Synthesis and antibiofilm evaluation of 3-hydroxy-2,3-dihydroquinazolin-4(1H)-one derivatives against opportunistic pathogen Acinetobacter baumannii.

The emergence of multidrug resistant microorganisms has triggered the impending need for new aitimicrobial strategies. The antivirulence strategy with the benefite of alleviating the drug resistance becomes the focus of research. In this study, 22 quorum sensing inhibitors were synthesized by mimicking the structure of autoinducer and acinetobactin and up to 34% biofilm inhibition was observed with 5u. The biofilm inhibition effect was further demonstrated with extracellular polysaccharides inhibition and synergism with Gentamycin sulphate.

Sugar-powered nanoantimicrobials for combating bacterial biofilms.

Bacterial biofilms cause chronic infections due to their inherent tolerance to antimicrobial therapies. We describe and compare the efficacy of two types of sugar (d-glucose and d-mannose)-modified cyclodextrin nanocarriers (CD-GLU and CD-MAN) loaded with antibacterial agents for preventing and eradicating bacterial biofilm. The antibacterial agents comprise a quorum sensing inhibitor (5,6-dimethyl-2-aminobenzimidazole, DMABI) and two antibiotics (erythromycin and rifampicin), and the cyclodextrin nanocarriers were tested on Pseudomonas aeruginosa (Gram-negative) and Staphylococcus aureus (Gram-positive). DMABI loaded in the CD-GLU carrier was significantly more effective at inhibiting the development of Pseudomonas aeruginosa biofilm when compared to either its free form or when it is loaded in CD without grafted sugar moieties. Antibiotics loaded in CD-GLU and CD-MAN carriers were similarly more effective at dispersing pre-formed Pseudomonas aeruginosa biofilms. These antibacterial compounds loaded in the CD-GLU and CD-MAN carriers were somewhat less effective in eradicating Staphylococcus aureus biofilm as compared to Pseudomonas aeruginosa biofilm. This difference is attributed to the different extent of penetration of the sugar-grafted carriers into the biofilms of these two species of bacterial cells. Although the sugar-grafted carrier-antibacterial agent complexes exhibit potent effects against bacterial biofilms, they are not cytotoxic to mammalian cells.

The Acinetobacter baumannii Two-Component System AdeRS Regulates Genes Required for Multidrug Efflux, Biofilm Formation, and Virulence in a Strain-Specific Manner.

UNLABELLED: The opportunistic pathogen Acinetobacter baumannii is able to persist in the environment and is often multidrug resistant (MDR), causing difficulties in the treatment of infections. Here, we show that the two-component system AdeRS, which regulates the production of the AdeABC multidrug resistance efflux pump, is required for the formation of a protective biofilm in an ex vivo porcine mucosal model, which mimics a natural infection of the human epithelium. Interestingly, deletion of adeB impacted only on the ability of strain AYE to form a biofilm on plastic and only on the virulence of strain Singapore 1 for Galleria mellonella RNA-Seq revealed that loss of AdeRS or AdeB significantly altered the transcriptional landscape, resulting in the changed expression of many genes, notably those associated with antimicrobial resistance and virulence interactions. For example, A. baumannii lacking AdeRS displayed decreased expression of adeABC, pil genes, com genes, and a pgaC-like gene, whereas loss of AdeB resulted in increased expression of pil and com genes and decreased expression of ferric acinetobactin transport system genes. These data define the scope of AdeRS-mediated regulation, show that changes in the production of AdeABC mediate important phenotypes controlled by AdeRS, and suggest that AdeABC is a viable target for antimicrobial drug and antibiofilm discovery [corrected].

Sugar-Grafted Cyclodextrin Nanocarrier as a "Trojan Horse" for Potentiating Antibiotic Activity.

PURPOSE: The use of "Trojan Horse" nanocarriers for antibiotics to enhance the activity of antibiotics against susceptible and resistant bacteria is investigated. METHODS: Antibiotic carriers (CD-MAN and CD-GLU) are prepared from ß-cyclodextrin grafted with sugar molecules (D-mannose and D-glucose, respectively) via azide-alkyne click reaction. The sugar molecules serve as a chemoattractant enticing the bacteria to take in higher amounts of the antibiotic, resulting in rapid killing of the bacteria. RESULTS: Three types of hydrophobic antibiotics, erythromycin, rifampicin and ciprofloxacin, are used as model drugs and loaded into the carriers. The minimum inhibitory concentration of the antibiotics in the CD-MAN-antibiotic and CD-GLU-antibiotic complexes for Gram-negative Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii strains, and a number of Gram-positive Staphylococcus aureus strains, including the methicillin-resistant strains (MRSA), are reduced by a factor ranging from 3 to >100. The CD-MAN-antibiotic complex is also able to prolong the stability of the loaded antibiotic and inhibit development of intrinsic antibiotic resistance in the bacteria. CONCLUSIONS: These non-cytotoxic sugar-modfied nanocarriers can potentiate the activity of existing antibiotics, especially against multidrug-resistant bacteria, which is highly advantageous in view of the paucity of new antibiotics in the pipeline.

Bifunctional Coating with Sustained Release of 4-Amide-piperidine-C12 for Long-Term Prevention of Bacterial Colonization on Silicone.

Bacterial colonization by nosocomial pathogens on medical device surface can cause serious and life-threatening infections. We showed that 4-amide-piperidine-C12 (4AP12), the base form of 4-dodecaneamidepiperidine HCl, has broad-spectrum antimicrobial activity against both Gram-negative and Gram-positive bacteria and fungi. Resistance assay confirmed that prolonged exposure of bacteria to subinhibitory concentrations of 4AP12 did not induce resistance to 4AP12. The possible antimicrobial mechanism of 4AP12 was investigated, and attributed to the disruption of the cell membrane of microorganisms and subsequent cell lysis. The hydrophobic 4AP12 was incorporated in Pluronic F127 diacrylate (F127DA) micelles, which were then graft-copolymerized with acrylic acid and cross-linked onto ozonized silicone surface. Sulfobetaine methacrylate and F127DA were then graft-copolymerized as an antifouling layer on top of the F127DA-AA hydrogel containing the 4AP12, thus forming a microscale two-layer bifunctional coating. Sustained release of 4AP12 at a rate of up to 1 µg/day per cm2 of hydrogel-coated silicone surface was achieved and this was sufficient to inhibit ∼97% of bacterial colonization by Acinetobacter baumannii in artificial urine medium under static condition over a 14-day period. Bacterial colonization by Escherichia coli and Pseudomonas aeruginosa under similar conditions was also significantly reduced. In addition, after 96 h exposure to flowing artificial urine (0.7 mL/min), Escherichia coli colonization on the 4AP12-loaded hydrogel-coated surface was reduced by ∼89% compared to the pristine surface. The concentration of 4AP12 that was released and was effective in inhibiting bacterial colonization did not result in significant cytotoxicity to human epithelial cells.

Disruption of biofilm formation by the human pathogen Acinetobacter baumannii using engineered quorum-quenching lactonases.

Acinetobacter baumannii is a major human pathogen associated with multidrug-resistant nosocomial infections; its virulence is attributed to quorum-sensing-mediated biofilm formation, and disruption of biofilm formation is an attractive antivirulence strategy. Here, we report the first successful demonstration of biofilm disruption in a clinical isolate of A. baumannii S1, using a quorum-quenching lactonase obtained by directed evolution; this engineered lactonase significantly reduced the biomass of A. baumannii-associated biofilms, demonstrating the utility of this antivirulence strategy.

A method for generating marker-less gene deletions in multidrug-resistant Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii is an important nosocomial pathogen that has become increasingly resistant to multiple antibiotics. Genetic manipulation of MDR A. baumannii is useful especially for defining the contribution of each active efflux mechanism in multidrug resistance. Existing methods rely on the use of an antibiotic selection marker and are not suited for multiple gene deletions. RESULTS: A tellurite-resistant (sacB⁺, xylE⁺) suicide vector, pMo130-TelR, was created for deleting the adeFGH and adeIJK operons in two clinical MDR A. baumannii, DB and R2 from Singapore. Using a two-step selection, plasmid insertion recombinants (first-crossover) were selected for tellurite resistance and the deletion mutants (second-crossover) were then selected for loss of sacB. The DNA deletions were verified by PCR while loss of gene expression in the ΔadeFGH, ΔadeIJK and ΔadeFGHΔadeIJK deletion mutants was confirmed using qRT-PCR. The contribution of AdeFGH and AdeIJK pumps to MDR was defined by comparing antimicrobial susceptibilities of the isogenic mutants and the parental strains. The deletion of adeIJK produced no more than eight-fold increase in susceptibility to nalidixic acid, tetracycline, minocycline, tigecycline, clindamycin, trimethoprim and chloramphenicol, while the deletion of adeL-adeFGH operon alone had no impact on antimicrobial susceptibility. Dye accumulation assays using H33342 revealed increased dye retention in all deletion mutants, except for the R2ΔadeFGH mutant, where a decrease was observed. Increased accumulation of ethidium bromide was observed in the parental strains and all pump deletion mutants in the presence of efflux inhibitors. The efflux pump deletion mutants in this study revealed that only the AdeIJK, but not the AdeFGH RND pump, contributes to antimicrobial resistance and dye accumulation in MDR A. baumannii DB and R2. CONCLUSIONS: The marker-less gene deletion method using pMo130-TelR is applicable for creating single and multiple gene deletions in MDR A. baumannii. The adeFGH and adeIJK operons were successfully deleted separately and together using this method and the impact of each efflux pump on antimicrobial resistance could be defined clearly.

N-Octanoylhomoserine lactone signalling mediated by the BpsI-BpsR quorum sensing system plays a major role in biofilm formation of Burkholderia pseudomallei.

The genome of Burkholderia pseudomallei encodes three acylhomoserine lactone (AHL) quorum sensing systems, each comprising an AHL synthase and a signal receptor/regulator. The BpsI-BpsR system produces N-octanoylhomoserine lactone (C8HL) and is positively auto-regulated by its AHL product. The products of the remaining two systems have not been identified. In this study, tandem MS was used to identify and quantify the AHL species produced by three clinical B. pseudomallei isolates - KHW, K96243 and H11 - three isogenic KHW mutants that each contain a null mutation in an AHL synthase gene, and recombinant Escherichia coli heterologously expressing each of the three B. pseudomallei AHL synthase genes. BpsI synthesized predominantly C8HL, which accounted for more than 95 % of the extracellular AHLs produced in stationary-phase KHW cultures. The major products of BpsI(2) and BpsI(3) were N-(3-hydroxy-octanoyl)homoserine lactone (OHC8HL) and N-(3-hydroxy-decanoyl)homoserine lactone, respectively, and their corresponding transcriptional regulators, BpsR(2) and BpsR(3), were capable of driving reporter gene expression in the presence of these cognate lactones. Formation of biofilm by B. pseudomallei KHW was severely impaired in mutants lacking either BpsI or BpsR but could be restored to near wild-type levels by exogenous C8HL. BpsI(2) was not required, and BpsI(3) was partially required for biofilm formation. Unlike the bpsI mutant, biofilm formation in the bpsI(3) mutant could not be restored to wild-type levels in the presence of OHC8HL, the product of BpsI(3). C8HL and OHC8HL had opposite effects on biofilm formation; exogenous C8HL enhanced biofilm formation in both the bpsI(3) mutant and wild-type KHW while exogenous OHC8HL suppressed the formation of biofilm in the same strains. We propose that exogenous OHC8HL antagonizes biofilm formation in B. pseudomallei, possibly by competing with endogenous C8HL for binding to BpsR.

Synthesis of cyclic di-nucleotidic acids as potential inhibitors targeting diguanylate cyclase.

Five analogs of cyclic di-nucleotidic acid including c-di-GMP were synthesized and evaluated for their biological activities on Slr1143, a diguanylate cyclase of Synechocystis sp. Slr1143 was overexpressed from the recombinant plasmid which contained the gene of interest and subsequently purified by affinity chromatography. A new HPLC method capable of separating the compound and product peaks with good resolution was optimized and applied to the analysis of the compounds. Results obtained show that cyclic di-inosinylic acid 1b demonstrates a stronger inhibition on Slr1143 than c-di-GMP and is a potential inhibitor for biofilm formation.

CdpA is a Burkholderia pseudomallei cyclic di-GMP phosphodiesterase involved in autoaggregation, flagellum synthesis, motility, biofilm formation, cell invasion, and cytotoxicity.

Cyclic diguanylic acid (c-di-GMP) is an intracellular signaling molecule involved in regulation of cellular functions such as motility, biofilm formation and virulence. Intracellular level of c-di-GMP is controlled through opposing diguanylate cyclase (DGC) and phosphodiesterase (PDE) activities of GGDEF and EAL domain proteins, respectively. We report the identification and characterization of cdpA, a gene encoding a protein containing an EAL domain in the Gram-negative soil bacillus and human pathogen Burkholderia pseudomallei KHW. Purified recombinant CdpA protein exhibited PDE activity in vitro. Evidence that CdpA is a major c-di-GMP-specific PDE in B. pseudomallei KHW was shown by an 8-fold-higher c-di-GMP level in the cdpA-null mutant as compared to the wild type and the complemented cdpA mutant. The presence of higher intracellular c-di-GMP levels in the cdpA-null mutant was associated with increased production of exopolysaccharides, increased cell-to-cell aggregation, absence of flagella and swimming motility, and increased biofilm formation. The relevance of CdpA in B. pseudomallei virulence was demonstrated by a 3-fold reduction in invasion of human lung epithelial cells and a 6-fold reduction in cytotoxicity on human macrophage cells infected with the cdpA mutant.

Growth-related changes in intracellular spermidine and its effect on efflux pump expression and quorum sensing in Burkholderia pseudomallei.

The Burkholderia pseudomallei BpeAB-OprB resistance-nodulation-division (RND) family pump effluxes aminoglycoside and macrolide antibiotics as well as acylhomoserine lactones (AHLs) involved in quorum sensing. Expression of bpeA-lacZ was cell density-dependent and was inducible in the presence of these compounds. Intracellular levels of spermidine and N-acetylspermidine increased with cell density in wild-type B. pseudomallei KHW, but were always lower in the bpeAB pump mutant at all growth phases. The significance of changes in intracellular spermidine on efflux pump expression was demonstrated by the disruption of the binding of the BpeR repressor protein to the bpeABoprB regulatory region in vitro in the presence of increasing spermidine concentrations. This was supported by dose-dependent activation of bpeA-lacZ transcription in vivo in the presence of exogenous spermidine and N-acetylspermidine, thus implicating the involvement of the BpeAB-OprB pump in spermidine homeostasis in B. pseudomallei. Consequently, inhibition of intracellular spermidine synthesis reduced the efflux of AHLs by BpeAB-OprB. Other potential therapeutic applications of spermidine synthase inhibitors include the reduction of swimming motility and biofilm formation by B. pseudomallei.

A multigene family of Heteractis magnificalysins (HMgs).

Sea anemones are passive predators. They use their specialized stinging cells (nematocysts) to immobilize any prey that blunders into them. A cnida fires, everting a tubule which delivers toxins that may stick to a prey. These toxins include neurotoxins, cytotoxins, cardiotoxins and haemolysins. Heteractis magnificalysins (HMgs) belong to a family of cytolysins from the sea anemone Heteractis magnifica. HMgs are 19.5kDa basic proteins of 177 amino acids with pI values ranging from 8 to 10. From 52 cloned HMg gene sequences, we showed that HMgs are encoded by a multigene family whose members are highly homologous to each other. HMg genes are intronless, and may have arisen by gene duplication, gene conversion or mutation. By modifying the extraction procedure, we purified more natural HMg proteins from H. magnifica, thus demonstrating that H. magnifica are naturally competent to produce a large number of HMg cytolysins. Native and recombinant HMg proteins differed from each other in their amino acid sequences and biological activities. In each H. magnifica, many cytolysin isoforms are produced. H. magnifica appeared to have evolved a survival mechanism whereby a large number of cytolysins of different biological properties are produced for defense and offence.

A role of Burkholderia pseudomallei flagella as a virulent factor.

Burkholderia pseudomallei is an agent of melioidosis and is closely related to avirulent B. thailandensis. Burkholderia thailandensis has a 15-bp deletion within the variable region of the flagellin gene fliC compared with B. pseudomallei. The difference in the fliC gene might be related to virulence. In the present study, the invasion, internalization and intracellular replication of both phagocytic (mouse macrophage cell line RAW264.7) and non-phagocytic cells (human lung epithelial cell line A549) of B. pseudomallei fliC knockout mutant (MM35) complemented with its own fliC (Cp) or with B. thailandensis fliC (Ct) was compared with those of the wild-type strains of B. pseudomallei (1026b) and B. thailandensis (E257). In phagocytic cells, there was no significant difference in bacterial uptake between Cp and Ct, but MM35 was internalized significantly less compared with 1026b, Cp, Ct and E257. The results suggest that flagella are involved in macrophage invasion. In non-phagocytic cells, Cp and Ct showed similar invasive capacities while 1026b, Cp and Ct showed significantly higher invasiveness than MM35, suggesting that flagella facilitate the non-phagocytic cell invasion. However, the invasive capacity of MM35 was significantly higher than that of E257, suggesting that in addition to the flagella, B. pseudomallei may need other factor(s) to facilitate invasion in non-phagocytic cells.

Control of quorum sensing by a Burkholderia pseudomallei multidrug efflux pump.

The Burkholderia pseudomallei KHW quorum-sensing systems produced N-octanoyl-homoserine lactone, N-decanoyl-homoserine lactone, N-(3-hydroxy)-octanoyl-homoserine lactone, N-(3-hydroxy)-decanoyl-homoserine lactone, N-(3-oxo)-decanoyl-homoserine lactone, and N-(3-oxo)-tetradecanoyl-homoserine lactone. The extracellular secretion of these acyl-homoserine lactones is dependent absolutely on the function of the B. pseudomallei BpeAB-OprB efflux pump.

Synergistic interaction between phenothiazines and antimicrobial agents against Burkholderia pseudomallei.

The gram-negative soil bacillus Burkholderia pseudomallei is the causative agent of melioidosis, a severe and potentially fatal septicemic disease that is endemic to Southeast Asia and northern Australia. Its intrinsic resistance to many antibiotics is attributed mainly to the presence of several drug efflux pumps, and therefore, inhibitors of such pumps are expected to restore the activities of many clinically important antimicrobial agents that are the substrates of these pumps. The phenothiazine antipsychotic and antihistaminic drugs prochlorperazine, chlorpromazine, and promazine have a synergistic interaction with a wide spectrum of antimicrobial agents, thereby enhancing their antimicrobial potency against B. pseudomallei. Antimicrobial agents that interacted synergistically with the phenothiazines include streptomycin, erythromycin, oleandomycin, spectinomycin, levofloxacin, azithromycin, and amoxicillin-clavulanic acid. The MICs of these antibiotics were reduced as much as 8,000-fold in the presence of the phenothiazines. Antimicrobial agents which did not interact synergistically with the phenothiazines include gentamicin, amoxicillin, and ampicillin. Omeprazole, a proton pump inhibitor, provided an augmentation of antimicrobial activities similar to that of the phenothiazines, suggesting that the phenothiazines might have interfered with the proton gradient at the inner membrane. B. pseudomallei cells accumulated more erythromycin in the presence of the phenothiazines, an effect similar to that of carbonyl cyanide m-chlorophenylhydrazone, a proton gradient uncoupler. In the presence of the phenothiazines, a much reduced concentration of erythromycin (0.06x MIC) also protected human lung epithelial cells and macrophage cells from B. pseudomallei infection and attenuated its cytotoxicity.

Method for regulated expression of single-copy efflux pump genes in a surrogate Pseudomonas aeruginosa strain: identification of the BpeEF-OprC chloramphenicol and trimethoprim efflux pump of Burkholderia pseudomallei 1026b.

Construction and integration of recombinant mini-Tn7 expression vectors into the chromosome of a surrogate, efflux-sensitized, and biosafe Pseudomonas aeruginosa host was validated as a generally applicable method for studies of uncharacterized bacterial efflux pumps. Using this method, the Burkholderia pseudomallei bpeEF-oprC operon was shown to encode a chloramphenicol and trimethoprim efflux pump.

The Burkholderia pseudomallei BpeAB-OprB efflux pump: expression and impact on quorum sensing and virulence.

BpeAB-OprB is a multidrug efflux pump of the bacterial pathogen Burkholderia pseudomallei and is responsible for conferring antimicrobial resistance to aminoglycosides and macrolides. Expression of bpeAB-oprB is inducible by its substrate erythromycin and upon entry into stationary phase. BpeR, a member of the TetR family, functions as a repressor of the bpeAB-oprB operon. bpeR expression was similarly induced at stationary phase but lagged behind the induction of bpeAB-oprB expression. The induction of bpeAB-oprB expression could be advanced to the early exponential phase by exogenous addition of the B. pseudomallei autoinducers N-octanoyl-homoserine lactone (C8HSL) and N-decanoyl-homoserine lactone (C10HSL), suggesting that the bpeAB-oprB operon may be quorum regulated. On the other hand, acyl-homoserine lactone (acyl-HSL) production was undetectable in the bpeAB-null mutant and strains which overexpress bpeR. The failure of these strains to produce acyl-HSLs seemed to be at the level of synthesis of acyl-HSLs, as growth-phase-dependent expression of the autoinducer synthase BpsI was abolished in the bpeAB-null mutant. bpsI expression remained growth phase dependent in the bpeR mutant which had functional BpeAB-OprB. BpeAB-OprB function is likewise necessary for optimal production of quorum-sensing-controlled virulence factors such as siderophore and phospholipase C and for biofilm formation. Cell invasion and cytotoxicity towards human lung epithelial (A549) and human macrophage (THP-1) cells were also significantly attenuated in both the bpeAB mutant and bpeR-overexpressing strains, thus suggesting the possibility of attenuating B. pseudomallei virulence using inhibitors of the BpeAB-OprB efflux pump.

Cloning and molecular characterization of the first aquatic hyaluronidase, SFHYA1, from the venom of stonefish (Synanceja horrida).

We report here for the first time the molecular characterization of a hyaluronidase from an aquatic source. SFHYA1 is the hyaluronidase found in the venom gland of stonefish, Synanceja horrida. Using a cDNA segment amplified with degenerate oligonucleotides based on the amino acid sequences of a conserved region in testicular-type hyaluronidases and a tryptic fragment of SFHYA1, clones encoding the precursor of this enzyme were isolated from a cDNA library prepared from stonefish venom glands. The deduced amino acid sequence of SFHYA1 shows that SFHYA1 is expressed as a precursor peptide with a 28-residue signal peptide for targeting it into endoplasmic reticulum. Mature SFHYA1 is a polypeptide composed of 449 residues containing three potential N-glycosylation sites, four putative hyaluronan-binding motifs [B(X)7B] and various residues implicated in substrate binding and catalysis. This cDNA was expressed in an active form in insect-cells but not in E. coli. Homology-based computational analyses suggested that SFHYA1 closely resembles the PH-20 family of hyaluronidases.