Active antibiotic resistome in soils unraveled by single-cell isotope probing and targeted metagenomics.

Antimicrobial resistance (AMR) in soils represents a serious risk to human health through the food chain and human-nature contact. However, the active antibiotic-resistant bacteria (ARB) residing in soils that primarily drive AMR dissemination are poorly explored. Here, single-cell Raman-D2O coupled with targeted metagenomics is developed as a culture-independent approach to phenotypically and genotypically profiling active ARB against clinical antibiotics in a wide range of soils. This method quantifies the prevalence (contamination degree) and activity (spread potential) of soil ARB and reveals a clear elevation with increasing anthropogenic activities such as farming and the creation of pollution, thereby constituting a factor that is critical for the assessment of AMR risks. Further targeted sorting and metagenomic sequencing of the most active soil ARB uncover several uncultured genera and a pathogenic strain. Furthermore, the underlying resistance genes, virulence factor genes, and associated mobile genetic elements (including plasmids, insertion sequences, and prophages) are fully deciphered at the single-cell level. This study advances our understanding of the soil active AMR repertoire by linking the resistant phenome to the genome. It will aid in the risk assessment of environmental AMR and guide the combat under the One Health framework.

Dysbiosis in the Gut Microbiota of Soil Fauna Explains the Toxicity of Tire Tread Particles.

Tread particles (TPs) from vehicle tires are widely distributed in soil ecosystems; therefore, there is an urgent need to evaluate their effects on soil biota. In the present study, the soil worm Enchytraeus crypticus was incubated for 21 days in soil microcosms containing increasing concentrations of TPs (0, 0.0048%, 0.024%, 0.12%, 0.6%, and 3% of dry soil weight). High concentrations of zinc (Zn, 9407.4 mg kg-1) and polycyclic aromatic hydrocarbons (PAHs, 46.8 mg kg-1) were detected in the TPs, which resulted in their increased concentrations in soils amended with TPs. We demonstrated that TPs had an adverse effect on the survival (decreased by more than 25%) and reproduction (decreased by more than 50%) of the soil worms. Moreover, TP exposure disturbed the microbiota of the worm guts and surrounding soil. In addition, a covariation between bacterial and fungal communities was observed in the worm guts after exposure to TPs. Further analysis showed that TP exposure caused an enrichment of microbial genera associated with opportunistic pathogenesis in the worm guts. The combined results from this study indicate that TPs might threaten the terrestrial ecosystem by affecting soil fauna and their gut microbiota.

RNA Stable Isotope Probing of Potential Feammox Population in Paddy Soil.

Anaerobic ammonium oxidation coupled to iron reduction (Feammox) is a recently discovered pathway contributing to nitrogen loss in various ecosystems such as paddy soils and sediments. However, little is known about the microbes driving Feammox in an agricultural ecosystem. Here, we demonstrated the occurrence of Feammox in paddy soils of Southern China using a 15N isotopic tracing technique, and examined the microbial communities associated with Feammox using RNA based stable isotope probing (RNA-SIP) combined with Illumina sequencing. Feammox was detected in all collected soils with direct N2 production as the dominant Feammox pathway. It was estimated that approximately 6.91% of the applied nitrogen fertilizers were lost through Feammox in the paddy soils. RNA-SIP results showed that the composition of enriched active microbial communities were dependent on soil properties, especially the soil pH and grain size. Geobacter were enriched in most soils across various properties. The abundance of enriched GOUTA19 were significantly higher in soils with low pH than those in soils with medium pH and high pH, and the relative abundance of active Nitrososphaeraceae and Pseudomonas only increased in soils with medium and high pH during 4-day of incubation. These results suggested Feammox is a ubiquitous and important process for N loss. Geobacter, GOUTA19, Nitrososphaeraceae and Pseudomonas were active during the incubation that favored Feammox and the growth of Feammox microbes, suggesting these microbes were potentially associated with Feammox in natural agricultural soils.

Mobile Incubator for Iron(III) Reduction in the Gut of the Soil-Feeding Earthworm Pheretima guillelmi and Interaction with Denitrification.

Diets of soil-feeding earthworms contain abundant nitrate and iron(III) oxides, which are potential electron acceptors for mineralization of organic compounds. The earthworm gut provides an ideal habitat for ingested iron(III)-reducing microorganisms. However, little is known about iron(III) reduction and its interaction with other processes in the guts of earthworms. Here, we determined the dynamics of iron(III) and revealed its interaction with the turnover of organic acids and nitrate in the gut of the earthworm Pheretima guillelmi. Samples from gut contents combined with anoxic incubation were used for chemical analysis and 16S rRNA based Illumina sequencing. Chemical analysis showed that higher ratios of iron(II)/iron(III), nitrite/nitrate, and more abundant organic acids were contained in the in vivo gut of the earthworm P. guillelmi than those in the in situ soil. A higher rate of iron(III) reduction was detected in treatments of microcosmic incubation with gut contents (IG gut) than that with soil (IG soil), and nitrate reduction occurred earlier than iron(III) reduction in both treatments. Potential iron(III) reducers were dominated by fermentative genera Clostridium, Bacillus, and Desulfotomaculum in the treatment of IG gut, while they were dominated by dissimilatory iron(III)-reducing genera Geobacter in the treatment of IG soil. The iron(III)-reducing microbial community shared several genera with denitrifers in the treatment of IG gut, revealing a close link between iron(III) reduction and denitrification in the gut of earthworms. Collectively, our findings demonstrated that iron(III) reduction occurred along the gut and provided novel insights into the great contribution of earthworm gut microbiota on Fe and the associated C and N cycling in soil environments.

Impacts of dietary copper on the swine gut microbiome and antibiotic resistome.

Restrictions on antibiotic growth promoters have prompted livestock producers to use alternative growth promoters, and dietary copper (Cu) supplementation is currently being widely used in pig production. However, elevated doses of dietary Cu constitute a risk for co-selection of antibiotic resistance and the risk may depend on the type of Cu-based feed additives being used. We here report the first controlled experiment investigating the impact of two contrasting Cu-based feed additives on the overall swine gut microbiome and antibiotic resistome. DNA was extracted from fecal samples (n = 96) collected at four time points during 116 days from 120 pigs allotted to three dietary treatments: control, divalent copper sulfate (CuSO4; 250 µg Cu g-1 feed), and monovalent copper oxide (Cu2O; 250 µg Cu g-1 feed). Bacterial community composition, antibiotic resistance genes (ARGs), and mobile genetic elements (MGEs) were assessed, and bioavailable Cu ([Cu]bio) was determined using whole-cell bacterial bioreporters. Cu supplementation to feed increased total Cu concentrations ([Cu]total) and [Cu]bio in feces 8-10 fold and at least 670-1000 fold, respectively, but with no significant differences between the two Cu sources. The swine gut microbiome harbored highly abundant and diverse ARGs and MGEs irrespective of the treatments throughout the experiment. Microbiomes differed significantly between pig growth stages and tended to converge over time, but only minor changes in the bacterial community composition and resistome could be linked to Cu supplementation. A significant correlation between bacterial community composition (i.e., bacterial taxa present) and ARG prevalence patterns were observed by Procrustes analysis. Overall, results of the experiment did not provide evidence for Cu-induced co-selection of ARGs or MGEs even at a Cu concentration level exceeding the maximal permitted level for pig diets in the EU (25 to 150 µg Cu g-1 feed depending on pig age).

Limited impacts of high doses of dietary copper on the gut bacterial metal resistome explain negligible co-selection of antibiotic resistance.

High dietary intake of Cu has previously been linked to the selection of Cu resistance and co-selection of antibiotic resistance in specific gut bacteria. Based on a novel HT-qPCR metal resistance gene chip as combined with 16S rRNA gene amplicon sequencing and phenotypic resistance typing of Escherichia coli isolates, we here report the impacts of two contrasting Cu-based feed additives on the swine gut bacterial metal resistome and community assembly. DNA was extracted from fecal samples (n = 80) collected at day 26 and 116 of the experiment from 200 pigs allotted to five dietary treatments: negative control (NC) diet with 20 µg CuSO4 g-1 and four diets added 125 or 250 µg CuSO4 g-1 feed or 125 or 250 µg Cu2O g-1 feed to the NC diet. Dietary Cu supplementation reduced the relative abundance of Lactobacillus, but it had negligible impacts on bacterial community composition relative to the gut microbiome maturation effect (time). The relative importance of different bacterial community assembly processes was not markedly affected by the dietary Cu treatments, and differences in swine gut metal resistome composition could be explained primarily by differences in bacterial community composition rather than by dietary Cu treatments. High dietary Cu intake (250 µg Cu g-1) selected for phenotypic Cu resistance in E. coli isolates, but surprisingly it did not result in increased prevalence of the Cu resistance genes targeted by the HT-qPCR chip. In conclusion, the lacking impacts of dietary Cu on the gut bacterial metal resistome explain results from a previous study showing that even high therapeutic doses of dietary Cu did not cause co-selection of antibiotic resistance genes and mobile genetic elements known to harbor these genes.

Prevalence of antibiotic resistance genes in Pangasianodon hypophthalmus and Oreochromis niloticus aquaculture production systems in Bangladesh.

Antibiotic resistance genes (ARGs) constitute emerging pollutants of significant public health concern. Antibiotics applied in aquaculture may stimulate the proliferation and dissemination of ARGs. This study investigated the prevalence and diversity of ARGs in Pangasianodon hypophthalmus (formerly Pangasius) and Oreochromis niloticus (formerly Tilapia) commercial aquaculture ponds from four economically important divisions (i.e. regions) of Bangladesh using a high-throughput qPCR ARG SmartChip and further aimed to explore effects of aquaculture pond management and water quality on the observed ARG prevalence patterns. A total of 160 ARGs and 10 mobile genetic elements (MGEs) were detected across all samples (n = 33), of which 76 ARGs and MGEs were shared between all regions. Multidrug resistance genes were the most frequently encountered ARGs, followed by ARGs conferring resistance to ß-lactams, aminoglycosides, tetracyclines, and macrolide-lincosamide-streptogramin B (MLSB). Research ponds managed by the Bangladesh Agricultural University had the lowest abundance and diversity of ARGs, suggesting that proper management such as regular water quality monitoring, fortnightly water exchange and use of probiotics instead of antibiotics may mitigate the dissemination of antibiotic resistance from aquaculture ponds. The Adonis test (R2 = 0.35, p < 0.001) and distance decay relationships revealed that the ARGs composition displayed a significant biogeographical pattern (i.e., separation based on geographic origin). However, this effect could possibly be due to feed type as different feed types were used in different regions. In conclusion, our results indicate that there is a vast potential for improving aquaculture pond management practices in Bangladesh to mitigate the environmental dissemination of ARGs and their subsequent transmission to humans.

Earthworm gut: An overlooked niche for anaerobic ammonium oxidation in agricultural soil.

Soil fauna takes an active part in accelerating turnover of nutrients in terrestrial ecosystems. Anaerobic ammonium oxidation (anammox) has been widely characterized, however, whether anammox is active in earthworm gut and the effect of earthworm on anammox in soil remain unknown. In this study, the activity, abundance and community of anammox bacteria in earthworm guts and soils from microcosms were determined using a 15N-tracing technique, quantitative PCR, and anammox bacterial 16S rRNA gene amplicon sequencing. Results showed that anammox rates in guts ranged between 5.81 and 14.19 nmol N g-1 dw gut content h-1, which were significantly (P < 0.01) higher than that in their surrounding soils during 30 day incubation. On the contrary, abundances of hzsB genes encoding subunit B hydrazine synthase in guts were significantly (P < 0.05) lower than those in their surrounding soils. Anammox rates, denitrification N2 production rates and hzsB genes in soils with earthworms were significantly (P < 0.05) lower than those in control soils. Anammox bacterial compositions differed significantly (P < 0.05) between gut and soil, and earthworm altered anammox bacterial communities in soils. Brocadia, Kuenenia and abundant unclassified anammox bacteria were detected in collected soils and gut contents, in which Brocadia was only detected in guts. These results suggested that microbes in earthworm gut increase, but present of earthworm reduces anammox and denitrification associated N loss by altering the anammox bacterial community compositions in soils.

Exposure to heavy metal and antibiotic enriches antibiotic resistant genes on the tire particles in soil.

The widespread occurrence of tire particles (TPs) in soils has attracted considerable attention due to their potential threats. The assemblage of bacteria and associated antibiotic resistant genes (ARGs) on TPs is yet largely unknown, especially under the stress of soil pollutants. In the present study, TPs were incubated in soils with or without the stress of heavy metal (Cu2+) or/and antibiotic (tetracycline), and bacterial community and ARG profile on TPs and in soils were explored using high-throughput sequencing and high-throughput quantitative PCR. Results indicated that bacterial community structure on TPs was significantly different from the surrounding soils, with a lower diversity, and significantly shifted by heavy metal and antibiotic exposure. Additionally, a diverse set of ARGs were detected on TPs, and their abundances were significantly increased under the stress of heavy metal and antibiotic, revealing a strong synergistic effect. Moreover, a good fit was observed for the correlation between bacterial community and ARG profile on TPs. Taken together, this study, for the first time, demonstrates that TPs can provide a novel niche for soil bacteria and soil resistome, and heavy metal and antibiotic exposure may potentially increase the abundance of ARGs on TPs, threatening soil ecosystems and human health.

Cadmium enhances conjugative plasmid transfer to a fresh water microbial community.

Co-selection of antibiotic resistance genes (ARGs) by heavy metals might facilitate the spread of ARGs in the environments. Cadmium contamination is ubiquitous, while, it remains unknown the extent to which cadmium (Cd2+) impact plasmid-mediated transfer of ARGs in aquatic bacterial communities. In the present study, we found that Cd2+ amendment at sub-inhibitory concentration significantly increased conjugation frequency of RP4 plasmid from Pseudomonas putida KT2442 to a fresh water microbial community by liquid mating method. Cd2+ treatment (1-100 mg/L) significantly increased the cell membrane permeability and antioxidant activities of conjugation mixtures. Amendments of 10 and 100 mg/L Cd2+ significantly enhanced the mRNA expression levels of mating pair formation gene (trbBp) and the DNA transfer and replication gene (trfAp) due to the repression of regulatory genes (korA, korB and trbA). Phylogenetic analysis of transconjugants indicated that Proteobacteria was the dominant recipients and high concentration of Cd2+ treatment resulted in expanded recipient taxa. This study suggested that sub-inhibitory Cd2+ contamination would facilitate plasmid conjugation and contributed to the maintenance and spread of plasmid associated ARGs, and highlighted the urgent need for effective remediation of Cd2+ in aquatic environments.

Exposure of CuO nanoparticles and their metal counterpart leads to change in the gut microbiota and resistome of collembolans.

The widespread use of copper oxide nanoparticles (CuONPs) has dramatically increased their concentrations in soils and severely affected the health of soil organisms. The gut microbiota critically contributes to the metabolism and immune system of its host and is sensitive to environmental pollution. The toxic effect of CuONPs on the gut microbiota, especially in soil fauna, still needs further research. In the present study, a comprehensive toxicological test was performed to reveal the effects of CuONPs and their metal counterpart on the gut microbiota of soil collembolans using Illumina high throughput sequencing. Furthermore, the concomitant changes in the collembolans gut-associated antibiotic resistance genes (ARGs) and metabolism were investigated using high-throughput quantitative PCR and carbon and nitrogen stable isotope compositions. Both CuONPs and ionic copper (Cu) exposure disturbed the collembolan gut microbial community structure while only CuONPs reduced the gut microbial diversity. A total of 66 ARGs were detected in the collembolan guts, and CuONPs exposure induced a reduction in both diversity and abundance of ARGs. Additionally, CuONPs and ionic Cu exposure altered the C and N stable isotope compositions of the collembolans, indicating a change in their metabolism. Moreover, structural equation modeling indicated that 85.5% of the carbon stable isotope variations and 73.3% of the nitrogen stable isotope variations were explained by changes in Cu bioaccumulation and the gut microbiota. The results of the present study extend our knowledge regarding the comprehensive toxicity of metal oxide NPs on soil fauna.

Long-term application of Swedish sewage sludge on farmland does not cause clear changes in the soil bacterial resistome.

The widespread practice of applying sewage sludge to arable land makes use of nutrients indispensable for crops and reduces the need for inorganic fertilizer, however this application also provides a potential route for human exposure to chemical contaminants and microbial pathogens in the sludge. A recent concern is that such practice could promote environmental selection and dissemination of antibiotic resistant bacteria or resistance genes. Understanding the risks of sludge amendment in relation to antibiotic resistance development is important for sustainable agriculture, waste treatment and infectious disease management. To assess such risks, we took advantage of an agricultural field trial in southern Sweden, where land used for growing different crops has been amended with sludge every four years since 1981. We sampled raw, semi-digested and digested and stored sludge together with soils from the experimental plots before and two weeks after the most recent amendment in 2017. Levels of selected antimicrobials and bioavailable metals were determined and microbial effects were evaluated using both culture-independent metagenome sequencing and conventional culturing. Antimicrobials or bioavailable metals (Cu and Zn) did not accumulate to levels of concern for environmental selection of antibiotic resistance, and no coherent signs, neither on short or long time scales, of enrichment of antibiotic-resistant bacteria or resistance genes were found in soils amended with digested and stored sewage sludge in doses up to 12 metric tons per hectare. Likewise, only very few and slight differences in microbial community composition were observed after sludge amendment. Taken together, the current study does not indicate risks of sludge amendment related to antibiotic resistance development under the given conditions. Extrapolations should however be done with care as sludge quality and application practices vary between regions. Hence, the antibiotic concentrations and resistance load of the sludge are likely to be higher in regions with larger antibiotic consumption and resistance burden than Sweden.

Effects of biochar amendments on antibiotic resistome of the soil and collembolan gut.

A diverse array of ARGs has been detected in the guts of soil fauna residing in farmland soil. Biochar has been widely used in farmland for soil remediation and improvement of soil quality; however, the effects of biochar amendment on the gut-associated ARGs of soil fauna remain unclear. In the present study, collembolans were cultivated in soils amended with 6 types of biochars. High-throughput qPCR was used to establish ARG profiles of the collembolan guts as well as the surrounding soils. A total of 73 and 162 subtypes of ARGs were detected in the collembolan guts and soils, respectively. Biochar amendment significantly altered the ARG compositions of the collembolan guts and soils, in a biochar quality-dependent manner. However, only manure-derived biochar, which contained elevated concentrations of heavy metals, increased the relative abundance of gut-associated ARGs. Changes in the gut microbial community, MGEs and biochar properties explained 84% of the total ARG variations in the collembolan guts. The findings of this study suggested that biochar properties should receive more attention, as high doses of heavy metals in biochar could increase the abundance of ARGs in collembolan guts, thereby contributing to the spread of ARGs in the environment through collembolan movement.

Heavy metal-induced co-selection of antibiotic resistance genes in the gut microbiota of collembolans.

Heavy metal induced co-selection of antibiotic resistance genes (ARGs) has become an emerging environmental issue. The guts of soil fauna offer a unique habitat in the terrestrial ecosystem and harbor a variety of microorganisms. However, the effects of heavy metals on the gut-associated ARGs of soil fauna are poorly understood. In the present study, collembolans were cultivated with four types of heavy metals (Zn, Cu, Cd, and Cr) and one antibiotic (oxytetracycline), to investigate their impact on the gut-associated ARGs. High-throughput quantitative PCR and 16S rRNA gene amplicon sequencing were used to examine changes in the gut-associated ARGs and microbial composition caused by the metals and antibiotic. The results showed that heavy metals alone induced co-selection of ARGs in the collembolan gut, but the effects were weaker than selection by oxytetracycline. When Zn or Cu was present together with oxytetracycline, there was a strong synergistic effect between the compounds, which increased the selection of ARGs in the collembolan guts. Furthermore, redundancy analysis revealed that the gut microbiota and mobile genetic elements (MGEs) were significantly correlated with the ARG composition. These results extend our understanding on effects of heavy metals on the dispersal of ARGs in the soil food web.

The gut microbiota of soil organisms show species-specific responses to liming.

Liming is a common agronomic practice used for alleviating soil acidification to improve plant growth. However, it is still unclear how liming can affect the gut microbiota composition of soil fauna, and subsequently the nutrient cycling and litter decomposition mediated by soil fauna. In the present study the effect of liming on the gut microbiota of two types of soil fauna, Folsomia candida, and Enchytraeus crypticus was investigated by using 16S rRNA gene high-throughput sequencing. The results revealed that there are differences between the gut microbial communities of the two types of soil fauna as well as between the gut microbiome of the soil fauna and the surrounding soil. Enterobacteriaceae and Bacillaceae were the predominant families in the gut microbiota of E. crypticus, while Rickettsiaceae and Moraxellaceae were the predominant families in the gut microbiota of F. candida. Liming affected the gut microbiota of E. crypticus at both the taxonomical and core microbiota level. The gut microbiota of F. candida was not affected by liming. Structural equation models suggest that 97% of the variation in the E. crypticus gut microbiota could be explained by liming-induced changes in soil properties and the soil microbial community. The indirect effects of liming, caused by a shift in the soil microbial community, contributed more in reshaping the gut microbiota of E. crypticus than the direct effects of the changed soil properties did. These findings suggest that the effects of liming on the gut microbiota composition in soil fauna are species-specific and are likely dependent on the response of the host to changes in soil properties and the soil microbial community.