulted in a dramatic decrease, in abundance of the CblA major pilin and the cblA transcript. Our results demonstrate that the B. cenocepacia cblS, cblT, and cblR genes are essential for cable pilus expression and that their effect is exerted at the level of transcription of the cblBACDS operon. These findings are consistent with the proposed function of the cblSTR gene products as a multicomponent signal transduction pathway controlling the expression of cable pilus biosynthetic genes in B. cenocepacia.

Transcriptional and Posttranscriptional Control of Cable Pilus Gene Expression in Burkholderia cenocepacia

Burkholderia cenocepacia is an important member of the Burkholderia cepacia complex, a group of closely related bacteria that inhabits a wide variety of environmental niches in nature and that also colonizes the lungs of compromised humans. Certain strains of B. cenocepacia express peritrichous adherence organelles known as cable pili, thought to be important in the colonization of the lower respiratory tract. The genetic locus required for cable pilus biogenesis is comprised of at least five genes, designated cblB, cblA, cblC, cblD, and cblS. In this study a transcriptional analysis of cbl gene expression was undertaken. The principal promoter, located upstream of the cbl locus, was identified and characterized. By using lacZ transcriptional fusions, the effects of multiple environmental cues on cbl gene expression were examined. High osmolarity, temperature of 37 degrees C, acidic pH, and low iron bioavailability were found to induce cbl gene expression. Northern hybridization analysis of the cbl locus identified a single, stable transcript corresponding to cblA, encoding the major pilin subunit. Transcriptional fusion studies combined with reverse transcription-PCR analysis indicated that the stable cblA transcript is the product of an mRNA processing event. This event may ensure high levels of expression of the major pilin, relative to other components of the assembly pathway. Our findings lend further insight into the control of cable pilus biogenesis in B. cenocepacia and provide evidence for regulation of cbl gene expression on both the transcriptional and posttranscriptional levels.

Adherence and autoaggregation phenotypes of a Burkholderia cenocepacia cable pilus mutant

Cable pili are unique peritrichous adherence organelles expressed by certain strains of the opportunistic human pathogen Burkholderia cenocepacia. Cable pili have been proposed to facilitate binding to human epithelial cells and mucin, and may play a role in the ability of B. cenocepacia to colonise the respiratory tract of compromised hosts. In this study, a genetic approach was undertaken to assess the role of cable pili in mediating adherence as well as bacterial cell–cell interactions. The cblA gene, encoding the major pilin subunit, was insertionally inactivated, and the resulting mutant was shown to be blocked in CblA expression and in cable pilus morphogenesis. Although non-piliated, the cblA mutant was not defective in adherence to either porcine mucin or to cultured A549 human respiratory epithelial cells. Microscopic and flow cytometric analyses of B. cenocepacia cultures revealed that cable pilus expression facilitated the formation of diffuse cell networks, whereas disruption of cable pilus biogenesis enhanced autoaggregation and the formation of compact cell aggregates. Autoaggregation was observed both in culture and during B. cenocepacia infection of A549 epithelial cell monolayers. These findings indicate that cable pilus expression plays an important role in mediating B. cenocepacia cell–cell interactions, and that both cable pilus-dependent and cable pilus-independent mechanisms may contribute to B. cenocepacia adherence to cellular and acellular surfaces.

Lack of cable pili expression by cblA-containing Burkholderia cepacia complex.

The Burkholderia cepacia complex consists of several closely related bacterial species (or genomovars) which although generally not pathogenic for healthy individuals, contribute significantly to morbidity and mortality among persons with cystic fibrosis (CF). Certain B. cepacia complex strains are more frequently recovered from CF sputum cultures than are others, and these typically reside in genomovar III. The ET12 clone is a genomovar III strain that predominates among CF patients in Canada and the United Kingdom and is characterized by distinctive cblA-encoded pili that have a cable-like morphology. In a previous survey of B. cepacia complex isolates recovered from 606 CF patients in the US, a single genomovar III ET12 isolate (isolate AU0007) was identified; several cblA-containing genomovar I isolates, however, were also detected. In the study reported here, analysis by PFGE revealed several distinct strain types among these genomovar I isolates, and sequence analysis of their cblA genes demonstrated 87.8-88.4% identity to the ET12 cblA sequence. Southern analysis indicated that the cblA variant from each genomovar I isolate resides on a 4 kbp EcoRI fragment, in contrast to ET12 isolates, in which cblA localizes to a 5 kbp EcoRI fragment. Western blot assay indicated expression of the 16 kDa major pilin subunit by ET12 isolates, including AU0007, but neither whole-cell nor surface-protein extracts of the genomovar I reacted. Electron microscopy revealed the complete absence of pili expression by the genomovar I isolates. In contrast to typical ET12 isolates, AU0007 appeared to be hyperpiliated with rigid pili that lacked the cable morphology and did not bind cytokeratin 13, which has been previously identified as the epithelial cell receptor for the ET12 cable-pili-associated adhesin.

Preventing Burkholderia cepacia complex infection in cystic fibrosis: Is there a middle ground?

Preventing Burkholderia cepacia complex infection in cystic fibrosis: Is there a middle ground?

Molecular epidemiology of cystic fibrosis-linked Burkholderia cepacia complex isolates from three national referral centres in Ireland.

Burkholderia cepacia is a Gram-negative bacterium associated with increasing morbidity and mortality and is readily transmitted among infected cystic fibrosis (CF) patients. The B. cepacia complex consists of five distinct subgroups, termed genomovars. A collection of 17 presumptive B. cepacia isolates, obtained from three national CF referral centres located in different geographical regions in Ireland, was studied. The aim of this study was to investigate these isolates using molecular subtyping protocols for evidence of genetic relationships and for the presence of antibiotic resistance-encoding class 1 integron structures. Genomovar classifications were assigned to each isolate based on HaeIII enzyme profiles of their recA locus. Genetic relationships among this collection were also assessed after restriction fragment length polymorphism (RFLP)-mediated analysis of the 16S rDNA locus and DNA amplification fingerprinting (DAF). The surface expression of the cable pilus gene (cblA) may facilitate an early step in the infection process. All isolates were tested by amplification strategies for this marker. Burkholderia cepacia is known to be resistant to several antimicrobial agents. Resistance typing showed that the majority were resistant to three or more common antimicrobial agents. Five of the 17 isolates were resistant to sulphonamide, a characteristic linked with the presence of class 1 integrons. Gene cassettes containing beta-lactamase (oxa) and aminoglycoside acetyltransferase (aac(6')-1a) encoding genes were identified by polymerase chain reaction amplification. Most of the isolates in this study were classified as genomovar III and were indistinguishable based on their corresponding 16S rDNA-RFLP profiles, whilst DAF further subtyped the collection. The cblA marker was identified in 47% of the isolates, many of which clustered in the genomovar III group. Class 1 integrons with recombined gene cassettes containing bla-OXA and aac(6')-1a genes were identified. This study demonstrates the application of molecular methods to investigate B. cepacia, a well-recognized human pathogen, cultured from Irish CF patients. Genomovar III was the most common genomic type identified. DNA fingerprinting further subtyped the latter isolates, facilitating a more detailed description of the molecular epidemiology. Drug resistance in these organisms can be explained, at least in part, by the presence of class 1 integrons. Development of targeted infection control strategies could be facilitated using these applied methods.

Differential invasion of respiratory epithelial cells by members of the Burkholderia cepacia complex

To investigate whether there are differences between members of the Burkholderia cepacia complex in their ability to invade human respiratory epithelial cells, 11 strains belonging to genomovars I-V were studied in an antibiotic protection assay using the A549 cell line. Strains belonging to genomovars II and III were more invasive than those of genomovars I, IV and V. There was also intra-genomovar variation in invasiveness. No correlation between invasiveness and other putative virulence factors of importance in B. cepacia infection in individuals with cystic fibrosis, cable pilus and B. cepacia epidemic strain marker was identified.

Immunolocalisation of Burkholderia cepacia in the lungs of cystic fibrosis patients.

Infection by Burkholderia cepacia is sometimes fatal in patients with cystic fibrosis (CF), as the organism can cause necrotising pneumonia and septicaemia (the cepacia syndrome), and is resistant to antibiotics. To increase knowledge of the pathogenesis of lung infection, the present study investigated the distribution of B. cepacia in lung explants from nine CF recipients of double lung transplants, of which six were colonised with both B. cepacia and Pseudomonas aeruginosa and the other three with P. aeruginosa only. In one case, explants of the donor lung (allograft) became available after the patient succumbed post-operatively to the cepacia syndrome. Further autopsy sections were examined from two patients who had chronic and then acute infection with B. cepacia. A specific antibody reactive with all five genomovars of the B. cepacia complex and another antibody specific for the 22-kDa adhesin of cable pili, were used to localise bacteria in situ. In chronic infection, the organisms were diffusely distributed, but most concentrated in hyperplastic bronchiolar epithelium, inflamed peribronchial and perivascular areas, between adjacent airway epithelial cells and in pathologically thickened alveolar septae and luminal macrophages. In acute infections the distribution was more focal, with B. cepacia on injured airway surfaces and in sites of pneumonia and abscess formation. In autopsy sections from one of the patients with chronic, then acute infection, B. cepacia was also observed in the lumen of blood capillaries. These results suggest that B. cepacia has the capacity to be highly invasive, migrating from the airways across the epithelial barrier to invade the lung parenchyma and capillaries, thereby initiating septicaemia.

Preferential adherence of cable-piliated burkholderia cepacia to respiratory epithelia of CF knockout mice and human cystic fibrosis lung explants.

The Burkholderia cepacia complex consists of at least five well-documented bacterial genomovars, each of which has been isolated from the sputum of different patients with cystic fibrosis (CF). Although the world-wide prevalence of this opportunist pathogen in CF patients is low (1-3%), 'epidemic' clusters occur in geographically isolated regions. Prevalence in some of these clusters is as high as 30-40%. The majority of CF B. cepacia isolates belong to genomovar III, but the relationship between genomovar and virulence has not yet been defined. Because the initial stage of infection involves bacterial binding to host tissues, the present study investigated differences in the binding of representative isolates of all five genomovars to fixed nasal sections of UNC cftr (-/-) and (+/+) mice and to human lung explants, biopsy and autopsy tissue of CF and non-CF patients. Binding was highest for isolates of genomovar III, subgroup RAPD type 2, but only if the isolates expressed the cable pili phenotype. Antibodies to the 22-kDa adhesin of cable pili virtually abolished binding. Binding occurred only to cftr (-/-) nasal sections or to CF lung sections and was negligible in cftr (+/+) or human non-CF, histologically normal lung sections. Unlike normal epithelia, the hyperplastic epithelia of CF bronchioles were enriched in cytokeratin 13, a 55-kDa protein that has previously been shown to act as a receptor in vitro for cable-piliated B. cepacia. These findings may help to explain the high transmissibility ofCbl-positive, genomovar III strains of B. cepacia among CF patients.