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.

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.

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.

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.

NMR-based metabolic profiling to follow the production of anti-phytopathogenic compounds in the culture of the marine strain Streptomyces sp. PNM-9.

Actinobacteria are the major source of bioactive secondary metabolites and are featured in the search for antimicrobial compounds. We used nuclear magnetic resonance (RMN)-metabolic profiling and multivariate data analysis (MVDA) to correlate the metabolites' production of Streptomyces sp. PNM-9 from the algae Dictyota sp. and their biological activity against the rice phytopathogenic bacteria Burkholderia spp. The compounds 2-methyl-N-(2'-phenylethyl)-butanamide (1) and 3-methyl-N-(2'-phenylethyl)-butanamide (2) were identified through MVDA and 2D NMR experiments in the organic extract of a 15-days LB media culture of Streptomyces sp. PNM-9. Compounds 1 and 2 were isolated and their structures confirmed by one- and two-dimensional NMR and mass spectrometry (MS) data. Compounds 1 and 2 were active against the rice pathogenic bacteria Burkholderia glumae (ATCC 33,617) displaying minimal inhibitory concentration (MIC) values of 2.43 mM and 1.21 mM, respectively. The metabolomics-guided approach employing NMR-metabolic profiling was useful for marine microbial bioprospecting and suggested Streptomyces sp. PNM-9 strain and its compounds as a potential control against phytopathogenic bacteria.

Profiling structural diversity and activity of 2-alkyl-4(1H)-quinolone N-oxides of Pseudomonas and Burkholderia.

We synthesized all major saturated and unsaturated 2-alkyl-4(1H)-quinolone N-oxides of Pseudomonas and Burkholderia, quantified their native production levels and characterized their antibiotic activities against competing Staphylococcus aureus. We demonstrate that quinolone core methylation and position of unsaturation in the alkyl-chain dictate antibiotic potency which supports the proposed mechanism of action.

Reciprocal antibiotic collateral sensitivity in Burkholderia multivorans.

Antibiotic collateral sensitivity (CS) occurs when a bacterium that acquires resistance to a treatment drug exhibits decreased resistance to a different drug. Here we identify reciprocal CS networks and candidate genes in Burkholderia multivorans. Burkholderia multivorans was evolved to become resistant to each of six antibiotics. The antibiogram of the evolved strain was compared with the immediate parental strain to determine CS and cross-resistance. The evolution process was continued for each resistant strain. CS interactions were observed in 170 of 279 evolved strains. CS patterns grouped into two clusters based on the treatment drug being a ß-lactam antibiotic or not. Reciprocal pairs of CS antibiotics arose in ≥25% of all evolved strains. A total of 68 evolved strains were subjected to whole-genome sequencing and the resulting mutation patterns were correlated with antibiograms. Analysis revealed there was no single gene responsible for CS and that CS seen in B. multivorans is likely due to a combination of specific and non-specific mutations. The frequency of reciprocal CS, and the degree to which resistance changed, suggests a long-term treatment strategy; when resistance to one drug occurs, switch to use of the other member of the reciprocal pair. This switching could theoretically be continued indefinitely, allowing life-long treatment of chronic infections with just two antibiotics.

Burkholderia ubonensis Meropenem Resistance: Insights into Distinct Properties of Class A ß-Lactamases in Burkholderia cepacia Complex and Burkholderia pseudomallei Complex Bacteria.

Burkholderia pseudomallei, the founding member of the B. pseudomallei complex (Bpc), is a biothreat agent and causes melioidosis, a disease whose treatment mainly relies on ceftazidime and meropenem. The concern is that B. pseudomallei could enhance its drug resistance repertoire by the acquisition of DNA from resistant near-neighbor species. Burkholderia ubonensis, a member of the B. cepacia complex (Bcc), is commonly coisolated from environments where B. pseudomallei is present. Unlike B. pseudomallei, in which significant primary carbapenem resistance is rare, it is not uncommon in B. ubonensis, but the underlying mechanisms are unknown. We established that carbapenem resistance in B. ubonensis is due to an inducible class A PenB ß-lactamase, as has been shown for other Bcc bacteria. Inducibility is not sufficient for high-level resistance but also requires other determinants, such as a PenB that is more robust than that present in susceptible isolates, as well as other resistance factors. Curiously and diagnostic for the two complexes, both Bpc and Bcc bacteria contain distinct annotated PenA class A ß-lactamases. However, the protein from Bcc bacteria is missing its essential active-site serine and, therefore, is not a ß-lactamase. Regulated expression of a transcriptional penB'-lacZ (ß-galactosidase) fusion in the B. pseudomallei surrogate B. thailandensis confirms that although Bpc bacteria lack an inducible ß-lactamase, they contain the components required for responding to aberrant peptidoglycan synthesis resulting from ß-lactam challenge. Understanding the diversity of antimicrobial resistance in Burkholderia species is informative about how the challenges arising from potential resistance transfer between them can be met.IMPORTANCEBurkholderia pseudomallei causes melioidosis, a tropical disease that is highly fatal if not properly treated. Our data show that, in contrast to B. pseudomallei, B. ubonensis ß-lactam resistance is fundamentally different because intrinsic resistance is mediated by an inducible class A ß-lactamase. This includes resistance to carbapenems. Our work demonstrates that studies with near-neighbor species are informative about the diversity of antimicrobial resistance in Burkholderia and can also provide clues about the potential of resistance transfer between bacteria inhabiting the same environment. Knowledge about potential adverse challenges resulting from the horizontal transfer of resistance genes between members of the two complexes enables the design of effective countermeasures.

Differences in Cystic Fibrosis-Associated Burkholderia spp. Bacteria Metabolomes after Exposure to the Antibiotic Trimethoprim.

The Burkholderia cepacia complex is a group of closely related bacterial species with large genomes that infect immunocompromised individuals and those living with cystic fibrosis. Some of these species are found more frequently and cause more severe disease than others, yet metabolomic differences between these have not been described. Furthermore, our understanding of how these species respond to antibiotics is limited. We investigated the metabolomics differences between three most prevalent Burkholderia spp. associated with cystic fibrosis: B. cenocepacia, B. multivorans, and B. dolosa in the presence and absence of the antibiotic trimethoprim. Using a combination of supervised and unsupervised metabolomics data visualization and analysis tools, we describe the overall differences between strains of the same species and between species. Specifically, we report, for the first time, the role of the pyomelanin pathway in the metabolism of trimethoprim. We also report differences in the detection of known secondary metabolites such as fragin, ornibactin, and N-acylhomoserine lactones and their analogs in closely related strains. Furthermore, we highlight the potential for the discovery of new secondary metabolites in clinical strains of Burkholderia spp. The metabolomics differences described in this study highlight the personalized nature of closely related Burkholderia strains.

Multi-Omic Analyses Provide Links between Low-Dose Antibiotic Treatment and Induction of Secondary Metabolism in Burkholderia thailandensis.

Low doses of antibiotics can trigger secondary metabolite biosynthesis in bacteria, but the underlying mechanisms are generally unknown. We sought to better understand this phenomenon by studying how the antibiotic trimethoprim activates the synthesis of the virulence factor malleilactone in Burkholderia thailandensis Using transcriptomics, quantitative multiplexed proteomics, and primary metabolomics, we systematically mapped the changes induced by trimethoprim. Surprisingly, even subinhibitory doses of the antibiotic resulted in broad transcriptional and translational alterations, with ∼8.5% of the transcriptome and ∼5% of the proteome up- or downregulated >4-fold. Follow-up studies with genetic-biochemical experiments showed that the induction of malleilactone synthesis can be sufficiently explained by the accumulation of methionine biosynthetic precursors, notably homoserine, as a result of inhibition of the folate pathway. Homoserine activated the malleilactone gene cluster via the transcriptional regulator MalR and gave rise to a secondary metabolome which was very similar to that generated by trimethoprim. Our work highlights the expansive changes that low-dose trimethoprim induces on bacterial physiology and provides insights into its stimulatory effect on secondary metabolism.IMPORTANCE The discovery of antibiotics ranks among the most significant accomplishments of the last century. Although the targets of nearly all clinical antibiotics are known, our understanding regarding their natural functions and the effects of subinhibitory concentrations is in its infancy. Stimulatory rather than inhibitory functions have been attributed to low-dose antibiotics. Among these, we previously found that antibiotics activate silent biosynthetic genes and thereby enhance the metabolic output of bacteria. The regulatory circuits underlying this phenomenon are unknown. We take a first step toward elucidating these circuits and show that low doses of trimethoprim (Tmp) have cell-wide effects on the saprophyte Burkholderia thailandensis Most importantly, inhibition of one-carbon metabolic processes by Tmp leads to an accumulation of homoserine, which induces the production of an otherwise silent cytotoxin via a LuxR-type transcriptional regulator. These results provide a starting point for uncovering the molecular basis of the hormetic effects of antibiotics.

Burkholderia multivorans Exhibits Antibiotic Collateral Sensitivity.

Burkholderia multivorans is a member of the Burkholderia cepacia complex whose members are inherently resistant to many antibiotics and can cause chronic lung infections in patients with cystic fibrosis. A possible treatment for chronic infections arises from the existence of collateral sensitivity (CS)-acquired resistance to a treatment antibiotic results in a decreased resistance to a nontreatment antibiotic. Determining CS patterns for bacteria involved in chronic infections may lead to sustainable treatment regimens that reduce development of multidrug-resistant bacterial strains. CS has been found to occur in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Here, we report that B. multivorans exhibits antibiotic CS, as well as cross-resistance (CR), describe CS and CR networks for six antibiotics (ceftazidime, chloramphenicol, levofloxacin, meropenem, minocycline, and trimethoprim-sulfamethoxazole), and identify candidate genes involved in CS. Characterization of CS and CR patterns allows antibiotics to be separated into two clusters based on the treatment drug to which the evolved strain developed primary resistance, suggesting an antibiotic therapy strategy of switching between members of these two clusters.

Influence of pH on the activity of finafloxacin against extracellular and intracellular Burkholderia thailandensis, Yersinia pseudotuberculosis and Francisella philomiragia and on its cellular pharmacokinetics in THP-1 monocytes.

OBJECTIVES: Burkholderia pseudomallei, Yersinia pestis and Francisella tularensis are facultative intracellular bacteria causing life-threatening infections. We have (a) compared the activity of finafloxacin (a fluoroquinolone in development showing improved activity at acidic pH) with that of ciprofloxacin, levofloxacin and imipenem against the extracellular and intracellular (THP-1 monocytes) forms of infection by attenuated surrogates of these species (B. thailandensis, Y. pseudotuberculosis, F. philomiragia) and (b) assessed finafloxacin cellular pharmacokinetics (accumulation, distribution, efflux). METHODS: Bacteria in broth or in infected monocytes were exposed to antibiotics at pH 7.4 or 5.5 for 24 hr. Maximal relative efficacies (Emax) and static concentrations (Cs) were calculated using the Hill equation (concentration-response curves). Finafloxacin pharmacokinetics in cells at pH 7.4 or 5.5 was investigated using 14C-labelled drug. RESULTS: Extracellularly, all drugs sterilized the cultures, with finafloxacin being two to six times more potent at acidic pH. Intracellularly, Emax reached the limit of detection (4-5 log10 cfu decrease) for finafloxacin against all species, but only against B. thailandensis and F. philomiragia for ciprofloxacin and levofloxacin, while imipenem caused less than 2 log10 cfu decrease for all species. At acid pH, Cs shifted to two to five times lower values for finafloxacin and to one to four times higher values for the other drugs. Finafloxacin accumulated in THP-1 cells by approximately fivefold at pH 7.4 but up to 20-fold at pH 5.5, and distributed in the cytosol. CONCLUSIONS: Fluoroquinolones have proven to be effective in reducing the intracellular reservoirs of B. thailandensis, Y. pseudotuberculosis and F. philomiragia, with finafloxacin demonstrating an additional advantage in acidic environments.

Natural variation in the expression and catalytic activity of a naringenin 7-O-methyltransferase influences antifungal defenses in diverse rice cultivars.

Phytoalexins play a pivotal role in plant-pathogen interactions. Whereas leaves of rice (Oryza sativa) cultivar Nipponbare predominantly accumulated the phytoalexin sakuranetin after jasmonic acid induction, only very low amounts accumulated in the Kasalath cultivar. Sakuranetin is synthesized from naringenin by naringenin 7-O-methyltransferase (NOMT). Analysis of chromosome segment substitution lines and backcrossed inbred lines suggested that NOMT is the underlying cause of differential phytoalexin accumulation between Nipponbare and Kasalath. Indeed, both NOMT expression and NOMT enzymatic activity are lower in Kasalath than in Nipponbare. We identified a proline to threonine substitution in Kasalath relative to Nipponbare NOMT as the main cause of the lower enzymatic activity. Expanding this analysis to rice cultivars with varying amounts of sakuranetin collected from around the world showed that NOMT induction is correlated with sakuranetin accumulation. In bioassays with Pyricularia oryzae, Gibberella fujikuroi, Bipolaris oryzae, Burkholderia glumae, Xanthomonas oryzae, Erwinia chrysanthemi, Pseudomonas syringae, and Acidovorax avenae, naringenin was more effective against bacterial pathogens and sakuranetin was more effective against fungal pathogens. Therefore, the relative amounts of naringenin and sakuranetin may provide protection against specific pathogen profiles in different rice-growing environments. In a dendrogram of NOMT genes, those from low-sakuranetin-accumulating cultivars formed at least two clusters, only one of which involves the proline to threonine mutation, suggesting that the low sakuranetin chemotype was acquired more than once in cultivated rice. Strains of the wild rice species Oryza rufipogon also exhibited differential sakuranetin accumulation, indicating that this metabolic diversity predates rice domestication.