Direct RNA-based detection and differentiation of CTX-M-type extended-spectrum β-lactamases (ESBL).
The current global spread of multi-resistant Gram-negatives, particularly extended spectrum β-lactamases expressing bacteria, increases the likelihood of inappropriate empiric treatment of critically ill patients with subsequently increased mortality. From a clinical perspective, fast detection of resistant pathogens would allow a pre-emptive correction of an initially inappropriate treatment. Here we present diagnostic amplification-sequencing approach as proof of principal based on the fast molecular detection and correct discrimination of CTX-M-β-lactamases, the most frequent ESBL family. The workflow consists of the isolation of total mRNA and CTX-M-specific reverse transcription (RT), amplification and pyrosequencing. Due to the high variability of the CTX-M-β-lactamase-genes, degenerated primers for RT, qRT as well as for pyrosequencing, were used and the suitability and discriminatory performance of two conserved positions within the CTX-M genes were analyzed, using one protocol for all isolates and positions, respectively. Using this approach, no information regarding the expected CTX-M variant is needed since all sequences are covered by these degenerated primers. The presented workflow can be conducted within eight hours and has the potential to be expanded to other β-lactamase families.
Center for Infectious Diseases and Infection's Control, Jena University Hospital, Jena, Germany.
Claudia
C
Stein
Oliwia
O
Makarewicz
Yvonne
Y
Pfeifer
Christian
C
Brandt
João Costa
JC
Ramos
Mareike
M
Klinger
Mathias W
MW
Pletz
D004926:Escherichia coli / Q000201:enzymology; D009154:Mutation; D012313:RNA / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D048348:Reverse Transcription; D001618:beta-Lactamases / Q000235:genetics*
D012313:RNA; D001618:beta-Lactamases