Genetic recombination-mediated evolutionary interactions between phages of potential industrial importance and prophages of their hosts within or across the domains of Escherichia, Listeria, Salmonella, Campylobacter, and Staphylococcus.

BACKGROUND: The in-depth understanding of the role of lateral genetic transfer (LGT) in phage-prophage interactions is essential to rationalizing phage applications for human and animal therapy, as well as for food and environmental safety. This in silico study aimed to detect LGT between phages of potential industrial importance and their hosts. METHODS: A large array of genetic recombination detection algorithms, implemented in SplitsTree and RDP4, was applied to detect LGT between various Escherichia, Listeria, Salmonella, Campylobacter, Staphylococcus, Pseudomonas, and Vibrio phages and their hosts. PHASTER and RAST were employed respectively to identify prophages across the host genome and to annotate LGT-affected genes with unknown functions. PhageAI was used to gain deeper insights into the life cycle history of recombined phages. RESULTS: The split decomposition inferences (bootstrap values: 91.3-100; fit: 91.433-100), coupled with the Phi (0.0-2.836E-12) and RDP4 (P being well below 0.05) statistics, provided strong evidence for LGT between certain Escherichia, Listeria, Salmonella, and Campylobacter virulent phages and prophages of their hosts. The LGT events entailed mainly the phage genes encoding for hypothetical proteins, while some of these genetic loci appeared to have been affected even by intergeneric recombination in specific E. coli and S. enterica virulent phages when interacting with their host prophages. Moreover, it is shown that certain L. monocytogenes virulent phages could serve at least as the donors of the gene loci, involved in encoding for the basal promoter specificity factor, for L. monocytogenes. In contrast, the large genetic clusters were determined to have been simultaneously exchanged by many S. aureus prophages and some Staphylococcus temperate phages proposed earlier as potential therapeutic candidates (in their native or modified state). The above genetic clusters were found to encompass multiple genes encoding for various proteins, such as e.g., phage tail proteins, the capsid and scaffold proteins, holins, and transcriptional terminator proteins. CONCLUSIONS: It is suggested that phage-prophage interactions, mediated by LGT (including intergeneric recombination), can have a far-reaching impact on the co-evolutionary trajectories of industrial phages and their hosts especially when excessively present across microbially rich environments.

Phenotypic and genotypic antimicrobial resistance of Listeria spp. in Spain.

Listeriosis is a zoonotic disease caused by Listeria monocytogenes and Listeria ivanovii. The genus Listeria currently includes 27 recognized species and is found throughout the environment. The number of systematic studies on antimicrobial resistance in L. monocytogenes isolates from domestic farms using antimicrobial substances is limited. Importantly, dairy ruminant farms are reservoir of hypervirulent lineage I L. monocytogenes isolates, previously associated with human clinical cases. Considering that the classes of antibiotics used in food-producing domestic animals are frequently the same or closely related to those used in human medicine, studies about the impact of antibiotic use on the acquisition of antibiotic resistance in Listeria spp. in domestic animal farms are, therefore, of high importance. Here, susceptibility to 25 antibiotics was determined. Eighty-one animal-related, 35 food and 21 human pathogenic Listeria spp. isolates and 114 animal-related non-pathogenic Listeria spp. isolates were tested. Whole genome sequencing data was used for molecular characterization. Regarding L. monocytogenes, 2 strains from the clinical-associated linage I showed resistance to erythromycin, both related to dairy ruminants. Acquired resistance to one antibiotic was exhibited in 1.5% of L. monocytogenes isolates compared with 14% of non-pathogenic Listeria spp. isolates. Resistance to tetracycline (7.9%), doxycycline (7.9%), penicillin (4.4%), and ampicillin (4.4%) were the most frequently observed in non-pathogenic Listeria spp. While resistance to two or more antibiotics (5.6%) was most common in Listeria spp., isolates, resistance to one antibiotic was also observed (1.6%). The present results show that non-pathogenic Listeria spp. harbour antimicrobial resistance genes.

High-throughput homogenous assay for the direct detection of Listeria monocytogenes DNA.

The Amplified Luminescent Proximity Homogenous Assay-linked Immunosorbent Assay (AlphaLISA) is known for detecting various protein targets; however, its ability to detect nucleic acid sequences is not well established. Here, the capabilities of the AlphaLISA technology were expanded to include direct detection of DNA (aka: oligo-Alpha) and was applied to the detection of Listeria monocytogenes. Parameters were defined that allowed the newly developed oligo-Alpha to differentiate L. monocytogenes from other Listeria species through the use of only a single nucleotide polymorphism within the 16S rDNA region. Investigations into the applicability of this assay with different matrices demonstrated its utility in both milk and juice. One remarkable feature of the oligo-Alpha is that greater sensitivity could be achieved through the use of multiple acceptor oligos compared to only a single acceptor oligo, even when only a single donor oligo was employed. Additional acceptor oligos were easily incorporated into the assay and a tenfold change in the detection limit was readily achieved, with detection limits of 250 attomole of target being recorded. In summary, replacement of antibodies with oligonucleotides allows us to take advantage of genotypic difference(s), which both expands its repertoire of biological markers and furthers its use as a diagnostic tool.

A comparative study on the occurrence, genetic characteristics, and factors associated with the distribution of Listeria species on cattle farms and beef abattoirs in Gauteng Province, South Africa.

These cross-sectional studies reported the occurrence, genetic characteristics, and factors associated with the distribution of Listeria species on cattle farms and beef abattoirs in Gauteng Province, South Africa. A total of 328 samples (faeces, feeds, silage, and drinking water) were collected from 23 cattle farms (communal, cow-calf, and feedlot), and 262 samples (faeces, carcass swabs, and effluents) from 8 beef abattoirs (low throughput and high throughput) were processed using standard bacteriological and molecular methods to detect Listeria species. The factors associated with the prevalence of Listeria species were investigated, and multiplex polymerase chain reaction (mPCR) was used to determine Listeria species, the pathogenic serogroups, and the carriage of eight virulence-associated genes by Listeria monocytogenes. The overall prevalence of Listeria species in cattle farms was 14.6%, comprising Listeria innocua (11.3%), Listeria monocytogenes (3.4%), Listeria welshimeri (0.0%) compared with 11.1%, comprising Listeria innocua (5.7%), Listeria monocytogenes (4.6%), Listeria welshimeri (0.8%) for beef abattoirs. Of the three variables (area, type of farm/abattoir, and sample type) investigated, only the sample types at abattoirs had a significant (P < 0.001) effect on the prevalence of L. innocua and L. welshimeri. The frequency of distribution of the serogroups based on 11 L. monocytogenes isolated from farms was 72.7% and 27.3% for the serogroup 1/2a-3a and 4b-4d-4e, respectively, while for the 12 L. monocytogenes isolates recovered from abattoirs, it was 25%, 8.3%, 50% and 16.7% for the serogroup 1/2a-3a, 1/2b-3b, 1/2c-3c, and 4b-4d-4e respectively (P < 0.05). All (100%) isolates of L. monocytogenes from the farms and abattoirs were positive for seven virulence genes (hlyA, inlB, plcA, iap, inlA, inlC, and inlJ). The clinical and food safety significance of the findings cannot be ignored.

Genomic characterization of Listeria monocytogenes and Listeria innocua isolated from milk and dairy samples in Ethiopia.

Listeriosis caused by Listeria monocytogenes often poses a significant threat to vulnerable populations. Dairy products have been implicated in outbreaks of listeriosis worldwide. In Ethiopia, studies have identified Listeria spp. and L. monocytogenes in various dairy products, but the genetic diversity and phylogenetic relationships of these bacteria remain largely unknown in the low- and middle-income countries. Therefore, we conducted whole-genome sequencing on 15 L. monocytogenes and 55 L. innocua isolates obtained from different levels of the dairy supply chains across three regions in Ethiopia. Genomes were assembled and used for MLST genotyping and single nucleotide polymorphism (SNP) analysis to infer phylogenetic relationships. We identified a total of 3 L. monocytogenes (i.e., 2, 145, and 18) and 12 L. innocua (i.e., 1489, 1619, 603, 537, 1010, 3186, 492, 3007, 1087, 474, 1008, and 637) MLST sequence types among the studied isolates. Some of these sequence types showed region-specific occurrence, while others were broadly distributed across regions. Through high-quality SNP analysis, we found that among 13 L. monocytogenes identified as ST 2, 11 of them were highly similar with low genetic variation, differing by only 1 to 10 SNPs, suggesting potential selection in the dairy food supply chain. The L. innocua isolates also exhibited low intra-ST genetic variation with only 0-10 SNP differences, except for the ST 1619, which displayed a greater diversity.

Diverse Listeria monocytogenes in-house clones are present in a dynamic frozen vegetable processing environment.

Listeria (L.) monocytogenes is of global concern for food safety as the listeriosis-causing pathogen is widely distributed in the food processing environments, where it can survive for a long time. Frozen vegetables contaminated with L. monocytogenes were recently identified as the source of two large listeriosis outbreaks in the EU and US. So far, only a few studies have investigated the occurrence and behavior of Listeria in frozen vegetables and the associated processing environment. This study investigates the occurrence of L. monocytogenes and other Listeria spp. in a frozen vegetable processing environment and in frozen vegetable products. Using whole genome sequencing (WGS), the distribution of sequence types (MLST-STs) and core genome sequence types (cgMLST-CT) of L. monocytogenes were assessed, and in-house clones were identified. Comparative genomic analyses and phenotypical characterization of the different MLST-STs and isolates were performed, including growth ability under low temperatures, as well as survival of freeze-thaw cycles. Listeria were widely disseminated in the processing environment and five in-house clones namely ST451-CT4117, ST20-CT3737, ST8-CT1349, ST8-CT6243, ST224-CT5623 were identified among L. monocytogenes isolates present in environmental swab samples. Subsequently, the identified in-house clones were also detected in product samples. Conveyor belts were a major source of contamination in the processing environment. A wide repertoire of stress resistance markers supported the colonization and survival of L. monocytogenes in the frozen vegetable processing facility. The presence of ArgB was significantly associated with in-house clones. Significant differences were also observed in the growth rate between different MLST-STs at low temperatures (4 °C and 10 °C), but not between in-house and non-in-house isolates. All isolates harbored major virulence genes such as full length InlA and InlB and LIPI-1, yet there were differences between MLST-STs in the genomic content. The results of this study demonstrate that WGS is a strong tool for tracing contamination sources and transmission routes, and for identifying in-house clones. Further research targeting the co-occurring microbiota and the presence of biofilms is needed to fully understand the mechanism of colonization and persistence in a food processing environment.

Taxonomy, ecology, and relevance to food safety of the genus Listeria with a particular consideration of new Listeria species described between 2010 and 2022.

Since 2010, the genus Listeria has had the addition of 22 new species that more than tripled the number of species identified until 2010. Sixteen of these 22 new species are distantly related to the type species, Listeria monocytogenes, and several of these present phenotypes that distinguish them from classical Listeria species (L. monocytogenes, Listeria innocua, Listeria ivanovii, Listeria seeligeri, Listeria welshimeri, and Listeria grayi). These 22 newly described species also show that Listeria is more genetically diverse than previously estimated. While future studies and surveys are needed to clarify the distribution of these species, at least some of these species may not be widely spread, while other species may be frequently found spread to human-related settings (e.g., farms and processing facilities), and others may be adapted to specific environmental habitats. Here, we review the taxonomic, phylogenetic, and ecological characteristics of these new Listeria species identified since 2010 and re-iterate the suggestion of re-classification of some species into three new genera: Murraya, Mesolisteria, and Paenilisteria. We also provide a review of current detection issues and the relevance to food safety related to the identification of these new species. For example, several new non-pathogenic species could be misidentified as the pathogen L. monocytogenes, based on methods that do not target L. monocytogenes-specific virulence genes/factors, leading to unnecessary product recalls. Moreover, eight species in the proposed new genus Mesolisteria are not good indicators of environmental conditions that could allow L. monocytogenes to grow since Mesolisteria species are unable to grow at low temperatures.

The distinct translational landscapes of gram-negative Salmonella and gram-positive Listeria.

Translational control in pathogenic bacteria is fundamental to gene expression and affects virulence and other infection phenotypes. We used an enhanced ribosome profiling protocol coupled with parallel transcriptomics to capture accurately the global translatome of two evolutionarily distant pathogenic bacteria-the Gram-negative bacterium Salmonella and the Gram-positive bacterium Listeria. We find that the two bacteria use different mechanisms to translationally regulate protein synthesis. In Salmonella, in addition to the expected correlation between translational efficiency and cis-regulatory features such as Shine-Dalgarno (SD) strength and RNA secondary structure around the initiation codon, our data reveal an effect of the 2nd and 3rd codons, where the presence of tandem lysine codons (AAA-AAA) enhances translation in both Salmonella and E. coli. Strikingly, none of these features are seen in efficiently translated Listeria transcripts. Instead, approximately 20% of efficiently translated Listeria genes exhibit 70 S footprints seven nt upstream of the authentic start codon, suggesting that these genes may be subject to a novel translational initiation mechanism. Our results show that SD strength is not a direct hallmark of translational efficiency in all bacteria. Instead, Listeria has evolved additional mechanisms to control gene expression level that are distinct from those utilised by Salmonella and E. coli.

Contrasting genes conferring short- and long-term biofilm adaptation in Listeria.

Listeria monocytogenes is an opportunistic food-borne bacterium that is capable of infecting humans with high rates of hospitalization and mortality. Natural populations are genotypically and phenotypically variable, with some lineages being responsible for most human infections. The success of L. monocytogenes is linked to its capacity to persist on food and in the environment. Biofilms are an important feature that allow these bacteria to persist and infect humans, so understanding the genetic basis of biofilm formation is key to understanding transmission. We sought to investigate the biofilm-forming ability of L. monocytogenes by identifying genetic variation that underlies biofilm formation in natural populations using genome-wide association studies (GWAS). Changes in gene expression of specific strains during biofilm formation were then investigated using RNA sequencing (RNA-seq). Genetic variation associated with enhanced biofilm formation was identified in 273 genes by GWAS and differential expression in 220 genes by RNA-seq. Statistical analyses show that the number of overlapping genes flagged by either type of experiment is less than expected by random sampling. This novel finding is consistent with an evolutionary scenario where rapid adaptation is driven by variation in gene expression of pioneer genes, and this is followed by slower adaptation driven by nucleotide changes within the core genome.

Tetracycline resistance in Listeria monocytogenes and L. innocua from wild black bears (Ursus americanus) in the United States is mediated by novel transposable elements.

IMPORTANCE: Listeria monocytogenes causes severe foodborne illness and is the only human pathogen in the genus Listeria. Previous surveys of AMR in Listeria focused on clinical sources and food or food processing environments, with AMR in strains from wildlife and other natural ecosystems remaining under-explored. We analyzed 185 sequenced strains from wild black bears (Ursus americanus) from the United States, including 158 and 27 L. monocytogenes and L. innocua, respectively. Tetracycline resistance was the most prevalent resistance trait. In L. monocytogenes, it was encountered exclusively in serotype 4b strains with the novel Tn916-like element Tn916.1039. In contrast, three distinct, novel tetracycline resistance elements (Tn5801.UAM, Tn5801.551, and Tn6000.205) were identified in L. innocua. Interestingly, Tn5801.551 was identical to elements in L. monocytogenes from a major foodborne outbreak in the United States in 2011. The findings suggest the importance of wildlife and non-pathogenic Listeria species as reservoir for resistance elements in Listeria.

Characterisation of the growth behaviour of Listeria monocytogenes in Listeria synthetic media.

The foodborne pathogen Listeria monocytogenes can grow in a wide range of environmental conditions. For the study of the physiology of this organism, several chemically defined media have been developed over the past decades. Here, we examined the ability of L. monocytogenes wildtype strains EGD-e and 10403S to grow under salt and pH stress in Listeria synthetic medium (LSM). Furthermore, we determined that a wide range of carbon sources could support the growth of both wildtype strains in LSM. However, for hexose phosphate sugars such as glucose-1-phosphate, both L. monocytogenes strains need to be pre-grown under conditions, where the major virulence regulator PrfA is active. In addition, growth of both L. monocytogenes strains was observed when LSM was supplemented with the amino acid sugar N-acetylmannosamine (ManNAc). We were able to show that some of the proteins encoded in the operon lmo2795-nanE, such as the ManNAc-6-phosphate epimerase NanE, are required for growth in the presence of ManNAc. The first gene of the operon, lmo2795, encodes a transcriptional regulator of the RpiR family. Using electrophoretic mobility shift assays and quantitative real-time PCR analysis, we were able to show that Lmo2795 binds to the promoter region of the operon lmo2795-nanE and activates its expression.

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.

Comprehensive genomic analysis of the potential limitations of several published PCR primers targeting prfA-virulence gene cluster in Listeria species.

Polymerase chain reaction (PCR) is commonly used to detect Listeria monocytogenes, foodborne pathogen. This study conducted in silico genomic analysis to investigate the specificity and binding efficacy of four published pairs of PCR primers targeting Listeria prfA-virulence gene cluster (pVGC) based on Listeria sequences available. We first performed comprehensive genomic analyses of the pVGC, the main pathogenicity island in Listeria spp. In total, 2961 prfA, 642 plcB, 629 mpl, and 1181 hlyA gene sequences were retrieved from the NCBI database. Multiple sequence alignments and phylogenetic trees were generated using unique (non-identical or not-shared) sequences of each represented genes, targeting four pairs of PCR primers published previously, namely 202 prfA, 82 plcB, 150 mpl, and 176 hlyA unique gene sequences. Only the hlyA gene showed strong (over 94%) primer mapping results, while prfA, plcB, and mpl genes showed weak (<50%) matching results. In addition, nucleotide variations were observed at the 3' end of the primers, indicating non-binding to the targets could potentially cause false-negative results. Thus, we propose designing degenerate primers or multiple PCR primers based on as many isolates as possible to minimize the false-negative risk and reach the aim of low tolerable limits of detection.

Prevalence and Biological Characteristics of Listeria Species Isolated from Livestock and Poultry Meat in Gansu Province, China.

Listeria monocytogenes is a widespread foodborne pathogen contaminating foods during their production or processing stages. Fresh meat is susceptible to such contamination if it is not properly preserved. Our study was conducted to reveal the level of contamination and prevalence of Listeria spp. present in livestock and poultry meat from Gansu province. A total of 1,387 samples were collected from five cities in Gansu Province according to standard sampling procedures, of which 174 samples (12.5%) were positive for Listeria species. Among them, 14 isolates of L. monocytogenes (1.0%), 150 isolates of Listeria innocua (10.8%), and ten isolates of Listeria welshimeri (0.7%) were identified by conventional bacteriological and molecular identification methods. All isolates were subjected to serological assays, antimicrobial susceptibility tests, growth curve assays, determination of biofilm-forming capacity, and cluster analysis of the 16S rRNA gene sequences. Four predominant serotypes of L. monocytogenes were identified, including 1/2a (35.7%, 5/14), 1/2b (14.3%, 2/14), 1/2c (42.9%, 6/14), and 4b (7.1%, 1/14). All L. monocytogenes isolates were resistant to tetracycline and cefoxitin. Most L. innocua isolates (63.6%, 14/22) and L. welshimeri (40%, 4/10) were resistant to tetracycline. The high biofilm-forming ability was observed among 1/2c and 1/2a serotype isolates. The cluster analysis of the 16S rRNA gene sequences revealed a close genetic relationship between the three Listeria species. This study fills the gap in the knowledge of livestock and poultry meat that carry Listeria in slaughterhouses and markets in Gansu Province.

Contrasting Genetic Diversity of Listeria Pathogenicity Islands 3 and 4 Harbored by Nonpathogenic Listeria spp.

Listeria monocytogenes causes the severe foodborne disease listeriosis. Several clonal groups of L. monocytogenes possess the pathogenicity islands Listeria pathogenicity island 3 (LIPI-3) and LIPI-4. Here, we investigated the prevalence and genetic diversity of LIPI-3 and LIPI-4 among 63 strains of seven nonpathogenic Listeria spp. from the natural environment, i.e., wildlife (black bears [Ursus americanus]) and surface water. Analysis of the whole-genome sequence data suggested that both islands were horizontally acquired but differed considerably in their incidence and genetic diversity. LIPI-3 was identified among half of the L. innocua strains in the same genomic location as in L. monocytogenes (guaA hot spot) in a truncated form, with only three strains harboring full-length LIPI-3, and a highly divergent partial LIPI-3 was observed in three Listeria seeligeri strains, outside the guaA hot spot. Premature stop codons (PMSCs) and frameshifts were frequently noted in the LIPI-3 gene encoding listeriolysin S. On the other hand, full-length LIPI-4 without any PMSCs was found in all Listeria innocua strains, in the same genomic location as L. monocytogenes and with ~85% similarity to the L. monocytogenes counterpart. Our study provides intriguing examples of genetic changes that pathogenicity islands may undergo in nonpathogenic bacterial species, potentially in response to environmental pressures that promote either maintenance or degeneration of the islands. Investigations of the roles that LIPI-3 and LIPI-4 play in nonpathogenic Listeria spp. are warranted to further understand the differential evolution of genetic elements in pathogenic versus nonpathogenic hosts of the same genus. IMPORTANCE Listeria monocytogenes is a serious foodborne pathogen that can harbor the pathogenicity islands Listeria pathogenicity island 3 (LIPI-3) and LIPI-4. Intriguingly, these have also been reported in nonpathogenic L. innocua from food and farm environments, though limited information is available for strains from the natural environment. Here, we analyzed whole-genome sequence data of nonpathogenic Listeria spp. from wildlife and surface water to further elucidate the genetic diversity and evolution of LIPI-3 and LIPI-4 in Listeria. While the full-length islands were found only in L. innocua, LIPI-3 was uncommon and exhibited frequent truncation and genetic diversification, while LIPI-4 was remarkable in being ubiquitous, albeit diversified from L. monocytogenes. These contrasting features demonstrate that pathogenicity islands in nonpathogenic hosts can evolve along different trajectories, leading to either degeneration or maintenance, and highlight the need to examine their physiological roles in nonpathogenic hosts.

Influence of temperature on regulation of key virulence and stress response genes in Listeria monocytogenes biofilms.

Temperature is a major determinant of Listeria (L.) monocytogenes adherence and biofilm formation on abiotic surfaces. However, its role on gene regulation of L. monocytogenes mature biofilms has not been investigated. In the present study, we aimed to evaluate the impact of temperature up- and down-shift on L. monocytogenes biofilms gene transcription. L. monocytogenes strain EGD-e biofilms were first developed on stainless steel surfaces in Brain Heart Infusion broth at 20 °C for 48 h. Then, nutrient broth was renewed, and mature biofilms were exposed to 10 °C, 20 °C or 37 °C for 24 h. Biofilm cells were harvested and RNA levels of plcA, prfA, hly, mpl, plcB, sigB, bapL, fbpA, fbpB, lmo2178, lmo0880, lmo0160, lmo1115, lmo 2089, lmo2576, lmo0159 and lmo0627 were evaluated by quantitative RT-PCR. The results revealed an over-expression of all genes tested in biofilm cells compared to planktonic cells. When biofilms were further allowed to proliferate at 20 °C for 24 h, the transcription levels of key virulence, stress response and putative binding proteins genes plcA, sigB, fbpA, fbpB, lmo1115, lmo0880 and lmo2089 decreased. A temperature-dependent transcription for sigB, plcA, hly, and lmo2089 genes was observed after biofilm proliferation at 10 °C or 37 °C. Our findings suggest that temperature differentially affects gene regulation of L. monocytogenes mature biofilms, thus modulating attributes such as virulence, stress response and pathogenesis.

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.

Genomic characterization of Listeria spp. isolated from tonsils, udder and feces of domestic dairy ruminants in Spain.

Two species of Listeria are pathogenic, Listeria monocytogenes and Listeria ivanovii. Although studies have shown that dairy ruminants shed Listeria spp. in feces, there is little information about ruminants that do not shed Listeria spp. in their feces but asymptomatically carry them in organs. We evidence that ruminants can asymptomatically carry L. ivanovii in udders and L. monocytogenes and L. ivanovii in tonsils without fecal shedding. Whole-genome sequence of L. monocytogenes and L. ivanovii contained known core genes involved in virulence and antibiotic resistance. This work highlights tonsils and udders as a Listeria intra-host site of colonization.

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Objective: To construct Listeria monocytogenes (LM) and Listeria ivanovii (LI) balanced lethal systems expressing cervical cancer antigens, to study their basic biological characteristics, and to provide reference data for the immunotherapy of cervical cancer. Methods: Through seamless cloning via in vitro ligation kit, the HPV16 E6E7 fusion protein antigen gene constructed in our lab was spliced to the complement plasmid pCWgfp-LM dal-Amp that contained the nutritional gene dal. Then, we replaced the ampicillin (Amp) resistance gene of the complement plasmid with the asd nutrition gene. The ligation reaction mixture was transformed into Escherichia coli (E. coli) recipient bacteria DH5αΔasd and the complement plasmid pCWgfp-E6E7-LM dal-Ampfree, which expressed cervical cancer antigens and had no Amp resistance, was obtained by nutrition screening from the E. coli DH5αΔasd. The plasmid pCWgfp-E6E7-LM dal-Ampfree was complemented into LMΔdd and LIΔdd, the attenuated nutrition-deficient Listeria strains with the virulence genes actA and plcB and nutrition genes dal and dat deleted by electroporation, thereby obtaining LM and LI balanced lethal systems expressing cervical cancer antigen genes. The in vitro growth of the strains was observed. Western blot was performed to examine the status of antigen protein expression. PCR was performed to measure the in vitro passage stability of complement plasmid pCWgfp-E6E7-LM dal-Ampfree. Their basic biological characteristics were examined by biochemical reaction tests and hemolysis assay. Results: Two Listeria balanced lethal systems expressing cervical cancer antigen were successfully constructed. The HPV16 type E6E7 fusion protein was successfully expressed in the two Listeria balanced lethal systems. pCWgfp-E6E7-LM dal-Ampfree, the positive plasmid expressing cervical cancer antigen, maintained stable existence in the two Listeria balanced lethal systems. The two Listeria balanced lethal systems expressing cervical cancer antigen showed significantly better recovery growth in comparison with Listeria nutrition deficiency strains. The results of biochemical reaction tests showed that most of the biochemical reaction of the two Listeria balanced lethal systems expressing cervical cancer antigen were consistent with those of Listeria attenuated strains. The two Listeria balanced lethal systems expressing cervical cancer antigen still maintained the hemolytic ability, although their hemolytic ability was slightly inferior to that of the Listeria balanced lethal systems not expressing cervical cancer antigen and the Listeria attenuated strains. Conclusion: The two Listeria balanced lethal systems expressing cervical cancer antigen genes are constructed successfully. They display normal in vitro growth. The complement plasmid pCWgfp-E6E7-LM dal-Ampfree can maintain stable existence in vitro, showing little change in its biochemical characteristics and hemolytic ability. Further research should be conducted to investigate the potential of these two recombinant strains to be used as candidate strains for cervical cancer therapeutic vaccine.

Hemolysin function of Listeria is related to biofilm formation: transcriptomics analysis.

Listeriolysin O (LLO) is the main virulence protein of Listeria monocytogenes (LM), that helps LM escape lysosomes. We previously found that the cellular immune response elicited by L.ivanovii (LI) is weaker than that elicited by LM. We speculated that this may be related to the function of ivanolysin O (ILO). Here, we constructed hemolysin gene deletion strain, LIΔilo, and a modified strain, LIΔilo::hly, in which ilo was replaced by hly. Prokaryotic transcriptome sequencing was performed on LI, LIΔilo, and LIΔilo::hly. Transcriptome differences between the three strains were compared, and genes and pathways with significant differences between the three strains were analyzed. Prokaryotic transcriptome sequencing results revealed the relationship of ilo to the ribosome, quorum sensing, and phosphotransferase system (PTS) pathways, etc. LIΔilo exhibited attenuated biofilm formation ability compared to LI. Biofilm formation was significantly recovered or even increased after replenishing hly. After knocking out ilo, the relative expression levels of some virulence genes, including sigB, prfA, actA, smcL, and virR, were up-regulated compared to LI. After replenishing hly, these genes were down-regulated compared to LIΔilo. The trend and degree of such variation were not completely consistent when cultured in media containing only monosaccharides or disaccharides. The results confirmed that hemolysin is related to some important biological properties of Listeria, including biofilm formation and virulence gene expression levels. This is the first comprehensive study on ILO function at the transcriptomic level and the first evidence of a relationship between Listeria hemolysin and biofilm formation.