Polymeric Composites with Silver (I) Cyanoximates Inhibit Biofilm Formation of Gram-Positive and Gram-Negative Bacteria.

Biofilms are surface-associated microbial communities known for their increased resistance to antimicrobials and host factors. This resistance introduces a critical clinical challenge, particularly in cases associated with implants increasing the predisposition for bacterial infections. Preventing such infections requires the development of novel antimicrobials or compounds that enhance bactericidal effect of currently available antibiotics. We have synthesized and characterized twelve novel silver(I) cyanoximates designated as Ag(ACO), Ag(BCO), Ag(CCO), Ag(ECO), Ag(PiCO), Ag(PICO) (yellow and red polymorphs), Ag(BIHCO), Ag(BIMCO), Ag(BOCO), Ag(BTCO), Ag(MCO) and Ag(PiPCO). The compounds exhibit a remarkable resistance to high intensity visible light, UV radiation and heat and have poor solubility in water. All these compounds can be well incorporated into the light-curable acrylate polymeric composites that are currently used as dental fillers or adhesives of indwelling medical devices. A range of dry weight % from 0.5 to 5.0 of the compounds was tested in this study. To study the potential of these compounds in preventing planktonic and biofilm growth of bacteria, we selected two human pathogens (Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus) and Gram-positive environmental isolate Bacillus aryabhattai. Both planktonic and biofilm growth was abolished completely in the presence of 0.5% to 5% of the compounds. The most efficient inhibition was shown by Ag(PiCO), Ag(BIHCO) and Ag(BTCO). The inhibition of biofilm growth by Ag(PiCO)-yellow was confirmed by scanning electron microscopy (SEM). Application of Ag(BTCO) and Ag(PiCO)-red in combination with tobramycin, the antibiotic commonly used to treat P. aeruginosa infections, showed a significant synergistic effect. Finally, the inhibitory effect lasted for at least 120 h in P. aeruginosa and 36 h in S. aureus and B. aryabhattai. Overall, several silver(I) cyanoximates complexes efficiently prevent biofilm development of both Gram-negative and Gram-positive bacteria and present a particularly significant potential for applications against P. aeruginosa infections.

Genetic and genomic insights into the role of benzoate-catabolic pathway redundancy in Burkholderia xenovorans LB400.

Transcriptomic and proteomic analyses of Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB) degrader, have implicated growth substrate- and phase-dependent expression of three benzoate-catabolizing pathways: a catechol ortho cleavage (ben-cat) pathway and two benzoyl-coenzyme A pathways, encoded by gene clusters on the large chromosome (boxC) and the megaplasmid (boxM). To elucidate the significance of this apparent redundancy, we constructed mutants with deletions of the ben-cat pathway (the DeltabenABCD::kan mutant), the boxC pathway (the DeltaboxABC::kan mutant), and both pathways (the DeltabenABCDDelta boxABC::kan mutant). All three mutants oxidized benzoate in resting-cell assays. However, the DeltabenABCD::kan and DeltabenABCD DeltaboxABC::kan mutants grew at reduced rates on benzoate and displayed increased lag phases. By contrast, growth on succinate, on 4-hydroxybenzoate, and on biphenyl was unaffected. Microarray and proteomic analyses revealed that cells of the DeltabenABCD::kan mutant growing on benzoate expressed both box pathways. Overall, these results indicate that all three pathways catabolize benzoate. Deletion of benABCD abolished the ability of LB400 to grow using 3-chlorobenzoate. None of the benzoate pathways could degrade 2- or 4-chlorobenzoate, indicating that the pathway redundancy does not directly contribute to LB400's PCB-degrading capacities. Finally, an extensive sigmaE-regulated oxidative stress response not present in wild-type LB400 grown on benzoate was detected in these deletion mutants, supporting our earlier suggestion that the box pathways are preferentially active under reduced oxygen tension. Our data further substantiate the expansive network of tightly interconnected and complexly regulated aromatic degradation pathways in LB400.

Calcium influences cellular and extracellular product formation during biofilm-associated growth of a marine Pseudoalteromonas sp.

Bacteria undergo a variety of physiological changes following a switch from planktonic growth to surface-associated biofilm growth. Here, it is shown that biofilm development of a marine isolate, Pseudoalteromonas sp. 1398, results in global changes in its cytosolic and extracellular proteomes. Calcium influences these proteome responses, and affects the amount of surface-associated biomass and extracellular matrix material produced by Pseudoalteromonas sp. 1398. Four extracellular proteins, characterized by N-terminal sequencing, showed increased abundances, while one protein, flagellin, showed reduced abundance at higher [Ca(2+)]. Immunoblotting and transmission-electron-microscopy analysis confirmed that higher [Ca(2+)] and surface-associated growth results in the repression of flagella production. Two-dimensional gel electrophoresis (2DGE) studies combined with cluster analysis of global proteome responses demonstrated that Ca(2+) had a greater regulatory influence on Pseudoalteromonas sp. growing in biofilms than on planktonic cultures. Approximately 22 % of the total cytosolic proteins resolved by 2DGE had differing abundances in response to a switch from planktonic growth to surface-associated growth when the cells were cultivated in 1 mM Ca(2+). At higher [Ca(2+)] this number increased to 38 %. Fifteen cellular proteins that were differentially expressed in response to biofilm growth and/or Ca(2+) were analysed by N-terminal sequencing and/or MS/MS. These proteins were identified as factors involved in cellular metabolic functions, putative proteases and transport proteins, although there were several proteins that had not been previously characterized. These results indicate that Ca(2+) causes global changes in matrix material, as well as in cellular and extracellular protein profiles of Pseudoalteromonas sp. 1398. These changes are more pronounced when the bacterium grows in biofilms than when it grows in planktonic culture.

Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa colonizes the pulmonary tissue of patients with cystic fibrosis (CF), leading to biofilm-associated infections. The pulmonary fluid of CF patients usually contains elevated concentrations of cations and may contain the P. aeruginosa redox-active pigment pyocyanin, which is known to disrupt calcium homeostasis of host cells. Since divalent cations are important bridging ions for bacterial polysaccharides and since they may play regulatory roles in bacterial gene expression, we investigated the effect of calcium ions on the extracellular matrix constituents of P. aeruginosa biofilms. For mucoid strain P. aeruginosa FRD1, calcium addition (1.0 and 10 mM as CaCl(2)) resulted in biofilms that were at least 10-fold thicker than biofilms without added calcium. Scanning confocal laser microscopy showed increased spacing between cells for the thick biofilms, and Fourier transform infrared spectroscopy revealed that the material between cells is primarily alginate. An algD transcriptional reporter demonstrated that calcium addition caused an eightfold increase in alg gene expression in FRD1 biofilms. Calcium addition also resulted in increased amounts of three extracellular proteases (AprA, LasB, and PrpL). Immunoblots of the biofilm extracellular material established that AprA was harbored within the biofilm extracellular matrix. An aprA deletion mutation and a mutation in gene for a putative P. aeruginosa calmodulin-like protein did not significantly affect calcium-induced biofilm structure. Two-dimensional gel electrophoresis showed increased amounts of phenazine biosynthetic proteins in FRD1 biofilms and in calcium-amended planktonic cultures. Spectrochemical analyses showed that the calcium addition causes a three- to fivefold increase in pyocyanin production. These results demonstrate that calcium addition affects the structure and extracellular matrix composition of mucoid P. aeruginosa biofilms, through increased expression and stability of bacterial extracellular products. The calcium-induced extracellular matrix of mucoid P. aeruginosa consists primarily of the virulence factor alginate and also harbors extracellular proteases and perhaps pyocyanin, a biomolecule that may further disrupt cellular calcium levels.

Growth substrate- and phase-specific expression of biphenyl, benzoate, and C1 metabolic pathways in Burkholderia xenovorans LB400.

Recent microarray experiments suggested that Burkholderia xenovorans LB400, a potent polychlorinated biphenyl (PCB)-degrading bacterium, utilizes up to three apparently redundant benzoate pathways and a C(1) metabolic pathway during biphenyl and benzoate metabolism. To better characterize the roles of these pathways, we performed quantitative proteome profiling of cells grown on succinate, benzoate, or biphenyl and harvested during either mid-logarithmic growth or the transition between the logarithmic and stationary growth phases. The Bph enzymes, catabolizing biphenyl, were approximately 16-fold more abundant in biphenyl- versus succinate-grown cells. Moreover, the upper and lower bph pathways were independently regulated. Expression of each benzoate pathway depended on growth substrate and phase. Proteins specifying catabolism via benzoate dihydroxylation and catechol ortho-cleavage (ben-cat pathway) were approximately an order of magnitude more abundant in benzoate- versus biphenyl-grown cells at the same growth phase. The chromosomal copy of the benzoyl-coenzyme A (CoA) (box(C)) pathway was also expressed during growth on biphenyl: Box(C) proteins were approximately twice as abundant as Ben and Cat proteins under these conditions. By contrast, proteins of the megaplasmid copy of the benzoyl-CoA (box(M)) pathway were only detected in transition-phase benzoate-grown cells. Other proteins detected at increased levels in benzoate- and biphenyl-grown cells included general stress response proteins potentially induced by reactive oxygen species formed during aerobic aromatic catabolism. Finally, C(1) metabolic enzymes were present in biphenyl-grown cells during transition phase. This study provides insights into the physiological roles and integration of apparently redundant catabolic pathways in large-genome bacteria and establishes a basis for investigating the PCB-degrading abilities of this strain.

Degradation of benzyldimethylalkylammonium chloride by Aeromonas hydrophila sp. K.

AIMS: The aim of this work was to study the biodegradation of benzyldimethylalkylammonium chloride (BAC) by Aeromonas hydrophila sp. K, an organism isolated from polluted soil and capable of utilizing BAC as sole source of carbon and energy. METHODS AND RESULTS: High performance liquid chromatography and gas chromatography-mass spectrometry (GC-MS) analysis was used to study BAC degradation pathway. It was shown that during BAC biodegradation, formation of benzyldimethylamine, benzylmethylamine, benzylamine, benzaldehyde and benzoic acid occurred. Formation of benzyldimethylamine as the initial metabolite suggested that the cleavage of Calkyl-N bond occurred as the first step of BAC catabolism. Liberation of benzylmethylamine and benzylamine likely resulted from subsequent demethylation reactions, followed by deamination with formation of benzaldehyde. Benzaldehyde was rapidly converted into benzoic acid, which was further degraded. CONCLUSIONS: Aer. hydrophila sp. K is able to degrade BAC. A degradation pathway for BAC and related compounds is proposed. SIGNIFICANCE AND IMPACT OF STUDY: These findings are significant for understanding biodegradation pathways of benzyl-containing quaternary ammonium compounds.