Advances in Phage Therapy: Targeting the Burkholderia cepacia Complex.

The increasing prevalence and worldwide distribution of multidrug-resistant bacterial pathogens is an imminent danger to public health and threatens virtually all aspects of modern medicine. Particularly concerning, yet insufficiently addressed, are the members of the Burkholderia cepacia complex (Bcc), a group of at least twenty opportunistic, hospital-transmitted, and notoriously drug-resistant species, which infect and cause morbidity in patients who are immunocompromised and those afflicted with chronic illnesses, including cystic fibrosis (CF) and chronic granulomatous disease (CGD). One potential solution to the antimicrobial resistance crisis is phage therapy-the use of phages for the treatment of bacterial infections. Although phage therapy has a long and somewhat checkered history, an impressive volume of modern research has been amassed in the past decades to show that when applied through specific, scientifically supported treatment strategies, phage therapy is highly efficacious and is a promising avenue against drug-resistant and difficult-to-treat pathogens, such as the Bcc. In this review, we discuss the clinical significance of the Bcc, the advantages of phage therapy, and the theoretical and clinical advancements made in phage therapy in general over the past decades, and apply these concepts specifically to the nascent, but growing and rapidly developing, field of Bcc phage therapy.

Of onions and men: Report of cavitary community acquired pneumonia due to Burkholderia cepacia complex in an immunocompetent patient and review of the literature.

Burkholderia cepacia complex consists of highly antibiotic resistant gram negative bacilli that are plant symbionts and also potential agents of human infection.  This bacterial family's claim to fame in clinical medicine is as the scourge of cystic fibrosis patients, in whom it is a notorious respiratory pathogen.  Outside of cystic fibrosis, it rarely comes to mind as an etiology of community acquired pneumonia with or without lung cavitation in immunocompetent hosts.  We describe a case of an otherwise healthy, community-dwelling man who presented with subacute cavitary lung disease, the causative organism of which turned out to be Burkholderia cepacia complex.  Our report is accompanied by a review of the literature, which identified an additional eleven cases in the same category.  We analyze all of the available cases for the emergence of any identifiable patterns or peculiarities.

Major Aspects of Burkholderia gladioli and Burkholderia cepacia Infections in Children.

BACKGROUND: Burkholderia cepacia complex is an aerobic, non-spore-forming, catalase-positive, nonfermentative, Gram-negative bacterium common in environment. It is a serious pathogen especially for patients with cystic fibrosis (CF). But pathogenicity of Burkholderia is not limited to patients with CF. Herein, we aimed to reveal clinical patterns and outcomes of Burkholderia infections in pediatric patients in our hospital and also antimicrobial susceptibility of the isolated strain. METHODS: This retrospective study was conducted in Ankara Hematology Oncology Children's Training and Research Hospital. Patients with isolates of Burkholderia spp. between January 6, 2013, and January 12, 2018, were included in the study. RESULTS: Burkholderia spp. was isolated from 55 patients. 94.6% of these patients had underlying diseases and had prior hospitalization within a year. Burkholderia gladioli grew in 15 patients' samples (27.3%); 38 patients grew B. cepacia (69.1%). None of the patients that B. gladioli was isolated was diagnosed as CF;. all had nosocomial infections. B. gladioli seemed to be more susceptible to aminoglycosides, piperacillin-tazobactam, carbapenems and ciprofloxacin than B. cepacia (P = 0.00), whereas B. cepacia seemed to be more susceptible to ceftazidime than B. gladioli (P = 0.032). In addition, B. cepacia was more susceptible to trimethoprim-sulfamethoxazole and levofloxacin than B. gladioli, but this difference was not statistically significant (P = 0.76). CONCLUSIONS: The incidence of nosocomial infections caused by Burkholderia spp. is rare especially in pediatric literature. In our study, nosocomial Burkholderia infections occurred mostly in intensive care unit patients. The surveillance of Burkholderia infections is still very important, and the clinicians should be aware of changing epidemiology and increasing resistance of the microorganism. Besides, there are no internationally agreed minimal inhibitory concentration breakpoints and disk-diffusion test thresholds for susceptibility testing for Burkholderia. Thus, the methods which were used for antibiotic susceptibility testing in our center might cause uncertainty about the results and internationally agreed minimal inhibitory concentration breakpoints and disk-diffusion test thresholds for susceptibility testing for Burkholderia is still a gap to fill for the current literature.

Intracellular survival and innate immune evasion of Burkholderia cepacia: Improved understanding of quorum sensing-controlled virulence factors, biofilm, and inhibitors.

Burkholderia cepacia complex (Bcc) are opportunistic pathogens implicated with nosocomial infections, and high rates of morbidity and mortality, especially in individuals with cystic fibrosis (CF). B. cepacia are naturally resistant to different classes of antibiotics, and can subvert the host innate immune responses by producing quorum sensing (QS) controlled virulence factors and biofilms. It still remains a conundrum as to how exactly the bacterium survives the intracellular environment within the host cells of CF patients and immunocompromised individuals although the bacterium can invade human lung epithelial cells, neutrophils, and murine macrophages. The mechanisms associated with intracellular survival in the airway epithelial cells and the role of QS and virulence factors in B. cepacia infections in cystic fibrosis remain largely unclear. The current review focuses on understanding the role of QS-controlled virulence factors and biofilms, and provides additional impetus to understanding the potentials of QS-inhibitory strategies against B. cepacia.

El controvertido complejo Burkholderia cepacia, un grupo de especies promotoras del crecimiento vegetal y patógenas de plantas, animales y humanos / The controversial Burkholderia cepacia complex, a group of plant growth promoting species and plant, animals and human pathogens

El complejo Burkholderia cepacia está formado por 22 especies conocidas como patógenos oportunistas en personas inmunocomprometidas, especialmente en aquellas con fibrosis quística. También se aíslan de infecciones nosocomiales y son difíciles de erradicar debido a su capacidad intrínseca para resistir una gran variedad de antibióticos. En general, estas especies presentan genomas de gran tamaño (hasta 9 Mpb) divididos en 2-5 replicones. Esta característica aporta una gran versatilidad metabólica, que se considera importante para habitar el suelo, el agua, las plantas, incluso los nódulos en leguminosas. Algunas especies del complejo B. cepacia exhiben actividades benéficas, como biorremediación, biocontrol y promoción del crecimiento vegetal. No obstante, debido a su papel en infecciones de humanos, su uso en la agricultura está restringido. El complejo B. cepacia es un tema constante de estudio debido a su impacto en el sector salud y su potencial en la agricultura. En este trabajo se examina la historia del complejo B. cepacia y se revisa la información reciente relacionada con este grupo de bacterias.

The Burkholderia cepacia complex is a group of 22 species, which are known as opportunistic pathogens in immunocompromised people, especially those suffering from cystic fibrosis. It is also found in nosocomial infections and is difficult to eradicate due to intrinsic resistance to several antibiotics. The species have large genomes (up to 9 Mbp), distributed into 2-5 replicons. These features significantly contribute to genome plasticity, which makes them thrive in different environments like soil, water, plants or even producing nodules in legume plants. Some B. cepacia complex species are beneficial in bioremediation, biocontrol and plant-growth promotion. However, because the B. cepacia complex is involved in human infection, its use in agriculture is restricted. B. cepacia complex is being constantly studied due to the health problems that it causes and because of its agricultural potential. In this review, the history of B. cepacia complex and the most recently published information related to this complex are revised.

Accurate identification and epidemiological characterization of Burkholderia cepacia complex: an update.

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are among the most important pathogens isolated from cystic fibrosis (CF) patients and in hospital acquired infections (HAI). Accurate identification of Bcc is questionable by conventional biochemical methods. Clonal typing of Burkholderia is also limited due to the problem with identification. Phenotypic identification methods such as VITEK2, protein signature identification methods like VITEK MS, Bruker Biotyper, and molecular targets such as 16S rRNA, recA, hisA and rpsU were reported with varying level of discrimination to identify Bcc. rpsU and/or 16S rRNA sequencing, VITEK2, VITEK MS and Bruker Biotyper could discriminate between Burkholderia spp. and non-Burkholderia spp. Whereas, Bcc complex level identification can be given by VITEK MS, Bruker Biotyper, and 16S rRNA/rpsU/recA/hisA sequencing. For species level identification within Bcc hisA or recA sequencing are reliable. Identification of Bcc is indispensable in CF patients and HAI to ensure appropriate antimicrobial therapy.

[The controversial Burkholderia cepacia complex, a group of plant growth promoting species and plant, animals and human pathogens]. / El controvertido complejo Burkholderia cepacia, un grupo de especies promotoras del crecimiento vegetal y patógenas de plantas, animales y humanos.

The Burkholderia cepacia complex is a group of 22 species, which are known as opportunistic pathogens in immunocompromised people, especially those suffering from cystic fibrosis. It is also found in nosocomial infections and is difficult to eradicate due to intrinsic resistance to several antibiotics. The species have large genomes (up to 9 Mbp), distributed into 2-5 replicons. These features significantly contribute to genome plasticity, which makes them thrive in different environments like soil, water, plants or even producing nodules in legume plants. Some B. cepacia complex species are beneficial in bioremediation, biocontrol and plant-growth promotion. However, because the B. cepacia complex is involved in human infection, its use in agriculture is restricted. B. cepacia complex is being constantly studied due to the health problems that it causes and because of its agricultural potential. In this review, the history of B. cepacia complex and the most recently published information related to this complex are revised.

Burkholderia multivorans septicemia in a pediatric liver transplant patient.

"Cepacia syndrome", caused by Burkholderia cepacia complex and often associated with cystic fibrosis, carries a high mortality rate. It is rare for Burkholderia multivorans, a species within the B. cepacia complex, to cause cepacia syndrome even among patients with cystic fibrosis. This is the first reported fatal case of cepacia syndrome caused by B. multivorans occurring in a pediatric liver transplant recipient who does not have cystic fibrosis. We describe the unique characteristics of this pathogen among the non-cystic fibrosis population and the importance of early recognition and treatment.

Small Noncoding Regulatory RNAs from Pseudomonas aeruginosa and Burkholderia cepacia Complex.

Cystic fibrosis (CF) is the most life-limiting autosomal recessive disorder in Caucasians. CF is characterized by abnormal viscous secretions that impair the function of several tissues, with chronic bacterial airway infections representing the major cause of early decease of these patients. Pseudomonas aeruginosa and bacteria from the Burkholderia cepacia complex (Bcc) are the leading pathogens of CF patients' airways. A wide array of virulence factors is responsible for the success of infections caused by these bacteria, which have tightly regulated responses to the host environment. Small noncoding RNAs (sRNAs) are major regulatory molecules in these bacteria. Several approaches have been developed to study P. aeruginosa sRNAs, many of which were characterized as being involved in the virulence. On the other hand, the knowledge on Bcc sRNAs remains far behind. The purpose of this review is to update the knowledge on characterized sRNAs involved in P. aeruginosa virulence, as well as to compile data so far achieved on sRNAs from the Bcc and their possible roles on bacteria virulence.

Three Distinct Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition by the Cystic Fibrosis Pathogen Burkholderia dolosa.

The respiratory tracts of individuals afflicted with cystic fibrosis (CF) harbor complex polymicrobial communities. By an unknown mechanism, species of the Gram-negative Burkholderia cepacia complex, such as Burkholderia dolosa, can displace other bacteria in the CF lung, causing cepacia syndrome, which has a poor prognosis. The genome of Bdolosa strain AU0158 (BdAU0158) contains three loci that are predicted to encode contact-dependent growth inhibition (CDI) systems. CDI systems function by translocating the toxic C terminus of a large exoprotein directly into target cells, resulting in growth inhibition or death unless the target cells produce a cognate immunity protein. We demonstrate here that each of the three bcpAIOB loci in BdAU0158 encodes a distinct CDI system that mediates interbacterial competition in an allele-specific manner. While only two of the three bcpAIOB loci were expressed under the in vitro conditions tested, the third conferred immunity under these conditions due to the presence of an internal promoter driving expression of the bcpI gene. One BdAU0158 bcpAIOB allele is highly similar to bcpAIOB in Burkholderia thailandensis strain E264 (BtE264), and we showed that their BcpI proteins are functionally interchangeable, but contact-dependent signaling (CDS) phenotypes were not observed in BdAU0158. Our findings suggest that the CDI systems of BdAU0158 may provide this pathogen an ecological advantage during polymicrobial infections of the CF respiratory tract.IMPORTANCE Human-associated polymicrobial communities can promote health and disease, and interbacterial interactions influence the microbial ecology of such communities. Polymicrobial infections of the cystic fibrosis respiratory tract impair lung function and lead to the death of individuals suffering from this disorder; therefore, a greater understanding of these microbial communities is necessary for improving treatment strategies. Bacteria utilize contact-dependent growth inhibition systems to kill neighboring competitors and maintain their niche within multicellular communities. Several cystic fibrosis pathogens have the potential to gain an ecological advantage during infection via contact-dependent growth inhibition systems, including Burkholderia dolosa Our research is significant, as it has identified three functional contact-dependent growth inhibition systems in Bdolosa that may provide this pathogen a competitive advantage during polymicrobial infections.

Targeting the Bacterial Cytoskeleton of the Burkholderia cepacia Complex for Antimicrobial Development: A Cautionary Tale.

Burkholderia cepacia complex (BCC) bacteria are a group of opportunistic pathogens that cause severe lung infections in cystic fibrosis (CF). Treatment of BCC infections is difficult, due to the inherent and acquired multidrug resistance of BCC. There is a pressing need to find new bacterial targets for antimicrobials. Here, we demonstrate that the novel compound Q22, which is related to the bacterial cytoskeleton destabilising compound A22, can reduce the growth rate and inhibit growth of BCC bacteria. We further analysed the phenotypic effects of Q22 treatment on BCC virulence traits, to assess its feasibility as an antimicrobial. BCC bacteria were grown in the presence of Q22 with a broad phenotypic analysis, including resistance to H2O2-induced oxidative stress, changes in the inflammatory potential of cell surface components, and in-vivo drug toxicity studies. The influence of the Q22 treatment on inflammatory potential was measured by monitoring the cytokine responses of BCC whole cell lysates, purified lipopolysaccharide, and purified peptidoglycan extracted from bacterial cultures grown in the presence or absence of Q22 in differentiated THP-1 cells. BCC bacteria grown in the presence of Q22 displayed varying levels of resistance to H2O2-induced oxidative stress, with some strains showing increased resistance after treatment. There was strain-to-strain variation in the pro-inflammatory ability of bacterial lysates to elicit TNFα and IL-1ß from human myeloid cells. Despite minimal toxicity previously shown in vitro with primary CF cell lines, in-vivo studies demonstrated Q22 toxicity in both zebrafish and mouse infection models. In summary, destabilisation of the bacterial cytoskeleton in BCC, using compounds such as Q22, led to increased virulence-related traits in vitro. These changes appear to vary depending on strain and BCC species. Future development of antimicrobials targeting the BCC bacterial cytoskeleton may be hampered if such effects translate into the in-vivo environment of the CF infection.

The OmpR Regulator of Burkholderia multivorans Controls Mucoid-to-Nonmucoid Transition and Other Cell Envelope Properties Associated with Persistence in the Cystic Fibrosis Lung.

Bacteria from the Burkholderia cepacia complex grow in different natural and man-made environments and are feared opportunistic pathogens that cause chronic respiratory infections in cystic fibrosis patients. Previous studies showed that Burkholderia mucoid clinical isolates grown under stress conditions give rise to nonmucoid variants devoid of the exopolysaccharide cepacian. Here, we determined that a major cause of the nonmucoid morphotype involves nonsynonymous mutations and small indels in the ompR gene encoding a response regulator of a two-component regulatory system. In trans complementation of nonmucoid variants (NMVs) with the native gene restored exopolysaccharide production. The loss of functional Burkholderia multivorans OmpR had positive effects on growth, adhesion to lung epithelial cells, and biofilm formation in high-osmolarity medium, as well as an increase in swimming and swarming motilities. In contrast, phenotypes such as antibiotic resistance, biofilm formation at low osmolarity, and virulence in Galleria mellonella were compromised by the absence of functional OmpR. Transcriptomic studies indicated that loss of the ompR gene affects the expression of 701 genes, many associated with outer membrane composition, motility, stress response, iron acquisition, and the uptake of nutrients, consistent with starvation tolerance. Since the stresses here imposed on B. multivorans may strongly resemble the ones found in the cystic fibrosis (CF) airways and mutations in the ompR gene from longitudinally collected CF isolates have been found, this regulator might be important for the production of NMVs in the CF environment.IMPORTANCE Within the cystic fibrosis (CF) lung, bacteria experience high-osmolarity conditions due to an ion unbalance resulting from defects in CF transmembrane conductance regulator (CFTR) protein activity in epithelial cells. Understanding how bacterial CF pathogens thrive in this environment might help the development of new therapeutic interventions to prevent chronic respiratory infections. Here, we show that the OmpR response regulator of one of the species found in CF respiratory infections, Burkholderia multivorans, is involved in the emergence of nonmucoid colony variants and is important for osmoadaptation by regulating several cell envelope components. Specifically, genetic, phenotypic, genomic, and transcriptomic approaches uncover OmpR as a regulator of cell wall remodeling under stress conditions, with implications in several phenotypes such as exopolysaccharide production, motility, antibiotic resistance, adhesion, and virulence.

Use of Synthetic Hybrid Strains To Determine the Role of Replicon 3 in Virulence of the Burkholderia cepacia Complex.

The Burkholderia cepacia complex (Bcc) displays a wealth of metabolic diversity with great biotechnological potential, but the utilization of these bacteria is limited by their opportunistic pathogenicity to humans. The third replicon of the Bcc, megaplasmid pC3 (0.5 to 1.4 Mb, previously chromosome 3), is important for various phenotypes, including virulence, antifungal, and proteolytic activities and the utilization of certain substrates. Approximately half of plasmid pC3 is well conserved throughout sequenced Bcc members, while the other half is not. To better locate the regions responsible for the key phenotypes, pC3 mutant derivatives of Burkholderia cenocepacia H111 carrying large deletions (up to 0.58 Mb) were constructed with the aid of the FLP-FRT (FRT, flippase recognition target) recombination system from Saccharomyces cerevisiae The conserved region was shown to confer near-full virulence in both Caenorhabditis elegans and Galleria mellonella infection models. Antifungal activity was unexpectedly independent of the part of pC3 bearing a previously identified antifungal gene cluster, while proteolytic activity was dependent on the nonconserved part of pC3, which encodes the ZmpA protease. To investigate to what degree pC3-encoded functions are dependent on chromosomally encoded functions, we transferred pC3 from Burkholderia cenocepacia K56-2 and Burkholderia lata 383 into other pC3-cured Bcc members. We found that although pC3 is highly important for virulence, it was the genetic background of the recipient that determined the pathogenicity level of the hybrid strain. Furthermore, we found that important phenotypes, such as antifungal activity, proteolytic activity, and some substrate utilization capabilities, can be transferred between Bcc members using pC3.IMPORTANCE The Burkholderia cepacia complex (Bcc) is a group of closely related bacteria with great biotechnological potential. Some strains produce potent antifungal compounds and can promote plant growth or degrade environmental pollutants. However, their agricultural potential is limited by their opportunistic pathogenicity, particularly for cystic fibrosis patients. Despite much study, their virulence remains poorly understood. The third replicon, pC3, which is present in all Bcc isolates and is important for pathogenicity, stress resistance, and the production of antifungal compounds, has recently been reclassified from a chromosome to a megaplasmid. In this study, we identified regions on pC3 important for virulence and antifungal activity and investigated the role of the chromosomal background for the function of pC3 by exchanging the megaplasmid between different Bcc members. Our results may open a new avenue for the construction of antifungal but nonpathogenic Burkholderia hybrids. Such strains may have great potential as biocontrol strains for protecting fungus-borne diseases of plant crops.

An Oxygen-Sensing Two-Component System in the Burkholderia cepacia Complex Regulates Biofilm, Intracellular Invasion, and Pathogenicity.

Burkholderia dolosa is a member of the Burkholderia cepacia complex (BCC), which is a group of bacteria that cause chronic lung infection in patients with cystic fibrosis (CF) and can be associated with outbreaks carrying high morbidity and mortality. While investigating the genomic diversity of B. dolosa strains collected from an outbreak among CF patients, we previously identified fixL as a gene showing signs of strong positive selection. This gene has homology to fixL of the rhizobial FixL/FixJ two-component system. The goals of this study were to determine the functions of FixLJ and their role in virulence in B. dolosa. We generated a fixLJ deletion mutant and complemented controls in B. dolosa strain AU0158. Using a fixK-lacZ reporter we found that FixLJ was activated in low oxygen in multiple BCC species. In a murine pneumonia model, the B. dolosa fixLJ deletion mutant was cleared faster from the lungs and spleen than wild-type B. dolosa strain AU0158 at 7 days post infection. Interestingly, the fixLJ deletion mutant made more biofilm, albeit with altered structure, but was less motile than strain AU0158. Using RNA-seq with in vitro grown bacteria, we found ~11% of the genome was differentially expressed in the fixLJ deletion mutant relative to strain AU0158. Multiple flagella-associated genes were down-regulated in the fixLJ deletion mutant, so we also evaluated virulence of a fliC deletion mutant, which lacks a flagellum. We saw no difference in the ability of the fliC deletion mutant to persist in the murine model relative to strain AU0158, suggesting factors other than flagella caused the phenotype of decreased persistence. We found the fixLJ deletion mutant to be less invasive in human lung epithelial and macrophage-like cells. In conclusion, B. dolosa fixLJ is a global regulator that controls biofilm formation, motility, intracellular invasion/persistence, and virulence.

Clinical and in vitro evidence for the antimicrobial therapy in Burkholderia cepacia complex infections.

Treatment of infections caused by Burkholderia cepacia complex (Bcc) in cystic fibrosis (CF) patients poses a complex problem. Bcc is multidrug-resistant due to innate and acquired mechanisms of resistance. As CF patients receive multiple courses of antibiotics, susceptibility patterns of strains from CF patients may differ from those noted in strains from non-CF patients. Thus, there was a need for assessing in vitro and clinical data to guide antimicrobial therapy in these patients. A systematic search of literature, followed by extraction and analysis of available information from human and in vitro studies was done. The results of the analysis are used to address various aspects like use of antimicrobials for pulmonary and non-pulmonary infections, use of combination versus monotherapy, early eradication, duration of therapy, route of administration, management of biofilms, development of resistance during therapy, pharmacokinetics-pharmacodynamics correlations, therapy in post-transplant patients and newer drugs in Bcc-infected CF patients.

The tyrosine kinase BceF and the phosphotyrosine phosphatase BceD of Burkholderia contaminans are required for efficient invasion and epithelial disruption of a cystic fibrosis lung epithelial cell line.

Bacterial tyrosine kinases and their cognate protein tyrosine phosphatases are best known for regulating the biosynthesis of polysaccharides. Moreover, their roles in the stress response, DNA metabolism, cell division, and virulence have also been documented. The aim of this study was to investigate the pathogenicity and potential mechanisms of virulence dependent on the tyrosine kinase BceF and phosphotyrosine phosphatase BceD of the cystic fibrosis opportunistic pathogen Burkholderia contaminans IST408. The insertion mutants bceD::Tp and bceF::Tp showed similar attenuation of adhesion and invasion of the cystic fibrosis lung epithelial cell line CFBE41o- compared to the parental strain B. contaminans IST408. In the absence of bceD or bceF genes, B. contaminans also showed a reduction in the ability to translocate across polarized epithelial cell monolayers, demonstrated by a higher transepithelial electrical resistance, reduced flux of fluorescein isothiocyanate-labeled bovine serum albumin, and higher levels of tight junction proteins ZO-1, occludin, and claudin-1 present in monolayers exposed to these bacterial mutants. Furthermore, bceD::Tp and bceF::Tp mutants induced lower levels of interleukin-6 (IL-6) and IL-8 release than the parental strain. In conclusion, although the mechanisms of pathogenicity dependent on BceD and BceF are not understood, these proteins contribute to the virulence of Burkholderia by enhancement of cell attachment and invasion, disruption of epithelial integrity, and modulation of the proinflammatory response.

Growth on mannitol-rich media elicits a genome-wide transcriptional response in Burkholderia multivorans that impacts on multiple virulence traits in an exopolysaccharide-independent manner.

In common with other members of the Burkholderia cepacia complex (BCC), Burkholderia multivorans is capable of producing exopolysaccharide (EPS) when grown on certain mannitol-rich media. The significance of the resulting mucoid phenotype and the genome-wide response to mannitol has never been characterized despite its clinical relevance following the approval of a dried-powder preparation of mannitol as an inhaled osmolyte therapy for cystic fibrosis (CF) patients. In the present study we defined the transcriptional response of B. multivorans ATCC 17616, a model genome-sequenced strain of environmental origin, to growth on mannitol-rich yeast extract media (MYEM). EPS-dependent and -independent impact of MYEM on virulence-associated traits was assessed in both strain ATCC 17616 and the CF isolate B. multivorans C1576. Our studies revealed a significant transcriptional response to MYEM encompassing approximately 23 % of predicted genes within the genome. Strikingly, this transcriptional response identified that EPS induction occurs in ATCC 17616 without the upregulation of the bce-I and bce-II EPS gene clusters, despite their pivotal role in EPS biosynthesis. Of approximately 20 differentially expressed putative virulence factors, 16 exhibited upregulation including flagella, ornibactin, oxidative stress proteins and phospholipases. MYEM-grown B. multivorans also exhibited enhanced motility, biofilm formation and epithelial cell invasion. In contrast to these potential virulence enhancements, MYEM-grown B. multivorans C1576 showed attenuated virulence in the Galleria mellonella infection model. All of the observed phenotypic responses occurred independently of EPS production, highlighting the profound impact that mannitol-based growth has on the physiology and virulence of B. multivorans.

Comparative metabolic systems analysis of pathogenic Burkholderia.

Burkholderia cenocepacia and Burkholderia multivorans are opportunistic drug-resistant pathogens that account for the majority of Burkholderia cepacia complex infections in cystic fibrosis patients and also infect other immunocompromised individuals. While they share similar genetic compositions, B. cenocepacia and B. multivorans exhibit important differences in pathogenesis. We have developed reconciled genome-scale metabolic network reconstructions of B. cenocepacia J2315 and B. multivorans ATCC 17616 in parallel (designated iPY1537 and iJB1411, respectively) to compare metabolic abilities and contextualize genetic differences between species. The reconstructions capture the metabolic functions of the two species and give insight into similarities and differences in their virulence and growth capabilities. The two reconstructions have 1,437 reactions in common, and iPY1537 and iJB1411 have 67 and 36 metabolic reactions unique to each, respectively. After curating the extensive reservoir of metabolic genes in Burkholderia, we identified 6 genes essential to growth that are unique to iPY1513 and 13 genes uniquely essential to iJB1411. The reconstructions were refined and validated by comparing in silico growth predictions to in vitro growth capabilities of B. cenocepacia J2315, B. cenocepacia K56-2, and B. multivorans ATCC 17616 on 104 carbon sources. Overall, we identified functional pathways that indicate B. cenocepacia can produce a wider array of virulence factors compared to B. multivorans, which supports the clinical observation that B. cenocepacia is more virulent than B. multivorans. The reconciled reconstructions provide a framework for generating and testing hypotheses on the metabolic and virulence capabilities of these two related emerging pathogens.

Biosafety and colonization of Burkholderia multivorans WS-FJ9 and its growth-promoting effects on poplars.

Burkholderia cepacia complex (Bcc) is a group of bacteria with conflicting biological characteristics, which make them simultaneously beneficial and harmful to humans. They have been exploited for biocontrol, bioremediation, and plant growth promotion. However, their capacity as opportunistic bacteria that infect humans restricts their biotechnological applications. Therefore, the risks of using these bacteria should be assessed. In this study, Burkholderia multivorans WS-FJ9 originally isolated from pine rhizosphere, which was shown to be efficient in solubilizing phosphate, was evaluated with respect to its biosafety, colonization in poplar rhizosphere, and growth-promoting effects on poplar seedlings. Pathogenicity of B. multivorans WS-FJ9 on plants was determined experimentally using onion and tobacco as model plants. Onion bulb inoculated with B. multivorans WS-FJ9 showed slight hypersensitive responses around the inoculation points, but effects were not detectable based on the inner color and odor of the onion. Tobacco leaves inoculated with B. multivorans WS-FJ9 exhibited slightly water-soaked spots around the inoculation points, which did not expand or develop into lesions even with repeated incubation. Pathogenicity of the strain in alfalfa, which has been suggested as an alternative Bcc model for mice, was not detectable. Results from gene-specific polymerase chain reactions showed that the tested B. multivorans WS-FJ9 strain did not possess the BCESM and cblA virulence genes. Scanning electron microscopy revealed that the colonization of the WS-FJ9 strain reached 1.4 × 10(4) colony forming units (cfu) g(-1) rhizosphere soil on day 77 post-inoculation. The B. multivorans WS-FJ9 strain could colonize the rhizosphere as well as the root tissues and cells of poplars. Greenhouse evaluations in both sterilized and non-sterilized soils indicated that B. multivorans WS-FJ9 significantly promoted growth in height, root collar diameter, and plant biomass of inoculated poplar seedlings compared with controls. Phosphorus contents of roots and stems of treated seedlings were 0.57 and 0.55 mg g(-1) higher than those of the controls, respectively. Phosphorus content was lower in the rhizosphere soils by an average of 1.03 mg g(-1) compared with controls. The results demonstrated that B. multivorans WS-FJ9 is a nonpathogenic strain that could colonize the roots and significantly promote the growth of poplar seedlings.

[Characterization of genotypes for Burkholderia cepacia complex strains isolated from patients in hospitals of Russian Federation].

88 cultures of microorganisms referred to the Burkholderia cepacia complex (Bcc) during initial identification were analyzed by multilocus sequencing (Multilocus Sequence Typing, MLST). 13 genotypes (sequence type, ST) were detected, 9 of them (708, 709, 710, 711, 712, 714, 727, 728, 729) were identified for the first time. Two new alleles for the gene trpB (357, 358), one of the genes atpD (306) and gltB (352) were detected and registered. It was found that strains of 2 genotypes (711, 712) belong to the species B. multivorans, 1 (ST102) - B. contaminans, 1 (ST51) - B. stabilis, 1 (ST729) - B. vietnamiensis. Most strains of the sample, representing 8 genotypes (208, 241, 728, 727, 708, 709, 710, 714), belong to the species B. cenocepacia. Identified genotypes differ in the global spread of the world: 4 genotype (51, 102, 208, 241) have intercontinental distribution, 1 (712) - intra. It is shown that strains causing nosocomial infections, in most cases refer to genotypes 728 and 708. Epidemiologically significant in respect of patients with cystic fibrosis should recognize genotype 709, detected in strains isolated from patients in seven federal districts (FD) of Russia. The Bcc strains of genotypes 241 (B. cenocepacia) and 729 (B. vietnamiensis) were isolated from the patients of the Far Eastern FD. They are not typical for other FD Russia. The possibility of concomitant infection in cystic fibrosis patient with two genotypes 709 - epidemiologically significant and 708 - nosocomial, was indicated. The long-termpersistence of a single genotype strain in the organism of patients with cystic fibrosis was demonstrated as for Bcc species B. cenocepacia (ST 709), so for B. multivorans (ST712). The possibility of transferring the strain Bcc, typical for nosocomial environment to patient with cystic fibrosis at surgery was observed.