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Persistence of recombinant bacteria to antimicrobial silver

Niu, Aping. Persistence of recombinant bacteria to antimicrobial silver. 2014, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_11067

Abstract

Silver, owing to its effective antimicrobial properties, has been used against a broad range of microorganisms. Silver is now utilized commonly in numerous consumer products, medical devices and clinical applications. However, the mechanism of action of the silver is not yet fully established and well-understood. In addition, it is also important to understand the biochemical and evolutionary pathways that give rise to resistance. Here, we report new genetic determinants for silver resistance in E. coli and explore aspects of their mechanism and laboratory evolution.
Initial exploration of the antimicrobial activity of silver showed that (1) antimicrobial ability of silver is time and dose-dependent; (2) Ag ions have much more antibiotic activity than silver nanoparticles (AgNPs) and (3) the antimicrobial ability of AgNPs is size-dependent. Further selection for resistance genes of E. coli using AgNO3 and AgNPs led to the identification of several candidates, including cysD and ycdB, which displayed cross-resistance to Ag ion and AgNPs as well as Cu+ and Cd2+. The genes cysD and ycdB conferred less resistance to metallic Ag(0) under anaerobic incubation than aerobic incubation. These results support that Ag+ ions are the main toxic agents of AgNPs. These novel anti-silver genes also endowed resistance to the antibiotics kanamycin and ampicillin; in these experiments, antibacterial synergy between kanamycin and silver, but not between ampicillin and silver, was also found. Quantification of oxygen radicals suggest that silver ion is bactericidal through production of reactive oxygen species and that silver-resistance genes prevent their generation.
The selected gene ycdB and control gene cueO, both of which led to increased silver resistance, encode Tat-dependent proteins, which are transported after folding from cytoplasm to periplasm. Chapter 2 focuses on several Tat-containing genes, which also gave more resistance to Ag ion. The 7 selected Tat sequence genes, including torA, yedY, sufI, ycdO and hybA, were recombinantly expressed in various truncated forms, showing that for ycdB and yedY deleting Tat sequences impaired export and silver-resistance ability, despite increased expression, but that for other Tat genes deleting Tat had little effect on either periplasmic translocation or resistance. In all cases, expression of the Tat export sequence alone or with the his-tag in absence of the gene led to suppression of resistance.
Finally, we explored the evolvability of selected genes, such as yeaO, ydgT, iscA and ycdB for silver-resistance. Evolved mutants of yeaO and ydgT were found that endowed increased resistance to silver compared to wildtypes. In these two cases, increased resistance to silver did not lead to increased antibiotic resistance. In short, several kinds of anti-silver genes were identified in our studies, showing various pathways rendering resistance to silver. Weak resistance functions for some genes were evolvable. Our studies provide a deeper insight into silver’s mechanism of action and of the possible resistance pathways in bacteria, which may in some cases lead also to cross-resistance to antibiotics.
Advisors:Ward, Thomas R.
Committee Members:Pfohl, Thomas
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie > Bioanorganische Chemie (Ward)
Item Type:Thesis
Thesis no:11067
Bibsysno:Link to catalogue
Number of Pages:77 p.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:56
Deposited On:23 Dec 2014 10:09

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