A rapid and accurate method for the detection of four aminoglycoside modifying enzyme drug resistance gene in clinical strains of Escherichia coli by a multiplex polymerase chain reaction

Abstract

Background Antibiotics are highly effective drugs used in the treatment of infectious diseases. Aminoglycoside antibiotics are one of the most common antibiotics in the treatment of bacterial infections. However, the development of drug resistance against those medicines is becoming a serious concern. Aim This study aimed to develop an efficient, rapid, accurate, and sensitive detection method that is applicable for routine clinical use. Methods Escherichia coli was used as a model organism to develop a rapid, accurate, and reliable multiplex polymerase chain reaction (M-PCR) for the detection of four aminoglycoside modifying enzyme (AME) resistance genes Aac(6′)-Ib, Aac(3)-II, Ant(3″)-Ia, and Aph(3′)-Ia. M-PCR was used to detect the distribution of AME resistance genes in 237 clinical strains of E. coli. The results were verified by simplex polymerase chain reaction (S-PCR). Results Results of M-PCR and S-PCR showed that the detection rates of Aac(6′)-Ib, Aac(3)-II, Ant(3″)-Ia, and Aph(3′)-Ia were 32.7%, 59.2%, 23.5%, and 16.8%, respectively, in 237 clinical strains of E. coli. Compared with the traditional methods for detection and identification, the rapid and accurate M-PCR detection method was established to detect AME drug resistance genes. This technique can be used for the clinical detection as well as the surveillance and monitoring of the spread of those specific antibiotic resistance genes.

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“…The amikacin plasmid resistance genes ( Aac(6′)-Ib , Aac(3)-II , Ant(3″)-Ia , and Aph(3′)-Ia) were detected in the resistant E. coli by Shi et al’s [ 14 ] multiplex PCR protocol with modifications including the Aac(6′)-Ib forward primer design (BLAST, NCBI) ( Table 1 ). To evaluate their co-presence, the AMEs genes were detected also in the isolates that harbored PMQR or meropenem plasmid-resistant genes.…”

“…The resistance mechanism developed against aminoglycosides is an enzymatic modification of the molecule by the aminoglycoside-modifying enzymes (AME), particularly, 6′- N -acetyltransferase type Ib [AAC(6′)-Ib], O -nucleotidyltransferases (ANT) and O -phosphotransferases (APH) [ 12 , 13 ]. Among the AME resistance genes, Aac(6′)-Ib , Aac(3)-II , Ant(3″)-Ia , and Aph(3′)-Ia are the most widely distributed ones [ 14 ].…”