[Real-time PCR for fast detection of plasmid-mediated qnr genes in extended spectrum beta-lactamase producing Enterobacteriaceae].
OBJECTIVE
To develop a fast and reliable real time PCR technique for detecting plasmid-mediated quinolone resistance genes qnrA, qnrB and qnrS.


METHODS
A real-time PCR assay using SYBR Green I and Roche LightCycler(®) was developed to detect qnr genes. Detection of qnr genes was based on comparison of melting temperature differences with a positive control of each qnr genes. This assay was performed to study 138 isolates collected from diagnostic and screening samples in the Champagne-Ardenne region in 2004 (France).


RESULTS
In optimized conditions, the three positive controls tested alone and with isolates confirmed the specificity of the PCR primers. Each PCR assay was able to test 30 strains in 60min for 1 qnr gene. Out of 138 isolates screened, 3.6 % isolates were positive for a qnrA1, 1.5 % for qnrS1 and no qnrB-like gene. Prevalence of qnr determinants was 5 % and reached 9.5 % in clinical isolates.


CONCLUSIONS
Real-time PCR is a fast and reliable technique for screening of qnr-positive strains. This study shows a relatively high prevalence of qnr determinants (5 %) among ESBL-producing Enterobacteriaceae.
Laboratoire de bactériologie-virologie-hygiène, CHU Robert-Debré, rue du Général-Koenig, 51092 Reims cedex, France. tguillard@chu-reims.fr
T
T
Guillard
J-D
JD
Cavallo
E
E
Cambau
V
V
Duval
O
O
Bajolet
L
L
Brasme
C
C
de Champs
V
V
Vernet-Garnier
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D052160:Benzothiazoles; D002954:Citrobacter / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D003199:Computer Systems; D003959:Diamines; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D004754:Enterobacter / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D004755:Enterobacteriaceae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D005456:Fluorescent Dyes; D024841:Fluoroquinolones / Q000494:pharmacology*; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D011815:R Factors / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics*; D047090:beta-Lactams / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D052160:Benzothiazoles; D003959:Diamines; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D024841:Fluoroquinolones; D009930:Organic Chemicals; C485384:Qnr protein, E coli; C485385:Qnr protein, Klebsiella pneumoniae; D011804:Quinolines; D047090:beta-Lactams; C098022:SYBR Green I; D001618:beta-Lactamases