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Antibiotic Resistance and Mobile Elements Group (ARMEG), Microbiology and Infectious Diseases Unit, School of Medicine, University of Western Sydney, New South Wales, AustraliaDepartment of Microbiology and Infectious Diseases, Sydney South West Pathology Service – Liverpool, New South Wales, Australia
Despite recent attention to surface cleaning and hand hygiene programmes, multiresistant organisms (MROs) continue to be isolated from the hospital environment. Biofilms, consisting of bacteria embedded in exopolymeric substances (EPS) are difficult to remove due to their increased resistance to detergents and disinfectants, and periodically release free-swimming planktonic bacteria back into the environment which may may act as an infection source.
To establish whether reservoirs of MROs exist in the environment as biofilms.
Following terminal cleaning, equipment and furnishings were removed aseptically from an intensive care unit (ICU) and subjected to culture and scanning electron microscopy (SEM). Samples were placed in 5 mL of tryptone soya broth, sonicated for 5 min before plate culture on horse blood agar, Brillance MRSA and Brilliance VRE agar plates. Samples for SEM were fixed in 3% glutaraldehyde and hexamethyldisilizane (HMDS) prior to sputter-coating with gold and examination in an electron microscope.
Biofilm was demonstrated visually on the sterile supply bucket, the opaque plastic door, the venetian blind cord, and the sink rubber, whereas EPS alone was seen on the curtain. Viable bacteria were grown from three samples, including MRSA from the venetian blind cord and the curtain.
Biofilm containing MROs persist on clinical surfaces from an ICU despite terminal cleaning, suggesting that current cleaning practices are inadequate to control biofilm development. The presence of MROs being protected within these biofilms may be the mechanism by which MROs persist within the hospital environment.
Contamination of the inanimate environment around patients constitutes an important reservoir of MRO with the risk of HAI increased by an average of 73% if the patient previously occupying the room had MRSA, vancomycin-resistant enterococcus (VRE), acinetobacter, Clostridium difficile or other pathogens.
We hypothesize that, despite the decreased moisture availability on dry surfaces, bacteria within the ICU environment also reside in biofilms, and that within these biofilms, MROs are protected from physical removal and chemical disinfection.
A biofilm is a structured community of organisms encased and attached to a surface by exopolymeric substances (EPS). The EPS makes up to 90% of the biofilm providing protection from environmental desiccation and this EPS is extremely difficult to remove using detergents.
These properties of biofilms result in decreased efficacy of cleaning and disinfection, thereby promoting the persistence of bacteria, including MROs, in the environment.
In this study we investigated whether biofilms can be found on furnishings in the ICU.
Following terminal cleaning in a 16-bed ICU, i.e initial cleaning with neutral detergent, followed by disinfection with 500 ppm chlorine (Diversol5000, Johnson Diversey, Smithfield, Australia), equipment and furnishings were aseptically removed from patient and common-use areas.
Items were destructively sampled using sterile gloves, forceps, pliers, scissors, or scalpel blades, depending on the material being sampled. Gloves and instruments were changed between each sample. Samples were then placed into sterile containers for transport to the laboratory. Small items, such as a sterile supply reagent box, were transported intact to the laboratory; larger items, such as the mattress and door, had sections removed (up to 8×10 cm in size) into sterile containers. Following transport to the laboratory, these large pieces were further sectioned into smaller pieces, using a sterile technique.
Scanning electron microscopy (SEM)
Samples up to 1 cm2 were fixed in 3% glutaraldehyde, dehydrated through ethanol, immersed in hexamethyldisilizane (HMDS; Polysciences Inc., Warrington, PA, USA) for 3 min before sputter-coating with 20 nm gold film and examined in an SEM microscope as previously described.
An item was classified as being biofilm positive if bacteria attached to a surface and surrounded by EPS could be visualized.
Sections of equipment or furnishings up to 2 cm2 were placed in 4 mL of tryptone soya broth, sonicated for 5 min and 100 μL spread over horse blood agar plates (HBA), Brilliance MRSA agar plates for the detection of multiresistant Staphylococcus aureus (MRSA) and Brillance VRE agar plates for the detection of vancomycin-resistant enterococcus (Oxoid, Adelaide, Australia). MRSA plates were incubated for 18–24 h and VRE and HBA plates up to 48 h.
Six samples were examined by SEM (Table I). We failed to demonstrate biofilm on only one sample. Four samples had principally coccoid-shaped bacteria encased in large amounts of EPS and the sample from the curtain had ‘strings’ of dehydrated EPS evident. (Figure 1).
Table IScanning electron microscopy (SEM) and culture results for environmental surfaces
Bacteria grew on HBA from four of the six samples, demonstrating the presence of culturable organisms. The venetian blind cord and curtain, positive for biofilm by SEM, also grew MRSA. The mattress grew MRSA and E. faecium but we were unable to demonstrate biofilm visually on this sample (Table I). Two samples positive for biofilm were culture negative, using the procedure described above.
Many studies have shown that contamination of the environment makes an important contribution to HAI and that enhanced cleaning protocols reduce environmental contamination, which translates into decreased incidence of HAI.
In Dancer et al.’s study, the addition of one extra member of cleaning staff, five days a week, resulted in a 32.5% reduction in microbial contamination of hand-touch sites and a 26.6% reduction in new MRSA infections, saving the hospital an estimated £30,000 to £70,000.
We hypothesize that surface condensation occurs, producing a thin film of water, or that the relative humidity in the ICU is high enough to allow biofilms to develop on ICU surfaces. Once formed, the EPS would protect the bacteria from desiccation and make them harder to remove.
We further hypothesize that MROs persist in the environment, in the face of enhanced cleaning, as biofilms. Although detergents are good at removing patient soil and planktonic bacteria, they are less effective at removing biofilm, rendering current cleaning protocols less efficient.
In industry, extreme measures including physical scraping and use of concentrated biocides are often required to remove biofilm, such as when removing legionella from water-cooling towers.
Of the six furnishings sampled bacteria were demonstrated to be embedded in EPS on four samples and residual EPS on one, whereas only the mattress sample was negative for biofilm by SEM. SEM of the non-porous covering of the hospital mattress shows that the surface is not completely level but has many microscopic dips. This is similar to the dips and imperfections that have been observed on new Teflon endoscope tubing.
A similar situation may exist with the hospital mattresses and, if a larger area were to be inspected, biofilm may be found.
Using destructive sampling followed by sonication and broth culture, bacteria were grown from three of these biofilm-positive samples. Both the venetian blind curtain cord and the curtain grew MRSA. Even the mattress, the sole sample for which we failed to visually demonstrate biofilm, grew MRSA and VRE. It is worrying that we demonstrated biofilm on the reagent bucket that was used to contain sterile supplies, such as catheters and bandages. Although we did not detect MRSA or VRE, we were able to show that viable bacteria were present in the biofilm. Additionally the rate of acquisition of new resistant determinants is increased in bacteria residing in biofilm.
Whether this occurs when water is limited is unknown.
Despite visual confirmation of biofilm, neither the wash basin nor the plastic door grew bacteria when aerobic culture and HBA were used. These bacteria could have been dead, or not culturable using the conditions used, or unculturable due to their state of growth in the biofilm. Bacteria growing as biofilm are notoriously difficult to culture, although sonication of the sample in broth increases the rate of recovery.
The combination of high antibiotic use and environmental biofilms in the ICU may be the mechanism whereby increased genetic exchange occurs between bacteria residing in biofilms, leading to persistence of antibiotic-resistant environmental bacteria, despite enhanced cleaning.
Using destructive sampling, followed by SEM and culture, we have demonstrated the presence of biofilm and biofilm containing MROs on clinical surfaces from an ICU despite terminal cleaning, suggesting that current cleaning practices are inadequate to control biofilm development. The presence of MROs being protected within these biofilms may be the mechanism by which MROs persist within the hospital environment.
The authors would like to acknowledge the scientific staff of Sydney South West Pathology Service – Liverpool, who supplied the chromogenic agar plates. We would like to thank Ms Debra Birch, Macquarie University Microscopy Unit for her expertise and help in obtaining the scanning electron micrographs.
Conflict of interest statements
Ponce de Leon Rosales S.
Rangel Frausto M.S.
The burden of nosocomial infection in the intensive care unit: effects on organ failure, mortality and costs. A nested case–control study.