A Burkholderia cenocepacia-like environmental isolate strongly inhibits the plant fungal pathogen Zymoseptoria tritici.

Fungal phytopathogens cause significant reductions in agricultural yields annually, and overusing chemical fungicides for their control leads to environmental pollution and the emergence of resistant pathogens. Exploring natural isolates with strong antagonistic effects against pathogens can improve our understanding of their ecology and develop new treatments for the future. We isolated and characterized a novel bacterial strain associated with the species Burkholderia cenocepacia, termed APO9, which strongly inhibits Zymoseptoria tritici, a commercially important pathogenic fungus causing Septoria tritici blotch in wheat. Additionally, this strain exhibits inhibitory activity against four other phytopathogens. We found that physical contact plays a crucial role for APO9's antagonistic capacity. Genome sequencing of APO9 and biosynthetic gene cluster (BGC) analysis identified nine classes of BGCs and three types of secretion systems (types II, III, and IV), which may be involved in the inhibition of Z. tritici and other pathogens. To identify genes driving APO9's inhibitory activity, we screened a library containing 1,602 transposon mutants and identified five genes whose inactivation reduced inhibition efficiency. One such gene encodes for a diaminopimelate decarboxylase located in a terpenoid biosynthesis gene cluster. Phylogenetic analysis revealed that while some of these genes are also found across the Burkholderia genus, as well as in other Betaproteobacteria, the combination of these genes is unique to the Burkholderia cepacia complex. These findings suggest that the inhibitory capacity of APO9 is complex and not limited to a single mechanism, and may play a role in the interaction between various Burkholderia species and various phytopathogens within diverse plant ecosystems. IMPORTANCE: The detrimental effects of fungal pathogens on crop yields are substantial. The overuse of chemical fungicides contributes not only to environmental pollution but also to the emergence of resistant pathogens. Investigating natural isolates with strong antagonistic effects against pathogens can improve our understanding of their ecology and develop new treatments for the future. We discovered and examined a unique bacterial strain that demonstrates significant inhibitory activity against several phytopathogens. Our research demonstrates that this strain has a wide spectrum of inhibitory actions against plant pathogens, functioning through a complex mechanism. This plays a vital role in the interactions between plant microbiota and phytopathogens.

Characterization of Volatile Organic Compounds Emitted from Endophytic Burkholderia cenocepacia ETR-B22 by SPME-GC-MS and Their Inhibitory Activity against Various Plant Fungal Pathogens.

The use of antagonistic microorganisms and their volatile organic compounds (VOCs) to control plant fungal pathogens is an eco-friendly and promising substitute for chemical fungicides. In this work, endophytic bacterium ETR-B22, isolated from the root of Sophora tonkinensis Gagnep., was found to exhibit strong antagonistic activity against 12 fungal pathogens found in agriculture. Strain ETR-B22 was identified as Burkholderia cenocepacia based on 16S rRNA and recA sequences. We evaluated the antifungal activity of VOCs emitted by ETR-B22. The VOCs from strain ETR-B22 also showed broad-spectrum antifungal activity against 12 fungal pathogens. The composition of the volatile profiles was analyzed based on headspace solid phase microextraction (HS-SPME) gas chromatography coupled to mass spectrometry (GC-MS). Different extraction strategies for the SPME process significantly affected the extraction efficiency of the VOCs. Thirty-two different VOCs were identified. Among the VOC of ETR-B22, dimethyl trisulfide, indole, methyl anthranilate, methyl salicylate, methyl benzoate, benzyl propionate, benzyl acetate, 3,5-di-tert-butylphenol, allyl benzyl ether and nonanoic acid showed broad-spectrum antifungal activity, and are key inhibitory compounds produced by strain ETR-B22 against various fungal pathogens. Our results suggest that the endophytic strain ETR-B22 and its VOCs have high potential for use as biological controls of plant fungal pathogens.

Various Evolutionary Trajectories Lead to Loss of the Tobramycin-Potentiating Activity of the Quorum-Sensing Inhibitor Baicalin Hydrate in Burkholderia cenocepacia Biofilms.

Combining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant bacteria. As potentiators do not interfere with essential processes, it has been hypothesized that they are less likely to induce resistance. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop reduced susceptibility toward one such adjuvant, baicalin hydrate (BH). Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to TOB and TOB+BH compared to the start population, and the potentiating effect of BH toward TOB was lost. Whole-genome sequencing was performed to probe which changes were involved in the reduced effect of BH, and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter). No changes in the MIC or MBC of TOB or changes in the number of persister cells were observed. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that B. cenocepacia J2315 biofilms rapidly lose susceptibility toward the antibiotic-potentiating activity of BH and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as mechanisms.

Disruption of Quorum Sensing and Virulence in Burkholderia cenocepacia by a Structural Analogue of the cis-2-Dodecenoic Acid Signal.

Quorum sensing (QS) signals are widely used by bacterial pathogens to control biological functions and virulence in response to changes in cell population densities. Burkholderia cenocepacia employs a molecular mechanism in which the cis-2-dodecenoic acid (named Burkholderiadiffusible signal factor [BDSF]) QS system regulates N-acyl homoserine lactone (AHL) signal production and virulence by modulating intracellular levels of cyclic diguanosine monophosphate (c-di-GMP). Thus, inhibition of BDSF signaling may offer a non-antibiotic-based therapeutic strategy against BDSF-regulated bacterial infections. In this study, we report the synthesis of small-molecule mimics of the BDSF signal and evaluate their ability to inhibit BDSF QS signaling in B. cenocepacia A novel structural analogue of BDSF, 14-Me-C16:Δ2 (cis-14-methylpentadec-2-enoic acid), was observed to inhibit BDSF production and impair BDSF-regulated phenotypes in B. cenocepacia, including motility, biofilm formation, and virulence, while it did not inhibit the growth rate of this pathogen. 14-Me-C16:Δ2 also reduced AHL signal production. Genetic and biochemical analyses showed that 14-Me-C16:Δ2 inhibited the production of the BDSF and AHL signals by decreasing the expression of their synthase-encoding genes. Notably, 14-Me-C16:Δ2 attenuated BDSF-regulated phenotypes in various Burkholderia species. These findings suggest that 14-Me-C16:Δ2 could potentially be developed as a new therapeutic agent against pathogenic Burkholderia species by interfering with their QS signaling.IMPORTANCEBurkholderia cenocepacia is an important opportunistic pathogen which can cause life-threatening infections in susceptible individuals, particularly in cystic fibrosis and immunocompromised patients. It usually employs two types of quorum sensing (QS) systems, including the cis-2-dodecenoic acid (BDSF) system and N-acyl homoserine lactone (AHL) system, to regulate virulence. In this study, we have designed and identified an unsaturated fatty acid compound (cis-14-methylpentadec-2-enoic acid [14-Me-C16:Δ2]) that is capable of interfering with B. cenocepacia QS signaling and virulence. We demonstrate that 14-Me-C16:Δ2 reduced BDSF and AHL signal production in B. cenocepacia It also impaired QS-regulated phenotypes in various Burkholderia species. These results suggest that 14-Me-C16:Δ2 could interfere with QS signaling in many Burkholderia species and might be developed as a new antibacterial agent.

Competitive Fitness of Essential Gene Knockdowns Reveals a Broad-Spectrum Antibacterial Inhibitor of the Cell Division Protein FtsZ.

To streamline the elucidation of antibacterial compounds' mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing. Here, we used Illumina sequencing of transposon insertions to track the competitive fitness of a Burkholderia cenocepacia library containing essential gene knockdowns. Using this method, we characterized a novel benzothiadiazole derivative, 10126109 (C109), with antibacterial activity against B. cenocepacia, for which whole-genome sequencing of low-frequency spontaneous drug-resistant mutants had failed to identify the drug target. By combining the identification of hypersusceptible mutants and morphology screening, we show that C109 targets cell division. Furthermore, fluorescence microscopy of bacteria harboring green fluorescent protein (GFP) cell division protein fusions revealed that C109 prevents divisome formation by altering the localization of the essential cell division protein FtsZ. In agreement with this, C109 inhibited both the GTPase and polymerization activities of purified B. cenocepacia FtsZ. C109 displayed antibacterial activity against Gram-positive and Gram-negative cystic fibrosis pathogens, including Mycobacterium abscessus C109 effectively cleared B. cenocepacia infection in the Caenorhabditis elegans model and exhibited additive interactions with clinically relevant antibiotics. Hence, C109 is an enticing candidate for further drug development.

Targeting the Nonmevalonate Pathway in Burkholderia cenocepacia Increases Susceptibility to Certain ß-Lactam Antibiotics.

The nonmevalonate pathway is the sole pathway for isoprenoid biosynthesis in Burkholderia cenocepacia and is possibly a novel target for the development of antibacterial chemotherapy. The goals of the present study were to evaluate the essentiality of dxr, the second gene of the nonmevalonate pathway, in B. cenocepacia and to determine whether interfering with the nonmevalonate pathway increases susceptibility toward antibiotics. To this end, a rhamnose-inducible conditional dxr knockdown mutant of B. cenocepacia strain K56-2 (B. cenocepacia K56-2dxr) was constructed, using a plasmid which enables the delivery of a rhamnose-inducible promoter in the chromosome. Expression of dxr is essential for bacterial growth; the growth defect observed in the dxr mutant could be complemented by expressing dxr in trans under the control of a constitutive promoter, but not by providing 2-C-methyl-d-erythritol-4-phosphate, the reaction product of DXR (1-deoxy-d-xylulose 5-phosphate reductoisomerase). B. cenocepacia K56-2dxr showed markedly increased susceptibility to the ß-lactam antibiotics aztreonam, ceftazidime, and cefotaxime, while susceptibility to other antibiotics was not (or was much less) affected; this increased susceptibility could also be complemented by in trans expression of dxr A similarly increased susceptibility was observed when antibiotics were combined with FR900098, a known DXR inhibitor. Our data confirm that the nonmevalonate pathway is essential in B. cenocepacia and suggest that combining potent DXR inhibitors with selected ß-lactam antibiotics is a useful strategy to combat B. cenocepacia infections.

Competitive Growth Enhances Conditional Growth Mutant Sensitivity to Antibiotics and Exposes a Two-Component System as an Emerging Antibacterial Target in Burkholderia cenocepacia.

Chemogenetic approaches to profile an antibiotic mode of action are based on detecting differential sensitivities of engineered bacterial strains in which the antibacterial target (usually encoded by an essential gene) or an associated process is regulated. We previously developed an essential-gene knockdown mutant library in the multidrug-resistant Burkholderia cenocepacia by transposon delivery of a rhamnose-inducible promoter. In this work, we used Illumina sequencing of multiplex-PCR-amplified transposon junctions to track individual mutants during pooled growth in the presence of antibiotics. We found that competition from nontarget mutants magnified the hypersensitivity of a clone underexpressing gyrB to novobiocin by 8-fold compared with hypersensitivity measured during clonal growth. Additional profiling of various antibiotics against a pilot library representing most categories of essential genes revealed a two-component system with unknown function, which, upon depletion of the response regulator, sensitized B. cenocepacia to novobiocin, ciprofloxacin, tetracycline, chloramphenicol, kanamycin, meropenem, and carbonyl cyanide 3-chlorophenylhydrazone, but not to colistin, hydrogen peroxide, and dimethyl sulfoxide. We named the gene cluster esaSR for enhanced sensitivity to antibiotics sensor and response regulator. Mutational analysis and efflux activity assays revealed that while esaS is not essential and is involved in antibiotic-induced efflux, esaR is an essential gene and regulates efflux independently of antibiotic-mediated induction. Furthermore, microscopic analysis of cells stained with propidium iodide provided evidence that depletion of EsaR has a profound effect on the integrity of cell membranes. In summary, we unraveled a previously uncharacterized two-component system that can be targeted to reduce antibiotic resistance in B. cenocepacia.

Synthetic cystic fibrosis sputum medium diminishes Burkholderia cenocepacia antifungal activity against Aspergillus fumigatus independently of phenylacetic acid production.

Phenylacetic acid (PAA), an intermediate of phenylalanine degradation, is emerging as a signal molecule in microbial interactions with the host. In this work, we explore the presence of phenylalanine and PAA catabolism in 3 microbial pathogens of the cystic fibrosis (CF) lung microbiome: Pseudomonas aeruginosa, Burkholderia cenocepacia, and Aspergillus fumigatus. While in silico analysis of B. cenocepacia J2315 and A. fumigatus Af293 genome sequences showed complete pathways from phenylalanine to PAA, the P. aeruginosa PAO1 genome lacked several coding genes for phenylalanine and PAA catabolic enzymes. High-performance liquid chromatography analysis of supernatants from B. cenocepacia K56-2 detected PAA when grown in Luria-Bertani medium but not in synthetic cystic fibrosis sputum medium (SCFM). However, we were unable to identify PAA production by A. fumigatus or P. aeruginosa in any of the conditions tested. The inhibitory effect of B. cenocepacia on A. fumigatus growth was evaluated using agar plate interaction assays. Inhibition of fungal growth by B. cenocepacia was lessened in SCFM but this effect was not dependent on bacterial production of PAA. In summary, while we demonstrated PAA production by B. cenocepacia, we were not able to link this metabolite with the B. cenocepacia - A. fumigatus microbial interaction in CF nutritional conditions.

Structural Features Essential to the Antimicrobial Functions of Human SPLUNC1.

SPLUNC1 is an abundantly secreted innate immune protein in the mammalian respiratory tract that exerts bacteriostatic and antibiofilm effects, binds to lipopolysaccharide (LPS), and acts as a fluid-spreading surfactant. Here, we unravel the structural elements essential for the surfactant and antimicrobial functions of human SPLUNC1 (short palate lung nasal epithelial clone 1). A unique α-helix (α4) that extends from the body of SPLUNC1 is required for the bacteriostatic, surfactant, and LPS binding activities of this protein. Indeed, we find that mutation of just four leucine residues within this helical motif to alanine is sufficient to significantly inhibit the fluid spreading abilities of SPLUNC1, as well as its bacteriostatic actions against Gram-negative pathogens Burkholderia cenocepacia and Pseudomonas aeruginosa. Conformational flexibility in the body of SPLUNC1 is also involved in the bacteriostatic, surfactant, and LPS binding functions of the protein as revealed by disulfide mutants introduced into SPLUNC1. In addition, SPLUNC1 exerts antibiofilm effects against Gram-negative bacteria, although α4 is not involved in this activity. Interestingly, though, the introduction of surface electrostatic mutations away from α4 based on the unique dolphin SPLUNC1 sequence, and confirmed by crystal structure, is shown to impart antibiofilm activity against Staphylococcus aureus, the first SPLUNC1-dependent effect against a Gram-positive bacterium reported to date. Together, these data pinpoint SPLUNC1 structural motifs required for the antimicrobial and surfactant actions of this protective human protein.

Intra- and Interspecies Effects of Outer Membrane Vesicles from Stenotrophomonas maltophilia on ß-Lactam Resistance.

The treatment ofStenotrophomonas maltophiliainfection with ß-lactam antibiotics leads to increased release of outer membrane vesicles (OMVs), which are packed with two chromosomally encoded ß-lactamases. Here, we show that these ß-lactamase-packed OMVs are capable of establishing extracellular ß-lactam degradation. We also show that they dramatically increase the apparent MICs of imipenem and ticarcillin for the cohabituating speciesPseudomonas aeruginosaandBurkholderia cenocepacia.

Clinafloxacin for Treatment of Burkholderia cenocepacia Infection in a Cystic Fibrosis Patient.

Respiratory infection with Burkholderia cenocepacia is associated with accelerated decline in lung function and increased mortality in cystic fibrosis (CF) patients (A. M. Jones, M. E. Dodd, J. R. W. Govan, V. Barcus, C. J. Doherty, J. Morris, and A. K. Webb, Thorax 59:948-951, 2004, http://dx.doi.org/10.1136/thx.2003.017210). B. cenocepacia often possesses innate resistance to multiple antimicrobial classes, making eradication uncommon in established infection (P. B. Davis, Am J Respir Crit Care Med 173:475-482, 2006, http://dx.doi.org/10.1164/rccm.200505-840OE). We report the use of clinafloxacin in a CF patient with advanced B. cenocepacia infection, present pharmacokinetic (PK) data, and discuss the potential therapeutic role of clinafloxacin in patients with this condition.

Broad-spectrum anti-biofilm peptide that targets a cellular stress response.

Bacteria form multicellular communities known as biofilms that cause two thirds of all infections and demonstrate a 10 to 1000 fold increase in adaptive resistance to conventional antibiotics. Currently, there are no approved drugs that specifically target bacterial biofilms. Here we identified a potent anti-biofilm peptide 1018 that worked by blocking (p)ppGpp, an important signal in biofilm development. At concentrations that did not affect planktonic growth, peptide treatment completely prevented biofilm formation and led to the eradication of mature biofilms in representative strains of both Gram-negative and Gram-positive bacterial pathogens including Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus, Salmonella Typhimurium and Burkholderia cenocepacia. Low levels of the peptide led to biofilm dispersal, while higher doses triggered biofilm cell death. We hypothesized that the peptide acted to inhibit a common stress response in target species, and that the stringent response, mediating (p)ppGpp synthesis through the enzymes RelA and SpoT, was targeted. Consistent with this, increasing (p)ppGpp synthesis by addition of serine hydroxamate or over-expression of relA led to reduced susceptibility to the peptide. Furthermore, relA and spoT mutations blocking production of (p)ppGpp replicated the effects of the peptide, leading to a reduction of biofilm formation in the four tested target species. Also, eliminating (p)ppGpp expression after two days of biofilm growth by removal of arabinose from a strain expressing relA behind an arabinose-inducible promoter, reciprocated the effect of peptide added at the same time, leading to loss of biofilm. NMR and chromatography studies showed that the peptide acted on cells to cause degradation of (p)ppGpp within 30 minutes, and in vitro directly interacted with ppGpp. We thus propose that 1018 targets (p)ppGpp and marks it for degradation in cells. Targeting (p)ppGpp represents a new approach against biofilm-related drug resistance.

A novel method for investigating Burkholderia cenocepacia infections in patients with cystic fibrosis and other chronic diseases of the airways.

BACKGROUND: Burkholderia cenocepacia is a Gram-negative, opportunistic pathogen that is a cause of morbidity and mortality in patients with cystic fibrosis (CF). Research efforts over the past few decades contributed to our understanding of these infections by identifying virulence factors. However, little is known about how this pathogen adapts to the harsh environment found inside the CF airways, which is characterized by a unique mucus containing high concentrations of inflammatory markers. The current study developed a novel model to further investigate this phenomenon. RESULTS: Monolayers of human A549 lung carcinoma cells (HLCCs) were exposed to a mixture of artificial CF sputum medium (ASMDM) in tissue culture growth medium, and subsequently infected with B. cenocepacia K56-2 for 24 h. The data showed that this model supported B. cenocepacia growth. In addition, consistent with similar studies using current models such as CF airway tissue samples, HLCC viability was reduced by more than 70 % when grown in 60 % ASMDM and infected with B. cenocepacia compared to mock-infected controls and medium alone. Furthermore, the amount of B. cenocepacia cells associated with the HLCC monolayer was more than 10 times greater in 60 % ASMDM when compared to medium controls. CONCLUSIONS: These findings suggest that HLCC monolayers in 60 % ASMDM serve as a valid alternative to study B. cenocepacia infections in patients with CF, and possibly other chronic diseases of the airways. Furthermore, the results obtained in this study suggest an important role for CF sputum in B. cenocepacia pathogenesis.

Cell Wall Recycling-Linked Coregulation of AmpC and PenB ß-Lactamases through ampD Mutations in Burkholderia cenocepacia.

In many Gram-negative pathogens, mutations in the key cell wall-recycling enzyme AmpD (N-acetyl-anhydromuramyl-L-alanine amidase) affect the activity of the regulator AmpR, which leads to the expression of AmpC ß-lactamase, conferring resistance to expanded-spectrum cephalosporin antibiotics. Burkholderia cepacia complex (Bcc) species also have these Amp homologs; however, the regulatory circuitry and the nature of causal ampD mutations remain to be explored. A total of 92 ampD mutants were obtained, representing four types of mutations: single nucleotide substitution (causing an amino acid substitution or antitermination of the enzyme), duplication, deletion, and IS element insertion. Duplication, which can go through reversion, was the most frequent type. Intriguingly, mutations in ampD led to the induction of two ß-lactamases, AmpC and PenB. Coregulation of AmpC and PenB in B. cenocepacia, and likely also in many Bcc species with the same gene organization, poses a serious threat to human health. This resistance mechanism is of evolutionary optimization in that ampD is highly prone to mutations allowing rapid response to antibiotic challenge, and many of the mutations are reversible in order to resume cell wall recycling when the antibiotic challenge is relieved.

Pseudomonas aeruginosa displays an altered phenotype in vitro when grown in the presence of mannitol.

D-mannitol has been approved in dry powder formulation as an effective antimucolytic agent in patients with cystic fibrosis. What is not known is the effect of adding a metabolisable sugar on the biology of chronic bacterial pathogens in the CF lung. Therefore, a series of simple in vitro experiments were performed to examine the effect of adding D-mannitol on the phenotype of the CF respiratory pathogens Pseudomonas aeruginosa and Burkholderia cenocepacia. Clinical isolates (n = 86) consisting of P. aeruginosa (n = 51), B. cenocepacia (n = 26), P. putida (n = 4), Stenotrophomonas maltophila (n = 3) and Pseudomonas spp. (n = 2) were examined by supplementing basal nutrient agar with varying concentrations of D-mannitol (0-20% [w/v]) and subsequently examining for any change in microbial phenotype. The effect of supplementation with mannitol was four-fold, namely i) To increase the proliferation and increase in cell density of all CF organisms examined, with an optimal concentration of 2-4% (w/v) D-mannitol. No such increase in cell proliferation was observed when mannitol was substituted with sodium chloride. ii) Enhanced pigment production was observed in 2/51 (3.9%) of the P. aeruginosa isolates examined, in one of the P. putida isolates, and in 3/26 (11.5%) of the B. cenocepacia isolates examined. iii). When examined at 4.0% (w/v) supplementation with mannitol, 11/51 (21.6%) P. aeruginosa isolates and 3/26 (11.5%) B. cenocepacia isolates were seen to exhibit the altered adhesion phenotype. iv). With respect to the altered mucoid phenotype, 5/51 (9.8%) P. aeruginosa produced this phenotype when grown at 4% mannitol. Mucoid production was greatest at 4%, was poor at 10% and absent at 20% (w/v) mannitol. The altered mucoid phenotype was not observed in the B. cenocepacia isolates or any of the other clinical taxa examined. Due consideration therefore needs to be given, where there is altered physiology within the small airways, leading to a potentially altered biological state of the colonising microorganisms in novel inhaled pharmaceutical interventions in CF, particularly those, which are not designated as antimicrobial agents.

Mechanism of resistance to an antitubercular 2-thiopyridine derivative that is also active against Burkholderia cenocepacia.

The discovery of new compounds that are able to inhibit the growth of Burkholderia cenocepacia is of primary importance for cystic fibrosis patients. Here, the mechanism of resistance to a new pyridine derivative already shown to be effective against Mycobacterium tuberculosis and to have good activity toward B. cenocepacia was investigated. Increased expression of a resistance-nodulation-cell division (RND) efflux system was detected in the resistant mutants, thus confirming their important roles in B. cenocepacia antibiotic resistance.

Putrescine reduces antibiotic-induced oxidative stress as a mechanism of modulation of antibiotic resistance in Burkholderia cenocepacia.

Communication of antibiotic resistance among bacteria via small molecules is implicated in transient reduction of bacterial susceptibility to antibiotics, which could lead to therapeutic failures aggravating the problem of antibiotic resistance. Released putrescine from the extremely antibiotic-resistant bacterium Burkholderia cenocepacia protects less-resistant cells from different species against the antimicrobial peptide polymyxin B (PmB). Exposure of B. cenocepacia to sublethal concentrations of PmB and other bactericidal antibiotics induces reactive oxygen species (ROS) production and expression of the oxidative stress response regulator OxyR. We evaluated whether putrescine alleviates antibiotic-induced oxidative stress. The accumulation of intracellular ROS, such as superoxide ion and hydrogen peroxide, was assessed fluorometrically with dichlorofluorescein diacetate, while the expression of OxyR and putrescine synthesis enzymes was determined in luciferase assays using chromosomal promoter-lux reporter system fusions. We evaluated wild-type and isogenic deletion mutant strains with defects in putrescine biosynthesis after exposure to sublethal concentrations of PmB and other bactericidal antibiotics. Exogenous putrescine protected against oxidative stress induced by PmB and other antibiotics, whereas reduced putrescine synthesis resulted in increased ROS generation and a parallel increased sensitivity to PmB. Of the 3 B. cenocepacia putrescine-synthesizing enzymes, PmB induced only BCAL2641, an ornithine decarboxylase. This study reveals BCAL2641 as a critical component of the putrescine-mediated communication of antibiotic resistance and as a plausible target for designing inhibitors that would block the communication of such resistance among different bacteria, ultimately reducing the window of therapeutic failure in treating bacterial infections.

Differential roles of RND efflux pumps in antimicrobial drug resistance of sessile and planktonic Burkholderia cenocepacia cells.

Burkholderia cenocepacia is notorious for causing respiratory tract infections in people with cystic fibrosis. Infections with this organism are particularly difficult to treat due to its high level of intrinsic resistance to most antibiotics. Multidrug resistance in B. cenocepacia can be ascribed to different mechanisms, including the activity of efflux pumps and biofilm formation. In the present study, the effects of deletion of the 16 operons encoding resistance-nodulation-cell division (RND)-type efflux pumps in B. cenocepacia strain J2315 were investigated by determining the MICs of various antibiotics and by investigating the antibiofilm effect of these antibiotics. Finally, the expression levels of selected RND genes in treated and untreated cultures were investigated using reverse transcriptase quantitative PCR (RT-qPCR). Our data indicate that the RND-3 and RND-4 efflux pumps are important for resistance to various antimicrobial drugs (including tobramycin and ciprofloxacin) in planktonic B. cenocepacia J2315 populations, while the RND-3, RND-8, and RND-9 efflux systems protect biofilm-grown cells against tobramycin. The RND-8 and RND-9 efflux pumps are not involved in ciprofloxacin resistance. Results from the RT-qPCR experiments on the wild-type strain B. cenocepacia J2315 suggest that there is little regulation at the level of mRNA expression for these efflux pumps under the conditions tested.

Pore-forming pyocin S5 utilizes the FptA ferripyochelin receptor to kill Pseudomonas aeruginosa.

Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to define the receptor-binding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other S-type pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain.

Deciphering the role of Burkholderia cenocepacia membrane proteins in antimicrobial properties of chitosan.

Chitosan, a versatile derivative of chitin, is widely used as an antimicrobial agent either alone or mixed with other natural polymers. Burkholderia cenocepacia is a multidrug-resistant bacteria and difficult to eradicate. Our previous studies shown that chitosan had strong antibacterial activity against B. cenocepacia. In the current study, we have investigated the molecular aspects for the susceptibility of B. cenocepacia in response to chitosan antibacterial activity. We have conducted RNA expression analysis of drug efflux system by RT-PCR, membrane protein profiling by SDS-PAGE, and by LC-MS/MS analysis following the validation of selected membrane proteins by real-time PCR analysis. By RT-PCR analysis, it was found that orf3, orf9, and orf13 were expressed at detectable levels, which were similar to control, while rest of the orf did not express. Moreover, shotgun proteomics analysis revealed 21 proteins in chitosan-treated cells and 16 proteins in control. Among them 4 proteins were detected as shared proteins under control and chitosan-treated cells and 17 proteins as uniquely identified proteins under chitosan-treated cells. Among the catalog of uniquely identified proteins, there were proteins involved in electron transport chain and ATP synthase, metabolism of carbohydrates and adaptation to atypical conditions proteins which indicate that utilization and pattern of chitosan is diverse which might be responsible for its antibacterial effects on bacteria. Moreover, our results showed that RND drug efflux system, which display the ability to transport a variety of structurally unrelated drugs from a cell and consequently are capable of conferring resistance to a diverse range of chemotherapeutic agents, was not determined to play its role in response to chitosan. It might be lipopolysaccharides interaction with chitosan resulted in the destabilization of membrane protein to membrane lyses to cell death. Membrane proteome analysis were also validated by RT-qPCR analysis, which corroborated our results that of membrane proteins.