Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae.
OBJECTIVE
To develop a rapid and reliable tool to detect by multiplex PCR assays the most frequently widespread beta-lactamase genes encoding the OXA-1-like broad-spectrum beta-lactamases, extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC beta-lactamases and class A, B and D carbapenemases.


METHODS
Following the design of a specific group of primers and optimization using control strains, a set of six multiplex PCRs and one simplex PCR was created. An evaluation of the set was performed using a collection of 31 Enterobacteriaceae strains isolated from clinical specimens showing a resistance phenotype towards broad-spectrum cephalosporins and/or cephamycins and/or carbapenems. Direct sequencing from PCR products was subsequently carried out to identify beta-lactamase genes.


RESULTS
Under optimized conditions, all positive controls confirmed the specificity of group-specific PCR primers. Except for the detection of carbapenemase genes, multiplex and simplex PCR assays were carried out using the same PCR conditions, allowing assays to be performed in a single run. Out of 31 isolates selected, 22 strains produced an ESBL, mostly CTX-M-15 but also CTX-M-1 and CTX-M-9, SHV-12, SHV-5, SHV-2, TEM-21, TEM-52 and a VEB-type ESBL, 6 strains produced a plasmid-mediated AmpC beta-lactamase (five DHA-1 and one CMY-2) and 3 strains produced both an ESBL (two SHV-12, one CTX-M-15) and a plasmid-mediated AmpC beta-lactamase (DHA-1).


CONCLUSIONS
We report here the development of a useful method composed of a set of six multiplex PCRs and one simplex PCR for the rapid screening of the most frequently encountered beta-lactamases. This method allowed direct sequencing from the PCR products.
Dallenne
Caroline
C
Université Pierre et Marie Curie Paris 6, Faculté de Médecine Pierre et Marie Curie, Département de Bactériologie, Paris, France.
Da Costa
Anaelle
A
Decré
Dominique
D
Favier
Christine
C
Arlet
Guillaume
G
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004755:Enterobacteriaceae / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D004756:Enterobacteriaceae Infections / Q000382:microbiology; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics*; D047090:beta-Lactams / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D047090:beta-Lactams; D001618:beta-Lactamases