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. 2020 Apr 10:8:e8944.
doi: 10.7717/peerj.8944. eCollection 2020.

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

Yaoqiang Shi #  1   2 , Chao Li #  1   2 , Guangying Yang  1   2 , Xueshan Xia  1   2 , Xiaoqin Mao  3 , Yue Fang  1   2 , A-Mei Zhang  1   2 , Yuzhu Song  1   2
Affiliations

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

Yaoqiang Shi et al. PeerJ. .

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.

Keywords: Aminoglycoside modifying enzyme drug resistance gene; Molecular detection; Multiplex polymerase chain reaction; Polymerase chain reaction.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. The sensitivity and accuracy evaluation of Aac(6′)-Ib, Ant(3″)-Ia, Aph(3′)-Ia, and Aac(3)-II resistance genes by S-PCR.
The serially diluted positive plasmids with resistance gene were used as the template in the sensitivity evaluation of S-PCR. (A) Aac(6′)-Ib, 3 × 109−3 × 100 copies/µL. (C) Ant(3″)-Ia, 4 × 109−4 × 100 copies/µL. (E) Aph(3′)-Ia, 5 × 109−5 × 100 copies/µL. (G) Aac(3)-II, 4 × 1010−4 × 100 copies/µL. Meanwhile, the four resistance genes were detected in the 237 clinical strains of E. coli respectively, the bacterial solution as the S-PCR template. (B) The accuracy evaluation of Aac(6′)-Ib resistance genes. (D) The accuracy evaluation of Ant(3″)-Ia resistance genes. (F) The accuracy evaluation of Aph(3′)-Ia resistance genes. (H) The accuracy evaluation of Aac(3)-II resistance genes. All experiments were repeated six times. The nuclease-free water used as template for NC (Negative control). M, marker.
Figure 2
Figure 2. Establishment of M-PCR reaction system.
Figure 3
Figure 3. The accuracy evaluation of M-PCR.
Figure 4
Figure 4. The sensitivity evaluation of M-PCR.
The gradient dilution of plasmids and bacterial solution was used in the sensitivity evaluation of M-PCR. (A) The four equal concentration plasmids with Aac(6′)-Ib, Ant(3″)-Ia, Aph(3′)-Ia, and Aac(3)-II resistance genes were mixed, and serially diluted as 10-fold (1 × 108−1 × 100), as the sensitivity evaluation of M-PCR. The nuclease-free water used in lane 10 as template for NC. (B) The E. coli 1611NY0004 strain with four resistance genes was used as the template for the the sensitivity evaluation of M-PCR. The bacterial solution was serially diluted as 10-fold (1 × 108−1 × 100). The nuclease-free water used in lane 10 as template for NC. NC, negative control; M, marker.
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