High-throughput Quantitative PCR Approach Research Articles

Co-proliferation of antimicrobial resistance genes in tilapia farming ponds associated with use of antimicrobials

The prevalence of antimicrobial resistance genes (ARGs) in aquaculture has raised serious public concerns for food safety and human health, but its relationships to the use of antimicrobials in aquacultural ponds and even to their residues in the whole aquatic environment remain unclear. In this study, a better coverage of 323 target ARGs and 40 mobile genetic elements (MGEs) was analyzed in sediment using a smart chip-based high-throughput quantitative PCR approach (HT-qPCR) in random 20 ponds of a tilapia farming base in southern China, whose antimicrobial residues were reported previously. In total, 159 ARGs and 29 MGEs were quantified in 58 surface sediment samples across the ponds. Absolute abundance of ARGs ranged from 0.2 to 13.5 × 106 copies g−1, dominated by the categories of multidrug and sulfonamides. The quantified ARGs abundance and the antimicrobial compound residues were significantly correlated instead with the antimicrobial categories, mainly compounds in categories of fluoroquinolones and sulfonamides and trimethoprim (TMP). Antimicrobial residues alone explained 30.6 % of the ARGs' variation quantified in sediment across the ponds, indicating the clear link between antimicrobials and the proliferation of ARGs in aquaculture. Co-proliferation of the ARGs with non-related antimicrobial compounds quantified in sediment was also observed, especially for aminoglycosides' ARGs, which were highly associated with integrons (intI 1) as argued being carried by the intI 1 gene cassette arrays. Physicochemical properties of sediment (pH, electric conductivity, and total sulfur content) highly contributed the variations of the quantified ARGs abundance (21 %) across all the sediment samples equaling to the MGEs (20 %), suggesting co-selection for ARGs' proliferation in the aquaculture environment. This study provides insights into the interactions between residual antimicrobials and ARGs, which would enhance the understandings on the use and management of antimicrobials in aquaculture worldwide to strategize mitigation of antimicrobial resistance in aquaculture.

Fumigation practice combined with organic fertilizer increase antibiotic resistance in watermelon rhizosphere soil

Chemical fumigants and organic fertilizer are commonly used in facility agriculture to control soil-borne diseases and promote soil health. However, there is a lack of evidence for the effect of non-antibiotic fumigants on the distribution of antibiotic resistance genes (ARGs) in plant rhizosphere soils. Here, the response of a wide spectrum of ARGs and mobile genetic elements (MGEs) to dazomet fumigation practice in the rhizosphere soil of watermelon was investigated along its branching, flowering and fruiting growth stages in plastic shelters using high-throughput quantitative PCR approach. Our results indicated that soil fumigation combined with organic fertilizer application significantly increased the relative abundance of ARGs and MGEs in the rhizosphere soil of watermelon plant. The positive correlations between the relative abundance of ARGs and MGEs suggested that soil fumigation might increase the horizontal gene transfer (HGT) potential of ARGs. This result was further confirmed by the enhanced associations between ARG and MGE subtypes in the networks of fumigation treatments. Moreover, bipartite associations between ARGs/MGEs and microbial communities (bacteria and fungi) revealed a higher percentage of linkage between MGEs and microbial taxa in the fumigated soils. Structural equation model analysis further suggested that the increases in antibiotic resistance after fumigation and organic fertilizer application were mainly driven by MGEs and fungal community. Together, our results provide vital evidence that dazomet fumigation process combined with organic fertilizer in plastic shelters has the great potential to promote ARGs' dissemination in the rhizosphere, and raise cautions of the acquired resistance by soil-borne fungal pathogen and the potential spreading of ARGs along soil-plant continuum.

High-throughput diagnosis of human pathogens and fecal contamination in marine recreational water

Waterborne pathogens and their associated diseases are major threats to public health, and surveillance of pathogens and identification of the sources of pollution are imperative for preventing infections. However, simultaneously quantitative detection of multiple pathogens and pollution sources in water environments is the major challenge. In this study, we developed and validated a highly sensitive (mostly >80%) and highly specific (>99%) high-throughput quantitative PCR (HT-qPCR) approach, which could simultaneously quantify 68 marker genes of 33 human pathogens and 23 fecal markers of 10 hosts. The HT-qPCR approach was then successfully used to investigate pathogens and fecal pollution in marine recreational water samples of Xiamen, China. Totally, seven pathogenic marker genes were found in 13 beach bathing waters, which targeted Acanthamoeba spp., Clostridium perfringens, enteropathogenic Escherichia coli, Klebsiella pneumoniae, Vibrio cholera/V. parahaemolyticus and Legionella spp.. Fecal markers from human and dog were the most frequently detected, indicating human and dog feces were the main contamination in the recreational waters. Nanopore sequencing of full-length 16S rRNA gene revealed that 28 potential human pathogens were detected and electrical conductivity, salinity, oxidation-reduction potential and dissolved oxygen were significantly correlated with the variation in bacterial community. Our results demonstrated that HT-qPCR approach had the potential rapid quantification of microbial contamination, providing useful data for assessment of microbial pathogen associated health risk and development of management practices to protect human health.

Large-scale patterns of soil antibiotic resistome in Chinese croplands

Soil is a vital reservoir of antibiotic resistance genes (ARGs), but we still know little about their distribution in cropland soils and the main driving forces. Here we performed an investigation for ARGs patterns in 105 cropland soils (planted with maize, peanut or soybean) along a 2, 200 km transect in China using high-throughput quantitative PCR approaches. Totally, 204 ARGs were detected, with a higher diversity found in central China than that in northeast and south China. The most abundant (top 50%) and highly shared (present in >50% samples) ARGs regarded as core resistome were dominated by multidrug resistance genes such as oprJ, acrA-05 and acrA-04. Regressive analyses revealed that the relative abundance of total ARGs and core resistome both had significant relationships with mobile genetic elements (MGEs). Anthropogenic factors including the consumption of plastic films and soil properties including heavy metals showed good correlations with the diversity of ARGs. Structural equation modelling analysis further explained that anthropogenic factors were the main forces shaping the ARGs patterns. These findings highlight the importance of human activities in shaping soil antibiotic resistome in the croplands, providing potential management strategies to mitigate the dissemination of ARGs to humans via food chain.

The impacts of different high-throughput profiling approaches on the understanding of bacterial antibiotic resistance genes in a freshwater reservoir

Bacterial antibiotic resistance genes (ARGs), a kind of emerging environmental pollutants, greatly threat human health through pathogenic bacteria. High-throughput quantitative PCR (HT-qPCR) and metagenomic approaches are two popular tools applied in aquatic environmental ARGs monitoring. However, current poor knowledge of different ARG profiling approaches' impacts on the understanding of the ARGs in aquatic environments greatly limit the further field application of these two approaches. For filling such knowledge gap, this study simultaneously employed these two approaches to examine and compare the ARGs in a freshwater reservoir across space and time. We found metagenomic approach detected more ARG subtypes and much higher bacitracin resistance genes' abundances than HT-qPCR. In general, HT-qPCR and metagenomics analyses both revealed similar ARG dynamic patterns and co-occurrence patterns between ARGs and bacterial taxa as well as the relationships between ARGs and environmental factors. Our results indicated the impacts of different ARG profiling approaches on the understanding of bacterial ARGs might be minor or negligible. HT-qPCR approach has the superiorities of time-saving, absolute quantification, low requirement for bioinformatics skills but also has some drawbacks including higher PCR amplification & primer bias, higher primer dependency and relative lower ARG subtype quantification capability compared to metagenomic approach. We suggest HT-qPCR approach can be employed for routine aquatic environmental monitoring, and metagenomic approach could be applied in comprehensive surveys for getting more ARG subtype information. Our data can be a useful reference for choosing right ARG profiling approaches for bacterial ARGs monitoring and risk assessment in aquatic 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.

High throughput profiling of antibiotic resistance genes in urban park soils with reclaimed water irrigation.

Reclaimed water irrigation (RWI) in urban environments is becoming popular, due to rapid urbanization and water shortage. The continuous release of residual antibiotics and antibiotic resistance genes (ARGs) from reclaimed water could result in the dissemination of ARGs in the downstream environment. This study provides a comprehensive profile of ARGs in park soils exposed to RWI through a high-throughput quantitative PCR approach. 147 ARGs encoding for resistance to a broad-spectrum of antibiotics were detected among all park soil samples. Aminoglycoside and beta-lactam were the two most dominant types of ARGs, and antibiotic deactivation and efflux pump were the two most dominant mechanisms in these RWI samples. The total enrichment of ARGs varied from 99.3-fold to 8655.3-fold compared to respective controls. Six to 60 ARGs were statistically enriched among these RWI samples. Four transposase genes were detected in RWI samples. TnpA-04 was the most enriched transposase gene with an enrichment was up to 2501.3-fold in Urumqi RWI samples compared with control soil samples. Furthermore, significantly positive correlation was found between ARGs and transposase abundances, indicating that transposase might be involved in the propagation of ARGs. This study demonstrated that RWI resulted in the enrichment of ARGs in urban park soils.