RESUMO
Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of blaIMP genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all blaIMP genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for blaIMP-81, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of blaIMP, which is useful for the surveillance of bacteria with blaIMP in clinical and public health settings or environmental fields.
Assuntos
Proteínas de Bactérias , Enterobacteriaceae , Reação em Cadeia da Polimerase Multiplex , beta-Lactamases , Humanos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , beta-Lactamases/genética , beta-Lactamases/isolamento & purificação , Primers do DNA/genética , Farmacorresistência Bacteriana Múltipla/genética , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Enterobacteriaceae/isolamento & purificação , Infecções por Enterobacteriaceae/microbiologia , Genes Bacterianos/genética , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase Multiplex/métodos , Análise de Sequência de DNARESUMO
A multiplex PCR assay in a single tube was developed for the detection of the carbapenemase genes of Enterobacteriaceae. Primers were designed to amplify the following six carbapenemase genes: blaKPC, blaIMP, blaNDM, blaVIM, blaOXA-48-like, and blaGES. Of 70 blaIMP variants, 67 subtypes were simulated to be PCR-positive based on in silico simulation and the primer-design strategy. After determining the optimal PCR conditions and performing in vitro assays, the performance of the PCR assay was evaluated using 51 and 91 clinical isolates with and without carbapenemase genes, respectively. In conclusion, the combination of multiplex PCR primers and QIAGEN Multiplex PCR Plus Kit was used to determine the best performance for the rapid and efficient screening of carbapenemase genes in Enterobacteriaceae. The assay had an overall sensitivity and specificity of 100%. This PCR assay compensates for the limitations of phenotypic testing, such as antimicrobial susceptibility testing and the modified carbapenem inactivation method, in clinical and public health settings.
Assuntos
Proteínas de Bactérias/genética , Enterobacteriaceae/enzimologia , Genes Bacterianos , Reação em Cadeia da Polimerase Multiplex/métodos , beta-Lactamases/genética , Primers do DNA/genética , Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/diagnóstico , Humanos , Sensibilidade e EspecificidadeRESUMO
Guiana extended-spectrum (GES) ß-lactamases are emerging in Japan. The GES family can be classified into 2 groups, one with extended-spectrum ß-lactamase (ESBL)-like activity, which hydrolyzes penicillins and cephalosporins, and the other with carbapenemase-like activity with an extended spectrum toward carbapenems. This difference is mediated by variations in a specific amino acid in the GES protein: G170â¯N or G170S substitutions. We developed an amplification refractory mutation system (ARMS) PCR assay that enabled rapid identification of these variant genes without sequencing.
