Carla P. Coutinho

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.

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.

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.

Burkholderia cenocepacia Phenotypic Clonal Variation during a 3.5-Year Colonization in the Lungs of a Cystic Fibrosis Patient

ABSTRACT 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 patients 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.

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

ABSTRACT 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.

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 patients 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.

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.

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)-β-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.

SNaPBceBcon: a Practical Tool for Identification and Genotyping of Burkholderia cepacia and Burkholderia contaminans

ABSTRACT We propose an optimized protocol for an extensive population analysis of Burkholderia cepacia and Burkholderia contaminans. Seven new polymorphisms were added to the recently proposed SNaPBcen assay, and a total of 18 markers ensured the clear identification and distinction of B. cepacia and B. contaminans isolates and high genotypic discrimination (Simpson index of 0.94) compared to those for multilocus sequence typing.