UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.
UNASSIGNED: 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.

Burkholderia spp. are opportunistic pathogens that cause infection in patients with disrupted immunity. The study intended to demonstrate the epidemiology and clinical features associated with Burkholderia spp. bacteremia. This retrospective study was performed to assess the clinical and laboratory characteristics of patients whose blood cultures were growing Burkholderia spp. and, based on their underlying comorbidities, were subjected to survival analysis from January 2022 to December 2022 at a university hospital in northern India. Three hundred patients with Burkholderia spp. bacteremia were included in this study conducted over 1 year. The mean age of the patients was 33.86 years with a male predominance of 56.67% (170/300, 56.67%). Underlying malignancies (207/300, 69.0%) were the most common clinical diagnosis, and catheter in situ (300/300, 100.0%) was the most common risk factor. Burkholderia cenocepacia (244/300, 81.33%) was the most common Burkholderia spp. isolated. All isolates were highly susceptible to minocycline. Kidney disease (P = 0.029), hypertension (P = 0.005), type 2 diabetes mellitus (P = 0.039), and respiratory disease (P <0.001) in patients were significantly associated with death owing to Burkholderia spp. bacteremia, whereas patients with malignancies (P <0.001) and undergoing treatment were significantly associated with a better outcome when the microorganism was susceptible to empirical antibiotics. The presence of indwelling devices, mechanical ventilation (P <0.001), and a hemodialysis catheter (P = 0.026) were statistically significant risk factors associated with poor outcomes.

Within cystic fibrosis microbiology, there is often mismatch between the antibiotic susceptibility result of an isolated bacterial pathogen and the clinical outcome, when the patient is treated with the same antibiotic. The reasoning for this remains largely elusive. Antibiotic susceptibility to four antibiotics (ceftazidime, meropenem, minocycline and trimethoprim-sulfamethoxazole) was determined in consecutive isolates (n = 11) from an adult cystic fibrosis patient, over a 63 month period. Each isolate displayed its own unique resistotype. The first isolate was sensitive to all four antibiotics, in accordance with Clinical and Laboratory Standards Institute methodology and interpretative criteria. Resistance was first detected at four months, showing resistance to ceftazidime and meropenen and intermediate resistance to minocycline and trimethoprim-sulfamethoxazole. Pan resistance was first detected at 18 months (resistotype IV), with three resistotypes (I, II and III) preceding this complete resistotype. The bacterium continued to display further antibiotic susceptibility heterogeneity for the next 45 months, with the description of an additional seven resistotypes (resistotypes V-XI). The Relative Resistance Index of this bacterium over the 63 month period showed no relationship between the development of antibiotic resistance and time. Adoption of mathematical modelling employing multinomial distribution demonstrated that large numbers of individual colony picks (>40/sputum), would be required to be 78% confident of capturing all 11 resistotypes present. Such a requirement for large numbers of colony picks combined with antibiotic susceptibility-related methodological problems creates a conundrum in biomedical science practice, in providing a robust assay that will capture antibiotic susceptibility variation, be pragmatic and cost-effective to deliver as a pathology service, but have the reliability to help clinicians select appropriate antibiotics for their patients. This study represents an advance in biomedical science as it demonstrates potential variability in antibiotic susceptibility testing with Burkholderia cenocepacia. Respiratory physicians and paediatricians need to be made aware of such variation by biomedical scientists at the bench, so that clinicians can contextualise the significance of the reported susceptibility result, when selecting appropriate antibiotics for their cystic fibrosis patient. Furthermore, consideration needs to be given in providing additional guidance on the laboratory report to highlight this heterogeneity to emphasise the potential for misalignment between susceptibility result and clinical outcome.

The Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis (CF) and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals; pigment production has been reported to enable Bcc strains to overcome the host cell oxidative burst. In this work, we investigated the role of pyomelanin in resistance to oxidative stress and virulence in strains J2315 and K56-2, two epidemic CF isolates belonging to the Burkholderia cenocepacia ET-12 lineage. We previously reported that a single amino acid change from glycine to arginine at residue 378 in homogentisate 1,2-dioxygenase (HmgA) affects the pigment production phenotype: pigmented J2315 has an arginine at position 378, while non-pigmented K56-2 has a glycine at this position. Herein, we performed allelic exchange to generate isogenic non-pigmented and pigmented strains of J2315 and K56-2, respectively, and tested these to determine whether pyomelanin contributes to the protection against oxidative stress in vitro as well as in a respiratory infection in CGD mice in vivo. Our results indicate that the altered pigment phenotype does not significantly impact these strains\' ability to resist oxidative stress with H2O2 and NO in vitro and did not change the virulence and infection outcome in CGD mice in vivo suggesting that other factors besides pyomelanin are contributing to the pathophysiology of these strains.IMPORTANCEThe Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic bacteria that are often associated with fatal pulmonary infections in patients with impaired immunity, particularly those with cystic fibrosis and chronic granulomatous disease (CGD). Some Bcc strains are known to naturally produce pyomelanin, a brown melanin-like pigment known for scavenging free radicals and overcoming the host cell oxidative burst. We investigated the role of pyomelanin in Burkholderia cenocepacia strains J2315 (pigmented) and K56-2 (non-pigmented) and performed allelic exchange to generate isogenic non-pigmented and pigmented strains, respectively. Our results indicate that the altered pigment phenotype does not significantly impact these strains\' ability to resist H2O2 or NO in vitro and did not alter the outcome of a respiratory infection in CGD mice in vivo. These results suggest that pyomelanin may not always constitute a virulence factor and suggest that other features are contributing to the pathophysiology of these strains.

Burkholderia semiarida was previously identified solely as a plant pathogen within the Burkholderia cepacia complex. We present a case in China involving recurrent pneumonia attributed to B. semiarida infection. Of note, the infection manifested in an immunocompetent patient with no associated primary diseases and endured for >3 years.

BACKGROUND: Burkholderia cepacia complex (BCC) are non-fermenting Gram-negative bacteria that can chronically colonize the lungs of people with cystic fibrosis (pwCF), causing a severe and progressive respiratory failure, post-transplant complications and epidemic outbreaks. Therefore, rapid and accurate identification of these bacteria is relevant for pwCF, in order to facilitate early eradication and prevent chronic colonization. However, BCCs are often quite difficult to detect on culture media as they have a slow growth rate and can be hidden by other fast-growing microorganisms, including Pseudomonas aeruginosa and filamentous fungi.
METHODS: We evaluated the sensitivity of CHROMagar™ B. cepacia agar using 11 isolates from a well-characterized BCC collection, using BCA agar (Oxoid, UK) as a gold standard. We also studied 180 clinical sputum samples to calculate positive (PPV) and negative (NPV) predictive values. Furthermore, we used three of the well-characterized BCC isolates to determine the limit of detection (LOD).
RESULTS: Eleven isolates grew on CHROMagar™ B. cepacia at 37ºC after 48 h. The NPV and PPV of CHROMagar™ B. cepacia were 100% and 87.5%, respectively. The LOD of CHROMagar™ B. cepacia was around 1 × 103 CFU/ml, requiring a ten-fold dilution lower bacterial load than BCA for BCC detection.
CONCLUSIONS: CHROMagar™ B. cepacia agar proved to have a very good sensitivity and specificity for the detection of clinical BCCs. Moreover, the chromogenic nature of the medium allowed us to clearly differentiate BCC from other Gram-negative species, filamentous fungi and yeasts, thereby facilitating the identification of contaminants.

We report a clinical isolate of Burkholderia thailandensis 2022DZh obtained from a patient with an infected wound in southwest China. Genomic analysis indicates that this isolate clusters with B. thailandensis BPM, a human isolate from Chongqing, China. We recommend enhancing monitoring and surveillance for B. thailandensis infection in both humans and livestock.

Long before COVID-19 made social distancing familiar, people with cystic fibrosis (CF) already practiced such behaviors. CF is held up as a classic example of genetic disease, yet people with CF are also susceptible to bacteria from the environment and from other CF patients. Starting in the 1980s, a bacterial epidemic in the CF population highlighted clashing priorities of connection, physical safety, and environmental protection. Policymakers ultimately called for the physical separation of people with CF from one another via recommendations that reconfigured the CF community. Simultaneously, medical researchers recognized that one highly transmissible CF pathogen called cepacia was being developed for environmental applications and got the EPA to limit cepacia\'s environmental deployment. Environmental regulations speak to the challenge of useful microbes that harm a minority, but CF cross-infection also involves legal implications for microbial and genetic discrimination, social consequences for CF communities, and ethical questions about balancing autonomy, harms, and benefits. As scientists increasingly study connections between host genetics, microbial genetics, and infectious risks, CF is a vital referent.

Burkholderia spp. are often resistant to antibiotics, and infections with these organisms are difficult to treat. A potential alternative treatment for Burkholderia spp. infections is bacteriophage (phage) therapy; however, it can be difficult to locate phages that target these bacteria. Prophages incorporated into the bacterial genome have been identified within Burkholderia spp. and may represent a source of useful phages for therapy. Here, we investigate whether prophages within Burkholderia spp. clinical isolates can kill conspecific and heterospecific isolates. Thirty-two Burkholderia spp. isolates were induced for prophage release, and harvested phages were tested for lytic activity against the same 32 isolates. Temperate phages were passaged and their host ranges were determined, resulting in four unique phages of prophage origin that showed different ranges of lytic activity. We also analyzed the prophage content of 35 Burkholderia spp. clinical isolate genomes and identified several prophages present in the genomes of multiple isolates of the same species. Finally, we observed that Burkholdera cenocepacia isolates were more phage-susceptible than Burkholderia multivorans isolates. Overall, our findings suggest that prophages present within Burkholderia spp. genomes are a potentially useful starting point for the isolation and development of novel phages for use in phage therapy.

Burkholderia cepacia complex bacteria have emerged as opportunistic pathogens in patients with cystic fibrosis and immunocompromised individuals, causing life-threatening infections. Because of the relevance of these microorganisms, genetic manipulation is crucial for explaining the genetic mechanisms leading to pathogenesis. Despite the availability of allelic exchange tools to obtain unmarked gene deletions in Burkholderia, these require a step of merodiploid formation and another of merodiploid resolution through two independent homologous recombination events, making the procedure long-lasting. The CRISPR/Cas9-based system could ease this constraint, as only one step is needed for allelic exchange. Here, we report the modification of a two-plasmid system (pCasPA and pACRISPR) for genome editing in Burkholderia multivorans. Several modifications were implemented, including selection marker replacement, the optimization of araB promoter induction for the expression of Cas9 and λ-Red system encoding genes, and the establishment of plasmid curing procedures based on the sacB gene or growth at a sub-optimal temperature of 18°C-20°C with serial passages. We have shown the efficiency of this CRISPR/Cas9 method in the precise and unmarked deletion of different genes (rpfR, bceF, cepR, and bcsB) from two strains of B. multivorans, as well as its usefulness in the targeted insertion of the gfp gene encoding the green fluorescence protein into a precise genome location. As pCasPA was successfully introduced in other Burkholderia cepacia complex species, this study opens up the possibility of using CRISPR/Cas9-based systems as efficient tools for genome editing in these species, allowing faster and more cost-effective genetic manipulation.IMPORTANCEBurkholderia encompasses different species of bacteria, some of them pathogenic to animals and plants, but others are beneficial by promoting plant growth through symbiosis or as biocontrol agents. Among these species, Burkholderia multivorans, a member of the Burkholderia cepacia complex, is one of the predominant species infecting the lungs of cystic fibrosis patients, often causing respiratory chronic infections that are very difficult to eradicate. Since the B. multivorans species is understudied, we have developed a genetic tool based on the CRISPR/Cas9 system to delete genes efficiently from the genomes of these strains. We could also insert foreign genes that can be precisely placed in a chosen genomic region. This method, faster than other conventional strategies based on allelic exchange, will have a major contribution to understanding the virulence mechanisms in B. multivorans, but it can likely be extended to other Burkholderia species.