Article

Mechanism of copper surface toxicity in Escherichia coli O157:H7 and Salmonella involves immediate membrane depolarization followed by slower rate of DNA destruction which differs from that observed for Gram-positive bacteria

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Abstract

We have reported previously that copper I and II ionic species, and superoxide but not Fenton reaction generated hydroxyl radicals, are important in the killing mechanism of pathogenic enterococci on copper surfaces. In this new work we determined if the mechanism was the same in non-pathogenic ancestral (K12) and laboratory (DH5α) strains, and a pathogenic strain (O157), of Escherichia coli. The pathogenic strain exhibited prolonged survival on stainless steel surfaces compared with the other E. coli strains but all died within 10 min on copper surfaces using a 'dry' inoculum protocol (with approximately 10(7)  cfu cm(-2) ) to mimic dry touch contamination. We observed immediate cytoplasmic membrane depolarization, not seen with enterococci or methicillin resistant Staphylococcus aureus, and loss of outer membrane integrity, inhibition of respiration and in situ generation of reactive oxygen species on copper and copper alloy surfaces that did not occur on stainless steel. Chelation of copper (I) and (II) ionic species still had the most significant impact on bacterial survival but protection by d-mannitol suggests hydroxyl radicals are involved in the killing mechanism. We also observed a much slower rate of DNA destruction on copper surfaces compared with previous results for enterococci. This may be due to protection of the nucleic acid by the periplasm and the extensive cell aggregation that we observed on copper surfaces. Similar results were obtained for Salmonella species but partial quenching by d-mannitol suggests radicals other than hydroxyl may be involved. The results indicate that copper biocidal surfaces are effective for Gram-positive and Gram-negative bacteria but bacterial morphology affects the mechanism of toxicity. These surfaces could not only help to prevent infection spread but also prevent horizontal gene transmission which is responsible for the evolution of virulent toxin producing and antibiotic resistant bacteria.

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... The first and crucial step consists in the dissolution of copper ions from the surface and their accumulation in the small aqueous space between the material surface and bacterial membrane, reaching the mM range [19,20]. These copper ions can lead to (i) the generation of reactive oxygen species (ROS) [21], (ii) the inhibition of the respiratory chain [22], (iii) lipid peroxidation [23,24]/damages to cell membrane [24][25][26][27], (iv) DNA degradation [28,29], (v) modified protein expression [30], and (vi) displacement of iron-sulfur clusters and inactivation of metalloproteins [31,32] (Figure 1). The actual mechanism(s) as well as the precise order in which these mechanisms are involved in copper-mediated contact killing is expected to be dependent on factors such as the bacterial physiology and environmental conditions (e.g., presence of moisture or buffering/proteinaceous agents). ...
... As cell wall/membrane is the bacterial part directly in contact with the surface, it is a likely target when one is looking to a rapid antimicrobial mechanism of action. Indeed, membrane damages have been described for a wide range of microorganisms in contact with copper-containing surfaces [25][26][27]42]. However, a few reports failed to register copper surface-induced membrane alterations [43]. ...
... The binding of cuprous and/or cupric ions to lipopolysaccharide, peptidoglycan and/or carboxylic groups in the bacterial membrane can also induce membrane depolarization [45]. Membrane depolarization is indeed thought to be the main mechanism of bactericidal activity by some authors [25,27]. This phenomenon leads to a leakage of cytoplasmic content and ultimately to the complete rupture of the membrane and cell death. ...
Article
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Copper has been used for its antimicrobial properties since Antiquity. Nowadays, touch surfaces made of copper-based alloys such as brasses are used in healthcare settings in an attempt to reduce the bioburden and limit environmental transmission of nosocomial pathogens. After a brief history of brass uses, the various mechanisms that are thought to be at the basis of brass antimicrobial action will be described. Evidence shows that direct contact with the surface as well as cupric and cuprous ions arising from brass surfaces are instrumental in the antimicrobial effectiveness. These copper ions can lead to oxidative stress, membrane alterations, protein malfunctions, and/or DNA damages. Laboratory studies back up a broad spectrum of activity of brass surfaces on bacteria with the possible exception of bacteria in their sporulated form. Various parameters influencing the antimicrobial activity such as relative humidity, temperature, wet/dry inoculation or wear have been identified, making it mandatory to standardize antibacterial testing. Field trials using brass and copper surfaces consistently report reductions in the bacterial bioburden but, evidence is still sparse as to a significant impact on hospital acquired infections. Further work is also needed to assess the long-term effects of chemical/physical wear on their antimicrobial effectiveness.
... With respect to the ionic forms of copper, Cu + (cuprous ion) is seen to be more bactericidal than the cupric ion (Cu 2+ ) due to its higher penetration ability into the bacterial membranes [2]. The most distinguishing feature of metallic copper or alloyed copper surfaces, including Cu-NPs, is their ability to achieve very high killing efficacy of microbes in close contact within a short time [75,82,95,119,177,178]. This phenomenon, called "contact-killing", is particularly accelerated under "dry" conditions (a few to several minutes) compared to the wet conditions (several hours) [95,179]. ...
... When the copper ions bind to negatively charged domains on the bacteria cell membrane, the potential difference decreases, and depolarisation can occur. When the potential difference reaches zero, membrane weakness or rupture can happen [95,177]. The bindings of copper ions at the peptidoglycans or on the lipopolysaccharide carboxyl In support of this, it is reported that when E. coli cells were applied on copper coupons, a large number of copper ions were taken by E. coli within 90 min, and the absorption is found to be more when the bacteria plated on the copper surface by dry method. ...
... Membrane polarisation is considered a mechanism by which copper ions can exert a toxic effect on bacteria [95,177]. It is thought that metabolically active bacteria have an electrical potential difference (~100-200 mV) between the inside and outside of the cell that can vary with species. ...
Article
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Pathogen transfer and infection in the built environment are globally significant events, leading to the spread of disease and an increase in subsequent morbidity and mortality rates. There are numerous strategies followed in healthcare facilities to minimize pathogen transfer, but complete infection control has not, as yet, been achieved. However, based on traditional use in many cultures, the introduction of copper products and surfaces to significantly and positively retard pathogen transmission invites further investigation. For example, many microbes are rendered unviable upon contact exposure to copper or copper alloys, either immediately or within a short time. In addition, many disease-causing bacteria such as E. coli O157:H7, hospital superbugs, and several viruses (including SARS-CoV-2) are also susceptible to exposure to copper surfaces. It is thus suggested that replacing common touch surfaces in healthcare facilities, food industries, and public places (including public transport) with copper or alloys of copper may substantially contribute to limiting transmission. Subsequent hospital admissions and mortality rates will consequently be lowered, with a concomitant saving of lives and considerable levels of resources. This consideration is very significant in times of the COVID-19 pandemic and the upcoming epidemics, as it is becoming clear that all forms of possible infection control measures should be practiced in order to protect community well-being and promote healthy outcomes.
... Another feature of the antibacterial activity of copper and its alloys is that only a few copper-resistant bacteria have been reported. This may be related to the degradation of bacterial nucleic acids observed on copper and its alloy surfaces [12][13][14][15][16][17][18]. Bacteria have the ability of horizontal gene transfer (HGT), which can occur on touch surfaces. ...
... For its further utilization and optimization, it is important to appropriately evaluate the nucleic acids degradability of materials as well as to investigate its mechanism. In order to evaluate the fragmentation of bacterial genes, electrophoresis of nucleic acids extracted from the bacteria applied onto the material surface is mostly performed [12,[14][15][16][17][18], but a quantitative evaluation is difficult since bacterial numbers will change at the material surface depending on its antibacterial activity. There is no quantitative evaluation method of a material's ability to degrade nucleic acids on its surface. ...
... Dominancy or sequences among these processes remains unclear; it may be different depending on the type of microorganisms [4]. As described before, the degradation of bacterial genes is considered as beneficial to inhibit HGT of antibiotic-resistant genes and confirmed by electrophoresis of nucleic acids extracted from bacteria applied to the copper surface [12][13][14][15][16][17][18]. However, this method has a limitation on its quantitativeness since the amount of extracted nucleic acids will be influenced by the difference in the number of viable cells recovered from the copper and the reference material. ...
Article
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Copper (Cu) and its alloys have bactericidal activity known as “contact killing” with degradation of nucleic acids inside the bacteria, which is beneficial to inhibit horizontal gene transfer (HGF). In order to understand the nucleic acid degradability of Cu and its alloy surfaces, we developed a new in vitro method to quantitatively evaluate it by a swab method under a “dry” condition and compared it with that of commercially available antibacterial materials such as antibacterial stainless steel, pure silver, and antibacterial resins. As a result, only Cu and its alloys showed continuous degradation of nucleic acids for up to 6 h of contact time. The nucleic acid degradability levels of the Cu alloys and other antibacterial materials correlate to their antibacterial activities evaluated by a film method referring to JIS Z 2801:2012 for Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. Nucleic acid degradation by copper (I) and (II) chlorides was confirmed at the ranges over 10 mM and 1–20 mM, respectively, suggesting that the copper ion release may be responsible for the degradation of the nucleic acids on Cu and its alloy surfaces. In conclusion, the higher Cu content in the alloys gave higher nucleic acid degradability and higher antibacterial activities.
... 82012-1 through -6). Dry copper surfaces showed an antimicrobial effect of up to 7 log 10 steps against several types of MoV in laboratory experiments (Molteni et al. 2010;Warnes et al. 2012;Bleichert et al. 2014). ...
... When titanium discs were coated with amorphous hydrocarbon film containing copper nanoparticles, the experiments yielded up to 4 log 10 steps reduction of E. coli and S. aureus after 24 h incubation (Thukkaram et al. 2021). Regardless of the respective mechanism, it was observed that continuous copper ion release is essential for the efficacy of copper surfaces and cleaning protocols must be able to remove any substances on coated surfaces that may chelate released ions (Warnes et al. 2012). ...
... To quantify the efficacy, the most common procedure is the plating of microorganisms on appropriate agar plates and counting the colony-forming units (cfu) (Warnes et al. 2012;Eichner et al. 2020), whereas a future application might be growth curves of bacteria allowing a high throughput of samples in solution or even of biomaterials itself in the form of so-called proliferation assays (Bechert et al. 2000). For the two mentioned unconventional methods, a bacterial culture with known cell density is applied to an AMC surface and a reference surface. ...
Article
Recent reports provide evidence that contaminated healthcare environments represent major sources for the acquisition and transmission of pathogens. Antimicrobial coatings (AMC) may permanently and autonomously reduce the contamination of such environmental surfaces complementing standard hygiene procedures. This review provides an overview of the current status of AMC and the demands to enable a rational application of AMC in health care settings. Firstly, a suitable laboratory test norm is required that adequately quantifies the efficacy of AMC. In particular, the frequently used wet testing (e.g. ISO 22196) must be replaced by testing under realistic, dry surface conditions. Secondly, field studies should be mandatory to provide evidence for antimicrobial efficacy under real-life conditions. The antimicrobial efficacy should be correlated to the rate of nosocomial transmission at least. Thirdly, the respective AMC technology should not add additional bacterial resistance development induced by the biocidal agents and co- or cross-resistance with antibiotic substances. Lastly, the biocidal substances used in AMC should be safe for humans and the environment. These measures should help to achieve a broader acceptance for AMC in healthcare settings and beyond. Technologies like the photodynamic approach already fulfil most of these AMC requirements.
... Therefore, search for a strong alternative candidate that can kill or inhibit multidrug resistance microbes is needed [88]. Metal nanoparticles (MNPs) are known to possess potent antimicrobial activity against a wide variety of microbes, including bacteria (Gram-negative and Gram-positive) and fungi, via their photodynamic effects and strong oxidative stress [89]. Furthermore, the direct physical contact of MNPs to bacterial membrane results in the release of intercellular material, loss of cell membrane integrity, and cell death [88]. ...
... In literature, several reports are offered on MNPs' (AgNPs, AuNPs, ZnSNPs) exhibiting bactericidal activity. However, only a few reports focus on the support of their antifungal and antibacterial activity with appropriate mechanisms [88][89][90][91]. Moreover, many reports available in literature that favor the mechanism involved in bacterial and fungi cells are almost similar. ...
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Research and innovation in nanoparticles (NPs) synthesis derived from biomaterials have gained much attention due to their unique characteristics, such as low-cost, easy synthesis methods, high water solubility, and eco-friendly nature. NPs derived from macrofungi, including various mushroom species, such as Agaricus bisporus, Pleurotus spp., Lentinus spp., and Ganoderma spp. are well known to possess high nutritional, immune-modulatory, antimicrobial (antibacterial, antifungal and antiviral), antioxidant, and anticancerous properties. Fungi have intracellular metal uptake ability and maximum wall binding capacity; because of which, they have high metal tolerance and bioaccumulation ability. Primarily, two methods have been comprehended in the literature to synthesize metal NPs from macrofungi, i.e., the intracellular method, which refers to NP synthesis inside fungal cells by transportation of ions in the presence of enzymes; and the extracellular method, which refers to the treatment of fungal biomolecules aqueous filtrate with a metal precursor. Pleurotus derived metal NPs are known to inhibit the growth of numerous foodborne pathogenic bacteria and fungi. To the best of our knowledge, there is no such review article reported in the literature describing the synthesis and complete application and mechanism of NPs derived from macrofungi. Herein, we intend to summarize the progressive research on macrofungi derived NPs regarding their synthesis as well as applications in the area of antimicrobial (antibacterial & antifungal), anticancer, antioxidant, catalytic and food preservation. Additionally, the challenges associated with NPs synthesis will also be discussed.
... Several studies have demonstrated the antimicrobial capacity of copper and its alloys to bacteria, fungi, and viruses [16,17,18,19,20]. The mechanisms by which this occurs are not fully understood, but some studies indicate that the copper surface eliminates bacteria by an attack consisting of three mechanisms: bacterial membrane damage, DNA degradation and intracellular damage inside the membrane plasma [21,22,20]. ...
... Several studies have demonstrated the antimicrobial capacity of copper and its alloys to bacteria, fungi, and viruses [16,17,18,19,20]. The mechanisms by which this occurs are not fully understood, but some studies indicate that the copper surface eliminates bacteria by an attack consisting of three mechanisms: bacterial membrane damage, DNA degradation and intracellular damage inside the membrane plasma [21,22,20]. The survival of E. coli O157 on seven 1 cm x 1 cm copper plates with different degrees of purity has recently been evaluated and compared with stainless steel. ...
Article
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— The consumption of sprouts in the human diet has grown during the last years, but great concern raised from public health institutions, food industry and consumers regarding their safety since foodborne diseases caused by microorganisms have been reported. Copper metal as a contact surface was studied during the germination of alfalfa seeds (Medicago sativa L.) inside a rotating drum on a laboratory scale and compared with a plastic surface of food-grade. A system of three rotating drums was used inside a thermo-regulated chamber to germinate seeds. To evaluate the antibacterial activity of copper sheets, alfalfa seeds were inoculated with 4.2 log cfu g-1 of Escherichia coli and after 84 hours of germination sprouts were evaluated for E. coli, mesophilic aerobic bacteria, the content of copper and other minerals (potassium, calcium, magnesium, sodium, iron, manganese, and zinc), total mass, unit mass and length, and color. The contact of alfalfa sprouts with copper sheets allowed to reduce the E. coli load from 6.54 to <0.1 log cfu g-1. However, all sprouts exceeded in copper (> 10 ppm) according to Food Sanitary Regulations. Germinated mass and length decreased after copper treatments. No statistically significant differences were observed between treatments for the remaining quality parameters. Finally, it is concluded that copper was very efficient in reducing the microbial load of E. coli in alfalfa sprouts, complying with the regulations established by the Chilean Ministry of Health.
... The absorption of Cu ions and the increasing development of ROS have been linked to the loss of bacterial cell viability. Surfaces of copper do not produce additional oxidative nucleic acid damage (Warnes et al., 2012). However, nucleic acids were released from damaged cells +that increases as the Cu content of the surface alloy decreases. ...
... Other metallic nanoparticles have antimicrobial potential, including (Al -Fe-Mg-Ni-Pa-Pl and Ti). Because most experiments have focused on bacterial activity, there are only a few reports on the antibacterial mechanism of metal nanoparticles (Ravishankar & Jamuna., 2011; Warnes et al., 2012;Chiriac et al., 2016). The role of these nanoparticles as antifungal agents were being debated at the moment. ...
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Nanotechnology has captured the attention of researchers in all basic sciences, including physics, chemistry, and biology. It is concerned with particle size range from 1-100 nanometers. In this review they give a brief description about Nanotechnology in Iraq, Types of synthesis, study the characterization and its application in various fields viz. medicine, industry, agriculture, electronic and in the oil field. Nanoparticles have been used in a variety of sectors of research and technology in recent years, ranging from material science to microbiology. As a result, nanoparticle synthesis can be considered an active area in nanoparticle research and application. Physical, chemical, and biological methods of nanoparticle synthesis are all available. The biological approach is the most effective of these.
... Copper ions cause damage to the outer or/and an inner membrane that can lead to the outflow of intracellular components like glutamate which affects cell apoptosis and potassium content [39]. Most of the studies agree that the primary attack of copper is the cell membrane in bacteria [16,40]. The microbial membranes contain polymers with electronegative chemical groups that serve as spots for the absorption of metal cations [41]. ...
... Copper can kill undesirable viruses and bacteria by doing physical contact with them (see Figure 2a). Most of the studies agree that the primary target of attack of copper is the cell wall in the bacteria [16,40]. This is attributed to the presence of polymers with highly electronegative chemical groups (e.g., peptidoglycan, phospholipid, teichoic or teichuronic acids, and lipoteichoic acid groups) on the bacterial membrane that serves as sites for absorption of metal cations. ...
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Microbial contamination of medical devices and treatment rooms leads to several detrimental hospital and device-associated infections. Antimicrobial copper coatings are a new approach to control healthcare-associated infections (HAI’s). This review paper focuses on the efficient methods for depositing highly adherent copper-based antimicrobial coatings onto a variety of metal surfaces. Antimicrobial properties of the copper coatings produced by various deposition methods including thermal spray technique, electrodeposition, electroless plating, chemical vapor deposition (CVD), physical vapor deposition (PVD), and sputtering techniques are compared. The coating produced using different processes did not produce similar properties. Also, process parameters often could be varied for any given coating process to impart a change in structure, topography, wettability, hardness, surface roughness, and adhesion strength. In turn, all of them affect antimicrobial activity. Fundamental concepts of the coating process are described in detail by highlighting the influence of process parameters to increase antimicrobial activity. The strategies for developing antimicrobial surfaces could help in understanding the mechanism of killing the microbes.
... CuONPs activity is known to be dependent on bacterial species, and its antibacterial effect also takes place by membrane damage and ROS production (Lemire et al., 2013;Beyth et al., 2015), which revealed that the loss of viability of bacterial cell has been related employing the uptake of copper ions and enlarged construction of reactive oxygen species. Other authors observed that the surfaces of copper do not make major oxidative DNA damage in vivo and various other proof recommend that DNA damage is not the main reason for copper-mediated surface killing (Warnes et al., 2012). ...
... CuONPs activity is known to be dependent on bacterial species, and its antibacterial effect also takes place by membrane damage and ROS production (Lemire et al., 2013;Beyth et al., 2015), which revealed that the loss of viability of bacterial cell has been related employing the uptake of copper ions and enlarged construction of reactive oxygen species. Other authors observed that the surfaces of copper do not make major oxidative DNA damage in vivo and various other proof recommend that DNA damage is not the main reason for copper-mediated surface killing (Warnes et al., 2012). DNA and RNA released from dead cells are slowly corrupted at a rate that enhances employing the copper content of the surface alloy. ...
... Fluorescence probes-microscopy or fluorometry: Live/Dead staining-SYTO9 (green) is able to penetrate the intact cell membrane and propidium iodide stains in red cells with a damaged envelope [37,38] Labeling intracellular copper ions (CS1 sensor) [29] Inductively coupled plasma measurement of intracellular copper [39] Membrane depolarization-using fluorescent probes as rhodamine and BacLight Bacterial Membrane Potential Kit containing oxa-(DiO) carbocyanine dye [40] Respiration activity-CTC (5-cyano-2,3-ditolyl tetrazolium chloride) redox dye [41] ...
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Biofilms are structures comprising microorganisms associated to surfaces and enclosed by an extracellular polymeric matrix produced by the colonizer cells. These structures protect microorganisms from adverse environmental conditions. Biofilms are typically associated with several negative impacts for health and industries and no effective strategy for their complete control/eradication has been identified so far. The antimicrobial properties of copper are well recognized among the scientific community, which increased their interest for the use of these materials in different applications. In this review the use of different copper materials (copper, copper alloys, nanoparticles and copper-based coatings) in medical settings, industrial equipment and plumbing systems will be discussed considering their potential to prevent and control biofilm formation. Particular attention is given to the mode of action of copper materials. The putative impact of copper materials in the health and/or products quality is reviewed taking into account their main use and the possible effects on the spread of antimicrobial resistance.
... Several studies have demonstrated the antimicrobial capacity of copper and its alloys to bacteria, fungi, and viruses (Wilks et al., 2005;Noyce et al., 2006;Wilks et al., 2006;Grass et al., 2011;Warnes et al., 2012). The mechanisms by which this occurs are not fully understood, but some studies indicate that the copper surface eliminates bacteria by an attack consisting of three mechanisms: bacterial membrane damage, DNA degradation and intracellular damage inside the membrane plasma (Santo et al., 2008;Santo et al., 2011). ...
Article
Full-text available
Copper metal as a contact surface was studied during the germination of alfalfa seeds (Medicago sativa L.) inside a rotating drum on a laboratory scale and compared with a plastic surface of food-grade. A system of three rotating drums was used inside a thermo-regulated chamber to germinate seeds. Alfalfa seeds were inoculated with 4.2 log cfu g-1 of Escherichia coli and after 84 h of germination sprouts were evaluated for E. coli, mesophilic aerobic bacteria, the content of copper and other minerals (potassium, calcium, magnesium, sodium, iron, manganese, and zinc), total mass, unit mass and length, and color. The contact of alfalfa sprouts with copper sheets allowed to reduce the E. coli load from 6.54 to <0.1 log cfu g-1. However, all sprouts exceeded in copper (> 10 ppm) according to Food Sanitary Regulations. Germinated mass and length decreased after copper treatments. No statistically significant differences were observed between treatments for the remaining quality parameters. Finally, it is concluded that copper was very efficient in reducing the microbial load of E. coli in alfalfa sprouts, complying with the regulations established by the Chilean Ministry of Health.
... When the bacteria were in contact with the microdomain of such a potential difference, the cathodic electrons would consume H + due to the electrochemical potential difference, which is responsible for the bacterial charge transfer process, blocking the respiratory metabolism and energy production of bacteria [51][52][53], as shown in Figure 1. In addition, the overproduction of ROS by the synergistic effect of H + depletion and Cu ions burst release due to the consumption of cathodic electrons would further affect the bio-activities of bacteria, such as membrane damage [54,55], DNA degradation and so on [56]. It is worth noting that since the charge transfer for H + depletion between the cell and substratum is a short-range interaction [57], a sufficient contact between a Cu-bearing stainless steel and bacteria is the key to its antibacterial performance. ...
Article
Metals play an essential role in biochemistry, and the use of metals for antimicrobial applications has been well studied. Biomaterials incorporating copper and silver demonstrated high antimicrobial activity while maintaining good cytocompatibility. In this overview, nanostruc-ture processing is underscored as a method to overcome the shortcomings of conventional bioceramics. Restructuring crystalline structures at the nanoscale make it less susceptible to brittle fracture. Furthermore, the bioactivity of the material is significantly improved through nanoscale surface modifications such as coatings. The effectiveness of nanostructured bioma-terials featuring copper and silver in combatting bacterial attachment and proliferation is described. Nanoscale copper-containing stainless steel achieved antimicrobial activity by disrupting bacterial metabolism and damaging bacterial membranes while providing a conducive environment for cellular proliferation. Copper-containing stainless steels can be used to prevent marine corrosion, food preservation and combat implant-related infections. It is envisaged that future antimicrobial strategies will continue to feature copper and silver nanostructured biomaterials prominently. ARTICLE HISTORY
... 3−6 Although not yet fully understood and dependent on several factors (physical form, Cu oxidation state, form of application, pH, among others), 7 the antibacterial activity of Cu is based on two main mechanisms. When the metal is delivered as Cu 2+ , the membrane depolarization mode of action prevails, 8 while in the presence of Cu-nanoparticles the generation of Reactive Oxygen Species (ROS) seems dominant. 9 The effectiveness of Cu-based compounds has led to their extensive use to control foliar pathogens worldwide, especially in organic farming, where the use of many conventional pesticides is forbidden. ...
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Despite its high chemical value, most of lignin is nowadays burnt as low value fuel. It is therefore important to find innovative applications for its use. Copper compounds are used as plant protection products for more than 50 different diseases in viticulture, arable crops, hops, and horticulture, and they have been used for more than 100 years. Minimization of copper in agriculture has become a fundamental issue due to its negative environmental impact. Here we present a series of hybrid organic-inorganic materials ([email protected]), deriving from the combination of lignin with brochantite, Cu4(OH)6SO4. Optimization of the synthetic procedures has allowed us to isolate lignin-based materials containing different percentages of copper, where the brochantite crystals are featured by different morphologies and dimensions. A more environmentally safe synthesis of [email protected] materials by mechanochemistry is also investigated, which reduces the amount of water used and makes easier and faster the isolation of the final materials. Tests on strawberry and tomato plants in a greenhouse have highlighted a significative efficacy of the [email protected] materials against different pathogens at a copper content much lower than the one of copper-based commercial pesticides. A crystal morphology-activity correlation is also traced out. The synergic activity of lignin and copper ions can be used to reduce the copper content for efficient pathogen control. Moreover, the mechanochemical approach ensures a greener synthetic approach, in a perspective of a more sustainable agriculture.
... However, heterojunction materials (C1BP, C3BP and C5BP) show antibacterial ability on both two bacteria [70], and the inhibition rate is directly proportional to the Cu 2 O content. The main suggested reason of J Mater Sci copper activity against pathogens relates to the ability of Cu 2 O to penetrate through the bacterial cell wall or outer membrane and bind to DNA, thereby blocking the cell replication process [71][72][73][74][75]. By comparing the antibacterial abilities, we can find that the inhibition rate of heterogeneous junction material to E. coli is generally higher than staphylococcus aureus, which shows that heterojunction material has a better inhibition effect on E. coli than S. aureus. ...
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Novel p–n heterojunction of Cu2O/Bi2O2CO3 photocatalysts with various proportion of Cu2O are synthesized by the precipitation method at room temperature, which are then combined with electrospinning method and electron beam irradiation to construct a photocatalyst-polymer structure. The results indicated that the Cu2O/Bi2O2CO3 photocatalyst obtained, in contrast to the pure Bi2O2CO3, enabled improved visible-light absorption, which can separate and migrate the charge carriers efficiently via solid p–n heterojunction interfacial effect. Accordingly, the Cu2O/Bi2O2CO3/PEO nanofibers allowed for the higher visible-light-responsive photocatalytic activity for the degradation of chloramphenicol (CAP), which the highest degradation efficiency is 98.2% for CAP in 30 min and obviously higher than that of Bi2O2CO3 membrane. In addition, combining cytotoxicity experiments with LC-IT-QTOF, it is found that intermediates such as DH-CAP and NO-CAP produced during the degradation of CAP are main possible reasons for the increase in the solution cytotoxicity. Besides, the obtained materials show significant inhibition against Escherichia coli and Staphylococcus aureus with the maximum inhibition rates of 81.5% and 75.6%, respectively.
... Mid log (OD 600~0 .5 i.e., 10 7 -10 8 cfu/mL) cells (0.5 mL) were suspended in same volume of 1% glucose-saline solution and treated with HC for 1 h. After 1 h cells were washed, resuspended in 1% glucose-saline solution and stained with 1 mg/mL Rhodamine 123 for 10 min in the dark [24]. Excess stain was removed by centrifugation and the fluorescence was quantified in a plate reader with Ex 488 nm and Em 530 nm. ...
Preprint
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Hydroxychavicol (HC), found abundantly in Piper betle leaves is credited with antimicrobial property. Previously we had shown HC induces reactive oxygen species mediated DNA damage in bacterial cells. HC also resulted in membrane compromise revealing its pleiotropic effects on cellular targets. The ki-netics and exact sequence of events leading to inhibition of growth and cell death in E. coli after HC treatment remains poorly understood. We show that sub-lethal concentration (125 mg/mL) of HC causes cellular filamentation within 1 h of treatment, while a higher concentration (750 mg/mL) induces cell breakage. HC-treated cells were found to experience oxidative stress as early as 10 min, while evidence of membrane damage was apparent at 30 min. DNA damage repair genes were found to be activated at 60 min. Interestingly, HC-induced cell permeabilization was inhibited and enhanced by external Mg 2þ and EDTA, respectively, suggesting that HC damages the outer membrane. Kinetic experiments revealed that HC-treated cells underwent oxidative stress, membrane damage and DNA damage in that order. Because gram negative bacteria such as E. coli are refractory to several antibiotics due to the presence of the outer membrane, we hypothesized that HC pretreatment would sensitize E. coli to hydrophobic antibiotics. Our study reveals for the first time that HC could sensitize bacteria to clinically used antibiotics due to its outer membrane damaging property.
... Damage to the membranes or to DNA accrues by oxidative stress events, depending on the presence of glutathione (27), or by massive overload of the cell by dissolved copper ions (11,28). Physiologically, respiration ceases due to depletion of concentration gradients across the compromised membranes (29), and replication is prevented because genomes become fragmented beyond repair (28). These events may be followed by a complete breakdown of cellular structural integrity (11,12). ...
Article
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.
... This indicates that the antibacterial mechanism of Cu should be different from that of the high pH value. It has previously been shown that Cu ions exert an antimicrobial activity, leading to increased influx of Cu 2+ into bacteria and generation of reactive oxygen species, which results in inhibition of respiration and degradation of DNA and loss of cytoplasm [43]. ...
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Periodontal diseases are mainly the results of infections and inflammation of the gum and bone that surround and support the teeth. In this study, the alveolar bone destruction in periodontitis is hypothesized to be treated with novel Mg-Cu alloy grafts due to their antimicrobial and osteopromotive properties. In order to study this new strategy using Mg-Cu alloy grafts as a periodontal bone substitute, the in vitro degradation and antibacterial performance were examined. The pH variation and Mg2+ and Cu2+ release of Mg-Cu alloy extracts were measured. Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), two common bacteria associated with periodontal disease, were cultured in Mg-Cu alloy extracts, and bacterial survival rate was evaluated. The changes of bacterial biofilm and its structure were revealed by scanning electron microscopy (SEM) and transmission electronic microscopy (TEM), respectively. The results showed that the Mg-Cu alloy could significantly decrease the survival rates of both P. gingivalis and A. actinomycetemcomitans. Furthermore, the bacterial biofilms were completely destroyed in Mg-Cu alloy extracts, and the bacterial cell membranes were damaged, finally leading to bacterial apoptosis. These results indicate that the Mg-Cu alloy can effectively eliminate periodontal pathogens, and the use of Mg-Cu in periodontal bone grafts has a great potential to prevent infections after periodontal surgery.
... Several modes of action have been put forward, although their sequence and interactions remain unclear. The production of reactive oxygen species can be catalyzed by Cu via a Fenton-like reaction in vitro, but contrasting data have been reported regarding its role in DNA degradation in vivo (Macomber et al., 2007;Tian et al., 2012;Warnes et al., 2012;Cui et al., 2014;San et al., 2015). The inactivation of Fe-S proteins by competition of Cu with Fe in an oxygen-independent manner has been reported (Macomber and Imlay, 2009;Arguello et al., 2013), as well as membrane damage via lipid peroxidation (Espirito Santo et al., 2011;Hong et al., 2012;Santo et al., 2012). ...
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Metallic copper to combat bacterial proliferation in drinking water systems is being investigated as an attractive alternative to existing strategies. A potential obstacle to this approach is the induction of metal resistance mechanisms in contaminating bacteria, that could severely impact inactivation efficacy. Thus far, the role of these resistance mechanisms has not been studied in conditions relevant to drinking water systems. Therefore, we evaluated the inactivation kinetics of Cupriavidus metallidurans CH34 in contact with metallic copper in drinking water. Viability and membrane permeability were examined for 9 days through viable counts and flow cytometry. After an initial drop in viable count, a significant recovery was observed starting after 48 h. This behavior could be explained by either a recovery from an injured/viable-but-non-culturable state or regrowth of surviving cells metabolizing lysed cells. Either hypothesis would necessitate an induction of copper resistance mechanisms, since no recovery was seen in a CH34 mutant strain lacking metal resistance mechanisms, while being more pronounced when copper resistance mechanisms were pre-induced. Interestingly, no biofilms were formed on the copper surface, while extensive biofilm formation was observed on the stainless steel control plates. When CH34 cells in water were supplied with CuSO 4 , a similar initial decrease in viable counts was observed, but cells recovered fully after 7 days. In conclusion, we have shown that long-term bacterial survival in the presence of a copper surface is possible upon the induction of metal resistance mechanisms. This observation may have important consequences in the context of the increasing use of copper as an antimicrobial surface, especially in light of potential co-selection for metal and antimicrobial resistance.
... With the emergence of multidrug-resistant pathogens and a reduced number of antibiotics being discovered, metals have received increased attention in recent years, especially as potent antimicrobial agents against antibiotic-resistant pathogens [17,18]. Numerous transition metals and metalloids, such as silver, copper, selenium and tellurium, have been identified as antimicrobial and antibiofilm agents that are effective against several bacterial pathogens, such as Staphylococcus aureus, Escherichia coli, Salmonella and Pseudomonas aeruginosa [18][19][20][21]. ...
Article
Introduction. Clostridioides difficile is an enteric pathogen that causes a serious toxin-mediated colitis in humans. Bacterial exotoxins and sporulation are critical virulence components that contribute to pathogenesis, and disease transmission and relapse, respectively. Therefore, reducing toxin production and sporulation could significantly minimize C. difficile pathogenicity and disease outcome in affected individuals. Aim. This study investigated the efficacy of a natural flavone glycoside, baicalin, in reducing toxin synthesis, sporulation and spore germination in C. difficile in vitro . Methodology. Hypervirulent C. difficile isolates BAA 1870 or 1803 were cultured in brain heart infusion broth with or without the subinhibitory concentration (SIC) of baicalin, and incubated at 37 °C for 24 h under strictly anaerobic conditions. The supernatant was harvested after 24 h for determining C. difficile toxin production by ELISA. In addition, a similar experiment was performed wherein samples were harvested for assessing total viable counts, and heat-resistant spore counts at 72 h of incubation. Furthermore, C. difficile spore germination and spore outgrowth kinetics, with or without baicalin treatment, was measured in a plate reader by recording optical density at 600 nm. Finally, the effect of baicalin on C. difficile toxin, sporulation and virulence-associated genes was investigated using real-time quantitative PCR. Results. The SIC of baicalin significantly reduced toxin synthesis, sporulation and spore outgrowth when compared to control. In addition, C. difficile genes critical for pathogenesis were significantly down-regulated in the presence of baicalin. Conclusion. Our results suggest that baicalin could potentially be used to control C. difficile , and warrant future studies in vivo .
... Membrane depolarization is the widely proposed mechanism responsible for killing of bacteria by Cu ions. 348,349 Active bacteria has of a membrane induced potential difference of nearly 100−200 mV between the inside and outside the cell, whereas the interior of the cell is at a less negative potential. These Cu ions reduce the potential difference by binding with bacterial cells' negatively charged domains on both the inside and outside of the cell which promotes membrane depolarization, thus making the membranes leaky or ruptured too if the potential difference becomes zero. ...
Article
The ongoing worldwide pandemic due to COVID-19 has created awareness toward ensuring best practices to avoid the spread of microorganisms. In this regard, the research on creating a surface which destroys or inhibits the adherence of microbial/viral entities has gained renewed interest. Although many research reports are available on the antibacterial materials or coatings, there is a relatively small amount of data available on the use of antiviral materials. However, with more research geared toward this area, new information is being added to the literature every day. The combination of antibacterial and antiviral chemical entities represents a potentially path-breaking intervention to mitigate the spread of disease-causing agents. In this review, we have surveyed antibacterial and antiviral materials of various classes such as small-molecule organics, synthetic and biodegradable polymers, silver, TiO2, and copper-derived chemicals. The surface protection mechanisms of the materials against the pathogen colonies are discussed in detail, which highlights the key differences that could determine the parameters that would govern the future development of advanced antibacterial and antiviral materials and surfaces.
... In the literature, "contact killing" abilities of copper are often described, but the underlying mechanisms are not yet fully understood [17,41,42]. The primary effect seems to be caused by damage to the cell membranes, while DNA damage occurs later [43][44][45]. This finding applies to both dry and moist copper surfaces [42,46]. ...
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Objectives Evidence about modifications of dental luting materials to minimize biological failure at the “marginal gap” between teeth and fixed prosthodontics is scarce. We compared a copper-modified (Co-ZOP) and a conventional zinc oxide phosphate cement (ZOP) in terms of antimicrobial and cytotoxic potentials in vitro and in vivo.Materials and methodsSpecimens of ZOP and Co-ZOP were characterized by the mean arithmetic roughness (Ra) and surface free energy (SFE). Powder components were examined using scanning electron microscopy (SEM). Energy-dispersive X-ray spectroscopy (EDX) showed elemental material compositions. In vitro microbial adhesion was shown using SEM, luminescence, and fluorescence assays. CCK-8 assays of mouse fibroblasts (L929) and human gingival fibroblasts (GF-1) were performed after 6, 24, and 48 h of specimen incubation. In vivo, ZOP and Co-ZOP specimens were applied intraorally for 12 h; biofilm accumulation was shown using SEM.ResultsRa of ZOP and Co-ZOP showed no significant differences; SFE was significantly higher for Co-ZOP. EDX exhibited minor copper radiation for Co-ZOP, none for ZOP. In vitro fungal adhesion to Co-ZOP was significantly higher than to ZOP; in vitro streptococcal adhesion, cytotoxicity, and in vivo biofilm formation were not significantly different.Conclusions Co-ZOP showed low surface allocations of copper with no improved antimicrobial properties compared with conventional ZOP in vitro or in vivo.Clinical relevanceAntimicrobial effects and low cytotoxicity of biomaterials are important for the clinical outcome. Based on our in vitro and in vivo results, no clinical recommendation can be given for the tested Co-ZOP.
... Recent research articles reported that metal complexes having copper(II) atom demonstrated efficient antibiofilm potential against gram positive and gram negative bacteria [36,37]. It has been observed that copper ions in the complexes exhibit antibiofilm potential through different mode of actions, remarkably through DNA destruction since intracellular and extracellular genetic materials assumes a crucial role at various levels of biofilm formation and maturation [38]. ...
Article
A polymeric tetrakis N-imidazole copper(II) sulphate complex [Cu(imidazole)4(SO4)]n 1, was synthesized and thoroughly characterized by various spectroscopic techniques, elemental analysis and single crystal X-ray crystallography. X-ray diffraction analysis confirmed that polymer complex 1 crystallized in a monoclinic C2/c space group possessing the lattice constants, a = 9.2213(1) Å, b = 17.6062(3) Å, c = 10.5710(2) Å, α = γ = 90˚ and β = 93.561˚ (1) per unit cell. Hirshfeld surface and DFT calculations were executed to simulate the electronic ground state energies in gas phase which showed that the HOMO and HOMO–1 were distributed on the axially coordinated SO42– moiety and considerable amount of electron density was present on Cu(II). Absorption and emission spectroscopic techniques were used to investigate the binding interaction of the copper(II) imidazole complex with the therapeutic target ct-DNA. The results suggested that the complex 1 binds to the ct-DNA through an electrostatic mode. The gel electrophoresis experiments of 1 were carried out with plasmid pBR322 which revealed that the polymer complex could unwind the plasmid DNA at 30 μM micromolar concentration via discernible oxidative cleavage pathway. Molecular docking studies were also carried out to support the results of DNA binding experiments. In addition, the antimicrobial potential of polymer complex was evaluated against various key pathogens viz., B. subtilis, E. coli, K. pneumonia and S. aureus. Antibiofilm behaviour of the complex 1 was examined against E. coli and S. aureus. The results demonstrated that 1 could act as an active antibacterial agent as it inhibited the bacterial growth in agar plates very efficiently.
... The treatment of C. albicans with Ag-phendione induced extensive smearing of DNA, indicating non-specific cleavage of the DNA [25]. In addition, several studies have reported that copper nanoparticles dramatically affect the bacterial redox systems that culminate with DNA fragmentation [49][50][51]. ...
Article
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Tackling microbial resistance requires continuous efforts for the development of new molecules with novel mechanisms of action and potent antimicrobial activity. Our group has previously identified metal-based compounds, [Ag(1,10-phenanthroline-5,6-dione)2]ClO4 (Ag-phendione) and [Cu(1,10-phenanthroline-5,6-dione)3](ClO4)2.4H2O (Cu-phendione), with efficient antimicrobial action against multidrug-resistant species. Herein, we investigated the ability of Ag-phendione and Cu-phendione to bind with double-stranded DNA using a combination of in silico and in vitro approaches. Molecular docking revealed that both phendione derivatives can interact with the DNA by hydrogen bonding, hydrophobic and electrostatic interactions. Cu-phendione exhibited the highest binding affinity to either major (-7.9 kcal/mol) or minor (-7.2 kcal/mol) grooves. In vitro competitive quenching assays involving duplex DNA with Hoechst 33258 or ethidium bromide demonstrated that Ag-phendione and Cu-phendione preferentially bind DNA in the minor grooves. The competitive ethidium bromide displacement technique revealed Cu-phendione has a higher binding affinity to DNA (Kapp=2.55×106 M-1) than Ag-phendione (Kapp=2.79×105 M-1) and phendione (Kapp=1.33×105 M-1). Cu-phendione induced topoisomerase-I-mediated DNA relaxation of supercoiled plasmid DNA. Moreover, Cu-phendione was able to induce oxidative DNA injuries with the addition of free radical scavengers inhibiting DNA damage. Ag-phendione and Cu-phendione avidly displaced propidium iodide bound to DNA in permeabilized Pseudomonas aeruginosa cells in a dose-dependent manner as judged by cytometry. The treatment of P. aeruginosa with bactericidal concentrations of Cu-phendione (15 µM) induced DNA fragmentation as visualized by either agarose gel or TUNEL assays. Altogether, these results highlight a possible novel DNA-targeted mechanism by which phendione-containing complexes, in part, elicit toxicity toward the multidrug-resistant pathogen P. aeruginosa.
... Copper transport in eukaryotes (e.g., S. cerevisiae, Aspergillus fumigatus) is carried out by the membrane-associated Cu transporting protein (Ctr) family, Cu binding or acquisition motifs (Mets) (Antsotegi-Uskola et al., 2020;Smith et al., 2017), or low-affinity uptake systems as the Fe, Cu, and Zn transporter Fet4 in S. cerevisiae (Smith et al., 2017). Modes for Cu toxicity include depolarization of the cytoplasmic membrane, loss of membrane integrity, inhibition of respiration and generation of reactive oxygen species that provoke damages in the DNA and other cell structures (Warnes et al., 2012). Copper also induces oxidative stress responses at higher concentrations that oversaturate homeostasis inside the cell. ...
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Mine wastes pollute the environment with metals and metalloids in toxic concentrations, causing problems for humans and wildlife. Microorganisms colonize and inhabit mine wastes, and can influence the environmental mobility of metals through metabolic activity, biogeochemical cycling and detoxification mechanisms. In this article we review the microbiology of the metals and metalloids most commonly associated with mine wastes: arsenic, cadmium, chromium, copper, lead, mercury, nickel and zinc. We discuss the molecular mechanisms by which bacteria, archaea, and fungi interact with contaminant metals and the consequences for metal fate in the environment, focusing on long‐term field studies of metal‐impacted mine wastes where possible. Metal contamination can decrease the efficiency of soil functioning and essential element cycling due to the need for microbes to expend energy to maintain and repair cells. However, microbial communities are able to tolerate and adapt to metal contamination, particularly when the contaminant metals are essential elements that are subject to homeostasis or have a close biochemical analog. Stimulating the development of microbially reducing conditions, for example in constructed wetlands, is beneficial for remediating many metals associated with mine wastes. It has been shown to be effective at low pH, circumneutral and high pH conditions in the laboratory and at pilot field‐scale. Further demonstration of this technology at full field‐scale is required, as is more research to optimize bioremediation and to investigate combined remediation strategies. Microbial activity has the potential to mitigate the impacts of metal mine wastes, and therefore lessen the impact of this pollution on planetary health.
... Although GE is much larger than the aforementioned ions, it is highly competitive for the negatively charged LPS sites consequently leading to ion displacement and outer membrane perturbance [45]. The copper ions might also replace Mg +2 and Ca 2+ ions increasing the outer membrane perturbance, but the Cu 2+ mode of action is also related to an increased intracellular ROS level, hydroxyl radical formation, and the impairment of the iron-sulfur dehydratase enzymes [46,47] (Figure 7a). The observed in vitro additive effect (FICI = 1) may come from the simultaneous activity of both antibacterials elevating the ROS level in the cell ( Figure 7a). ...
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In this study, we investigated the anti-pseudomonal activity of cupric ions (Cu2+), strawberry furanone (HDMF), gentamicin (GE), and three lytic Pseudomonas aeruginosa bacteriophages (KT28, KTN4, LUZ19), separately and in combination. HDMF showed an anti-virulent effect but only when applied with Cu2+ or GE. GE, at a sub-minimal inhibitory concentration, slowed down phage progeny production due to protein synthesis inhibition. Cu2+ significantly reduced both the bacterial cell count and the number of infective phage particles, likely due to its genotoxicity or protein inactivation and cell membrane disruption effects. Furthermore, Cu2+‘s probable sequestration by phage particles led to the reduction of free toxic metal ions available in the solution. An additive antibacterial effect was only observed for the combination of GE and Cu2+, potentially due to enhanced ROS production or to outer membrane permeabilization. This study indicates that possible interference between antibacterial agents needs to be carefully investigated for the preparation of effective therapeutic cocktails.
... Finally, log reductions were calculated by subtracting the viable counts of bacteria exposed to Cu surfaces from those of bacteria recovered from glass slides. This is an EPA validated procedure [35] and a similar antibacterial testing technique and incubation time has been used by other researchers [1,17,36,37]. ...
Article
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Contact surfaces have been identified as one of the main routes for pathogen transmission. The efficacy to kill both viruses and bacteria on touch surfaces is critical to reducing the rampant spread of harmful pathogens. Copper is one such material that has been traditionally used for its antimicrobial properties. However, most contact/touch surfaces are made up of steel or aluminum due to their structural properties. Therefore, coating high-touch components with copper is one possible solution to improve antibacterial efficacy. In this study, copper was coated on both stainless steel and aluminum substrates using a cold spray process which is a fast and economic coating technique. The coated samples in both as-deposited and heat-treated states were exposed to Escherichia coli and Staphylococcus aureus bacteria, and their efficacy was compared with bulk copper plate. It was found that both bacterial cells responded differently to the different coating properties such as coating thickness, porosity, hardness, surface roughness, oxide content, and galvanic coupling effect. These correlations were elucidated in light of various results obtained from antibacterial and bacterial attachment tests, and materials characterizations of the coatings. It is possible to tailor copper coating characteristics to render them more effective against targeted bacteria.
... Although the antibacterial mechanism of the solid copper surface has yet to be clearly understood, several studies have investigated the result of the so-called contact killing [8,9]. When bacteria are directly in contact with metallic copper, copper ions accumulate inside the cell because the bacteria recognize the copper ions as essential nutrients [10,11]. The cell and DNA are then damaged and destroyed by the depolarization effect and reactive oxygen species (ROS) [12]. ...
Article
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In this work, a copper coating is developed on a carbon steel substrate by exploiting the superwetting properties of liquid copper. We characterize the surface morphology, chemical composition, roughness, wettability, ability to release a copper ion from surfaces, and antibacterial efficacy (against Escherichia coli and Staphylococcus aureus). The coating shows a dense microstructure and good adhesion, with thicknesses of approximately 20–40 µm. X-ray diffraction (XRD) analysis reveals that the coated surface structure is composed of Cu, Cu2O, and CuO. The surface roughness and contact angle measurements suggest that the copper coating is rougher and more hydrophobic than the substrate. Inductively coupled plasma atomic emission spectroscopy (ICP-AES) measurements reveal a dissolution of copper ions in chloride-containing environments. The antibacterial test shows that the copper coating achieves a 99.99% reduction of E. coli and S. aureus. This study suggests that the characteristics of the copper-coated surface, including the chemical composition, high surface roughness, good wettability, and ability for copper ion release, may result in surfaces with antibacterial properties.
... As shown in several studies, nanoscale size allows for the nanoparticles to penetrate microbial cell walls and biofilms layers, causing several events such as irreversible damage of microbial membranes [9,117,118]. Several hypotheses for nanoparticles mechanisms of action have been explored, including cell membrane alterations and disruption [119,120], ROS generation, and lipid peroxidation [121][122][123]. Metabolic pathway disruption is also described as a mechanism of NPs [124,125]. ...
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Bacterial biofilms are defined as complex aggregates of bacteria that grow attached to surfaces or are associated with interfaces. Bacteria within biofilms are embedded in a self-produced extracellular matrix made of polysaccharides, nucleic acids, and proteins. It is recognized that bacterial biofilms are responsible for the majority of microbial infections that occur in the human body, and that biofilm-related infections are extremely difficult to treat. This is related with the fact that microbial cells in biofilms exhibit increased resistance levels to antibiotics in comparison with planktonic (free-floating) cells. In the last years, the introduction into the market of novel compounds that can overcome the resistance to antimicrobial agents associated with biofilm infection has slowed down. If this situation is not altered, millions of lives are at risk, and this will also strongly affect the world economy. As such, research into the identification and eradication of biofilms is important for the future of human health. In this sense, this article provides an overview of techniques developed to detect and imaging biofilms as well as recent strategies that can be applied to treat biofilms during the several biofilm formation steps.
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In aquaculture of the swimming crab Portunus trituberculatus, massive deaths have been recorded in the winter months due to infection with a novel emerging parasite, Mesanophrys sp. However, no information was available regarding the prevention and control of this particular parasite. Therefore, the present study was conducted to evaluate the anti-parasitic efficacy and toxicity of formalin against the Mesanophrys sp. In vitro results showed that the anti-parasitic efficacy of formalin improved with concentration increasing from 0 to 20 ppm within 24 h. In particular, when treated with formalin at 16.0, 15.0, 11.0, 10.0, 9.0, and 6.0 ppm for 0.5, 1, 2, 4, 6, 12, and 24 h respectively, the Mesanophrys sp. mortality rate reached 100%. To gain insights into the effects the formalin treatment had on the parasite, cell micro- and ultra-structure were investigated. It was determined that the cells contracted gradually and became rounded, intracellular vacuoles were observed at early time points (Ф≤4.83 ± 1.26 μm) and then disappeared. Cilia were shed and macronuclear chromatin became condensed and agglutinated. Small holes and bubbles appeared on surface of the parasites. In an in vivo trial, formalin was applied prior to Mesanophrys sp. artificial infection as prophylaxis to P. trituberculatus. The results showed that formalin prophylactic treatment effectively prevented P. trituberculatus from Mesanophrys sp. infection, thus remarkably reducing the mortality of crabs compared with the non-formalin-exposed and infected crabs. Furthermore, the normal behavior and survival of P. trituberculatus were not impacted by the prophylactic treatment.
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Salmonella enteritidis is an important food-borne pathogen. The use of antibiotics is a serious threat to animal and human health, owing to the existence of resistant strains and drug residues. Lactic acid bacteria, as a new alternative to antibiotics, has attracted much attention. In this study, we investigated the antibacterial potential and underlying mechanism of Lactobacillus rhamnosus SQ511 against S. enteritidis ATCC13076. The results revealed that L. rhamnosus SQ511 significantly inhibited S. enteritidis ATCC13076 growth or even caused death. Laser confocal microscopic imaging revealed that the cell-free supernatant (CFS) of L. rhamnosus SQ511 elevated the reactive oxygen species level and bacterial membrane depolarization in S. enteritidis ATCC13076, leading to cell death. Furthermore, L. rhamnosus SQ511 CFS had severely deleterious effects on S. enteritidis ATCC13076, causing membrane destruction and the release of cellular materials. In addition, L. rhamnosus SQ511 CFS significantly reduced the expression of virulence, motility, adhesion, and invasion genes in S. enteritidis ATCC13076 (P < 0.05), and considerably inhibited motility and biofilm formation capacity (P < 0.05). Thus, antimicrobial compounds produced by L. rhamnosus SQ511 strongly inhibited S. enteritidis growth, mobility, biofilm formation, membrane disruption, and reactive oxygen species generation, and regulated virulence-related gene expressions, presenting promising applications as a probiotic agent.
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Overwhelming growth of bacterial biofilms on different metal-based pipeline materials are intractable and pose a serious threat to public health when tap water flows though these pipelines. Indeed, the underlying mechanism of biofilm growth on the surface of different pipeline materials deserves detailed exploration to provide subsequent implementation strategies for biofilm control. Thus, in this study, how bacteria response to their encounters was explored, when they inhabit different metal-based pipeline substrates. Results revealed that bacteria proliferated when they grew on stainless steel (SS) and titanium sheet (Ti), quickly developing into bacterial biofilms. In contrast, the abundance of bacteria on copper (Cu) and nickel foam (Ni) substates decreased sharply by 4–5 logs within 24 h. The morphological shrinkage and shortening of bacterial cells, as well as a sudden 64-fold increase of carbohydrate content in extracellular polymeric substances (EPS), were observed on Cu substrate. Furthermore, generation of reactive oxygen species and fluctuation of enzymatic activity demonstrated the destruction of redox equilibrium in bacteria. Bacteria cultured on Cu substrate showed the strongest response, followed by Ni, SS and Ti. The oxidative stress increased quickly during the growth of bacterial biofilm, and almost all tested metal transporter-related genes were upregulated by 2–11 folds on Cu, which were higher than on other substrates (1–2 folds for SS and Ti, 2–9 folds for Ni). Finally, these behaviors were compared under the biofilm regulatory molecular network. This work may facilitate better understanding different response mechanisms during bacterial biofilm colonization on metal-based pipelines and provide implications for subsequent biofilm control.
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This study investigates the interaction between E. coli bacteria and various surface states of Cu. Pure Cu was oxidized at 298 K, 673 K, and 1273 K, and its surface chemical state was characterized using X-ray photoelectron spectroscopy. The oxidized specimens were immersed in both bacteria-free and bacteria-containing solutions, and the release of Cu ions from each specimen was evaluated. As a result, the specimens, which existed as mainly Cu⁰ or Cu⁺ on the surface, exhibited the same corrosion behavior and promoted the elution of Cu ions due to the presence of E. coli in the immersion solution. In contrast, the release of Cu ions from the specimen that existed mainly as Cu²⁺ did not change in the presence of bacteria. According to the XPS analyses before and after immersion in bacteria-free and bacteria-containing solutions, the generation of Cu²⁺ on the surface was inhibited by the presence of E. coli, and promoted Cu ion elution. This study proposes that the specific corrosion behavior of Cu due to bacteria is beneficial for developing its antibacterial properties. Fullsize Image
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Background: The introduction of antimicrobial surfaces into healthcare environments is believed to impact positively on the rate of healthcare-associated infections by significantly decreasing pathogen presence on surfaces. Aim: To report on a novel efficacy test that uses a dry bacterial inoculum to measure the microbicidal efficacy of antimicrobial surfaces. Methods: An aerosolized dry inoculum of Staphylococcus aureus or Acinetobacter baumannii was deposited on copper alloy surfaces or a hospital-grade stainless-steel surface. Surviving bacteria were enumerated following incubation of the inoculated surfaces at an environmentally relevant temperature and relative humidity. Damage caused to bacteria by the aerosolization process and by the different surfaces was investigated. Findings: Dry inoculum testing showed a <2-log10 reduction in S. aureus or A. baumannii on the copper alloy surfaces tested after 24 h at 20°C and 40% relative humidity. Potential mechanisms of action included membrane damage, DNA damage and arrested cellular respiration. The aerosolization process caused some damage to bacterial cells. Once this effect was taken into account, the antimicrobial activity of copper surfaces was evident. Conclusions: Our test provided a realistic deposition of a bacterial inoculum to a surface and, as such, a realistic protocol to assess the efficacy of dry antimicrobial environmental surfaces in vitro.
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The transmission of bacterial infections through contaminated surfaces is nowadays an increasing source of concern, also related to the current pandemic situation. Functional materials that prevent the adhesion of microorganisms and/or induce their eradication thus avoiding fomite transmission are highly needed. In this work, a highly antimicrobial hybrid with sensorial capability is developed to be further applied as interactive high traffic surface coatings. The nanocomposite is composed of polyvinylidene fluoride (PVDF), a highly stable fluorinated polymer, incorporating copper core‐shell nanowires (NWs). The NWs comprised of copper and shelled with silver is highly antimicrobial, inducing a full kill effect against Escherichia coli and Staphylococcus epidermidis strains but biocompatible towards mammalian cells at concentrations below 0.5 mg mL−1. Further NWs incorporation on PVDF matrix retains its antimicrobial activity reducing in 6.5 logs the E. coli and 4.5 logs the S. epidermidis. NW/PVDF composites demonstrate suitable mechanical and electrical characteristics for the development of capacitive sensing surfaces, allowing for the fabrication of an antimicrobial capacitive touch sensing matrix for interactive surfaces. Multifunctional polymer composites are developed for high‐traffic touch surfaces, allowing them to manage viruses or bacteria transmission and, simultaneously, work as interactive surfaces. The antimicrobial capability is provided using synthesized core/shell metallic nanowires which are then embedded into polyvinylidene fluoride polymer, with a high dielectric response. The antimicrobial and sensorial capability is demonstrated in an interactive touch sensing surface.
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Cryptocaryonosis, caused by the ciliate parasite Cryptocaryon irritans, results in large economic losses in large yellow croaker (Larimichthys crocea) culture. In order to explore the effect of copper plates on C. irritans tomonts, incubation rate, morphological characteristics, copper concentration, and transcriptome changes were studied. After copper plate treatment for 6 h, the tomonts showed erosion in the internal structure and the tomonts wall exhibited reduced transparency. Compared with the control group, the hatching rate of tomonts in the treatment group was significantly lower. Copper concentrations of tomonts in the treatment group was 340.67 ± 0.94 mg/kg, while the copper value in the control group was 22.40 ± 0.91 mg/kg. Transcriptome analysis revealed that 21 GO terms were significantly enriched by differential expression genes (DEGs). Among them, the regulation of cellular respiration and protein-containing complexes were highly enriched (q-value<0.001). The results of KEGG analysis showed that the ribosome pathway and the systemic lupus erythematosus pathway were the most significantly enriched pathways. In addition, most of the annotated DEGs on these two pathways were down-regulated indicating the killing effect was mainly mediated by ribosomal proteins, histones, and small nuclear ribonucleoproteins.
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The aim of this work was the green synthesis of copper nanoparticles (Cu-NPs) using aqueous extracts of (i) bilberry (Vaccinium myrtillus L.) waste residues from the production of fruit juices and (ii) non-edible “false bilberry” fruits (Vaccinium uliginosum L. subsp. gaultherioides). Different cupric salts (CuCl2, Cu(CH3COO)2 and Cu(NO3)2) were used for the synthesis. The formation of stable nanoparticles (CuNPs) was assessed by transmission electron microscopy and the oxidation state of copper in these aggregates was followed by X-ray photoelectron spectroscopy. The polyphenol composition of the extracts was characterised, before and after the synthesis, using spectrophotometric methods (i.e. total soluble polyphenols and total monomeric anthocyanins) and high-performance liquid chromatography coupled with tandem mass spectrometry (i.e. individual anthocyanins). Polyphenol concentration in the extracts was found to decrease after the synthesis, indicating their active participation to the processes, which led to the formation of Cu-NPs. The antimicrobial activity of Cu-NPs, berry extracts, and cupric ion solutions were analysed by broth microdilution and time-kill assays, on prokaryotic and eukaryotic models. The antimicrobial activity of Cu-NPs, especially those derived from bilberry waste residues, appeared to be higher for both Gram-negative and Gram-positive bacteria, and for fungi, compared to the ones of its single components (cupric salts and berry extracts). Therefore, Cu-NPs from the green synthesis here proposed can be considered as a cost-effective sanitization tool with a wide spectrum of action.
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Objective To evaluate 3 formulations of copper (Cu)-based self-sanitizing surfaces for antimicrobial efficacy and durability over 1 year in inpatient clinical areas and laboratories. Design Randomized control trial. Setting We assessed 3 copper formulations: (1) solid alloy 80% Cu–20% Ni (integral copper), (2) spray-on 80% Cu–20% Ni (spray-on) and (3) 16% composite copper-impregnated surface (CIS). In total, 480 coupons (1 cm ² ) of the 3 products and control surgical grade (AISI 316) stainless steel were inserted into gaskets and affixed to clinical carts used in patient care areas (including emergency and maternity units) and on microbiology laboratory bench work spaces (n = 240). The microbial burden and assessment of resistance to wear, corrosion, and material compatibility were determined every 3 months. Participants included 3 tertiary-care Canadian adult hospital and 1 pediatric-maternity hospital. Results Copper formulations used on inpatient units statistically significantly reduced bacterial bioburden compared to stainless steel at months 3 and 6. Only the integral copper product had significantly less bacteria than stainless steel at month 12. No statistically significant differences were detected in microbial burden between copper formulations and stainless-steel coupons on microbiology laboratory benches where bacterial counts were low overall. All mass changes and corrosion rates of the formulations were acceptable by engineering standards. Conclusions Copper surfaces vary in their antimicrobial efficacy after 1 year of hospital use. Frequency of cleaning and disinfection influence the impact of copper; the greatest reduction in microbial bioburden occurred in clinical areas compared to the microbiology laboratory where cleaning and disinfection were performed multiple times daily.
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Antimicrobial resistance is a major global threat to human health due to the rise, spread and persistence of multidrug-resistant bacteria or 'superbugs'. There is an urgent need to develop novel chemotherapeutics to overcome this overarching challenge. We have derivatised a clinically used fluoroquinolone antibiotic ciprofloxacin and complexed it to a copper phenanthrene framework. This has resulted in the development of two novel metallo-antibiotics of general formula [Cu(N,N)(CipHA)]NO3 where N,N represents a phenanthrene ligand and CipHA stands for a hydroxamic acid of a ciprofloxacin (Cip) derivative. Comprehensive studies, including a detailed proteomic study in which S. aureus cells were exposed to the complexes, were undertaken to gain an insight into their mode of action. These new complexes possess potent anti-bacterial activity against Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA). In addition, they were found to be well tolerated in vivo in Galleria mellonella larvae, which has both functional and structural similarities to that of the innate immune system of mammals. Our findings suggest that proteins involved in virulence, pathogenesis and the synthesis of nucleotides and DNA repair mechanisms are most affected. In addition, both complexes affected similar cell pathways when compared to the clinically used Cip, including CAMP resistance. The Cu-DPPZ-CipHA (DPPZ = dipyrido[3,2-a:2',3'-c]phenazine) analogue also induces cell leakage, which leads to altered proteome indicative of reduced virulence and increased stress.
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Frequent food poisoning and food-borne diseases outbreaking in recent years have caused people to attach great attention to food safety, especially the food contact materials that are essential in the food industrial chains and daily lives, ensuring their clean sanitation are of great importance in blocking microbial contamination and spread of food-borne pathogens. Stainless steel (SS) is one of the most accepted and widely used food contact material, and the Cu-bearing SS possesses excellent antibacterial performance and maintains the original mechanical properties of SS, maybe making it a better substitute for the conventional SS in the food area. Taking advantages of bactericidal and antifouling properties of Cu-bearing SS, this study simulated a variety of food contact scenarios, explored a new strategy for food preservation and food safety by using Cu-bearing SS as a food contact material. The results showed that the Cu-bearing SS could not only delay the spoilage of different foods by inhibiting the activity of microorganisms in foods, but also reduce the expressions of spoilage traits of bacteria as well as the formation of biofilms by quenching the quorum-sensing signals, and further creating a good bacteriostatic atmosphere for the contacted food and its surrounding environment. In addition, the remarkable antifouling property of Cu-bearing SS would give the material a self-cleaning feature for food applications, which can avoid secondary contamination of food as a source of contamination. This study well demonstrates that the Cu-bearing SS has broad application potentials and prospects in the food area.
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Copper alloys surfaces have been proposed as a promising solution to avoid microbial contamination of surfaces but their antibiofilm effect is still unclear. All copper alloys tested have demonstrated efficient antibiofilm activity against Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, with differences between alloys. The artificial aging of copper alloys led to variable effects on the bacterial adhesion, depending on alloy. The results highlighted the promising antibiofilm activity of various copper alloys and suggest that the alloy composition is an essential parameter in this activity.
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Wound healing is a highly dynamic process and innovative therapeutic approaches are currently developed to address challenges of providing optimal wound care. In this study, phosphate-based glasses in the (CuO)x·(KPO3)79.5-x·(ZnO)20·(Ag2O)0.5 system (CuKPO3ZnAg), with different CuO/ KPO3 ratios were prepared by melt-quenching technique. Constant Cu concentrations were released from the samples during immersion in Simulated Body Fluid (SBF), while Zn concentrations were slightly decreased over time. Glass surface phosphatation leading to formation of Zn crystalline salts was revealed through spectroscopic techniques. This finding was supported by SEM images that illustrated new compound formation. Subsequent cytotoxicity evaluation on HaCaT Keratinocytes using the indirect MTT cell viability assay revealed a CuO concentration-dependent cytotoxicity profile and excellent biocompatibility at low CuO concentrations, in all CuKPO3ZnAg glasses. Furthermore, the (CuO)5·(KPO3)74.5·(ZnO)20·(Ag2O)0.5 sample (5CuKPO3ZnAg), demonstrated superior antibacterial potency against S. aureus (ATCC 25923) strain compared to amoxicillin and ciprofloxacin. In vivo full-thickness wound healing evaluation showed a significantly higher regenerative effect of the 5CuKPO3ZnAg sample, in terms of angiogenesis, collagen synthesis and re-epithelialization compared to non-treated wounds. These findings advance our understanding of the therapeutic perspectives of phosphate-based glasses, showing promising potential for wound-healing applications. This article is protected by copyright. All rights reserved
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From its uses in ancient civilisations, copper has an established history as an antimicrobial agent. Extensive research has determined the efficacy and mechanism of copper's antimicrobial activity against microorganisms. The process is multifaceted with the main mechanism of bactericidal activity being the generation of reactive oxygen species (ROS), which irreversibly damages membranes. Copper ions released from surfaces lead to RNA degradation and membrane disruption of enveloped viruses. For fungi, the mechanism involves the physical deterioration of the membrane and copper ion influx. Due to variations in the experimental parameters, it is difficult to compare studies directly. In this review article, we outline the importance of the experimental conditions currently employed and how they bear little resemblance to real-world conditions. We endorse previous recommendations calling for an update to industrial standard tests.
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Numerous studies have found that the surface topography affects the material antibacterial properties by reducing the attachment of bacteria on the surfaces without influencing the viability of the adhered cells. For Cu-bearing alloys with excellent contact-killing properties, bacterial adhesion on the surface is also accompanied by short-range interactions which regulate the toxic effects of the material surface against bacterial cells. Thus, the surface topography of Cu-bearing alloys, as an important factor dominating the exposure level of bacteria on the surfaces, should affect the subsequent contact-killing efficiency. In this work, our major focus was on the regulation mechanism of the surface features on the material-bacterial interactions. We correlated the surface properties including different surface roughnesses of Cu-bearing stainless steel (SS) with the bacterial damage pattern and attempted to clarify the role of surface roughness in mediating the contact-killing behavior of Cu-bearing SS. The results of both atomic force microscopy and scanning electron microscopy investigations showed that E. coli cells experienced the most rapid physical and mechanical damages after incubating with the diamond-polished Cu-bearing SS surface. The bacterial cells noticeably stiffened and the adhesion force significantly increased, as evidenced by force-distance curve measurements. Because of the enhanced hydrophobicity and higher surface potential of the diamond-polished surface, which strengthened the Lewis acid-base attractive forces and weakened the electrostatic barrier between the bacteria and the surface, a higher exposure surface for bacteria was generated. Furthermore, the contact-induced charge transfer, manifested by Cu ion burst release, and reactive oxygen species overexpression contribute to an efficient contact-killing process.
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Lignin is a natural polymer produced in huge amounts by the paper industry. Innovative applications of lignin, especially in agriculture, represent a valuable way to develop a more sustainable economy. Its antioxidant and antimicrobial properties, combined with its biodegradability, make it particularly attractive for the development of plant protection products. Copper is an element that has long been used as a pesticide in agriculture. Despite its recognized antimicrobial activity, the concerns derived from its negative environmental impact is forcing research to move toward the development of more effective and sustainable copper‐based pesticides. Here a simple and sustainable way of synthesizing a new hybrid material composed of Cu2O nanocrystals embedded into lignin, named Lignin@Cu2O is presented. The formation of cuprite nanocrystals leaves the biopolymer intact, as evidenced by infrared spectroscopy, gel permeation chromatography, and Pyrolysis‐GC analysis. The combined activity of lignin and cuprite make Lignin@Cu2O effective against Listeria monocytogenes and Rhizoctonia solani at low copper dosage, as evidenced by in vitro and in vivo tests conducted on tomato plants. The reaction between a basic solution of lignin and copper sulfate leads to the formation of the hybrid material Lignin@Cu2O, where cuprite nanocrystals are embedded into the biopolymeric framework. The material is an effective antimicrobial agent at low copper dosage against several pathogens, such as Listeria monocytogenes and Rhizoctonia solani, as demonstrated by in vitro and in vivo tests.
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Electrocatalytic metals and microorganisms can be combined for CO2 conversion in microbial electrosynthesis (MES). However, a systematic investigation on the nature of interactions between metals and MES is still lacking. To investigate this nature, we integrated a copper electrocatalyst, converting CO2 to formate, with microorganisms, converting CO2 to acetate. A co‐catalytic (i.e. metabolic) relationship was evident, as up to 140 mg·L‐1 of formate was produced by sole copper oxide, while formate was also evidently produced by copper and consumed by microorganisms producing acetate. Due to non‐metabolic interactions, current density decreased by over 4 times, though acetate yield increased by 3.3 times. Despite the antimicrobial role of copper, biofilm formation was possible on a pure copper surface. Overall, we show for the first time that a CO2‐reducing copper electrocatalyst can be combined with MES under biological conditions, resulting in metabolic and non‐metabolic interactions.
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Escherichia coli O157:H7 is a serious pathogen causing haemorrhagic colitis. It has been responsible for several large-scale outbreaks in recent years. E. coli O157:H7 is able to survive in a range of environments, under various conditions. The risk of infection from contaminated surfaces is recognised, especially due to the low infectious dose required. In this study, a high concentration (107 cells) of E. coli O157 was placed onto different metals and survival time measured. Results showed E. coli O157 to survive for over 28 days at both refrigeration and room temperatures on stainless steel. Copper, in contrast, has strong antibacterial properties (no bacteria can be recovered after only 90 min exposure at 20 °C, increasing to 270 min at 4 °C) but its poor corrosion resistance and durability make it unsuitable for use as a surface material. Other copper-containing alloys, such as copper nickels and copper silvers, have improved durability and anticorrosion properties and greatly reduce bacterial survival times at these two temperatures (after 120 min at 20 °C and 360 min at 4 °C, no E. coli could be detected on a copper nickel with a 73% copper content). Use of a surface material with antibacterial properties could aid in preventing cross-contamination events in food processing and domestic environments, if standard hygiene measures fail.
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The acquisition of microbes with the subsequent development of an infection while hospitalized continues to challenge healthcare worldwide. The CDC estimates the overall risk, mortality and cost to the USA to be ~5%, 100,000 deaths and ~45 billion additional dollars; rates for Medical Intensive Care Units (MICU) are higher where the risk often exceeds 25%. At issue is whether reducing the microbial burden of the environment can lead to an effective method to limit the risk of acquiring an infection while hospitalized.
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Aims: As copper has been previously suggested as an antimicrobial surface, we tested the effectiveness of copper as an antifungal surface which could be used in air-conditioning systems as an alternative to aluminium. Methods and results: Coupons of copper (C11000) and aluminium were inoculated with fungal isolates (Aspergillus spp., Fusarium spp., Penicillium chrysogenum and Candida albicans) for various time periods. Culture on potato dextrose agar and an in situ viability assay using the fluorochrome FUN-1 were used to determine whether spores had survived. The results showed increased die off of fungal isolates tested compared to aluminium. In addition, copper also prevented the germination of spores present, thereby reducing the risk of the release of spores. Conclusions: Copper offered an antifungal surface and prevented subsequent germination of spores present. FUN-1 demonstrated that fungal spores entered into a viable but not culturable (VBNC) state on copper indicating the importance of using such methods when assessing the effect of an antifungal as culture alone may give false results. Significance and impact of study: Copper offers a valuable alternative to aluminium which could be used in air-conditioning systems in buildings, particularly in hospital environments where patients are more susceptible to fungal infections.
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Contaminated touch surfaces have been implicated in the spread of hospital-acquired infections, and the use of biocidal surfaces could help to reduce this cross-contamination. In a previous study we reported the death of aqueous inocula of pathogenic Enterococcus faecalis or Enterococcus faecium isolates, simulating fomite surface contamination, in 1 h on copper alloys, compared to survival for months on stainless steel. In our current study we observed an even faster kill of over a 6-log reduction of viable enterococci in less than 10 min on copper alloys with a "dry" inoculum equivalent to touch contamination. We investigated the effect of copper(I) and copper(II) chelation and the quenching of reactive oxygen species on cell viability assessed by culture and their effects on genomic DNA, membrane potential, and respiration in situ on metal surfaces. We propose that copper surface toxicity for enterococci involves the direct or indirect action of released copper ionic species and the generation of superoxide, resulting in arrested respiration and DNA breakdown as the first stages of cell death. The generation of hydroxyl radicals by the Fenton reaction does not appear to be the dominant instrument of DNA damage. The bacterial membrane potential is unaffected in the early stages of wet and dry surface contact, suggesting that the membrane is not compromised until after cell death. These results also highlight the importance of correct surface cleaning protocols to perpetuate copper ion release and prevent the chelation of ions by contaminants, which could reduce the efficacy of the surface.
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Biofilms are slimy aggregates of microbes that are likely responsible for many chronic infections as well as for contamination of clinical and industrial environments. Pseudomonas aeruginosa is a prevalent hospital pathogen that is well known for its ability to form biofilms that are recalcitrant to many different antimicrobial treatments. We have devised a high-throughput method for testing combinations of antimicrobials for synergistic activity against biofilms, including those formed by P. aeruginosa. This approach was used to look for changes in biofilm susceptibility to various biocides when these agents were combined with metal ions. This process identified that Cu2+ works synergistically with quaternary ammonium compounds (QACs; specifically benzalkonium chloride, cetalkonium chloride, cetylpyridinium chloride, myristalkonium chloride, and Polycide) to kill P. aeruginosa biofilms. In some cases, adding Cu2+ to QACs resulted in a 128-fold decrease in the biofilm minimum bactericidal concentration compared to that for single-agent treatments. In combination, these agents retained broad-spectrum antimicrobial activity that also eradicated biofilms of Escherichia coli, Staphylococcus aureus, Salmonella enterica serovar Cholerasuis, and Pseudomonas fluorescens. To investigate the mechanism of action, isothermal titration calorimetry was used to show that Cu2+ and QACs do not interact in aqueous solutions, suggesting that each agent exerts microbiological toxicity through independent biochemical routes. Additionally, Cu2+ and QACs, both alone and in combination, reduced the activity of nitrate reductases, which are enzymes that are important for normal biofilm growth. Collectively, the results of this study indicate that Cu2+ and QACs are effective combinations of antimicrobials that may be used to kill bacterial biofilms.
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Copper is both an essential nutrient and a toxic element able to catalyze free radicals formation which damage lipids and proteins. Although the available copper redox species in aerobic environment is Cu(II), proteins that participate in metal homeostasis use Cu(I). With isolated Escherichia coli membranes, we have previously shown that electron flow through the respiratory chain promotes cupric ions reduction by NADH dehydrogenase-2 and quinones. Here, we determined Cu(II)-reductase activity by whole cells using strains deficient in these respiratory chain components. Measurements were done by the appearance of Cu(I) in the supernatants of cells exposed to sub-lethal Cu(II) concentrations. In the absence of quinones, the Cu(II)-reduction rate decreased ~70% in respect to the wild-type strain, while this diminution was about 85% in a strain lacking both NDH-2 and quinones. The decrease was ~10% in the absence of only NDH-2. In addition, we observed that quinone deficient strains failed to grow in media containing either excess or deficiency of copper, as we have described for NDH-2 deficient mutants. Thus, the Cu(II)-reduction by E. coli intact cells is mainly due to quinones and to a lesser extent to NDH-2, in a quinone-independent way. To our knowledge, this is the first in vivo demonstration of the involvement of E. coli respiratory components in the Cu(II)-reductase activity which contributes to the metal homeostasis.
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Metallic copper surfaces have strong antimicrobial properties and kill bacteria, such as Escherichia coli, within minutes in a process called contact killing. These bacteria are exposed to acute copper stress under dry conditions which is different from chronic copper stress in growing liquid cultures. Currently, the physiological changes of E. coli during the acute contact killing process are largely unknown. Here, a label-free, quantitative proteomic approach was employed to identify the differential proteome profiles of E. coli cells after sub-lethal and lethal exposure to dry metallic copper. Of the 509 proteins identified, 110 proteins were differentially expressed after sub-lethal exposure, whereas 136 proteins had significant differences in their abundance levels after lethal exposure to copper compared to unexposed cells. A total of 210 proteins were identified only in copper-responsive proteomes. Copper surface stress coincided with increased abundance of proteins involved in secondary metabolite biosynthesis, transport and catabolism, including efflux proteins and multidrug resistance proteins. Proteins involved in translation, ribosomal structure and biogenesis functions were down-regulated after contact to metallic copper. The set of changes invoked by copper surface-exposure was diverse without a clear connection to copper ion stress but was different from that caused by exposure to stainless steel. Oxidative posttranslational modifications of proteins were observed in cells exposed to copper but also from stainless steel surfaces. However, proteins from copper stressed cells exhibited a higher degree of oxidative proline and threonine modifications.
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Bacteria, yeasts, and viruses are rapidly killed on metallic copper surfaces, and the term "contact killing" has been coined for this process. While the phenomenon was already known in ancient times, it is currently receiving renewed attention. This is due to the potential use of copper as an antibacterial material in health care settings. Contact killing was observed to take place at a rate of at least 7 to 8 logs per hour, and no live microorganisms were generally recovered from copper surfaces after prolonged incubation. The antimicrobial activity of copper and copper alloys is now well established, and copper has recently been registered at the U.S. Environmental Protection Agency as the first solid antimicrobial material. In several clinical studies, copper has been evaluated for use on touch surfaces, such as door handles, bathroom fixtures, or bed rails, in attempts to curb nosocomial infections. In connection to these new applications of copper, it is important to understand the mechanism of contact killing since it may bear on central issues, such as the possibility of the emergence and spread of resistant organisms, cleaning procedures, and questions of material and object engineering. Recent work has shed light on mechanistic aspects of contact killing. These findings will be reviewed here and juxtaposed with the toxicity mechanisms of ionic copper. The merit of copper as a hygienic material in hospitals and related settings will also be discussed.
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Metallic copper surfaces rapidly and efficiently kill bacteria. Cells exposed to copper surfaces accumulated large amounts of copper ions, and this copper uptake was faster from dry copper than from moist copper. Cells suffered extensive membrane damage within minutes of exposure to dry copper. Further, cells removed from copper showed loss of cell integrity. Acute contact with metallic copper surfaces did not result in increased mutation rates or DNA lesions. These findings are important first steps for revealing the molecular sensitive targets in cells lethally challenged by exposure to copper surfaces and provide a scientific explanation for the use of copper surfaces as antimicrobial agents for supporting public hygiene.
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Surfaces 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 inactivate Candida albicans and Saccharomyces cerevisiae within 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 hyperactive S. cerevisiae Ctr1p (ScCtr1p) copper uptake transporter in Saccharomyces resulted in faster inactivation of mutant cells than of wild-type cells. Similarly, lack of the C. albicans Crp1p (CaCrp1p) copper-efflux P-type ATPase or the metallothionein CaCup1p caused more-rapid killing of Candida mutant cells than of wild-type cells. Candida and Saccharomyces took 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 with Saccharomyces. 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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The increasing incidence of nosocomial infections caused by glycopeptide-resistant enterococci is a global concern. Enterococcal species are also difficult to eradicate with existing cleaning regimens; they can survive for long periods on surfaces, thus contributing to cases of reinfection and spread of antibiotic-resistant strains. We have investigated the potential use of copper alloys as bactericidal surfaces. Clinical isolates of vancomycin-resistant Enterococcus faecalis and Enterococcus faecium were inoculated onto copper alloy and stainless steel surfaces. Samples were assessed for the presence of viable cells by conventional culture, detection of actively respiring cells, and assessment of cell membrane integrity. Both species survived for up to several weeks on stainless steel. However, no viable cells were detected on any alloys following exposure for 1 h at an inoculum concentration of <or=10(4) CFU/cm(2). Analysis of genomic and plasmid DNA from bacterial cells recovered from metal surfaces indicates substantial disintegration of the DNA following exposure to copper surfaces that is not evident in cells recovered from stainless steel. The DNA fragmentation is so extensive, and coupled with the rapid cell death which occurs on copper surfaces, that it suggests that mutation is less likely to occur. It is therefore highly unlikely that genetic information can be transferred to receptive organisms recontaminating the same area. A combination of effective cleaning regimens and contact surfaces containing copper could be useful not only to prevent the spread of viable pathogenic enterococci but also to mitigate against the occurrence of potential resistance to copper, biocides, or antibiotics and the spread of genetic determinants of resistance to other species.
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The ability of many bacteria to adhere to surfaces and to form biofilms has major implications in a variety of industries including the food industry, where biofilms create a persistent source of contamination. The formation of a biofilm is determined not only by the nature of the attachment surface, but also by the characteristics of the bacterial cell and by environmental factors. This review focuses on the features of the bacterial cell surface such as flagella, surface appendages and polysaccharides that play a role in this process, in particular for bacteria linked to food-processing environments. In addition, some aspects of the attachment surface, biofilm control and eradication will be highlighted.
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A meat factory commensal bacterium, Acinetobacter calcoaceticus, affected the spatial distribution of Escherichia coli O157:H7 surface colonization. The biovolume of E. coli O157:H7 was 400-fold higher (1.2 x 10(6) microm(3)) in a dynamic cocultured biofilm than in a monoculture (3.0 x 10(3) microm(3)), and E. coli O157:H7 colonized spaces between A. calcoaceticus cell clusters.
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Bacteria are rapidly killed on copper surfaces. However, the mechanism of this process remains unclear. Using Enterococcus hirae, the effect of inactivation of copper homeostatic genes and of medium compositions on survival and copper dissolution was tested. The results support a role for dissolved copper ions in killing.
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Enterohemorrhagic Escherichia coli O157:H7 is a major food-borne pathogen causing severe disease in humans worldwide. Healthy cattle are a reservoir of E. coli O157:H7 and bovine food products and fresh produce contaminated with bovine waste are the most common sources for disease outbreaks in the United States. E. coli O157:H7 also survives well in the environment. The ability to cause human disease, colonize the bovine gastrointestinal tract, and survive in the environment, requires that E. coli O157:H7 adapt to a wide variety of conditions. Three major virulence factors of E. coli O157:H7 have been identified including Shiga toxins, a pathogenicity island called the locus of enterocyte effacement, and an F-like plasmid, pO157. Among these virulence factors, the role of the pO157 is least understood. This review provides a board overview of E. coli O157:H7 with an emphasis on the pO157.
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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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To compare silver and copper, metals with known antimicrobial properties, by evaluating the effects of temperature and humidity on efficacy by challenging with methicillin resistant Staphylococcus aureus (MRSA). Using standard methodology described in a globally used Japanese Industrial Standard, JIS Z 2801, a silver ion-containing material exhibited >5 log reduction in MRSA viability after 24 h at >90% relative humidity (RH) at 20 degrees C and 35 degrees C but only a <0.3 log at approximately 22% RH and 20 degrees C and no reduction at approximately 22% RH and 35 degrees C. Copper alloys demonstrated >5 log reductions under all test conditions. While the high humidity (>90% RH) and high temperature (35 degrees C) utilized in JIS Z 2801 produce measurable efficacy in a silver ion-containing material, it showed no significant response at lower temperature and humidity levels typical of indoor environments. The high efficacy levels displayed by the copper alloys, at temperature and humidity levels typical of indoor environments, compared to the low efficacy of the silver ion-containing material under the same conditions, favours the use of copper alloys as antimicrobial materials in indoor environments such as hospitals.
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The ability of metal ions to damage DNA and cause mutagenesis has been analyzed with reversion and forward mutation assays using single-stranded DNA templates. We previously reported that incubation of phi X174 am3 DNA with Fe2+ in vitro results in mutagenesis when the treated DNA is transfected into Escherichia coli spheroplasts (Loeb, L. A., James, E. A., Waltersdorph, A. M., and Klebanoff, S. J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3918-3922, 1988). We now extend these studies to other metal ions. Of the metal ions tested, copper ions were the most mutagenic; the frequency of mutants produced was equal to or greater than that produced by Fe2+. Mutagenesis by Cu+ was diminished by catalase, mannitol, and superoxide dismutase suggesting the involvement of H2O2, hydroxyl ions, and superoxide, respectively. However, the findings that Cu+ and Cu2+ are nearly equally mutagenic and that the mutagenic activities are not completely inhibited by oxygen free radical scavengers make it unlikely that the mechanism for mutagenesis is simply the production of hydroxyl free radicals. The spectra of mutations produced by either copper ion using the lacZ gene as a target are very similar and differ from those reported with other agents. The predominant mutagenic sequence changes are single-base substitutions, the most frequent being replacement of a template C by a T. This transition presumably results from mispairing of an altered C with deoxyadenosine. Copper-induced mutations are not randomly distributed. Instead, they are found predominantly in clusters suggesting direct interaction of copper ions with specific nucleotide sequences in DNA. Evidence is considered that the high frequency of C----T transitions may be a common manifestation of DNA damage by oxygen radicals.
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The site specificity of metallic ion binding in Escherichia coli K-12 lipopolysaccharide was assessed by collecting high-resolution phosphorus nuclear magnetic resonance spectra in the presence of manganese, a paramagnetic divalent cation. This technique revealed high-affinity interactions between the cation and all of the lipopolysaccharide phosphoryl groups. To ascertain whether the carboxyl groups of 2-keto-3-deoxyoctonate contributed to the metal cation binding, lipopolysaccharide was chemically modified using a glycine ethyl ester - carbodiimide reaction. Of the three available carboxyl groups, only one was neutralized by the exogenously added ligand; the others appeared to be cross-linked within the molecule. By analogy, only one carboxyl group should be freely available for binding metallic ions, while the others are probably neutralized by the close proximity of endogenous amino substituents. Although high-resolution phosphorus nuclear magnetic resonance showed that an intermolecular conformational change had occurred after the carboxyl groups were neutralized, titration with manganese revealed no differences in the apparent strength of the interactions between the cation and the phosphoryl groups. Together, these data suggest that the high affinity of lipopolysaccharide for divalent metallic ions can be attributed primarily to the phosphoryl substituents and not free carboxyl groups.
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Although flow cytometry has been used to study antibiotic effects on bacterial membrane potential (MP) and membrane permeability, flow cytometric results are not always well correlated to changes in bacterial counts. Using new, precise techniques, we simultaneously measured MP, membrane permeability, and particle counts of antibiotic-treated and untreated Staphylococcus aureus and Micrococcus luteus cells. MP was calculated from the ratio of red and green fluorescence of diethyloxacarbocyanine [DiOC(2)(3)]. A normalized permeability parameter was calculated from the ratio of far red fluorescence of the nucleic acid dye TO-PRO-3 and green DiOC(2)(3) fluorescence. Bacterial counts were calculated by the addition of polystyrene beads to the sample at a known concentration. Amoxicillin increased permeability within 45 min. At concentrations of <1 microg/ml, some organisms showed increased permeability but normal MP; this population disappeared after 4 h, while bacterial counts increased. At amoxicillin concentrations above 1 microg/ml, MP decreased irreversibly and the particle counts did not increase. Tetracycline and erythromycin caused smaller, dose- and time-dependent decreases in MP. Tetracycline concentrations of <1 microg/ml did not change permeability, while a tetracycline concentration of 4 microg/ml permeabilized 50% of the bacteria; 4 microg of erythromycin per ml permeabilized 20% of the bacteria. Streptomycin decreased MP substantially, with no effect on permeability; chloramphenicol did not change either permeability or MP. Erythromycin pretreatment of bacteria prevented streptomycin and amoxicillin effects. Flow cytometry provides a sensitive means of monitoring the dynamic cellular events that occur in bacteria exposed to antibacterial agents; however, it is probably simplistic to expect that changes in a single cellular parameter will suffice to determine the sensitivities of all species to all drugs.
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Plasmids harboring multiple antimicrobial-resistance determinants (R plasmids) were transferred in simulated natural microenvironments from various bacterial pathogens of human, animal, or fish origin to susceptible strains isolated from a different ecological niche. R plasmids in a strain of the human pathogen Vibrio cholerae O1 E1 Tor and a bovine Escherichia coli strain were conjugated to a susceptible strain of the fish pathogenic bacterium Aeromonas salmonicida subsp. salmonicida in marine water. Conjugations of R plasmids between a resistant bovine pathogenic E. coli strain and a susceptible E. coli strain of human origin were performed on a hand towel contaminated with milk from a cow with mastitis. A similar conjugation event between a resistant porcine pathogenic E. coli strain of human origin was studied in minced meat on a cutting board. Conjugation of R plasmids between a resistant strain of the fish pathogenic bacterium A. salmonicida subsp. salmonicida and a susceptible E. coli strain of human origin was performed in raw salmon on a cutting board. R plasmids in a strain of A. salmonicida subsp. salmonicida and a human pathogenic E. coli strain were conjugated to a susceptible porcine E. coli strain in porcine feces. Transfer of the different R plasmids was confirmed by plasmid profile analyses and determination of the resistance pattern of the transconjugants. The different R plasmids were transferred equally well under simulated natural conditions and under controlled laboratory conditions, with median conjugation frequencies ranging from 3 x 10(-6) to 8 x 10(-3). The present study demonstrates that conjugation and transfer of R plasmids is a phenomenon that belongs to the environment and can occur between bacterial strains of human, animal, and fish origins that are unrelated either evolutionarily or ecologically even in the absence of antibiotics. Consequently, the contamination of the environment with bacterial pathogens resistant to antimicrobial agents is a real threat not only as a source of disease but also as a source from which R plasmids can easily spread to other pathogens of diverse origins.
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Knowledge of biofilm structure and function has changed significantly in the last few years due to advances in light microscopy. One pertinent example is the use of scanning confocal laser microscopy (SCLM) to visualise corrosion pits caused by the biofilm mosaic footprint on corroding metal surfaces. Nevertheless, SCLM has some limitations as to its widespread use, including cost, inability to observe motile bacteria and eukaryotic grazers within biofilms, and difficulty to scan a curved surface. By contrast, episcopic differential interference contrast (EDIC) microscopy has provided a rapid, real time analysis of biofilms on opaque, curved, natural or man-made surfaces without the need for cover slips and oil. EDIC, coupled with epi-fluorescence (EDIC/EF), microscopy has been used successfully to visualise the 3-D biofilm structure, physiological niches, protozoal grazing and iron biomineralization, and the location of specific pathogens such as Legionella pneumophila, Campylobacter jejuni and Cryptosporidium parvum. These species were identified using gold nanoparticles or fluorophores coupled to monoclonal antibodies or 16S rRNA probes, respectively. Among its many potential uses, the EDIC technique will provide a rapid procedure to facilitate the calibration of the modern generation of biofilm-sensing electrodes.
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