Establishment of a Simple and Quick Method for Detecting Extended-Spectrum β-Lactamase (ESBL) Genes in Bacteria.
Extended-spectrum β-lactamase (ESBL) genes that render bacteria resistant to antibiotics are commonly detected using phenotype testing, which is time consuming and not sufficiently accurate. To establish a better method, we used phenotype testing to identify ESBL-positive bacterial strains and conducted PCR to screen for TEM (named after the patient Temoneira who provided the first sample), sulfhydryl reagent variable (SHV), cefotaxime (CTX)-M-1, and CTX-M-9, the 4 most common ESBL types and subtypes. We then performed multiplex PCR with 1 primer containing a biotin and hybridized the PCR products with gene-specific probes that were coupled with microbeads and coated with a specific fluorescence. The hybrids were linked to streptavidin-R-phycoerythrins (SA-PEs) and run through a flow cytometer, which sorted the fluorescently dyed microbeads and quantified the PEs. The results from single PCR, multiplex PCR, and cytometry were consistent with each other. We used this method to test 169 clinical specimens that had been determined for phenotypes and found 154 positive for genotypes, including 30 of the 45 samples that were negative for phenotypes. The CTX-M genotype tests alone, counting both positive and negative cases, showed 99.41% (168/169) consistency with the ESBL phenotype test. Thus, we have established a multiplex-PCR system as a simple and quick method that is high throughput and accurate for detecting 4 common ESBL types and subtypes.
Clinical Laboratory, Baiyun Hospital affiliated to Guizhou Medical University Hospital, Guiyang 550058, P.R. China;; Central Laboratory, Guizhou Medical University Hospital, Guiyang 550004, P.R. China.
Song-Tao
ST
Han
Department of Microbiology and Immunology, Guizhou Medical University Hospital, Guiyang 550004, P.R. China.
Ying
Y
Fei
Clinical Laboratory, Baiyun Hospital affiliated to Guizhou Medical University Hospital, Guiyang 550058, P.R. China.
Jin-You
JY
Huang
Clinical Laboratory, Baiyun Hospital affiliated to Guizhou Medical University Hospital, Guiyang 550058, P.R. China.
Mei
M
Xu
The Hormel Institute, University of Minnesota, Austin, Minnesota 55912, USA; and.
Li-Chan
LC
Chen
Department of Pathology, Guizhou Medical University Hospital, Guiyang 550004, P.R. China.
D Joshua
DJ
Liao
Clinical Laboratory, Baiyun Hospital affiliated to Guizhou Medical University Hospital, Guiyang 550058, P.R. China;; Central Laboratory, Guizhou Medical University Hospital, Guiyang 550004, P.R. China.
Yu-Jie
YJ
Tan
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D004927:Escherichia coli Infections / Q000175:diagnosis* / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000235:genetics*; D005798:Genes, Bacterial; D006801:Humans; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics; D025202:Molecular Diagnostic Techniques; D060885:Multiplex Polymerase Chain Reaction; D015203:Reproducibility of Results; D018440:beta-Lactam Resistance / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics*
D029968:Escherichia coli Proteins; D001618:beta-Lactamases
antibiotics; drug-resistance gene; flow fluorescence; multiplex PCR
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