Control of Citrus Canker in Greenhouse and Field with a Zinc, Urea, and Peroxide Ternary Solution.

Accumulation of toxic copper in soil and development of copper-resistant pests are emerging challenges currently faced by the agricultural community worldwide. As an alternative, we have developed a ternary zinc chelate solution (TSOL) pesticide where zinc ions are the primary active ingredient. The material is composed of zinc, urea, and hydrogen peroxide. Urea was chosen as it is widely used as a plant fertilizer and can also bind to both zinc and hydrogen peroxide. No phytotoxicity was observed with TSOL on Meyer lemon (Citrus × meyeri) seedlings at a field spray rate of 800 µg/mL Zn metal concentration. Antimicrobial studies showed that TSOL exhibited improved killing efficacy against Escherichia coli and Xanthomonas alfalfae compared to Zn ions alone. Citrus canker field trials in a grapefruit (Chrysopelea paradisi) grove over three years showed that TSOL provided comparable disease protection to copper products at an equivalent or lower metal content.

N-acetyl Cysteine Coated Gallium Particles Demonstrate High Potency against Pseudomonas aeruginosa PAO1.

Nosocomial infections pose serious health concerns with over 2 million reported annually in the United States. Many of these infections are associated with bacterial resistance to antibiotics and hence, alternative treatments are critically needed. The objective of this study was to assess the antimicrobial efficacy of a gallium (Ga)-based particle coated with N-Acetyl Cysteine (Ga-NAC) against Pseudomonas aeruginosa PAO1. Our studies showed the Minimum Inhibitory Concentration (MIC) of PAO1 treated with Ga-NAC was 1 µg/mL. Cytotoxicity of Ga-NAC against multiple cell lines was determined with no cytotoxicity observed up to concentrations of 2000 µg/mL (metal concentration), indicating a high therapeutic window. To elucidate potential antibacterial modes of action, Inductively Coupled Plasma-Mass Spectrometry (ICP-MS), infrared spectroscopy, and atomic force microscopy (AFM) were used. The results suggest improved Ga3+ interaction with PAO1 through Ga-NAC particles. No significant change in cell membrane chemistry or roughening was detected. As cell membrane integrity remained intact, the antimicrobial mode of action was linked to cellular internalization of Ga and subsequent iron metabolic disruption. Furthermore, Ga-NAC inhibited and disrupted biofilms seen with crystal violet assay and microscopy. Our findings suggest the Ga-NAC particle can potentially be used as an alternative to antibiotics for treatment of Pseudomonas aeruginosa infections.

Antimicrobial Zn-Based "TSOL" for Citrus Greening Management: Insights from Spectroscopy and Molecular Simulation.

Huanglongbing (HLB), also known as citrus greening, is a bacterial disease that poses a devastating threat to the citrus industry worldwide. To manage this disease efficiently, we developed and characterized a ternary aqueous solution (TSOL) that contains zinc nitrate, urea, and hydrogen peroxide. We report that TSOL exhibits better antimicrobial activity than commercial bactericides for growers. X-ray fluorescence analysis demonstrates that zinc is delivered to citrus leaves, where the bacteria reside. FTIR and Raman spectroscopy, molecular dynamics simulations, and density functional theory calculations elucidate the solution structure of TSOL and reveal a water-mediated interaction between Zn2+ and H2O2, which may facilitate the generation of highly reactive hydroxyl radicals contributing to superior antimicrobial activity of TSOL. Our results not only suggest TSOL as a potent antimicrobial agent to suppress bacterial growth in HLB-infected trees, but also provide a structure-property relationship that explains the superior performance of TSOL.

Nanoparticles in mitigating gaseous emissions from liquid dairy manure stored under anaerobic condition.

A number of mitigation techniques exist to reduce the emissions of pollutant gases and greenhouse gases (GHGs) from anaerobic storage of livestock manure. Nanoparticle (NP) application is a promising mitigating treatment option for pollutant gases, but limited research is available on the mode of NP application and their effectiveness in gaseous emission reduction. In this study, zinc silica nanogel (ZnSNL), copper silica nanogel (CuSNL), and N-acetyl cysteine (NACL) coated zinc oxide quantum dot (Qdot) NPs were compared to a control lacking NPs. All three NPs tested significantly reduced gas production and concentrations compared to non-treated manure. Overall, cumulative gas volumes were reduced by 92.73%-95.83%, and concentrations reduced by 48.98%-99.75% for H2S, and 20.24%-99.82% for GHGs. Thus, application of NPs is a potential treatment option for mitigating pollutant and GHG emissions from anaerobically stored manure.

Fixed-Quat: An Attractive Nonmetal Alternative to Copper Biocides against Plant Pathogens.

In this paper, we report a nonphytotoxic bactericide and fungicide formulation containing a composite of silica and quaternary ammonium compound (quat). The composite material was prepared using an acid-catalyzed sol-gel method. Positively charged quat was associated with a negatively charged silica-gel matrix, producing a stable suspension of fixed-quat gel (FQ-G). The morphology of FQ-G and the interaction of quat with silica were characterized using SEM and FTIR, respectively. Silica gel significantly reduced quat phytotoxicity when tested at 500 and 1000 µg/mL foliar-application rates. The in vitro antimicrobial efficacy of FQ-G was evaluated against Xanthomonas alfalfae, Pseudomonas syringae, and Clavibacter michiganensis, showing comparable efficacies to that of quat itself. In field conditions, its efficacy in controlling the bacterial and fungal diseases citrus canker, scab, and melanose on 'Ray Ruby' red grapefruit was evaluated. Foliar application rates at 100 and 200 µg/mL provided comparable disease control to those of several copper standards, demonstrating the potential for use as an alternative agricultural biocide.

Antimicrobial Magnesium Hydroxide Nanoparticles As an Alternative to Cu Biocide for Crop Protection.

In agriculture, prolonged use of copper biocides increases the risk of development of Cu resistance and its accumulation in soil, demanding an alternative. In this paper, we report antimicrobial magnesium hydroxide nanoparticles (NPs) as an alternative to Cu biocides with low cytotoxicity. To improved bioavailability, Mg hydroxide NPs were synthesized followed by coating with water-soluble capping agents, trisodium citrate (zeta potential, ξ = -22 mV) or betaine (ξ = +35 mV). Electron microscopy study confirmed the formation of ∼10-nm-sized cubical NPs with citrate and ∼100-nm-sized lamellar NPs with betaine. As-synthesized Mg hydroxide NPs inhibited bacterial growth of X. alfalfae, P. syringae, and E. coli within 4 h. Significant bacterial growth inhibition and killing were observed at 24 h post-treatment. Phytotoxicity studies on tomato plants showed no significant tissue injury. Therefore, Mg hydroxide NPs have the potential to serve as a Cu alternative.

Hydrothermally Treated Chitosan Hydrogel Loaded with Copper and Zinc Particles as a Potential Micronutrient-Based Antimicrobial Feed Additive.

Large-scale use of antibiotics in food animal farms as growth promoters is considered as one of the driving factors behind increasing incidence of microbial resistance. Several alternatives are under investigation to reduce the amount of total antibiotics used in order to avoid any potential transmission of drug resistant microbes to humans through food chain. Copper sulfate and zinc oxide salts are used as feed supplement as they exhibit antimicrobial properties in addition to being micronutrients. However, higher dosage of copper and zinc (often needed for growth promoting effect) to animals is not advisable because of potential environmental toxicity arising from excreta. Innovative strategies are needed to utilize the complete potential of trace minerals as growth promoting feed supplements. To this end, we describe here the development and preliminary characterization of hydrothermally treated chitosan as a delivery vehicle for copper and zinc nanoparticles that could act as a micronutrient-based antimicrobial feed supplement. Material characterization studies showed that hydrothermal treatment makes a chitosan hydrogel that rearranged to capture the copper and zinc metal particles. Systemic antimicrobial assays showed that this chitosan biopolymer matrix embedded with copper (57.6 µg/ml) and zinc (800 µg/ml) reduced the load of model gut bacteria (target organisms of growth promoting antibiotics), such as Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, and Lactobacillus fermentum under in vitro conditions. Particularly, the chitosan/copper/zinc hydrogel exhibited significantly higher antimicrobial effect against L. fermentum, one of the primary targets of antibiotic growth promoters. Additionally, the chitosan matrix ameliorated the cytotoxicity levels of metal supplements when screened against a murine macrophage cell line RAW 264.7 and in TE-71, a murine thymic epithelial cell line. In this proof-of-concept study, we show that by using chitosan as a delivery platform, micronutrient-based metal feed additives could be used to minimize the undesirable levels of microbial population without causing significant cytotoxic effect under in vitro conditions. These findings provide the platform for further studies in target animal models to quantify the required physiological concentrations of copper and zinc when delivered via a chitosan hydrogel platform to elicit a growth promoting effect without causing any toxicity.

Peptide nucleic acids inhibit growth of Brucella suis in pure culture and in infected murine macrophages.

Peptide nucleic acids (PNAs) are single-stranded, synthetic nucleic acid analogues containing a pseudopeptide backbone in place of the phosphodiester sugar-phosphate. When PNAs are covalently linked to cell-penetrating peptides (CPPs) they readily penetrate the bacterial cell envelope, inhibit expression of targeted genes and cause growth inhibition both of Gram-positive and Gram-negative bacteria. However, the effectiveness of PNAs against Brucella, a facultative intracellular bacterial pathogen, was unknown. The susceptibility of a virulent Brucella suis strain to a variety of PNAs was assessed in pure culture as well as in murine macrophages. The studies showed that some of the PNAs targeted to Brucella genes involved in DNA (polA, dnaG, gyrA), RNA (rpoB), cell envelope (asd), fatty acid (kdtA, acpP) and protein (tsf) synthesis inhibit the growth of B. suis in culture and in macrophages after 24 h of treatment. PNA treatment inhibited Brucella growth by interfering with gene expression in a sequence-specific and dose-dependent manner at micromolar concentrations. The most effective PNA in broth culture was that targeting polA at ca. 12 µM. In contrast, in B. suis-infected macrophages, the most effective PNAs were those targeting asd and dnaG at 30 µM; both of these PNAs had little inhibitory effect on Brucella in broth culture. The polA PNA that inhibits wild-type B. suis also inhibits the growth of wild-type Brucella melitensis 16M and Brucella abortus 2308 in culture. This study reveals the potential usefulness of antisense PNA constructs as novel therapeutic agents against intracellular Brucella.

Over-expression of homologous antigens in a leucine auxotroph of Brucella abortus strain RB51 protects mice against a virulent B. suis challenge.

Infection by members of the Gram-negative bacterial genus Brucella causes brucellosis in a variety of mammals. Brucellosis in swine remains a challenge, as there is no vaccine in the USA approved for use in swine against brucellosis. Here, we developed an improved recombinant Brucella abortus vaccine strain RB51 that could afford protection against Brucella suis infection by over-expressing genes encoding homologous proteins: L7/L12 ribosomal protein, Cu/Zn superoxide dismutase [SOD] and glycosyl-transferase [WboA]. Using strain RB51leuB as a platform and an antibiotic-resistance marker free plasmid, strains RB51leuB/SOD, RB51leuB/SOD/L7/L12 and RB51leuB/SOD/WboA were constructed to over-express the antigens: SOD alone, SOD and ribosomal protein L7/L12 or SOD and glycosyl-transferase, respectively. The ability of these vaccine candidates to protect against a virulent B. suis challenge were evaluated in a mouse model. All vaccine groups protected mice significantly (P<0.05) when compared to the control group. Within the vaccine groups, the mice vaccinated with strain RB51leuB/SOD/WboA were significantly better protected than those that were vaccinated with either strain RB51leuB/SOD or RB51leuB/SOD/L7/L12. These results suggest that Brucella antigens can be over-expressed in strain RB51leuB and elicit protective immune responses against brucellosis. Since the plasmid over-expressing homologous antigens does not carry an antibiotic resistance gene, it complies with federal regulations and therefore could be used to develop safer multi-species vaccines for prevention of brucellosis caused by other species of Brucella.

Brucella abortus strain RB51 leucine auxotroph as an environmentally safe vaccine for plasmid maintenance and antigen overexpression.

To avoid potentiating the spread of an antibiotic resistance marker, a plasmid expressing a leuB gene and a heterologous antigen, green fluorescent protein (GFP), was shown to complement a leucine auxotroph of cattle vaccine strain Brucella abortus RB51, which protected CD1 mice from virulent B. abortus 2308 and elicited GFP antibodies.

Brucella suis urease encoded by ure1 but not ure2 is necessary for intestinal infection of BALB/c mice.

BACKGROUND: In prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure1 and ure2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing. RESULTS: The deduced amino acid sequence of urease-alpha subunits of operons-1 and -2 exhibited substantial identity with the structural ureases of alpha- and beta-proteobacteria, Gram-positive and Gram-negative bacteria, fungi, and higher plants. Four ure deficient strains were generated by deleting one or more of the genes encoding urease subunits of B. suis strain 1330 by allelic exchange: strain 1330Deltaure1K (generated by deleting ureD and ureA in ure1 operon), strain 1330Deltaure2K (ureB and ureC in ure2 operon), strain 1330Deltaure2C (ureA, ureB, and ureC in ure2 operon), and strain 1330Deltaure1KDeltaure2C (ureD and ureA in ure1 operon and ureA, ureB, and ureC in ure2 operon). When grown in urease test broth, strains 1330, 1330Deltaure2K and 1330Deltaure2C displayed maximal urease enzyme activity within 24 hours, whereas, strains 1330Deltaure1K and 1330Deltaure1KDeltaure2C exhibited zero urease activity even 96 h after inoculation. Strains 1330Deltaure1K and 1330Deltaure1KDeltaure2C exhibited slower growth rates in tryptic soy broth relative to the wild type strain 1330. When the BALB/c mice were infected intraperitoneally with the strains, six weeks after inoculation, the splenic recovery of the ure deficient strains did not differ from the wild type. In contrast, when the mice were inoculated by gavage, one week after inoculation, strain 1330Deltaure1KDeltaure2C was cleared from livers and spleens while the wild type strain 1330 was still present. All B. suis strains were killed when they were incubated in-vitro at pH 2.0. When the strains were incubated at pH 2.0 supplemented with 10 mM urea, strain 1330Deltaure1K was completely killed, strain 1330Deltaure2C was partially killed, but strains 1330 and 1330Deltaure2K were not killed. CONCLUSION: These findings suggest that the ure1 operon is necessary for optimal growth in culture, urease activity, resistance against low-pH killing, and in vivo persistence of B. suis when inoculated by gavage. The ure2 operon apparently enhances the resistance to low-pH killing in-vitro.