Bactericidal Activity of Copper and Niobium–Alloyed Austenitic Stainless Steel

Biofouling and microbiologically influenced corrosion are processes of material deterioration that originate from the attachment of microorganisms as quickly as the material is immersed in a nonsterile environment. Stainless steels, despite their wide use in different industries and as appliances and implant materials, do not possess inherent antimicrobial properties. Changes in hygiene legislation and increased public awareness of product quality makes it necessary to devise control methods that inhibit biofilm formation or to act at an early stage of the biofouling process and provide the release of antimicrobial compounds on a sustainable basis and at effective level. These antibacterial stainless steels may find a wide range of applications in fields, such as kitchen appliances, medical equipment, home electronics, and tools and hardware. The purpose of this study was to obtain antibacterial stainless steel and thus mitigate the microbial colonization and bacterial infection. Copper is known as an antibacterial agent; in contrast, niobium has been demonstrated to improve the antimicrobial effect of copper by stimulating the formation of precipitated copper particles and its distribution in the matrix of the stainless steel. Thus, we obtained slides of 3.8% copper and 0.1% niobium alloyed stainless steel; subjected them to three different heat treatment protocols (550°C, 700°C, and 800°C for 100, 200, 300, and 400 hours); and determined their antimicrobial activities by using different initial bacterial cell densities and suspending solutions to apply the bacteria to the stainless steels. The bacterial strain used in these experiments was Escherichia coli CCM 4517. The best antimicrobial effects were observed in the slides of stainless steel treated at 700°C and 800°C using an initial cell density of approximately 10⁵ cells ml⁻¹ and phosphate-buffered saline as the solution in which the bacteria came into contact with copper and niobium–containing steel.     

Biomolecules in multilayer film for antimicrobial and easy-cleaning stainless steel surface applications

Microorganisms are able to attach to, grow on, and ultimately form biofilms on a large variety of surfaces, such as industrial equipment, food contact surfaces, medical implants, prostheses and operating rooms. Once organized into biofilms, bacteria are difficult to remove and kill, which increases the risk of cross-contamination and infection. One way to address the problem may thus be to develop antibacterial, anti-adhesion, ‘easy cleaning’ surfaces. In this study, stainless steel (SS) surfaces with antibacterial properties were created by embedding several antimicrobial peptides in a multilayer film architecture. The biocidal effect of these surfaces was demonstrated against both Gram-positive and Gram-negative bacteria according to two ISO tests. Also, coating SS surfaces with either mucin or heparin led to a reduction of S. epidermidis adhesion of almost 95% vs the bare substratum. Finally, by combining both antibacterial and anti-adhesion biomolecules in the same multilayer film, SS surfaces with better cleanability were produced. This surface coating property may help to delay the buildup of a dead bacterial layer which is known to progressively reduce exposure of the coating, leading to an undesirable decrease in the antibacterial effect of the surface.     

Development of a CrN/Cu nanocomposite coating on titanium-modified stainless steel for antibacterial activity against Pseudomonas aeruginosa

A relatively simple method was developed to fabricate CrN/Cu nanocomposite coatings using pulsed DC magnetron sputtering for application in antibacterial activity. These nanocomposite coatings were applied on titanium (Ti)-modified stainless steel substrata (D-9 alloy) and the antibacterial activity of these coating with respect to the Gram-negative bacterium Pseudomonas aeruginosa was investigated qualitatively and quantitatively. Scanning electron microscopy, epifluorescence microscope analyses, and total viable counts confirmed that inclusion of copper in the CrN/Cu nanocomposite coatings provided antibacterial activity against P. aeruginosa. The quantitative examination of the bacterial activity of P. aeruginosa was estimated by the survival ratio as calculated from the number of viable cells which formed colonies on nutrient agar plates.     

Bacterial Community Structure of Biofilms on Artificial Surfaces in an Estuary

This study examined bacterial community structure of biofilms on stainless steel and polycarbonate in seawater from the Delaware Bay. Free-living bacteria in the surrounding seawater were compared to the attached bacteria during the first few weeks of biofilm growth. Surfaces exposed to seawater were analyzed by using 16S rDNA libraries, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE). Community structure of the free-living bacterial community was different from that of the attached bacteria according to FISH and DGGE. In particular, alpha-proteobacteria dominated the attached communities. Libraries of 16S rRNA genes revealed that representatives of the Rhodobacterales clade were the most abundant members of biofilm communities. Changes in community structure during biofilm growth were also examined by DGGE analysis. We hypothesized that bacterial communities on dissimilar surfaces would initially differ and become more similar over time. In contrast, the compositions of stainless steel and polycarbonate biofilms were initially the same, but differed after about 1 week of biofilm growth. These data suggest that the relationship between surface properties and biofilm community structure changes as biofilms grow on surfaces such as stainless steel and polycarbonate in estuarine water.     

In situ detection of bacteria involved in cathodic depolarization and stainless steel surface corrosion using microautoradiography

Aims: To examine the activity of bacteria involved in cathodic depolarization and surface corrosion on stainless steel in an in situ model system. Methods and Results: The microautoradiographic technique (MAR) was used to evaluate the activity of bacterial populations on stainless steel surfaces with a single cell resolution. Anaerobic uptake and fixation of ¹⁴C‐labelled bicarbonate occurred within corrosion sites in the absence of atmospheric hydrogen or other external electron donors, whereas it was taken up and fixed by bacteria at all other stainless steel surfaces in the presence of atmospheric hydrogen. This indicates that the bacteria utilized electrons originating from the corrosion sites due to the ongoing corrosion (cathodic depolarization). Conclusion: Under in situ conditions, bacteria were fixating ¹⁴C‐labelled bicarbonate at corrosion sites in the absence of atmospheric hydrogen. This indicates that electrons transferred to the bacteria provided energy for bicarbonate fixation due to cathodic depolarization. Significance and Impact of the Study: Application of the MAR method showed ongoing biocorrosion in the applied in situ model system and allowed in situ examination of bacterial activity on a single cell level directly on a metal surface providing information about potential corrosion mechanisms. Furthermore, application of fluorescence in situ hybridization in combination with MAR allows for identification of the active bacteria.     

Growth phase-dependent surface properties of Legionella pneumophila and their role in adhesion to stainless steel coated QCM-D sensors

Legionella pneumophila cell surface hydrophobicity and charge are important determinants of their mobility and persistence in engineered water systems (EWS). These surface properties may differ depending on the growth phase of L. pneumophila resulting in variable adhesion and persistence within EWS. We describe the growth-dependent variations in L. pneumophila cell surface hydrophobicity and surface charge using the microbial adhesion to hydrocarbon assay and microelectrophoresis, respectively, and their role in cell adhesion to stainless steel using a quartz crystal microbalance with dissipation (QCM-D) monitoring instrument. We observed a steady increase in L. pneumophila hydrophobicity during their lifecycle in culture media. Cell surfaces of stationary phase L. pneumophila were significantly more hydrophobic than their lag and midexponential counterparts. No significant changes in L. pneumophila cell surface charge were noted. Morphology of L. pneumophila remained relatively constant throughout their lifecycle. In the QCM-D study, lag and exponential phase L. pneumophila weakly adhered to stainless steel surfaces resulting in viscoelastic layers. In contrast, stationary phase bacteria were tightly and irreversibly bound to the surfaces, forming rigid layers. Our results suggest that the stationary phase of L. pneumophila would highly favour their adhesion to plumbing surfaces and persistence in EWS.     

Use of a quartz crystal microbalance to investigate the antiadhesive potential of N-acetyl-L-cysteine

The reduction of bacterial biofilm formation on stainless steel surfaces by N-acetyl-L-cysteine (NAC) is attributed to effects on bacterial growth and polysaccharide production, as well as an increase in the wettability of steel surfaces. In this report, we show that NAC-coated stainless steel and polystyrene surfaces affect both the initial adhesion of Bacillus cereus and Bacillus subtilis and the viscoelastic properties of the interaction between the adhered bacteria and the surface. A quartz crystal microbalance with dissipation was shown to be a powerful and sensitive technique for investigating changes in the applied NAC coating for initial cell surface interactions of bacteria. The kinetics of frequency and dissipation shifts were dependent on the bacteria, the life cycle stage of the bacteria, and the surface. We found that exponentially grown cells gave rise to a positive frequency shift as long as their cell surface hydrophobicity was zero. Furthermore, when the characteristics of binding between the cell and the surface for different growth phases were compared, the rigidity increased from exponentially grown cells to starved cells. There was a trend in which an increase in the viscoelastic properties of the interaction, caused by the NAC coating on stainless steel, resulted in a reduction in irreversibly adhered cells. Interestingly, for B. cereus that adhered to polystyrene, the viscoelastic properties decreased, while there was a reduction in adhered cells, regardless of the life cycle stage. Altogether, NAC coating on surfaces was often effective and could both decrease the initial adhesion and increase the detachment of adhered cells and spores. The most effective reduction was found for B. cereus spores, for which the decrease was caused by a combination of these two parameters.     

Copper as an antimicrobial agent against opportunistic pathogenic and multidrug resistant Enterobacter bacteria

Infections by Enterobacter species are common and are multidrug resistant. The use of bactericidal surface materials such as copper has lately gained attention as an effective antimicrobial agent due to its deadly effects on bacteria, yeast, and viruses. The aim of the current study was to assess the antibacterial activity of copper surfaces against Enterobacter species. The antibacterial activity of copper surfaces was tested by overlying 5×10(6) CFU/ml suspensions of representative Enterobacter strains and comparing bacterial survival counts on copper surfaces at room temperature. Iron, stainless steel, and polyvinylchloride (PVC) were used as controls. The mechanisms responsible for bacterial killing on copper surfaces were investigated by a mutagenicity assay of the D-cycloserin (cyclA gene), single cell gel electrophoresis, a staining technique, and inductively coupled plasma mass spectroscopy. Copper yielded a significant decrease in the viable bacterial counts at 2 h exposure and a highly significant decrease at 4 h. Loss of cell integrity and a significantly higher influx of copper into bacterial cells exposed to copper surfaces, as compared to those exposed to the controls, were documented. There was no increase in mutation rate and DNA damage indicating that copper contributes to bacterial killing by adversely affecting cellular structure without directly targeting the genomic DNA. These findings suggest that copper's antibacterial activity against Enterobacter species could be utilized in health care facilities and in food processing plants to reduce the bioburden, which would increase protection for susceptible members of the community.     

Reduction of bacterial adhesion on titanium-doped diamond-like carbon coatings

A range of titanium doped diamond-like carbon (Ti-DLC) coatings with different Ti contents were prepared on stainless steel substrates using a plasma-enhanced chemical vapour deposition technique. It was found that both the electron donor surface energy and the surface roughness of the Ti-DLC coatings increased with increasing Ti contents in the coatings. Bacterial adhesion to the coatings was evaluated against Escherichia coli WT F1693 and Pseudomonas aeruginosa ATCC 33347. The experimental data showed that bacterial adhesion decreased with the increases of the Ti content, the electron donor surface energy and surface roughness of the coatings, while the bacterial removal percentage increased with the increases of these parameters. The Ti-DLC coatings reduced bacterial attachment by up to 75% and increased bacterial detachment from 15 to 45%, compared with stainless steel control.     

Cell surface properties as factors involved in Proteus vulgaris adhesion to stainless steel under starvation conditions

The purpose of these investigations was to evaluate the influence of limited nutrient availability in the culture medium on Proteus vulgaris biofilm formation on surfaces of stainless steel. The relationship between the P. vulgaris adhesion to the abiotic surfaces, the cellular ATP levels, cell surface hydrophobicity and changes in the profiles of extracellular proteins and lipopolysaccharides was examined. In all experimental variants the starvation conditions induced the bacterial cells to adhere to the surfaces of stainless steel. Higher ATP content and lower cell surface hydrophobicity of P. vulgaris cells was observed upon nutrient-limited conditions. Under starvation conditions a reduction in the levels of extracellular low molecular weight proteins was noticed. High molecular weight proteins formed the conditioning layer on stainless steel plates, making the bacteria adhesion process more favorable. The production of low molecular weight carbohydrates promoted more advanced stages of P. vulgaris biofilm formation process on the surfaces of stainless steel upon starvation.     

A novel co-culture model of murine K12 osteosarcoma cells and S. aureus on common orthopedic implant materials: ‘the race to the surface’ studied in vitro

Infection is a major cause of orthopedic implant failure. There are few studies assessing both tissue cell and bacterial adherence on common orthopedic implant materials in a co-culture environment. An in vitro co-culture model was created using K12 osteosarcoma cells and Staphylococcus aureus in a medium incubated over metal disks for 48 h. The results showed that, in the presence of S. aureus, there were fewer osteosarcoma cells attached to the disks for all substrata tested. There were significantly more osteosarcoma cells adhering to the cobalt chrome than the stainless steel and titanium disks. Overall, in the presence of osteosarcoma cells, there were more bacteria adhering to the disks for all the substrata tested, with significantly more bacteria adhering to the stainless steel disks compared to cobalt chrome and titanium disks. Scanning electron microscopy verified that osteosarcoma cells and bacteria were adherent to the metal disks after incubation for 48 h. Furthermore, the observation that more bacteria were in the co-culture than in the control sample suggests that the osteosarcoma cells serve as a nutrient source for the bacteria. Future models assessing the interaction of osteogenic cells with bacteria on a substratum would be improved if the model accounted for the role of the immune system in secondary bone healing.     

The effect of turbulent flow and surface roughness on biofilm formation in drinking water

There is considerable interest in both Europe and the USA in the effects of microbiological fouling on stainless steels in potable water. However, little is known about the formation and effects of biofilms, on stainless steel in potable water environments, particularly in turbulent flow regimes. Results are presented on the development of biofilms on stainless steel grades 304 and 316 after exposure to potable water at velocities of 0.32, 0.96 and 1.75 m s⁻¹. Cell counts on slides of stainless steel grades 304 and 316 with both 2B (smooth) and 2D (rough) finishes showed viable and total cell counts were higher at the higher flow rates of 0.96 and 1.75 m s⁻¹, compared to a flow rate of 0.32 m s⁻¹. Extracellular polysaccharide levels were not significantly different (P< 0.05) between each flow rate on all stainless steel surfaces studied. higher levels were found at the higher water velocities. the biofilm attached to stainless steel was comprised of a mixed bacterial flora including Acinetobacter sp, Pseudomonas spp, Methylobacterium sp, and Corynebacterium/Arthrobacter spp. Epifluorescence microscopy provided evidence of rod-shaped bacteria and the formation of stands, possibly of extracellular material attached to stainless steel at high flow rates but not at low flow rates. Received 04 February 1998/ Accepted in revised form 12 February 1999     

N-acetyl-L-cysteine affects growth,extracellular polysaccharide production,and bacterial biofilm formation on solid surfaces

N-Acetyl-L-cysteine (NAC) is used in medical treatment of patients with chronic bronchitis. The positive effects of NAC treatment have primarily been attributed to the mucus-dissolving properties of NAC, as well as its ability to decrease biofilm formation, which reduces bacterial infections. Our results suggest that NAC also may be an interesting candidate for use as an agent to reduce and prevent biofilm formation on stainless steel surfaces in environments typical of paper mill plants. Using 10 different bacterial strains isolated from a paper mill, we found that the mode of action of NAC is chemical, as well as biological, in the case of bacterial adhesion to stainless steel surfaces. The initial adhesion of bacteria is dependent on the wettability of the substratum. NAC was shown to bind to stainless steel, increasing the wettability of the surface. Moreover, NAC decreased bacterial adhesion and even detached bacteria that were adhering to stainless steel surfaces. Growth of various bacteria, as monocultures or in a multispecies community, was inhibited at different concentrations of NAC. We also found that there was no detectable degradation of extracellular polysaccharides (EPS) by NAC, indicating that NAC reduced the production of EPS, in most bacteria tested, even at concentrations at which growth was not affected. Altogether, the presence of NAC changes the texture of the biofilm formed and makes NAC an interesting candidate for use as a general inhibitor of formation of bacterial biofilms on stainless steel surfaces.     

Insights into discharge argon-mediated biofilm inactivation

Formation of bacterial biofilms at solid–liquid interfaces creates numerous problems in biomedical sciences. Conventional sterilization and decontamination methods are not suitable for new and more sophisticated biomaterials. In this paper, the efficiency and effectiveness of gas discharges in the inactivation and removal of biofilms on biomaterials were studied. It was found that although discharge oxygen, nitrogen and argon all demonstrated excellent antibacterial and antibiofilm activity, gases with distinct chemical/physical properties underwent different mechanisms of action. Discharge oxygen- and nitrogen-mediated decontamination was associated with strong etching effects, which can cause live bacteria to relocate thus spreading contamination. On the contrary, although discharge argon at low powers maintained excellent antibacterial ability, it had negligible etching effects. Based on these results, an effective decontamination approach using discharge argon was established in which bacteria and biofilms were killed in situ and then removed from the contaminated biomaterials. This novel procedure is applicable for a wide range of biomaterials and biomedical devices in an in vivo and clinical setting.     

Antibacterial properties of nine pure metals: a laboratory study using Staphylococcus aureus and Escherichia coli

Bacterial attachment and growth on material surfaces are considered to be the primary steps leading to the formation of biofilm. Biofilms in hospital and food processing settings can result in bacterial infection and food contamination, respectively. Prevention of bacterial attachment, therefore, is considered to be the best strategy for abating these menaces and therefore the development of antibacterial metals becomes important. In this study, nine pure metals, viz. titanium, cobalt, nickel, copper, zinc, zirconium, molybdenum, tin, and lead have been tested for their antibacterial properties against two bacterial strains, Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. This was accomplished using two assay methods, the film contact method and the shaking flask method. The results show that the antibacterial properties varied significantly with different metals and the effectiveness of metals to resist bacterial attachment varied with the bacterial strain. Among the metals tested, titanium and tin did not exhibit antibacterial properties. TEM images showed that metal accumulation resulted in the disruption of the bacterial cell wall and other cellular components.     

Antibacterial isoeugenol coating on stainless steel and polyethylene surfaces prevents biofilm growth

Aims

Pathogenic bacteria can spread between individuals or between food items via the surfaces they share. Limiting the survival of pathogens on surfaces, therefore, presents an opportunity to limit at least one route of how pathogens spread. In this study, we propose that a simple coating with the essential oil isoeugenol can be used to circumvent the problem of bacterial transfer via surfaces.

Methods and Results

Two commonly used materials, stainless steel and polyethylene, were coated by physical adsorption, and the coatings were characterized by Raman spectroscopy, atomic force microscopy and water contact angle measurements. We quantified and visualized the colonization of coated and uncoated surfaces by three bacteria: Staphylococcus aureus, Listeria monocytogenes and Pseudomonas fluorescens. No viable cells were detected on surfaces coated with isoeugenol.

Conclusions

The isoeugenol coating prepared with simple adsorption proved effective in preventing biofilm formation on stainless steel and polyethylene surfaces. The result was caused by the antibacterial effect of isoeugenol, as the coating did not diminish the adhesive properties of the surface.

Significance and Impact of the Study

Our study demonstrates that a simple isoeugenol coating can prevent biofilm formation of S. aureus, L. monocytogenes and P. fluorescens on two commonly used surfaces.     

Adsorption of flavobacterium breve and pseudomonas fluorescens p17 on different metals: Electrochemical polarization effect

The electrochemical polarization effect on early adsorption of Flavobacterium breve and Pseudomonas fluorescens P17 to platinum, titanium, stainless steel, copper, aluminum alloy and mild steel was studied. A well‐defined peak characterized the bacterial adsorption dependence on externally applied potential. Maximal adsorption occurred in the potential range of ‐0.5 to 0.5 V (SCE) for all tested metals. A shift of applied potential towards both a positive and a negative direction from the maximal adsorption potential (Emax,ad) was accompanied by a gradual decrease in bacterial adsorption. The extent of bacterial adsorption strongly depended on the nature of the metallic substratum and decreased accordingly as follows: platinum > titanium > stainless steel > aluminum alloy > carbon steel > copper. Adsorption on all tested metals was approximately two orders of magnitude higher with the relatively more hydrophobic F. breve compared to the less hydrophobic P. fluorescens P17. The effect of electrochemical polarization on the initial stages of bacterial adsorption onto metallic substrata is further discussed.     

Metal corrosion by aerobic bacteria isolated from stimulated corrosion systems: Effects of additional nitrate sources

Many microorganisms are reported to influence the corrosive behaviour of mild steel and stainless steel in different habitats. In this study, 40 bacterial strains were isolated from corroded mild steel and stainless steel coupons in the nitrate supplemented environments. The corrosion abilities of the isolates against the mild steel and stainless steel coupons were tested with or without additional nitrate sources. The presence of bacterial isolates alone stimulated the corrosion of mild steel coupons. Most of the bio-corrosion processes of mild steel coupons were mitigated by adding nitrate supplement with bacterial isolates. The effects of bacterial isolates and additional nitrogen sources on corrosion of stainless steels were varied. Not all bacterial isolates stimulated the corrosion on stainless steel during the study period. Unlike the effects on mild steel coupons, additional NaNO₃ might stimulate, retard the corrosion rate by the bacterial isolates or have limited effects. Similar results were obtained when NH₄NO₃ was used. Phylogenetic analysis demonstrated that all isolates were closely related. The majority of the bacterial isolates from corroded metal coupons were identified as Bacillus species. Others were identified as Pseudomonas sp., Marinobacter sp., and Halomonas species. The results prove that the isolated aerobic microorganisms do play a role in the corrosion process of stainless and mild steel. Adding additional nitrate sources might be a tool to mitigate corrosion of mild steel which was stimulated by the presence of bacteria. However, to prevent the corrosion of stainless steels, it might need a trial and errors approach in each case.     

电解海水的抑菌活性及对食品加工表面材料的消毒效果

为了考察直接电解海水消除细菌污染的可能性,本文将海水及海水稀释成不同浓度后通过氧化电解水装置进行电解不同时间后,所得酸性电解海水、碱性电解海水和中性电解海水对病原菌[埃希氏大肠杆菌(Escherichina coli)、沙门氏菌(Salmonella)、单核细胞增生李斯特菌(Listeria moncytogene)、摩化摩根(Morganella morganii)、副溶血性弧菌(Vibrio parahaemolyticus)]以及食品加工表面接触材料(地板砖、不锈钢板、瓷砖、手套、抹布)的消毒效果进行分析研究.结果表明,酸性电解海水具有良好的杀菌效果,能将107 CFU/mL的病原菌悬液在1 min内几乎全部杀死.碱性电解海水和中性水无明显的杀菌效果.通过模拟食品加工过程,对食品加工表面接触材料人为染菌,研究电解海水对表面材料的消毒效果,结果表明酸性电解海水仍能将表面材料含有的107CFU/cm2病原菌在5 min之内几乎全部杀灭.由此说明电解海水对食品加工表面接触材料具有明显的消毒效果,能取代以淡水为原料的电解水杀菌效果是高效廉价和不浪费淡水资源的一种理想消毒剂.     

Antibacterial potential of hGlyrichin encoded by a human gene

Emerging multidrug‐resistant (MDR) bacteria are an enormous threat to human life because of their resistance to currently available antibiotics. The genes encoding antibacterial peptides have been studied extensively and are excellent candidates for a new generation of antibiotic drugs to fight MDR bacteria. In contrast to traditional antibiotics, antibacterial peptides, which do not cause drug resistance, have an unparalleled advantage. However, because most antibacterial peptides originate in species other than humans, the hetero‐immunological rejection of antibacterial peptides is a key disadvantage that limits their clinical application. In this study, we identify hGlyrichin as a potential human antibacterial polypeptide. The hGlyrichin polypeptide kills a variety of bacteria including the MDR bacteria methicillin‐resistant Staphylococcus aureus, MDR Pseudomonas aeruginosa, and MDR tubercle bacillus. A 19 amino acid peptide (pCM19) at positions 42–60 of hGlyrichin is crucial for its antibacterial activity. The hGlyrichin polypeptide kills bacteria through the destruction of the bacterial membrane. In addition, all peptides that are homologous to hGlyrichin have antibacterial activity and can penetrate the bacterial membrane. Importantly, hGlyrichin does not cause hemolytic side effects in vitro or in vivo. Therefore, based on the virtues of hGlyrichin, i.e., the absence of hetero‐immunological rejection and hemolytic side effects and the unambiguous efficacy of killing pathogenic MDR bacteria, we propose hGlyrichin as a potential human antibacterial polypeptide. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.