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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.

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-PI1). We find that the vaginal microbiome of pregnancies that ended preterm exhibits unique genetic profiles. It is more genetically diverse at the species level and harbors a higher richness and diversity of antimicrobial resistance genes, likely promoted by transduction. Interestingly, we find thatGardnerellaspecies, a group of central vaginal pathobionts, are driving this higher genetic diversity, particularly during the first half of the pregnancy. We further present evidence thatGardnerellaspp. 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.

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.

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. A population genomic analysis of 1,854 Listeria soil isolates collected across the contiguous United States identifies geographically prevalent phylogroups with increased pangenome openness and recombination, as a result of adaptation to variable environments.

CRISPR-Cas systems provide their prokaryotic hosts with sequence-specific immunity to foreign genetic elements, including bacteriophages and plasmids. While most interfere with phage infection though cleavage of viral DNA, type VI CRISPR systems use the RNA-guided nuclease Cas13 to recognize mRNA targets. Upon engaging with target RNA, Cas13 cleaves both phage and host transcripts nonspecifically, leading to a state of cell dormancy that is incompatible with phage propagation. However, whether and how infected cells recover from dormancy is not clear. Here we show that type VI CRISPR systems frequently co-occur with DNA-cleaving restriction modification (RM) systems. Using genetics and microscopy, we show that Cas13 and RM systems synergize in anti-phage defense in the natural type VI CRISPR host Listeria seeligeri. Cleavage of the phage genome by RM removes the source of phage transcripts, enabling cells to recover from Cas13-induced cellular dormancy. We find that Cas13 and RM systems operating simultaneously eliminate phage DNA and neutralize infection more effectively than either defense alone. Thus, cells harboring both defense systems exhibit robust anti-phage immunity and survive infection. Our work therefore reveals that type VI CRISPR immunity is cell-autonomous and non-abortive, if paired with RM or similar DNA-targeting defenses. The ability of an abortive response to be resolved by the actions of another anti-phage defense has implications for the roles of diverse host-directed immune systems in bacteria.

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.

State-of-the-art nanopore sequencing enables rapid and real-time identification of novel pathogens, which has wide application in various research areas and is an emerging diagnostic tool for infectious diseases including COVID-19. Nanopore translocation enables de novo sequencing with long reads (> 10 kb) of novel genomes, which has advantages over existing short-read sequencing technologies. Biological nanopore sequencing has already achieved success as a technology platform but it is sensitive to empirical factors such as pH and temperature. Alternatively, ångström- and nano-scale solid-state nanopores, especially those based on two-dimensional (2D) membranes, are promising next-generation technologies as they can surpass biological nanopores in the variety of membrane materials, ease of defining pore morphology, higher nucleotide detection sensitivity, and facilitation of novel and hybrid sequencing modalities. Since the discovery of graphene, atomically-thin 2D materials have shown immense potential for the fabrication of nanopores with well-defined geometry, rendering them viable candidates for nanopore sequencing membranes. Here, we review recent progress and future development trends of 2D materials and their ångström- and nano-scale pore-based nucleic acid (NA) sequencing including fabrication techniques and current and emerging sequencing modalities. In addition, we discuss the current challenges of translocation-based nanopore sequencing and provide an outlook on promising future research directions.

L. swaminathanii is a novel sensu stricto species that originated from a US National Park and it will be the first Listeria identified to date without official standing in the nomenclature. Validation was impeded by the National Park’s requirements for strain access, ultimately deemed too restrictive by the International Committee on Systematics of Prokaryotes.

Soil samples collected in the Great Smoky Mountains National Park yielded a

Soil samples collected in the Great Smoky Mountains National Park yielded aListeriaisolate that could not be classified to the species level. Whole-genome sequence-based average nucleotide identity BLAST andin silicoDNA-DNA Hybridization analyses confirmed this isolate to be a novelListeria sensu strictospecies with the highest similarity toL. marthii(ANI=93.9%, isDDH=55.9%). Additional whole-genome-based analysis using the Genome Taxonomy Database Toolkit, an automated program for classifying bacterial genomes, further supported delineation as a novelListeria sensu strictospecies, as this tool failed to assign a species identification but identifiedL. marthiias the closest match. Phenotypic and genotypic characterization results indicate that this species is nonpathogenic. Specifically, the novelListeriaspecies described here is phenotypically (i) non-hemolytic and (ii) negative for phosphatidylinositol-specific phospholipase C activity; the draft genome lacks all virulence genes found in theListeriapathogenicity island 1 (LIPI-1), as well as the internalin genesinlAandinlB. While the type strain for the new species is phenotypically catalase-negative (an unusual characteristic forListeria sensu strictospecies), its genome contained an apparently intact catalase gene (kat); hence assessment of this phenotype with future isolates will be important. Rapid species identification systems (ListeriaAPI, VITEK 2, VITEK MS) misidentified this novel species as eitherL. monocytogenes, L. innocua, orL. marthii. We propose the nameL. swaminathanii, and the type strain is FSL L7-0020T(=ATCC TSD-239T).IMPORTANCEL. swaminathaniiis a novelsensu strictospecies that originated from a US National Park, and its place of origin is ultimately preventing this species from achieving valid status. The US National Park Service restricts strain accessibility and open access is currently a prerequisite for species validation. Essentially the only criteria that was not met forL. swaminathaniivalidation is accessibility of the type strain, therefore nomenclature status should not negate the significance of this discovery. As a novelsensu strictospecies,L. swaminathaniiexpands the group of species whose presence is associated with an increased risk of anL. monocytogenescontamination, and therefore could play an important role in public health. While developers ofListeriaspp. detection methods historically only included validly publish species in their validation studies,L. swaminathaniiis unequivocally asensu strictospecies and should be included as well.

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.

Spontaneous preterm birth (sPTB) is a leading cause of maternal and neonatal morbidity and mortality, yet both its prevention and early risk stratification are limited. The vaginal microbiome has been associated with PTB risk, possibly via metabolic or other interactions with its host. Here, we performed untargeted metabolomics on 232 vaginal samples, in which we have previously profiled the microbiota using 16S rRNA gene sequencing. Samples were collected at 20-24 weeks of gestation from women with singleton pregnancies, of which 80 delivered spontaneously before 37 weeks of gestation. We find that the vaginal metabolome correlates with the microbiome and separates into six clusters, three of which are associated with spontaneous preterm birth (sPTB) in Black women. Furthermore, while we identify five metabolites that associate with sPTB, another five associate with sPTB only when stratifying by race. We identify multiple microbial correlations with metabolites associated with sPTB, including intriguing correlations between vaginal bacteria that are considered sub-optimal and metabolites that were enriched in women who delivered at term. We propose that several sPTB-associated metabolites may be exogenous, and investigate another using metabolic models. Notably, we use machine learning models to predict sPTB risk using metabolite levels, weeks to months in advance, with high accuracy. We show that these predictions are more accurate than microbiome-based and maternal covariates-based models. Altogether, our results demonstrate the potential of vaginal metabolites as early biomarkers of sPTB and highlight exogenous exposures as potential risk factors for prematurity.

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 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.

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 coli. E. 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.

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.

High-quality habitats for wildlife (e.g., forest) provide essential ecosystem services while increasing species diversity and habitat connectivity. Unfortunately, presence of such habitats adjacent to produce fields may increase risk for contamination of fruits and vegetables by enteric bacteria, including Escherichia coli. E. coli survives in extra-host environments (e.g., soil) and could disperse across landscapes by wildlife. Understanding how terrestrial landscapes impact the distribution of soil E. coli is of importance in assessing the contamination risk of agricultural products. Here, using multi-locus 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 of two ecological forces (environmental selection and dispersal) 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 was 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 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.IMPORTANCEUnderstanding the ecology of enteric bacteria in extra-host environments is important to allow for development and implementation of strategies to minimize pre-harvest 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. For watersheds with widespread produce fields, E. coli appears to experience local adaptation, possibly due to exposure to environmental stresses associated with agricultural activities. In contrast, for watersheds with high forest coverage we found evidence for wildlife-driven dispersal of E. coli, which might facilitate more frequent genetic exchange in this environment. Agricultural areas in such watersheds may have a higher risk of produce contamination due to less environmental constraints and higher potential of dispersal of enteric bacteria between locations. The significance of our research lies in exploring ecological principles underlying the biogeographic pattern of enteric bacteria at the regional level, which can inform agricultural, environmental and public health scientists that aim to reduce the risk of food contamination by enteric bacteria.

The revolution of molecular techniques in the field of microbial ecology not only allows an in-depth exploration of abundant bacteria, but also confirms the existence of “rare biosphere.” However, the mechanisms underlying the assembly process of abundant and rare bacteria in plateau lakes are still poorly understood. In the present study, distribution of abundant and rare bacteria in 21 lakes on Yungui Plateau, China and the relative importance of deterministic and neutral factors to their biogeographic patterns were examined. Results surprisingly showed that similar biogeographic patterns of rare and abundant taxa were generally shaped via similar assembly mechanisms. For both bacterial groups, deterministic process strongly influenced the distribution, while neutral process was not at play. In addition, they both exhibited a weak distance–decay relationship. Nevertheless, environmental drivers of rare and abundant taxa and their importance were not exactly the same. Water physicochemical property was the most dominant driver shaping the biogeographic pattern of rare taxa, followed by lake morphology and watershed land-use, while for abundant taxa, watershed land-use was the most dominant driver and lake morphology did not play a significant role. This study suggested that rare bacteria in high-altitude aquatic ecosystems might experience equally low extinction risk and respond to environmental changes in a similar manner to abundant ones, but their ecological niches and functions were not identical. To fully understand the assembly mechanism of bacterial community, it is necessary to differentiate the community by traits of taxa.

Aeromonas schubertii is a conditional pathogen widely distributed in aquatic environments, yet it was only recently reported as a causative pathogen in fish. This study investigated antioxidant capacity, non-specific immunity, histopathology and immune-related gene expression profiles in Nile tilapia Oreochromis niloticus injected with A. schubertii. The results show that A. schubertii infection stimulated the activation of antioxidant activity and non-specific immunity in Nile tilapia. Histopathological analysis revealed that inflammation existed in the liver, spleen, head kidney, brain and intestine of the infected fish. Quantitative real-time PCR was performed to measure mRNA expression levels of six immune-related genes (interleukin-1β, tumor necrosis factor-α, cyclooxygenase-2, transforming growth factor beta, interleukin-10 and heat shock protein 70) in the intestine and head kidney at different timepoints after experimental infection. The results demonstrated clear transcriptional activation of these immune-related genes in the intestine and head-kidney tissues. To our knowledge, this is the first study to investigate the host response to A. schubertii infection in a fish. These results will contribute to further understanding of the pathogenesis and host defense system in fishes following infection with A. schubertii.

Figure S1. The distribution of AMR gene numbers of AMR S. Dublin, AMR S. Newport, and AMR S. Typhimurium isolates. The AMR gene number of AMR S. Dublin is significantly different from that of AMR S. Newport and AMR S. Typhimurium. Figure S2. The distribution of PPG gene numbers of AMR S. Dublin, AMR S. Newport, and AMR S. Typhimurium isolates. Figure S3. The distribution of pseudogene numbers in S. Dublin, S. Newport, and S. Typhimurium. Figure S4. Gene tree of AAC (6′)-Iaa inferred by maximum likelihood method. Tree is rooted by midpoint. Bootstrap values > 70% are presented on the tree. S. Dublin is indicated by blue, S. Newport by blue, and S. Typhimurium by red. Figure S5. Maximum likelihood tree of AMR S. Newport isolates, and Lineage II (sub-lineages - IIA, IIB and IIC) and Lineage III reference isolates. Tree is rooted by midpoint. Bootstrap values of major clades are presented on the tree. Reference isolates are indicated by red. (DOCX 681 kb)

Excessive nutrient loads resulted in cascading trophic effects and ecosystem responses. Aims of this study were to determine if the thresholds in nutrient gradient related to phytoplankton community composition could be identified in eutrophic lake, and further to analyze the change of phytoplankton assemblage along the nutrient concentration based on Threshold Indicator Taxa ANalysis (TITAN). The results presented the significant community thresholds estimate for negative taxa declining at 1.650 mg/L TN and 131.5 μg/L TP, as well as simultaneously increasing for positive taxa at 1.665 mg/L TN and 151.5 μg/L TP along nutrient enrichment gradient. However, there was unremarkable change point determined for TN:TP ratios in Lake Dianchi. Elevated TN and TP altered the phytoplankton assemblage, even may induce the fade of algal blooms across the threshold in the hypertrophic lake. The findings could provide implications for deeply deciphering abrupt transitions for phytoplankton assemblage and developing nutrient tactics to protect the lake ecosystems.

Naphthenic acids (NAs) have been gaining recognition in recent years as potentially harmful environmental contaminants. Few studies have focused on the potential ecotoxicity of NAs to terrestrial environment. In this study, the responses of antioxidant system and lipid peroxidation and DNA damage were investigated after exposing Eisenia fetida to soil contaminated with NAs. The results indicated that NAs induced a significant increase (p < 0.05) in superoxide dismutase and catalase enzyme activities. The glutathione peroxidase enzyme activities were significantly inhibited (p < 0.05) in the medium and high dose treatments. An increase in malondialidehyde indicated that NAs could cause cellular lipid peroxidation in the tested earthworms. The percentage of DNA in the tail of comet assay of coelomocytes as an indication of DNA damage increased after treatment with different doses of NAs, and a dose-dependent DNA damage of coelomocytes was found. In conclusion, oxidative stress caused by NAs exposure induces physiological responses and genotoxicity on earthworms. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1337-1343, 2016.

Geographic location and physicochemical properties are thought to represent major factors that shape soil bacterial community abundance and diversity. Crude oil contamination is becoming a notable concern with respect to soil property variation; however, the quantifiable influences of geographic location, physicochemical properties, and oil contamination are still poorly understood. In this study, the 16S ribosomal RNA genes of bacteria in the four oil fields in China were analyzed by using pyrosequencing. Results showed that physicochemical properties were the most dominant factor of bacterial community distribution, followed by geographical location. Oil contamination was a driving factor whose indirect influence was stronger than its direct influence. Under the impact of these three factors, different oil fields presented diversified and distinguishable bacterial community features. The soil of sites with the highest total petroleum hydrocarbon content (HB), nitrogen content (DQ), and phosphorus content (XJ) contained the largest proportion of functional groups participating in hydrocarbon degradation, nitrogen turnover, and phosphorus turnover, respectively. The first dominant phylum of the site with loam soil texture (HB) was Actinobacteria instead of Proteobacteria in other sites with sandy or sandy loam soil texture (DQ, SL, XJ). The site with the highest salinization and alkalization (SL) exhibited the largest proportion of unique local bacteria. The site that was located in the desert with extremely low precipitation (XJ) had the most diversified bacteria distribution. The bacterial community diversity was strongly influenced by soil physicochemical properties.

Naphthenic acids (NAs) are a toxic complex mixture of carboxylic acids occurring naturally in petroleum. Due to the serious potential risks of NAs towards terrestrial ecology during crude oil exploration and production processes, and the lack of efficient methodologies for extraction and analysis of these compounds, the goal of this study is to detail the development of a routine method for extraction, and quantitative and qualitative analysis of NAs in oil contaminated soils. Solid phase extraction using a MAX cartridge was employed in combination with GC/MS. Ethyl acetate with 2% formic acid used as an elution solvent showed the best recoveries of NAs (98.36–112.35%). The total NA concentration and NA profiles of oil contaminated soil samples from 4 oil fields in China were examined. High concentrations of NAs (maximum, 132.91 mg kg−1) were detected in soils, which implied toxic and estrogenic risks for human and terrestrial organisms. Different profiles of NA mixtures were observed in soils from 4 different oil fields, and evaporation and biodegradation could influence the compounds of NAs. The authors present the first feasible method for analysis of NAs in soil.

Sequencing of single genes remains an important tool that allows the rapid classification of bacteria. Sequencing of a portion of sigB , which encodes a stress-responsive alternative sigma factor, has emerged as a commonly used molecular tool for the initial characterization of diverse Listeria isolates. In this study, evolutionary approaches were used to assess the validity of sigB allelic typing for Listeria . For a data set of 4,280 isolates, sigB allelic typing showed a Simpson's index of diversity of 0.96. Analyses of 164 sigB allelic types (ATs) found among the 6 Listeria sensu stricto species, representing these 4,280 isolates, indicate that neither frequent homologous recombination nor positive selection significantly contributed to the evolution of sigB , confirming its genetic stability. The molecular clock test provided evidence for unequal evolution rates across clades; Listeria welshimeri displayed the lowest sigB diversity and was the only species in which sigB evolved in a clocklike manner, implying a unique natural history. Among the four L. monocytogenes lineages, sigB evolution followed a molecular clock only in lineage IV. Moreover, sigB displayed a significant negative Tajima D value in lineage II, suggesting a recent population bottleneck followed by lineage expansion. The absence of positive selection along with the violation of the molecular clock suggested a nearly neutral mechanism of Listeria sensu stricto sigB evolution. While comparison with a whole-genome sequence-based phylogeny revealed that the sigB phylogeny did not correctly reflect the ancestry of L. monocytogenes lineage IV, the availability of a large sigB AT database allowed accurate species classification. IMPORTANCE sigB allelic typing has been widely used for species delineation and subtyping of Listeria . However, an informative evaluation of this method from an evolutionary perspective was missing. Our data indicate that the genetic stability of sigB is affected by neither frequent homologous recombination nor positive selection, which supports that sigB allelic typing provides reliable subtyping and classification of Listeria sensu stricto strains. However, multigene data are required for accurate phylogeny reconstruction of Listeria . This study thus contributes to a better understanding of the evolution of sigB and confirms the robustness of the sigB subtyping system for Listeria .

Aquatic ecosystems are ecologically important, but continuously threatened by a growing number of human induced changes. This study evaluates the research trends of "aquatic ecosystem" between 1992 and 2011 in journals of all subject categories of the science citation index and social sciences citation index. The analyzed parameters include publication output, cited publication, document type, language, distributions of journal, author, country and institutes, and analysis of author keywords and keywords plus. The results showed that over the past two decades, there was a consistent growth in publication output with involvement of increasing number of countries and institutions, and North America was still the leading region in the subject. Classification of the top 30 author keywords indicated that more research attentions were paid to the study on aquatic organism, water environment and aquatic ecosystem condition. Aquatic ecosystem, water quality, and fish were the top three most frequently used author keywords. In addition, owing to its significant impact on aquatic ecosystems, climate change has been placed crucial emphasis recently. Aquatic ecosystem research trend was shifting from water environment to aquatic ecosystem wide issues.

Sequencing of single genes remains an important tool that allows the rapid classification of bacteria. Sequencing of a portion of sigB, which encodes a stress-responsive alternative sigma factor, has emerged as a commonly used molecular tool for the initial characterization of diverse Listeria isolates. In this study, evolutionary approaches were used to assess the validity of sigB allelic typing for Listeria. For a data set of 4,280 isolates, sigB allelic typing showed a Simpson's index of diversity of 0.96. Analyses of 164 sigB allelic types (ATs) found among the 6 Listeria sensu stricto species, representing these 4,280 isolates, indicate that neither frequent homologous recombination nor positive selection significantly contributed to the evolution of sigB, confirming its genetic stability. The molecular clock test provided evidence for unequal evolution rates across clades; Listeria welshimeri displayed the lowest sigB diversity and was the only species in which sigB evolved in a clocklike manner, implying a unique natural history. Among the four L. monocytogenes lineages, sigB evolution followed a molecular clock only in lineage IV. Moreover, sigB displayed a significant negative Tajima D value in lineage II, suggesting a recent population bottleneck followed by lineage expansion. The absence of positive selection along with the violation of the molecular clock suggested a nearly neutral mechanism of Listeria sensu stricto sigB evolution. While comparison with a whole-genome sequence-based phylogeny revealed that the sigB phylogeny did not correctly reflect the ancestry of L. monocytogenes lineage IV, the availability of a large sigB AT database allowed accurate species classification.

The mechanisms of community assembly are a central focus in the field of microbial ecology. However, to what extent these mechanisms differ in importance by traits of groups is poorly understood. Here we quantified the importance of neutral and niche processes in community assembly for bacteria, habitat specialists and generalists in 21 plateau lakes of China. Results showed that both neutral and niche processes played a critical role in the assembly of entire bacterial communities, shaping a unique biogeographical pattern. A few habitat generalists and many specialists were identified. Interestingly, habitat specialists were only governed by niche process, with seven significant environmental variables—salinity, dissolved oxygen, water transparency, total phosphorus, ammonium-nitrogen, temperature and total nitrogen—independently explaining 40.3% of the biological variation. By contrast, habitat generalists were strongly driven by neutral process, with 50.9% of the variation of detection frequency explained in neutral community model. Only three environmental variables—salinity, total nitrogen and dissolved oxygen—significantly affected the distribution of habitat generalists, independently explaining 13.6% of the variation. Governed by different assembly mechanisms, habitat specialists and generalists presented disparate biogeographical patterns. Our result emphasizes the importance of investigating the bacterial community assembly at more refined levels than entire communities.

Elevated environmental pressures negatively affect the bacterial community structure. However, little knowledge about the nonlinear responses of spatially related environmental variable across multiple plateau lake ecosystems on bacterioplankton communities has been gathered. Here, we used 454 pyrosequencing of 16S rRNA genes to study the associations of bacterial communities in terms of environmental characteristics as well as the potentially ecological threshold-inducing shifts of the bacterial community structure along the key environmental variables based on hypothesized structural equation models and the SEGMENTED method in 21 plateau lakes. Our results showed that water transparency was the major driving force and that total nitrogen was more significant than total phosphorus in determining the taxon composition of the bacterioplankton community. Significant community threshold estimates for bacterioplankton were observed at 7.36 for pH and 25.6% for the percentage of the agricultural area, while the remarkable change point of the cyanobacteria community structure responding to pH was at 7.74. Furthermore, the findings indicated that increasing nutrient loads can induce a distinct shift in dominance from Proteobacteria to Cyanobacteria, as well as a sharp decrease and adjacent increase when crossing the change point for Actinobacteria and Bacteroidetes along the gradient of the agricultural area.

Since total petroleum hydrocarbons (TPH) are toxic and persistent in environments, studying the impact of oil contamination on microbial communities in different soils is vital to oil production engineering, effective soil management and pollution control. This study analyzed the impact of oil contamination on the structure, activity and function in carbon metabolism of microbial communities of Chernozem soil from Daqing oil field and Cinnamon soil from Huabei oil field through both culture-dependent techniques and a culture-independent technique-pyrosequencing. Results revealed that pristine microbial communities in these two soils presented disparate patterns, where Cinnamon soil showed higher abundance of alkane, (polycyclic aromatic hydrocarbons) PAHs and TPH degraders, number of cultivable microbes, bacterial richness, bacterial biodiversity, and stronger microbial activity and function in carbon metabolism than Chernozem soil. It suggested that complicated properties of microbes and soils resulted in the difference in soil microbial patterns. However, the changes of microbial communities caused by oil contamination were similar in respect of two dominant phenomena. Firstly, the microbial community structures were greatly changed, with higher abundance, higher bacterial biodiversity, occurrence of Candidate_division_BRC1 and TAO6, disappearance of BD1-5 and Candidate_division_OD1, dominance of Streptomyces, higher percentage of hydrocarbon-degrading groups, and lower percentage of nitrogen-transforming groups. Secondly, microbial activity and function in carbon metabolism were significantly enhanced. Based on the characteristics of microbial communities in the two soils, appropriate strategy for in situ bioremediation was provided for each oil field. This research underscored the usefulness of combination of culture-dependent techniques and next-generation sequencing techniques both to unravel the microbial patterns and understand the ecological impact of contamination.

Naphthenic acids (NAs) have been gaining recognition in recent years as potentially harmful environmental contaminants. Few studies have focused on the potential ecotoxicity of NAs to terrestrial environment. In this study, the responses of antioxidant system and lipid peroxidation and DNA damage were investigated after exposing Eisenia fetida to soil contaminated with NAs. The results indicated that NAs induced a significant increase (p 0.05) in superoxide dismutase and catalase enzyme activities. The glutathione peroxidase enzyme activities were significantly inhibited (p 0.05) in the medium and high dose treatments. An increase in malondialidehyde indicated that NAs could cause cellular lipid peroxidation in the tested earthworms. The percentage of DNA in the tail of comet assay of coelomocytes as an indication of DNA damage increased after treatment with different doses of NAs, and a dose-dependent DNA damage of coelomocytes was found. In conclusion, oxidative stress caused by NAs exposure induces physiological responses and genotoxicity on earthworms. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1337-1343, 2016.

Alkanes and polycyclic aromatic hydrocarbons (PAHs) are the commonly detected petroleum hydrocarbon contaminants in soils in oil exploring areas. Hydrocarbon-degrading genes are useful biomarks for estimation of the bioremediation potential of contaminated sites. However, the links between environmental factors and the distribution of alkane and PAH metabolic genes still remain largely unclear. The present study investigated the abundances and diversities of soil n-alkane and PAH-degrading bacterial communities targeting both alkB and nah genes in two oil exploring areas at different geographic regions. A large variation in the abundances and diversities of alkB and nah genes occurred in the studied soil samples. Various environmental variables regulated the spatial distribution of soil alkane and PAH metabolic genes, dependent on geographic location. The soil alkane-degrading bacterial communities in oil exploring areas mainly consisted of Pedobacter, Mycobacterium, and unknown alkB-harboring microorganisms. Moreover, the novel PAH-degraders predominated in nah gene clone libraries from soils of the two oil exploring areas. This work could provide some new insights towards the distribution of hydrocarbon-degrading microorganisms and their biodegradation potential in soil ecosystems.

Polycyclic aromatic hydrocarbons (PAHs) are one of the major pollutants in soils in oil exploring areas. Biodegradation is the major process for natural elimination of PAHs from contaminated soils. Functional genes can be used as biomarkers to assess the biodegradation potential of indigenous microbial populations. However, little is known about the distribution of PAH-degrading genes in the environment. The links between environmental parameters and the distribution of PAH metabolic genes remain essentially unclear. The present study investigated the abundance and diversity of naphthalene dioxygenase genes in the oil-contaminated soils in the Shengli Oil Field (China). Spatial variations in the density and diversity of naphthalene dioxygenase genes occurred in this area. Four different sequence genotypes were observed in the contaminated soils, with the predominance of novel PAH-degrading genes. Pearson’s correlation analysis illustrated that gene abundance had positive correlations with the levels of total organic carbon and aromatic hydrocarbons, while gene diversity showed a negative correlation with the level of polar aromatics. This work could provide some new insights toward the distribution of PAH metabolic genes and PAH biodegradation potential in oil-contaminated ecosystems.