scholarly journals A Quantitative Metagenomic Sequencing Approach for High Throughput Gene Quantification and Demonstration with Environmental Antibiotic Resistance Genes

2021 ◽  
Author(s):  
Bo Li ◽  
Xu Li ◽  
Tao Yan
Keyword(s):  
Antibiotic Resistance ◽  
High Throughput ◽  
Environmental Samples ◽  
Target Genes ◽  
Antibiotic Resistance Genes ◽  
Metagenomic Sequencing ◽  
Absolute Concentration ◽  
Gene Quantification ◽  
Microbial Risks ◽  
Generation Sequencing

Comprehensive microbial risk assessment requires high-throughput quantification of diverse microbial risks in the environment. Current metagenomic next-generation sequencing approaches can achieve high-throughput detection of genes indicative of microbial risks, but lacks quantitative capabilities. This study developed and tested a quantitative metagenomic next-generation sequencing (qmNGS) approach. Numerous xenobiotic synthetic internal DNA standards were used to determine the sequencing yield (Y seq ) of the qmNGS approach, which can then be used to calculate absolute concentration of target genes in environmental samples based on metagenomic sequencing results. The qmNGS approach exhibited excellent linearity as indicated by a strong linear correlation (r 2 = 0.98) between spiked and detected concentrations of internal standards. High-throughput capability of the qmNGS approach was demonstrated with artificial E. coli mixtures and cattle manure samples, for which 95 ± 3 and 208 ± 4 types of antibiotic resistance genes (ARGs) were detected and quantified simultaneously. The qmNGS approach was further compared with qPCR and demonstrated comparable levels of accuracy and less variation for the quantification of six target genes (16S, tetO , sulI , tetM , ermB and qnrS ). IMPORTANCE Monitoring and comprehensive assessment of microbial risks in the environment requires high-throughput gene quantification. The quantitative mNGS (qmNGS) approach developed in this study incorporated numerous xenobiotic and synthetic DNA internal standard fragments into metagenomic NGS workflow, which are used to determine a new parameter called sequencing yield that relates sequence base reads to absolute concentration of target genes in the environmental samples. The qmNGS approach demonstrated excellent method linearity and comparable performance as the qPCR approach with high-throughput capability. This new qmNGS approach can achieve high-throughput and accurate gene quantification in environmental samples, and has the potential to become a useful tool in monitoring and comprehensively assessing microbial risks in the environment.

scholarly journals Combined effects of composting and antibiotic administration on cattle manure–borne antibiotic resistance genes

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Ishi Keenum ◽  
Robert K. Williams ◽  
Partha Ray ◽  
Emily D. Garner ◽  
Katharine F. Knowlton ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Land Application ◽  
Multidimensional Analysis ◽  
Small Scale ◽  
Antibiotic Administration ◽  
Metagenomic Sequencing ◽  
Combined Effects ◽  
Bacterial Microbiota

Abstract Background Research is needed to delineate the relative and combined effects of different antibiotic administration and manure management practices in either amplifying or attenuating the potential for antibiotic resistance to spread. Here, we carried out a comprehensive parallel examination of the effects of small-scale (> 55 °C × 3 days) static and turned composting of manures from dairy and beef cattle collected during standard antibiotic administration (cephapirin/pirlimycin or sulfamethazine/chlortetracycline/tylosin, respectively), versus from untreated cattle, on “resistomes” (total antibiotic resistance genes (ARGs) determined via shotgun metagenomic sequencing), bacterial microbiota, and indicator ARGs enumerated via quantitative polymerase chain reaction. To gain insight into the role of the thermophilic phase, compost was also externally heated to > 55 °C × 15 days. Results Progression of composting with time and succession of the corresponding bacterial microbiota was the overarching driver of the resistome composition (ANOSIM; R = 0.424, p = 0.001, respectively) in all composts at the small-scale. Reduction in relative abundance (16S rRNA gene normalized) of total ARGs in finished compost (day 42) versus day 0 was noted across all conditions (ANOSIM; R = 0.728, p = 0.001), except when externally heated. Sul1, intI1, beta-lactam ARGs, and plasmid-associated genes increased in all finished composts as compared with the initial condition. External heating more effectively reduced certain clinically relevant ARGs (blaOXA, blaCARB), fecal coliforms, and resistome risk scores, which take into account putative pathogen annotations. When manure was collected during antibiotic administration, taxonomic composition of the compost was distinct according to nonmetric multidimensional analysis and tet(W) decayed faster in the dairy manure with antibiotic condition and slower in the beef manure with antibiotic condition. Conclusions This comprehensive, integrated study revealed that composting had a dominant effect on corresponding resistome composition, while little difference was noted as a function of collecting manure during antibiotic administration. Reduction in total ARGs, tet(W), and resistome risk suggested that composting reduced some potential for antibiotic resistance to spread, but the increase and persistence of other indicators of antibiotic resistance were concerning. Results indicate that composting guidelines intended for pathogen reduction do not necessarily provide a comprehensive barrier to ARGs or their mobility prior to land application and additional mitigation measures should be considered.

scholarly journals Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM): a method for detection of antimicrobial resistance in environmental samples

2021 ◽  
Author(s):  
Schuyler D. Smith ◽  
Jin Choi ◽  
Nicole Ricker ◽  
Fan Yang ◽  
Shannon Hinsa-Leasure ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Test Performance ◽  
Environmental Samples ◽  
High Throughput Sequencing ◽  
Antibiotic Resistance Genes ◽  
Full Spectrum ◽  
Antibiotic Resistant ◽  
Quantitative Monitoring ◽  
The Cost

Effective monitoring of antibiotic resistance genes and their dissemination in environmental ecosystems has been hindered by the cost and efficiency of methods available for the task. We developed a method entitled the Diversity of Antibiotic Resistance genes and Transfer Elements-Quantitative Monitoring (DARTE-QM), a system implementing high-throughput sequencing to simultaneously sequence thousands of antibiotic resistant genes representing a full-spectrum of antibiotic resistance classes commonly seen in environmental systems. In this study, we demonstrated DARTE-QM by screening 662 antibiotic resistance genes within environmental samples originated from manure, soil, and animal feces, in addition to a mock-community used as a control to test performance. DARTE-QM offers a new approach to studying antibiotic resistance in environmental microbiomes, showing advantages in efficiency and the ability to scale for many samples. This method provides a means of data acquisition that will alleviate the obstacles that many researchers in this area currently face.

Abundance, diversity and mobility potential of antibiotic resistance genes in pristine Tibetan Plateau soil as revealed by soil metagenomics

2020 ◽  
Vol 96 (10) ◽  
Author(s):  
Bo Li ◽  
Zeng Chen ◽  
Fan Zhang ◽  
Yongqin Liu ◽  
Tao Yan
Keyword(s):  
Antibiotic Resistance ◽  
Microbial Diversity ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Soil Samples ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Metagenomic Sequencing ◽  
Clinical Issue ◽  
Abundance And Diversity

ABSTRACT Widespread occurrence of antibiotic resistance genes (ARGs) has become an important clinical issue. Studying ARGs in pristine soil environments can help to better understand the intrinsic soil resistome. In this study, 10 soil samples were collected from a high elevation and relatively pristine Tibetan area, and metagenomic sequencing and bioinformatic analyses were conducted to investigate the microbial diversity, the abundance and diversity of ARGs and the mobility potential of ARGs as indicated by different mobile genetic elements (MGEs). A total of 48 ARG types with a relative abundance of 0.05–0.28 copies of ARG/copy of 16S rRNA genes were detected in Tibetan soil samples. The observed ARGs were mainly associated with antibiotics that included glycopeptide and rifamycin; the most abundant ARGs were vanRO and vanSO. Low abundance of MGEs and potentially plasmid-related ARGs indicated a low horizontal gene transfer risk of ARGs in the pristine soil. Pearson correlation and redundancy analyses showed that temperature and total organic carbon were the major environmental factors controlling both microbial diversity and ARG abundance and diversity.

scholarly journals Effects of Antibiotics on the Bacterial Community, Metabolic Functions and Antibiotic Resistance Genes in Mariculture Sediments during Enrichment Culturing

2020 ◽  
Vol 8 (8) ◽  
pp. 604
Author(s):  
Meng-Qi Ye ◽  
Guan-Jun Chen ◽  
Zong-Jun Du
Keyword(s):  
Antibiotic Resistance ◽  
Bacterial Community ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
High Throughput ◽  
Antibiotic Resistance Genes ◽  
Control Group ◽  
Rrna Gene ◽  
Metabolic Functions ◽  
Culture Dependent

The effect of antibiotics on the diversity and functioning of indigenous microorganisms in the environment has attracted much attention. In this study, effects of exposure to six different antibiotics on the bacterial community, metabolic functions and antibiotic resistance genes (ARGs) in marine sediments during enrichment culturing were investigated. Classical culture-dependent method and high-throughput 16S rRNA gene sequencing method were both applied. In the culture-dependent analysis, the obtained 1549 isolates belonged to four phyla (Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria) and 155 genera. Proteobacteria and Firmicutes were the dominant phyla. The diversity and abundance of obtained bacteria after antibiotic processing exhibited different degrees of decrease. Enrichment culturing for different time could also affect the bacterial community composition. Some genera of bacteria were not isolated in the control group, but they could be isolated in the antibiotic-treated groups. In high-throughput 16S rRNA gene amplicon sequencing analyses, all the effective reads were clustered into 2822 OTUs at 97% similarity cutoff; they were annotated to 49 phyla, 103 class, 220 orders, 347 families, 624 genera and 1122 species. An alpha diversity analysis indicated that the community diversity and richness decreased under antibiotic exposure. The changes at the genus level were much more obvious. Only 48 genera of 129 genera were shared by all the samples. A total of 29 genera which were not detected in the initial control sample could be detected in at least one antibiotic-treated group. SIMPER analysis showed that OTU2543 and OTU1450 were the most common taxa to the dissimilarity of bacterial community between antibiotic-treated groups and the control group. OTU2034 and OUT2543 were the most contributive taxa to dissimilarity of groups incubating for different time. Metabolism was the predominant bacterial function. A total of 30 ARGs were detected in the samples. This study mainly focused on the changes of microbiota under the selective pressure of antibiotics for different time and the results demonstrated that the antibiotic could affect the bacterial diversity and richness in the marine ecosystem.

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