Molecular Detection of Antibiotic Resistance

Template DNA for PCRs was prepared by boiling bacterial suspensions in 10 mM Tris–EDTA pH 8.0 (Sigma-Aldrich, St. Louis, MO, USA) according to Aldous et al.^{61 (link)}. Enterobacterales with phenotypic resistance to 3rd-generation cephalosporins were screened by PCR for β-lactamase (bla) genes encoding enzymes SHV, TEM, and CTX-M (groups 1, 2, 8 and 9) as previously described^{62 (link)–64 (link)}. Isolates of the A. calcoaceticus-baumannii complex and P. aeruginosa were examined for the presence of bla_PER, bla_GES, and bla_VEB by PCR as described^{65 (link)}. Colistin-resistant isolates (MIC > 2 mg/L) were screened for mcr-1 to mcr-5 as well as mcr-6 to mcr-9 genes using multiplex PCR protocols as described by Rebelo et al.^{66 (link)} and Borowiak et al.^{67 (link)}, respectively. The obtained PCR amplicons were purified with the innuPREP DOUBLEpure Kit (Analytik Jena AG, Jena, Germany) and subjected to Sanger sequencing at Microsynth Seqlab (Göttingen, Germany).

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Savin M., Alexander J., Bierbaum G., Hammerl J.A., Hembach N., Schwartz T., Schmithausen R.M., Sib E., Voigt A, & Kreyenschmidt J. (2021). Antibiotic-resistant bacteria, antibiotic resistance genes, and antibiotic residues in wastewater from a poultry slaughterhouse after conventional and advanced treatments. Scientific Reports, 11, 16622.

Publication 2021

innuPREP DOUBLEpure Kit

Bacterial Cephalosporins Colistin Edta Enzymes Genes Multiplex pcr P aeruginosa Phenotypic Tris β lactamase

Corresponding Organization : University of Bonn

Other organizations : Karlsruhe Institute of Technology, Federal Institute for Risk Assessment, Hochschule Geisenheim University

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Variable analysis

independent variables

Presence of β-lactamase (bla) genes encoding enzymes SHV, TEM, and CTX-M (groups 1, 2, 8 and 9) in Enterobacterales with phenotypic resistance to 3rd-generation cephalosporins
Presence of bla PER, bla GES, and bla VEB in Acinetobacter calcoaceticus-baumannii complex and Pseudomonas aeruginosa isolates
Presence of mcr-1 to mcr-5 as well as mcr-6 to mcr-9 genes in colistin-resistant isolates (MIC > 2 mg/L)

dependent variables

Identification of β-lactamase (bla) genes in Enterobacterales with phenotypic resistance to 3rd-generation cephalosporins
Identification of bla PER, bla GES, and bla VEB in Acinetobacter calcoaceticus-baumannii complex and Pseudomonas aeruginosa isolates
Identification of mcr-1 to mcr-5 as well as mcr-6 to mcr-9 genes in colistin-resistant isolates (MIC > 2 mg/L)

control variables

Template DNA for PCRs was prepared by boiling bacterial suspensions in 10 mM Tris–EDTA pH 8.0 (Sigma-Aldrich, St. Louis, MO, USA) according to Aldous et al.

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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