SmartChip Real-Time PCR System applications
- Antibiotic resistance genes
- mRNA, miRNA, and lncRNA as disease biomarkers
- Pathogen detection in human samples and food
- Genotyping using animal and blood samples
Antibiotic resistance genes
The discovery and use of antibiotics are two of the most significant breakthroughs in twentieth-century medicine—leading to dramatic reductions in human morbidity and mortality. Antibiotics and other antimicrobial agents are also used extensively in agriculture. Typically, livestock are given antibiotics to encourage growth and prevent illness, thereby increasing the amount of food produced. There are multiple classes of antibiotics, categorized based on their mechanism of action. Antibiotics can damage the cell wall of a bacterium, block DNA, RNA, or protein synthesis, or even inhibit the metabolic growth of bacteria. Some antibiotics are specific to certain species of bacteria (narrow-spectrum), whereas others can affect a wide range of bacteria (broad-spectrum).
One emerging problem with antibiotics is the development of antibiotic-resistant bacteria. Bacteria can acquire antibiotic resistance genes (ARGs) over time due to selective pressure. Excessive antibiotic use can lead to the development of spontaneous ARGs in certain strains. As these strains grow and expand, different antibiotics have to be developed to combat them. Ultimately, the current strategy is to prevent overuse of antibiotics, thereby limiting the exposure of bacteria to these agents and minimizing selective pressures. For human patients, overuse can result from incorrect dosing, over- or mis-prescribing, or improper disposal. Critically, antibiotic use in agriculture can also contribute to bacteria developing ARGs. Not only can antibiotic-resistant bacteria develop in livestock, but manure can act as a vector to transmit antibiotic-resistant strains into the environment via soil, water, and produce. Thus, antibiotic-resistant bacteria can be spread widely, and recent efforts have focused on identifying ARGs and tracking their prevalence in a variety of environments and sample types.
There are currently over 1,000 types of ARGs that have been identified to confer resistance to the hundreds of available antibiotics. Consequently, monitoring human and environmental samples for ARGs requires the ability to perform high-throughput real-time PCR (qPCR) screens for many different target sequences in numerous sample types. Further, because bacteria are constantly developing new ARGs, it is critical to be able to modify and add new targets to the screen. Thus, ARG research requires a flexible, high-throughput qPCR system to monitor gene expression in a wide range of sample types.
The SmartChip Real-Time PCR System has been used in numerous studies for profiling and tracking ARGs in a variety of samples. Initially, high-throughput screens were performed using panels of 296 targets, which supported the ability to screen up to 16 soil samples per chip. In recent years, the panels have evolved to contain 384 targets (see Table I), as well as being run on soil, water, sediment, manure, lettuce, fish, and sludge sample types. The flexibility and throughput of the SmartChip system have been instrumental in enabling these and future studies of the antibiotic resistome. In fact, the SmartChip system has been utilized in 75% of the publications using high-throughput qPCR for antibiotic resistance research (Waseem et al. 2019).
|Target gene category||Number of primer sets|
|Mobile genetic elements||52|
Table I. Antibiotic resistance gene categories that have been analyzed on the SmartChip system.
An, X.-L. et al. Tracking antibiotic resistome during wastewater treatment using high throughput quantitative PCR. Environ. Int. 117, 146–153 (2018).
Chen, Q. et al. Long-term field application of sewage sludge increases the abundance of antibiotic resistance genes in soil. Environ. Int. 92-93, 1–10 (2016).
Chen, Q.-L., An, X.-L., Zheng, B.-X., Ma, Y.-B. & Su, J.-Q. Long-term organic fertilization increased antibiotic resistome in phyllosphere of maize. Sci. Total Environ. 645, 1230–1237 (2018).
Chen, Q.-L. et al. Application of struvite alters the antibiotic resistome in soil, rhizosphere, and phyllosphere. Environ. Sci. Technol. 51, 8149–8157 (2017).
Chen, Q.-L. et al. Effect of biochar amendment on the alleviation of antibiotic resistance in soil and phyllosphere of Brassica chinensis L. Soil Biol. Biochem. 119, 74–82 (2018).
Chen, Y. et al. High-throughput profiling of antibiotic resistance gene dynamic in a drinking water river-reservoir system. Water Res. 149, 179–189 (2019).
Chen, Z. et al. Antibiotic resistance genes and bacterial communities in cornfield and pasture soils receiving swine and dairy manures. Environ. Pollut. 248, 947–957 (2019).
Cheng, J.-H., Tang, X.-Y. & Cui, J.-F. Effect of long-term manure slurry application on the occurrence of antibiotic resistance genes in arable purple soil (entisol). Sci. Total Environ. 647, 853–861 (2019).
Choi, J. et al. Practical implications of erythromycin resistance gene diversity on surveillance and monitoring of resistance. FEMS Microbiol. Ecol. 94, (2018).
Cui, E.-P. et al. Amendment soil with biochar to control antibiotic resistance genes under unconventional water resources irrigation: Proceed with caution. Environ. Pollut. 240, 475–484 (2018).
Ding, J. et al. Long-term application of organic fertilization causes the accumulation of antibiotic resistome in earthworm gut microbiota. Environ. Int. 124, 145–152 (2019).
Do, T. T. et al. Antibiotic resistance gene detection in the microbiome context. Microb. Drug Resist. 24, 542–546 (2018).
Gao, J.-F., Liu, X.-H., Fan, X.-Y. & Dai, H.-H. Effects of triclosan on performance, microbial community and antibiotic resistance genes during partial denitrification in a sequencing moving bed biofilm reactor. Bioresour. Technol. 281, 326–334 (2019).
Gou, M. et al. Aerobic composting reduces antibiotic resistance genes in cattle manure and the resistome dissemination in agricultural soils. Sci. Total Environ. 612, 1300–1310 (2018).
Gu, J. et al. High-throughput analysis of the effects of different fish culture methods on antibiotic resistance gene abundances in a lake. Environ. Sci. Pollut. Res. 26, 5445–5453 (2019).
Guo, X. et al. Antibiotic resistome associated with small-scale poultry production in rural Ecuador. Environ. Sci. Technol. 52, 8165–8172 (2018).
Guo, Y. et al. The antibiotic resistome of free-living and particle-attached bacteria under a reservoir cyanobacterial bloom. Environ. Int. 117, 107–115 (2018).
Han, X.-M. et al. Antibiotic resistance genes and associated bacterial communities in agricultural soils amended with different sources of animal manures. Soil Biol. Biochem. 126, 91–102 (2018).
Han, Y. et al. Combined impact of fishmeal and tetracycline on resistomes in mariculture sediment. Environ. Pollut. 242, 1711–1719 (2018).
Hu, H.-W. et al. Diversity of herbaceous plants and bacterial communities regulates soil resistome across forest biomes. Environ. Microbiol. 20, 3186–3200 (2018).
Huang, X. et al. Higher temperatures do not always achieve better antibiotic resistance gene removal in anaerobic digestion of swine manure. Appl. Environ. Microbiol. 85, e02878-18 (2019).
Jiao, Y.-N. et al. Biomarkers of antibiotic resistance genes during seasonal changes in wastewater treatment systems. Environ. Pollut. 234, 79–87 (2018).
Jong, M.-C. et al. Co-optimization of sponge-core bioreactors for removing total nitrogen and antibiotic resistance genes from domestic wastewater. Sci. Total Environ. 634, 1417–1423 (2018).
Kang, W., Zhang, Y.-J., Shi, X., He, J.-Z. & Hu, H.-W. Short-term copper exposure as a selection pressure for antibiotic resistance and metal resistance in an agricultural soil. Environ. Sci. Pollut. Res. 25, 29314–29324 (2018).
Kanger, K. et al. Antibiotic resistome and microbial community structure during anaerobic co-digestion of food waste, paper and cardboard. bioRxiv 564823 (2019). doi:10.1101/564823
Karkman, A. et al. High-throughput quantification of antibiotic resistance genes from an urban wastewater treatment plant. FEMS Microbiol. Ecol. 92, 1–7 (2016).
Li, Y. et al. Prevalence of antibiotic resistance genes in air-conditioning systems in hospitals, farms, and residences. Int. J. Environ. Res. Public Health 16, 683 (2019).
Lin, W., Zhang, M., Zhang, S. & Yu, X. Can chlorination co-select antibiotic-resistance genes? Chemosphere 156, 412–419 (2016).
Liu, L. et al. Large-scale biogeographical patterns of bacterial antibiotic resistome in the waterbodies of China. Environ. Int. 117, 292–299 (2018).
Lu, X.-M., Li, W.-F. & Li, C.-B. Characterization and quantification of antibiotic resistance genes in manure of piglets and adult pigs fed on different diets. Environ. Pollut. 229, 102–110 (2017).
McCann, C. M. et al. Understanding drivers of antibiotic resistance genes in High Arctic soil ecosystems. Environ. Int. 125, 497–504 (2019).
Muurinen, J. et al. Influence of manure application on the environmental resistome under Finnish agricultural practice with restricted antibiotic use. Environ. Sci. Technol. 51, 5989–5999 (2017).
Muziasari, W. I. et al. Aquaculture changes the profile of antibiotic resistance and mobile genetic element associated genes in Baltic Sea sediments. FEMS Microbiol. Ecol. 92, fiw052 (2016).
Muziasari, W. I. et al. The resistome of farmed fish feces contributes to the enrichment of antibiotic resistance genes in sediments below Baltic Sea fish farms. Front. Microbiol. 7, 1–10 (2017).
Ouyang, W. Y., Huang, F. Y., Zhao, Y., Li, H. & Su, J. Q. Increased levels of antibiotic resistance in urban stream of Jiulongjiang River, China. Appl. Microbiol. Biotechnol. 99, 5697–5707 (2015).
Pärnänen, K. M. M. et al. Antibiotic resistance in European wastewater treatment plants mirrors the pattern of clinical antibiotic resistance prevalence. Sci. Adv. 5, eaau9124 (2019).
Qian, X. et al. Diversity, abundance, and persistence of antibiotic resistance genes in various types of animal manure following industrial composting. J. Hazard. Mater. 344, 716–722 (2018).
Stedtfeld, R. D. et al. Antimicrobial resistance Dashboard application for mapping environmental occurrence and resistant pathogens. FEMS Microbiol. Ecol. 92, 1–9 (2016).
Stedtfeld, R. D. et al. TCDD influences reservoir of antibiotic resistance genes in murine gut microbiome. FEMS Microbiol. Ecol. 93, 1–8 (2017).
Stedtfeld, R. D. et al. Isothermal assay targeting class 1 integrase gene for environmental surveillance of antibiotic resistance markers. J. Environ. Manage. 198, 213–220 (2017).
Stedtfeld, R. D. et al. Primer set 2.0 for highly parallel qPCR array targeting antibiotic resistance genes and mobile genetic elements. FEMS Microbiol. Ecol. 94, (2018).
Strieder-Barboza, C., de Souza, J., Raphael, W., Lock, A. L. & Contreras, G. A. Fetuin-A: A negative acute-phase protein linked to adipose tissue function in periparturient dairy cows. J. Dairy Sci. 101, 2602–2616 (2018).
Su, J. Q. et al. Antibiotic resistome and its association with bacterial communities during sewage sludge composting. Environ. Sci. Technol. 49, 7356–7363 (2015).
Tang, M. et al. Abundance and distribution of antibiotic resistance genes in a full-scale anaerobic-aerobic system alternately treating ribostamycin, spiramycin and paromomycin production wastewater. Environ. Geochem. Health (2017). doi:10.1007/s10653-017-9987-5
Tian, Z., Chi, Y., Yu, B., Yang, M. & Zhang, Y. Thermophilic anaerobic digestion reduces ARGs in excess sludge even under high oxytetracycline concentrations. Chemosphere 222, 305–313 (2019).
Tian, Z., Zhang, Y. & Yang, M. Chronic impacts of oxytetracycline on mesophilic anaerobic digestion of excess sludge: Inhibition of hydrolytic acidification and enrichment of antibiotic resistome. Environ. Pollut. 238, 1017–1026 (2018).
Wan, K. et al. Organic carbon: An overlooked factor that determines the antibiotic resistome in drinking water sand filter biofilm. Environ. Int. 125, 117–124 (2019).
Wang, B., Li, G., Cai, C., Zhang, J. & Liu, H. Assessing the safety of thermally processed penicillin mycelial dreg following the soil application: Organic matter's maturation and antibiotic resistance genes. Sci. Total Environ. 636, 1463–1469 (2018).
Wang, F. et al. Long-term effect of different fertilization and cropping systems on the soil antibiotic Resistome. Environ. Sci. Technol. 52, 13037–13046 (2018).
Wang, F. et al. Influence of soil characteristics and proximity to Antarctic research stations on abundance of antibiotic resistance genes in soils. Environ. Sci. Technol. 50, 12621–12629 (2016).
Wang, F.-H. et al. High throughput profiling of antibiotic resistance genes in urban park soils with reclaimed water irrigation. Environ. Sci. Technol. 48, 9079–9085 (2014).
Wang, H. et al. The antibiotic resistome of swine manure is significantly altered by association with the Musca domestica larvae gut microbiome. ISME J. 11, 100–111 (2017).
Wang, H.-T. et al. Effects of arsenic on gut microbiota and its biotransformation genes in earthworm Metaphire sieboldi. Environ. Sci. Technol. acs.est.8b06695 (2019). doi:10.1021/acs.est.8b06695
Wang, Q., Wang, P. & Yang, Q. Occurrence and diversity of antibiotic resistance in untreated hospital wastewater. Sci. Total Environ. 621, 990–999 (2018).
Waseem, H. et al. Contributions and challenges of high throughput qPCR for determining antimicrobial resistance in the environment: a critical review. Molecules 24, 163 (2019).
Wolters, B. et al. Soil amendment with sewage sludge affects soil prokaryotic community composition, mobilome and resistome. FEMS Microbiol. Ecol. 95, (2018).
Xiang, Q. et al. Spatial and temporal distribution of antibiotic resistomes in a peri-urban area is associated significantly with anthropogenic activities. Environ. Pollut. 235, 525–533 (2018).
Xie, W.-Y. et al. Long-term impact of field applications of sewage sludge on soil antibiotic resistome. Environ. Sci. Technol. 50, 12602–12611 (2016).
Xie, W.-Y. et al. Changes in antibiotic concentrations and antibiotic resistome during commercial composting of animal manures. Environ. Pollut. 219, 182–190 (2016).
Xie, W.-Y. et al. Long-term effects of manure and chemical fertilizers on soil antibiotic resistome. Soil Biol. Biochem. 122, 111–119 (2018).
Xu, L. et al. High-throughput profiling of antibiotic resistance genes in drinking water treatment plants and distribution systems. Environ. Pollut. 213, 119–126 (2016).
Yan, W. et al. The changes of bacterial communities and antibiotic resistance genes in microbial fuel cells during long-term oxytetracycline processing. Water Res. 142, 105–114 (2018).
Yang, L. et al. Application of biosolids drives the diversity of antibiotic resistance genes in soil and lettuce at harvest. Soil Biol. Biochem. 122, 131–140 (2018).
Zhang, M., Chen, L., Ye, C. & Yu, X. Co-selection of antibiotic resistance via copper shock loading on bacteria from a drinking water bio-filter. Environ. Pollut. 233, 132–141 (2018).
Zhang, Q. et al. Species-specific response of the soil collembolan gut microbiome and resistome to soil oxytetracycline pollution. Sci. Total Environ. 668, 1183–1190 (2019).
Zhang, Y.-J. et al. Salinity as a predominant factor modulating the distribution patterns of antibiotic resistance genes in ocean and river beach soils. Sci. Total Environ. 668, 193–203 (2019).
Zhao, Y. et al. Feed additives shift gut microbiota and enrich antibiotic resistance in swine gut. Sci. Total Environ. 621, 1224–1232 (2018).
Zhao, Y. et al. Evidence for co-selection of antibiotic resistance genes and mobile genetic elements in metal polluted urban soils. Sci. Total Environ. 656, 512–520 (2019).
Zhao, Y. et al. AsChip: A High-Throughput qPCR Chip for Comprehensive Profiling of Genes Linked to Microbial Cycling of Arsenic. Environ. Sci. Technol. 53, 798–807 (2019).
Zheng, H.-S., Guo, W.-Q., Wu, Q.-L., Ren, N.-Q. & Chang, J.-S. Electro-peroxone pretreatment for enhanced simulated hospital wastewater treatment and antibiotic resistance genes reduction. Environ. Int. 115, 70–78 (2018).
Zheng, J., Chen, T. & Chen, H. Antibiotic resistome promotion in drinking water during biological activated carbon treatment: Is it influenced by quorum sensing? Sci. Total Environ. 612, 1–8 (2018).
Zheng, J. et al. High-throughput profiling and analysis of antibiotic resistance genes in East Tiaoxi River, China. Environ. Pollut. 230, 648–654 (2017).
Zheng, J. et al. High-throughput profiling of seasonal variations of antibiotic resistance gene transport in a peri-urban river. Environ. Int. 114, 87–94 (2018).
Zhou, X. et al. Turning pig manure into biochar can effectively mitigate antibiotic resistance genes as organic fertilizer. Sci. Total Environ. 649, 902–908 (2019).
Zhou, X., Qiao, M., Su, J.-Q. & Zhu, Y.-G. High-throughput characterization of antibiotic resistome in soil amended with commercial organic fertilizers. J. Soils Sediments 19, 641–651 (2019).
Zhou, Z.-C. et al. Prevalence and transmission of antibiotic resistance and microbiota between humans and water environments. Environ. Int. 121, 1155–1161 (2018).
Zhu, B., Chen, Q., Chen, S. & Zhu, Y. G. Does organically produced lettuce harbor higher abundance of antibiotic resistance genes than conventionally produced? Environ. Int. 98, 152–159 (2017).
Zhu, D. et al. Antibiotics disturb the microbiome and increase the incidence of resistance genes in the gut of a common soil collembolan. Environ. Sci. Technol. 52, 3081–3090 (2018).
Zhu, D. et al. Exposure of a soil collembolan to Ag nanoparticles and AgNO3 disturbs its associated microbiota and lowers the incidence of antibiotic resistance genes in the Gut. Environ. Sci. Technol. 52, 12748–12756 (2018).
Zhu, D. et al. Land use influences antibiotic resistance in the microbiome of soil collembolans Orchesellides sinensis. Environ. Sci. Technol. 52, 14088–14098 (2018).
Zhu, Y.-G. et al. Continental-scale pollution of estuaries with antibiotic resistance genes. Nat. Microbiol. 2, 16270 (2017).
Takara Bio USA, Inc.
United States/Canada: +1.800.662.2566 • Asia Pacific: +1.650.919.7300 • Europe: +33.(0)1.3904.6880 • Japan: +81.(0)77.565.6999
FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. © 2022 Takara Bio Inc. All Rights Reserved. All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at takarabio.com.