Contribution of Copper Ion Resistance to Survival of Escherichia coli on Metallic Copper Surfaces

ABSTRACT Bacterial contamination of touch surfaces poses a serious threat for public health. The use of bactericidal surface materials, such as copper and its alloys, might constitute a way to aid the use of antibiotics and disinfectants, thus minimizing the risk of emergence and spread of multiresistant germs. The survival of Escherichia coli on metallic copper surfaces has been studied previously; however, the mechanisms underlying bacterial inactivation on copper surfaces have not been elucidated. Data presented in this study suggest that bacteria are killed rapidly on dry copper surfaces. Several factors, such as copper ion toxicity, copper chelators, cold, osmotic stress, and reactive oxygen species, but not anaerobiosis, influenced killing rates. Strains deleted in copper detoxification systems were slightly more sensitive than was the wild type. Preadaptation to copper enhanced survival rates upon copper surface exposure. This study constitutes a first step toward understanding the reasons for metallic copper surface-mediated killing of bacteria.

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Isolation and Characterization of Bacteria Resistant to Metallic Copper Surfaces

Applied and Environmental Microbiology ◽

10.1128/aem.01952-09 ◽

2010 ◽

Vol 76 (5) ◽

pp. 1341-1348 ◽

Cited By ~ 93

Author(s):

Christophe EspÃ¯Â¿Â½rito Santo ◽

Paula Vasconcelos Morais ◽

Gregor Grass

Keyword(s):

Copper Ions ◽

Copper Alloys ◽

Metallic Copper ◽

Copper Ion ◽

Copper Surface ◽

Bacterial Strains ◽

Copper Surfaces ◽

Isolation And Characterization ◽

Detoxification Systems

ABSTRACT Metallic copper alloys have recently attracted attention as a new antimicrobial weapon for areas where surface hygiene is paramount. Currently it is not understood on a molecular level how metallic copper kills microbes, but previous studies have demonstrated that a wide variety of bacteria, including Escherichia coli, Staphylococcus aureus, and Clostridium difficile, are inactivated within minutes or a few hours of exposure. In this study, we show that bacteria isolated from copper alloy coins comprise strains that are especially resistant against the toxic properties exerted by dry metallic copper surfaces. The most resistant of 294 isolates were Gram-positive staphylococci and micrococci, Kocuria palustris, and Brachybacterium conglomeratum but also included the proteobacterial species Sphingomonas panni and Pseudomonas oleovorans. Cells of some of these bacterial strains survived on copper surfaces for 48 h or more. Remarkably, when these dry-surface-resistant strains were exposed to moist copper surfaces, resistance levels were close to those of control strains and MICs for copper ions were at or below control strain levels. This suggests that mechanisms conferring resistance against dry metallic copper surfaces in these newly isolated bacterial strains are different from well-characterized copper ion detoxification systems. Furthermore, staphylococci on coins did not exhibit increased levels of resistance to antibiotics, arguing against coselection with copper surface resistance traits.

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Mechanisms of Contact-Mediated Killing of Yeast Cells on Dry Metallic Copper Surfaces

Applied and Environmental Microbiology ◽

10.1128/aem.01704-10 ◽

2010 ◽

Vol 77 (2) ◽

pp. 416-426 ◽

Cited By ~ 82

Author(s):

Davide Quaranta ◽

Travis Krans ◽

Christophe Espírito Santo ◽

Christian G. Elowsky ◽

Dylan W. Domaille ◽

...

Keyword(s):

Copper Ions ◽

Metallic Copper ◽

Membrane Damage ◽

Ion Homeostasis ◽

Genetic Material ◽

Copper Ion ◽

Yeast Cells ◽

Wild Type ◽

Copper Surfaces ◽

Mutant Cells

ABSTRACTSurfaces made of copper or its alloys have strong antimicrobial properties against a wide variety of microorganisms. However, the molecular mode of action responsible for the antimicrobial efficacy of metallic copper is not known. Here, we show that dry copper surfaces inactivateCandida albicansandSaccharomyces cerevisiaewithin minutes in a process called contact-mediated killing. Cellular copper ion homeostasis systems influenced the kinetics of contact-mediated killing in both organisms. Deregulated copper ion uptake through a hyperactiveS. cerevisiaeCtr1p (ScCtr1p) copper uptake transporter inSaccharomycesresulted in faster inactivation of mutant cells than of wild-type cells. Similarly, lack of theC. albicansCrp1p (CaCrp1p) copper-efflux P-type ATPase or the metallothionein CaCup1p caused more-rapid killing ofCandidamutant cells than of wild-type cells.CandidaandSaccharomycestook up large quantities of copper ions as soon as they were in contact with copper surfaces, as indicated by inductively coupled plasma mass spectroscopy (ICP-MS) analysis and by the intracellular copper ion-reporting dye coppersensor-1. Exposure to metallic copper did not cause lethality through genotoxicity, deleterious action on a cell's genetic material, as indicated by a mutation assay withSaccharomyces. Instead, toxicity mediated by metallic copper surfaces targeted membranes in both yeast species. With the use of Live/Dead staining, onset of rapid and extensive cytoplasmic membrane damage was observed in cells from copper surfaces. Fluorescence microscopy using the indicator dye DiSBaC2(3) indicated that cell membranes were depolarized. Also, during contact-mediated killing, vacuoles first became enlarged and then disappeared from the cells. Lastly, in metallic copper-stressed yeasts, oxidative stress in the cytoplasm and in mitochondria was elevated.

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Reduced Virulence of an fliC Mutant of Shiga-Toxigenic Escherichia coli O113:H21

Infection and Immunity ◽

10.1128/iai.74.3.1962-1966.2006 ◽

2006 ◽

Vol 74 (3) ◽

pp. 1962-1966 ◽

Cited By ~ 16

Author(s):

Trisha J. Rogers ◽

James C. Paton ◽

Hui Wang ◽

Ursula M. Talbot ◽

Adrienne W. Paton

Keyword(s):

Escherichia Coli ◽

Polymorphonuclear Leukocytes ◽

Immunofluorescence Microscopy ◽

Close Association ◽

Survival Rates ◽

Fatal Outcome ◽

Wild Type ◽

Potent Inducer ◽

Gut Colonization ◽

Overall Survival Rates

ABSTRACT The contribution of flagellin to the virulence of the O113:H21 Shiga-toxigenic Escherichia coli (STEC) strain 98NK2 was investigated in the streptomycin-treated mouse model. Groups of mice were challenged with either the wild-type STEC or a fliC deletion derivative thereof. There was no difference in the level of gut colonization by the two strains, but the fliC mutant was significantly less virulent than its parent; the overall survival rates were 43.7% and 81.2%, respectively (P < 0.025). This is the first report of a nontoxic accessory virulence factor contributing to a fatal outcome of STEC infection in this model. Although H21 FliC is known to be a potent inducer of CXC chemokines, including interleukin 8, there was no obvious difference in the recruitment of polymorphonuclear leukocytes to the intestinal epithelium of mice challenged with either strain. However, immunofluorescence microscopy suggested that the fliC mutant was less capable of forming a close association with the colonic epithelium. This may have reduced the uptake of Stx2 by mice infected with the mutant.

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Atomic layer deposition of zirconium oxide on copper patterned silicon substrate

The Journal of Undergraduate Research at the University of Illinois at Chicago ◽

10.5210/jur.v7i1.7526 ◽

2014 ◽

Vol 7 (1) ◽

Author(s):

J. Parulekar ◽

S. Selvaraj ◽

C.G. Takoudis

Keyword(s):

Growth Rate ◽

Atomic Layer Deposition ◽

Copper Oxide ◽

Metallic Copper ◽

Atomic Layer ◽

Copper Surface ◽

Silicon Surfaces ◽

Silicon Substrates ◽

Copper Surfaces ◽

Layer Deposition

Atomic layer deposition (ALD) was performed on copper patterned silicon substrates using zirconium precursor and ethanol as both an oxygen source and reducing agent. Ethanol targeted copper oxide formed on the copper surface, reverting it back to metallic copper. Selective ALD (SALD) of metal oxides on silicon surfaces over copper surfaces has been demonstrated up to 2-3 nm, though the process seems to lose its selectivity afterwards. We strive to maintain selectivity to thicker films by stepping away from conventional ALD processes utilizing oxidants. From previous studies with HfO2 and TiO2 SALD, we speculate that the oxidation of copper to copper oxide spoils selectivity. In this present study, we carried out oxidant-free ALD by using ethanol as a co-reactant solely on the silicon portion of these substrates. This process will occur in-situ every 20-30 ALD cycles for ALD of ZrO2. As expected, reduced ALD growth rate was observed with ethanol compared to that of water or ozone, with a growth rate of about 0.04 nm/cycle on the silicon portion of the substrate.

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Glucose-6-phosphate dehydrogenase and ferredoxin-NADP(H) reductase contribute to damage repair during the soxRS response of Escherichia coli

Microbiology ◽

10.1099/mic.0.28612-0 ◽

2006 ◽

Vol 152 (4) ◽

pp. 1119-1128 ◽

Cited By ~ 51

Author(s):

Mariana Giró ◽

Néstor Carrillo ◽

Adriana R. Krapp

Keyword(s):

Escherichia Coli ◽

Survival Rates ◽

Multicopy Plasmid ◽

Wild Type ◽

Iron Sulfur Clusters ◽

Iron Sulfur ◽

Oxidative Challenge ◽

Glucose 6 Phosphate Dehydrogenase

The NADP(H)-dependent enzymes glucose-6-phosphate dehydrogenase (G6PDH) and ferredoxin(flavodoxin)-NADP(H) reductase (FPR), encoded by the zwf and fpr genes, respectively, are committed members of the soxRS regulatory system involved in superoxide resistance in Escherichia coli. Exposure of E. coli cells to the superoxide propagator methyl viologen (MV) led to rapid accumulation of G6PDH, while FPR was induced after a lag period of several minutes. Bacteria expressing G6PDH from a multicopy plasmid accumulated higher NADPH levels and displayed a protracted soxRS response, whereas FPR build-up had the opposite effects. Inactivation of either of the two genes resulted in enhanced sensitivity to MV killing, while further increases in the cellular content of FPR led to higher survival rates under oxidative conditions. In contrast, G6PDH accumulation over wild-type levels of expression failed to increase MV tolerance. G6PDH and FPR could act concertedly to deliver reducing equivalents from carbohydrates, via NADP+, to the FPR acceptors ferredoxin and/or flavodoxin. To evaluate whether this electron-transport system could mediate reductive repair reactions, the pathway was reconstituted in vitro from purified components; the reconstituted system was found to be functional in reactivation of oxidatively damaged iron–sulfur clusters of hydro-lyases such as aconitase and 6-phosphogluconate dehydratase. Recovery of these activities after oxidative challenge was faster and more extensive in transformed bacteria overexpressing FPR than in wild-type cells, indicating that the reductase could sustain hydro-lyase repair in vivo. However, FPR-deficient mutants were still able to fix iron–sulfur clusters at significant rates, suggesting that back-up routes for ferredoxin and/or flavodoxin reduction might be called into action to rescue inactivated enzymes when FPR is absent.

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Mutant Strains of Escherichia coli and Methicillin-Resistant Staphylococcus aureus Obtained by Laboratory Selection To Survive on Metallic Copper Surfaces

Applied and Environmental Microbiology ◽

10.1128/aem.01788-20 ◽

2020 ◽

Vol 87 (1) ◽

Author(s):

Pauline Bleichert ◽

Lucy Bütof ◽

Christian Rückert ◽

Martin Herzberg ◽

Romeu Francisco ◽

...

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Metallic Copper ◽

Laboratory Evolution ◽

Methicillin Resistant ◽

Content Type ◽

Copper Surfaces ◽

Mutant Strains ◽

Contact Killing ◽

Solid Copper

ABSTRACT Artificial laboratory evolution was used to produce mutant strains of Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) able to survive on antimicrobial metallic copper surfaces. These mutants were 12- and 60-fold less susceptible to the copper-mediated contact killing process than their respective parent strains. Growth levels of the mutant and its parent in complex growth medium were similar. Tolerance to copper ions of the mutants was unchanged. The mutant phenotype remained stable over about 250 generations under nonstress conditions. The mutants and their respective parental strains accumulated copper released from the metallic surfaces to similar extents. Nevertheless, only the parental strains succumbed to copper stress when challenged on metallic copper surfaces, suffering complete destruction of the cell structure. Whole-genome sequencing and global transcriptome analysis were used to decipher the genetic alterations in the mutant strains; however, these results did not explain the copper-tolerance phenotypes on the systemic level. Instead, the mutants shared features with those of stressed bacterial subpopulations entering the early or “shallow” persister state. In contrast to the canonical persister state, however, the ability to survive on solid copper surfaces was adopted by the majority of the mutant strain population. This indicated that application of solid copper surfaces in hospitals and elsewhere has to be accompanied by strict cleaning regimens to keep the copper surfaces active and prevent evolution of tolerant mutant strains. IMPORTANCE Microbes are rapidly killed on solid copper surfaces by contact killing. Copper surfaces thus have an important role to play in preventing the spread of nosocomial infections. Bacteria adapt to challenging natural and clinical environments through evolutionary processes, for instance, by acquisition of beneficial spontaneous mutations. We wish to address the question of whether mutants can be selected that have evolved to survive contact killing on solid copper surfaces. We isolated such mutants from Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA) by artificial laboratory evolution. The ability to survive on solid copper surfaces was a stable phenotype of the mutant population and not restricted to a small subpopulation. As a consequence, standard operation procedures with strict hygienic measures are extremely important to prevent the emergence and spread of copper-surface-tolerant persister-like bacterial strains if copper surfaces are to be sustainably used to limit the spread of pathogenic bacteria, e.g., to curb nosocomial infections.

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Role ofrpoSin Escherichia coli O157:H7 Strain H32 Biofilm Development and Survival

Applied and Environmental Microbiology ◽

10.1128/aem.02149-12 ◽

2012 ◽

Vol 78 (23) ◽

pp. 8331-8339 ◽

Cited By ~ 16

Author(s):

Jessica R. Sheldon ◽

Mi-Sung Yim ◽

Jessica H. Saliba ◽

Wai-Hong Chung ◽

Kwok-Yin Wong ◽

...

Keyword(s):

Escherichia Coli ◽

Lake Water ◽

Biofilm Formation ◽

Survival Rates ◽

Confocal Scanning Laser Microscopy ◽

Wild Type ◽

Minimal Growth ◽

Content Type ◽

Biofilm Development

ABSTRACTThe protein RpoS is responsible for mediating cell survival during the stationary phase by conferring cell resistance to various stressors and has been linked to biofilm formation. In this study, the role of therpoSgene inEscherichia coliO157:H7 biofilm formation and survival in water was investigated. Confocal scanning laser microscopy of biofilms established on coverslips revealed a nutrient-dependent role ofrpoSin biofilm formation, where the biofilm biomass volume of therpoSmutant was 2.4- to 7.5-fold the size of itsrpoS+wild-type counterpart in minimal growth medium. The enhanced biofilm formation of therpoSmutant did not, however, translate to increased survival in sterile double-distilled water (ddH2O), filter-sterilized lake water, or unfiltered lake water. TherpoSmutant had an overall reduction of 3.10 and 5.30 log10in sterile ddH2O and filter-sterilized lake water, respectively, while only minor reductions of 0.53 and 0.61 log10in viable counts were observed for the wild-type form in the two media over a 13-day period, respectively. However, the survival rates of the detached biofilm-derivedrpoS+andrpoSmutant cells were comparable. Under the competitive stress conditions of unfiltered lake water, the advantage conferred by the presence ofrpoSwas lost, and both the wild-type and knockout forms displayed similar declines in viable counts. These results suggest thatrpoSdoes have an influence on both biofilm formation and survival ofE. coliO157:H7 and that the advantage conferred byrpoSis contingent on the environmental conditions.

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