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.

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.

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.

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.

Mapping the Efficacy and Mode of Action of Ethylzingerone [4-(3-Ethoxy-4-Hydroxyphenyl) Butan-2-One] as an Active Agent against Burkholderia Bacteria.

Burkholderia cepacia complex (Bcc) bacteria are intrinsically antimicrobial-resistant opportunistic pathogens and key risk species in the contamination of nonfood industrial products. New agents and formulations to prevent growth of Burkholderia in home care (cleaning agents) and personal-care (cosmetics and toiletries) products are required. We characterized how ethylzingerone [4-(3-ethoxy-4-hydroxyphenyl) butan-2-one] (HEPB) acts as a preservative with activity against Burkholderia species encountered in industry. Burkholderia (n = 58) and non-Burkholderia (n = 7) bacteria were screened for susceptibility to HEPB, and its mode of action and resistance were determined for a model Burkholderia vietnamiensis strain using transposon mutagenesis, transcriptomics, and genome resequencing analysis. The susceptibility of Burkholderia spp. to HEPB (MIC = 0.45% ± 0.11% [wt/vol]; MBC = 0.90% ± 0.3% [wt/vol]) was characterized, with limited inter- and intraspecies differences. HEPB (1% [wt/vol]) was rapidly bactericidal, producing a 6-log reduction in viability within 4 h. Spontaneous resistance to HEPB did not develop, but transient phenotypes with altered growth characteristics and susceptibility to antibiotics were identified after prolonged exposure to sublethal HEPB concentrations. Transposon mutagenesis and RNA-sequencing analysis identified multiple genetic pathways associated with HEPB exposure, including stress response mechanisms, altered permeability, regulation of intracellular pH, damage and repair of intracellular components, and alteration and repair of lipopolysaccharides. Key pathways included the stringent response, homeostasis of intracellular pH by the kdp operon, protection against electrophiles by KefC, and repair of oxidized proteins by methionine sulfoxide reductase enzymes. In summary, we show that HEPB has potent, targeted efficacy against Burkholderia bacteria without promoting wider stable antimicrobial resistance. The mode of action of HEPB against Burkholderia is multifactorial, but killing by intracellular oxidation is a key mechanism of this promising agent.IMPORTANCEBurkholderia bacteria are opportunistic pathogens that can overcome preservatives used in the manufacture of nonsterile industrial products and occasionally cause contamination. Consequently, new preservatives to prevent the growth of key risk Burkholderia cepacia complex bacteria in nonfood industrial products are urgently required. Here, we show that ethylzingerone is active against these problematic bacteria, killing them via a multifactorial mode of action which involves intracellular oxidation.

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.

Discovery and Biosynthesis of Bolagladins: Unusual Lipodepsipeptides from Burkholderia gladioli Clinical Isolates*.

Two Burkholderia gladioli strains isolated from the lungs of cystic fibrosis patients were found to produce unusual lipodepsipeptides containing a unique citrate-derived fatty acid and a rare dehydro-ß-alanine residue. The gene cluster responsible for their biosynthesis was identified by bioinformatics and insertional mutagenesis. In-frame deletions and enzyme activity assays were used to investigate the functions of several proteins encoded by the biosynthetic gene cluster, which was found in the genomes of about 45 % of B. gladioli isolates, suggesting that its metabolic products play an important role in the growth and/or survival of the species. The Chrome Azurol S assay indicated that these metabolites bind ferric iron, which suppresses their production when added to the growth medium. Moreover, a gene encoding a TonB-dependent ferric-siderophore receptor is adjacent to the biosynthetic genes, suggesting that these metabolites may function as siderophores in B. gladioli.

Reclassification of the Specialized Metabolite Producer Pseudomonas mesoacidophila ATCC 31433 as a Member of the Burkholderia cepacia Complex.

Pseudomonas mesoacidophila ATCC 31433 is a Gram-negative bacterium, first isolated from Japanese soil samples, that produces the monobactam isosulfazecin and the ß-lactam-potentiating bulgecins. To characterize the biosynthetic potential of P. mesoacidophila ATCC 31433, its complete genome was determined using single-molecule real-time DNA sequence analysis. The 7.8-Mb genome comprised four replicons, three chromosomal (each encoding rRNA) and one plasmid. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 was misclassified at the time of its deposition and is a member of the Burkholderia cepacia complex, most closely related to Burkholderia ubonensis The sequenced genome shows considerable additional biosynthetic potential; known gene clusters for malleilactone, ornibactin, isosulfazecin, alkylhydroxyquinoline, and pyrrolnitrin biosynthesis and several uncharacterized biosynthetic gene clusters for polyketides, nonribosomal peptides, and other metabolites were identified. Furthermore, P. mesoacidophila ATCC 31433 harbors many genes associated with environmental resilience and antibiotic resistance and was resistant to a range of antibiotics and metal ions. In summary, this bioactive strain should be designated B. cepacia complex strain ATCC 31433, pending further detailed taxonomic characterization.IMPORTANCE This work reports the complete genome sequence of Pseudomonas mesoacidophila ATCC 31433, a known producer of bioactive compounds. Large numbers of both known and novel biosynthetic gene clusters were identified, indicating that P. mesoacidophila ATCC 31433 is an untapped resource for discovery of novel bioactive compounds. Phylogenetic analysis demonstrated that P. mesoacidophila ATCC 31433 is in fact a member of the Burkholderia cepacia complex, most closely related to the species Burkholderia ubonensis Further investigation of the classification and biosynthetic potential of P. mesoacidophila ATCC 31433 is warranted.

Discovery and Biosynthesis of Gladiolin: A Burkholderia gladioli Antibiotic with Promising Activity against Mycobacterium tuberculosis.

An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites.

Molecular epidemiology of Pseudomonas aeruginosa in an unsegregated bronchiectasis cohort sharing hospital facilities with a cystic fibrosis cohort.

While Pseudomonas aeruginosa (PA) cross-infection is well documented among patients with cystic fibrosis (CF), the equivalent risk among patients with non-CF bronchiectasis (NCFB) is unclear, particularly those managed alongside patients with CF. We performed analysis of PA within a single centre that manages an unsegregated NCFB cohort alongside a segregated CF cohort. We found no evidence of cross-infection between the two cohorts or within the segregated CF cohort. However, within the unsegregated NCFB cohort, evidence of cross-infection was found between three (of 46) patients. While we do not presently advocate any change in the management of our NCFB cohort, longitudinal surveillance is clearly warranted.

Burkholderia: an update on taxonomy and biotechnological potential as antibiotic producers.

Burkholderia is an incredibly diverse and versatile Gram-negative genus, within which over 80 species have been formally named and multiple other genotypic groups likely represent new species. Phylogenetic analysis based on the 16S rRNA gene sequence and core genome ribosomal multilocus sequence typing analysis indicates the presence of at least three major clades within the genus. Biotechnologically, Burkholderia are well-known for their bioremediation and biopesticidal properties. Within this review, we explore the ability of Burkholderia to synthesise a wide range of antimicrobial compounds ranging from historically characterised antifungals to recently described antibacterial antibiotics with activity against multiresistant clinical pathogens. The production of multiple Burkholderia antibiotics is controlled by quorum sensing and examples of quorum sensing pathways found across the genus are discussed. The capacity for antibiotic biosynthesis and secondary metabolism encoded within Burkholderia genomes is also evaluated. Overall, Burkholderia demonstrate significant biotechnological potential as a source of novel antibiotics and bioactive secondary metabolites.

Phenotypic characterization of an international Pseudomonas aeruginosa reference panel: strains of cystic fibrosis (CF) origin show less in vivo virulence than non-CF strains.

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) and acute opportunistic infections in people without CF. Forty-two P. aeruginosa strains from a range of clinical and environmental sources were collated into a single reference strain panel to harmonise research on this diverse opportunistic pathogen. To facilitate further harmonized and comparable research on P. aeruginosa, we characterized the panel strains for growth rates, motility, virulence in the Galleria mellonella infection model, pyocyanin and alginate production, mucoid phenotype, LPS pattern, biofilm formation, urease activity, and antimicrobial and phage susceptibilities. Phenotypic diversity across the P. aeruginosa panel was apparent for all phenotypes examined, agreeing with the marked variability seen in this species. However, except for growth rate, the phenotypic diversity among strains from CF versus non-CF sources was comparable. CF strains were less virulent in the G. mellonella model than non-CF strains (P = 0.037). Transmissible CF strains generally lacked O-antigen, produced less pyocyanin and had low virulence in G. mellonella. Furthermore, in the three sets of sequential CF strains, virulence, O-antigen expression and pyocyanin production were higher in the earlier isolate compared to the isolate obtained later in infection. Overall, this full phenotypic characterization of the defined panel of P. aeruginosa strains increases our understanding of the virulence and pathogenesis of P. aeruginosa and may provide a valuable resource for the testing of novel therapies against this problematic pathogen.

Rapid Detection of Emerging Pathogens and Loss of Microbial Diversity Associated with Severe Lung Disease in Cystic Fibrosis.

Respiratory infection in cystic fibrosis (CF) is polymicrobial, but standard sputum microbiology does not account for the lung microbiome or detect changes in microbial diversity associated with disease. As a clinically applicable CF microbiome surveillance scheme, total sputum nucleic acids isolated by a standard high-throughput robotic method for accredited viral diagnosis were profiled for bacterial diversity using ribosomal intergenic spacer analysis (RISA) PCR. Conventional culture and RISA were performed on 200 paired sputum samples from 93 CF adults; pyrosequencing of the 16S rRNA gene was applied to 59 patients to systematically determine bacterial diversity. Compared to the microbiology data, RISA profiles clustered into two groups: the emerging nonfermenting Gram-negative organisms (eNFGN) and Pseudomonas groups. Patients who were culture positive for Burkholderia, Achromobacter, Stenotrophomonas, and Ralstonia clustered within the eNFGN group. Pseudomonas group RISA profiles were associated with Pseudomonas aeruginosa culture-positive patients. Sequence analysis confirmed the abundance of eNFGN genera and Pseudomonas within these respective groups. Low bacterial diversity was associated with severe lung disease (P < 0.001) and the presence of Burkholderia (P < 0.001). An absence of Streptococcus (P < 0.05) occurred in individuals with lung function in the lowest quartile. In summary, nucleic acids isolated from CF sputum can serve as a single template for both molecular virology and bacteriology, with a RISA PCR rapidly detecting the presence of dominant eNFGN pathogens or P. aeruginosa missed by culture (11% of cases). We provide guidance for how this straightforward CF microbiota profiling scheme may be adopted by clinical laboratories.

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

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

Arabidopsis thaliana and Pisum sativum models demonstrate that root colonization is an intrinsic trait of Burkholderia cepacia complex bacteria.

Burkholderia cepacia complex (Bcc) bacteria possess biotechnologically useful properties that contrast with their opportunistic pathogenicity. The rhizosphere fitness of Bcc bacteria is central to their biocontrol and bioremediation activities. However, it is not known whether this differs between species or between environmental and clinical strains. We investigated the ability of 26 Bcc strains representing nine different species to colonize the roots of Arabidopsis thaliana and Pisum sativum (pea). Viable counts, scanning electron microscopy and bioluminescence imaging were used to assess root colonization, with Bcc bacteria achieving mean (±sem) levels of 2.49±0.23×10(6) and 5.16±1.87×10(6) c.f.u. per centimetre of root on the A. thaliana and P. sativum models, respectively. The A. thaliana rhizocompetence model was able to reveal loss of colonization phenotypes in Burkholderia vietnamiensis G4 transposon mutants that had only previously been observed in competition experiments on the P. sativum model. Different Bcc species colonized each plant model at different rates, and no statistical difference in root colonization was observed between isolates of clinical or environmental origin. Loss of the virulence-associated third chromosomal replicon (>1 Mb DNA) did not alter Bcc root colonization on A. thaliana. In summary, Bcc bacteria possess intrinsic root colonization abilities irrespective of their species or source. As Bcc rhizocompetence does not require their third chromosomal replicon, the possibility of using synthetic biology approaches to engineer virulence-attenuated biotechnological strains is tractable.

Emerging cystic fibrosis pathogens and the microbiome.

Cystic fibrosis (CF) respiratory infection is characterised by the presence of typical human bacterial pathogens such as Pseudomonas aeruginosa, Haemophilus influenzae and Staphylococcus aureus. Less typical pathogens such as Burkholderia, Stenotrophomonas, Achromobacter, Pandorea and Ralstonia have emerged as problematic infections which are largely unique to people with CF. Using molecular methods, two groups of anaerobic bacteria Prevotella species and the Streptococcus milleri group have also recently been shown to be highly prevalent in CF sputum. Collectively, the diversity of microorganisms present in respiratory specimens has been designated the CF microbiome. The challenges posed by emerging CF pathogens and a microbiome-based view of CF infection are discussed in terms of their impact on clinical outcome, diagnosis and therapy.

Key role for efflux in the preservative susceptibility and adaptive resistance of Burkholderia cepacia complex bacteria.

Bacteria from the Burkholderia cepacia complex (Bcc) are encountered as industrial contaminants, and little is known about the species involved or their mechanisms of preservative resistance. Multilocus sequence typing (MLST) revealed that multiple Bcc species may cause contamination, with B. lata (n = 17) and B. cenocepacia (n = 11) dominant within the collection examined. At the strain level, 11 of the 31 industrial sequence types identified had also been recovered from either natural environments or clinical infections. Minimal inhibitory (MIC) and minimum bactericidal (MBC) preservative concentrations varied across 83 selected Bcc strains, with industrial strains demonstrating increased tolerance for dimethylol dimethyl hydantoin (DMDMH). Benzisothiazolinone (BIT), DMDMH, methylisothiazolinone (MIT), a blend of 3:1 methylisothiazolinone-chloromethylisothiazolinone (M-CMIT), methyl paraben (MP), and phenoxyethanol (PH), were all effective anti-Bcc preservatives; benzethonium chloride (BC) and sodium benzoate (SB) were least effective. Since B. lata was the dominant industrial Bcc species, the type strain, 383(T) (LMG 22485(T)), was used to study preservative tolerance. Strain 383 developed stable preservative tolerance for M-CMIT, MIT, BIT, and BC, which resulted in preservative cross-resistance and altered antibiotic susceptibility, motility, and biofilm formation. Transcriptomic analysis of the B. lata 383 M-CMIT-adapted strain demonstrated that efflux played a key role in its M-CMIT tolerance and elevated fluoroquinolone resistance. The role of efflux was corroborated using the inhibitor l-Phe-Arg-ß-napthylamide, which reduced the MICs of M-CMIT and ciprofloxacin. In summary, intrinsic preservative tolerance and stable adaptive changes, such as enhanced efflux, play a role in the ability of Bcc bacteria to cause industrial contamination.

Intravenous antibiotics reduce the presence of Aspergillus in adult cystic fibrosis sputum.

BACKGROUND: Pseudomonas aeruginosa and Aspergillus fumigatus frequently co-colonise the airways of patients with cystic fibrosis (CF). This study aimed to assess the impact of short-term administration of intravenous antipseudomonal antibiotics during CF exacerbations on the presence of Aspergillus. METHODS: Pre- and post-antibiotic sputum samples from 26 adult patients with CF and chronic Pseudomonas colonisation were analysed for the presence of Aspergillus by fungal culture, real-time PCR and galactomannan antigen (GM). Lung function (forced expiratory volume in 1 s and forced vital capacity % predicted) and blood levels of total IgE, specific A fumigatus IgE and specific A fumigatus IgG were measured at the start and end of antibiotics. Respiratory viral real-time PCR and bacterial community profiling using ribosomal intergenic spacer analysis (RISA) were performed to estimate concurrent changes in the lung microbiome. RESULTS: Aspergillus PCR and GM were more sensitive than culture in detecting Aspergillus species (culture 8%, GM 31%, PCR 77%). There was a significant decline in the presence of Aspergillus, measured both by PCR and GM index, following antibacterial therapy (PCR: median increase in crossing threshold 1.7 (IQR 0.5-3.8), p<0.001; GM: median fall in GM index 0.7 (IQR 0.4-1.6), p=0.016). All patients improved clinically with a significant increase in lung function (p<0.0001). RISA community analysis showed large changes in bacterial community similarity in 67% of patients following antibiotics. Viral RT-PCR demonstrated the presence of a concurrent respiratory virus in 27% of patients. CONCLUSIONS: Intravenous antibiotics targeting Pseudomonas during CF pulmonary exacerbations have a negative impact on the presence of Aspergillus in sputum samples.

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.