Computational protocol: Occurrence of the mcr-1 Colistin Resistance Gene and other Clinically Relevant Antibiotic Resistance Genes in Microbial Populations at Different Municipal Wastewater Treatment Plants in Germany

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Protocol publication

[…] All primers and references are listed in Table , which do also contains the accuracy (R2) as well as efficiency (%) values for each qPCR detection system. More specifically, the Escherichia coli NRZ-14408 reference strain is carrying the mcr-1 colistin resistance gene and was kindly provided from National Reference Center in Bochum, Germany. For mcr-1 primer design the NCBI Primer BLAST software was used. For the quantification of the mcr-1 gene in environmental water samples a colistin-resistant E. coli NRZ-14408 (hospital isolate, mcr-1 positive) reference bacterium was used to create a calibration curve, which is shown in . An R2 value of 0.999 and an efficiency of 97.6% were determined, indicating the high specificity of the mcr-1 detection system.The antibiotic resistance genes blaTEM, CTX-M, CTX-M-32, and CMY-2 are directed against ß-lactam antibiotics, whereas the tetM resistance gene is coding for the resistance against tetracycline and the ermB gene mediates the resistance against erythromycin. All primers, reference strains, and quality values of each detection system are listed in Table . In addition calibration and melting curves are given in . The abundances of these ARGs were quantified in all WWTP effluent wastewater samples via qPCR approach using different reference strains carrying the mentioned resistance genes.The yccT gene present in E. coli DSM 1103 was used as taxonomic gene marker as well as the gltA gene from K. pneumoniae DSM 30104 (Clifford et al., ). Total DNA of pure culture was extracted with the DNA extraction kit for soil (MP Biomedical, Illkrich, France) and subsequently used for the generation of target specific calibration curves. 16S rRNA primers were used to quantify eubacterial rDNA in the water samples for normalization. Here, an already cloned fragment of the eubacterial 16S rRNA gene in the pNORM plasmid of E. coli DH5α was used (Stalder et al., ). Plasmid-DNA was extracted with the GeneJet Plasmid Miniprep Kit (Thermo Scientific, Waldham, USA) and was used for the generation of the calibration curve which in turn was used for the calculation of the 16S rRNA gene copy number in water samples.The mcr-1, ermB, tetM resistance genes, and the four ß-lactamase genes (CTX-M. CTX-M-32, CMY-2, blaTEM), the ribosomal 16S rRNA gene for Eubacteria, and the specific taxonomic gene markers of E. coli, K. pneumonia, and Acinetobacter baumannii were quantified in a SYBR Green qPCR approach. Reactions were run in volumes of 20 μL, containing 10 μL Maxima SYBR Green/ROX qPCR Master Mix (2x) (Thermo Scientific), 8.2 μL of nuclease-free water (Ambion, Life technologies, Karlsbad, Germany), 0.4 μL of the respective primers (stock concentration 10 μM, Table ), and 1 μL of template DNA (20 ng μL−1). The qPCR protocol comprised 10 min at 95°C for activation of the DNA polymerase followed by 40 cycles of 15 s at 95°C and 1 min at appropriate temperature for primer annealing and elongation (see ). Each water sample was analyzed in technical triplicates. To determine the specificity of the amplification, a melting curve was recorded by raising the temperature from 60 to 95°C (1°C every 10 s). Data analysis was performed by using the Bio-Rad CFX Manager software. […]

Pipeline specifications

Software tools Primer-BLAST, CFX Manager
Application qPCR
Organisms Acinetobacter baumannii, Escherichia coli
Diseases Pneumonia
Chemicals Colistin