Development of phenyl-urea-based small molecules that target penicillin-binding protein 4.

Staphylococcus aureus has the ability to invade cortical bone osteocyte lacuno-canalicular networks (OLCNs) and cause osteomyelitis. It was recently established that the cell wall transpeptidase, penicillin-binding protein 4 (PBP4), is crucial for this function, with pbp4 deletion strains unable to invade OLCNs and cause bone pathogenesis in a murine model of S. aureus osteomyelitis. Moreover, PBP4 has recently been found to modulate S. aureus resistance to ß-lactam antibiotics. As such, small molecule inhibitors of S. aureus PBP4 may represent dual functional antimicrobial agents that limit osteomyelitis and/or reverse antibiotic resistance. A high throughput screen recently revealed that the phenyl-urea 1 targets PBP4. Herein, we describe a structure-activity relationship (SAR) study on 1. Leveraging in silico docking and modeling, a set of analogs was synthesized and assessed for PBP4 inhibitory activities. Results revealed a preliminary SAR and identified lead compounds with enhanced binding to PBP4, more potent antibiotic resistance reversal, and diminished PBP4 cell wall transpeptidase activity in comparison to 1.

Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides.

Helicobacter pylori is the primary cause of 78% of gastric cancer cases, providing an opportunity to prevent cancer by controlling a single bacterial pathogen within the complex gastric microbiota. We developed highly selective antimicrobial agents against H. pylori by fusing an H. pylori-binding guide peptide (MM1) to broad-spectrum antimicrobial peptides. The common dairy probiotic Lactococcus lactis was then engineered to secrete these guided antimicrobial peptides (gAMPs). When co-cultured in vitro with H. pylori, the gAMP probiotics lost no toxicity compared to unguided AMP probiotics against the target, H. pylori, while losing >90% of their toxicity against two tested off-target bacteria. To test binding to H. pylori, the MM1 guide was fused to green fluorescent protein (GFP), resulting in enhanced binding compared to unguided GFP as measured by flow cytometry. In contrast, MM1-GFP showed no increased binding over GFP against five different off-target bacteria. These highly selective gAMP probiotics were then tested by oral gavage in mice infected with H. pylori. As a therapy, the probiotics outperformed antibiotic treatment, effectively eliminating H. pylori in just 5 days, and also protected mice from challenge infection as a prophylactic. As expected, the gAMP probiotics were as toxic against H. pylori as the unguided AMP probiotics. However, a strong rebound in gastric species diversity was found with both the selective gAMP probiotics and the non-selective AMP probiotics. Eliminating the extreme microbial dysbiosis caused by H. pylori appeared to be the major factor in diversity recovery. IMPORTANCE Alternatives to antibiotics in the control of Helicobacter pylori and the prevention of gastric cancer are needed. The high prevalence of H. pylori in the human population, the induction of microbial dysbiosis by antibiotics, and increasing antibiotic resistance call for a more sustainable approach. By selectively eliminating the pathogen and retaining the commensal community, H. pylori control may be achieved without adverse health outcomes. Antibiotics are typically used as a therapeutic post-infection, but a more targeted, less disruptive approach could be used as a long-term prophylactic against H. pylori or, by extension, against other gastrointestinal pathogens. Furthermore, the modular nature of the guided antimicrobial peptide (gAMP) technology allows for the substitution of different guides for different pathogens and the use of a cocktail of gAMPs to avoid the development of pathogen resistance.

Genetic Determinants of Acinetobacter baumannii Serum-Associated Adaptive Efflux-Mediated Antibiotic Resistance.

Acinetobacter baumannii is a nosocomial pathogen of serious healthcare concern that is becoming increasingly difficult to treat due to antibiotic treatment failure. Recent studies have revealed that clinically defined antibiotic-susceptible strains upregulate the expression of a repertoire of putative drug efflux pumps during their growth under biologically relevant conditions, e.g., in human serum, resulting in efflux-associated resistance to physiologically achievable antibiotic levels within a patient. This phenomenon, termed Adaptive Efflux Mediated Resistance (AEMR), has been hypothesized to account for one mechanism by which antibiotic-susceptible A. baumannii fails to respond to antibiotic treatment. In the current study, we sought to identify genetic determinants that contribute to A. baumannii serum-associated AEMR by screening a transposon mutant library for members that display a loss of the AEMR phenotype. Results revealed that mutation of a putative pirin-like protein, YhaK, results in a loss of AEMR, a phenotype that could be complemented by a wild-type copy of the yhaK gene and was verified in a second strain background. Ethidium bromide efflux assays confirmed that the loss of AEMR phenotype due to pirin-like protein mutation correlated with reduced overarching efflux capacity. Further, flow cytometry and confocal microscopy measures of a fluorophore 7-(dimethylamino)-coumarin-4-acetic acid (DMACA)-tagged levofloxacin isomer, ofloxacin, further verified that YhaK mutation reduces AEMR-mediated antibiotic efflux. RNA-sequencing studies revealed that YhaK may be required for the expression of multiple efflux-associated systems, including MATE and ABC families of efflux pumps. Collectively, the data indicate that the A. baumannii YhaK pirin-like protein plays a role in modulating the organism's adaptive efflux-mediated resistance phenotype.

Identification of Staphylococcus aureus Penicillin Binding Protein 4 (PBP4) Inhibitors.

Methicillin-resistant Staphylococcus aureus (MRSA) is a global healthcare concern. Such resistance has historically been attributed to the acquisition of mecA (or mecC), which encodes an alternative penicillin binding protein, PBP2a, with low ß-lactam affinity. However, recent studies have indicated that penicillin binding protein 4 (PBP4) is also a critical determinant of S. aureus methicillin resistance, particularly among community-acquired MRSA strains. Thus, PBP4 has been considered an intriguing therapeutic target as corresponding inhibitors may restore MRSA ß-lactam susceptibility. In addition to its role in antibiotic resistance, PBP4 has also recently been shown to be required for S. aureus cortical bone osteocyte lacuno-canalicular network (OLCN) invasion and colonization, providing the organism with a niche for re-occurring bone infection. From these perspectives, the development of PBP4 inhibitors may have tremendous impact as agents that both reverse methicillin resistance and inhibit the organism's ability to cause chronic osteomyelitis. Accordingly, using a whole-cell high-throughput screen of a 30,000-member small molecule chemical library and secondary assays we identified putative S. aureus PBP4 inhibitors. Quantitative reverse transcriptase mediated PCR and PBP4 binding assays revealed that hits could be further distinguished as compounds that reduce PBP4 expression versus compounds that are likely to affect the protein's function. We also showed that 6.25 µM (2.5 µg/mL) of the lead candidate, 9314848, reverses the organism's PBP4-dependent MRSA phenotype and inhibits its ability to traverse Microfluidic-Silicon Membrane-Canalicular Arrays (µSiM-CA) that model the OLCN orifice. Collectively, these molecules may represent promising potential as PBP4-inhibitors that can be further developed as adjuvants for the treatment of MRSA infections and/or osteomyelitis prophylactics.

Multifunctional Surface, Subsurface, and Systemic Therapeutic (MS3T) Formulation for the Control of Citrus Canker.

A multifunctional surface, subsurface and systemic therapeutic (MS3T) formulation comprised of two bactericides, both didecyldimethylammonium chloride (DDAC) and a zinc (Zn)-chelate, was developed as an alternative to copper pesticides for crop protection. Agricultural grade chemicals were used to prepare MS3T formulations. Minimal inhibitory concentration (MIC) was determined to be tested in vitro against Xanthomonas alfalfae subsp. citrumelonis (herein called Xa), Escherichia coli (E. coli), and Pseudomonas syringae (Ps). Assessment of the phytotoxic potential was carried out on tomato under greenhouse conditions. Moreover, field trials were conducted during three consecutive years on grapefruit (Chrysopelea paradise) groves to evaluate efficacy against citrus canker (Xanthomonas citri subsp. citri), scab (Elsinoe fawcetti), and melanose (Diaporthe citri). In addition to disease control, improvements to both fruit yield and quality were observed likely due to the nutritional activity of MS3T via the sustained release of plant nutrients (Zn and nitrogen). Zn residues of leaf tissues were analyzed via atomic absorption spectroscopy (AAS) at various time points before and after MS3T foliar applications throughout the duration of the 2018 field trial. Field trial results demonstrated MS3T to be an effective alternative to copper (Cu)-based formulations for the control of citrus canker.

Copper-fixed quat: a hybrid nanoparticle for application as a locally systemic pesticide (LSP) to manage bacterial spot disease of tomato.

The development of bacterial tolerance against pesticides poses a serious threat to the sustainability of food production. Widespread use of copper (Cu)-based products for plant disease management has led to the emergence of copper-tolerant pathogens such as Xanthomonas perforans (X. perforans) strains in Florida, which is very destructive to the tomato (Solanum lycopersicum) industry. In this study, we report a hybrid nanoparticle (NP)-based system, coined Locally Systemic Pesticide (LSP), which has been designed for improved efficacy compared to conventional Cu-based bactericides against Cu-tolerant X. perforans. The silica core-shell structure of LSP particles makes it possible to host ultra-small Cu NPs (<10 nm) and quaternary ammonium (Quat) molecules on the shell. The morphology, release of Cu and Quat, and subsequent in vitro antimicrobial properties were characterized for LSP NPs with core diameters from 50 to 600 nm. A concentration of 4 µg mL-1 (Cu): 1 µg mL-1 (Quat) was found to be sufficient to inhibit the growth of Cu-tolerant X. perforans compared to 100 µg mL-1 (metallic Cu) required with standard Kocide 3000. Wetting properties of LSP exhibited contact angles below 60°, which constitutes a significant improvement from the 90° and 85° observed with water and Kocide 3000, respectively. The design was also found to provide slow Cu release to the leaves upon water washes, and to mitigate the phytotoxicity of water-soluble Cu and Quat agents. With Cu and Quat bound to the LSP silica core-shell structure, no sign of phytotoxicity was observed even at 1000 µg mL-1 (Cu). In greenhouse and field experiments, LSP formulations significantly reduced the severity of bacterial spot disease compared to the water control. Overall, the study highlights the potential of using LSP particles as a candidate for managing tomato bacterial spot disease and beyond.

Optimization of 4-Substituted Benzenesulfonamide Scaffold To Reverse Acinetobacter baumannii Serum-Adaptive Efflux Associated Antibiotic Tolerance.

Acinetobacter baumannii is a nosocomial pathogen of urgent concern for public health due to rising rates of multidrug and pandrug resistance. In the context of environmental cues such as growth in human serum, A. baumannii is known to display adaptive efflux, in which a multitude of efflux-associated genes are upregulated, resulting in efflux-mediated drug tolerance in strains that are otherwise susceptible to antibiotic therapy. Previously, we identified a sulfonamide-containing scaffold molecule (ABEPI1) that reversed serum-associated antibiotic tolerance in A. baumannii. Herein, we present structure-activity relationship studies on 29 newly synthesized analogues. These molecules were characterized for their ability to potentiate multiple antibiotics in serum, reduce serum-associated ethidium bromide efflux and depolarize bacterial cell membranes. In addition, they were assessed for toxicity to mammalian cells. Collectively, these molecules may represent promising potential adjuvants for use in combination with new and existing antibiotics to treat A. baumannii bacterial infections.

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.

Silica-quaternary ammonium "Fixed-Quat" nanofilm coated fiberglass mesh for water disinfection and harmful algal blooms control.

Intensification of pollution loading worldwide has promoted an escalation of different types of disease-causing microorganisms, such as harmful algal blooms (HABs), instigating detrimental impacts on the quality of receiving surface waters. Formation of unwanted disinfection by-products (DBPs) resulting from conventional disinfection technologies reveals the need for the development of new sustainable alternatives. Quaternary Ammonium Compounds (QACs) are cationic surfactants widely known for their effective biocidal properties at the ppm level. In this study, a novel silica-based antimicrobial nanofilm was developed using a composite of silica-modified QAC (Fixed-Quat) and applied to a fiberglass mesh as an active surface via sol-gel technique. The synthesized Fixed-Quat nanocoating was found to be effective against E. coli with an inactivation rate of 1.3 × 10-3 log reduction/cm min. The Fixed-Quat coated fiberglass mesh also demonstrated successful control of Microcystis aeruginosa with more than 99% inactivation after 10 hr of exposure. The developed antimicrobial mesh was also evaluated with wild-type microalgal species collected in a water body experiencing HABs, obtaining a 97% removal efficiency. Overall, the silica-functionalized Fixed-Quat nanocoating showed promising antimicrobial properties for water disinfection and HABs control, while decreasing concerns related to DBPs formation and the possible release of toxic nanomaterials into the environment.

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.

Multimodal Generally Recognized as Safe ZnO/Nanocopper Composite: A Novel Antimicrobial Material for the Management of Citrus Phytopathogens.

Copper (Cu) bactericides/fungicides are used extensively for crop protection in agriculture. Concerns for Cu accumulation in soil, Cu leaching into the surrounding ecosystem, and development of Cu resistance in phytopathogenic bacteria are evident. While there is no suitable alternative to Cu available to date for agricultural uses, it is possible to reduce Cu per application by supplementing with Zn and improving Cu bioavailability using nanotechnology. We have prepared a non-phytotoxic composite material consisting of generally recognized as safe ZnO 800 particles and nanocopper-loaded silica gel (ZnO-nCuSi). The morphology of the ZnO-nCuSi material was characterized using scanning electron microscopy, showing ZnO particles dispersed in the silica gel matrix. ZnO-nCuSi demonstrated strong in vitro antimicrobial properties against several model plant bacterial species. Two consecutive year field efficacy results showed that agri-grade ZnO-nCuSi was effective in controlling citrus canker disease at less than half the metallic rate of the commercial cuprous oxide/zinc oxide pesticide.

Copper (Cu)-silica nanocomposite containing valence-engineered Cu: a new strategy for improving the antimicrobial efficacy of Cu biocides.

Copper (Cu) compounds are widely used as antibacterial/antifungal agents for protecting food crops. Prolonged use of Cu biocides would lead to undesirable Cu levels in agricultural soil. In the absence of a suitable alternative, prudent use of Cu biocides is required. This paper reports for the first time a composite material of sol-gel silica host matrix loaded with mixed-valence Cu as an alternative to conventional biocides. In this composite material, Cu is present in different oxidation states. The hydrophilic silica matrix serves as a water-dispersible delivery vehicle for antimicrobial Cu. It is hypothesized that a mixed-valence Cu system, specifically enriched with Cu(0) and Cu(I), will exhibit enhanced antimicrobial efficacy over traditional Cu(II) compounds. Materials were characterized by high-resolution transmission electron microscopy and X-ray photoelectron spectroscopy for the determination of particle size, morphology, crystallinity, and Cu oxidation states. Antimicrobial studies against Xanthomonas alfalfae and Escherichia coli (minimum inhibitory concentration) showed improved efficacy in MV-CuSiNG treatment compared to CuSiNG and other controls. Phytotoxicity studies performed (in Vinca sp. and Hamlin orange) under greenhouse conditions showed that the newly prepared nanocomposite is safe for plants, demonstrating potential usefulness of the material in agricultural biocides.