Burkholderia Cepacia Research Articles

Draft genome of hydrocarbon-degrader Burkholderia cepacia BCTT, isolated from soil chronically polluted with crude oil in Trinidad.

Burkholderia cepacia BCTT, isolated from chronically polluted soil in Trinidad, shows a capacity to survive in crude oil as a sole carbon source. Here, we report its high-quality draft genome sequence and highlight those pathways and genes involved in xenobiotic degradation. These data give a clearer insight into this organism's biotechnological potential.

Enhanced Plant Growth Through Composite Inoculation of Phosphate-Solubilizing Bacteria: Insights from Plate and Soil Experiments

Excessive application of phosphorus (P) fertilizers does not alleviate P deficiency in soils and may cause water eutrophication. The available P in acidic soils is bound to minerals, such as iron and aluminum, in forms that are difficult to utilize by plants. The low availability of P is detrimental to soil health and crop growth. To address the P imbalance in the soil, different bioremediation techniques, such as phosphate-solubilizing bacteria (PSB) application, have been employed. However, the systematic analysis of the effects of composite inoculation of PSB on crops remains elusive. In this study, the effects of composite-inoculated PSB on plant growth were systematically evaluated by two scales: plate experiment and soil test. This study employed six different strains of PSB including Lelliottia amnigena 1-1 (A), Kluyvera intermedia 1-2 (B), Pseudomonas tolaasii 1-6 (C), Burkholderia cepacia 2-5 (D), Pseudomonas frederiksbergensis 2-11 (E), and Pseudomonas rhodesiae 2-47 (F). Among the 57 different combinations of these strains, four combinations (AE, AF, ADF, and AEF) indicated higher phosphate-solubilizing abilities than the single strains. These combinations were used for subsequent experiments. The plate experiment revealed that composite strains were more effective than single strains in promoting the growth and development of seedlings and roots of oilseed rape. Furthermore, AE, AF, and AEF combinations indicated excellent growth-promoting effects. Moreover, the soil test revealed that the composite inoculation of AE and AEF significantly enhanced biomass accumulation and root development in oilseed rape. The increased growth-promoting effects of the composite strains were observed to be associated with to their phosphate-solubilizing capacities. Both scales confirmed that compared to single inoculation, composite inoculation of PSB is more beneficial for plant growth. This study provides composite inoculation materials and foundational data to support the bioremediation of P imbalance in soil.

Phage therapy to treat cystic fibrosis Burkholderia cepacia complex lung infections: perspectives and challenges

Burkholderia cepacia complex is a cause of serious lung infections in people with cystic fibrosis, exhibiting extremely high levels of antimicrobial resistance. These infections are difficult to treat and are associated with high morbidity and mortality. With a notable lack of new antibiotic classes currently in development, exploring alternative antimicrobial strategies for Burkholderia cepacia complex is crucial. One potential alternative seeing renewed interest is the use of bacteriophage (phage) therapy. This review summarises what is currently known about Burkholderia cepacia complex in cystic fibrosis, as well as challenges and insights for using phages to treat Burkholderia cepacia complex lung infections.

A Burkholderia contaminans outbreak in an Intensive Care Unit associated with contaminated bath solution: control and microbiological findings

BackgroundThe Burkholderia cepacia complex (BCC) comprises a group of bacteria with a growing threat as a contaminant of non-sterile solutions. We describe an outbreak of a BCC involving patients at intensive care unit related to a no-rinse bathing solution (NRBS). MethodsWe carried out patients, environmental and laboratory investigation performing analyses of cases, pulsed-field-gel electrophoresis and whole genome sequence (WGS) of isolates. ResultsWe investigated 32 cases and 16 isolates that were identified as Burkholderia contaminans, belonging to two different clones. One clone (new ST2175) was identified in 6 sequences (4 from patients; 2 from bath cart samples) and for the remaining 10 isolates (7 isolates from patients; 3 from NRBS) we identified the ST762. The investigation demonstrated that NRBS was the source of the outbreak caused by ST762 clone of B. contaminans. DiscussionEarly suspicion of a common source, rapid implementation of control measures and laboratory support are vital in an outbreak investigation. We also highlight the role of WGS which was very important to conclude our investigation regarding environmental samples and bacterial typing. ConclusionWe highlight the need to regulation water-based products and the role of WGS for investigate environmental samples.

Improvement of substrate specificity of the direct electron transfer type FAD-dependent glucose dehydrogenase catalytic subunit

The heterotrimeric flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase derived from Burkholderia cepacia (BcGDH) has many exceptional features for its use in glucose sensing—including that this enzyme is capable of direct electron transfer with an electrode in its heterotrimeric configuration. However, this enzyme’s high catalytic activity towards not only glucose but also galactose presents an engineering challenge. To increase the substrate specificity of this enzyme, it must be engineered to reduce its activity towards galactose while maintaining its activity towards glucose. To aid in these mutagenesis studies, the crystal structure composed of BcGDH’s small subunit and catalytic subunit (BcGDHγα), in complex with D-glucono-1,5-lactone was elucidated and used to construct the three-dimensional model for targeted, site-directed mutagenesis. BcGDHγα was then mutated at three different residues, glycine 322, asparagine 474 and asparagine 475. The single mutations that showed the greatest glucose selectivity were combined to create the resulting mutant, α-G322Q-N474S-N475S. The α-G322Q-N474S-N475S mutant and BcGDHγα wild type were then characterized with dye-mediated dehydrogenase activity assays to determine their kinetic parameters. The α-G322Q-N474S-N475S mutant showed more than a 2-fold increase in Vmax towards glucose and this mutant showed a lower activity towards galactose in the physiological range (5 mM) of 4.19 U mg−1, as compared to the wild type, 86.6 U mg−1. This resulting increase in specificity lead to an 81.7 gal/glc % activity for the wild type while the α-G322Q-N474S-N475S mutant had just 10.9 gal/glc % activity at 5 mM. While the BcGDHγα wild type has high specificity towards galactose, our engineering α-G322Q-N474S-N475S mutant showed concentration dependent response to glucose and was not affected by galactose.

Growth Behaviours of Two Rhizospheric Bacterial Strains in the Presence of Different Cadmium Concentrations and Temperatures

Understanding the interaction of Cd with plants, as well as the search for alternatives to minimize its effects, has caught the interest of the scientific community, due to the accelerated growth of contamination with this metal and its high toxicity. The aim of the present study was to evaluate the ability of two rhizospheric bacterial strains LC7504001.1 Burkholderia cepacia (C65RLIM) and FJ972527.1 Pseudomonas aeruginosa (C85RLIM) to three concentrations of cadmium (100, 300 and 500 mg/L) and to compare the growth behaviour with increasing temperature from 32 to 45°C. The results show different growth patterns for each strain tested against exposure to the three cadmium concentrations and the four temperatures tested. The Pseudomonas aeruginosa strain (C85RLIM) had the best adaptive behaviour to the three cadmium concentrations tested, and also showed stability when subjected to 500 mg/L CdCl2 and temperatures between 40 and 45°C. The results predict the possible behaviour of rhizospheric bacteria in the face of global warming and heavy metal remediation processes.

Activity of antibiotics against Burkholderia cepacia complex in artificial sputum medium.

Burkholderia cepacia complex (Bcc) is a collection of intrinsically drug-resistant Gram-negative bacteria that cause life-threatening disease in people with cystic fibrosis (CF). Standard antimicrobial susceptibility testing methods have poor predictive value for clinical outcomes in Bcc infections, probably due in part to differences between in vitro testing conditions and the environment in which Bcc grow in the lungs of people with CF. To compare the activity of commonly used antibiotics under standard in vitro testing conditions with activity in conditions mimicking those found in vivo. Two Bcc strains were grown alone and with six different antibiotics (minocycline, ceftazidime, meropenem, tobramycin, levofloxacin, trimethoprim-sulfamethoxazole) in two different media: standard cation-adjusted Mueller-Hinton broth and an artificial sputum medium designed to simulate the environment in the lungs of people with CF through addition of components including mucin, free DNA and amino acids. Two different starting conditions were used for time-kill assays: a standard ∼5 × 106 cfu/mL inoculum, and a high-density inoculum in which bacteria were grown for 72 hours before addition of antibiotics. Growth detection was performed by colony enumeration and by detection of resazurin reduction. There were major discrepancies between standard susceptibility results and activity in our models. Some antibiotics, including ceftazidime, showed minimal activity in all time-kill assays despite low minimal inhibitory concentrations, while others, notably tobramycin, were more active in high-density growth conditions than in standard time-kill assays. This work underscores the urgent need to develop more clinically relevant susceptibility testing approaches for Bcc.

Practical Guidance for Clinical Microbiology Laboratories: Updated guidance for processing respiratory tract samples from people with cystic fibrosis.

SUMMARYThis guidance presents recommendations for clinical microbiology laboratories for processing respiratory samples from people with cystic fibrosis (pwCF). Appropriate processing of respiratory samples is crucial to detect bacterial and fungal pathogens, guide treatment, monitor the epidemiology of cystic fibrosis (CF) pathogens, and assess therapeutic interventions. Thanks to CF transmembrane conductance regulator modulator therapy, the health of pwCF has improved, but as a result, fewer pwCF spontaneously expectorate sputum. Thus, the collection of sputum samples has decreased, while the collection of other types of respiratory samples such as oropharyngeal and bronchoalveolar lavage samples has increased. To optimize the detection of microorganisms, including Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Burkholderia cepacia complex; other less common non-lactose fermenting Gram-negative bacilli, e.g., Stenotrophomonas maltophilia, Inquilinus, Achromobacter, Ralstonia, and Pandoraea species; and yeasts and filamentous fungi, non-selective and selective culture media are recommended for all types of respiratory samples, including samples obtained from pwCF after lung transplantation. There are no consensus recommendations for laboratory practices to detect, characterize, and report small colony variants (SCVs) of S. aureus, although studies are ongoing to address the potential clinical impact of SCVs. Accurate identification of less common Gram-negative bacilli, e.g., S. maltophilia, Inquilinus, Achromobacter, Ralstonia, and Pandoraea species, as well as yeasts and filamentous fungi, is recommended to understand their epidemiology and clinical importance in pwCF. However, conventional biochemical tests and automated platforms may not accurately identify CF pathogens. MALDI-TOF MS provides excellent genus-level identification, but databases may lack representation of CF pathogens to the species-level. Thus, DNA sequence analysis should be routinely available to laboratories for selected clinical circumstances. Antimicrobial susceptibility testing (AST) is not recommended for every routine surveillance culture obtained from pwCF, although selective AST may be helpful, e.g., for unusual pathogens or exacerbations unresponsive to initial therapy. While this guidance reflects current care paradigms for pwCF, recommendations will continue to evolve as CF research expands the evidence base for laboratory practices.

Challenges in Managing Newly Diagnosed Granulomatosis With Polyangiitis and Concurrent Respiratory Infections: A Retrospective Case Series.

Granulomatosis with polyangiitis (GPA), formerly termed Wegener's granulomatosis, is an autoimmune disease marked by necrotizing granulomatous inflammation and vasculitis affecting small-sized vessels. It commonly impacts the renal and respiratory systems. This retrospective case series sampling conductedin a tertiary care hospital between May 2023 and April 2024 examined six newly diagnosed GPA patients who wereproteinase 3 cytoplasmic-antinuclear cytoplasmic antibody (PR3 c-ANCA) positive and had concurrent respiratory infections. None of them had any prior immunosuppressive conditions.The age range was 18-47 years with a mean of 35.0 (standard deviation: 11.83). All the patients had pneumonia (N=6, 100%). Out of all, five hadbacterial pneumonia (N=5, 83.3%) and one had tuberculous pneumonia (N=1, 16.7%). A high levelof PR3 c-ANCA (>150 RU/mL) was noted in four patients (N=4, 66.7%).Common symptoms included dry cough (N=5, 83.3%), loss of weight and appetite(N=2, 33.3%), and fever (N=2, 33.3%). Three patients had otitis mediaand/or nasal polyposis (N=3, 50%). Two patients (N=2, 33.3%) with life-threatening organ dysfunction were given concurrent antibiotics and steroids; the antibiotics were later modified based on culture and sensitivity results. One of these patients received antituberculosis therapy as Mycobacterium tuberculosis (MTB) was detected after 27 days of incubation in mycobacterial growth indicator tube broth. The remaining four patients (N=4, 66.7%)receivedantibiotics initially for 5-7 days until clinical resolution of pneumonia. Ultimately, they all showed clinical and radiological resolution (N=6, 100%) within 3-6 months of treatment. The patients exhibited constitutional symptoms such as fever and weight loss; lower airway disease symptoms including dry cough and hemoptysis; nasal and ear disease symptoms like epistaxis, ear pain, and ear discharge; and a renal disease symptom, hematuria.Computed tomography of the thorax revealed bilateral consolidations, most of which were cavitating. Bronchoalveolar lavagecultures grew Escherichia coli, Burkholderia cepacia, Pseudomonas aeruginosa, Klebsiella pneumoniae, and MTB, whereas pus swab cultures from otitis media grew Pseudomonas aeruginosa, Staphylococcus aureus,and coagulase-negative staphylococci. This study highlights the therapeutic challenges of GPA complicated by concurrent infections. Patients exhibited typical GPA signs, confirmed by PR3 c-ANCA levels. Concurrent infections require cautious antibiotic treatment before starting immunosuppressive therapy, except in life-threatening organ dysfunction. A unique case presented with both tuberculosis and GPA. Tailored treatment regimens combining antibiotics and immunosuppressives, including corticosteroids, methotrexate, and rituximab, resulted in clinical and radiological improvement in all the patients within 3-6 months. The addition of co-trimoxazole reduced the incidence of non-severe GPA relapses. Tailored treatment plans addressing both infectious and autoimmune aspects are essential for optimal care in GPA complicated by concurrent infections. This study highlights the need for a multidisciplinary approachinvolving pulmonologist, rheumatologist, microbiologist, and pathologistin the diagnosis and treatment of GPA, emphasizing the importance of individualized treatment plans tailored to the specific clinical scenario.

Substrate specificity of commercial lipases activated by a hydration–aggregation pretreatment in anhydrous esterification reactions

Substrate specificity in non-aqueous esterification catalyzed by commercial lipases activated by hydration–aggregation pretreatment was investigated. Four microbial lipases from Rhizopus japonicus, Burkholderia cepacia, Rhizomucor miehei, and Candida antarctica (fraction B) were used to study the effect of the carbon chain length of saturated fatty acid substrates on the esterification activity with methanol in n-hexane. Hydration–aggregation pretreatment had an activation effect on all lipases used, and different chain length dependencies of esterification activity for lipases from different origins were demonstrated. The effects of various acidic substrates with different degrees of unsaturation, aromatic rings, and alcohol substrates with different carbon chain lengths on esterification activity were examined using R. japonicus lipase, which demonstrated the most remarkable activity enhancement after hydration–aggregation pretreatment. Furthermore, in the esterification of myristic acid with methanol catalyzed by the hydrated–aggregated R. japonicus lipase, maximum reaction rate (5.43 × 10−5 mmol/(mg-biocat min)) and Michaelis constants for each substrate (48.5 mM for myristic acid, 24.7 mM for methanol) were determined by kinetic analysis based on the two-substrate Michaelis-Menten model.

Immobilized Burkholderia cepacia lipase and its application in selective enrichment of EPA by hydrolysis of fish oil

Eicosapentaenoic acid (EPA), an orally active Omega-3 long-chain polyunsaturated fatty acid, has attracted much attention for its medical and healthcare value. Due to a large amount of polyunsaturated fatty acids (PUFA) including docosahexanoic acid (DHA) in fish oil, the highly specific enrichment of EPA from fish oil is difficult to achieve. In our study, we conducted immobilization studies on Burkholderia cepacia lipase (BCL) and its application in the selective enrichment of EPA in fish oil. BCL was immobilized on resin LX201A by covalent crosslinking, and immobilization conditions including glutaraldehyde concentration, pH, time were optimized. The optimal immobilization conditions were 0.5% glutaraldehyde, 4 h, and pH 8.0. The loading rate of BCL reached to 93.5%, and its activity was up to 68263 U/g under the optimal immobilization conditions. After hydrolysis reaction of fish oil for 15h catalyzed by immobilized BCL, EPA content showed a more increase by 33.2%, while a slight increase by 0.78% in DHA content. Moreover, after recycling use for 10 times, the catalytic efficiency of immobilized BCL remained at 93.0%. These indicated that immobilized BCL possessed good selectivity, reusability and stability, providing great application potential in EPA selective enrichment in fish oil industry.

Phage Therapy: An Alternative Approach to Combating Multidrug-Resistant Bacterial Infections in Cystic Fibrosis.

Patients with cystic fibrosis (CF) are prone to developing life-threatening lung infections with a variety of pathogens that are difficult to eradicate, such as Burkholderia cepacia complex (Bcc), Hemophilus influenzae, Mycobacterium abscessus (Mab), Pseudomonas aeruginosa, and Staphylococcus aureus. These infections still remain an important issue, despite the therapy for CF having considerably improved in recent years. Moreover, prolonged exposure to antibiotics in combination favors the development and spread of multi-resistant bacteria; thus, the development of alternative strategies is crucial to counter antimicrobial resistance. In this context, phage therapy, i.e., the use of phages, viruses that specifically infect bacteria, has become a promising strategy. In this review, we aim to address the current status of phage therapy in the management of multidrug-resistant infections, from compassionate use cases to ongoing clinical trials, as well as the challenges this approach presents in the particular context of CF patients.

A CRISPR-Cas-associated transposon system for genome editing in Burkholderia cepacia complex species.

Species of the Burkholderia cepacia complex (Bcc) have great biotechnological potential but are also opportunistic pathogens. Genetic manipulation of Bcc species is necessary to understand gene-to-function links. However, limited genetic tools are available to manipulate Bcc, hindering our understanding of their pathogenic traits and their potential in biotechnological applications. We developed a genetic tool based on CRISPR-associated transposase to increase the genetic tools available for Bcc species. The genetic tool we developed in this study can be used for loss and gain of function in Bcc species. The significance of our work is in expanding currently available tools to manipulate Bcc.

A Comparative Metagenomic Analysis of Specified Microorganisms in Groundwater for Non-Sterilized Pharmaceutical Products

In pharmaceutical manufacturing, ensuring product safety involves the detection and identification of microorganisms with human pathogenic potential, including Burkholderia cepacia complex (BCC), Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, Staphylococcus aureus, Clostridium sporogenes, Candida albicans, and Mycoplasma spp., some of which may be missed or not identified by traditional culture-dependent methods. In this study, we employed a metagenomic approach to detect these taxa, avoiding the limitations of conventional cultivation methods. We assessed the groundwater microbiome’s taxonomic and functional features from samples collected at two locations in the spring and summer. All datasets comprised 436–557 genera with Proteobacteria, Bacteroidota, Firmicutes, Actinobacteria, and Cyanobacteria accounting for > 95% of microbial DNA sequences. The aforementioned species constituted less than 18.3% of relative abundance. Escherichia and Salmonella were mainly detected in Hot Springs, relative to Jefferson, while Clostridium and Pseudomonas were mainly found in Jefferson relative to Hot Springs. Multidrug resistance efflux pumps and BlaR1 family regulatory sensor-transducer disambiguation dominated in Hot Springs and in Jefferson. These initial results provide insight into the detection of specified microorganisms and could constitute a framework for the establishment of comprehensive metagenomic analysis for the microbiological evaluation of pharmaceutical-grade water and other non-sterile pharmaceutical products, ensuring public safety.

Consortium of Phosphorus-Solubilizing Bacteria Promotes Maize Growth and Changes the Microbial Community Composition of Rhizosphere Soil

Phosphorus deficiency severely limits crop yields and hinders sustainable agricultural development. Phosphate-solubilizing bacteria (PSB) are beneficial for crop growth because they enhance the uptake and utilization of phosphorus. This study explored the phosphorus-solubilizing, IAA-producing, nitrogen-fixing, potassium-solubilizing, and siderophore-producing abilities of three bacterial strains (Pantoea sp. J-1, Burkholderia cepacia Z-7, and Acinetobacter baumannii B-6) screened from the maize rhizosphere. A pot experiment was also conducted to explore the role of screened PSB in the growth of maize. Finally, the effects of the PSB on the physicochemical properties, enzyme activities, and microbial community structure of maize rhizosphere soil were analyzed. The results showed that strain Z-7 had the strongest abilities phosphorus solubilization, nitrogen fixation, potassium solubilization, and siderophore production, while strain J-1 exhibited the highest yield of IAA. The application of PSB promoted the growth of maize plants to different extents. Among the different treatments, the mixed bacterial treatment (J-1 + Z-7 + B-6) had the most potent growth promotion effect, and the consortium treatment significantly enhanced the activity of soil phosphatase. Soil pH, total phosphorus (TP), total potassium (TK), available phosphorus (AP), NH4+-N, and NO3−-N are key factors for the growth of maize plants. In addition, PSB significantly altered the microbial community structure in the maize rhizosphere soil, and the relative abundance of Proteobacteria increased by 16.07–69.10% compared to the control. These PSB have obvious growth-promoting abilities, with the potential to enhance crop productivity as excellent candidate strains for the development of biological fertilizers.

Onion‐pathogenic Burkholderia species: Role and regulation of characterized virulence determinants

AbstractMembers of the bacterial genus Burkholderia are a routine threat to onion production worldwide. In addition to the common onion‐pathogenic species, Burkholderia cepacia, Burkholderia orbicola and Burkholderia gladioli, other Burkholderia species have the potential to cause onion disease. Despite their impacts and long‐known association with onion disease, the virulence mechanisms of onion‐pathogenic Burkholderia are far less well understood than Burkholderia in human and murine infection models. In this review, we will focus on genetically characterized virulence factors in species that contribute to symptom production in onion and other plant hosts. Specifically, we will focus on the variable roles of specialized protein secretion systems (T2SS, T3SS and T4SS) and secreted proteins, thiosulphinate tolerance gene (TTG) clusters and the well‐characterized phytotoxin toxoflavin in virulence. The regulation and roles of LuxI/LuxS quorum‐sensing system and IclR‐type transcriptional regulator, qsmR, as master regulators of secondary metabolite production and virulence factors will also be discussed. The TTG clusters, involved in bacterial tolerance to thiosulphinate defence compounds, exhibit onion tissue‐specific contributions to virulence. This suggests that Burkholderia onion pathogens have tissue‐specific virulence strategies for causing disease.

Mutations Affecting Cellular Levels of Cobalamin (Vitamin B12) Confer Tolerance to Bactericidal Antibiotics in Burkholderia cenocepacia.

The Burkholderia cepacia complex (Bcc) consists of opportunistic pathogens known to cause pneumonia in immunocompromised individuals, especially those with cystic fibrosis. Treating Bcc pneumonia is challenging due to the pathogens' high multidrug resistance. Therefore, inhalation therapy with tobramycin powder, which can achieve high antibiotic concentrations in the lungs, is a promising treatment option. In this study, we investigated potential mechanisms that could compromise the effectiveness of tobramycin therapy. By selecting for B. cenocepacia survivors against tobramycin, we identified three spontaneous mutations that disrupt a gene encoding a key enzyme in the biosynthesis of cobalamin (Vitamin B12). This disruption may affect the production of succinyl-CoA by methylmalonyl-CoA mutase, which requires adenosylcobalamin as a cofactor. The depletion of cellular succinyl-CoA may impact the tricarboxylic acid (TCA) cycle, which becomes metabolically overloaded upon exposure to tobramycin. Consequently, the mutants exhibited significantly reduced reactive oxygen species (ROS) production. Both the wild-type and mutants showed tolerance to tobramycin and various other bactericidal antibiotics under microaerobic conditions. This suggests that compromised ROS-mediated killing, due to the impacted TCA cycle, underlies the mutants' tolerance to bactericidal antibiotics. The importance of ROS-mediated killing and the potential emergence of mutants that evade it through the depletion of cobalamin (Vitamin B12) provide valuable insights for developing strategies to enhance antibiotic treatments of Bcc pneumonia.

Comparative Analysis of the Mass Spectra of Mycobacterium abscessus Complex Strains Isolated on Various Nutrient Media.

Mycobacterium abscessus complex (MABSc) causes chronic infection in patients with concomitant structural changes in the respiratory tract, which is especially important for patients with cystic fibrosis. To isolate an MABSc culture from clinical material, a variety of nutrient media are used. For species determination of microorganisms isolated on these media, additional identification methods are used, for example, polymerase chain reaction, sequencing, or mass spectrometry. The latter method is relatively easy to implement but requires improvement, due to the identification inaccuracy of nontuberculosis mycobacterias in general. Consequently, a set of nutrient media may be important for subsequent identification by mass spectrometry. The study was conducted on 64 strains of MABSc representatives: 56 strains were obtained from patients with cystic fibrosis and 8 strains from patients with pulmonary pathology unrelated to cystic fibrosis. The obtained MABSc strains were transplanted to the universal chromogenic medium and the selective medium for the Burkholderia cepacia complex (BCC) isolation. Species identification was carried out by mass spectrometry based on matrix-activated laser time-of-flight desorption/ionization (MALDI-ToF MS). Microbial identification is based on a comparison of the obtained mass spectra with reference spectra from the database. Microorganisms were identified based on the coincidence degree (Score value). Sample preparation for microbial identification by mass spectrometry was carried out by an extended direct application method. Fragments of the rpoB and hsp65 genes with lengths of 752 bp and 441 bp, respectively, were used as molecular markers for subspecific identification of MABSc strains. A comparison of the peaks obtained after mass spectrometry of MABSc strains isolated on the studied nutrient media showed significant differences between these indicators selective medium for the BCC isolation with the supplement of iron polymaltose hydroxide (III) and universal chromogenic medium (P < 0.001) and selective medium for the BCC isolation with universal chromogenic medium (P < 0.001). Twenty-five strains of MABSc representatives were sequenced: results of subspecies determination in strains isolated on the universal chromogenic medium coincided with the results sequencing in 13 (86.6%) strains out of 15. MALDI-ToF mass spectrometry allows microbial identification in a short time and with minimal cost, but it does not yet allow the proper identification of the subspecies of certain microbial groups, such as MABSc. Cultivation methods need optimization and new approaches to the extraction process of the bacterial protein fraction.

Burkholderia cepacia in cystic fibrosis children and adolescents: overall survival and immune alterations.

In current literature there are only scarce data on the host inflammatory response during Burkholderia cepacia complex (Bcc) persistence. The primary objective of the present research was to carry out cross-sectional analyses of biomarkers and evaluate disease progression in cystic fibrosis (CF) patients with chronic Bcc infection and pathogen-free ones. The secondary aim was to assess prospectively overall survival of the study participants during up to 8 years of follow-up. The study included 116 paediatric patients with CF; 47 CF patients were chronically infected with Bcc, and 69 individuals were Bcc free. Plasma and sputum biomarkers (neutrophil elastase, MMP-8, MMP-9, MMP-12, IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, IL-22, IL-23, IL-17, IFN-γ, TGFβ1, TNF-α) were analysed using commercially available kits. Besides, inhibitory effect of dexamethasone on proliferative response of PHA-stimulated peripheral blood lymphocytes had been assessed. Bcc infected patients did not differ from Bcc free ones in demographic and clinical parameters, but demonstrated an increased rate of glucose metabolism disturbances and survival disadvantage during prolong follow-up period. Biomarkers analyses revealed elevated TNF-α and reduced IL-17F levels in sputum samples of Bcc infected patients. These patients also demonstrated improvement of peripheral blood lymphocyte sensitivity to steroid treatment and reduction in plasma pro-inflammatory (IL-17F and IL-18) and anti-inflammatory (TGFβ1 and IL-10) cytokine concentrations. Reduction in IL-17F levels may have several important consequences including increase in steroid sensitivity and glycemic control disturbances. Further investigations are needed to clarify the role of IL-17 cytokines in CF complication development. Low plasma TGFβ1 and IL-10 levels in Bcc infected group may be a sign of subverted activity of regulatory T cells. Such immune alterations may be one of the factors contributing to the development of the cepacia syndrome.

Tracking melioidosis in Iran: Utilizing abattoir-based surveillance as a One Health approach.

Burkholderia pseudomallei, an environmental saprophyte bacterium, causes melioidosis in humans and animals. It was first discovered in Iran between 1967 and 1976 in small ruminants, equines, environments and humans. No subsequent studies have been conducted to determine the existence and prevalence of this pathogen in the country. The present study aims to monitor the presence of B. pseudomallei in the ruminant population of the Golestan province of Iran, which largely depends on pastures. The ruminants can serve as sentinels to indicate the presence of the bacteria in the environment and its potential impact on human health in the One Health triad. Liver and lung abscesses from domestic sheep, cattle and goats in three industrial and three conventional slaughterhouses were sampled and analysed using 23S ribosomal DNA polymerase chain reaction (rDNA PCR) with primers CVMP 23-1 and CVP-23-2 for B. pseudomallei, Burkholderia cepacia and Burkholderia vietnamiensis, as well as B. pseudomallei-specific TTS1 real-time PCR, along with microbiological and biochemical assays. Out of the 97 animals sampled, only 14 (15%) tested positive for 23S rDNA PCR. However, the follow-up evaluation using TTS1 real-time PCR and microbiological and biochemical assays did not confirm the presence of B. pseudomallei in the samples. Although B. pseudomallei was not detected in the current survey, conducting abattoir-based surveillance of ruminants is a cost-effective One Health approach to monitor pathogenic Burkholderia. Developing standards of clinical and laboratory good practices for Burkholderia infections is crucial for One Health surveillance.