Combatting melioidosis with chemical synthetic lethality.

Burkholderia thailandensis has emerged as a nonpathogenic surrogate for Burkholderia pseudomallei, the causative agent of melioidosis, and an important Gram-negative model bacterium for studying the biosynthesis and regulation of secondary metabolism. We recently reported that subinhibitory concentrations of trimethoprim induce vast changes in both the primary and secondary metabolome of B. thailandensis. In the current work, we show that the folate biosynthetic enzyme FolE2 is permissive under standard growth conditions but essential for B. thailandensis in the presence of subinhibitory doses of trimethoprim. Reasoning that FolE2 may serve as an attractive drug target, we screened for and identified ten inhibitors, including dehydrocostus lactone (DHL), parthenolide, and ß-lapachone, all of which are innocuous individually but form a chemical-synthetic lethal combination with subinhibitory doses of trimethoprim. We show that DHL is a mechanism-based inhibitor of FolE2 and capture the structure of the covalently inhibited enzyme using X-ray crystallography. In vitro, the combination of subinhibitory trimethoprim and DHL is more potent than Bactrim, the current standard of care against melioidosis. Moreover, unlike Bactrim, this combination does not affect the growth of most commensal and beneficial gut bacteria tested, thereby providing a degree of specificity against B. pseudomallei. Our work provides a path for identifying antimicrobial drug targets and for utilizing binary combinations of molecules that form a toxic cocktail based on metabolic idiosyncrasies of specific pathogens.

Effects of calcium phosphate and phosphorus-dissolving bacteria on microbial structure and function during Torreya Grandis branch waste composting.

BACKGROUND BURKHOLDERIA: is a phosphorus solubilizing microorganism discovered in recent years, which can dissolve insoluble phosphorus compounds into soluble phosphorus. To investigate the effects of Burkholderia and calcium phosphate on the composting of Torreya grandis branches and leaves, as well as to explain the nutritional and metabolic markers related to the composting process. METHODS: In this study, we employed amplicon sequencing and untargeted metabolomics analysis to examine the interplay among phosphorus (P) components, microbial communities, and metabolites during T. grandis branch and leaf waste composting that underwent treatment with calcium phosphate and phosphate-solubilizing bacteria (Burkholderia). There were four composting treatments, 10% calcium phosphate (CaP) or 5 ml/kg (1 × 108/ml Burkholderia) microbial inoculum (WJP) or both (CaP + WJP), and the control group (CK). RESULTS: The results indicated that Burkholderia inoculation and calcium phosphate treatment affected the phosphorus composition, pH, EC, and nitrogen content. Furthermore, these treatments significantly affected the diversity and structure of bacterial and fungal communities, altering microbial and metabolite interactions. The differential metabolites associated with lipids and organic acids and derivatives treated with calcium phosphate treatment are twice as high as those treated with Burkholderia in both 21d and 42d. The results suggest that calcium phosphate treatment alters the formation of some biological macromolecules. CONCLUSION: Both Burkholderia inoculation and calcium phosphate treatment affected the phosphorus composition, nitrogen content and metabolites of T. grandis branch and leaf waste compost.These results extend our comprehension of the coupling of matter transformation and community succession in composting with the addition of calcium phosphate and phosphate-solubilizing bacteria.

Bacterial persistence to antibiotics activated by tRNA mutations.

OBJECTIVES: Bacterial persistence is a significant cause of the intractability of chronic and relapsing infections. Despite its importance, many of the underlying mechanisms are still not well understood. METHODS: Antibiotic-tolerant mutants of Burkholderia thailandensis were isolated through exposure to lethal doses of AMP or MEM, followed by whole-genome sequencing to identify mutations. Subsequently, these mutants underwent comprehensive characterization via killing curves, growth curves, and persistence-fraction plots. Northern blot analysis was employed to detect uncharged tRNA, while the generation of relA and spoT null mutations served to confirm the involvement of the stringent response in this persistence mechanism. Phenotypic reversion of the persistence mutation was demonstrated by incubating the mutants without antibiotics for 2 weeks. RESULTS: We have discovered a novel mechanism of persistence triggered by specific mutations at positions 32 or 38 within the anticodon loop of tRNAAsp. This leads to heightened persistence through a RelA-dependent stringent response. Notably, this persistence can be easily reverted to wild-type physiology by losing the mutant tRNA allele within the tRNA gene cluster when persistence is no longer essential for survival. CONCLUSIONS: This distinct form of persistence underscores the novel function of tRNA mutations at positions 32 or 38 within the anticodon loop, as well as the significance of the tRNA gene cluster in conferring adaptability to regulate persistence for enhanced survival.

Drug Resistance in Biofilm and Planktonic Cells of Achromobacter spp., Burkholderia spp., and Stenotrophomonas maltophilia Clinical Isolates.

Background: Biofilm production in nonfermenting Gram-negative bacteria influences drug resistance. The aim of this work was to evaluate the effect of different antibiotics on biofilm eradication of clinical isolates of Achromobacter, Burkholderia, and Stenotrophomonas maltophilia. Methods: Clinical isolates were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry in a third-level hospital in Monterrey, Mexico. Crystal violet staining was used to determine biofilm production. Drug susceptibility testing was determined by broth microdilution in planktonic cells and biofilm cells. Results: Resistance in planktonic cells was moderate to trimethoprim-sulfamethoxazole, and low to chloramphenicol, minocycline, levofloxacin (S. maltophilia and Burkholderia), ceftazidime, and meropenem (Burkholderia and Achromobacter). Biofilm eradication required higher drug concentrations of ceftazidime, chloramphenicol, levofloxacin, and trimethoprim-sulfamethoxazole than planktonic cells (p < 0.05). Levofloxacin showed biofilm eradication activity in S. maltophilia, minocycline and meropenem in Burkholderia, and meropenem in Achromobacter. Conclusions: Drug resistance increased due to biofilm production for some antibiotics, particularly ceftazidime and trimethoprim-sulfamethoxazole for all three pathogens, chloramphenicol for S. maltophilia and Burkholderia, and levofloxacin for Burkholderia. Some antibiotics could be used for the treatment of biofilm-associated infections in our population, such as levofloxacin for S. maltophilia, minocycline and meropenem for Burkholderia, and meropenem for Achromobacter.

Repurposing promethazine hydrochloride to inhibit biofilm formation against Burkholderia thailandensis.

Melioidosis is a severe infectious disease caused by Burkholderia pseudomallei, an intracellular pathogen with a high mortality rate and significant antibiotic resistance. The high mortality rate and resistance to antibiotics have drawn considerable attention from researchers studying melioidosis. This study evaluated the effects of various concentrations (75, 50, and 25 µg/mL) of promethazine hydrochloride (PTZ), a potent antihistamine, on biofilm formation and lipase activity after 24 h of exposure to B. thailandensis E264. A concentration-dependent decrease in both biofilm biomass and lipase activity was observed. RT-PCR analysis revealed that PTZ treatment not only made the biofilm structure loose but also reduced the expression of btaR1, btaR2, btaR3, and scmR. Single gene knockouts of quorum sensing (QS) receptor proteins (∆btaR1, ∆btaR2, and ∆btaR3) were successfully constructed. Deletion of btaR1 affected biofilm formation in B. thailandensis, while deletion of btaR2 and btaR3 led to reduced lipase activity. Molecular docking and biological performance results demonstrated that PTZ inhibits biofilm formation and lipase activity by suppressing the expression of QS-regulated genes. This study found that repositioning PTZ reduced biofilm formation in B. thailandensis E264, suggesting a potential new approach for combating melioidosis.

Phage-induced efflux down-regulation boosts antibiotic efficacy.

The interactions between a virus and its host vary in space and time and are affected by the presence of molecules that alter the physiology of either the host or the virus. Determining the molecular mechanisms at the basis of these interactions is paramount for predicting the fate of bacterial and phage populations and for designing rational phage-antibiotic therapies. We study the interactions between stationary phase Burkholderia thailandensis and the phage ΦBp-AMP1. Although heterogeneous genetic resistance to phage rapidly emerges in B. thailandensis, the presence of phage enhances the efficacy of three major antibiotic classes, the quinolones, the beta-lactams and the tetracyclines, but antagonizes tetrahydrofolate synthesis inhibitors. We discovered that enhanced antibiotic efficacy is facilitated by reduced antibiotic efflux in the presence of phage. This new phage-antibiotic therapy allows for eradication of stationary phase bacteria, whilst requiring reduced antibiotic concentrations, which is crucial for treating infections in sites where it is difficult to achieve high antibiotic concentrations.

A novel Burkholderia pyrrocinia strain effectively inhibits Fusarium graminearum growth and deoxynivalenol (DON) production.

BACKGROUND: Fusarium graminearum is a devastating fungal pathogen that poses a significant threat to global wheat production and quality. Control of this toxin-producing pathogen remains a major challenge. This study aimed to isolate strains with antagonistic activity against F. graminearum and at the same time to analyze the synthesis of deoxynivalenol (DON), in order to provide a new basis for the biological control of FHB. RESULTS: Total of 69 microorganisms were isolated from the soil of a wheat-corn crop rotation field, and an antagonistic bacterial strain F12 was identified as Burkholderia pyrrocinia by molecular biology and carbon source utilization. F. graminearum control by strain F12 showed excellent biological activities under laboratory conditions (95.8%) and field testing (63.09%). Meanwhile, the DON content of field-treated wheat grains was detected the results showed that F12 have significantly inhibited of DON, which was further verified by qPCR that F12 produces secondary metabolites that inhibit the expression of DON and pigment-related genes. In addition, the sterile fermentation broth of F12 not only inhibited mycelial growth and spore germination, but also prevented mycelia from producing spores. CONCLUSION: In this study B. pyrrocinia was reported to have good control of FHB and inhibition of DON synthesis. This novel B. pyrrocinia F12 is a promising biological inoculant, providing possibilities for controlling FHB, and a theoretical basis for the development of potential biocontrol agents and biofertilizers for agricultural use. © 2024 Society of Chemical Industry.

Thiosulfinate Tolerance Gene Clusters Are Common Features of Burkholderia Onion Pathogens.

Burkholderia gladioli pv. alliicola, B. cepacia, and B. orbicola are common bacterial pathogens of onion. Onions produce organosulfur thiosulfinate defensive compounds after cellular decompartmentalization. Using whole-genome sequencing and in silico analysis, we identified putative thiosulfinate tolerance gene (TTG) clusters in multiple onion-associated Burkholderia species similar to those characterized in other Allium-associated bacterial endophytes and pathogens. Sequence analysis revealed the presence of three Burkholderia TTG cluster types, with both Type A and Type B being broadly distributed in B. gladioli, B. cepacia, and B. orbicola in both the chromosome and plasmids. Based on isolate natural variation and generation of isogenic strains, we determined the in vitro and in vivo contribution of TTG clusters in B. gladioli, B. cepacia, and B. orbicola. The Burkholderia TTG clusters contributed to enhanced allicin tolerance and improved growth in filtered onion extracts by all three species. TTG clusters also made clear contributions to B. gladioli foliar necrosis symptoms and bacterial populations. Surprisingly, the TTG cluster did not contribute to bacterial populations in onion bulb scales by these three species. Based on our findings, we hypothesize onion-associated Burkholderia may evade or inhibit the production of thiosulfinates in onion bulb tissues. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genomic insights into an extensively drug-resistant and hypervirulent Burkholderia dolosa N149 isolate of a novel sequence type (ST2237) from a Vietnamese patient hospitalised for stroke.

OBJECTIVES: Burkholderia dolosa is a clinically important opportunistic pathogen in inpatients. Here we characterised an extensively drug-resistant and hypervirulent B. dolosa isolate from a patient hospitalised for stroke. METHODS: Resistance to 41 antibiotics was tested with the agar disc diffusion, minimum inhibitory concentration, or broth microdilution method. The complete genome was assembled using short-reads and long-reads and the hybrid de novo assembly method. Allelic profiles obtained by multilocus sequence typing were analysed using the PubMLST database. Antibiotic-resistance and virulence genes were predicted in silico using public databases and the 'baargin' workflow. B. dolosa N149 phylogenetic relationships with all available B. dolosa strains and Burkholderia cepacia complex strains were analysed using the pangenome obtained with Roary. RESULTS: B. dolosa N149 displayed extensive resistance to 31 antibiotics and intermediate resistance to 4 antibiotics. The complete genome included three circular chromosomes (6 338 630 bp in total) and one plasmid (167 591 bp). Genotypic analysis revealed various gene clusters (acr, amr, amp, emr, ade, bla and tet) associated with resistance to 35 antibiotic classes. The major intrinsic resistance mechanisms were multidrug efflux pump alterations, inactivation and reduced permeability of targeted antibiotics. Moreover, 91 virulence genes (encoding proteins involved in adherence, formation of capsule, biofilm and colony, motility, phagocytosis inhibition, secretion systems, protease secretion, transmission and quorum sensing) were identified. B. dolosa N149 was assigned to a novel sequence type (ST2237) and formed a mono-phylogenetic clade separated from other B. dolosa strains. CONCLUSIONS: This study provided insights into the antimicrobial resistance and virulence mechanisms of B. dolosa.

Insecticide resistance by a host-symbiont reciprocal detoxification.

Insecticide resistance is one of the most serious problems in contemporary agriculture and public health. Although recent studies revealed that insect gut symbionts contribute to resistance, the symbiont-mediated detoxification process remains unclear. Here we report the in vivo detoxification process of an organophosphorus insecticide, fenitrothion, in the bean bug Riptortus pedestris. Using transcriptomics and reverse genetics, we reveal that gut symbiotic bacteria degrade this insecticide through a horizontally acquired insecticide-degrading enzyme into the non-insecticidal but bactericidal compound 3-methyl-4-nitrophenol, which is subsequently excreted by the host insect. This integrated "host-symbiont reciprocal detoxification relay" enables the simultaneous maintenance of symbiosis and efficient insecticide degradation. We also find that the symbiont-mediated detoxification process is analogous to the insect genome-encoded fenitrothion detoxification system present in other insects. Our findings highlight the capacity of symbiosis, combined with horizontal gene transfer in the environment, as a powerful strategy for an insect to instantly eliminate a toxic chemical compound, which could play a critical role in the human-pest arms race.

Chemical or Genetic Alteration of Proton Motive Force Results in Loss of Virulence of Burkholderia glumae, the Cause of Rice Bacterial Panicle Blight.

Rice is an important source of food for more than half of the world's population. Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. B. glumae synthesizes toxoflavin, an essential virulence factor that is required for symptoms of the disease. The products of the tox operons, ToxABCDE and ToxFGHI, are responsible for the synthesis and the proton motive force (PMF)-dependent secretion of toxoflavin, respectively. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Our previous work has demonstrated that absence of certain DedA family members results in pleiotropic effects, impacting multiple pathways that are energized by PMF. We have demonstrated that a member of the DedA family from Burkholderia thailandensis, named DbcA, is required for the extreme polymyxin resistance observed in this organism. B. glumae encodes a homolog of DbcA with 73% amino acid identity to Burkholderia thailandensis DbcA. Here, we created and characterized a B. glumae ΔdbcA strain. In addition to polymyxin sensitivity, the B. glumae ΔdbcA strain is compromised for virulence in several BPB infection models and secretes only low amounts of toxoflavin (∼15% of wild-type levels). Changes in membrane potential in the B. glumae ΔdbcA strain were reproduced in the wild-type strain by the addition of subinhibitory concentrations of sodium bicarbonate, previously demonstrated to cause disruption of PMF. Sodium bicarbonate inhibited B. glumae virulence in rice, suggesting a possible non-toxic chemical intervention for bacterial panicle blight. IMPORTANCE Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Here, we constructed a B. glumae mutant with a deletion in a DedA family member named dbcA and report a loss of virulence in models of BPB. Physiological analysis of the mutant shows that the proton motive force is disrupted, leading to reduction of secretion of the essential virulence factor toxoflavin. The mutant phenotypes are reproduced in the virulent wild-type strain without an effect on growth using sodium bicarbonate, a nontoxic buffer that has been reported to disrupt the PMF. The results presented here suggest that bicarbonate may be an effective antivirulence agent capable of controlling BPB without imposing an undue burden on the environment.

A link between pH homeostasis and colistin resistance in bacteria.

Colistin resistance is complex and multifactorial. DbcA is an inner membrane protein belonging to the DedA superfamily required for maintaining extreme colistin resistance of Burkholderia thailandensis. The molecular mechanisms behind this remain unclear. Here, we report that ∆dbcA displays alkaline pH/bicarbonate sensitivity and propose a role of DbcA in extreme colistin resistance of B. thailandensis by maintaining cytoplasmic pH homeostasis. We found that alkaline pH or presence of sodium bicarbonate displays a synergistic effect with colistin against not only extremely colistin resistant species like B. thailandensis and Serratia marcescens, but also a majority of Gram-negative and Gram-positive bacteria tested, suggesting a link between cytoplasmic pH homeostasis and colistin resistance across species. We found that lowering the level of oxygen in the growth media or supplementation of fermentable sugars such as glucose not only alleviated alkaline pH stress, but also increased colistin resistance in most bacteria tested, likely by avoiding cytoplasmic alkalinization. Our observations suggest a previously unreported link between pH, oxygen, and colistin resistance. We propose that maintaining optimal cytoplasmic pH is required for colistin resistance in a majority of bacterial species, consistent with the emerging link between cytoplasmic pH homeostasis and antibiotic resistance.

Piperacillin triggers virulence factor biosynthesis via the oxidative stress response in Burkholderia thailandensis.

Natural products have been an important source of therapeutic agents and chemical tools. The recent realization that many natural product biosynthetic genes are silent or sparingly expressed during standard laboratory growth has prompted efforts to investigate their regulation and develop methods to induce their expression. Because it is difficult to intuit signals that induce a given biosynthetic locus, we recently implemented a forward chemical-genetic approach to identify such inducers. In the current work, we applied this approach to nine silent biosynthetic loci in the model bacterium Burkholderia thailandensis to systematically screen for elicitors from a library of Food and Drug Administration-approved drugs. We find that ß-lactams, fluoroquinolones, antifungals, and, surprisingly, calcimimetics, phenothiazine antipsychotics, and polyaromatic antidepressants are the most effective global inducers of biosynthetic genes. Investigations into the mechanism of stimulation of the silent virulence factor malleicyprol by the ß-lactam piperacillin allowed us to elucidate the underlying regulatory circuits. Low-dose piperacillin causes oxidative stress, thereby inducing redox-sensing transcriptional regulators, which activate malR, a pathway-specific positive regulator of the malleicyprol gene cluster. Malleicyprol is thus part of the OxyR and SoxR regulons in B. thailandensis, allowing the bacterium to initiate virulence in response to oxidative stress. Our work catalogs a diverse array of elicitors and a previously unknown regulatory input for secondary metabolism in B. thailandensis.

A ß-lactamase gene of Fusarium oxysporum alters the rhizosphere microbiota of soybean.

The rhizosphere is a multitrophic environment, and for soilborne pathogens such as Fusarium oxysporum, microbial competition in the rhizosphere is inevitable before reaching and infecting roots. This study established a tritrophic interaction among the plant growth-promoting rhizobacterium Burkholderia ambifaria, F. oxysporum and Glycine max (soybean) to study the effects of F. oxysporum genes on shaping the soybean microbiota. Although B. ambifaria inhibited mycelial growth and increased bacterial propagation in the presence of F. oxysporum, F. oxysporum still managed to infect soybean in the presence of B. ambifaria. RNA-Seq identified a putative F. oxysporum secretory ß-lactamase-coding gene, FOXG_18438 (abbreviated as Fo18438), that is upregulated during soybean infection in the presence of B. ambifaria. The ∆Fo18438 mutants displayed reduced mycelial growth towards B. ambifaria, and the complementation of full Fo18438 and the Fo18438 ß-lactamase domain restored mycelial growth. Using the F. oxysporum wild type, ∆Fo18438 mutants and complemented strains with full Fo18438, Fo18438 ß-lactamase domain or Fo18438 RTA1-like domain for soil inoculation, 16S rRNA amplicon sequencing revealed that the abundance of a Burkholderia operational taxonomic unit (OTU) was increased in the rhizosphere microbiota infested by the strains with Fo18438 ß-lactamase domain. Non-metric multidimensional scaling and PICRUSt2 functional analysis revealed differential abundance for the bacterial ß-lactam-related functions when contrasting the genotypes of F. oxysporum. These results indicated that the Fo18438 ß-lactamase domain provides F. oxysporum with the advantage of growing into the soybean rhizosphere, where ß-lactam antibiosis is involved in microbial competition. Accordingly, this study highlights the capability of an F. oxysporum gene for altering the soybean rhizosphere and taproot microbiota.

Drug screening to identify compounds to act as co-therapies for the treatment of Burkholderia species.

Burkholderia pseudomallei is a soil-dwelling organism present throughout the tropics. It is the causative agent of melioidosis, a disease that is believed to kill 89,000 people per year. It is naturally resistant to many antibiotics, requiring at least two weeks of intravenous treatment with ceftazidime, imipenem or meropenem followed by 6 months of orally delivered co-trimoxazole. This places a large treatment burden on the predominantly middle-income nations where the majority of disease occurs. We have established a high-throughput assay for compounds that could be used as a co-therapy to potentiate the effect of ceftazidime, using the related non-pathogenic bacterium Burkholderia thailandensis as a surrogate. Optimization of the assay gave a Z' factor of 0.68. We screened a library of 61,250 compounds and identified 29 compounds with a pIC50 (-log10(IC50)) greater than five. Detailed investigation allowed us to down select to six "best in class" compounds, which included the licensed drug chloroxine. Co-treatment of B. thailandensis with ceftazidime and chloroxine reduced culturable cell numbers by two orders of magnitude over 48 hours, compared to treatment with ceftazidime alone. Hit expansion around chloroxine was performed using commercially available compounds. Minor modifications to the structure abolished activity, suggesting that chloroxine likely acts against a specific target. Finally, an initial study demonstrates the utility of chloroxine to act as a co-therapy to potentiate the effect of ceftazidime against B. pseudomallei. This approach successfully identified potential co-therapies for a recalcitrant Gram-negative bacterial species. Our assay could be used more widely to aid in chemotherapy to treat infections caused by these bacteria.

Impaired purine homeostasis plays a primary role in trimethoprim-mediated induction of virulence genes in Burkholderia thailandensis.

One of the most commonly prescribed antibiotics against Burkholderia infections is co-trimoxazole, a cocktail of trimethoprim and sulfamethoxazole. Trimethoprim elicits an upregulation of the mal gene cluster, which encodes proteins involved in synthesis of the cytotoxic polyketide malleilactone; trimethoprim does so by increasing expression of the malR gene, which encodes the activator MalR. We report that B. thailandensis grown on trimethoprim exhibited increased virulence against Caenorhabditis elegans. This enhanced virulence correlated with an increase in expression of the mal gene cluster. Notably, inhibition of xanthine dehydrogenase by addition of allopurinol led to similar upregulation of malA and malR, with addition of trimethoprim or allopurinol also resulting in an equivalent intracellular accumulation of xanthine. Xanthine is a ligand for the transcription factor MftR that leads to attenuated DNA binding, and we show using chromatin immunoprecipitation that MftR binds directly to malR. Our gene expression data suggest that malR expression is repressed by both MftR and by a separate transcription factor, which also responds to a metabolite that accumulates on exposure to trimethoprim. Since allopurinol elicits a similar increase in malR/malA expression as trimethoprim, we suggest that impaired purine homeostasis plays a primary role in trimethoprim-mediated induction of malR and in turn malA.

Safer In Vitro Drug Screening Models for Melioidosis Therapy Development.

Melioidosis is a neglected tropical disease caused by the Gram-negative soil bacterium Burkholderia pseudomallei. Current antibiotic regimens used to treat melioidosis are prolonged and expensive, and often ineffective because of intrinsic and acquired antimicrobial resistance. Efforts to develop new treatments for melioidosis are limited by the risks associated with handling pathogenic B. pseudomallei, which restricts research to facilities with biosafety level three containment. Closely related nonpathogenic Burkholderia can be investigated under less stringent biosafety level two containment, and we hypothesized that they could be used as model organisms for developing therapies that would also be effective against B. pseudomallei. We used microbroth dilution assays to compare drug susceptibility profiles of three B. pseudomallei strains and five nonpathogenic Burkholderia strains. Burkholderia humptydooensis, Burkholderia thailandensis, and Burkholderia territorii had similar susceptibility profiles to pathogenic B. pseudomallei that support their potential as safer in vitro models for developing new melioidosis therapies.

Dietary antioxidant seleno-L-methionine protects macrophages infected with Burkholderia thailandensis.

Burkholderia pseudomallei is a facultative intracellular pathogen and the causative agent of melioidosis, a potentially life-threatening disease endemic in Southeast Asia and Northern Australia. Treatment of melioidosis is a long and costly process and the pathogen is inherently resistant to several classes of antibiotics, therefore there is a need for new treatments that can help combat the pathogen. Previous work has shown that the combination of interferon-gamma, an immune system activator, and the antibiotic ceftazidime synergistically reduced the bacterial burden of RAW 264.7 macrophages that had been infected with either B. pseudomallei or Burkholderia thailandensis. The mechanism of the interaction was found to be partially dependent on interferon-gamma-induced production of reactive oxygen species inside the macrophages. To further confirm the role of reactive oxygen species in the effectiveness of the combination treatment, we investigated the impact of the antioxidant and reactive oxygen species scavenger, seleno-L-methionine, on intracellular and extracellular bacterial burden of the infected macrophages. In a dose-dependent manner, high concentrations of seleno-L-methionine (1000 µM) were protective towards infected macrophages, resulting in a reduction of bacteria, on its own, that exceeded the reduction caused by the antibiotic alone and rivaled the effect of ceftazidime and interferon-gamma combined. Seleno-L-methionine treatment also resulted in improved viability of infected macrophages compared to untreated controls. We show that the protective effect of seleno-L-methionine was partly due to its inhibition of bacterial growth. In summary, our study shows a role for high dose seleno-L-methionine to protect and treat macrophages infected with B. thailandensis.

Cloning of a novel topramezone-resistant 4-hydroxyphenylpyruvate dioxygenase gene and improvement of its resistance through pressure acclimation.

Topramezone is a new 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide that is widely used on corn to control annual grass weeds and broadleaf weeds. Due to its broad-spectrum weed control capacity, improved activity, excellent crop selectivity, low mammalian toxicity and high environmental safety, topramezone is considered an ideal target herbicide for transgenic engineering of herbicide tolerance. In this study, a topramezone-resistant strain, Burkholderia sp. BW-1, was screened from soil, and a novel topramezone-resistant HPPD gene (Bkhppd) was cloned from this strain. Purified BkHPPD displayed relatively high HPPD activity and topramezone resistance with a half-maximal inhibitory concentration (IC50) of 572.0 nM. Two BkHPPD mutants designated as BkHPPDt31 and BkHPPDt76 were screened through pressure acclimation. BkHPPDt31 contained three amino acid substitutions (H65D, N160 T and N258S), whereas BkHPPDt76 contained four amino acid substitutions (H65D, N160 T, N258S and N343 T). The topramezone IC50 values of BkHPPDt31 and BkHPPDt76 were 1.1- and 2.3-fold higher, respectively, than that of wild-type BkHPPD. In addition, site-directed mutagenesis indicated that the increased resistance conferred by BkHPPDt31 resulted from the synergistic effects of the three site mutations rather than a single site mutation, and that substitution of asparagine 343 with threonine significantly decreased catalytic efficiency and affinity but increased topramezone resistance. In summary, this study provides a novel topramezone-resistant HPPD gene for the engineering of genetically modified herbicide-resistant crops and facilitates further elucidation of the resistance mechanism of BkHPPD and improvement of resistance through directed evolution.

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.