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Listeria spp. are widely distributed environmental bacteria associated with human foodborne illness. The ability to detect and characterize Listeria strains in the natural environment will contribute to improved understanding of transmission routes of contamination. The current standard for surveillance and outbreak source attribution is whole-genome sequencing (WGS) of Listeria monocytogenes clinical isolates. Recently, metagenomic sequencing has also been explored as a tool for the detection of Listeria spp. in environmental samples. This study evaluated soil samples from four locations across altitudes ranging from 1,500 to 4,500 ft in the Nantahala National Forest in North Carolina, USA. Forty-two Listeria isolates were cultured and sequenced, and 12 metagenomes of soil bacterial communities were generated. These isolates comprised 14 distinct strains from five species, including Listeria cossartiae subsp. cayugensis ( n = 8; n represents the number of distinct strains), L. monocytogenes ( n = 3), " Listeria swaminathanii " (Lsw) ( n = 1), Listeria marthii ( n = 1), and Listeria booriae ( n = 1). Most strains ( n = 13) were isolated from lower altitudes (1,500 or 2,500 ft), while the L. swaminathanii strain was isolated from both higher (4,500 ft) and lower (1,500 ft) altitudes. Metagenomic analysis of soil described a reduction in both bacterial community diversity and relative abundance of Listeria spp. as the altitude increased. Soil pH and cation exchange capacity were positively correlated ( P < 0.05) with the abundance of Listeria spp. as detected by metagenomics. By integrating culture-independent metagenomics with culture-based WGS, this study advances current knowledge regarding distribution of Listeria spp. in the natural environment and suggests the potential for future use of culture-independent methods in tracking the transmission of foodborne pathogens., Importance: As a foodborne pathogen, Listeria continues to cause numerous illnesses in humans and animals. Studying the diversity and distribution of Listeria in soil is crucial for understanding potential sources of contamination and developing effective strategies to prevent foodborne outbreaks of listeriosis. Additionally, examining the ecological niches and survival mechanisms of Listeria in natural habitats provides insights into its persistence and adaptability, informing risk assessments and public health interventions. This research contributes to a broader understanding of microbial ecology and the factors influencing foodborne pathogen emergence, ultimately enhancing food safety and protecting public health. Moreover, using a metagenomic approach provides a detailed understanding of the soil microbial ecosystems, leading to more effective monitoring and control of foodborne pathogens. This study also highlights the potential for integrating metagenomics into routine surveillance systems for food safety in the near future., Competing Interests: The authors declare no conflict of interest.

Bacteriophage ("Phage") products are gaining interest in controlling foodborne pathogens as they are natural, specific, and can replicate at the site of contamination. One challenge in determining the efficacy of phage biocontrol is accounting for residual phages that may impact the recovery and the enumeration of surviving bacteria downstream from the treatment on food surface (FS) or food contact surface (FCS). Typically, the efficacy of a phage formulation is tested by applying it to a FS or FCS that has been pre-inoculated with the target pathogen and incubating the treatment for a set period of time. The food sample is transferred into a liquid buffer and stomached to release the surviving bacteria for their enumeration. During these final steps, there is a potential for residual phage to interact with bacterial survivors, which could affect the calculated efficacy of the phage. Limited studies demonstrated that bacterial reductions in these experiments occur specifically during treatment and not during sample recovery. This review highlights the importance of including appropriate controls to determine if residual phages are impacting bacterial recovery and the methods used to mitigate those impacts, which involves either neutralizing residual phages or separating residual phages from the surviving bacteria.

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 m 2 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 PM 0.3 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.

Poultry is the primary source of Campylobacter infections and severe campylobacteriosis cases are treated with macrolides and fluoroquinolones. However, these drugs are less effective against antimicrobial-resistant strains. Here, we investigated the prevalence of phenotypic antimicrobial resistance and associated resistance genetic determinants in Campylobacter isolates collected from human clinical (N = 123) and meat (N = 80) sources in Pennsylvania in 2017 and 2018. Our goal was to assess potential differences in the prevalence of antimicrobial resistance in Campylobacter isolated from human and poultry meat sources in Pennsylvania and to assess the accuracy of predicting antimicrobial resistance phenotypes based on resistance genotypes. We whole genome sequenced isolates and identified genetic resistance determinants using the National Antimicrobial Resistance Monitoring System Campylobacter AMR workflow v2.0 in GalaxyTrakr. Phenotypic antimicrobial susceptibility testing was carried out using the E-Test and Sensititre CAMPYCMV methods for human clinical and poultry meat isolates, respectively, and the results were interpreted using the EUCAST epidemiological cutoff values. The 193 isolates were represented by 85 MLST sequence types and 23 clonal complexes, suggesting high genetic diversity. Resistance to erythromycin was confirmed in 6% human and 4% meat isolates. Prevalence of ciprofloxacin resistance was significantly higher in human isolates as compared to meat isolates. A good concordance was observed between phenotypic resistance and the presence of the corresponding known resistance genetic determinants., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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&#95;108 and LP125&#95;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&#95;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., Competing Interests: The authors declare no conflict of interest.

This paper presents ClustFinder, a command line tool designed to automate clustering of genomes based on genomic distance. This tool will aid researchers and public health professionals in the identification of epidemiological clusters. Here, we demonstrate the usage of ClustFinder with example datasets. ClustFinder is available at github.com/Denes-Lab/ClustFinder., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

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 ., Competing Interests: No competing financial interests exist., (© Daniel W. Bryan et al., 2023; Published by Mary Ann Liebert, Inc.)

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.

Salmonella enterica subsp. enterica serovar Newport ( S . Newport) is a clinically and epidemiologically significant serovar in the United States. It is the second most prevalent clinically isolated Salmonella serovar in the United States, and it can contaminate a wide variety of food products. In this study, we evaluated the population structure of S . Newport clinical isolates obtained by the Tennessee Department of Health during routine surveillance ( n  = 346), along with a diverse set of other global clinical isolates obtained from EnteroBase ( n  = 271). Most of these clinical isolates belonged to established lineages II and III. Additionally, we performed lineage-specific phylogenetic analyses and were able to identify 18 potential epidemiological clusters among the isolates from Tennessee, which represented a greater proportion of Tennessee isolates belonging to putative epidemiological clusters than the proportion of isolates of this serovar that are outbreak related. IMPORTANCE This study provides insight on the genomic diversity of one of the Salmonella serovars that most frequently cause human illness. Specifically, we explored the diversity of human clinical isolates from a localized region (Tennessee) and compared this level of diversity with the global context. Additionally, we showed that a greater proportion of isolates were associated with potential epidemiological clusters (based on genomic relatedness) than historical estimates. We also identified that one potential cluster was predicted to be multidrug resistant. Taken together, these findings provide insight on Salmonella enterica serovar Newport that can impact public health surveillance and responses and serve as a foundational context for the Salmonella research community.

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., (© 2022. The Author(s).)

Soil samples collected in the Great Smoky Mountains National Park yielded a Listeria isolate that could not be classified to the species level. Whole-genome sequence-based average nucleotide identity BLAST and in silico DNA-DNA Hybridization analyses confirmed this isolate to be a novel Listeria sensu stricto species with the highest similarity to L. marthii (ANI = 93.9%, isDDH = 55.9%). Additional whole-genome-based analysis using the Genome Taxonomy Database Toolkit further supported delineation as a novel Listeria sensu stricto species, as this tool failed to assign a species identification. Phenotypic and genotypic characterization results indicate that this species is nonpathogenic. Specifically, the novel Listeria species described here is phenotypically (i) nonhemolytic and (ii) negative for phosphatidylinositol-specific phospholipase C activity; the draft genome lacks all virulence genes found in the Listeria pathogenicity islands 1, 2, 3, and 4 as well as the internalin genes inlA and inlB . While the type strain contains an apparently intact catalase gene ( kat ), this strain is phenotypically catalase-negative (an unusual characteristic for Listeria sensu stricto species). Additional analyses identified a nonsynonymous mutation in a conserved codon of kat that is likely linked to the catalase-negative phenotype. Rapid species identification systems, including two biochemical and one matrix-assisted laser desorption/ionization, misidentified this novel species as either L. monocytogenes , L. innocua, or L. marthii . We propose the name L. swaminathanii, and the type strain is FSL L7-0020 T (=ATCC TSD-239 T ). IMPORTANCE L. swaminathanii is a novel sensu stricto species that originated from a US National Park and it will be the first Listeria identified to date without official standing in the nomenclature. Validation was impeded by the National Park's requirements for strain access, ultimately deemed too restrictive by the International Committee on Systematics of Prokaryotes. However, lack of valid status should not detract from the significance of adding a novel species to the Listeria sensu stricto clade. Notably, detection of non -monocytogenes sensu stricto species in a food processing environment indicate conditions that could facilitate the presence of the pathogen L. monocytogenes. If isolated, our data show a potential for L. swaminathanii to be misidentified as another sensu stricto, notably L. monocytogenes. Therefore, developers of Listeria spp. detection and identification methods, who historically only include validly published species in their validation studies, should include L. swaminathanii to ensure accurate results.

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., (© 2022. The Author(s).)

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., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Salmonellosis is one of the most frequently reported zoonotic foodborne diseases worldwide, and poultry is the most important reservoir of Salmonella enterica serovar Enteritidis. The use of lytic bacteriophages (phages) to reduce foodborne pathogens has emerged as a promising biocontrol intervention for Salmonella spp. Here, we describe and evaluate the newly isolated Salmonella phage STGO-35-1, including: (i) genomic and phenotypic characterization, (ii) an analysis of the reduction of Salmonella in chicken meat, and (iii) genome plasticity testing. Phage STGO-35-1 represents an unclassified siphovirus, with a length of 47,483 bp, a G + C content of 46.5%, a headful strategy of packaging, and a virulent lifestyle. Phage STGO-35-1 reduced S. Enteritidis counts in chicken meat by 2.5 orders of magnitude at 4 °C. We identified two receptor-binding proteins with affinity to LPS, and their encoding genes showed plasticity during an exposure assay. Phenotypic, proteomic, and genomic characteristics of STGO-35-1, as well as the Salmonella reduction in chicken meat, support the potential use of STGO-35-1 as a targeted biocontrol agent against S. Enteritidis in chicken meat. Additionally, computational analysis and a short exposure time assay allowed us to predict the plasticity of genes encoding putative receptor-binding proteins.