Soil exposure modulates the immune response to an influenza challenge in a mouse model.

There is increasing evidence that early life microbial exposure aids in immune system maturation, more recently known as the "old friends" hypothesis. To test this hypothesis, 4-week-old mice were exposed to soils of increasing microbial diversity for four weeks followed by an intranasal challenge with either live or heat inactivated influenza A virus and monitored for 7 additional days. Perturbations of the gut and lung microbiomes were explored through 16S rRNA amplicon sequencing. RNA-sequencing was used to examine the host response in the lung tissue through differential gene expression. We determined that compared to the gut microbiome, the lung microbiome is more susceptible to changes in beta diversity following soil exposure with Lachnospiraceae ASVs accounting for most of the differences between groups. While several immune system genes were found to be significantly differentially expressed in lung tissue due to soil exposures, there were no differences in viral load or weight loss. This study shows that exposure to diverse microbial communities through soil exposure alters the gut and lung microbiomes resulting in differential expression of specific immune system related genes within the lung following an influenza challenge.

Complete Genome Sequence of NAH7-Harboring Pseudomonas putida Strain G7.

Pseudomonas putida G7 (111) is an aerobic bacterial species discovered in soil known to harbor the naphthalene-degrading NAH7 plasmid. Here, we report the genome sequence of G7. The genome was assembled by Nanopore sequencing and consisted of a chromosome of 6.4 Mbp with a G+C content of 62.13%.

Predictive values of time-dense SARS-CoV-2 wastewater analysis in university campus buildings.

Wastewater-based SARS-CoV-2 surveillance on college campuses has the ability to detect individual clinical COVID-19 cases at the building-level. High concordance of wastewater results and clinical cases has been observed when calculated over a time window of four days or longer and in settings with high incidence of infection. At Duke University, twice a week clinical surveillance of all resident undergraduates was carried out in the spring 2021 semester. We conducted simultaneous wastewater surveillance with daily frequency on selected residence halls to assess wastewater as an early warning tool during times of low transmission with the hope of scaling down clinical test frequency. We evaluated the temporal relationship of the two time-dense data sets, wastewater and clinical, and sought a strategy to achieve the highest wastewater predictive values using the shortest time window to enable timely intervention. There were 11 days with clinical cases in the residence halls (80-120 occupants) under wastewater surveillance with 5 instances of a single clinical case and 3 instances of two clinical cases which also corresponded to a positive wastewater SARS-CoV-2 signal. While the majority (71%) of our wastewater samples were negative for SARS-CoV-2, 29% resulted in at least one positive PCR signal, some of which did not correlate with an identified clinical case. Using a criteria of two consecutive days of positive wastewater signals, we obtained a positive predictive value (PPV) of 75% and a negative predictive value of 87% using a short 2 day time window for agreement. A conventional concordance over a much longer 4 day time window resulted in PPV of only 60%. Our data indicated that daily wastewater collection and using a criteria of two consecutive days of positive wastewater signals was the most predictive approach to timely early warning of COVID-19 cases at the building level.

Evaluation of developing maize microbiomes and associations among nitrogen cyclers and key fungal taxa.

More sustainable approaches to agriculture are urgently needed to protect existing resources and optimize crop yields and to provide food for a growing global human population. More sustainable agricultural practices that utilize plant-microbe relationships across cultivation are urgently needed. The main objectives of this study were to track the prokaryotic and fungal microbiomes associated with key growth stages of developing maize to evaluate the relationships among nitrogen cycling bacteria and major fungal genera including those known to contain arbuscular mycorrhizal fungi and other important taxa. Prokaryotic and fungal microbiomes associated with bulk soils, rhizosphere soils and tissues of developing maize were characterized using Illumina MiSeq sequencing. Similarities in microbiome diversity and abundance were compared to sample metadata to explore the influence of external factors on microbiome development. Correlations among target fungal taxa, bulk bacteria and nitrogen cycling bacteria were determined using non-parametric Spearman correlations. Important maize-associated fungal taxa were detected in all samples across growth stages, with Fusarium, Penicillium and Aspergillus fungi comprising up to 4.21, 4.26 and 0.28% of all fungal genera, respectively. Thirteen statistically significant correlations between nitrogen cycling genera and targeted fungal genera were also identified (rS≥0.70 or rS≤-0.70; P<0.05). This study is the first to note a strong positive association among several nitrifying bacteria and Fusarium (R=0.71; P=0.0046), Aspergillus (R=0.71; P=0.0055) and Cladosporium spcies (R=0.74; P=0.0038), suggesting the levels of soil nitrate, nitrite or nitrification intermediates may have large roles in the proliferation of important maize-associated fungi.

Alterations of endophytic microbial community function in Spartina alterniflora as a result of crude oil exposure.

The 2010 Deepwater Horizon disaster remains one of the largest oil spills in history. This event caused significant damage to coastal ecosystems, the full extent of which has yet to be fully determined. Crude oil contains toxic heavy metals and substances such as polycyclic aromatic hydrocarbons that are detrimental to some microbial species and may be used as food and energy resources by others. As a result, oil spills have the potential to cause significant shifts in microbial communities. This study assessed the impact of oil contamination on the function of endophytic microbial communities associated with saltmarsh cordgrass (Spartina alterniflora). Soil samples were collected from two locations in coastal Louisiana, USA: one severely affected by the Deepwater Horizon oil spill and one relatively unaffected location. Spartina alterniflora seedlings were grown in both soil samples in greenhouses, and GeoChip 5.0 was used to evaluate the endophytic microbial metatranscriptome shifts in response to host plant oil exposure. Oil exposure was associated with significant shifts in microbial gene expression in functional categories related to carbon cycling, virulence, metal homeostasis, organic remediation, and phosphorus utilization. Notably, significant increases in expression were observed in genes related to metal detoxification with the exception of chromium, and both significant increases and decreases in expression were observed in functional gene subcategories related to hydrocarbon metabolism. These findings show that host oil exposure elicits multiple changes in gene expression from their endophytic microbial communities, producing effects that may potentially impact host plant fitness.

Identifying bioaugmentation candidates for bioremediation of polycyclic aromatic hydrocarbons in contaminated estuarine sediment of the Elizabeth River, VA, USA.

Estuarine sediments near former creosoting facilities along the Elizabeth River (Virginia, USA) are contaminated by polycyclic aromatic hydrocarbons (PAHs). In this study, we interrogated the bacterial community of the Elizabeth River with both culture-based and culture-independent methods to identify potential candidates for bioremediation of these contaminants. DNA-based stable isotope probing (SIP) experiments with phenanthrene and fluoranthene using sediment from the former Republic Creosoting site identified relevant PAH-degrading bacteria within the Azoarcus, Hydrogenophaga, and Croceicoccus genera. Targeted cultivation of PAH-degrading bacteria from the same site recovered 6 PAH-degrading strains, including one strain highly similar to Hydrogenophaga sequences detected in SIP experiments. Other isolates were most similar to organisms within the Novosphingobium, Sphingobium, Stenotrophomonas, and Alcaligenes genera. Lastly, we performed 16S rRNA gene amplicon microbiome analyses of sediment samples from four sites, including Republic Creosoting, with varying concentrations of PAHs. Analysis of these data showed a striking divergence of the microbial community at the highly contaminated Republic Creosoting site from less contaminated sites with the enrichment of several bacterial clades including those affiliated with the Pseudomonas genus. Sequences within the microbiome libraries similar to SIP-derived sequences were generally found at high relative abundance, while the Croceicoccus sequence was present at low to moderate relative abundance. These results suggest that Azoarcus and Hydrogenophaga strains might be good target candidates for biostimulation, while Croceicoccus spp. might be good targets for bioaugmentation in these sediments. Furthermore, this study demonstrates the value of culture-based and culture-independent methods in identifying promising bacterial candidates for use in a precision bioremediation scheme. KEY POINTS: • This study highlights the importance of using multiple strategies to identify promising bacterial candidates for use in a precision bioremediation scheme. • We used both selective cultivation techniques and DNA-based stable isotope probing to identify bacterial degraders of prominent PAHs at a historically contaminated site in the Elizabeth River, VA, USA. • Azoarcus and Hydrogenophaga strains might be good target candidates for biostimulation in Elizabeth River sediments, while Croceicoccus spp. might be good targets for bioaugmentation.

The effect of bacterial growth strategies on plasmid transfer and naphthalene degradation for bioremediation.

Mobilizable plasmids are extra-chromosomal, circular DNA that have contributed to the rapid evolution of bacterial genomes and have been used in environmental, biotechnological, and medicinal applications. Degradative plasmids with genetic capabilities to degrade organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs), have the potential to be useful for more environmentally friendly and cost-effective remediation technologies compared to existing physical remediation methods. Genetic bioaugmentation, the addition of catabolic genes into well-adapted communities via plasmid transfer (conjugation), is being explored as a remediation approach that is sustainable and long-lasting. Here, we explored the effect of the ecological growth strategies of plasmid donors and recipients on conjugation and naphthalene degradation of two PAH-degrading plasmids, pNL1 and NAH7. Overall, both pNL1 and NAH7 showed conjugation preferences towards a slow-growing ecological growth strategy, except when NAH7 was in a mixed synthetic community. These conjugation preferences were partially described by a combination of growth strategy, GC content, and phylogenetic relatedness. Further, removal of naphthalene via plasmid-mediated degradation was consistently higher in a community consisting of recipients with a slow-growing ecological growth strategy compared to a mixed community or a community consisting of fast-growing ecological growth strategy. Understanding plasmid conjugation and degradative preferences has the capacity to influence future remediation technology design and has broad implications in biomedical, environmental, and health fields.

Properties affecting transfer and expression of degradative plasmids for the purpose of bioremediation.

Plasmids, circular DNA that exist and replicate outside of the host chromosome, have been important in the spread of non-essential genes as well as the rapid evolution of prokaryotes. Recent advances in environmental engineering have aimed to utilize the mobility of plasmids carrying degradative genes to disseminate them into the environment for cost-effective and environmentally friendly remediation of harmful contaminants. Here, we review the knowledge surrounding plasmid transfer and the conditions needed for successful transfer and expression of degradative plasmids. Both abiotic and biotic factors have a great impact on the success of degradative plasmid transfer and expression of the degradative genes of interest. Properties such as ecological growth strategies of bacteria may also contribute to plasmid transfer and may be an important consideration for bioremediation applications. Finally, the methods for detection of conjugation events have greatly improved and the application of these tools can help improve our understanding of conjugation in complex communities. However, it remains clear that more methods for in situ detection of plasmid transfer are needed to help detangle the complexities of conjugation in natural environments to better promote a framework for precision bioremediation.

The Gut/Lung Microbiome Axis in Obesity, Asthma, and Bariatric Surgery: A Literature Review.

Mounting evidence suggests that obesity, parameters of metabolic syndrome, and asthma are significantly associated. Interestingly, these conditions are also associated with microbiome dysbiosis, notably in the airway microbiome for patients with asthma and in the gut microbiome for patients with obesity and/or metabolic syndrome. Considering that improvements in asthma control, lung function, and airway hyperresponsiveness are often reported after bariatric surgery, this review investigated the potential role of bacterial gut and airway microbiome changes after bariatric surgery in ameliorating asthma symptoms. Rapid and persistent gut microbiota alterations were reported following surgery, some of which can be sustained for years. The gut microbiome is thought to modulate airway cellular responses via short-chain fatty acids and inflammatory mediators, such that increased propionate and butyrate levels following surgery may aid in reducing asthma symptoms. In addition, increased prevalence of Akkermansia muciniphila after Roux-en-Y gastric bypass and sleeve gastrectomy may confer protection against airway hyperreactivity and inflammation. Metabolic syndrome parameters also improved following bariatric surgery, and whether weight-loss-independent metabolic changes affect airway processes and asthma pathobiology merits further research. Fulfilling knowledge gaps outlined in this review could facilitate the development of new therapeutic options for patients with obesity and asthma.

The role of gut microbial community and metabolomic shifts in adaptive resistance of Atlantic killifish (Fundulus heteroclitus) to polycyclic aromatic hydrocarbons.

Altered gut microbiomes may play a role in rapid evolution to anthropogenic change but remain poorly understood. Atlantic killifish (Fundulus heteroclitus) in the Elizabeth River, VA have evolved resistance to polycyclic aromatic hydrocarbons (PAHs) and provide a unique opportunity to examine the links between shifts in the commensal microbiome and organismal physiology associated with evolved resistance. Here, 16S rRNA sequence libraries derived from fish guts and sediments sampled from a highly PAH contaminated site revealed significant differences collected at similar samples from an uncontaminated site. Phylogenetic groups enriched in the libraries derived from PAH-resistant fish were dissimilar to their associated sediment libraries, suggesting the specific environment within the PAH-resistant fish intestine influence the gut microbiome composition. Gut metabolite analysis revealed shifts between PAH-resistant and non-resistant subpopulations. Notably, PAH-resistant fish exhibited reduced levels of tryptophan and increased levels of sphingolipids. Exposure to PAHs appears to impact several bacterial in the gut microbiome, particularly sphingolipid containing bacteria. Bacterial phylotypes known to include species containing sphingolipids were generally lower in the intestines of fish subpopulations exposed to high concentrations of PAHs, inferring a complex host-microbiome relationship. Overall, killifish microbial community shifts appear to be related to a suppression of overall metabolite level, indicating a potential role of the gut in organismal response to anthropogenic environmental change. These results on microbial and metabolomics shifts are potentially linked to altered bioenergetic phenotype observed in the same PAH-resistant killifish populations in other studies.

The environment shapes swine lung bacterial communities.

Previous studies of the lung microbiome have focused on characterizing the community and attempts to understand the role of community membership concerning disease or exposures such as cigarette smoke. However, we still lack an understanding of two critical aspects of the lung microbiome: the origin of the community members and their fate. As we continue to better understand how the lung microbiome influences human health, it is essential to determine how the environment shapes the lung microbiome membership. Using a pig model, we explored the relationship that the surrounding environment has on the resident lung bacteria by collecting environmental samples (soil, air, water, feed) to compare with lung samples (swab, lavage, and tissue). Results suggest that airborne bacteria make up the highest portion of the lung microbiome. Furthermore, bacteria from samples taken from the bronchioles can be correctly identified by which farm they originated, whereas those from alveolar samples are indistinguishable. The findings suggest that while the environment may shape the microbiome of the bronchioles, a distinct community exists within the alveoli. Our findings expand upon the current understanding of the lung microbiome and provide a model of how microbial communities within the lung relate to their surrounding environment.

A novel PCR-clamping assay reducing plant host DNA amplification significantly improves prokaryotic endo-microbiome community characterization.

Due to the sequence homology between the bacterial 16S rRNA gene and plant chloroplast and mitochondrial DNA, the taxonomic characterization of plant microbiome using amplicon-based high throughput sequencing often results in the overwhelming presence of plant-affiliated reads, preventing the thorough description of plant-associated microbial communities. In this work we developed a PCR blocking primer assay targeting the taxonomically informative V5-V6 region of the 16S rRNA gene in order to reduce plant DNA co-amplification, and increase diversity coverage of associated prokaryotic communities. Evaluation of our assay on the characterization of the prokaryotic endophytic communities of Zea mays, Pinus taeda and Spartina alternifora leaves led to significantly reducing the proportion of plant reads, yielded 20 times more prokaryotic reads and tripled the number of detected OTUs compared to a commonly used V5-V6 PCR protocol. To expand the application of our PCR-clamping assay across a wider taxonomic spectrum of plant hosts, we additionally provide an alignment of chloroplast and mitochondrial DNA sequences encompassing more than 200 terrestrial plant families as a supporting tool for customizing our blocking primers.

Long-Term Effects of Copper Nanopesticides on Soil and Sediment Community Diversity in Two Outdoor Mesocosm Experiments.

The use of novel pesticides containing nanomaterials (nanopesticides) is growing and is considered a promising approach to reduce the impacts of agriculture on the environment and human health. However, the environmental effects of these novel agrochemicals are not fully characterized, and more research is needed to determine the benefits and risks they confer. Here, we assessed the impacts of repeated exposures to a Cu(OH)2 nanopesticide on the soil and sediment biodiversity of target (terrestrial) and nontarget (wetland) ecosystems by performing long-term outdoor mesocosm experiments. As pesticides are often used concomitantly with other agrochemicals, we also tested for interactive effects between nanopesticide exposure and fertilization treatments in both ecosystems. We used high-throughput sequencing on three marker genes to characterize effects on bacterial, fungal, and total eukaryotic community structure and diversity. Interestingly, we found limited effects of nanopesticide exposure on the terrestrial soil communities. Conversely, we found significant shifts in the sediment communities of the wetland mesocosms, especially for eukaryotes (protists, fungi, and algae). In the absence of fertilization, fungal and total eukaryotic community compositions exposed to nanopesticides for long periods of time were distinct from unexposed communities. We identified 60 taxa that were significantly affected by nanopesticide exposure, most of which were microeukaryotes affiliated to cercozoans, Gastrotricha, or unicellular algal taxa. Our study suggests that this nanopesticide has limited effects on the soil biodiversity of a target terrestrial agroecosystem, while nontarget aquatic communities are more sensitive, particularly among protists which are not targeted by this bactericide/fungicide.

Investigating the mycobiome of the Holcomb Creosote Superfund Site.

Even though many fungi are known to degrade a range of organic chemicals and may be advantageous for targeting hydrophobic chemicals with low bioavailability due to their ability to secrete extracellular enzymes, fungi are not commonly leveraged in the context of bioremediation. Here we sought to examine the fungal microbiome (mycobiome) at a model creosote polluted site to determine if fungi were prevalent under high PAH contamination conditions as well as to identify potential mycostimulation targets. Several significant positive associations were detected between OTUs and mid-to high-molecular weight PAHs. Several OTUs were closely related to taxa that have previously been identified in culture-based studies as PAH degraders. In particular, members belonging to the Ascomycota phylum were the most diverse at higher PAH concentrations suggesting this phylum may be promising biostimulation targets. There were nearly three times more positive correlations as compared to negative correlations, suggesting that creosote-tolerance is more common than creosote-sensitivity in the fungal community. Future work including shotgun metagenomic analysis would help confirm the presence of specific degradation genes. Overall this study suggests that mycobiome and bacterial microbiome analyses should be performed in parallel to devise the most optimal in situ biostimulation treatment strategies.

Higher normalized concentrations of tetracycline resistance found in ballast and harbor water compared to ocean water.

Although ballast water is a known vector for the global transport of microorganisms, the Ballast Water Management Convention only sets limits for indicator organisms and does not consider antibiotic resistance genes (ARGs). Herein, we examined the concentration of indicator organisms and prevalence of three ARGs (sul1, tet (M), and vanA) in a total of 53 ballast, 21 harbor, and 8 ocean samples collected in Singapore, China, South Africa, and California. E. coli was found in significantly higher concentrations in ballast samples obtained in Singapore and China compared to South Africa (Singapore, p = 0.040) and California (Singapore, p < 0.001; China, p = 0.038). Harbor samples from China had significantly higher concentrations of E. coli than Singapore (p = 0.049) and California (p = 0.001). When compared to ocean samples, there were significantly higher concentrations of normalized tet(M) in ballast samples from California (p = 0.011) and Singapore (p = 0.019) and in harbor samples from California (p = 0.018), Singapore (p = 0.010), and South Africa (p = 0.008). These findings suggest that microbial loads significantly differ among ports. Furthermore, certain ARGs are enriched in ballast and harbor waters when compared to ocean water, which suggests that ballast waters have the potential to either transport higher concentrations of certain ARGs or that ballast tank conditions may exert selective pressure for some ARGs.

Modeling bacteriophage-induced inactivation of Escherichia coli utilizing particle aggregation kinetics.

Targeted inactivation of bacteria using bacteriophages has been proposed in applications ranging from bioengineering and biofuel production to medical treatments. The ability to differentiate between desirable and undesirable organisms, such as in targeting filamentous bacteria in activated sludge, is a potential advantage over conventional disinfectants. Like conventional disinfectants, bacteriophages exhibit non-linear concentration-time (Ct) dynamics in achieving bacterial inactivation. However, there is currently no workable model for predicting these observed non-linear inactivation rates. This work considers an approach to predicting bacteriophage-induced inactivation rates by utilizing classical particle aggregation theory. Bacteriophage-bacteria interactions are represented as a two-step process of transport by Brownian motion, differential settling, and shear, followed by attachment. Modifying classical expressions for particle-particle aggregation to include bacterial growth, death, and bacteriophage reproduction, the model was calibrated and validated using literature data. The calibrated model captures much of the observed non-linearity in inactivation rates and reasonably predicts the final host concentration. This model was shown to be most useful in systems more likely to reflect an industrial setting, where the initial multiplicity of infection, or MOI (the ratio of bacteriophage to host organisms), was 1 or greater. For systems of an initial MOI of less than 1 the model showed increased sensitivity to changes in input parameters and a less pronounced ability to reasonably predict inactivation rates.

Impacts of silver-coated antimicrobial screen covers on the cell-phone microbiome of resident physicians.

An antimicrobial screen was applied to the cell phones of 26 resident physicians to determine its effects on the phone microbiome and its potential to serve as a selective agent for antibiotic or silver resistance genes. No increase of these genes was observed now was there a shift in the overall microbial community.

Examining the behavior of crop-derived antibiotic resistance genes in anaerobic sludge batch reactors under thermophilic conditions.

The consumption of transgenic crops and their by-products has become increasingly common in the United States. Yet, uncertainty remains regarding the fate and behavior of DNA within food matrices once it exits the digestive track and enters into wastewater treatment plants (WWTPs). Because many transgenic crops have historically contained antibiotic resistance genes as selection markers, understanding the behavior and uptake of these transgenes by environmental microbes is of critical importance. To investigate the behavior of free transgenic crop DNA, thermophilic anaerobic batch reactors were amended with varying concentrations of transgenic crop genes (i.e., LUG, nptII, and bla) and the persistence of those genes was monitored over 60 days using quantitative PCR. Significant levels of nptII and bla were detected in extracellular DNA (eDNA). Furthermore, LUG maize marker genes were also detected in the control reactors, suggesting that other crop-derived transgenes contained within digested transgenic foods may also enter WWTPs. Possible bacterial transformation events were detected within the highest dose treatments at Days 30 and 60 of incubation. These findings suggest that within the average conventional digester residence times in the United States (30 days), there is a potential for bacterial transformation events to occur with crop-derived transgenes found in eDNA.

A new framework for approaching precision bioremediation of PAH contaminated soils.

Bioremediation is a sustainable treatment strategy which remains challenging to implement especially in heterogeneous environments such as soil and sediment. Herein, we present a novel precision bioremediation framework that integrates amplicon based metagenomic analysis and chemical profiling. We applied this approach to samples obtained at a site contaminated with polycyclic aromatic hydrocarbons (PAHs). Geobacter spp. were identified as biostimulation targets because they were one of the most abundant genera and previously identified to carry relevant degradative genes. Mycobacterium and Sphingomonads spp. were identified as bioaugmentation and genetic bioaugmentation targets, respectively, due to their positive associations with PAHs and their high abundance and species diversity at all sampling locations. Overall, this case study suggests this framework can help identify bacterial targets for precision bioremediation. However, it is imperative that we continue to build our databases as the power of metagenomic based approaches remains limited to microorganisms currently in our databases.

Microbiome composition and implications for ballast water classification using machine learning.

Ballast water is a vector for global translocation of microorganisms, and should be monitored to protect human and environmental health. This study utilizes high throughput sequencing (HTS) and machine learning to examine the bacterial and fungal microbiomes of ballast water to identify associations between 16S and 18S rRNA genes and the fungal ITS region. These sequencing regions were examined using the SILVA v132 and UNITE reference databases. The highest correlation was found between the communities in Silva_16S and UNITE_ITS (0.74). There was a higher proportion of positive inter-kingdom correlations than positive intra-kingdom interactions (p = 0.032). Understanding the reasons for this difference requires additional research under more controlled conditions. Finally, a machine learning model was used to examine the classification accuracy when using each sequencing region and reference database to identify ballast residence time and ballast sample location. There was significantly higher accuracy using SILVA (0.843) compared to UNITE (0.614) (p < 0.001). In the short term, future research with the goal of classifying ballast water samples based on location or ballast water residence time should be performed using the 16S rRNA gene and SILVA reference database. Research to curate other sequencing regions or the UNITE reference database in the aquatic ecosystem may improve the utility of these tools.