Real-time PCR methods for quantitative monitoring of streptomycin and tetracycline resistance genes in agricultural ecosystems

Walsh, Fiona; Ingenfeld, Anke; Zampicolli, M.; Hilber-Bodmer, Maja; Frey, Jürg E.; Duffy, Brion

doi:10.1016/j.mimet.2011.04.011

Cited by 71 publications

(38 citation statements)

References 33 publications

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“…Molecular tools, such as PCR and quantitative real-time PCR, have been developed for many ARGs (Heuer and Smalla, 2007;Walsh et al, 2011). A limited number of studies have applied molecular methods to aquatic environments.…”

Section: Introductionmentioning

confidence: 99%

Wastewater as a point source of antibiotic-resistance genes in the sediment of a freshwater lake

2014

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Antibiotic-resistance genes (ARGs) are currently discussed as emerging environmental contaminants. Hospital and municipal sewage are important sources of ARGs for the receiving freshwater bodies. We investigated the spatial distribution of different ARGs (sul1, sul2, tet(B), tet(M), tet(W) and qnrA) in freshwater lake sediments in the vicinity of a point source of treated wastewater. ARG contamination of Vidy Bay, Lake Geneva, Switzerland was quantified using real-time PCR and compared with total mercury (THg), a frequently particle-bound inorganic contaminant with known natural background levels. Two-dimensional mapping of the investigated contaminants in lake sediments with geostatistical tools revealed total and relative abundance of ARGs in close proximity of the sewage discharge point were up to 200-fold above levels measured at a remote reference site (center of the lake) and decreased exponentially with distance. Similar trends were observed in the spatial distribution of different ARGs, whereas distributions of ARGs and THg were only moderately correlated, indicating differences in the transport and fate of these pollutants or additional sources of ARG contamination. The spatial pattern of ARG contamination and supporting data suggest that deposition of particle-associated wastewater bacteria rather than co-selection by, for example, heavy metals was the main cause of sediment ARG contamination.

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Section: Introductionmentioning

confidence: 99%

Wastewater as a point source of antibiotic-resistance genes in the sediment of a freshwater lake

2014

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“…Valuable insight into the manure resistomes of pigs, cattle, and chickens has been gained from direct culturing of antibiotic-resistant bacteria (10, 16, 17), quantitative PCR (qPCR) amplification of AR genes (5, 18–20), and the exogenous isolation of plasmids carrying AR genes (21, 22). These approaches have shown that animal microbiomes are a substantial source of AR genes, especially of those conferring resistance to tetracycline and sulfonamide, which are often carried on plasmids (18, 21–26).…”

Section: Introductionmentioning

confidence: 99%

Diverse Antibiotic Resistance Genes in Dairy Cow Manure

Wichmann

Udikovic-Kolic

Andrew

et al. 2014

mBio

241

179

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Application of manure from antibiotic-treated animals to crops facilitates the dissemination of antibiotic resistance determinants into the environment. However, our knowledge of the identity, diversity, and patterns of distribution of these antibiotic resistance determinants remains limited. We used a new combination of methods to examine the resistome of dairy cow manure, a common soil amendment. Metagenomic libraries constructed with DNA extracted from manure were screened for resistance to beta-lactams, phenicols, aminoglycosides, and tetracyclines. Functional screening of fosmid and small-insert libraries identified 80 different antibiotic resistance genes whose deduced protein sequences were on average 50 to 60% identical to sequences deposited in GenBank. The resistance genes were frequently found in clusters and originated from a taxonomically diverse set of species, suggesting that some microorganisms in manure harbor multiple resistance genes. Furthermore, amid the great genetic diversity in manure, we discovered a novel clade of chloramphenicol acetyltransferases. Our study combined functional metagenomics with third-generation PacBio sequencing to significantly extend the roster of functional antibiotic resistance genes found in animal gut bacteria, providing a particularly broad resource for understanding the origins and dispersal of antibiotic resistance genes in agriculture and clinical settings.

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“…Relative quantification using the ⌬⌬C T method is the most common method used in gene expression analysis (48) but has also been applied in environmental microbiology (51). The method determines the gene expression ratio (or abundance ratio) of a target gene in a sample compared to a control, normalizing with the expression ratio of a reference gene.…”

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confidence: 99%

Simple Absolute Quantification Method Correcting for Quantitative PCR Efficiency Variations for Microbial Community Samples

Brankatschk

Bodenhausen

Zeyer

et al. 2012

Appl Environ Microbiol

187

126

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ABSTRACTReal-time quantitative PCR (qPCR) is a widely used technique in microbial community analysis, allowing the quantification of the number of target genes in a community sample. Currently, the standard-curve (SC) method of absolute quantification is widely employed for these kinds of analysis. However, the SC method assumes that the amplification efficiency (E) is the same for both the standard and the sample target template. We analyzed 19 bacterial strains and nine environmental samples in qPCR assays, targeting thenifHand 16S rRNA genes. TheEvalues of the qPCRs differed significantly, depending on the template. This has major implications for the quantification. If the sample and standard differ in theirEvalues, quantification errors of up to orders of magnitude are possible. To address this problem, we propose and test the one-point calibration (OPC) method for absolute quantification. The OPC method corrects for differences inEand was derived from the ΔΔCTmethod with correction forE, which is commonly used for relative quantification in gene expression studies. The SC and OPC methods were compared by quantifying artificial template mixtures fromGeobacter sulfurreducens(DSM 12127) andNostoc commune(Culture Collection of Algae and Protozoa [CCAP] 1453/33), which differ in theirEvalues. While the SC method deviated from the expectednifHgene copy number by 3- to 5-fold, the OPC method quantified the template mixtures with high accuracy. Moreover, analyzing environmental samples, we show that even small differences inEbetween the standard and the sample can cause significant differences between the copy numbers calculated by the SC and the OPC methods.

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Real-time PCR methods for quantitative monitoring of streptomycin and tetracycline resistance genes in agricultural ecosystems

Cited by 71 publications

References 33 publications

Wastewater as a point source of antibiotic-resistance genes in the sediment of a freshwater lake

Wastewater as a point source of antibiotic-resistance genes in the sediment of a freshwater lake

Diverse Antibiotic Resistance Genes in Dairy Cow Manure

Simple Absolute Quantification Method Correcting for Quantitative PCR Efficiency Variations for Microbial Community Samples

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