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Presence of biofilm containing viable multiresistant organisms despite terminal cleaning on clinical surfaces in an intensive care unit

  • K. Vickery
    Correspondence
    Corresponding author. Address: Australian School of Advanced Medicine, Macquarie University, North Ryde, NSW 2109, Australia. Tel.: +61 2 9812 3559; fax: +61 2 9812 3610.
    Affiliations
    Surgical Infection Research Group, Australian School of Advanced Medicine, Macquarie University, New South Wales, Australia
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  • A. Deva
    Affiliations
    Surgical Infection Research Group, Australian School of Advanced Medicine, Macquarie University, New South Wales, Australia
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  • A. Jacombs
    Affiliations
    Surgical Infection Research Group, Australian School of Advanced Medicine, Macquarie University, New South Wales, Australia
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  • J. Allan
    Affiliations
    Surgical Infection Research Group, Australian School of Advanced Medicine, Macquarie University, New South Wales, Australia
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  • P. Valente
    Affiliations
    Surgical Infection Research Group, Australian School of Advanced Medicine, Macquarie University, New South Wales, Australia
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  • I.B. Gosbell
    Affiliations
    Antibiotic Resistance and Mobile Elements Group (ARMEG), Microbiology and Infectious Diseases Unit, School of Medicine, University of Western Sydney, New South Wales, Australia

    Department of Microbiology and Infectious Diseases, Sydney South West Pathology Service – Liverpool, New South Wales, Australia
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Published:September 07, 2011DOI:https://doi.org/10.1016/j.jhin.2011.07.007

      Summary

      Background

      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.

      Aim

      To establish whether reservoirs of MROs exist in the environment as biofilms.

      Methods

      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.

      Findings

      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.

      Conclusion

      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.

      Keywords

      Introduction

      Healthcare-associated infections (HAIs) are a widespread problem, affecting 5–10% of all patients.
      • Sanchez-Velazquez L.D.
      • 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.
      In the intensive care unit (ICU), the presence of very sick, elderly and immunocompromised patients results in a disproportionate percentage (20%) of patients developing HAI.
      • Rosenthal V.D.
      • Maki D.G.
      • Mehta A.
      • et al.
      International Nosocomial Infection Control Consortium report, data summary for 2002–2007, issued January 2008.
      This problem is compounded by the spread of multiresistant organisms (MROs), making treatment difficult or ineffective.
      • Dancer S.J.
      Importance of the environment in meticillin-resistant Staphylococcus aureus acquisition: the case for hospital cleaning.
      HAIs add considerable morbidity, increase hospital stay times, increase mortality, and add costs to patient care.
      • Sanchez-Velazquez L.D.
      • 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.
      • Rosenthal V.D.
      • Maki D.G.
      • Mehta A.
      • et al.
      International Nosocomial Infection Control Consortium report, data summary for 2002–2007, issued January 2008.
      • Shannon R.P.
      • Patel B.
      • Cummins D.
      • Shannon A.H.
      • Ganguli G.
      • Lu Y.
      Economics of central line-associated bloodstream infections.
      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.
      • Dancer S.J.
      Importance of the environment in meticillin-resistant Staphylococcus aureus acquisition: the case for hospital cleaning.
      • Boyce J.M.
      Environmental contamination makes an important contribution to hospital infection.
      • Carling P.C.
      • Bartley J.M.
      Evaluating hygienic cleaning in health care settings: what you do not know can harm your patients.
      Numerous studies have shown persistence of these organisms in the environment even in the face of enhanced terminal cleaning.
      • Dancer S.J.
      • White L.F.
      • Lamb J.
      • Girvan E.K.
      • Robertson C.
      Measuring the effect of enhanced cleaning in a UK hospital: a prospective cross-over study.
      • Hota B.
      • Blom D.W.
      • Lyle E.A.
      • Weinstein R.A.
      • Hayden M.K.
      Interventional evaluation of environmental contamination by vancomycin-resistant enterococci: failure of personnel, product, or procedure?.
      • Hayden M.K.
      • Bonten M.J.M.
      • Blom D.W.
      • Lyle E.A.
      • van de Vijver D.A.M.C.
      • Weinstein R.A.
      Reduction in acquisition of vancomycin-resistant enterococcus after enforcement of routine environmental cleaning measures.
      Biofilms are generally found in moist environments, causing infection on implantable medical devices such as catheters and breast implants or on instruments routinely immersed in fluid.
      • Bryers J.D.
      Medical biofilms.
      • Pajkos A.
      • Deva A.K.
      • Vickery K.
      • Cope C.
      • Chang L.
      • Cossart Y.E.
      Detection of subclinical infection in significant breast implant capsules.
      • Pajkos A.
      • Vickery K.
      • Cossart Y.
      Is biofilm accumulation on endoscope tubing a contributor to the failure of cleaning and decontamination?.
      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.
      • Fux C.A.
      • Costerton J.W.
      • Stewart P.S.
      • Stoodley P.
      Survival strategies of infectious biofilms.
      • Vickery K.
      • Pajkos A.
      • Cossart Y.
      Removal of biofilm from endoscopes: evaluation of detergent efficiency.
      • Hadi R.
      • Vickery K.
      • Deva A.
      • Charlton T.
      Biofilm removal by medical device cleaners: comparison of two bioreactor detection assays.
      Additionally, bacteria within biofilms are up to 1500 times (typically 100–250 times) more resistant to biocides than the same ‘planktonic’ bacteria growing in liquid culture.
      • Fux C.A.
      • Costerton J.W.
      • Stewart P.S.
      • Stoodley P.
      Survival strategies of infectious biofilms.
      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.

      Methods

      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.

      Sample collection

      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.
      • Pajkos A.
      • Vickery K.
      • Cossart Y.
      Is biofilm accumulation on endoscope tubing a contributor to the failure of cleaning and decontamination?.
      An item was classified as being biofilm positive if bacteria attached to a surface and surrounded by EPS could be visualized.

      Microbiology

      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.

      Results

      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
      SampleSEMCulture plates
      HBAMRSAVRE
      CurtainPositive EPSGrowthPositiveNegative
      Venetian blind cordPositive biofilmGrowthPositiveNegative
      Mattress bayNegativeGrowthPositiveE. faecium
      See-through plastic doorPositive biofilmNegativeNegativeNegative
      Wash basin rubberPositive biofilmNegativeNegativeNegative
      Sterile supply reagent bucketPositive biofilmGrowthNegativeNegative
      HBA, horse blood agar; MRSA, multiresistant Staphylococcus aureus; VRE, vancomycin-resistant enterococcus.
      Figure thumbnail gr1
      Figure 1Scanning electron micrographs of: (a) blind cord (original magnification ×2500); (b) see-through ward door (original magnification ×5000); (c) red reagent box (original magnification ×7500); (d) curtain (original magnification ×2500). Horizontal arrows indicate coccoid bacteria embedded in exopolymeric substance (EPS). Vertical arrows indicate residual strings of EPS dehydrated during processing.
      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.

      Discussion

      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.
      • Boyce J.M.
      Environmental contamination makes an important contribution to hospital infection.
      • Carling P.C.
      • Bartley J.M.
      Evaluating hygienic cleaning in health care settings: what you do not know can harm your patients.
      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.
      • Dancer S.J.
      • White L.F.
      • Lamb J.
      • Girvan E.K.
      • Robertson C.
      Measuring the effect of enhanced cleaning in a UK hospital: a prospective cross-over study.
      Termination of the extra cleaner resulted in new clusters of MRSA infection within two to four weeks. However, even with enhanced cleaning, MROs can still be isolated from the environment.
      • Dancer S.J.
      • White L.F.
      • Lamb J.
      • Girvan E.K.
      • Robertson C.
      Measuring the effect of enhanced cleaning in a UK hospital: a prospective cross-over study.
      • Hota B.
      • Blom D.W.
      • Lyle E.A.
      • Weinstein R.A.
      • Hayden M.K.
      Interventional evaluation of environmental contamination by vancomycin-resistant enterococci: failure of personnel, product, or procedure?.
      • Hayden M.K.
      • Bonten M.J.M.
      • Blom D.W.
      • Lyle E.A.
      • van de Vijver D.A.M.C.
      • Weinstein R.A.
      Reduction in acquisition of vancomycin-resistant enterococcus after enforcement of routine environmental cleaning measures.
      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.
      • Vickery K.
      • Pajkos A.
      • Cossart Y.
      Removal of biofilm from endoscopes: evaluation of detergent efficiency.
      • Hadi R.
      • Vickery K.
      • Deva A.
      • Charlton T.
      Biofilm removal by medical device cleaners: comparison of two bioreactor detection assays.
      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.
      • Pajkos A.
      • Vickery K.
      • Cossart Y.
      Is biofilm accumulation on endoscope tubing a contributor to the failure of cleaning and decontamination?.
      With use, many of these dips or imperfections in endoscope tubing became contaminated with biofilm.
      • Pajkos A.
      • Vickery K.
      • Cossart Y.
      Is biofilm accumulation on endoscope tubing a contributor to the failure of cleaning and decontamination?.
      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.
      • Gillings M.R.
      • Holley M.P.
      • Stokes H.W.
      Evidence for dynamic exchange of qac gene cassettes between class 1 integrons and other integrons in freshwater biofilms.
      A significant correlation has been shown to exist between class 1 integron resistance genes, biocide resistance and biofilm formation in clinical strains of Acinetobacter baumannii.
      • Rajamohan G.
      • Srinivasan V.B.
      • Gebreyes W.A.
      Biocide-tolerant multidrug-resistant Acinetobacter baumannii clinical strains are associated with higher biofilm formation.
      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.
      • Fux C.A.
      • Costerton J.W.
      • Stewart P.S.
      • Stoodley P.
      Survival strategies of infectious biofilms.
      Dancer et al. found that antibiotic-resistant environmental bacteria were more prevalent in wards with a high level of antibiotic prescribing.
      • Dancer S.J.
      • Coyne M.
      • Robertson C.
      • Thomson A.
      • Guleri A.
      • Alcock S.
      Antibiotic use is associated with resistance of environmental organisms in a teaching hospital.
      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.

      Acknowledgements

      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

      None declared.

      Funding sources

      None.

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