Polyyne-producing Burkholderia suppress Globisporangium ultimum damping-off disease of Pisum sativum (pea).

Extensive crop losses are caused by oomycete and fungal damping-off diseases. Agriculture relies heavily on chemical pesticides to control disease, but due to safety concerns multiple agents have been withdrawn. Burkholderia were successfully used as commercial biopesticides because of their fungicidal activity and plant protective traits. However, their potential for opportunistic pathogenicity led to a moratorium on their registration as biopesticides. Subsequently, Burkholderia were shown to produce multiple specialised metabolites including potent antimicrobial polyynes. Cepacin A, a polyyne produced by Burkholderia ambifaria biopesticide strains was shown to be an important metabolite for the protection of germinating peas against Globisporangium ultimum (formerly Pythium) damping-off disease. Recently, there has been an expansion in bacterial polyyne discovery, with the metabolites and their biosynthetic gene pathways found in several bacterial genera including Burkholderia, Collimonas, Trinickia, and Pseudomonas. To define the efficacy of these bacterial polyyne producers as biopesticidal agents, we systematically evaluated metabolite production, in vitro microbial antagonism, and G. ultimum biocontrol across a panel of 30 strains representing four bacterial genera. In vitro polyyne production and antimicrobial activity was demonstrated for most strains, but only Burkholderia polyyne producers were protective within the in vivo G. ultimum damping-off pea protection model. B. ambifaria was the most effective cepacin-expressing biopesticide, and despite their known potential for plant pathogenicity Burkholderia gladioli and Burkholderia plantarii were uniquely shown to be protective as caryoynencin-producing biopesticides. In summary, Burkholderia are effective biopesticides due to their suite of antimicrobials, but the ability to deploy polyyne metabolites, caryoynencin and cepacin, is strain and species dependent. Graphical Abstract.

Identification of two distinct phylogenomic lineages and model strains for the understudied cystic fibrosis lung pathogen Burkholderia multivorans.

Burkholderia multivorans is the dominant Burkholderia pathogen recovered from lung infection in people with cystic fibrosis. However, as an understudied pathogen there are knowledge gaps in relation to its population biology, phenotypic traits and useful model strains. A phylogenomic study of B. multivorans was undertaken using a total of 283 genomes, of which 73 were sequenced and 49 phenotypically characterized as part of this study. Average nucleotide identity analysis (ANI) and phylogenetic alignment of core genes demonstrated that the B. multivorans population separated into two distinct evolutionary clades, defined as lineage 1 (n=58 genomes) and lineage 2 (n=221 genomes). To examine the population biology of B. multivorans, a representative subgroup of 77 B. multivorans genomes (28 from the reference databases and the 49 novel short-read genome sequences) were selected based on multilocus sequence typing (MLST), isolation source and phylogenetic placement criteria. Comparative genomics was used to identify B. multivorans lineage-specific genes - ghrB_1 in lineage 1 and glnM_2 in lineage 2 - and diagnostic PCRs targeting them were successfully developed. Phenotypic analysis of 49 representative B. multivorans strains showed considerable inter-strain variance, but the majority of the isolates tested were motile and capable of biofilm formation. A striking absence of B. multivorans protease activity in vitro was observed, but no lineage-specific phenotypic differences were demonstrated. Using phylogenomic and phenotypic criteria, three model B. multivorans CF strains were identified, BCC0084 (lineage 1), BCC1272 (lineage 2a) and BCC0033 lineage 2b, and their complete genome sequences determined. B. multivorans CF strains BCC0033 and BCC0084, and the environmental reference strain, ATCC 17616, were all capable of short-term survival within a murine lung infection model. By mapping the population biology, identifying lineage-specific PCRs and model strains, we provide much needed baseline resources for future studies of B. multivorans.

A 2.8 Å Structure of Zoliflodacin in a DNA Cleavage Complex with Staphylococcus aureus DNA Gyrase.

Since 2000, some thirteen quinolones and fluoroquinolones have been developed and have come to market. The quinolones, one of the most successful classes of antibacterial drugs, stabilize DNA cleavage complexes with DNA gyrase and topoisomerase IV (topo IV), the two bacterial type IIA topoisomerases. The dual targeting of gyrase and topo IV helps decrease the likelihood of resistance developing. Here, we report on a 2.8 Å X-ray crystal structure, which shows that zoliflodacin, a spiropyrimidinetrione antibiotic, binds in the same DNA cleavage site(s) as quinolones, sterically blocking DNA religation. The structure shows that zoliflodacin interacts with highly conserved residues on GyrB (and does not use the quinolone water-metal ion bridge to GyrA), suggesting it may be more difficult for bacteria to develop target mediated resistance. We show that zoliflodacin has an MIC of 4 µg/mL against Acinetobacter baumannii (A. baumannii), an improvement of four-fold over its progenitor QPT-1. The current phase III clinical trial of zoliflodacin for gonorrhea is due to be read out in 2023. Zoliflodacin, together with the unrelated novel bacterial topoisomerase inhibitor gepotidacin, is likely to become the first entirely novel chemical entities approved against Gram-negative bacteria in the 21st century. Zoliflodacin may also become the progenitor of a new safer class of antibacterial drugs against other problematic Gram-negative bacteria.

Discovery, mode of action and secretion of Burkholderia sensu lato key antimicrobial specialised metabolites.

Burkholderia sensu lato bacteria have genomes rich in biosynthetic gene clusters (BGCs) encoding for multiple bioactive specialised metabolites. Diverse classes of antimicrobial natural products have been isolated from Burkholderia, including polyynes, shikimate pathway derivatives, polyketides, non-ribosomal peptides and hybrid polyketide non-ribosomal peptides. We highlight examples of Burkholderia metabolites, overviewing their biosynthesis, bioactivity, mechanisms of action and secretion.

A pilot study investigating the effects of a manuka honey sinus rinse compared to a standard sinus rinse on sino-nasal outcome test scores in cystic fibrosis patients.

BACKGROUND: People with cystic fibrosis (CF) are prone to bacterial respiratory infections; these are often antibiotic resistant, are difficult to treat, and impact on the quality of life and lung function. The upper respiratory tract can act as a reservoir for these pathogens, and as part of clinical care, sinus rinses are used to alleviate symptoms in the upper airway. We have developed a sinus rinse containing manuka honey, to identify whether it can help improve symptoms or reduce the bacterial load. METHODS: We will undertake a randomised controlled trial where 30 adults with CF will be recruited and randomised to either the control or intervention group. Both groups will follow a sinus rinse protocol for 30 days (± 7 days); the control group will use the standard of care rinse, and the intervention group will use a manuka honey rinse. Both groups will provide samples at day 0 and day 30. The primary outcome measure will be a change in the 22-item Sino-Nasal Outcome Test (SNOT-22) score. Secondary outcomes will include changes to quality of life (questionnaire), bacterial load/community composition, and sputum viscosity. DISCUSSION: This trial will look at the use of a manuka honey-infused sinus rinse solution on patients diagnosed with cystic fibrosis (CF) suffering with sinusitis; it will allow us to determine the efficacy of the manuka honey sinus rinse compared to standard rinse and will allow us to determine if molecular bacterial diversity analysis will provide in-depth information beyond the usual conventional microbiological. It will allow us to determine the feasibility of recruiting participants to this type of trial, allow us to check participant compliance with the protocol, and inform future studies. TRIAL REGISTRATION: Approval was obtained from the Research Ethics Committee Wales REC7 reference 18/WA/0319. Results of this study will be published at international conferences and in peer-reviewed journals; they will also be presented to the relevant stakeholders and research networks. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier NCT04589897 (retrospectively registered).

Cloning and expression of Burkholderia polyyne biosynthetic gene clusters in Paraburkholderia hosts provides a strategy for biopesticide development.

Burkholderia have potential as biocontrol agents because they encode diverse biosynthetic gene clusters (BGCs) for a range of antimicrobial metabolites. Given the opportunistic pathogenicity associated with Burkholderia species, heterologous BGC expression within non-pathogenic hosts is a strategy to construct safe biocontrol strains. We constructed a yeast-adapted Burkholderia-Escherichia shuttle vector (pMLBAD_yeast) with a yeast replication origin 2 µ and URA3 selection marker and optimised it for cloning BGCs using the in vivo recombination ability of Saccharomyces cerevisiae. Two Burkholderia polyyne BGCs, cepacin (13 kb) and caryoynencin (11 kb), were PCR-amplified as three overlapping fragments, cloned downstream of the pBAD arabinose promoter in pMLBAD_yeast and mobilised into Burkholderia and Paraburkholderia heterologous hosts. Paraburkholderia phytofirmans carrying the heterologous polyyne constructs displayed in vitro bioactivity against a variety of fungal and bacterial plant pathogens similar to the native polyyne producers. Thirteen Paraburkholderia strains with preferential growth at 30°C compared with 37°C were also identified, and four of these were amenable to genetic manipulation and heterologous expression of the caryoynencin construct. The cloning and successful heterologous expression of Burkholderia biosynthetic gene clusters within Paraburkholderia with restricted growth at 37°C opens avenues for engineering non-pathogenic biocontrol strains.

The lung microbiota in children with cystic fibrosis captured by induced sputum sampling.

BACKGROUND: Spatial topography of the cystic fibrosis (CF) lung microbiota is poorly understood in childhood. How best to sample the respiratory tract in children for microbiota analysis, and the utility of microbiota profiling in clinical management of early infection remains unclear. By comparison with bronchoalveolar lavage (BAL), we assessed the ability of induced sputum (IS) sampling to characterise the lower airway microbiota. METHODS: Sample sets from IS and two or three matched BAL compartments were obtained for microbiota analysis as part of the CF-Sputum Induction Trial (UKCRN_14615, ISRCTNR_12473810). Microbiota profiles and pathogen detection were compared between matched samples. RESULTS: Twenty-eight patients, aged 1.1-17.7 years, provided 30 sample sets. Within-patient BAL comparisons revealed spatial heterogeneity in 8/30 (27%) sample sets indicating that the lower airway microbiota from BAL is frequently compartmentalised in children with CF. IS samples closely resembled one or more matched BAL compartments in 15/30 (50%) sets, and were related in composition in a further 9/30 (30%). IS detected 86.2% of the Top 5 genera found across matched BAL samples. The sensitivity of IS to detect specific CF-pathogens identified in matched BAL samples at relative abundance ≥5% varied between 43 and 100%, with negative predictive values between 73 and 100%. CONCLUSIONS: Spatial heterogeneity of the lower airway microbiota was observed in BAL samples and presents difficulties for consistent lung sampling. IS captured a microbiota signature representative of the lower airway in 80% of cases, and is a straightforward, non-invasive intervention that can be performed frequently to aid pathogen diagnosis and understand microbiota evolution in children with CF.

Evaluating the alginate oligosaccharide (OligoG) as a therapy for Burkholderia cepacia complex cystic fibrosis lung infection.

OligoG has previously shown potentiation of aztreonam against Burkholderia cepacia complex (Bcc) through biofilm disruption. A randomized, double-blind, placebo-controlled cross-over design was used to evaluate safety and efficacy of inhaled OligoG as a therapy for Bcc-infected CF patients taking aztreonam. Subjects received OligoG (1050 mg daily) or matching placebo for 28-days. Of 14 subjects completing the study, 8 showed a mean decrease in total bacterial CFU's (0.82 log10) after OligoG treatment. There was a reduction in mean Bcc CFU's (2.19 log10) after OligoG treatment but this was not statistically significant. Rheology analysis showed improvements in phase-angle after OligoG, but there was no statistically significant improvement in lung function parameters. Six out of 12 QoL summary scores showed relative improvement after OligoG treatment compared to placebo. There was a favourable safety profile for OligoG. Potential for reducing Bcc warrants further investigation of OligoG for the treatment of infection in CF.

Novel application of metagenomics for the strain-level detection of bacterial contaminants within non-sterile industrial products - a retrospective, real-time analysis.

The home and personal care (HPC) industry generally relies on initial cultivation and subsequent biochemical testing for the identification of microorganisms in contaminated products. This process is slow (several days for growth), labour intensive, and misses organisms which fail to revive from the harsh environment of preserved consumer products. Since manufacturing within the HPC industry is high-throughput, the process of identification of microbial contamination could benefit from the multiple cultivation-independent methodologies that have developed for the detection and analysis of microbes. We describe a novel workflow starting with automated DNA extraction directly from a HPC product, and subsequently applying metagenomic methodologies for species and strain-level identification of bacteria. The workflow was validated by application to a historic microbial contamination of a general-purpose cleaner (GPC). A single strain of Pseudomonas oleovorans was detected metagenomically within the product. The metagenome mirrored that of a contaminant isolated in parallel by a traditional cultivation-based approach. Using a dilution series of the incident sample, we also provide evidence to show that the workflow enables detection of contaminant organisms down to 100 CFU/ml of product. To our knowledge, this is the first validated example of metagenomics analysis providing confirmatory evidence of a traditionally isolated contaminant organism, in a HPC product.

The Hidden Genomic Diversity, Specialized Metabolite Capacity, and Revised Taxonomy of Burkholderia Sensu Lato.

Burkholderia sensu lato is a collection of closely related genera within the family Burkholderiaceae that includes species of environmental, industrial, biotechnological, and clinical importance. Multiple species within the complex are the source of diverse specialized metabolites, many of which have been identified through genome mining of their biosynthetic gene clusters (BGCs). However, the full, true genomic diversity of these species and genera, and their biosynthetic capacity have not been investigated. This study sought to cluster and classify over 4000 Burkholderia sensu lato genome assemblies into distinct genomic taxa representing named and uncharacterized species. We delineated 235 species groups by average nucleotide identity analyses that formed seven distinct phylogenomic clades, representing the genera of Burkholderia sensu lato: Burkholderia, Paraburkholderia, Trinickia, Caballeronia, Mycetohabitans, Robbsia, and Pararobbisa. A total of 137 genomic taxa aligned with named species possessing a sequenced type strain, while 93 uncharacterized species groups were demarcated. The 95% ANI threshold proved capable of delineating most genomic species and was only increased to resolve several closely related species. These analyses enabled the assessment of species classifications of over 4000 genomes, and the correction of over 400 genome taxonomic assignments in public databases into existing and uncharacterized genomic species groups. These species groups were genome mined for BGCs, their specialized metabolite capacity calculated per species and genus, and the number of distinct BGCs per species estimated through kmer-based de-replication. Mycetohabitans species dedicated a larger proportion of their relatively small genomes to specialized metabolite biosynthesis, while Burkholderia species harbored more BGCs on average per genome and possessed the most distinct BGCs per species compared to the remaining genera. Exploring the hidden genomic diversity of this important multi-genus complex contributes to our understanding of their taxonomy and evolutionary relationships, and supports future efforts toward natural product discovery.

Discovery of the Pseudomonas Polyyne Protegencin by a Phylogeny-Guided Study of Polyyne Biosynthetic Gene Cluster Diversity.

Natural products that possess alkyne or polyyne moieties have been isolated from a variety of biological sources and possess a broad a range of bioactivities. In bacteria, the basic biosynthesis of polyynes is known, but their biosynthetic gene cluster (BGC) distribution and evolutionary relationship to alkyne biosynthesis have not been addressed. Through comprehensive genomic and phylogenetic analyses, the distribution of alkyne biosynthesis gene cassettes throughout bacteria was explored, revealing evidence of multiple horizontal gene transfer events. After investigation of the evolutionary connection between alkyne and polyyne biosynthesis, a monophyletic clade was identified that possessed a conserved seven-gene cassette for polyyne biosynthesis that built upon the conserved three-gene cassette for alkyne biosynthesis. Further diversity mapping of the conserved polyyne gene cassette revealed a phylogenetic subclade for an uncharacterized polyyne BGC present in several Pseudomonas species, designated pgn. Pathway mutagenesis and high-resolution analytical chemistry showed the Pseudomonas protegens pgn BGC directed the biosynthesis of a novel polyyne, protegencin. Exploration of the biosynthetic logic behind polyyne production, through BGC mutagenesis and analytical chemistry, highlighted the essentiality of a triad of desaturase proteins and a thioesterase in both the P. protegens pgn and Trinickia caryophylli (formerly Burkholderia caryophylli) caryoynencin pathways. We have unified and expanded knowledge of polyyne diversity and uniquely demonstrated that alkyne and polyyne biosynthetic gene clusters are evolutionarily related and widely distributed within bacteria. The systematic mapping of conserved biosynthetic genes across the available bacterial genomic diversity proved to be a fruitful method for discovering new natural products and better understanding polyyne biosynthesis. IMPORTANCE Natural products bearing alkyne (triple carbon bond) or polyyne (multiple alternating single and triple carbon bonds) moieties exhibit a broad range of important biological activities. Polyyne metabolites have been implicated in important ecological roles such as cepacin mediating biological control of plant pathogens and caryoynencin protecting Lagriinae beetle eggs against pathogenic fungi. After further phylogenetic exploration of polyyne diversity, we identified a novel gene cluster in Pseudomonas bacteria with known biological control abilities and proved it was responsible for synthesizing a new polyyne metabolite, protegencin. The evolutionary analysis of polyyne pathways showed that multiple biosynthetic genes were conserved, and using mutagenesis, their essentiality was demonstrated. Our research provides a foundation for the future modification of polyyne metabolites and has identified a novel polyyne, protegencin, with potential bioactive roles of ecological and agricultural importance.

Construction and evaluation of a bioluminescent Pseudomonas aeruginosa reporter for use in preservative efficacy testing.

Preservative efficacy testing (PET) is a fundamental practice in industrial microbiology used to ensure product shelf-life and quality. To improve on current growth-based PET, bioluminescence was evaluated as a real-time bacterial viability indicator using Pseudomonas aeruginosa. Random mutagenesis of an industrial P. aeruginosa strain with a promoter-less luxCDABE mini-Tn5 was used to select a stable reporter (LUX12H5) with an un-altered growth and preservative susceptibility phenotype. Bioluminescence and viability were measured with and without preservatives (isothiazolinones, phenoxyethanol, and dimethyl dimethylol hydantoin) and an antibiotic comparator (ciprofloxacin). In the absence of antimicrobials, a good correlation between bioluminescence and viability (r2=0.92) was established. However, metabolic inhibition by isothiazolinone preservatives caused a rapid decline in light output that did not correlate to a reduced viability. Conversely, after ciprofloxacin exposure, the decline in viability was greater than that of bioluminescence. A positive attribute of the bioluminescence was the early detection of metabolic recovery and re-growth of preservative injured bacteria. Overall, while initial bioluminescence read-outs were less suited to current PET requirements, it shows promise as an early, direct indicator of bacterial regrowth in the context of long-term evaluation of preservative efficacy.

Genomics reveals the novel species placement of industrial contaminant isolates incorrectly identified as Burkholderia lata.

The Burkholderia cepacia complex (Bcc) is a closely related group of bacteria, composed of at least 20 different species, the accurate identification of which is essential in the context of infectious diseases. In industry, they can contaminate non-food products, including home and personal care products and cosmetics. The Bcc are problematic contaminants due to their ubiquitous presence and intrinsic antimicrobial resistance, which enables them to occasionally overcome preservation systems in non-sterile products. Burkholderia lata and Burkholderia contaminans are amongst the Bcc bacteria encountered most frequently as industrial contaminants, but their identification is not straightforward. Both species were historically established as a part of a group known collectively as taxon K, based upon analysis of the recA gene and multilocus sequence typing (MLST). Here, we deploy a straightforward genomics-based workflow for accurate Bcc classification using average nucleotide identity (ANI) and core-gene analysis. The workflow was used to examine a panel of 23 Burkholderia taxon K industrial strains, which, based on MLST, comprised 13 B. lata, 4 B. contaminans and 6 unclassified Bcc strains. Our genomic identification showed that the B. contaminans strains retained their classification, whilst the remaining strains were reclassified as Burkholderia aenigmatica sp. nov. Incorrect taxonomic identification of industrial contaminants is a problematic issue. Application and testing of our genomic workflow allowed the correct classification of 23 Bcc industrial strains, and also indicated that B. aenigmatica sp. nov. may have greater importance than B. lata as a contaminant species. Our study illustrates how the non-food manufacturing industry can harness whole-genome sequencing to better understand antimicrobial-resistant bacteria affecting their products.

New benzo(a)pyrene-degrading strains of the Burkholderia cepacia complex prospected from activated sludge in a petrochemical wastewater treatment plant.

The prospection of bacteria that are resistant to polyaromatic hydrocarbons (PAH) of activated sludge from a Petrochemical Wastewater Treatment Plant (WWTP) allows investigating potential biodegraders of PAH. For this purpose, sludge samples were cultured with benzo(a)pyrene and/or naphthalene as carbon sources. The recovered isolates were characterized by biochemical methods and identified based on the analysis of the sequence of three genes: 16S, recA and gyrB. The isolated strains were shown to be capable of producing surfactants, which are important for compound degradation. The ability to reduce benzo(a)pyrene in vitro was tested by gas chromatography. After 20 days of experiment, the consortium that was enriched with 1 mg/L of benzo(a)pyrene was able to reduce 30% of the compound when compared to a control without bacteria. The four isolated strains that significantly reduced benzo(a)pyrene belong to the Burkholderia cepacia complex and were identified within the consortium as the species B. cenocepacia IIIa, B. vietnamiensis, B. cepacia, and B. multivorans. This finding demonstrates the biotechnological potential of the B. cepacia complex strains for use in wastewater treatment and bioremediation. Previous studies on hydrocarbon-degrading strains focused mainly on contaminated soil or marine areas. In this work, the strains were prospected from activated sludge in a WWTP and showed the potential of indigenous samples to be used in both improving treatment systems and bioremediation of areas contaminated with petrochemical waste.

Phenotypic and Genotypic Adaptations in Pseudomonas aeruginosa Biofilms following Long-Term Exposure to an Alginate Oligomer Therapy.

Chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) evolve to generate environmentally adapted biofilm communities, leading to increased patient morbidity and mortality. OligoG CF-5/20, a low-molecular-weight inhaled alginate oligomer therapy, is currently in phase IIb/III clinical trials in CF patients. Experimental evolution of P. aeruginosa in response to OligoG CF-5/20 was assessed using a bead biofilm model allowing continuous passage (45 days; ∼245 generations). Mutants isolated after OligoG CF-5/20 treatment typically had a reduced biofilm-forming ability and altered motility profile. Genotypically, OligoG CF-5/20 provided no selective pressure on genomic mutations within morphotypes. Chronic exposure to azithromycin, a commonly prescribed antibiotic in CF patients, with or without OligoG CF-5/20 in the biofilm evolution model also had no effect on rates of resistance acquisition. Interestingly, however, cross-resistance to other antibiotics (e.g., aztreonam) was reduced in the presence of OligoG CF-5/20. Collectively, these findings show no apparent adverse effects from long-term exposure to OligoG CF-5/20, instead resulting in both fewer colonies with multidrug resistance (MDR)-associated phenotypes and improved antibiotic susceptibility of P. aeruginosaIMPORTANCE The emergence of multidrug-resistant (MDR) pathogens within biofilms in the cystic fibrosis lung results in increased morbidity. An inhalation therapy derived from alginate, OligoG CF-5/20, is currently in clinical trials for cystic fibrosis patients. OligoG CF-5/20 has been shown to alter sputum viscoelasticity, disrupt mucin polymer networks, and disrupt MDR pseudomonal biofilms. Long-term exposure to inhaled therapeutics may induce selective evolutionary pressures on bacteria within the lung biofilm. Here, a bead biofilm model with repeated exposure of P. aeruginosa to OligoG CF-5/20 (alone and in combination with azithromycin) was conducted to study these long-term effects and characterize the phenotypic and genotypic adaptations which result. These findings, over 6 weeks, show that long-term use of OligoG CF-5/20 does not lead to extensive mutational changes and may potentially decrease the pathogenicity of the bacterial biofilm and improve the susceptibility of P. aeruginosa to other classes of antibiotics.

Kill and cure: genomic phylogeny and bioactivity of Burkholderia gladioli bacteria capable of pathogenic and beneficial lifestyles.

Burkholderia gladioli is a bacterium with a broad ecology spanning disease in humans, animals and plants, but also encompassing multiple beneficial interactions. It is a plant pathogen, a toxin-producing food-poisoning agent, and causes lung infections in people with cystic fibrosis (CF). Contrasting beneficial traits include antifungal production exploited by insects to protect their eggs, plant protective abilities and antibiotic biosynthesis. We explored the genomic diversity and specialized metabolic potential of 206 B. gladioli strains, phylogenomically defining 5 clades. Historical disease pathovars (pv.) B. gladioli pv. allicola and B. gladioli pv. cocovenenans were distinct, while B. gladioli pv. gladioli and B. gladioli pv. agaricicola were indistinguishable; soft-rot disease and CF infection were conserved across all pathovars. Biosynthetic gene clusters (BGCs) for toxoflavin, caryoynencin and enacyloxin were dispersed across B. gladioli, but bongkrekic acid and gladiolin production were clade-specific. Strikingly, 13 % of CF infection strains characterized were bongkrekic acid-positive, uniquely linking this food-poisoning toxin to this aspect of B. gladioli disease. Mapping the population biology and metabolite production of B. gladioli has shed light on its diverse ecology, and by demonstrating that the antibiotic trimethoprim suppresses bongkrekic acid production, a potential therapeutic strategy to minimize poisoning risk in CF has been identified.

Implementation of microbiota analysis in clinical trials for cystic fibrosis lung infection: Experience from the OligoG phase 2b clinical trials.

Culture-independent microbiota analysis is widely used in research and being increasingly used in translational studies. However, methods for standardisation and application of these analyses in clinical trials are limited. Here we report the microbiota analysis that accompanied two phase 2b clinical trials of the novel, non-antibiotic therapy OligoG CF-5/20 for cystic fibrosis (CF) lung infection. Standardised protocols (DNA extraction, PCR, qPCR and 16S rRNA gene sequencing analysis) were developed for application to the Pseudomonas aeruginosa (NCT02157922) and Burkholderia cepacia complex (NCT02453789) clinical trials involving 45 and 13 adult trial participants, respectively. Microbiota analysis identified that paired sputum samples from an individual participant, taken within 2 h of each other, had reproducible bacterial diversity profiles. Although culture microbiology had identified patients as either colonised by P. aeruginosa or B. cepacia complex species at recruitment, microbiota analysis revealed patient lung infection communities were not always dominated by these key CF pathogens. Microbiota profiles were patient-specific and remained stable over the course of both clinical trials (6 sampling points over the course of 140 days). Within the Burkholderia trial, participants were infected with B. cenocepacia (n = 4), B. multivorans (n = 6), or an undetermined species (n = 3). Colonisation with either B. cenocepacia or B. multivorans influenced the overall bacterial community structure in sputum. Overall, we have shown that sputum microbiota in adults with CF is stable over a 2 h time-frame, suggesting collection of a single sample on a collection day is sufficient to capture the microbiota diversity. Despite the uniform pathogen culture-positivity status at recruitment, trial participants were highly heterogeneous in their lung microbiota. Understanding the microbiota profiles of individuals with CF ahead of future clinical trials would be beneficial in the context of patient stratification and trial design.

Reclassification of the biocontrol agents Bacillus subtilis BY-2 and Tu-100 as Bacillus velezensis and insights into the genomic and specialized metabolite diversity of the species.

The genomes of two historical Bacillus species strains isolated from the roots of oilseed rape and used routinely in PR China as biocontrol agents to suppress Sclerotinia disease were sequenced. Average nucleotide identity (ANI) and digital DNA-DNA hybridization analyses demonstrated that they were originally misclassified as Bacillus subtilis and now belong to the bacterial species Bacillus velezensis. A broader ANI analysis of available Bacillus genomes identified 292 B. velezensis genomes that were then subjected to core gene analysis and phylogenomics. Prediction and dereplication of specialized metabolite biosynthetic gene clusters (BGCs) defined the prevalence of multiple antimicrobial-associated BGCs and highlighted the natural product potential of B. velezensis. By defining the core and accessory antimicrobial biosynthetic capacity of the species, we offer an in-depth understanding of B. velezensis natural product capacity to facilitate the selection and testing of B. velezensis strains for use as biological control agents.

Genomic Assemblies of Members of Burkholderia and Related Genera as a Resource for Natural Product Discovery.

The genomes of 450 members of Burkholderiaceae, isolated from clinical and environmental sources, were sequenced and assembled as a resource for genome mining. Genomic analysis of the collection has enabled the identification of multiple metabolites and their biosynthetic gene clusters, including the antibiotics gladiolin, icosalide A, enacyloxin, and cepacin A.

Genomics-Driven Discovery of a Novel Glutarimide Antibiotic from Burkholderia gladioli Reveals an Unusual Polyketide Synthase Chain Release Mechanism.

A gene cluster encoding a cryptic trans-acyl transferase polyketide synthase (PKS) was identified in the genomes of Burkholderia gladioli BCC0238 and BCC1622, both isolated from the lungs of cystic fibrosis patients. Bioinfomatics analyses indicated the PKS assembles a novel member of the glutarimide class of antibiotics, hitherto only isolated from Streptomyces species. Screening of a range of growth parameters led to the identification of gladiostatin, the metabolic product of the PKS. NMR spectroscopic analysis revealed that gladiostatin, which has promising activity against several human cancer cell lines and inhibits tumor cell migration, contains an unusual 2-acyl-4-hydroxy-3-methylbutenolide in addition to the glutarimide pharmacophore. An AfsA-like domain at the C-terminus of the PKS was shown to catalyze condensation of 3-ketothioesters with dihydroxyacetone phosphate, thus indicating it plays a key role in polyketide chain release and butenolide formation.