Analysis of the MRSA DNA genotype

Chromosomal DNA analysis was carried out for MRSA strains isolated from the surrounding environment and the skin of the patient from June 24 to July 25, 2008, and their homology was determined. The results revealed that all MRSA strains isolated from the bed sheets and the skin of the MRSA-carrying/infected patient had 98% or higher homology, demonstrating that they had the same DNA genotype (Fig. 3A). It is noteworthy that the MRSA strains detected again after having been eliminated once through microbial substitution follow­ing antibiotic therapy had the same DNA genotype as that of the MRSA strains detected before the microbial substitution (lanes 7, 8 of Fig. 3A).

The MRSA strains isolated on 25 July from the patient’s skin and her surroundings, including her bed sheets, also showed 98% or higher homology. These findings indicate that MRSA strains that colonized the skin and those contaminating the surrounding environment had the same DNA genotype (Fig. 3B). Thus, it is inferred that MRSA strains isolated from the surroundings of the patient were derived from direct contact with the patient’s hands and the gloves of the healthcare workers, and exfoliation of the scaly skin lesions.

Fig. 3. DNA genotype analysis of Methicillin-resistant S. aureus (MRSA) strains isolated from the patient’s skin and bed sheet between June 24 and July 25, 2008. (A) shows that MRSA which was transmitted again after having disappeared once is genetically the same as MRSA detected early in infection. Lanes 1 and 8: MRSA strains isolated from the patient skin on 24 June and 25 July 2008, respectively. Lanes 2–7: MRSA strains isolated from the bed sheet on June 24, June 26, June 27, June 30, July 15 and July 25, 2008, respectively. (B) shows that MRSA isolated from the surroundings of the patient is also the same as MRSA from the patient. Lane 1: MRSA strain isolated from the patient skin on July 25, 2008. Lanes 2–5: MRSA strains isolated from mobile phone, bedside table, bedside rail and disposable gloves (used) on July 25, 2008.

Bactericidal effect of copper film

Whether copper film might also exert a bactericidal effect on MRSA and P. aeruginosa, like metallic copper, was examined using the patient MRSA, standard MRSA, standard S. aureus, and standard PA strains. The results revealed that, similar to copper plates, the copper film caused a gradual decrease in the viable cell count of each strain in proportion to the length of the reaction time. Patient and standard MRSA strains and the standard S. aureus strain were killed completely within 180 min (Fig. 4), and the standard PA strain within 10 min (Fig. 5).

Fig. 4. Bactericidal effects of copper on (a) standard Staphylococcus aureus, (b) standard Methicillin-resistant S. aureus (MRSA), and (c) patient MRSA strains.

Fig. 5. Bactericidal effect of copper film on Pseudomonas aeruginosa NBRC 12732.

To elucidate the bactericidal mechanism of the copper surface against S. aureus, the influences of several reagents (the metal chelators EDTA and BPSA, the hydroxyl radical remover mannitol, the superoxide remover SOD, and the hydrogen peroxide-degrading enzyme catalase) on the bactericidal effect of metallic copper against standard S. aureus strains were examined. The results are shown in Fig. 6. The bactericidal effect of copper was annulled over time by EDTA and BPSA. This suggests that the presence of ionized Cu+ and Cu2+ on the copper surface (particularly Cu+) is indispensable for the bactericidal effect of the copper surface. The bactericidal effect of copper was also eliminated by mannitol, SOD and catalase, and these findings show that the reactive oxygen species (ROS), including hydroxyl radicals, superoxide and hydrogen peroxide produced as a result of ionization via oxidation of the copper surface, was involved.

Fig. 6. Results of the disinfection performance test of copper plates in the presence of ethylenediaminetetraacetic acid (EDTA), bathocuproine disulphonic acid, disodium salt (BPSA), mannitol, superoxide dismutase (SOD) and catalase.

Preventive effect of the copper film on the spread of MRSA contamination of the environment

The copper film was attached to the bed sheets of the MRSA carrier, where contamination with MRSA and other Staphylococcus spp. was prominent, and the possible preventive effect of copper on the spread of MRSA contamination from the MRSA carrier to the surrounding environment was examined during a period of approximately 1 month from June 26 to July 16, 2008. The results are shown in Table SI (available from http://www.medicaljournals.se/acta/content/?doi=10.2340/00015555-1472). The cell counts of MRSA and other Staphylococcus spp. on the copper film surface were considerably lower than those on the non-film-coated control sheet surface. Although contamination with P. aeruginosa was confirmed on July 4 and 16, hardly any P. aeruginosa was detected on the copper film surface.

DISCUSSION

MRSA has been detected in various environments in the hospital. Contamination vehicles reported frequently include the indoor floors, indoor air, door handles, and bed linen (18). In the event of development of nosocomial MRSA infections in patients, MRSA is frequently isolated from the surrounding environment, the frequency of isolation from indoor floors being second only to that from the linen and gowns of the patients (3, 4). In this study, contamination with high colony counts of MRSA derived from an MRSA-infected patient with erythroderma was confirmed in the patient’s bed sheets, bed rail, and the floor and other surroundings of the bed. This corroborates the findings of previous reports (6, 7), that there is a high risk of MRSA contamination of the hospital environment, including of the bed sheets and floor, through scales and exudates from patients with exfoliative dermatitis. In addition, our study revealed limited MRSA isolation from the over-bed table, mobile phone, bedside table, and wash stand, showing a broader range of MRSA contamination than in previous reports. Thus, it is suggested that erythroderma patients who are infected with/carriers of MRSA can be an important source of serious and extensive MRSA contamination of the environment.

Similar to staphylococci, MRSA cells scatter in the air together with dust and skin scales when the bed is being made. Although the probability of airborne inoculation is low, it is reported as a cause of new carriers and infected patients (19). In our patient, who showed prominent production of scales, it is highly likely that these scales were scattered in the air. We also measured the MRSA cell counts scattering in the air at the time of bed sheet change in another MRSA-infected patient with psoriatic erythroderma who had prominent scales. The results showed that the cell count in the air increased to approximately 300 cfu/300 l at the time of bed sheet change, a nearly 9-fold higher level than usual, and MRSA accounted for a maximum of 70% of the cells in the air. Furthermore, within 30 min, the bacterial count returned to the baseline level (approximately 30 cfu) (data not shown). Similar to that reported for general bacterial cells floating in the waiting room of the outpatient clinic (20), MRSA cells scattering in the air during bed sheet changes are also attached mainly to floating dust particles measuring more than 5 mm (range 4–8 mm) in diameter, and most of the floating particles settle on the floor within 30 min (21). Therefore, to prevent airborne infection from MRSA-infected patients with erythroderma during treatment and nursing activities, nurses and other care staff should wear masks and caps during bed sheet changes and not enter the patient’s room for at least 30 min after a bed sheet change.

In this study, MRSA strains that were identical to those apparently eliminated by microbial substitution due to antibiotic therapy were isolated from the patient’s bed sheet after an interval of 16 days (Fig. 2). Therefore, it is presumed that the MRSA survived for a certain period in the environment around the patient’s bed, and then colonized and infected the patient again. Because it has been reported that the survival period of MRSA in the hospital room is approximately 14 days (22, 23), we believe that our estimation of the survival period of MRSA is appropriate.

The basic policy for control of nosocomial infection with MRSA is early detection of the infectious source (patient) and interruption of the pathway of contact transmission of the aetiological agent. To prevent contact transmission of infection via the hands, skin, contaminated materials, etc., it is important to carry out hygiene control of the environmental surfaces that come in direct or indirect contact with infectious agents, in addition to thorough hygiene of hands, including hand washing and disinfection after work operations. However, the status of extensive MRSA contamination of the environment determined in this study indicates that when patients with skin disease accompanied by prominent scales and exudates are infected with, or become carriers of, MRSA, complete prevention of MRSA transmission to the environment is extremely difficult. In 2003, the Society for Healthcare Epidemiology of America (SHEA) emphasized the need for active surveillance culture, and recommended implementation of surveillance culture in high-risk patients prior to their admission and skin culture in patients admitted with skin injury (24). However, at present, institutions practicing such measures are limited.

It has been pointed out that metallic copper and copper alloys may induce reduction in the risk of MRSA contamination of the environment in hospitals (3, 8, 9). We reported previously, for the first time, that when a metallic copper plate was set on the MRSA-contaminated floor around the bed of a patient with MRSA skin infection in the dermatology ward, the MRSA cell count was lower on the surface of the copper plate (14). In the present study, we found that the copper film exerted bactericidal activity, not only against S. aureus, including MRSA, but also against P. aeruginosa, and that the cell counts of the contaminant MRSA and P. aeruginosa derived from a patient with scaling dermatosis decreased on the surface of the copper film taped to the patient’s bed sheet. Exfoliative dermatitis is known to be associated with a high risk of MRSA contamination of the hospital environment (6, 7). These findings indicate that metallic copper may be useful for preventing the spread of MRSA contamination of the environment. Because bed linen is most susceptible to MRSA contamination from patients infected with MRSA (25), much is expected from the future development of linen with copper-derived bactericidal properties.

It has been speculated that the bactericidal effects of metallic copper on Escherichia coli and enterococci are exerted through the production of ROS, such as hydroxyl radicals and superoxide via the Haber-Weiss reaction and Fenton reaction between copper ions and hydrogen peroxide, or through the binding of copper ions (Cu+/Cu2+) to amino-acid side chains with cysteine or histidine, which leads to DNA damage, oxidation and degeneration of structural or functional proteins or lipids, suppression of the energy metabolic system, and osmotic stress in the bacterial cells (26, 27). In this study, the bactericidal effect against S. aureus on the copper surface was inhibited over time by metal-chelating drugs (BPSA and EDTA), hydroxyl radical removers (mannitol), superoxide remover (SOD), and hydrogen peroxide-degrading enzymes (catalase) (Fig. 6). These findings indicate that, as for E. coli (26), enterococci (27), and cryptosporidium (11), the bactericidal effect of copper is attributable to the activity of ROS. In addition, considering the finding that the bacterial component targeted by ROS is DNA in cases of enterococci (27), it is presumed that the bactericidal effect on S. aureus observed in this study may also be attributable to DNA damage of the bacterial cells. Bacteria have self-repair ability through the SOS reaction when their DNA is damaged by ultraviolet rays or quinolone antibacterial agents (28). It has been reported that E. coli cells that are lacking in the RecA gene, which controls this reaction, are susceptible to DNA damage by hydroxyl radicals produced via the Fenton reaction occurring endogenously after the administration of quinolone antibacterial agents (29). This finding provides strong support for our presumption that the target of the bactericidal activity of metallic copper is the bacterial cell DNA.

The bactericidal properties of metallic copper, which depend on the ionization of copper in the presence of water molecules, seem to be influenced by physical conditions (temperature and humidity). It is reported that metallic copper and copper alloys kill MRSA rapidly within 90 min at a temperature of 20–35ºC and humidity of 20–95%, whereas the bactericidal time is prolonged to 6 h at a temperature of 4ºC (8, 30, 31). This emphasizes the need to control the room temperature and humidity, particularly in the winter in Japan, when both parameters tend to be low, when using metallic copper or copper alloy in the indoor environments for their bactericidal properties.

A comparison of the bactericidal time of metallic copper for various bacterial species revealed that it was 10 min for P. aeruginosa, including multidrug resistant P. aeruginosa (Fig. 5), 90–180 min for S. aureus, including MRSA (Fig. 4), Legionella, and other major bacteria, including E. coli, and approximately 360 min for Methylobacterium mesophilicum (data not shown). In addition, the bactericidal time for multidrug resistant Mycobacterium tuberculosis was reported to be 5–15 days (32). This raises questions about the reasons for such differences in the bactericidal effect of metallic copper against various species. It is possible that the bactericidal time of metallic copper is proportional to the length of the generation time, considering the fact that the generation time is shortest (20 min) for P. aeruginosa, and longest (15–20 h) for M. tuberculosis, and that the longer generation time of S. aureus makes it less susceptible to ROS (33). In this study, contamination of bed linen by P. aeruginosa, an important cause of nosocomial infection, second only to MRSA, was completely inhibited by metallic copper (Table SI). Therefore, it seems promising to use metallic copper as a material to prevent P. aeruginosa contamination of the hospital environment.

This study has shown that contamination of the environment arising from an erythroderma patient with MRSA infection/carrying MRSA spread extensively to the surroundings in proportion to the frequency of contact with the patient, and that contamination by MRSA and P. aeruginosa was the most frequent. In addition, we utilized the excellent bactericidal properties of metallic copper for the bed sheets, which are not easy to disinfectant, and observed a marked decrease in the contamination rate. We conclude that metallic copper is a useful material for preventing the spread of environmental MRSA/P. aeruginosa contamination. It may be possible to effectively inhibit transmission of MRSA infection in the environment if various medical implements and materials were developed using metallic copper and used in the hospital with an accurate understanding of the sites of MRSA contamination, along with proper practice of disinfection and cleaning procedures.

The authors declare no conflict of interest.

REFERENCES