What's on a prophage: analysis of Salmonella spp. prophages identifies a diverse range of cargo with multiple virulence- and metabolism-associated functions.

The gain of mobile elements, such as prophages, can introduce cargo to the recipient bacterium that could facilitate its persistence in or expansion to a new environment, such as a host. While previous studies have focused on identifying and characterizing the genetic diversity of prophages, analyses characterizing the cargo that prophages carry have not been extensively explored. We characterized prophage regions from 303 Salmonella spp. genomes (representing 254 unique serovars) to assess the distribution of prophages in diverse Salmonella. On average, prophages accounted for 3.7% (0.1%-8.8%) of the total genomic content of each isolate. Prophage regions annotated as Gifsy 1 and Salmon Fels 1 were the most commonly identified intact prophages, suggesting that they are common throughout the Salmonella genus. Among 21,687 total coding sequences (CDSs) from intact prophage regions in subsp. enterica genomes, 7.5% (median; range: 1.1%-47.6%) were categorized as having a function not related to prophage integration or phage structure, some of which could potentially provide a functional attribute to the host Salmonella cell. These predicted functions could be broadly categorized into CDSs involved in: (i) modification of cell surface structures (i.e., glycosyltransferases); (ii) modulation of host responses (e.g., SodC/SodA, SopE, ArtAB, and typhoid toxin); (iii) conferring resistance to heavy metals and antimicrobials; (iv) metabolism of carbohydrates, amino acids, and nucleotides; and (v) DNA replication, repair, and regulation. Overall, our systematic analysis of prophage cargo highlights a broader role for prophage cargo in influencing the metabolic, virulence, and resistance characteristics of Salmonella. IMPORTANCE: Lysogenic bacteriophages (phages) can integrate their genome into a bacterial host's genome, potentially introducing genetic elements that can affect the fitness of the host bacterium. The functions of prophage-encoded genes are important to understand as these genes could be mobilized and transferred to a new host. Using a large genomic dataset representing >300 isolates from all known subspecies and species of Salmonella, our study contributes important new findings on the distribution of prophages and the types of cargo that diverse Salmonella prophages carry. We identified a number of coding sequences (CDSs) annotated as having cell surface-modifying attributes, suggesting that prophages may have played an important role in shaping Salmonella's diverse surface antigen repertoire. Furthermore, our characterization of prophages suggests that they play a broader role in facilitating the acquisition and transfer of CDSs associated with metabolism, DNA replication and repair, virulence factors, and to a lesser extent, antimicrobial resistance.

Adaptive strategies and ecological roles of phages in habitats under physicochemical stress.

Bacteriophages (phages) play a vital role in ecosystem functions by influencing the composition, genetic exchange, metabolism, and environmental adaptation of microbial communities. With recent advances in sequencing technologies and bioinformatics, our understanding of the ecology and evolution of phages in stressful environments has substantially expanded. Here, we review the impact of physicochemical environmental stress on the physiological state and community dynamics of phages, the adaptive strategies that phages employ to cope with environmental stress, and the ecological effects of phage-host interactions in stressful environments. Specifically, we highlight the contributions of phages to the adaptive evolution and functioning of microbiomes and suggest that phages and their hosts can maintain a mutualistic relationship in response to environmental stress. In addition, we discuss the ecological consequences caused by phages in stressful environments, encompassing biogeochemical cycling. Overall, this review advances an understanding of phage ecology in stressful environments, which could inform phage-based strategies to improve microbiome performance and ecosystem resilience and resistance in natural and engineering systems.

Microplastics and chemical leachates from plastic pipes are associated with increased virulence and antimicrobial resistance potential of drinking water microbial communities.

There is increasing recognition of the potential impacts of microplastics (MPs) on human health. As drinking water is the most direct route of human exposure to MPs, there is an urgent need to elucidate MPs source and fate in drinking water distribution system (DWDS). Here, we showed polypropylene random plastic pipes exposed to different water quality (chlorination and heating) and environmental (freeze-thaw) conditions accelerated MPs generation and chemical leaching. MPs showed various morphology and aggregation states, and chemical leaches exhibited distinct profiles due to different physicochemical treatments. Based on the physiological toxicity of leachates, oxidative stress level was negatively correlated with disinfection by-products in the leachates. Microbial network analysis demonstrated exposure to leachates (under three treatments) undermined microbial community stability and increased the relative abundance and dominance of pathogenic bacteria. Leachate physical and chemical properties (i.e., MPs abundance, hydrodynamic diameter, zeta potential, total organic carbon, dissolved ECs) exerted significant (p < 0.05) effects on the functional genes related to virulence, antibiotic resistance and metabolic pathways. Notably, chlorination significantly increased correlations among pathogenic bacteria, virulence genes, and antibiotic resistance genes. Overall, this study advances the understanding of direct and indirect risks of these MPs released from plastic pipes in the DWDS.

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.

Long-Term Phellodendri Cortex Supplementation in the Tiger Grouper (Epinephelus fuscoguttatus): Dual Effects on Intestinal Health Revealed by Transcriptome Analysis.

The tiger grouper (Epinephelus fuscoguttatus), an important mariculture fish in Southeast Asia, faces increasing health issues in recent years. Phellodendri Cortex (PC) is a traditional Chinese herbal medicine that exhibits a variety of beneficial effects on tiger groupers. The effects of PC, however, varies with the period of dietary intervention. This study aims to investigate the long-term effects of 1% PC supplementation on tiger groupers, focusing on growth, immunity, disease resistance, and intestinal gene expression. The tiger groupers (with an initial mean weight of 27.5 ± 0.5 g) were fed with a diet of Phellodendri Cortex supplementation and a control diet for 8 weeks. Our results indicate that the long-term PC supplementation did not affect growth or Vibrio disease resistance in tiger groupers. However, the transcriptome analysis revealed potential damage to the structural and functional integrity of the groupers' intestines. On the other hand, anti-inflammatory and cathepsin inhibition effects were also observed, offering potential benefits to fish enteritis prevention and therapy. Therefore, long-term PC supplementation in grouper culture should be applied with caution.

ARGem: a new metagenomics pipeline for antibiotic resistance genes: metadata, analysis, and visualization.

Antibiotic resistance is of crucial interest to both human and animal medicine. It has been recognized that increased environmental monitoring of antibiotic resistance is needed. Metagenomic DNA sequencing is becoming an attractive method to profile antibiotic resistance genes (ARGs), including a special focus on pathogens. A number of computational pipelines are available and under development to support environmental ARG monitoring; the pipeline we present here is promising for general adoption for the purpose of harmonized global monitoring. Specifically, ARGem is a user-friendly pipeline that provides full-service analysis, from the initial DNA short reads to the final visualization of results. The capture of extensive metadata is also facilitated to support comparability across projects and broader monitoring goals. The ARGem pipeline offers efficient analysis of a modest number of samples along with affordable computational components, though the throughput could be increased through cloud resources, based on the user's configuration. The pipeline components were carefully assessed and selected to satisfy tradeoffs, balancing efficiency and flexibility. It was essential to provide a step to perform short read assembly in a reasonable time frame to ensure accurate annotation of identified ARGs. Comprehensive ARG and mobile genetic element databases are included in ARGem for annotation support. ARGem further includes an expandable set of analysis tools that include statistical and network analysis and supports various useful visualization techniques, including Cytoscape visualization of co-occurrence and correlation networks. The performance and flexibility of the ARGem pipeline is demonstrated with analysis of aquatic metagenomes. The pipeline is freely available at https://github.com/xlxlxlx/ARGem.

Microdiversity of the vaginal microbiome is associated with preterm birth.

Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality. The vaginal microbiome has been associated with PTB, yet the mechanisms underlying this association are not fully understood. Understanding microbial genetic adaptations to selective pressures, especially those related to the host, may yield insights into these associations. Here, we analyze metagenomic data from 705 vaginal samples collected during pregnancy from 40 women who delivered preterm spontaneously and 135 term controls from the Multi-Omic Microbiome Study-Pregnancy Initiative. We find that the vaginal microbiome of pregnancies that ended preterm exhibited unique genetic profiles. It was more genetically diverse at the species level, a result which we validate in an additional cohort, and harbored a higher richness and diversity of antimicrobial resistance genes, likely promoted by transduction. Interestingly, we find that Gardnerella species drove this higher genetic diversity, particularly during the first half of the pregnancy. We further present evidence that Gardnerella spp. underwent more frequent recombination and stronger purifying selection in genes involved in lipid metabolism. Overall, our population genetics analyses reveal associations between the vaginal microbiome and PTB and suggest that evolutionary processes acting on vaginal microbes may play a role in adverse pregnancy outcomes such as PTB.

Comparative genomics unveils extensive genomic variation between populations of Listeria species in natural and food-associated environments.

Comprehending bacterial genomic variation linked to distinct environments can yield novel insights into mechanisms underlying differential adaptation and transmission of microbes across environments. Gaining such insights is particularly crucial for pathogens as it benefits public health surveillance. However, the understanding of bacterial genomic variation is limited by a scarcity of investigations in genomic variation coupled with different ecological contexts. To address this limitation, we focused on Listeria, an important bacterial genus for food safety that includes the human pathogen L. monocytogenes, and analyzed a large-scale genomic dataset collected by us from natural and food-associated environments across the United States. Through comparative genomics analyses on 449 isolates from the soil and 390 isolates from agricultural water and produce processing facilities representing L. monocytogenes, L. seeligeri, L. innocua, and L. welshimeri, we find that the genomic profiles strongly differ by environments within each species. This is supported by the environment-associated subclades and differential presence of plasmids, stress islands, and accessory genes involved in cell envelope biogenesis and carbohydrate transport and metabolism. Core genomes of Listeria species are also strongly associated with environments and can accurately predict isolation sources at the lineage level in L. monocytogenes using machine learning. We find that the large environment-associated genomic variation in Listeria appears to be jointly driven by soil property, climate, land use, and accompanying bacterial species, chiefly representing Actinobacteria and Proteobacteria. Collectively, our data suggest that populations of Listeria species have genetically adapted to different environments, which may limit their transmission from natural to food-associated environments.

Restriction endonuclease cleavage of phage DNA enables resuscitation from Cas13-induced bacterial dormancy.

Type VI CRISPR systems protect against phage infection using the RNA-guided nuclease Cas13 to recognize viral messenger RNA. Upon target recognition, Cas13 cleaves phage and host transcripts non-specifically, leading to cell dormancy that is incompatible with phage propagation. However, whether and how infected cells recover from dormancy is unclear. Here we show that type VI CRISPR and DNA-cleaving restriction-modification (RM) systems frequently co-occur and synergize to clear phage infections and resuscitate cells. In the natural type VI CRISPR host Listeria seeligeri, we show that RM cleaves the phage genome, thus removing the source of phage transcripts and enabling cells to recover from Cas13-induced cellular dormancy. We find that phage infections are neutralized more effectively when Cas13 and RM systems operate together. Our work reveals that type VI CRISPR immunity is cell-autonomous and non-abortive when paired with RM, and hints at other synergistic roles for the diverse host-directed immune systems in bacteria.

Microdiversity of the Vaginal Microbiome is Associated with Preterm Birth.

Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality. The vaginal microbiome has been associated with PTB, yet the mechanisms underlying this association are not fully understood. Understanding microbial genetic adaptations to selective pressures, especially those related to the host, may yield new insights into these associations. To this end, we analyzed metagenomic data from 705 vaginal samples collected longitudinally during pregnancy from 40 women who delivered preterm spontaneously and 135 term controls from the Multi-Omic Microbiome Study-Pregnancy Initiative (MOMS-PI). We find that the vaginal microbiome of pregnancies that ended preterm exhibits unique genetic profiles. It is more genetically diverse at the species level, a result which we validate in an additional cohort, and harbors a higher richness and diversity of antimicrobial resistance genes, likely promoted by transduction. Interestingly, we find that Gardnerella species, a group of central vaginal pathobionts, are driving this higher genetic diversity, particularly during the first half of the pregnancy. We further present evidence that Gardnerella spp. undergoes more frequent recombination and stronger purifying selection in genes involved in lipid metabolism. Overall, our results reveal novel associations between the vaginal microbiome and PTB using population genetics analyses, and suggest that evolutionary processes acting on the vaginal microbiome may play a vital role in adverse pregnancy outcomes such as preterm birth.

Preterm birth is associated with xenobiotics and predicted by the vaginal metabolome.

Spontaneous preterm birth (sPTB) is a leading cause of maternal and neonatal morbidity and mortality, yet its prevention and early risk stratification are limited. Previous investigations have suggested that vaginal microbes and metabolites may be implicated in sPTB. Here we performed untargeted metabolomics on 232 second-trimester vaginal samples, 80 from pregnancies ending preterm. We find multiple associations between vaginal metabolites and subsequent preterm birth, and propose that several of these metabolites, including diethanolamine and ethyl glucoside, are exogenous. We observe associations between the metabolome and microbiome profiles previously obtained using 16S ribosomal RNA amplicon sequencing, including correlations between bacteria considered suboptimal, such as Gardnerella vaginalis, and metabolites enriched in term pregnancies, such as tyramine. We investigate these associations using metabolic models. We use machine learning models to predict sPTB risk from metabolite levels, weeks to months before birth, with good accuracy (area under receiver operating characteristic curve of 0.78). These models, which we validate using two external cohorts, are more accurate than microbiome-based and maternal covariates-based models (area under receiver operating characteristic curve of 0.55-0.59). Our results demonstrate the potential of vaginal metabolites as early biomarkers of sPTB and highlight exogenous exposures as potential risk factors for prematurity.

Salmonella enterica serovar Cerro displays a phylogenetic structure and genomic features consistent with virulence attenuation and adaptation to cattle.

Salmonella enterica subsp. enterica (S.) serovar Cerro is rarely isolated from human clinical cases of salmonellosis but represents the most common serovar isolated from cattle without clinical signs of illness in the United States. In this study, using a large, diverse set of 316 isolates, we utilized genomic methods to further elucidate the evolutionary history of S. Cerro and to identify genomic features associated with its apparent virulence attenuation in humans. Phylogenetic analyses showed that within this polyphyletic serovar, 98.4% of isolates (311/316) represent a monophyletic clade within section Typhi and the remaining 1.6% of isolates (5/316) form a monophyletic clade within subspecies enterica Clade A1. Of the section Typhi S. Cerro isolates, 93.2% of isolates (290/311) clustered into a large clonal clade comprised of predominantly sequence type (ST) 367 cattle and environmental isolates, while the remaining 6.8% of isolates (21/311), primarily from human clinical sources, clustered outside of this clonal clade. A tip-dated phylogeny of S. Cerro ST367 identified two major clades (I and II), one of which overwhelmingly consisted of cattle isolates that share a most recent common ancestor that existed circa 1975. Gene presence/absence and rarefaction curve analyses suggested that the pangenome of section Typhi S. Cerro is open, potentially reflecting the gain/loss of prophage; human isolates contained the most open pangenome, while cattle isolates had the least open pangenome. Hypothetically disrupted coding sequences (HDCs) displayed clade-specific losses of intact speC and sopA virulence genes within the large clonal S. Cerro clade, while loss of intact vgrG, araH, and vapC occurred in all section Typhi S. Cerro isolates. Further phenotypic analysis suggested that the presence of a premature stop codon in speC does not abolish ornithine decarboxylase activity in S. Cerro, likely due to the activity of the second ornithine decarboxylase encoded by speF, which remained intact in all isolates. Overall, our study identifies specific genomic features associated with S. Cerro's infrequent isolation from humans and its apparent adaptation to cattle, which has broader implications for informing our understanding of the evolutionary events facilitating host adaptation in Salmonella.

Soil Collected in the Great Smoky Mountains National Park Yielded a Novel Listeria sensu stricto Species, L. swaminathanii.

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 L7-0020T (=ATCC TSD-239T). IMPORTANCEL. 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.

A non-optimal cervicovaginal microbiota in pregnancy is associated with a distinct metabolomic signature among non-Hispanic Black individuals.

Biomechanical and molecular processes of premature cervical remodeling preceding spontaneous preterm birth (sPTB) likely result from interactions between the cervicovaginal microbiota and host immune responses. A non-optimal cervicovaginal microbiota confers increased risk of sPTB. The cervicovaginal space is metabolically active in pregancy; microbiota can produce, modify, and degrade metabolites within this ecosystem. We establish that cervicovaginal metabolomic output clusters by microbial community in pregnancy among Black individuals, revealing increased metabolism within the amino acid and dipeptide pathways as hallmarks of a non-optimal microbiota. Few differences were detected in metabolomic profiles when stratified by birth outcome. The study raises the possibility that metabolites could distinguish women with greater risk of sPTB among those with similar cervicovaginal microbiota, and that metabolites within the amino acid and carbohydrate pathways may play a role in this distinction.

Nationwide genomic atlas of soil-dwelling Listeria reveals effects of selection and population ecology on pangenome evolution.

Natural bacterial populations can display enormous genomic diversity, primarily in the form of gene content variation caused by the frequent exchange of DNA with the local environment. However, the ecological drivers of genomic variability and the role of selection remain controversial. Here, we address this gap by developing a nationwide atlas of 1,854 Listeria isolates, collected systematically from soils across the contiguous United States. We found that Listeria was present across a wide range of environmental parameters, being mainly controlled by soil moisture, molybdenum and salinity concentrations. Whole-genome data from 594 representative strains allowed us to decompose Listeria diversity into 12 phylogroups, each with large differences in habitat breadth and endemism. 'Cosmopolitan' phylogroups, prevalent across many different habitats, had more open pangenomes and displayed weaker linkage disequilibrium, reflecting higher rates of gene gain and loss, and allele exchange than phylogroups with narrow habitat ranges. Cosmopolitan phylogroups also had a large fraction of genes affected by positive selection. The effect of positive selection was more pronounced in the phylogroup-specific core genome, suggesting that lineage-specific core genes are important drivers of adaptation. These results indicate that genome flexibility and recombination are the consequence of selection to survive in variable environments.

Listeria cossartiae sp. nov., Listeria farberi sp. nov., Listeria immobilis sp. nov., Listeria portnoyi sp. nov. and Listeria rustica sp. nov., isolated from agricultural water and natural environments.

A total of 27 Listeria isolates that could not be classified to the species level were obtained from soil samples from different locations in the contiguous United States and an agricultural water sample from New York. Whole-genome sequence-based average nucleotide identity blast (ANIb) showed that the 27 isolates form five distinct clusters; for each cluster, all draft genomes showed ANI values of <95 % similarity to each other and any currently described Listeria species, indicating that each cluster represents a novel species. Of the five novel species, three cluster with the Listeria sensu stricto clade and two cluster with sensu lato. One of the novel sensu stricto species, designated L. cossartiae sp. nov., contains two subclusters with an average ANI similarity of 94.9%, which were designated as subspecies. The proposed three novel sensu stricto species (including two subspecies) are Listeria farberi sp. nov. (type strain FSL L7-0091T=CCUG 74668T=LMG 31917T; maximum ANI 91.9 % to L. innocua), Listeria immobilis sp. nov. (type strain FSL L7-1519T=CCUG 74666T=LMG 31920T; maximum ANI 87.4 % to L. ivanovii subsp. londoniensis) and Listeria cossartiae sp. nov. [subsp. cossartiae (type strain FSL L7-1447T=CCUG 74667T=LMG 31919T; maximum ANI 93.4 % to L. marthii) and subsp. cayugensis (type strain FSL L7-0993T=CCUG 74670T=LMG 31918T; maximum ANI 94.7 % to L. marthii). The two proposed novel sensu lato species are Listeria portnoyi sp. nov. (type strain FSL L7-1582T=CCUG 74671T=LMG 31921T; maximum ANI value of 88.9 % to L. cornellensis and 89.2 % to L. newyorkensis) and Listeria rustica sp. nov. (type strain FSL W9-0585T=CCUG 74665T=LMG 31922T; maximum ANI value of 88.7 % to L. cornellensis and 88.9 % to L. newyorkensis). L. immobilis is the first sensu stricto species isolated to date that is non-motile. All five of the novel species are non-haemolytic and negative for phosphatidylinositol-specific phospholipase C activity; the draft genomes lack the virulence genes found in Listeria pathogenicity island 1 (LIPI-1), and the internalin genes inlA and inlB, indicating that they are non-pathogenic.

Effects of a Potential Host Gut-Derived Probiotic, Bacillus subtilis 6-3-1, on the Growth, Non-specific Immune Response and Disease Resistance of Hybrid Grouper (Epinephelus fuscoguttatus♀ × Epinephelus lanceolatus♂).

A potential host-derived probiotic, Bacillus subtilis 6-3-1, was successfully screened from 768 isolates from the intestines of healthy hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) based on multiple probiotic characteristics in vitro assays, such as, non-hemolytic activity, extracellular enzyme activity, inhibitory activity against pathogens, tolerance to gastrointestinal stress, cell surface hydrophobicity, autoaggregation, and antibiotic susceptibility. Eight weeks of feeding trial revealed that dietary supplementation of B. subtilis 6-3-1 at all three concentrations (1 × 106 CFU g-1 as BS6; 1 × 107 CFU g-1 as BS7; 1 × 108 CFU g-1 as BS8) could promote the growth performance of hybrid groupers to a certain extent at different time points. At the end of 8th week, BS6 and BS8 significantly promoted the weight gain rate (WGR), specific growth rate (SGR) of hybrid groupers. The digestive enzyme activities were also increased in BS6 and BS8 groups comparing with those in control group, except that the increase of amylase activities in BS6 was not significant (P > 0.05). However, BS7 showed the best non-specific immunity stimulating effects among the three concentration groups. While BS7 significantly boosted serum total protein contents, lysozyme (LZM), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and acid phosphatase (ACP) levels, BS6 significantly enhanced serum total protein, LZM activity, and BS8 significantly improved LZM, respiratory bursts activity. B. subtilis 6-3-1 up-regulated the expression of MyD88 in head kidney and intestine and increased villi length (VL) in intestine of BS7 group. It also up-regulated the expression of IgM in head kidney in BS6 group and IgM and TLR1 in intestine of BS8 group. Though all B. subtilis 6-3-1 supplemented groups reduced the cumulative mortality rate post-Vibro harveyi-challenge, BS7 showed the best protection effects among the three concentration groups. In conclusion, with its immune promoting, intestine health enhancing, and V. harveyi resisting effects, BS7 show great potential to be used as a probiotic in hybrid grouper culture.

Adjacent Terrestrial Landscapes Impact the Biogeographical Pattern of Soil Escherichia coli Strains in Produce Fields by Modifying the Importance of Environmental Selection and Dispersal.

High-quality habitats for wildlife (e.g., forest) provide essential ecosystem services while increasing species diversity and habitat connectivity. Unfortunately, the presence of such habitats adjacent to produce fields may increase risk for contamination of fruits and vegetables by enteric bacteria, including Escherichia coliE. coli survives in extrahost environments (e.g., soil) and could be dispersed across landscapes by wildlife. Understanding how terrestrial landscapes impact the distribution of soil E. coli strains is of importance in assessing the contamination risk of agricultural products. Here, using multilocus sequence typing, we characterized 938 E. coli soil isolates collected from two watersheds with different landscape patterns in New York State, USA, and compared the distribution of E. coli and the influence that environmental selection and dispersal have on the distribution between these two watersheds. Results showed that for the watershed with widespread produce fields, sparse forests, and limited interaction between the two land use types, E. coli composition was significantly different between produce field sites and forest sites; this distribution appears to be shaped by relatively strong environmental selection, likely from soil phosphorus, and slight dispersal limitation. For the watershed with more forested areas and stronger interaction between produce field sites and forest sites, E. coli composition between these two land use types was relatively homogeneous; this distribution appeared to be a consequence of wildlife-driven dispersal, inferred by competing models. Collectively, our results suggest that terrestrial landscape attributes could impact the biogeographic pattern of enteric bacteria by adjusting the importance of environmental selection and dispersal.IMPORTANCE Understanding the ecology of enteric bacteria in extrahost environments is important for the development and implementation of strategies to minimize preharvest contamination of produce with enteric pathogens. Our findings suggest that watershed landscape is an important factor influencing the importance of ecological drivers and dispersal patterns of E. coli Agricultural areas in such watersheds may have a higher risk of produce contamination due to fewer environmental constraints and higher potential of dispersal of enteric bacteria between locations. Thus, there is a perceived trade-off between priorities of environmental conservation and public health in on-farm food safety, with limited ecological data supporting or refuting the role of wildlife in dispersing pathogens under normal operating conditions. By combining field sampling and spatial modeling, we explored ecological principles underlying the biogeographic pattern of enteric bacteria at the regional level, which can benefit agricultural, environmental, and public health scientists who aim to reduce the risk of food contamination by enteric bacteria while minimizing negative impacts on wildlife habitats.

A phage-encoded anti-CRISPR enables complete evasion of type VI-A CRISPR-Cas immunity.

The CRISPR RNA (crRNA)-guided nuclease Cas13 recognizes complementary viral transcripts to trigger the degradation of both host and viral RNA during the type VI CRISPR-Cas antiviral response. However, how viruses can counteract this immunity is not known. We describe a listeriaphage (ϕLS46) encoding an anti-CRISPR protein (AcrVIA1) that inactivates the type VI-A CRISPR system of Listeria seeligeri Using genetics, biochemistry, and structural biology, we found that AcrVIA1 interacts with the guide-exposed face of Cas13a, preventing access to the target RNA and the conformational changes required for nuclease activation. Unlike inhibitors of DNA-cleaving Cas nucleases, which cause limited immunosuppression and require multiple infections to bypass CRISPR defenses, a single dose of AcrVIA1 delivered by an individual virion completely dismantles type VI-A CRISPR-mediated immunity.