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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.

Background Cystic fibrosis (CF) is characterised by chronic respiratory infections, involving opportunistic pathogens, including Burkholderia cenocepacia . The CF lung comprises hypoxic niches that drives bacterial adaptation, and the adaptability of pathogens to this environment is key to their successful colonisation. We previously identified several proteins encoded on a low-oxygen activated (Lxa) locus that were significantly increased in abundance in late chronic infection B. cenocepacia isolates. However, the impact of long-term hypoxia exposure on B. cenocepacia adaptation remains unclear. Results To investigate the role of hypoxia in driving traits associated with chronic infection, we exposed an early infection B. cenocepacia isolate to low (6% O₂) or atmospheric oxygen (21% O₂) over 22 days. By day 22, 364 proteins were significantly increased in abundance in hypoxia-adapted cultures relative to the early infection isolate (Day 0). Overall, 1066 individual proteins were significantly increased in abundance in the hypoxia-adapted cultures relative to normoxia-adapted cultures, across four different timepoints from day 1 to day 22. Comparative proteome analysis identified 81 proteins with consistent changes in abundance both in hypoxia-adapted cultures and the respective late infection isolate relative to the early infection isolate, including lxa -encoded proteins and the FixK transcriptional regulator. Proteins associated with shikimate pathways were also significantly changed in abundance. Importantly, hypoxia-adapted cultures showed increased survival in CF macrophages, increased attachment to CF lung cells, elevated protease activity, greater resistance to ceftazidime and ciprofloxacin, all of which are consistent with adaptations observed in late chronic infection isolates. Hypoxia-adapted cultures also displayed enhanced virulence in Galleria mellonella larvae, as did the late infection isolate. Conclusions The changes in phenotype and proteome of B. cenocepacia observed after long-term hypoxia suggest that hypoxia may drive the adaptation to chronic infection, promoting survival in macrophages, host-cell attachment, antibiotic resistance and protease activity. Therapeutic strategies that modulate oxygen availability or target hypoxia-sensing may hold promise in preventing or mitigating chronic infection in CF.

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.

Burkholderia cenocepacia , an opportunistic pathogen, poses a significant threat to human health, necessitating the discovery of effective quorum sensing inhibitors (QSIs). In this study, the quorum sensing inhibitory effects of deferiprone (DFP) on the B. cenocepacia 162,638 were validated. Notably, DFP demonstrated an ability to inhibit and disrupt bacterial biofilms, reducing biofilm formation by 44.59% at 1/4 MIC (minimum inhibitory concentration) and 24.32% at 1/8 MIC concentrations. The study also investigated DFP’s impact on motility, virulence, and QS signal levels. LC-MS/MS analysis showed a gradual reduction in the QS molecule C6-HSL as DFP concentrations increased. Additionally, DFP’s non-hemolytic properties and safety profile, as verified in Galleria mellonella infection models, highlighted its biocompatibility. RT-qPCR results further indicated that DFP downregulated QS-related gene expression, particularly those involved in ferric uptake regulation protein (Fur). Molecular docking studies identified Fur as a key target for DFP’s inhibitory action. Collectively, DFP was shown as a potential QSI with practical applications for controlling B. cenocepacia infections.

Abstract Bacterial biofilms mediate chronic and recurrent bacterial infections that are extremely difficult to treat by currently available standards of care. In nature, these encased bacterial communities are typically comprised of more than one genus or species. Specifically, in the airway, nontypeable Haemophilus influenzae (NTHI) predominates and is commonly isolated with one or more of the following co-pathogens with which it forms unique relationships: methicillin-resistant Staphylococcus aureus, Burkholderia cenocepacia, Pseudomonas aeruginosa, Streptococcus pneumoniae, and Moraxella catarrhalis. We recently showed that dual-genera biofilms comprised of NTHI plus a co-pathogen are disrupted when the biofilm matrix is destabilized by a pathogen-directed strategy that uses a humanized monoclonal antibody directed against the protective domains of bacterial DNABII proteins found at vertices of crossed strands of eDNA within the biofilm matrix. We also recently showed that a peptide synthesized from the host innate immune effector High Mobility Group Box 1 (HMGB1), called mB Box-97syn, competitively inhibits binding of the bacterial DNABII proteins to eDNA, which thereby also destabilizes single-species biofilms to release biofilm-resident bacteria into a transient yet highly vulnerable state that is more effectively cleared by the host innate immune system and/or antibiotics. Here, we expanded upon these studies to assess the ability of host-augmenting mB Box-97syn to both disrupt two-genera biofilms formed by NTHI plus a common co-pathogen, and prevent their formation. Disruption of established two-genera biofilms ranged from 57% to 88%, whereas prevention of two-genera biofilm formation ranged from 65% to 80% (P = .002 to P < .0001). The sobering recalcitrance of chronic and recurrent respiratory tract infections, combined with growing global concern of antimicrobial resistance (AMR), demands development of more effective management and prevention options. Ideally, novel treatment strategies would both target the pathogens and augment the host’s natural abilities to eradicate them. Herein, we provide additional data to support continued development of the latter concept via demonstration of mB Box-97syn’s efficacy against polymicrobial biofilms. A 97-mer peptide engineered from the host innate immune effector high-mobility group box 1 called ‘mB Box-97syn’ disrupts established biofilms formed by respiratory tract pathogens and also prevents their formation.

The overall antibiotic resistance of a bacterial population results from the combination of a wide range of susceptibilities displayed by subsets of bacterial cells. Bacterial heteroresistance to antibiotics has been documented for several opportunistic Gram-negative bacteria, but the mechanism of heteroresistance is unclear. We use Burkholderia cenocepacia as a model opportunistic bacterium to investigate the implications of heterogeneity in the response to the antimicrobial peptide polymyxin B (PmB) and also other bactericidal antibiotics. Here, we report that B. cenocepacia is heteroresistant to PmB. Population analysis profiling also identified B. cenocepacia subpopulations arising from a seemingly homogenous culture that are resistant to higher levels of polymyxin B than the rest of the cells in the culture, and can protect the more sensitive cells from killing, as well as sensitive bacteria from other species, such as Pseudomonas aeruginosa and Escherichia coli. Communication of resistance depended on upregulation of putrescine synthesis and YceI, a widely conserved low-molecular weight secreted protein. Deletion of genes for the synthesis of putrescine and YceI abrogate protection, while pharmacologic inhibition of putrescine synthesis reduced resistance to polymyxin B. Polyamines and YceI were also required for heteroresistance of B. cenocepacia to various bactericidal antibiotics. We propose that putrescine and YceI resemble \"danger\" infochemicals whose increased production by a bacterial subpopulation, becoming more resistant to bactericidal antibiotics, communicates higher level of resistance to more sensitive members of the population of the same or different species.

Burkholderia cenocepacia , an opportunistic respiratory pathogen particularly relevant for cystic fibrosis patients, is difficult to eradicate due to its high level of resistance to most clinically relevant antimicrobials. Consequently, the discovery of new antimicrobials as well as molecules capable of inhibiting its virulence is mandatory. In this regard quorum sensing (QS) represents a good target for anti-virulence therapies, as it has been linked to biofilm formation and is important for the production of several virulence factors, including proteases and siderophores. Here, we report the discovery of new diketopiperazine inhibitors of the B. cenocepacia acyl homoserine lactone synthase CepI, and report their anti-virulence properties. Out of ten different compounds assayed against recombinant CepI, four were effective inhibitors, with IC 50 values in the micromolar range. The best compounds interfered with protease and siderophore production, as well as with biofilm formation, and showed good in vivo activity in a Caenorhabditis elegans infection model. These molecules were also tested in human cells and showed very low toxicity. Therefore, they could be considered for in vivo combined treatments with established or novel antimicrobials, to improve the current therapeutic strategies against B . cenocepacia .

Burkholderia cenocepacia J2315 is representative of a highly problematic group of cystic fibrosis (CF) pathogens. Eradication of B. cenocepacia is very difficult with the antimicrobial therapy being ineffective due to its high resistance to clinically relevant antimicrobial agents and disinfectants. RND (Resistance-Nodulation-Cell Division) efflux pumps are known to be among the mediators of multidrug resistance in gram-negative bacteria. Since the significance of the 16 RND efflux systems present in B. cenocepacia (named RND-1 to -16) has been only partially determined, the aim of this work was to analyze mutants of B. cenocepacia strain J2315 impaired in RND-4 and RND-9 efflux systems, and assess their role in the efflux of toxic compounds. The transcriptomes of mutants deleted individually in RND-4 and RND-9 (named D4 and D9), and a double-mutant in both efflux pumps (named D4-D9), were compared to that of the wild-type B. cenocepacia using microarray analysis. Microarray data were confirmed by qRT-PCR, phenotypic experiments, and by Phenotype MicroArray analysis. The data revealed that RND-4 made a significant contribution to the antibiotic resistance of B. cenocepacia, whereas RND-9 was only marginally involved in this process. Moreover, the double mutant D4-D9 showed a phenotype and an expression profile similar to D4. The microarray data showed that motility and chemotaxis-related genes appeared to be up-regulated in both D4 and D4-D9 strains. In contrast, these gene sets were down-regulated or expressed at levels similar to J2315 in the D9 mutant. Biofilm production was enhanced in all mutants. Overall, these results indicate that in B. cenocepacia RND pumps play a wider role than just in drug resistance, influencing additional phenotypic traits important for pathogenesis.

The greatest obstacle for the treatment of cystic fibrosis patients infected with the Burkholderia species is their intrinsic antibiotic resistance. For this reason, there is a need to develop new effective compounds. Glutamate racemase, an essential enzyme for the biosynthesis of the bacterial cell wall, is an excellent candidate target for the design of new antibacterial drugs. To this aim, we recombinantly produced and characterized glutamate racemase from Burkholderia cenocepacia J2315. From the screening of an in-house library of compounds, two Zn (II) and Mn (III) 1,3,5-triazapentadienate complexes were found to efficiently inhibit the glutamate racemase activity with IC50 values of 35.3 and 10.0 μM, respectively. Using multiple biochemical approaches, the metal complexes have been shown to affect the enzyme activity by binding to the enzyme-substrate complex and promoting the formation of an inhibited dimeric form of the enzyme. Our results corroborate the value of glutamate racemase as a good target for the development of novel inhibitors against Burkholderia.

Within cystic fibrosis microbiology, there is often mismatch between the antibiotic susceptibility result of an isolated bacterial pathogen and the clinical outcome, when the patient is treated with the same antibiotic. The reasoning for this remains largely elusive. Antibiotic susceptibility to four antibiotics (ceftazidime, meropenem, minocycline and trimethoprim-sulfamethoxazole) was determined in consecutive isolates ( n = 11) from an adult cystic fibrosis patient, over a 63 month period. Each isolate displayed its own unique resistotype. The first isolate was sensitive to all four antibiotics, in accordance with Clinical and Laboratory Standards Institute methodology and interpretative criteria. Resistance was first detected at four months, showing resistance to ceftazidime and meropenen and intermediate resistance to minocycline and trimethoprim-sulfamethoxazole. Pan resistance was first detected at 18 months (resistotype IV), with three resistotypes (I, II and III) preceding this complete resistotype. The bacterium continued to display further antibiotic susceptibility heterogeneity for the next 45 months, with the description of an additional seven resistotypes (resistotypes V–XI). The Relative Resistance Index of this bacterium over the 63 month period showed no relationship between the development of antibiotic resistance and time. Adoption of mathematical modelling employing multinomial distribution demonstrated that large numbers of individual colony picks (>40/sputum), would be required to be 78% confident of capturing all 11 resistotypes present. Such a requirement for large numbers of colony picks combined with antibiotic susceptibility-related methodological problems creates a conundrum in biomedical science practice, in providing a robust assay that will capture antibiotic susceptibility variation, be pragmatic and cost-effective to deliver as a pathology service, but have the reliability to help clinicians select appropriate antibiotics for their patients. This study represents an advance in biomedical science as it demonstrates potential variability in antibiotic susceptibility testing with Burkholderia cenocepacia . Respiratory physicians and paediatricians need to be made aware of such variation by biomedical scientists at the bench, so that clinicians can contextualise the significance of the reported susceptibility result, when selecting appropriate antibiotics for their cystic fibrosis patient. Furthermore, consideration needs to be given in providing additional guidance on the laboratory report to highlight this heterogeneity to emphasise the potential for misalignment between susceptibility result and clinical outcome.