Liverpool University- Testing the effect of PIP cleaning products on disease causing bacteria
Testing the effect of PIP cleaning products on viability of Staphylococcus aureus (MRSA) and Clostridium difficile.
28th April – 12th May 2009
Report generated for BactaClean LLP, Envirosystems (UK) Ltd.
Work Request
‘To provide a solution to nosocomial in healthcare facilities as well as the reduction in the risk of infections in schools, homes and all other type facilities, we are wishing to introduce an innovative range of cleaning products into the UK including the Health Service, Resthomes and public places. Containing friendly probiotic bacteria, these cleaners leave behind the safe bacteria after a normal cleaning process has taken place thus populating the area. In doing so they carry on the cleaning process in places where brushes, mops and cloths cannot reach and also compete in numbers with pathogenic bacteria to restrict their growth through competitive exclusion. We need to test in a laboratory the effectiveness of this process using our PIP range of cleaners and measure how well they address the pathogenic bacteria load over time specifically looking at MRSA and Clostridium difficile. Conditions similar to those in hospital wards where different levels of disease challenge will be replicated in a laboratory situation in order to measure effects. Need to be doing sufficient replicated trials to produce meaningful data. Specific disease challenges off MRSA & C. Diff to be tested at varying levels of infection. Produce a dossier of the trial work and analysis of the findings.’
Results
PIP cleaning solutions were tested for their effect upon Staphylococcus aureus using the Methicillin-resistant strain MRSA252, a hospital epidemic strain isolated in the UK. In addition the solutions were tested for efficacy against Clostridium difficile MHCR strains 107 and 029. These isolates are two common clones causing epidemic outbreaks in UK hospitals.
Effect upon S. aureus
S. aureus MRSA 252 was cultured in BHI broth (Lab M) from stored frozen cultures and grown overnight at 37°C. Cells were diluted in water or water containing 6.5% BHI to approx. 108 cfu per ml before addition of PIP cleaning solution to 1% (v/v) (Chrisal). At time points 0, 4 and 24 h samples were removed and serially diluted before plating on BHI agar. Viability was assessed after overnight growth at 37°C and the effect of PIP was plotted. A representative dataset for 0 and 24 h time points is shown below (figure 1) and is typical of triplicate experiments. No difference was observed between PIP floor cleaning solution and sanitary solution under the conditions tested (data not shown).
Typically both cleaning solutions reduced viability around 10-fold over a 24 h period, producing a 2-3 fold reduction in viability at 4 h. PIP cleaning solution was also tested on desiccated cells of S. aureus on sterile plastic surfaces after drying at 37°C and maintained at 25°C overnight. The aim of this was to mimic dried cells of S. aureus in a hospital ward. Drying reduced viability of S. aureus markedly; the addition of floor cleaning solution produced a similar reduction in viability of approx 10-fold to that observed in the liquid assays detailed above and shown in figure 1.
Figure 1: Effect of PIP solution on viability of S. aureus MRSA 252. Viability tested using 1% (v/v) floor cleaning solution upon overnight culture cells. Error bars represent SE of mean from 4 separate samples.
Conclusion
Cleaning solutions diluted to 1% (v/v) reduce viability of S. aureus MRSA252 by approx 10-fold after 24 h incubation compared to an untreated control.
Effect upon C. difficile
Hospital outbreak strains of C. difficile were obtained from Royal Liverpool and Broadgreen University Hospital Trust. This study used MHCR109 and MHCR027. These isolates were cultured on fastidious anaerobe agar supplemented with 10%( v/v) blood in anaerobic containers at 37°C for 72 h to ensure the bacteria had sporulated. Spores were scraped from agar plates and washed in sterile water. Oil immersion microscopy revealed preparations were >90% spores. The spores were not heat treated prior to the assays. PIP cleaning solutions were tested for their effect upon the spores using the floor cleaning solution at 1% (v/v) in water with approx 105 cfu per ml in an aerobic assay. Samples were removed at 0, 4 and 24 h and serially diluted to calculate viable cells counts after overnight growth on FAA under anaerobic conditions at 37°C (figure 2). PIP floor cleaner diluted to 1% reduced viability of S. aureus MRSA252 by approx. 10-fold after 24 h incubation compared to an untreated control.
Figure 2: Representative experiment showing the effect of PIP floor cleaning solution (1% v/v) on survival of C. difficile spores of MHCR 109. Error bars represent standard error of the mean generated from quadruplicate samples. Experiments were repeated in triplicate.
Conclusion
Conclusion: PIP floor cleaning solution diluted to 1% (v/v) markedly reduces viability of C. difficile compared to the untreated control, as judged by viable counts of treated spore suspensions after 24 h of incubation under the assay conditions tested here. The proposed effect of the enzymes in the product is to induce germination of C. difficile, which will be deleterious to the survival of this anaerobe in the aerobic conditions of the assay. The results obtained here are entirely consistent with this claim given the observed reduction in viability.
Results generated by:
Dr Malcolm Horsburgh
Bacterial Pathogenesis Group
School of Biological Sciences
University of Liverpool
Liverpool, L69 7ZB
