Qing-Lin Chen

Long-term field application of sewage sludge increases the abundance of antibiotic resistance genes in soil

Sewage sludge and manure are common soil amendments in crop production; however, their impact on the abundance and diversity of the antibiotic resistome in soil remains elusive. In this study, by using high-throughput sequencing and high-throughput quantitative PCR, the patterns of bacterial community and antibiotic resistance genes (ARGs) in a long-term field experiment were investigated to gain insights into these impacts. A total of 130 unique ARGs and 5 mobile genetic elements (MGEs) were detected and the long-term application of sewage sludge and chicken manure significantly increased the abundance and diversity of ARGs in the soil. Genes conferring resistance to beta-lactams, tetracyclines, and multiple drugs were dominant in the samples. Sewage sludge or chicken manure applications caused significant enrichment of 108 unique ARGs and MGEs with a maximum enrichment of up to 3845 folds for mexF. The enrichment of MGEs suggested that the application of sewage sludge or manure may accelerate the dissemination of ARGs in soil through horizontal gene transfer (HGT). Based on the co-occurrence pattern of ARGs subtypes revealed by network analysis, aacC, oprD and mphA-02, were proposed to be potential indicators for quantitative estimation of the co-occurring ARGs subtypes abundance by power functions. The application of sewage sludge and manure resulted in significant increase of bacterial diversity in soil, Proteobacteria, Acidobacteria, Actinobacteria and Chloroflexi were the dominant phyla (>10% in each sample). Five bacterial phyla (Chloroflexi, Planctomycetes, Firmicutes, Gemmatimonadetes and Bacteroidetes) were found to be significantly correlated with the ARGs in soil. Mantel test and variation partitioning analysis (VPA) suggested that bacterial community shifts, rather than MGEs, is the major driver shaping the antibiotic resistome. Additionally, the co-occurrence pattern between ARGs and microbial taxa revealed by network analysis indicated that four bacterial families might be potential hosts of ARGs. These results may shed light on the mechanism underlining the effects of amendments of sewage sludge or manure on the occurrence and dissemination of ARGs in soil.

Does organically produced lettuce harbor higher abundance of antibiotic resistance genes than conventionally produced

The demand for organic food products, especially for organic vegetables has been growing rapidly in the last few decades. However, the risk of introducing more antibiotic resistant genes (ARGs) and antibiotic resistant bacteria (ARB) to the vegetables by organic production procedures has long been overlooked. In our study, by using high-throughput quantitative PCR and 16sRNA Illumina sequencing technology, we investigated the abundance and diversity of ARGs and the microbial communities in conventionally (CPL) and organically produced lettuce (OPL). A total of 134 ARGs were detected in the phyllosphere and leaf endophyte of the samples. Absolute copy numbers of ARGs in phyllosphere were 8-fold higher in the OPL than in CPL. We also observed a significant difference in the microbial communities between OPL and CPL, and a lower diversity of both phyllosphere and leaf endophytic bacteria in OPL than in CPL. The Mantel test and variation partitioning analysis (VPA) suggested that the profile of ARGs is strongly affected by bacterial community compositions. Network analysis between ARGs and bacterial taxa indicated that eight bacterial families were implicated to be the potential hosts of ARGs. These results provide insights into the impacts of organic farming on the profiles of bacterial and ARG compositions in vegetables.

An underappreciated hotspot of antibiotic resistance: The groundwater near the municipal solid waste landfill

Landfills are so far the most common practice for the disposals of municipal solid waste (MSW) worldwide. Since MSW landfill receives miscellaneous wastes, including unused/expired antibiotics and bioactive wastes, it gradually becomes a huge potential bioreactor for breeding antibiotic resistance. Antibiotic resistance genes (ARGs) in landfill can flow to the environment through leakage of landfill leachate and pose a risk to public health. Using high throughput quantitative Polymerase Chain Reaction (HT-qPCR), we investigated the prevalence, diversity of ARGs and its association with various mobile genetic elements (MGEs) in MSW landfill groundwater. Totally 171 unique ARGs (belonging to 9 ARG types, encompassing 3 major resistance mechanisms) and 8 MGEs (6 transposase genes, and 2 integron-integrase genes) were identified. The normalized abundance of ARG was ranging from 0.24 to 5.66 copies/cell with multidrug, beta-lactams and tetracycline resistance genes being the most abundant ARG types. The co-occurrence pattern and significant correlation between MGEs and ARGs, indicated that MGEs may play an important role in the persistence and proliferation of ARGs. A Mantel test and Procrustes analysis suggested that ARG profiles were significantly correlated with bacterial community. Variation partitioning analysis (VPA) further demonstrated that bacterial community shifts contribute 65.8% of the total ARG variations. Additionally network analysis revealed that 15 bacterial taxa at family level might be the potential hosts of ARGs. These findings provide evidence that groundwater near MSW landfill is an underappreciated hotspot of antibiotic resistance and contribute to the spread of ARGs via the flowing contaminated groundwater.

Application of genomic technologies to measure and monitor antibiotic resistance in animals

One of the richest reservoirs of antibiotic‐resistant bacteria and genes, animal intestinal microbiota contributes to the spread of antibiotic resistance in the environment and, potentially, to human pathogens. Both culture‐based genomic technology and culture‐independent metagenomics have been developed to investigate the abundance and diversity of antibiotic resistance genes. The characteristics, strengths, limitations, and challenges of these genomic approaches are discussed in this review in the context of antibiotic resistance in animals. We also discuss the advances in single‐cell genomics and its potential for surveillance of antibiotic resistance in animals.

Long-term nitrogen fertilization decreased the abundance of inorganic phosphate solubilizing bacteria in an alkaline soil

Inorganic phosphate solubilizing bacteria (iPSB) are essential to facilitate phosphorus (P) mobilization in alkaline soil, however, the phylogenetic structure of iPSB communities remains poorly characterized. Thus, we use a reference iPSB database to analyze the distribution of iPSB communities based on 16S rRNA gene illumina sequencing. Additionally, a noval pqqC primer was developed to quantify iPSB abundance. In our study, an alkaline soil with 27-year fertilization treatment was selected. The percentage of iPSB was 1.10~2.87% per sample, and the dominant iPSB genera were closely related to Arthrobacter, Bacillus, Brevibacterium and Streptomyces. Long-term P fertilization had no significant effect on the abundance of iPSB communities. Rather than P and potassium (K) additions, long-term nitrogen (N) fertilization decreased the iPSB abundance, which was validated by reduced relative abundance of pqqC gene (pqqC/16S). The decreased iPSB abundance was strongly related to pH decline and total N increase, revealing that the long-term N additions may cause pH decline and subsequent P releases relatively decreasing the demands of the iPSB community. The methodology and understanding obtained here provides insights into the ecology of inorganic P solubilizers and how to manipulate for better P use efficiency.

Spatial and temporal distribution of antibiotic resistomes in a peri-urban area is associated significantly with anthropogenic activities

With the rapid development of urbanization and industrialization, the peri-urban areas are often the sites for waste dumps, which may exacerbate the occurrence and spread of antibiotic resistance from waste to soil bacteria. However, the profiles of antibiotic resistomes and the associated factors influencing their dissemination in peri-urban areas have not been fully explored. Here, we characterized the antibiotic resistance genes (ARGs) in peri-urban arable and pristine soils in four seasons at the watershed scale, by using high-throughput qPCR. ARGs in peri-urban soils were diverse and abundant, with a total of 222 genes were detected in the peri-urban soil samples. The arable soil harbored more diverse ARGs compared to the pristine soils, and nearly all the ARGs detected in the pristine soils were also detected in the farmlands. A random forest prediction showed that the overall patterns of ARGs clustered closely with the landuse type. Mantel test and partial redundancy analysis indicated that bacterial community variation is a major contributor to antibiotic resistome alteration. Significant positive correlation was found between the abundance of ARGs and mobile genetic elements (MGEs), suggesting potential mobility of ARGs in peri-urban areas. Our results extend knowledge of the resistomes compositions in peri-urban areas, and suggest that anthropogenic activities driving its spatial and temporal distribution.

Trophic predator-prey relationships promote transport of microplastics compared with the single Hypoaspis aculeifer and Folsomia candida

Although the roles of earthworms and soil collembolans in the transport of microplastics have been studied previously, the effects of the soil biota at different trophic levels and interspecific relationships remain poorly understood. Here, we examine three soil microarthropod species to explore their effects on the transport of microplastics. The selected Folsomia candida and Hypoaspis aculeifer are extensively used model organisms, and Damaeus exspinosus is a common and abundant indigenous species in China. A model food chain (prey-collembolan and predator-mite) was structured to test the role of the predator-prey relationship in the transport of microplastics. Commercial Polyvinyl chloride (PVC) particles (Diameter: 80-250 μm) were selected as the test microplastics, because large amounts of PVC have persisted and accumulated in the environment. Synchronized soil microarthropods were held in plates for seven days to determine the movement of microplastics. The 5000 microplastic particles were carefully placed in the center of each plate prior to the introduction of the animals. Our results clearly show that all three microarthropod species moved and dispersed the microplastics in the plates. The 0.54%, 1.8% and 4.6% of the added microplastic particles were moved by collembolan, predatory mite and oribatid mite, respectively. Soil microarthropods (<0.2 cm) transported microplastic particles up to 9 cm. The avoidance behavior was observed in the collembolans in respect of the microplastics. The predatory -prey relationship did promote the transport of microplastics in the plates, increasing transport by 40% compared with the effects of adding single species (P < .05). Soil microarthropods commonly occur in surface soils (0-5 cm) and, due to their small body size, they can enter soil pores. Our results therefore suggest that the movement of microplastics by soil microarthropods may influence the exposure of other soil biota to microplastics and change the physical properties of soils.

Impact of wastewater treatment on the prevalence of integrons and the genetic diversity of integron gene cassettes

ABSTRACT The integron platform allows the acquisition, expression, and dissemination of antibiotic resistance genes within gene cassettes. Wastewater treatment plants (WWTPs) contain abundant resistance genes; however, knowledge about the impacts of wastewater treatment on integrons and their gene cassettes is limited. In this study, by using clone library analysis and high-throughput sequencing, we investigated the abundance of class 1, 2, and 3 integrons and their corresponding gene cassettes in three urban WWTPs. Our results showed that class 1 integrons were most abundant in WWTPs and that wastewater treatment significantly reduced the abundance of all integrons. The WWTP influents harbored the highest diversity of class 1 integron gene cassettes, whereas class 3 integron gene cassettes exhibited highest diversity in activated sludge. Most of the gene cassette arrays detected in class 1 integrons were novel. Aminoglycoside, beta-lactam, and trimethoprim resistance genes were highly prevalent in class 1 integron gene cassettes, while class 3 integrons mainly carried beta-lactam resistance gene cassettes. A core class 1 integron resistance gene cassette pool persisted during wastewater treatment, implying that these resistance genes could have high potential to spread into environments through WWTPs. These data provide new insights into the impact of wastewater treatment on integron pools and highlight the need for surveillance of resistance genes within both class 1 and 3 integrons. IMPORTANCE Wastewater treatment plants represent a significant sink and transport medium for antibiotic resistance bacteria and genes spreading into environments. Integrons are important genetic elements involved in the evolution of antibiotic resistance. To better understand the impact of wastewater treatment on integrons and their gene cassette contexts, we conducted clone library construction and high-throughput sequencing to analyze gene cassette contexts for class 1 and class 3 integrons during the wastewater treatment process. This study comprehensively profiled the distribution of integrons and their gene cassettes (especially class 3 integrons) in influents, activated sludge, and effluents of conventional municipal wastewater treatment plants. We further demonstrated that while wastewater treatment significantly reduced the abundance of integrons and the diversity of associated gene cassettes, a large fraction of integrons persisted in wastewater effluents and were consequentially discharged into downstream natural environments.

Distinct effects of struvite and biochar amendment on the class 1 integron antibiotic resistance gene cassettes in phyllosphere and rhizosphere

Struvite recovered from wastewater is promising for recycling phosphorus into soil as fertilizers. However, struvite application may prompt the proliferation of antibiotic resistance in soil and plant. This study examined the impacts of struvite application and biochar amendment on integrons abundance and gene cassette contexts in rhizosphere soil and phyllosphere using quantitative PCR and clone library analysis. Microcosm experiments revealed that class 1 integron was the most prevalent in all samples, with higher concentration and higher relative abundance in rhizosphere than those in phyllosphere. The majority of resistance gene cassettes were associated with genes encoding resistance to aminoglycosides, beta-lactams and chloramphenicols. Struvite application significantly increased the genetic diversity of antibiotic resistance gene cassettes in both rhizosphere and phyllosphere. However, biochar amendment attenuated the increasing effect of struvite application exerting on the class 1 integron antibiotic resistance gene cassette pool in phyllosphere. These findings highlighted human activities to be the source of integron gene cassette pool and raised the possibility of using biochar amendment as an alternative mean for mitigating antibiotic resistance in environments.

Long-term organic fertilization increased antibiotic resistome in phyllosphere of maize

Phyllosphere contains various microorganisms that may harbor diverse antibiotic resistance genes (ARGs). However, we know little about the composition of antibiotic resistome and the factors influencing the diversity and abundance of ARGs in the phyllosphere. In this study, 16S rRNA gene amplicon sequencing and high-throughput quantitative PCR approaches were employed to investigate the effects of long-term (over 10 years) organic fertilization on the phyllosphere bacterial communities and antibiotic resistome. Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes dominated in the phyllosphere bacterial communities. Long-term application of sewage sludge and chicken manure altered the phyllosphere bacterial community composition, with a remarkable decrease in bacterial alpha-diversity. A total of 124 unique ARGs were detected in the phyllosphere. The application of sewage sludge and chicken manure significantly increased the abundance of ARGs, with a maximum 2638-fold enrichment. Variation partitioning analysis (VPA) together with network analysis indicated that the profile of ARGs is strongly correlated with bacterial community compositions. These results improve the knowledge about the diversity of plant-associated antibiotic resistome and factors influencing the profile of ARGs in the phyllosphere.