Metallic copper corrosion rates, moisture content, and growth medium influence survival of copper ion-resistant bacteria

The rapid killing of various bacteria in contact with metallic copper is thought to be influenced by the influx of copper ions into the cells, but the exact mechanism is not fully understood. This study showed that the kinetics of contact killing of copper surfaces depended greatly on the amount of moisture present, copper content of alloys, type of medium used, and type of bacteria. We examined antibiotic- and copper ion-resistant strains of Escherichia coli and Enterococcus faecium isolated from pig farms following the use of copper sulfate as feed supplement. The results showed rapid killing of both copper ion-resistant E. coli and E. faecium strains when samples in rich medium were spread in a thin, moist layer on copper alloys with 85% or greater copper content. E. coli strains were rapidly killed under dry conditions, while E. faecium strains were less affected. Electroplated copper surface corrosion rates were determined from electrochemical polarization tests using the Stern–Geary method and revealed decreased corrosion rates with benzotriazole and thermal oxide coating. Copper ion-resistant E. coli and E. faecium cells suspended in 0.8% NaCl showed prolonged survival rates on electroplated copper surfaces with benzotriazole coating and thermal oxide coating compared to surfaces without anti-corrosion treatment. Control of surface corrosion affected the level of copper ion influx into bacterial cells, which contributed directly to bacterial killing.     

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

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

Antimicrobial mortar surfaces for the improvement of hygienic conditions

Aims:  To evaluate the effectiveness of various antimicrobial mortar formulations in inhibiting the growth of a selection of pathogens of environmental and hygienic concern.
Methods and Results:  Mortar prisms containing triclosan-incorporated fibres or different concentrations of silver copper zeolites were incubated with Escherichia coli , Listeria monocytogenes , Salmonella enterica or Staphylococcus aureus at 4 or 20°C for 24 h. From plate counting, a substantial bactericidal effect (>4 log units) could only be observed for the mortar specimens containing more than 3% zeolites on cement weight base, the effect being more pronounced at 20°C compared to 4°C. No inhibitory effect could be observed for mortar specimens containing antimicrobial fibres. Adenosinetriphosphate (ATP) measurements allowed for a rapid indication of the occurrence of antimicrobial activity.
Conclusions:  In order to obtain a bactericidal effect on mortar surfaces, concentrations of silver copper zeolites of more then 3% are required.
Significance and Impact of the Study:  To our knowledge, this is the first study in which the effectiveness of various antimicrobial mortar mixtures towards the inhibition of pathogens has been evaluated in a quantitative way. Antimicrobial concrete mixtures can be used for the improvement of the hygienic conditions in a variety of environments.     

不同类型铜合金板的杀菌特性

【目的】微生物对可接触表面的污染给公共卫生带来了极大的威胁。利用具有杀菌特性的铜及铜合金代替不锈钢等制品,可以降低消毒剂的使用和细菌的传播。【方法】通过分析3株金黄色葡萄球菌和2株大肠杆菌在铜及铜合金平板上的存活时间,对不同类型铜合金的杀菌特性进行了探索。【结果】铜合金平板的杀菌能力与其铜含量成正比;铜合金对同属细菌的杀菌能力相近,对不同属细菌则有一定差异;铜合金的杀菌效率与细菌对Cu2+抗性没有直接联系;铜合金杀菌的效率与细菌的细胞壁结构可能有很大关联。【结论】铜及铜合金是较好的杀菌材料。     

Fabrication of silver nanoparticles by Phoma glomerata and its combined effect against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus

Aims:  We report extracellular synthesis of silver nanoparticles (Ag-NPs) from Phoma glomerata and its efficacy against Escherichia coli , Staphylococcus aureus and Pseudomonas aeruginosa . The bacteria exhibiting resistance to various antibiotics showed remarkable sensitivity, when used in combination of antibiotics and Ag-NPs.
Methods and Results:  Biosynthesis of Ag-NPs was carried out by challenging the fungal cell filtrate with 1 mmol l⁻¹ silver nitrate. The Ag-NPs were characterized with the help of UV–Visible spectrophotometer and Fourier transform infrared spectroscopy. Scanning electron microscopy was carried out to detect the size of Ag-NPs. Evaluation of the combined effect(s) was studied by disc diffusion method against E. coli , Staph. aureus and Ps. aeruginosa .
Conclusions:  The biosynthesis route seems to be eco-friendly and easy to scale up the process. Thus, these Ag-NPs may prove as a better candidate for drugs and can potentially eliminate the problem of chemical agents because of their biogenic nature.
Significance and Impact of the Study:  The bacterial resistance against antibiotics has been increasing with alarming rate. To overcome this problem, there is a pressing need to develop bactericidal agents. Ag-NPs may prove to be an answer to drug-resistant bacteria.     

Rapid inactivation of <Emphasis Type="Italic">Cronobacter sakazakii</Emphasis> on copper alloys following periods of desiccation stress

Cronobacter spp. have been identified as the causative agent in meningitis and necrotizing enterocolitis in premature infants which can be linked to the bacterium's desiccation resistance and persistence in powdered infant formula. In this study we examined the efficacy of copper cast alloys in contact killing of Cronobacter sakazakii following periods of desiccation stress. Cronobacter sakazakii cells suspended in Tryptic Soy Broth (TSB) were killed within 10 min while kept moist on 99.9% copper alloys and within 1 min of drying on 99.9% copper alloys. Survival times were unchanged after cells suspended in TSB were desiccated for 33 days. Cronobacter sakazakii cells suspended in infant formula were killed within 30 min under moist conditions and within 3 min of drying on 99.9% copper alloys. However, when desiccated in infant formula for 45 days, survival times decreased to 10 and 1 min in moist and dry conditions, respectively. In contrast, no decrease in viable cells was noted on stainless steel surfaces under the experimental conditions employed in this study. Cronobacter sakazakii was rapidly killed on copper alloys under all testing conditions of this study indicating that desiccation and copper ion resistance do not prolong survival. These results could have important implications for the utilization of copper in the production and storage of powdered infant formula.     

Synergistic interactions between cefalexin and kanamycin in Mueller–Hinton broth medium and in milk

Aims:  This study investigated the in vitro bactericidal activity of an intramammary drug product by comparing the kill kinetics of cefalexin and kanamycin, alone and in fixed ratio combination, against Streptococcus uberis , Staphylococcus aureus and Escherichia coli strains isolated from field cases of bovine mastitis. The effect of milk as a diluent on the rate of bacterial killing was also assessed.
Methods and Results:  Antibacterial kill kinetics was determined against each bacterial strain in Mueller–Hinton broth (MHB) and in milk. In MHB, the fixed cefalexin : kanamycin combination (1·5 : 1 w/w) exhibited a clear synergistic bactericidal activity against the strains tested. The combination also showed an enhanced killing activity in milk, as compared to either agent alone.
Conclusions:  The data show the occurrence of synergistic interactions between cefalexin and kanamycin, resulting in a faster and enhanced bactericidal activity against major mastitis pathogens.
Significance and Impact of the Study:  The study demonstrated that the combination exhibited a larger and faster rate of kill of S. aureus , S. uberis and E. coli compared to either cefalexin or kanamycin alone, while using a lower total amount of antibiotic. Synergistic and additive effects were also observed when milk was used as a medium. The results support the use of this combination of narrow spectrum antibiotics to treat clinical mastitis via the intramammary route and provide data on its killing kinetics.     

Comparison of bacteriostatic and bactericidal activity of 13 essential oils against strains with varying sensitivity to antibiotics

Aims:  To compare the bacteriostatic and bactericidal activity of 13 chemotyped essential oils (EO) on 65 bacteria with varying sensitivity to antibiotics.
Methods and Results:  Fifty-five bacterial strains were tested with two methods used for evaluation of antimicrobial activity (CLSI recommendations): the agar dilution method and the time-killing curve method. EO containing aldehydes ( Cinnamomum verum bark and Cymbopogon citratus ), phenols ( Origanum compactum , Trachyspermum ammi , Thymus satureioides , Eugenia caryophyllus and Cinnamomum verum leaf) showed the highest antimicrobial activity with minimum inhibitory concentration (MIC) <2% (v/v) against all strains except Pseudomonas aeruginosa . Alcohol-based EO ( Melaleuca alternifolia , Cymbopogon martinii and Lavandula angustifolia ) exhibited varying degrees of activity depending on Gram status. EO containing 1·8-cineole and hydrocarbons ( Eucalyptus globulus , Melaleuca cajeputii and Citrus sinensis ) had MIC_90% ≥ 10% (v/v). Against P. aeruginosa , only C. verum bark and O. compactum presented MIC ≤2% (v/v). Cinnamomum verum bark, O. compactum , T. satureioides , C. verum leaf and M. alternifolia were bactericidal against Staphylococcus aureus and Escherichia coli at concentrations ranging from to 0·31% to 10% (v/v) after 1 h of contact. Cinnamomum verum bark and O. compactum were bactericidal against P. aeruginosa within 5 min at concentrations <2% (v/v).
Conclusions:  Cinnamomum verum bark had the highest antimicrobial activity, particularly against resistant strains.
Significance and Impact of the Study:  Bacteriostatic and bactericidal activity of EO on nosocomial antibiotic-resistant strains.     

Evaluation of efficacy of disinfectants against Salmonella from the feed industry

Aims:  To evaluate disinfectants against Salmonella under conditions relevant for the feed industry.
Materials and Results:  A survey on the use of disinfectants in feed industry showed that a range of different types was used. Nine disinfectants, reflecting the most commonly used active ingredients, were tested for bactericidal activity on Salmonella isolated from the feed industry. All disinfectants were efficient against Salmonella in suspension. The bactericidal effect varied considerably between different types of active compounds on bacteria dried on surfaces or grown as biofilm. Tenside-based disinfectants and hypochlorite were found to have low bactericidal activity and the efficiency was significantly reduced when the ratio of amount disinfectant per cell decreased. It was shown that concentrations of 70–80% ethanol were effective against Salmonella. Among the disinfectants tested a product containing 70% ethanol was most efficient followed by Virkon S.
Conclusions:  Many disinfectants had low bactericidal activity against Salmonella at surfaces while Virkon S and a product containing 70% ethanol were most effective. Another advantage of ethanol-based disinfectants is evaporation of ethanol, resulting in low residual water after use.
Significance and Impact of the Study:  Use of the disinfectants found to be efficient against surface associated Salmonella , may assist the industry in combating Salmonella .     

Membrane lipid peroxidation in copper alloy-mediated contact killing of Escherichia coli

Copper alloy surfaces are passive antimicrobial sanitizing agents that kill bacteria, fungi, and some viruses. Studies of the mechanism of contact killing in Escherichia coli implicate the membrane as the target, yet the specific component and underlying biochemistry remain unknown. This study explores the hypothesis that nonenzymatic peroxidation of membrane phospholipids is responsible for copper alloy-mediated surface killing. Lipid peroxidation was monitored with the thiobarbituric acid-reactive substances (TBARS) assay. Survival, TBARS levels, and DNA degradation were followed in cells exposed to copper alloy surfaces containing 60 to 99.90% copper or in medium containing CuSO(4). In all cases, TBARS levels increased with copper exposure levels. Cells exposed to the highest copper content alloys, C11000 and C24000, exhibited novel characteristics. TBARS increased immediately at a very rapid rate but peaked at about 30 min. This peak was associated with the period of most rapid killing, loss in membrane integrity, and DNA degradation. DNA degradation is not the primary cause of copper-mediated surface killing. Cells exposed to the 60% copper alloy for 60 min had fully intact genomic DNA but no viable cells. In a fabR mutant strain with increased levels of unsaturated fatty acids, sensitivity to copper alloy surface-mediated killing increased, TBARS levels peaked earlier, and genomic DNA degradation occurred sooner than in the isogenic parental strain. Taken together, these results suggest that copper alloy surface-mediated killing of E. coli is triggered by nonenzymatic oxidative damage of membrane phospholipids that ultimately results in the loss of membrane integrity and cell death.     

Effects of temperature and humidity on the efficacy of methicillin-resistant Staphylococcus aureus challenged antimicrobial materials containing silver and copper

Aims:  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) .
Methods and Results:  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°C and 35°C but only a <0·3 log at ∼22% RH and 20°C and no reduction at ∼22% RH and 35°C. Copper alloys demonstrated >5 log reductions under all test conditions.
Conclusions:  While the high humidity (>90% RH) and high temperature (35°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.
Significance and Impact of the Study:  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.     

Survival of bacteria on metallic copper surfaces in a hospital trial

Basic chemistry of copper is responsible for its Janus-faced feature: on one hand, copper is an essential trace element required to interact efficiently with molecular oxygen. On the other hand, interaction with reactive oxygen species in undesired Fenton-like reactions leads to the production of hydroxyl radicals, which rapidly damage cellular macromolecules. Moreover, copper cations strongly bind to thiol compounds disturbing redox-homeostasis and may also remove cations of other transition metals from their native binding sites in enzymes. Nature has learned during evolution to deal with the dangerous yet important copper cations. Bacterial cells use different efflux systems to detoxify the metal from the cytoplasm or periplasm. Despite this ability, bacteria are rapidly killed on dry metallic copper surfaces. The mode of killing likely involves copper cations being released from the metallic copper and reactive oxygen species. With all this knowledge about the interaction of copper and its cations with cellular macromolecules in mind, experiments were moved to the next level, and the antimicrobial properties of copper-containing alloys in an “everyday” hospital setting were investigated. The alloys tested decreased the number of colony-forming units on metallic copper-containing surfaces by one third compared to control aluminum or plastic surfaces. Moreover, after disinfection, repopulation of the surfaces was delayed on copper alloys. This study bridges a gap between basic research concerning cellular copper homeostasis and application of this knowledge. It demonstrates that the use of copper-containing alloys may limit the spread of multiple drug-resistant bacteria in hospitals.     

The assessment of the antibacterial and antifungal activities of aspirin, EDTA and aspirin–EDTA combination and their effectiveness as antibiofilm agents

Aims:  To evaluate the antimicrobial activities of aspirin, EDTA and an aspirin-EDTA (A-EDTA) combination against Pseudomonas aeruginosa , Escherichia coli and Candida albicans in planktonic and biofilm cultures.
Methods and Results:  Minimal inhibitory concentrations (MIC) and minimal biocidal concentrations (MBC) were determined using twofold broth microdilution and viable counting methods, respectively. Aspirin's recorded MIC values ranged from 1·2 to 2·7 mg ml⁻¹. Checkerboard assay demonstrated a synergism in antimicrobial activity upon combination. Aspirin's minimal biofilm eradication concentration values (MBEC) against the established biofilms ranged between 1·35 and 3·83 mg ml⁻¹. A complete eradication of bacterial biofilms was achieved after a 4-h treatment with the A-EDTA combination.
Conclusion:  Both aspirin and EDTA possess broad-spectrum antimicrobial activity for both planktonic and biofilm cultures. Aspirin used at the MBEC for 24 h was successful in eradicating P. aeruginosa , E. coli and C. albicans biofilms established on abiotic surfaces. Moreover, the exposure to the A-EDTA combination (4 h) effected complete bacterial biofilm eradication.
Significance and Impact of the Study:  There is a continuous need for the discovery of new antimicrobial agents. Aspirin and EDTA are 'nonantibiotic drugs', the combination of which can be used successfully to treat and eradicate biofilms established on abiotic surfaces.     

Potential for preventing spread of fungi in air-conditioning systems constructed using copper instead of aluminium

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.     

Multiple roles of Pseudomonas aeruginosa TBCF10839 PilY1 in motility, transport and infection

Polymorphonuclear neutrophils are the most important mammalian host defence cells against infections with Pseudomonas aeruginosa. Screening of a signature tagged mutagenesis library of the non-piliated P. aeruginosa strain TBCF10839 uncovered that transposon inactivation of its pilY1 gene rendered the bacterium more resistant against killing by neutrophils than the wild type and any other of the more than 3000 tested mutants. Inactivation of pilY1 led to the loss of twitching motility in twitching-proficient wild-type PA14 and PAO1 strains, predisposed to autolysis and impaired the secretion of quinolones and pyocyanin, but on the other hand promoted growth in stationary phase and bacterial survival in murine airway infection models. The PilY1 population consisted of a major full-length and a minor shorter PilY1* isoform. PilY1* was detectable in small extracellular quinolone-positive aggregates, but not in the pilus. P. aeruginosa PilY1 is not an adhesin on the pilus tip, but assists in pilus biogenesis, twitching motility, secretion of secondary metabolites and in the control of cell density in the bacterial population.     

How much ozone bactericidal activity is compromised by plasma components?

Aims:  Evaluation of bactericidal effect of different concentrations of ozone when used (a) as a gas, or (b) dissolved in saline. The addition of hydrogen peroxide or 4-hydroxynonenal dissolved in saline was also tested, as well as the effect of human plasma.
Methods and Results:  Staphylococcus aureus , methicillin-resistant Staph. aureus (MRSA), and Pseudomonas aeruginosa , suspended in their culture media were tested. While all bacteria suspended in protein-free saline were killed at high ozone concentrations, they survived when as little as 5% human plasma was present. Hydrogen peroxide was 100-fold less active than ozone and needed to remain in contact with bacteria for at least 60 min. 4-hydroxynonenal (2 μmol l⁻¹) was inhibitory for proliferation of both Staph. aureus and MRSA, but not for Ps. aeruginosa .
Conclusions:  Ozone and the cascade of its derivative products are potent bactericidal agents, but even small amounts of human plasma, hence of hydro- and liposoluble antioxidants, in bacterial suspensions inhibit oxidation and protect bacteria.
Significance and Impact of the Study:  Any substantial in vivo cytocidal effect of ozone and its derivatives can be excluded. On the other hand, topical and continuous action of various ozone preparations remains valuable in a variety of skin and mucosal infections.     

Chelated iron sources are inhibitors of Pseudomonas aeruginosa biofilms and distribute efficiently in an in vitro model of drug delivery to the human lung

Aims:  To determine whether chelated sources of ferric iron were efficient inhibitors of biofilm formation in Pseudomonas aeruginosa and might be suitable for drug delivery to the lungs of cystic fibrosis (CF) patients via nebulization.
Methods and Results:  The response of P. aeruginosa biofilms to elevated iron concentrations in the form of eight structurally varied iron chelators in a microtitre plate assay for biofilm production was examined in the lab. Among these iron chelates, picolinic acid and acetohydroxamic acid-chelated iron were able to effectively thwart biofilm production in P. aeruginosa PA14 and in 20 clinical isolates of P. aeruginosa from a local hospital. The chelated iron sources showed excellent distribution in an Anderson cascade impactor model of particle size distribution in the human lung.
Conclusions:  Ferric picolinate and ferric acetohydroxamate are effective anti-biofilm compounds against both lab and clinical strains of P. aeruginosa and are readily nebulized into particles of suitable size for lung delivery.
Significance and Impact of the Study:  The data herein serve both to solidify the growing base of literature correlating high iron levels with biofilm inhibition in P. aeruginosa and to highlight the potential of these chelators as nebulized agents to combat biofilms of P. aeruginosa in CF patients.     

Bactericidal and sporicidal performance of a polymer-encapsulated chlorine dioxide-coated surface

Aims:  To investigate the physical characteristics and the bactericidal and sporicidal potential of a polymer-encapsulated ClO₂ coating.
Methods and Results:  An antimicrobial coating based on polymer-encapsulated ClO₂ was developed. A low viscosity, water/oil/water double emulsion coating was formulated for easy on-site application. Escherichia coli , Pseudomonas aeruginosa , Bacillus subtilis and Staphylococcus aureus were applied onto the coating to study the bactericidal capabilities of the coating. The bactericidal performance of the coating increased when the contact time with the tested bacteria increased. Over 99% of the E. coli , Ps. aeruginosa , B. subtilis were killed with a contact time of 30 min. Although endospores of B. subtilis are more resistant, about 75% of the spores were killed after 72 h on the coating. Moreover, a sustained release of gaseous ClO₂ was achieved to maintain about 90% removal of B. subtilis with a 10-min contact time during a 28-day study period. The coating also exhibits antiadhesive properties against bacteria.
Conclusions:  A polymer-encapsulated ClO₂ coating with sustained release of ClO₂ and promising bactericidal and sporicidal features was tested for 28 days.
Significance and Impact of the Study:  This study provides a new direction for developing polymer-encapsulated ClO₂ coatings that possess persistent bactericidal and sporicidal properties.