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*** — Jesus said, “If you hold to my teaching, you are really my disciples. Then you will know the truth, and the truth will set you free” (John 8:32) - You will know the truth and the truth will set you free. Discover the TRUTH ... More real truth! Read and heed! ** From laboratory studies, it has emerged that bacteria on copper surfaces suffer rapid membrane damage and DNA degradation, in addition to other, less well-defined cell damage

Date:   8/30/2021 2:15:56 AM ( 3 y ago)

With the use of copper in hospitals and other facilities, it has become important to understand the mechanism of the so-called “contact killing” of bacteria, as it may bear on the possibility of the emergence of resistant organisms, on cleaning procedures, and on material and object engineering.

From laboratory studies, it has emerged that bacteria on copper surfaces suffer rapid membrane damage and DNA degradation, in addition to other, less well-defined cell damage (16–21). The order in which these processes take place and which one is the primary killing mechanism remain issues of debate. In fact, the sequence of events may depend on the type of microorganism (18). It is also clear that copper ions released from the surface play a role in contact killing, but bacterial copper resistance systems are not able to cope with the released copper (2, 22, 23).

A question which has not yet been addressed in detail is the role of physical contact of bacteria with the copper surface in contact killing. We thus engineered special copper surfaces, so-called “contact arrays”: copper surfaces were covered by an inert polymer into which arrays of holes less than 1 μm in diameter were etched by a photolithographic process, using laser interference (24). Enterococcus hirae was used as a model organism because Gram-positive organisms are frequent pathogens and the contact killing behavior of E. hirae had previously been studied (23). Also, its robust cell wall helped to preserve the shape of the bacteria during electron microscopy. The holes in the contact arrays were smaller than E. hirae, so the grid effectively prevented contact of the bacteria with the copper surface. It was found that contact killing on these contact arrays was reduced by 7 orders of magnitude compared to copper coupons, while the release of ionic copper was not significantly altered. Metallic iron did not appear to be active in contact killing, unless copper ions were also present. These experiments demonstrate the importance of both copper ions and bacterial-metal contact for efficient contact killing.

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MATERIALS AND METHODS

Bacterial strains and growth conditions.

Wild-type E. hirae ATCC 9790 was grown anaerobically (air-saturated medium was transferred to sealed tubes; these cultures became anaerobic after approximately 1 h) to stationary phase at 37°C in 10 ml of N medium (25). Cells were centrifuged for 5 min at 5,000 × g, washed twice with 20 ml of 100 mM Tris-Cl (pH 7), and resuspended in 10 ml of the same buffer. The average cell density was 2 × 108 to 8 × 108 CFU/ml. All handling of cells was performed aerobically.

Coupons and contact arrays.

Control C1 copper coupons were 15- by 15-mm squares of highly polished (root mean square roughness < 50 nm), 99.99% pure copper and were cleaned by ultrasonication in chloroform and ethanol for 10 min each, followed by air drying. CA contact arrays were prepared by spin coating C1 coupons with cresol resin AZ 1518 (MicroChemicals GmbH, Ulm, Germany), diluted 1:4 with 2-methoxy-1-methylethylacetate, under clean-room conditions. Spin coating was conducted at 3,500 rpm for 60 s with a ramp of 3,000 rpm/s, resulting in an average film thickness of 1.74 ± 0.05 μm. Coated samples were immediately dried on a hot plate at 100°C for 60 s. Laser interference patterning (26) was accomplished by illumination with an Nd:YAG nanosecond laser (Quanta-Ray PRO210; frequency of 10 Hz) with one pulse (10 ns) at an average fluency of 33 mJ/cm2. Exposed coupons were developed with a boric acid-based solution (AZ 351 B; MicroChemicals GmbH) for 60 s, followed by rinsing with distilled water and drying in an ambient atmosphere. Control coupons (CL1) were developed similarly but without prior exposure. Iron coupons were of polished 98.3% Fe, 1.4% Mn, 0.17% C (DIN 5512-1) and were cleaned like the copper coupons. Following preparation and/or cleaning, all coupons used in this study were stored under nitrogen until use.

Measurement of contact killing.

To assess contact killing, a wet plating technique was used essentially as described in reference 23. Briefly, 25-μl volumes of cells suspended in 100 mM Tris-Cl were applied to plain or modified copper or iron coupons. Following incubation for various times in a water-saturated atmosphere, 20-μl samples were withdrawn and serial dilutions in phosphate-buffered saline (PBS) were spread on N agar plates. Following growth for 24 h, survival was calculated from the CFU. Contact arrays with a porous surface were subjected to a reduced pressure of 2 kPa for 5 s right after application of cells to remove air from the pores.

Copper and iron determinations.

Copper or iron release from coupons during wet-plating incubations was assessed by removing 20-μl aliquots, diluting them 50-fold with 0.065% HNO3, and measuring the copper content by inductively coupled plasma atomic emission (ICP-AE) on a Jobin Yvon JY 24 instrument (HORIBA Jobin Yvon GmbH, Munich, Germany) at 324.754 nm.

SEM.

Cells suspended in water at approximately 2 × 108 cells/ml were applied to copper coupons or contact arrays and air dried. Scanning electron microscopy (SEM) images were acquired on a high-resolution dual-beam microscope (FE Strata DB 235) at 5 kV using the secondary electron detector (SED) mode.

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RESULTS

Design of contact arrays.

Laser interference patterning is a tool to generate topographic surface patterns on micrometer and submicrometer scales (26). We thus designed a microstructured polymer grid on top of a copper surface which would allow E. hirae cells to come close to the copper surface (<2 μm) yet prevent direct bacterial-metal contact.

To prepare such contact arrays, copper coupons were spin coated with a positive photoresist, which was then exposed by a short laser pulse to a specific light intensity distribution with a lateral spacing parameter of 770 nm. The utilized, unique intensity distribution was designed by splitting and recombining the initial laser beam in a three-beam laser interference setup.

Highly exposed areas of the photoresist were removed by photographic development, resulting in a honeycomb-like pattern of holes which extended down to the copper surface (Fig. 1). Preparation of the contact arrays required the optimization of a number of parameters, such as resist viscosity, spin coating parameters, interference pattern, laser fluency, etc. These engineering aspects of the work are not further discussed here.


 

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