title: Development and evaluation of a novel universal β-lactamase gene subtyping assay for blaSHV, blaTEM and blaCTX-M using clinical and livestock-associated Escherichia coli.
abstract: OBJECTIVE
  Antibiotic resistance among Escherichia coli is globally an increasing problem in public healthcare. Understanding the spread of plasmid-mediated ESBL genes is of great importance in elucidating their molecular epidemiology. However, differentiation of subtypes and alleles is frequently hampered by the lack of comprehensive diagnostic tools. We therefore developed a novel universal blaSHV, blaTEM and blaCTX-M subtyping assay based on PCR and Sanger sequencing that results in large amplicons of >700 bp, enabling differentiation of bla alleles as precisely as possible.
  
  
  METHODS
  The assay was established using 10 reference strains with known bla genotypes that represent all examined primer groups and 101 uncharacterized ESBL-producing E. coli of clinical and livestock-associated origins from different German regions. All isolates were tested in parallel with established blaSHV, blaTEM and blaCTX-M subtyping assays for the respective β-lactamases and their alleles.
  
  
  RESULTS
  The novel assay yielded equal (n = 92) or better (n = 47) subtyping results compared with established subtyping methods and reliably detected all expected enzymes in the reference strains. Overall, the occurring enzymes could be differentiated into groups representing one (n = 9), two (n = 5) or three (n = 4) highly similar alleles. Moreover, ESBL and non-ESBL allelic variants of blaSHV and blaTEM occurring in the same isolate were distinguished reliably.
  
  
  CONCLUSIONS
  We established a highly discriminatory assay for the subtyping of clinically important ESBL genes that can easily be used in epidemiological analyses.
authors.affiliation: Institute of Hygiene, University Hospital Münster, Münster, Germany.
authors.forename: Lena Marie
authors.initials: LM
authors.lastname: Strauß
authors.affiliation: Institute of Hygiene and Environmental Medicine, University of Medicine, Greifswald, Germany.
authors.forename: Carmen
authors.initials: C
authors.lastname: Dahms
authors.affiliation: Institute of Medical Microbiology, University Hospital Münster, Münster, Germany.
authors.forename: Karsten
authors.initials: K
authors.lastname: Becker
authors.affiliation: Institute of Hygiene and Environmental Medicine, University of Medicine, Greifswald, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Kramer
authors.affiliation: National Reference Laboratory for Multidrug-Resistant Gram-Negative Bacteria, Bochum, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Kaase
authors.affiliation: Institute of Hygiene, University Hospital Münster, Münster, Germany mellmann@uni-muenster.de.
authors.forename: Alexander
authors.initials: A
authors.lastname: Mellmann
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D004927:Escherichia coli Infections / Q000382:microbiology* / Q000662:veterinary*; D005838:Genotype; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D058751:Livestock; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D001618:beta-Lactamases / Q000145:classification* / Q000235:genetics*
chemical_list: D001618:beta-Lactamases
keywords: E. coli; ESBLs; PCR; typing