Adaptation and Survival of Burkholderia cepacia and B. contaminans During Long-Term Incubation in Saline Solutions Containing Benzalkonium Chloride.

The Burkholderia cepacia complex (Bcc) is a group of opportunistic pathogenic bacteria with a remarkable metabolic capacity and broad genotypic/phenotypic plasticity, allowing their adaptation to hostile conditions, including nutrient depleted solutions containing antimicrobial agents. Bcc bacteria are feared contaminants in pharmaceutical industries and cause nosocomial outbreaks, posing health threats to immunocompromised individuals and cystic fibrosis (CF) patients. In this study, the adaptation and survival of B. cepacia and B. contaminans isolates was investigated after long-term incubation in nutrient depleted saline solutions supplemented with increasing concentrations of the biocidal preservative benzalkonium chloride (BZK), recreating the storage conditions of pharmaceutical products. These epidemiologically related isolates were recovered from intrinsically contaminated saline solutions for nasal application and from two CF patients. Long-term incubation in saline solutions containing BZK led to the development of bacterial sub-populations that survived for at least 16 months, despite an initial 2-3 log decrease in viability, displaying a progressive dose-dependent decrease of colony and cell size, including the appearance of small colony variants (SCVs). Bacterial colonies lost pigmentation, changed the morphotype from rough to smooth and produced more spherical cells during extended incubation with BZK. The development of macroscopically visible cellular aggregates, rich in polysaccharide and harboring viable cells in their interior was triggered by BZK. The existence of a metabolic pathway for BZK degradation was confirmed through genome analysis. This study reveals mechanisms underlying the prevalence of Bcc bacteria as contaminants of pharmaceutical products containing BZK, which often lead to false-negative results during quality control and routine testing.

Burkholderia cepacia Complex Species Differ in the Frequency of Variation of the Lipopolysaccharide O-Antigen Expression During Cystic Fibrosis Chronic Respiratory Infection.

Burkholderia cepacia complex (Bcc) bacteria can adapt to the lung environment of cystic fibrosis (CF) patients resulting in the emergence of a very difficult to eradicate heterogeneous population leading to chronic infections associated with rapid lung function loss and increased mortality. Among the important phenotypic modifications is the variation of the lipopolysaccharide (LPS) structure at level of the O-antigen (OAg) presence, influencing adherence, colonization and the ability to evade the host defense mechanisms. The present study was performed to understand whether the loss of OAg expression during CF infection can be considered a general phenomenon in different Bcc species favoring its chronicity. In fact, it is still not clear why different Bcc species/strains differ in their ability to persist in the CF lung and pathogenic potential. The systematic two-decade-retrospective-longitudinal-screening conducted covered 357 isolates retrieved from 19 chronically infected patients receiving care at a central hospital in Lisbon. The study involved 21 Bcc strains of six/seven Bcc species/lineages, frequently or rarely isolated from CF patients worldwide. Different strains/clonal variants obtained during infection gave rise to characteristic OAg-banding patterns. The two most prevalent and feared species, B. cenocepacia and B. multivorans, showed a tendency to lose the OAg along chronic infection. B. cenocepacia recA lineage IIIA strains known to lead to particularly destructive infections exhibit the most frequent OAg loss, compared with lineage IIIB. The switch frequency increased with the duration of infection and the level of lung function deterioration. For the first time, it is shown that the rarely found B. cepacia and B. contaminans, whose representation in the cohort of patients examined is abnormally high, keep the OAg even during 10- or 15-year infections. Data from co-infections with different Bcc species reinforced these conclusions. Concerning the two other rarely found species examined, B. stabilis exhibited a stable OAg expression phenotype over the infection period while for the single clone of the more distantly related B. dolosa species, the OAg-chain was absent from the beginning of the 5.5-year infection until the patient dead. This work reinforces the relevance attributed to the OAg-expression switch suggesting marked differences in the various Bcc species.

Structure of O-Antigen and Hybrid Biosynthetic Locus in Burkholderia cenocepacia Clonal Variants Recovered from a Cystic Fibrosis Patient.

Burkholderia cenocepacia is an opportunistic pathogen associated with chronic lung infections and increased risk of death in patients with cystic fibrosis (CF). In this work, we investigated the lipopolysaccharide (LPS) of clinical variants of B. cenocepacia that were collected from a CF patient over a period of 3.5 years, from the onset of infection until death by necrotizing pneumonia (cepacia syndrome). We report the chemical structure of the LPS molecule of various sequential isolates and the identification of a novel hybrid O-antigen (OAg) biosynthetic cluster. The OAg repeating unit of the LPS from IST439, the initial isolate, is a [→2)-ß-D-Ribf-(1→4)-α-D-GalpNAc-(1→] disaccharide, which was not previously described in B. cenocepacia. The IST439 OAg biosynthetic gene cluster contains 7 of 23 genes that are closely homologous to genes found in B. multivorans, another member of the Burkholderia cepacia complex. None of the subsequent isolates expressed OAg. Genomic sequencing of these isolates enabled the identification of mutations within the OAg cluster, but none of these mutations could be associated with the loss of OAg. This study provides support to the notion that OAg LPS modifications are an important factor in the adaptation of B. cenocepacia to chronic infection and that the heterogeneity of OAgs relates to variation within the OAg gene cluster, indicating that the gene cluster might have been assembled through multiple horizontal transmission events.

Variation of Burkholderia cenocepacia virulence potential during cystic fibrosis chronic lung infection.

During long-term lung infection in cystic fibrosis (CF) patients, Burkholderia cenocepacia faces multiple selective pressures in this highly stressful and fluctuating environment. As a consequence, the initial infecting strain undergoes genetic changes that result in the diversification of genotypes and phenotypes. Whether this clonal expansion influences the pathogenic potential is unclear. The virulence potential of 39 sequential B. cenocepacia (recA lineage IIIA) isolates, corresponding to 3 different clones retrieved from 3 chronically infected CF patients was compared in this study using the non-mammalian infection hosts Galleria mellonella and Caenorhabditis elegans. The isolates used in this retrospective study were picked randomly from selective agar plates as part of a CF Center routine, from the onset of infection until patients' death after 3.5 and 7.5 y or the more recent isolation date after 12.5 y of chronic infection. The infection models proved useful to assess virulence potential diversification, but for some isolates the relative values diverged in C. elegans and G. mellonella. Results also reinforce the concept of the occurrence of clonal diversification and co-existence of multiple phenotypes within the CF lungs, also with respect to pathogenicity. No clear trend of decrease (or increase) of the virulence potential throughout long-term infection was found but there is an apparent tendency for a clone/patient-dependent decrease of virulence when the G. mellonella model was used. The sole avirulent variant in both infection hosts was found to lack the small third replicon previously associated to virulence. Although possible, the in vivo loss of this nonessential megaplasmid was found to be a rare event (1 among a total of 64 isolates examined).

Incidence of Burkholderia contaminans at a cystic fibrosis centre with an unusually high representation of Burkholderia cepacia during 15 years of epidemiological surveillance.

The Burkholderia cepacia complex (Bcc) is a heterogeneous group of bacteria comprising around 20 related species. These bacteria are important opportunistic pathogens, especially in cystic fibrosis (CF) patients, and are associated with a worse prognosis and decreased life expectancy. The taxonomic position of 20 Bcc isolates retrieved from CF patients receiving care at Hospital Santa Maria (HSM), in Lisbon, from 1995 to 2006, was re-examined in the present work. These isolates, formerly classified as Burkholderia cepacia (taxon K), are here reclassified as Burkholderia contaminans, including the former B. cepacia IST408, which was the focus of previous studies regarding the biosynthesis of the exopolysaccharide 'cepacian'. The CF population examined has been previously described as having an exceptionally high representation of B. cepacia, presumably due to a contamination arising from saline solutions for nasal application. Twenty-one additional isolates, obtained from a chronically infected patient, from 2006 to 2010, were also identified as B. contaminans. This study also provides insight into the potential clinical impact of B. contaminans, a species that is rarely associated with CF infections. Isolates belonging to this species were shown to be involved in chronic and transient respiratory infections, and were associated with severe lung function deterioration and with a case of death with cepacia syndrome. However, since the patients were co-infected with Burkholderia cenocepacia and other non-Burkholderia bacteria, the role played by B. contaminans is unclear. Nevertheless, B. contaminans isolates were found to prevail over B. cenocepacia isolates during co-infection of at least one chronically infected patient.

Burkholderia dolosa phenotypic variation during the decline in lung function of a cystic fibrosis patient during 5.5 years of chronic colonization.

Although rarely isolated from cystic fibrosis (CF) patients, Burkholderia dolosa is associated with accelerated lung function decline. During 18 years of epidemiological surveillance in the major Portuguese CF centre in Lisbon, only one patient was infected with B. dolosa. Pulmonary deterioration, associated with the evolution of forced expiratory volume in 1 s, occurred during 5.5 years of colonization with this B. dolosa clone (with the new sequence type ST-668). Transient co-colonization with Burkholderia cenocepacia and other bacterial and fungal pathogens occurred, but B. dolosa prevailed until the patient's death. The systematic assessment of relevant phenotypes for the sequential clonal isolates examined in this retrospective study (14 of B. dolosa and four of B. cenocepacia) showed that they were variants, although in general no isolation time-dependent pattern of alteration was identified. However, the first B. dolosa isolate retrieved was more susceptible to gentamicin, imipenem and tobramycin, and exhibited a higher swarming motility compared with most of the isolates obtained during the later stages of disease progression and antimicrobial therapy.

Proteomic profiling of Burkholderia cenocepacia clonal isolates with different virulence potential retrieved from a cystic fibrosis patient during chronic lung infection.

Respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. In this work, we assessed the virulence potential of three B. cenocepacia clonal isolates obtained from a CF patient between the onset of infection (isolate IST439) and before death with cepacia syndrome 3.5 years later (isolate IST4113 followed by IST4134), based on their ability to invade epithelial cells and compromise epithelial monolayer integrity. The two clonal isolates retrieved during late-stage disease were significantly more virulent than IST439. Proteomic profiling by 2-D DIGE of the last isolate recovered before the patient's death, IST4134, and clonal isolate IST439, was performed and compared with a prior analysis of IST4113 vs. IST439. The cytoplasmic and membrane-associated enriched fractions were examined and 52 proteins were found to be similarly altered in the two last isolates compared with IST439. These proteins are involved in metabolic functions, nucleotide synthesis, translation and protein folding, cell envelope biogenesis and iron homeostasis. Results are suggestive of the important role played by metabolic reprogramming in the virulence potential and persistence of B. cenocepacia, in particular regarding bacterial adaptation to microaerophilic conditions. Also, the content of the virulence determinant AidA was higher in the last 2 isolates. Significant levels of siderophores were found to be secreted by the three clonal isolates in an iron-depleted environment, but the two late isolates were more tolerant to low iron concentrations than IST439, consistent with the relative abundance of proteins involved in iron uptake.

SNaPBcen: a novel and practical tool for genotyping Burkholderia cenocepacia.

Burkholderia cenocepacia is the most prevalent and feared member of the Burkholderia cepacia complex in lung infections of cystic fibrosis (CF). Genotyping and monitoring of long-term colonization are critical at clinical units; however, the differentiation of specific lineages performed by multilocus sequence typing (MLST) is still limited to a small number of isolates due to the high cost and time-consuming procedure. The aim of this study was to optimize a protocol (the SNaPBcen assay) for extensive bacterial population studies. The strategy used for the SNaPBcen assay is based on targeting single nucleotide polymorphisms (SNPs) located in MLST genes instead of sequencing full MLST sequences. Nonpolymorphic and redundant MLST positions were eliminated, and a set of 24 polymorphisms included in the SNaPBcen assay ensures a high-resolution genomic characterization. These polymorphisms were identified based on the comparative analysis of 137 B. cenocepacia MLST profiles available online (http://pubmlst.org/bcc/). The group of 81 clinical isolates of B. cenocepacia examined in this study using the SNaPBcen assay revealed 51 distinct profiles, and a final discriminatory power of 0.9997 compared with MLST was determined. The SNaPBcen assay was able to reveal isolates with microvariations and the presence of multiple clonal variants in patients chronically colonized with B. cenocepacia. Main phylogenetic subgroups IIIA, IIIB, and IIIC of B. cenocepacia could be separated by the Gl94R polymorphism included in the panel. The SNaPBcen assay proved to be a rapid and robust alternative to the standard MLST for B. cenocepacia, allowing the simultaneous analysis of multiple polymorphisms following amplification and mini-sequencing reactions.

Chemistry and biology of the potent endotoxin from a Burkholderia dolosa clinical isolate from a cystic fibrosis patient.

This is the first report of the chemical and biological properties of the lipooligosaccharide (LOS) endotoxin isolated from Burkholderia dolosa IST4208, an isolate recovered from a cystic fibrosis (CF) patient in a Portuguese CF center. B. dolosa is a member of the Burkholderia cepacia complex, a group of closely related species that are highly problematic and opportunistic pathogens in CF. B. dolosa infection leads to accelerated loss of lung function and decreased survival. The structural determination of its endotoxin was achieved using a combination of chemistry and spectroscopy, and has revealed a novel endotoxin structure. The purified LOS was tested for its immunostimulatory activity on human HEK 293 cells expressing TLR-4, MD-2, and CD-14. In these assays, the LOS showed strong proinflammatory activity.

Burkholderia cenocepacia phenotypic clonal variation during a 3.5-year colonization in the lungs of a cystic fibrosis patient.

Chronic lung infection is the major cause of morbidity and premature mortality in cystic fibrosis (CF) patients. Bacteria of the Burkholderia cepacia complex are the most threatening pathogens in CF, and a better understanding of how these bacteria adapt to the CF airway environment and resist the host defense mechanisms and therapeutically administered antibiotics is crucial. To provide clues to the adaptive strategies adopted by Burkholderia cenocepacia during long-term colonization, we carried out a phenotypic assessment of 11 clonal variants obtained at the major Portuguese CF Center in Lisbon from sputa of the same CF patient during 3.5 years of colonization of the lungs, until the patient's death with cepacia syndrome. Phenotypic characterization included susceptibility assays against different classes of antimicrobials and characterization of cell motility, cell hydrophobicity and zeta potential, colony and cell morphology, fatty acid composition, growth under iron limitation/load conditions, exopolysaccharide production, and size of the biofilms formed. The results suggest the occurrence of clonal expansion during long-term colonization. For a number of the characteristics tested, no isolation time-dependent consistent alteration pattern could be identified. However, the values for antimicrobial susceptibility and swarming motility for the first B. cenocepacia isolate, thought to have initiated the infection, were consistently above those for the clonal variants obtained during the course of infection, and the opposite was found for the zeta potential. The adaptive strategy for long-term colonization, described here for the first time, involved the alteration of membrane fatty acid composition, in particular a reduction of the degree of fatty acid saturation, in the B. cenocepacia variants retrieved, along with the deterioration of pulmonary function and severe oxygen limitation.

Quantitative proteomics (2-D DIGE) reveals molecular strategies employed by Burkholderia cenocepacia to adapt to the airways of cystic fibrosis patients under antimicrobial therapy.

Chronic respiratory infections caused by Burkholderia cenocepacia in patients with cystic fibrosis (CF) are characterized by low responsiveness to antibiotic therapy and, in general, to a more rapid decline of lung function. To get clues into the molecular mechanisms underlying the adaptive strategies employed to deal with the stressing conditions of the CF lung including antibiotic therapy, quantitative proteomics (2-D DIGE) was used to compare the expression programs of two clonal isolates retrieved from a chronically infected CF patient. Isolate IST439 was the first bacterium recovered while the clonal variant IST4113 was obtained after 3 years of persistent infection and intravenous therapy with ceftazidime/gentamicin. This isolate exhibits higher resistance levels towards different classes of antimicrobials. Proteins of the functional categories Energy metabolism, Translation, Nucleotide synthesis, Protein folding and stabilization are more abundant in IST4113, compared with IST439, suggesting an increased protein synthesis, DNA repair and stress resistance in IST4113. The level of proteins involved in peptidoglycan, membrane lipids and lipopolysaccharide synthesis is also altered and proteins involved in iron binding and transport are more abundant in IST4113. The quantitative comparison of the two proteomes suggests a genetic adaptation leading to increased antimicrobial resistance and bacterial persistence in the CF airways.

Long-term colonization of the cystic fibrosis lung by Burkholderia cepacia complex bacteria: epidemiology, clonal variation, and genome-wide expression alterations.

Long-term respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) patients generally lead to a more rapid decline in lung function and, in some cases, to a fatal necrotizing pneumonia known as the "cepacia syndrome." Bcc bacteria are ubiquitous in the environment and are recognized as serious opportunistic pathogens that are virtually impossible to eradicate from the CF lung, posing a serious clinical threat. The epidemiological survey of Bcc bacteria involved in respiratory infections at the major Portuguese CF Treatment Center at Santa Maria Hospital, in Lisbon, has been carried out by our research group for the past 16 years, covering over 500 clinical isolates where B. cepacia and B. cenocepacia are the predominant species, with B. stabilis, B. contaminans, B. dolosa, and B. multivorans also represented. The systematic and longitudinal study of this CF population during such an extended period of time represents a unique case-study, comprehending 41 Bcc-infected patients (29 pediatric and 12 adult) of whom around 70% have been persistently colonized between 7 months and 9 years. During chronic infection, the CF airways represent an evolving ecosystem, with multiple phenotypic variants emerging from the clonal population and becoming established in the patients' airways as the result of genetic adaptation. Understanding the evolutionary mechanisms involved is crucial for an improved therapeutic outcome of chronic infections in CF. This review focuses on our contribution to the understanding of these adaptive mechanisms based on extensive phenotypic, genotypic, and genome-wide expression approaches of selected Bcc clonal variants obtained during long-term colonization of the CF airways.

Genomic expression analysis reveals strategies of Burkholderia cenocepacia to adapt to cystic fibrosis patients' airways and antimicrobial therapy.

Pulmonary colonization of cystic fibrosis (CF) patients with Burkholderia cenocepacia or other bacteria of the Burkholderia cepacia complex (Bcc) is associated with worse prognosis and increased risk of death. During colonization, the bacteria may evolve under the stressing selection pressures exerted in the CF lung, in particular, those resulting from challenges of the host immune defenses, antimicrobial therapy, nutrient availability and oxygen limitation. Understanding the adaptive mechanisms that promote successful colonization and long-term survival of B. cenocepacia in the CF lung is essential for an improved therapeutic outcome of chronic infections. To get mechanistic insights into these adaptive strategies a transcriptomic analysis, based on DNA microarrays, was explored in this study. The genomic expression levels in two clonal variants isolated during long-term colonization of a CF patient who died from the cepacia syndrome were compared. One of the isolates examined, IST439, is the first B. cenocepacia isolate retrieved from the patient and the other isolate, IST4113, was obtained three years later and is more resistant to different classes of antimicrobials. Approximately 1000 genes were found to be differently expressed in the two clonal variants reflecting a marked reprogramming of genomic expression. The up-regulated genes in IST4113 include those involved in translation, iron uptake (in particular, in ornibactin biosynthesis), efflux of drugs and in adhesion to epithelial lung tissue and to mucin. Alterations related with adaptation to the nutritional environment of the CF lung and to an oxygen-limited environment are also suggested to be a key feature of transcriptional reprogramming occurring during long-term colonization, antibiotic therapy and the progression of the disease.