Liquid Biphasic Flotation (LBF) is an advanced recovery method that has been effectively applied for biomolecules extraction. The objective of this investigation is to incorporate the fermentation and extraction process of lipase from Burkholderia cepacia using flotation system. Initial study was conducted to compare the performance of bacteria growth and lipase production using flotation and shaker system. From the results obtained, bacteria shows quicker growth and high lipase yield via flotation system. Integration process for lipase separation was investigated and the result showed high efficiency reaching 92.29% and yield of 95.73%. Upscaling of the flotation system exhibited consistent result with the lab-scale which are 89.53% efficiency and 93.82% yield. The combination of upstream and downstream processes in a single system enables the acceleration of product formation, improves the product yield and facilitates downstream processing. This integration system demonstrated its potential for biomolecules fermentation and separation that possibly open new opportunities for industrial production.
Depolymerase is an enzyme that plays an important role in the hydrolysis of polyhydroxyalkanoates [PHAs]. In the current study, Burkholderia cepacia DP1 was obtained from Penang, Malaysia in which the enzyme was purified using ion exchange and gel filtration (Superdex-75) column chromatography. The molecular mass of the enzyme was estimated to be 53.3 kDa using SDS-PAGE. The enzyme activity was increased to 36.8 folds with the recovery of 16.3% after purification. The enzyme activity was detected between pH 6.0-10 and at 35-55 °C with pH 6.0 and 45 °C facilitating the maximum activity. Depolymerase was inactivated by Tween-20, Tween-80, SDS and PMSF, but insensitive to metal ions (Mg2+, Ca2+, K+, Na2+, Fe3+) and organic solvents (methanol, ethanol, and acetone). The apparent Km values of the purified P(3HB) depolymerase enzyme for P(3HB) and P(3HB-co-14%3HV) were 0.7 mg/ml and 0.8 mg/ml, respectively. The Vmax values of the purified enzyme were 10 mg/min and 8.89 mg/min for P(3HB) and P(3HB-co-14%3HV), respectively. The current study discovered a new extracellular poly(3-hydroxybutyrate) [P(3HB)] depolymerase enzyme from Burkholderia cepacia DP1 isolated and purified to homogeneity from the culture supernatant. To the best of our knowledge, this is the first report demonstrating the purification and biochemical characterization of P(3HB) depolymerase enzyme from genus Burkholderia.
An extractive fermentation technique was developed using a thermoseparating reagent to form a two-phase system for simultaneous cell cultivation and downstream processing of extracellular Burkholderia cepacia lipase. A 10% (w/w) solution of ethylene oxide-propylene oxide (EOPO) with a molecular mass of 3900 g/mol and pH 8.5, a 200 rpm speed, and 30 °C were selected as the optimal conditions for lipase production (55 U/ml). Repetitive batch fermentation was performed by continuous replacement of the top phase every 24h, which resulted in an average cell growth mass of 4.7 g/L for 10 extractive batches over 240 h. In scaling-up the process, a bench-scale bioreactor was tested under the conditions that had been optimized in flasks. The production rate and recovery yield were higher in the bioreactor compared to fermentation performed in flasks.
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.
Burkholderia cepacia is a Gram-negative pathogen that causes serious respiratory infections in immunocompromised patients and individuals with cystic fibrosis. This bacterium is known to release extracellular proteins that may be involved in virulence.
PCR and restriction fragment length polymorphism (RFLP) typing of flagellin genes (fliC) from 57 clinical isolates of Burkholderia cepacia indicated that only type 11 flagellins were present. Twenty-two isolates previously identified as the epidemic UK cystic fibrosis strain were indistinguishable by this method, as were 11 isolates from a pseudo-outbreak in Senegal. Other clinical isolates, including 19 from disparate sources in Malaysia, were separated into nine fliC RFLP groups, exhibiting a large degree of divergence. When isolates were indistinguishable by fliC genotyping, their similarity was confirmed by whole genome macro-restriction analysis with pulsed-field gel electrophoresis following XbaI digestion. The variation in fliC sequences of B. cepacia was far greater than that with B. pseudomallei, supporting the view that 'B. cepacia', as currently defined, may comprise several different genomic species.
The contribution of microbial depolymerase has received much attention because of its potential in biopolymer degradation. In this study, the P(3HB) depolymerase enzyme of a newly isolated Burkholderia cepacia DP1 from soil in Penang, Malaysia, was optimized using response surface methodology (RSM). The factors affecting P(3HB) depolymerase enzyme production were studied using one-variable-at-a-time approach prior to optimization. Preliminary experiments revealed that the concentration of nitrogen source, concentration of carbon source, initial pH and incubation time were among the main factors influencing the enzyme productivity. An increase of 9.4 folds in enzyme production with an activity of 5.66 U/mL was obtained using optimal medium containing 0.028% N of di-ammonium hydrogen phosphate and 0.31% P(3HB-co-21%4HB) as carbon source at the initial pH of 6.8 for 38 h of incubation. Moreover, the RSM model showed great similarity between predicted and actual enzyme production indicating a successful model validation. This study warrants the ability of P(3HB) degradation by B. cepacia DP1 in producing higher enzyme activity as compared to other P(3HB) degraders being reported. Interestingly, the production of P(3HB) depolymerase was rarely reported within genus Burkholderia. Therefore, this is considered to be a new discovery in the field of P(3HB) depolymerase production.
This paper demonstrates Pseudomonas cepacia lipase catalyzed hydrolysis of p-nitrophenyl palmitate under irradiation of light with wavelengths of 250-750 nm. The reaction follows Michaelis-Menten Kinetics and the light irradiation increases the overall rate of hydrolysis. Using Lineweaver-Burk plot K M and V max values for the reaction in presence of light are found to be 39.07 and 66.67 mM/min/g, respectively; while for the same reaction under dark condition, the values are 7.08 and 10.21 mM/min/g. The linear form of enzyme dependent rate of reaction confirms that no mass-transfer limitations are present and the reaction is a kinetically controlled enzymatic reaction.
We have developed a multiplex PCR assay for rapid identification and differentiation of cultures for Burkholderia pseudomallei, Burkholderia thailandensis, Burkholderia mallei and Burkholderia cepacia complex. The assay is valuable for use in clinical and veterinary laboratories, and in a deployable laboratory during outbreaks.
Burkholderia cepacia (B. cepacia) is an aerobic Gram-negative bacillus found in various aquatic environments and can cause food contamination. We investigated the photodynamic antibacterial effects of food additive curcumin combined with EDTA on B. cepacia. We found a ~4-log reduction in B. cepacia viability when photo-irradiated with curcumin at 50 μM by blue LED light (16 mW/cm2) for 30 min with 0.4% (w/v) EDTA. Moreover, the bacterial morphological alterations and the leakage of intracellular contents were observed after photodynamic treatment. There were also obvious genomic DNA cleavage and a general loss of bacterial proteins assigned to large-scale protein degradation after photodynamic inactivation treatment. Collectively, curcumin in combination with EDTA illuminated by blue LED is a potential candidate for photodynamic inactivation of B. cepacia.
The feasibility of using waste glycerol from the biodiesel industry for biosynthesis of polyhydroxyalkanoate (PHA) by Burkholderia cepacia BPT1213 was evaluated. Culture conditions were optimized by growing B. cepacia BPT1213 in mineral salt medium supplemented with 2% waste glycerol in a 2.5 L bioreactor. Response surface methodology was used to determine the influence of aeration rate (0.6-1.8 vvm), agitation speed (100-300 rpm), and cultivation period (48-72 hr) on PHA production. The optimum conditions for the growth and PHA accumulation were 1.5 vvm, 300 rpm, and 72 hr, with predicted values of 5.08 g/L cell dry weight (CDW), 66.07% PHA content, and 3.35 g/L total PHA concentration. Using these conditions, the experimental system produced 5.63 g/L of CDW with 64.00% wt/wt PHA content, which is threefold higher PHA concentration (3.60 g/L) compared to the non-optimized conditions. The melting temperature (Tm ) of purified PHA was 173.45 ± 1.05°C. In conclusion, the statistical approach was significantly increased the PHA production using waste glycerol as the sole carbon source.
Burkholderia cepacia is an opportunistic human pathogen associated with lung infections. Secretory proteins of B. cepacia are known to be involved in virulence and may mediate important host-pathogen interactions. In the present study, secretory proteins isolated from B. cepacia culture supernatant were separated using two-dimensional gel electrophoresis, followed by Western blot analysis to identify the immunogenic proteins. Mice antibodies raised to B. cepacia inactivated whole bacteria, outer membrane protein and culture filtrate antigen detected 74, 104 and 32 immunogenic proteins, respectively. Eighteen of these immunogenic proteins which reacted with all three antibodies were identified and might be potential molecules as a diagnostic marker or a putative candidate vaccine against B. cepacia infections.
This study was conducted to screen the endophytic bacteria as a biological control agent (BCA) against Ganoderma boninense. A total of 581 endophytic bacteria were successfully isolated from symptomless oil palm root tissues at Teluk Intan, Perak, Malaysia. Three endophytic bacteria, Pseudomonas aeruginosa GanoEB1, Burkholderia cepacia GanoEB2, and Pseudomonas syringae GanoEB3 were found to have a potential as BCA based on their percentage inhibition of radial growth (PIRG) in dual culture and culture filtrate tests. Two nursery trials were conducted to evaluate the capability of these bacteria to suppress Ganoderma disease in oil palm seedlings that were artificially infected with G. boninense using rubber wood block (RWB) sitting technique. The percentage of disease incidence (DI), severity of foliar symptoms (SFS) and dead seedlings were used as the assessment tools. As a result, DI and SFS have developed much slower in the seedlings that were pre-treated with bacteria compared to untreated seedlings. After 6 months of inoculation, Ganoderma disease incidence was reduced from 62-75% in the seedlings treated with P. aeruginosa GanoEB1, followed by B. cepacia GanoEB2 (31-59%) and P. syringae GanoEB3 (30-31%). Among these three endophytic bacteria, P. aeruginosa GanoEB1 was the most effective in controlling Ganoderma disease and the dead seedlings were in the range of 13.3-26.7%, followed by B. cepacia GanoEB2 (33.3% for both trials) and P. syringae GanoEB3 (33.3-40.0%) compared to untreated seedlings at 60% for both trials. A field study needs to be conducted to verify their effectiveness in controlling Ganoderma in oil palm.
The effect of physical shearing on the attachment of six Pseudomonas aeruginosa strains and six Burkholderia cepacia strains to glass, stainless steel, polystyrene and Teflon® was determined. A significant (p < 0.05) decrease in hydrophobicity was apparent for all P. aeruginosa strains (17-36%) and B. cepacia, MS 5 (20%) after shearing. A significant (p < 0.05) decrease in attachment of some P. aeruginosa (0.2-0.5 log CFU/cm2) and B. cepacia (0.2-0.4 log CFU/cm2) strains to some surface types was apparent after shearing. Significant (p < 0.05) correlation was observed for both numbers of flagellated cells and hydrophobicity against attachment to glass, stainless steel and polystyrene for P. aeruginosa while only hydrophobicity showed significant correlation against the same surfaces for B. cepacia. Scanning electron microscopy and protein analysis showed that shearing removed surface proteins from the cells and may have led to the observed changes in hydrophobicity and attachment to abiotic surfaces.
Burkholderia cepacia is an opportunistic human pathogen associated with life-threatening pulmonary infections in immunocompromised individuals. Pathogenesis of B. cepacia infection involves adherence, colonisation, invasion, survival and persistence in the host. In addition, B. cepacia are also known to secrete factors, which are associated with virulence in the pathogenesis of the infection. In this study, the host factor that may be the cause of the infection was elucidated in human epithelial cell line, A549, that was exposed to live B. cepacia (mid-log phase) and its secretory proteins (mid-log and early-stationary phases) using the Illumina Human Ref-8 microarray platform. The non-infection A549 cells were used as a control. Expression of the host genes that are related to apoptosis, inflammation and cell cycle as well as metabolic pathways were differentially regulated during the infection. Apoptosis of the host cells and secretion of pro-inflammatory cytokines were found to be inhibited by both live B. cepacia and its secretory proteins. In contrast, the host cell cycle and metabolic processes, particularly glycolysis/glycogenesis and fatty acid metabolism were transcriptionally up-regulated during the infection. Our microarray analysis provided preliminary insights into mechanisms of B. cepacia pathogenesis. The understanding of host response to an infection would provide novel therapeutic targets both for enhancing the host's defences and repressing detrimental responses induced by the invading pathogen.
Immobilized PS-C 'Amano' II lipase was used to catalyze the interesterification of palm olein (POo) with 30, 50, and 70% stearic acid in n-hexane at 60 degrees C. The catalytic performance of the immobilized lipase was evaluated by determining the composition change of fatty acyl groups and triacylglycerol (TAG) by gas liquid chromatography and high-performance liquid chromatography, respectively. The interesterification process resulted in the formation of new TAGs, mainly tripalmitin and dipalmitostearin, both of which were absent in the original oil. These changes in TAG composition resulted in an increase in slip melting point, from the original 25.5 degrees C to 36.3, 37.0, and 40.0 degrees C in the modified POo with 30, 50, and 70% stearic acid, respectively. All the reactions attained steady state in about 6 h. This type of work will find great applications in food industries, such as confectionery.
We describe the first clinical case of Burkholderia cepacia keratitis registered in Southeast Asia. A man in his mid-70s with underlying poorly controlled diabetes mellitus came with complaints of painful red left eye for 4 days. This was accompanied with photophobia and blurring of vision after being injured by a wooden particle while cutting grass. Slit-lamp examination showed a paracentral anterior corneal stromal infiltrates with overlying epithelial defect. Culture of the corneal smear isolated B. cepacia that was sensitive to ceftazidime, meropenem and bactrim (trimethorprim and sulfomethoxazole). Topical ceftazidime was given intensively to the patient and the infection resolved after 6 weeks of treatment.
A previously healthy Chinese male returned from working in the Malaysian jungle with a fever. A blood culture grew Gram-negative bacilli that were initially identified as Burkholderia cepacia by the VITEK 2 system but were subsequently found to be Burkholderia pseudomallei by partial sequencing of the 16S rRNA gene. The identification of B. pseudomallei using commercially available automated systems is problematic and clinicians in non-endemic areas should be aware of the possibility of melioidosis in patients with a relevant travel history and blood cultures growing Burkholderia spp.
The genus Burkholderia consists of diverse species which includes both "friends" and "foes." Some of the "friendly" Burkholderia spp. are extensively used in the biotechnological and agricultural industry for bioremediation and biocontrol. However, several members of the genus including B. pseudomallei, B. mallei, and B. cepacia, are known to cause fatal disease in both humans and animals. B. pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively, while B. cepacia infection is lethal to cystic fibrosis (CF) patients. Due to the high rate of infectivity and intrinsic resistance to many commonly used antibiotics, together with high mortality rate, B. mallei and B. pseudomallei are considered to be potential biological warfare agents. Treatments of the infections caused by these bacteria are often unsuccessful with frequent relapse of the infection. Thus, we are at a crucial stage of the need for Burkholderia vaccines. Although the search for a prophylactic therapy candidate continues, to date development of vaccines has not advanced beyond research to human clinical trials. In this article, we review the current research on development of safe vaccines with high efficacy against B. pseudomallei, B. mallei, and B. cepacia. It can be concluded that further research will enable elucidation of the potential benefits and risks of Burkholderia vaccines.
The increase in prevalence of antimicrobial-resistant bacteria (ARB) is currently a serious threat, thus there is a need for new antimicrobial compounds to combat infections caused by these ARB. An antimicrobial-producing bacterium, Burkholderia paludis was recently isolated and was able to produce a type of siderophore with antimicrobial properties, later identified as pyochelin. The chelating ability of pyochelin has been well-characterized but not for its antimicrobial characteristics. It was found that pyochelin had MIC values (MBC values) of 3.13 µg/mL (6.26 µg/mL) and 6.26 µg/mL (25.00 µg/mL) against three Enterococcus strains and four Staphylococcus strains. Pyochelin was able to inhibit E. faecalis ATCC 700802 (a vancomycin-resistant strain) in a time and dose dependent manner via killing kinetics assay. It was demonstrated that pyochelin enhanced the production of intracellular reactive oxygen species (ROS) over time, which subsequently caused a significant increase in malondialdehyde (MDA) production (a marker for lipid peroxidation) and ultimately led to cell death by disrupting the integrity of the bacterial membrane (validated via BacLight assay). This study has revealed the mechanism of action of pyochelin as an antimicrobial agent for the first time and has shown that pyochelin might be able to combat infections caused by E. faecalis in the future.