Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance.
BACKGROUND
The evolution of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) has been accelerated recently by the indiscriminate application of antibiotics. Antibiotic resistance has challenged the success of medical interventions and therefore is considered a hazardous threat to human health.


OBJECTIVE
The present study aimed to describe the use of zinc finger nuclease (ZFN) technology to target and disrupt a plasmid-encoded β-lactamase, which prevents horizontal gene transfer-mediated evolution of ARBs.


METHODS
An engineered ZFN was designed to target a specific sequence in the ampicillin resistance gene (amp(R)) of the pTZ57R plasmid. The Escherichia coli bacteria already contained the pZFN kanamycin-resistant (kana(R)) plasmid as the case or the pP15A, kana(R) empty vector as the control, were transformed with the pTZ57R; the ability of the designed ZFN to disrupt the β-lactamase gene was evaluated with the subsequent disturbed ability of the bacteria to grow on ampicillin (amp) and ampicillin-kanamycin (amp-kana)-containing media. The effect of mild hypothermia on the ZFN gene targeting efficiency was also evaluated.


RESULTS
The growth of bacteria in the case group on the amp and amp-kana-containing media was significantly lower compared with the control group at 37°C (P < 0.001). Despite being more efficient in hypothermic conditions at 30°C (P < 0.001), there were no significant associations between the incubation temperature and the ZFN gene targeting efficiency.


CONCLUSIONS
Our findings revealed that the ZFN technology could be employed to overcome ampicillin resistance by the targeted disruption of the ampicillin resistance gene, which leads to inactivation of β-lactam synthesis. Therefore, ZFN technology could be engaged to decrease the antibiotic resistance issue with the construction of a ZFN archive against different ARGs. To tackle the resistance issue at the environmental level, recombinant phages expressing ZFNs against different ARGs could be constructed and released into both hospital and urban wastewater systems.
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran.
Mansoureh
M
Shahbazi Dastjerdeh
Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran.
Shirin
S
Kouhpayeh
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran.
Faezeh
F
Sabzehei
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran.
Hossein
H
Khanahmad
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran.
Mansour
M
Salehi
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran.
Zahra
Z
Mohammadi
Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, IR Iran.
Laleh
L
Shariati
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran.
Zahra
Z
Hejazi
Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran.
Parisa
P
Rabiei
Department of Immunology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, IR Iran.
Mostafa
M
Manian
Ampicillin Resistance; Beta-Lactamases; Gene Targeting; Genome Editing; Microbial Drug Resistance; Zinc Finger Nuclease