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.

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.

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.

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.

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.

Complete Genome Sequence of a Serotype 7 Listeria monocytogenes Strain, FSL R9-0915.

Listeria monocytogenes serotype 7 lacks glycosidic constituents in wall teichoic acids. Here, we present the complete genome sequence of L. monocytogenes serotype 7 strain FSL R9-0915 and an analysis of genes known to affect L. monocytogenes antigenicity. This strain is used as a control strain in Listeria phage host range analyses.

Complete Genome Sequences of Three Listeria monocytogenes Bacteriophage Propagation Strains.

Bacteriophages can be used as a biocontrol for the foodborne pathogen Listeria monocytogenes Propagation of phages is a necessary step for their use in experimental studies and biocontrol applications. Here, we present the complete genomes of three Listeria monocytogenes strains commonly used as propagation hosts for Listeria phages.

Mutant and Recombinant Phages Selected from In Vitro Coevolution Conditions Overcome Phage-Resistant Listeria monocytogenes.

Bacteriophages (phages) are currently available for use by the food industry to control the foodborne pathogen Listeria monocytogenes Although phage biocontrols are effective under specific conditions, their use can select for phage-resistant bacteria that repopulate phage-treated environments. Here, we performed short-term coevolution experiments to investigate the impact of single phages and a two-phage cocktail on the regrowth of phage-resistant L. monocytogenes and the adaptation of the phages to overcome this resistance. We used whole-genome sequencing to identify mutations in the target host that confer phage resistance and in the phages that alter host range. We found that infections with Listeria phages LP-048, LP-125, or a combination of both select for different populations of phage-resistant L. monocytogenes bacteria with different regrowth times. Phages isolated from the end of the coevolution experiments were found to have gained the ability to infect phage-resistant mutants of L. monocytogenes and L. monocytogenes strains previously found to be broadly resistant to phage infection. Phages isolated from coinfected cultures were identified as recombinants of LP-048 and LP-125. Interestingly, recombination events occurred twice independently in a locus encoding two proteins putatively involved in DNA binding. We show that short-term coevolution of phages and their hosts can be utilized to obtain mutant and recombinant phages with adapted host ranges. These laboratory-evolved phages may be useful for limiting the emergence of phage resistance and for targeting strains that show general resistance to wild-type (WT) phages.IMPORTANCEListeria monocytogenes is a life-threatening bacterial foodborne pathogen that can persist in food processing facilities for years. Phages can be used to control L. monocytogenes in food production, but phage-resistant bacterial subpopulations can regrow in phage-treated environments. Coevolution experiments were conducted on a Listeria phage-host system to provide insight into the genetic variation that emerges in both the phage and bacterial host under reciprocal selective pressure. As expected, mutations were identified in both phage and host, but additionally, recombination events were shown to have repeatedly occurred between closely related phages that coinfected L. monocytogenes This study demonstrates that in vitro evolution of phages can be utilized to expand the host range and improve the long-term efficacy of phage-based control of L. monocytogenes This approach may also be applied to other phage-host systems for applications in biocontrol, detection, and phage therapy.

Homburgvirus LP-018 Has a Unique Ability to Infect Phage-Resistant Listeria monocytogenes.

Listeria phage LP-018 is the only phage from a diverse collection of 120 phages able to form plaques on a phage-resistant Listeria monocytogenes strain lacking rhamnose in its cell wall teichoic acids. The aim of this study was to characterize phage LP-018 and to identify what types of mutations can confer resistance to LP-018. Whole genome sequencing and transmission electron microscopy revealed LP-018 to be a member of the Homburgvirus genus. One-step-growth curve analysis of LP-018 revealed an eclipse period of ~60-90 min and a burst size of ~2 PFU per infected cell. Despite slow growth and small burst size, LP-018 can inhibit the growth of Listeria monocytogenes at a high multiplicity of infection. Ten distinct LP-018-resistant mutants were isolated from infected Listeria monocytogenes 10403S and characterized by whole genome sequencing. In each mutant, a single mutation was identified in either the LMRG_00278 or LMRG_01613 encoding genes. Interesting, LP-018 was able to bind to a representative phage-resistant mutant with a mutation in each gene, suggesting these mutations confer resistance through a mechanism independent of adsorption inhibition. Despite forming plaques on the rhamnose deficient 10403S mutant, LP-018 showed reduced binding efficiency, and we did not observe inhibition of the strain under the conditions tested. Two mutants of LP-018 were also isolated and characterized, one with a single SNP in a gene encoding a BppU domain protein that likely alters its host range. LP-018 is shown to be a unique Listeria phage that, with additional evaluation, may be useful in biocontrol applications that aim to reduce the emergence of phage resistance.

Cross-resistance to phage infection in Listeria monocytogenes serotype 1/2a mutants.

Bacteriophage-based biocontrols are one of several tools available to control Listeria monocytogenes in food and food processing environments. The objective of this study was to determine if phage-resistance that has been characterized with a select few Listeria phages would also confer resistance to a diverse collection of over 100 other Listeria phages. We show that some mutations that are likely to emerge in bacteriophage-treated populations of serotype 1/2a L. monocytogenes can lead to cross-resistance against almost all types of characterized Listeria phages. Out of the 120 phages that showed activity against the parental strain, only one could form visible plaques on the mutant strain of L. monocytogenes lacking rhamnose in its wall teichoic acids. An additional two phages showed signs of lytic activity against this mutant strain; although no visible plaques were observed. The findings presented here are consistent with other studies showing mutations conferring phage resistance through loss of rhamnose likely pose the greatest challenge for phage-based biocontrol in serotype 1/2a strains.