Microstructure-dependent particulate filtration using multifunctional metallic nanowire foams.

The COVID-19 pandemic has shown the urgent need for the development of efficient, durable, reusable and recyclable filtration media for the deep-submicron size range. Here we demonstrate a multifunctional filtration platform using porous metallic nanowire foams that are efficient, robust, antimicrobial, and reusable, with the potential to further guard against multiple hazards. We have investigated the foam microstructures, detailing how the growth parameters influence the overall surface area and characteristic feature size, as well as the effects of the microstructures on the filtration performance. Nanogranules deposited on the nanowires during electrodeposition are found to greatly increase the surface area, up to 20 m2 g-1. Surprisingly, in the high surface area regime, the overall surface area gained from the nanogranules has little correlation with the improvement in capture efficiency. However, nanowire density and diameter play a significant role in the capture efficiency of PM0.3 particles, as do the surface roughness of the nanowire fibers and their characteristic feature sizes. Antimicrobial tests on the Cu foams show a >99.9995% inactivation efficiency after contacting the foams for 30 seconds. These results demonstrate promising directions to achieve a highly efficient multifunctional filtration platform with optimized microstructures.

Selection of mutant Listeria phages under food-relevant conditions can enhance application potential.

Bacteriophages are viruses that infect and kill bacteria. Currently, phage products are available for the control of the pathogen Listeria monocytogenes in food products in the United States. In this study, we explore whether experimental evolution can be used to generate phages with improved abilities to function under specific food-relevant conditions. Ultra-pasteurized oat and whole milk were chosen as test matrices as they represent different food groups, yet have similar physical traits and macronutrient composition. We showed that (i) wild-type phage LP-125 infection kinetics are different in the two matrices and (ii) LP-125 has a significantly higher burst size in oat milk. From this, we attempted to evolve LP-125 to have improved infection kinetics in whole milk. Ancestral LP-125 was passaged through 10 rounds of amplification in milk conditions. Plaque-purified DNA samples from milk-selected phages were isolated and sequenced, and mutations present in the isolated phages were identified. We found two nonsynonymous substitutions in LP125_108 and LP125_112 genes, which encode putative baseplate-associated glycerophosphoryl diester phosphodiesterase and baseplate protein, respectively. Protein structural modeling showed that the substituted amino acids in the mutant phages are predicted to localize to surface-exposed helices on the corresponding structures, which might affect the surface charge of proteins and their interaction with the bacterial cell. The phage containing the LP125_112 mutation adsorbed significantly faster than the ancestral phage in both oat and whole milk. Follow-up experiments suggest that fat content may be a key factor for the expression of the phenotype of this mutation. IMPORTANCE Bacteriophages are one of the tools available to control the foodborne pathogen, Listeria monocytogenes. Phage products must work under a broad range of food conditions to be an effective control for L. monocytogenes. Here, we show that the experimental evolution of phages can be used to generate new phages with phenotypes useful under specific conditions. We used this approach to select for a mutant phage that more efficiently binds to L. monocytogenes that is grown in whole milk and oat milk. We show that the fat content of these milks is necessary for the expression of this phenotype. Our findings show that experimental evolution can be used to select for improved phages with better performance under specific conditions. This approach has the potential to support the development of condition-specific phage-based biocontrols in the food industry.

BMaps: A Web Application for Fragment-Based Drug Design and Compound Binding Evaluation.

Fragment-based drug design uses data about where, and how strongly, small chemical fragments bind to proteins, to assemble new drug molecules. Over the past decade, we have been successfully using fragment data, derived from thermodynamically rigorous Monte Carlo fragment-protein binding simulations, in dozens of preclinical drug programs. However, this approach has not been available to the broader research community because of the cost and complexity of doing simulations and using design tools. We have developed a web application, called BMaps, to make fragment-based drug design widely available with greatly simplified user interfaces. BMaps provides access to a large repository (>550) of proteins with 100s of precomputed fragment maps, druggable hot spots, and high-quality water maps. Users can also employ their own structures or those from the Protein Data Bank and AlphaFold DB. Multigigabyte data sets are searched to find fragments in bondable orientations, ranked by a binding-free energy metric. The designers use this to select modifications that improve affinity and other properties. BMaps is unique in combining conventional tools such as docking and energy minimization with fragment-based design, in a very easy to use and automated web application. The service is available at https://www.boltzmannmaps.com.

Characterization of a Diverse Collection of Salmonella Phages Isolated from Tennessee Wastewater.

Background: Salmonella enterica is one of the most prevalent bacterial foodborne pathogens. Salmonella phages are currently used in biocontrol applications and have potential for use as therapeutics. Materials and Methods: Phages were enriched and purified from a diversity of Salmonella host isolates. Morphology was determined with transmission electron microscopy, host ranges were characterized using an efficiency of plaquing assay, and comparative genomic analysis was performed to determine taxonomy. Results: Ten phages were isolated and characterized. Phages showed activity against 23 out of the 24 Salmonella serovars evaluated. Two phages also showed activity against Escherichia coli strain B. Phages belonged to five different genera (Ithacavirus, Gelderlandvirus, Kuttervirus, Tlsvirus, and Epseptimavirus), two established species, and eight novel species. Conclusions: The phages described here further demonstrate the diversity of S. enterica phages present in wastewater effluent. This work contributes a collection of characterized phages from eastern Tennessee that may be of use in future phage-based applications targeting S. enterica.

Soil Collected from a Single Great Smoky Mountains Trail Contains a Diversity of Listeria monocytogenes and Listeria spp.

Listeria monocytogenes, a foodborne pathogen, and other Listeria spp. are present in natural environments. Isolating and characterizing strains from natural reservoirs can provide insight into the prevalence and diversity of Listeria spp. in these environments, elucidate their contribution to contamination of agricultural and food processing environments and food products, and lead to the discovery of novel species. In this study, we evaluated the diversity of Listeria spp. isolated from soil in a small region of the Great Smoky Mountains National Park, the most biodiverse national park in the U.S. National Park system. Of the 17 Listeria isolates recovered, whole-genome sequencing revealed that 14 were distinct strains. The strains represented a diversity of Listeria species (L. monocytogenes [n = 9], L. cossartiae subsp. cossartiae [n = 1], L. marthii [n = 1], L. booriae [n = 1], and a potentially novel Listeria sp. [n = 2]), as well as a diversity of sequence types based on multilocus sequence typing (MLST) and core genome MLST, including many novel designations. The isolates were not closely related (≥99.99% average nucleotide identity) to any isolates in public databases (NCBI, PATRIC), which also indicated novelty. The Listeria samples isolated in this study were collected from high-elevation sites near a creek that ultimately leads to the Mississippi River; thus, Listeria present in this natural environment could potentially travel downstream to a large region that includes portions of nine southeastern and midwestern U.S. states. This study provides insight into the diversity of Listeria spp. in the Great Smoky Mountains and indicates that this environment is a reservoir of novel Listeria spp. IMPORTANCE Listeria monocytogenes is a foodborne pathogen that can cause serious systemic illness that, although rare, usually results in hospitalization and has a relatively high mortality rate compared to other foodborne pathogens. Identification of novel and diverse Listeria spp. can provide insights into the genomic evolution, ecology, and evolution and variance of pathogenicity of this genus, especially in natural environments. Comparing L. monocytogenes and Listeria spp. isolates from natural environments, such as those recovered in this study, to contamination and/or outbreak strains may provide more information about the original natural sources of these strains and the pathways and mechanisms that lead to contamination of food products and agricultural or food processing environments.

Antimicrobial properties of a multi-component alloy.

High traffic touch surfaces such as doorknobs, countertops, and handrails can be transmission points for the spread of pathogens, emphasizing the need to develop materials that actively self-sanitize. Metals are frequently used for these surfaces due to their durability, but many metals also possess antimicrobial properties which function through a variety of mechanisms. This work investigates metallic alloys comprised of several metals which individually possess antimicrobial properties, with the target of achieving broad-spectrum, rapid sanitation through synergistic activity. An entropy-motivated stabilization paradigm is proposed to prepare scalable alloys of copper, silver, nickel and cobalt. Using combinatorial sputtering, thin-film alloys were prepared on 100 mm wafers with ≈50% compositional grading of each element across the wafer. The films were then annealed and investigated for alloy stability. Antimicrobial activity testing was performed on both the as-grown alloys and the annealed films using four microorganisms-Phi6, MS2, Bacillus subtilis and Escherichia coli-as surrogates for human viral and bacterial pathogens. Testing showed that after 30 s of contact with some of the test alloys, Phi6, an enveloped, single-stranded RNA bacteriophage that serves as a SARS-CoV-2 surrogate, was reduced up to 6.9 orders of magnitude (> 99.9999%). Additionally, the non-enveloped, double-stranded DNA bacteriophage MS2, and the Gram-negative E. coli and Gram-positive B. subtilis bacterial strains showed a 5.0, 6.4, and 5.7 log reduction in activity after 30, 20 and 10 min, respectively. Antimicrobial activity in the alloy samples showed a strong dependence on the composition, with the log reduction scaling directly with the Cu content. Concentration of Cu by phase separation after annealing improved activity in some of the samples. The results motivate a variety of themes which can be leveraged to design ideal antimicrobial surfaces.

Analysis of Derivatized Wall Teichoic Acids Confirms that a Mutation in Phage-Resistant Listeria monocytogenes Impacts Rhamnose Decoration.

Listeria monocytogenes is a Gram-positive foodborne pathogen that causes listeriosis, an illness that may result in serious health consequences or death. Wall teichoic acids (WTAs) are external cell wall glycopolymers that play many biological roles. Here, the WTA composition was determined for several phage-resistant mutant strains of L. monocytogenes. The strains included wild-type (WT) L. monocytogenes 10403S, and three phage-resistant mutant strains derived from 10403S, consisting of two well-characterized strains and one with unknown impact on cell physiology. Several WTA monomers were prepared from WT 10403S, as analytical standards. The WTA monomer fraction was then isolated from the mutant strains and the corresponding per-trimethylsilylated derivatives were analyzed by gas chromatography-flame ionization detection. WTA monomer, GlcNAc-Rha-Rbo, was detected in 10403S, and not detected in the phage-resistant strains known to lack rhamnose and N-acetylglucosamine; although the expected monomers GlcNAc-Rbo and Rha-Rbo were detected, respectively. GlcNAc-Rha-Rbo was also detected in strain UTK P1-0001, which is known to impact phage adsorption through an undetermined mechanism, albeit at a lower intensity than the WT 10403S, which is consistent with partial loss of function through truncation in RmlC protein. WTA monomers were also detected in an unpurified cell pellet, demonstrating that the method employed in this study can be used to rapidly screen L. monocytogenes without laborious WTA purification. This study lays the groundwork for future studies on WTA compositional analysis to support genomic data, and serves as a foundation for the development of new rapid methods for WTA compositional analysis.

Phenotypic characterization and analysis of complete genomes of two distinct strains of the proposed species "L. swaminathanii".

Recently, a new Listeria species, "Listeria swaminathanii", was proposed. Here, we phenotypically and genotypically characterize two additional strains that were previously obtained from soil samples and compare the results to the type strain. Complete genomes for both strains were assembled from hybrid Illumina and Nanopore sequencing reads and annotated. Further genomic analysis including average nucleotide identity (ANI) and detection of mobile genetic elements and genes of interest (e.g., virulence-associated) were conducted. The strains showed 98.7-98.8% ANI with the type strain. The UTK C1-0015 genome contained a partial monocin locus and a plasmid, while the UTK C1-0024 genome contained a full monocin locus and a prophage. Phenotypic characterization consistent with those performed on the proposed type strain was conducted to assess consistency of phenotypes across a greater diversity of the proposed species (n = 3 instead of n = 1). Only a few findings were notably different from those of the type strain, such as catalase activity, glycerol metabolism, starch metabolism, and growth at 41 °C. This study further expands our understanding of this newly proposed sensu stricto Listeria species.

Phylogeny of the Bacillus altitudinis Complex and Characterization of a Newly Isolated Strain with Antilisterial Activity.

ABSTRACT: Bacillus strain UTK D1-0055 was isolated from a laboratory environment and appeared to have antilisterial activity. The genome was sequenced, the strain was identified as Bacillus altitudinis, and a high-quality complete annotated genome was produced. The taxonomy was evaluated for this and related Bacillus species (B. aerophilus, B. pumilus, B. safensis, B. stratosphericus, and B. xiamenensis) because the taxonomy is unclear and contains errors in public databases such as NCBI. The included strains grouped into seven clusters based on average nucleotide identity. Strains designated as B. aerophilus, B. altitudinis, and B. stratosphericus grouped together in the cluster containing the B. altitudinis type strain, suggesting that these three species should be considered a single species, B. altitudinis. The antimicrobial activity of UTK D1-0055 was evaluated against a panel of 15 Listeria strains (nine Listeria monocytogenes serotypes, Listeria innocua, and Listeria marthii), other foodborne pathogens (six Salmonella enterica serotypes and Escherichia coli), and three representative fungi (Saccharomyces cerevisiae, Botrytis cinerea, and Hyperdermium pulvinatum). Antibacterial activity was observed against all Listeria strains, but no antibacterial effects were found against the other tested bacterial and fungal strains. Biosynthetic gene clusters were identified in silico that may be related to the observed antibacterial activity, and these clusters included genes that putatively encode bacteriocins and nonribosomally synthesized peptides. The B. altitudinis strain identified in this investigation had a broad range of antilisterial activity, suggesting that it and other related strains may be useful for biocontrol in the food industry.

Characterization of a Novel Group of Listeria Phages That Target Serotype 4b Listeria monocytogenes.

Listeria monocytogenes serotype 4b strains are the most prevalent clinical isolates and are widely found in food processing environments. Bacteriophages are natural viral predators of bacteria and are a promising biocontrol agent for L. monocytogenes. The aims of this study were to characterize phages that specifically infect serotype 4b strains and to assess their ability to inhibit the growth of serotype 4b strains. Out of 120 wild Listeria phages, nine phages were selected based on their strong lytic activity against the model serotype 4b strain F2365. These nine phages can be divided into two groups based on their morphological characteristics and host range. Comparison to previously characterized phage genomes revealed one of these groups qualifies to be defined as a novel species. Phages LP-020, LP-027, and LP-094 were selected as representatives of these two groups of phages for further characterization through one-step growth curve and inhibition of serotype 4b L. monocytogenes experiments. Listeria phages that target serotype 4b showed an inhibitory effect on the growth of F2365 and other serotype 4 strains and may be useful for biocontrol of L.monocytogenes in food processing environments.