Abstract
The accurate identification of Extended-Spectrum β-Lactamase (ESBL) genes in Gram-negative bacteria is necessary for surveillance and epidemiological studies of transmission through foods. We report a novel rapid, cheap, and accurate closed tube molecular diagnostic tool based on two multiplex HRM protocols for analysis of the predominant ESBL families encountered in foods. The first multiplex PCR assay targeted blaCTX-M including phylogenetic groups 1 (CTX-M-1-15, including CTX-M-1, CTX-M-3 and CTX-M-15), 2 (CTX-M-2), and 9 (CTX-M-9-14, including CTX-M-9 and CTX-M-14). The second assay involved blaTEM /bla CTX-M /blaSHV, including TEM variants (TEM-1 and TEM-2), SHV-1-56 (SHV-1, SHV-2 and SHV-56), and CTX-M-8-41 (CTX-M-8, CTX-M-25, CTX-M-26 and CTX-M-39 to CTX-M-41). The individual melting curves were differentiated by a temperature shift according to the type of ESBL gene. The specificity and sensitivity of the first assay were 100% and 98%, respectively. For the second assay, the specificity and sensitivity were 87% and 89%, respectively. The detection of ESBL variants or mutations in existing genes was also demonstrated by the subtyping of a variant of the CTXM-1-15. The HRM is a potential tool for the rapid detection of present β-lactamase genes and their characterization in a highly sensitive, closed-tube, inexpensive method that is applicable in high throughput studies.
Highlights
Antibiotics belonging to the class β-lactams have been instrumental in both human and veterinary medicine for the treatment of infections by Gram-negative pathogens [1]
We report a novel rapid, cheap, and accurate closed tube molecular diagnostic tool based on two multiplex High-resolution DNA melting analysis (HRM) protocols for analysis of the predominant ESBL families encountered in foods
ESBL detection in foodborne Gram-negative bacteria relies on phenotypic tests based on disc diffusion or dilution tests, which involves a significant delay in results due to manual steps and incubation time
Summary
Antibiotics belonging to the class β-lactams have been instrumental in both human and veterinary medicine for the treatment of infections by Gram-negative pathogens [1]. Several Gram-negative pathogens developed the ability to produce mutant forms of the “older” β-lactamases, called extended-spectrum β-lactamases (ESBLs) These mutant forms of β-lactamases are able to hydrolyze the new-generation cephalosporins and aztreonam [4]. ESBLs can be classified as class A β-lactamases mainly from the three main families, TEM, SHV, and CTX-M [5] Another closely related but distinct group of β-lactamases includes the AmpC β-lactamases, which confer resistance to additional β-lactams and β-lactamase inhibitors [6]. Β-lactams that are commonly hydrolyzed by ESBL and AmpC β-lactamases belong to a group of critically important antimicrobials in both human and animal medicine [7] This has resulted in increased morbidity, mortality, and health care costs all over the world [8]
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