A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System

A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System December, 2014 By John Edwards Source Probiotics Ltd.

The Chrisal Cleaning System for Hospitals and Health Care Facilities Hospital Acquired Infections and Antibiotic Resistant Pathogens While hospitals and health care facilities are challenged with hospital acquired infections that are proving to be difficult and expensive to treat; the implications for patients can be much greater, resulting in morbidity and death. Clearly the cleaning regimens now in place are not coping with the increased resistance of organisms such as MRSA, VRE, Clostridium difficile, E. coli, Salmonella sp., Streptococcus sp., and others. The Chrisal probiotic products represent a new paradigm in cleaning by using specific, naturally occurring, Type 1 bacteria in combination with cleaning agents such as surfactants and enzymes for their mechanism of action. They have an efficacy that has been well demonstrated in a number of hospital studies as well as in active use over the past six years, and Chrisal products are used globally in a range of facilities from acute care hospitals and medical clinics, to long term health care facilities for the aged. The majority of these are in Europe; however there are now locations in the US. The Mechanism of Action Bacteria have a strong tendency to develop resistance to any substance that might be detrimental or lethal to them. This phenomenon is well demonstrated for antibiotics and disinfectants. In order to avoid the development of resistance, none of the Chrisal cleaning products has any direct biocidal action towards other organisms; instead the mechanism of action is based on the principle of competitive exclusion, combined with an influence on the quorum sensing communication between organisms. Disinfectants have an unspecific biocidal action; killing both harmful and harmless microorganisms. This results in a surface actually more conducive to rapid colonization in a very limited time frame. Each disinfection procedure leaves organic matter behind that serves as protein and carbohydrate sources, creating fuel to sustain the fast re-colonization of the surface. Disinfection also removes the normal beneficial bacteria that hold back pathogen growth through competitive exclusion. Using the energy resources created by disinfectants, pathogens can multiply from one to a population of one million cells within 8 hours. While disinfection results in a fast reduction in the total number of micro-organisms, the effect is very short and unstable. The resistance of pathogens like MRSA to higher concentrations and frequencies of disinfectant/antibiotic applications has resulted in an increased risk to people and the environment from both the pathogens and the aggressive chemicals meant to kill them. Disinfectants and Biofilms The effectiveness of disinfectants is determined by standardized tests, and the Government of Canada has released a Guidance Document for Disinfectant Drugs which may be found at http://www.hc-sc.gc.ca/dhp-mps/prodpharma/applic-demande/guide-ld/disinfectdesinfect/disinf_desinf-eng.php. The problem with using the information above to determine if disinfectants are effective is that laboratory tests are often not an indicator of what happens in true life. Many species of bacteria build biofilm around themselves which can drastically reduce the effectiveness of the disinfectant. In a paper published in 2011 in the journal Biofouling: The Journal of Bioadhesion and Biofilm Reasearch, Bridier et al reviewed the past research and stated: It is now generally accepted that the biofilm growth mode induces microbial resistance to disinfection that can lead to substantial economic and health concerns. A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 2

The paper cited that there are a variety of factors at play here: 1....The formation and maintenance of mature biofilms are intimately linked to the production of an extracellular matrix (Branda et al. 2005; Ma et al. 2009). The multiple layers of cells and EPS may constitute a complex and compact structure within which biocides find it difficult to penetrate and reach internal layers, thus hampering their efficacy. 2....But because biocides are often highly chemically reactive molecules, the presence of organic matter such as proteins, nucleic acids or carbohydrates can profoundly impair their efficacy (Lambert and Johnston 2001) and potential interactions between antimicrobials and biofilm components seem more likely to explain the limitations of penetration into the biofilm. 3....some results have shown that despite an effective penetration of a biocide into a biofilm, only a low level of inactivation was achieved (Stewart et al. 2001). Moreover, the resistance of a S.aureus biofilm to a QAC could, to a great extent, be attributed to phenotypic modifications to cells rather than the protective presence of an EPS matrix (Campanac et al. 2002). These findings highlight the existence of additional mechanisms involved in biofilm resistance. During a disinfection process, the reaction-diffusion limited penetration of biocides into a biofilm may result in only low levels of exposure to the antimicrobial agent in deeper regions of the biofilm. Biofilm cells will therefore develop adaptive responses to sub-lethal concentrations of the disinfectant. Increased survival following the same QAC shock was reported in adapted Pseudomonas aeruginosa, alongside concomitant modifications to membrane composition (Jones et al. 1989; Mechin et al. 1999). 4. Moreover, cross-resistance to other QACs (Mechin et al. 1999) or to antibiotics (Braoudaki and Hilton 2004) has been reported for adapted cells. This resistance is very significant in that research has shown that bacteria such as E. Coli, S aureus, P. aeruginosa in their biofilms may exhibit resistance coefficients up to 1000 times that of their planktonic bretheren. To access the paper go to http://www.tandfonline.com/doi/full/10.1080/08927014.2011.626899. Why are these problems not relevant to the Chrisal products? First, the principle behind competitive exclusion is that during the cleaning procedure a layer of beneficial bacteria is placed on the treated surface, immediately occupying the surface with "good" bacteria. They consume all remaining food sources, leaving nothing behind for potential pathogenic invaders looking for space and food. The beneficial Chrisal bacteria are much more active and out-compete all other organisms. Second, "Quorum Sensing" is an extremely fast means of communication between bacteria, using species-specific signal molecules. When the Chrisal bacteria are applied to the surface, an overwhelming competition for space, food and moisture is established, along with a flood of Chrisal's signalling molecules. This immediately results in the pathogenic bacteria, informing each other through quorum sensing that conditions have become unfavourable for growth, leading them to enter a dormant phase and allow for their subsequent removal. A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 3

The third major and key advantage is that Chrisal immediately starts to remove the biofilm that was created by pathogens and other bacteria and it is this very biofilm that serves to protect colonies of harmful bacteria, as well as hold dirt and support viruses and other problematic substances. The Chrisal approach has several main advantages; it provides a stable solution to problems with pathogens and no resistance can be developed. The only demand set by this method is that the frequency of cleaning is kept constant. This allows for up to several days between each cleaning, which is far more time than any other cleaning methodology. It is important to realize that the total number of micro-organisms will tend to remain the same after Chrisal treatment; however it is the harmless bacteria simply replacing the harmful ones. The Properties of Chrisal Bacteria All of the Chrisal products contain only Class 1 species of bacteria that are listed on the Canadian Domestic Substances List, as well as approved on the Association of American Feed Control Officials List. This list is found in the 2009 AAFCO Official Publication, Chapter 5 (Official Feed Definitions), Part 36 (Fermentation Products), and sub part 14 (Direct-Fed Microorganisms). All bacteria belong to the genus Bacillus, and originate from the natural environment. The species used are the same as those commonly employed in the biotechnology industry over the last 40 years. No strains have been genetically altered. Class 1 is the lowest risk category, according to all International classification schemes, and none are considered pathogenic, nor hazardous, according to Council Directive 2000/54/EC. Individual strains are studied to determine their precise identification using the latest biochemical and fingerprinting techniques (e.g. 1500 bp, 16S rdna sequencing). This identity is then compared with National databases for monitoring changes in microbial nomenclature. None of the Bacillus strains used in Chrisal NV/SA products produced B. cereus-like toxins, confirming the view that such species are safe to use in the microbial products. No strain is known to induce any acute oral, dermal and inhalation toxicity, dermal irritation, eye irritation and skin sensitivity. Testing in FDA licensed laboratories has further confirmed this. Supplier records indicate that there are no incidences of respiratory illness or skin infection traceable to production cultures. A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 4

Results of Trials Performed in Hospitals, Universities, and Medical Clinics 1. Lokeren Hospital, Lokeren, Belgium In 2006 an eighteen month study of the use of Chrisal cleaning products commenced at Lokeren Hospital in conjunction with the University of Gent. The study was comprised of two phases: Phase 1: The entire third floor of the AZ Lokeren hospital was treated with Chrisal s cleaning products and compared to the first Floor where conventional cleaning chemicals were used. The patient types on Floors 1 and 3 are similar. Phase 2: Following the first phase, a buffer period of one month with overall regular cleaning was inserted. Subsequently, the entire hospital was cleaned with the Chrisal products, except for the third Floor now served as a control. The study involved the taking of thousands of test samples during a rotation of cleaning regimens that included both conventional chemical cleaners and Chrisal probiotic cleaning products. Some of the important results from that study were: i. A Reduction in the Occurrence of Coliform Bacteria Cleaning with Chrisal products resulted in areas that had lower coliform counts than the control floor. On average, the reduction of coliform counts in those areas cleaned with Chrisal products was 60%. A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 5

ii. A Reduction in the occurrence of Staphylococcus aureus Cleaning with Chrisal products resulted in areas that had lower S. aureus counts than the control floor. On average, the reduction of S. aureus counts in those areas cleaned with Chrisal products was 74%. This efficacy was irrespective of the antibiotic resistance of the S. aureus strain. iii. Changes in the occurrence of Staphylococcus aureus when cleaning regimens changed. Cleaning with Chrisal products decreases the number of S. aureus. From the moment cleaning stops, the number of S. aureus increases again to values within the same range as those prior to Chrisal cleaning. These observations demonstrate that the observed reductions in S. aureus are the result of the Chrisal cleaning. A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 6

As a result of the study, Lokeren Hospital decided to use Chrisal cleaning products throughout the hospital. Following is a letter dated November 2009 describing the effects of that decision. Their use continues to the present in all areas of the hospital including the operating rooms. There are also other hospitals in Belgium that have been using Chrisal products for periods of years. A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 7

2. Miami Jewish Home and Hospital An extensive study of the efficacy of Chrisal was undertaken in a 740 bed hospital and long term care facility in Miami, FL in 2008 that led to the adoption of Chrisal products in the hospital. Some of the important results of that study were: i. A Dramatic Reduction in Pathogens as seen from Petri-Film testing The following image shows the change in the number of pathogens observed by Petri-Film testing. Standard Products Used before Chrisal applied Week 1 after the start of Chrisal use. Week 2 after the start of Chrisal use. Week 3 after the start of Chrisal use. A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 8

Following is the reference letter from Miami Jewish Home and Hospital dated November 24, 2009. A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 9

3. St. Anne di Ferrara Hospital, Ferrara, Italy The preliminary study was undertaken in 2011 to compare the Chrisal cleaning products to traditional chemical cleaning products in both a laboratory and hospital environment. The study was funded by an Italian company COPMA SCRL and conducted through the University of Ferrara at the Saint Anne di Ferrara Hospital. As of 2014 the hospital is using Chrisal products through all parts of the hospital including the ICU. The study involved both in vitro and in vivo studies, In the in vitro studies the effect of Chrisal products upon three species of pathogenic bacteria were studied. They were Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The in vivo studies demonstrated that 1 hour after application of the Chrisal products on the sample surfaces that had been previously contaminated with various microbial strains, the reduction in the concentration of pathogens was 6 logs, or 1,000,000 times lower than the initial counts. The in vivo studies were conducted over several months at the St. Anne di Ferrara Hospital and demonstrated the following results: i. Percentage Reduction of Pathogens when Comparing Traditional Cleaning Procedures to Chrisal Cleaning Sampling Points Pathogen General Outpatient Mean value of Medicine Ward General Medicine Phase 1 & 2 Phase 1&2 Ward Phase 3 (*) Entry and end Staphylococcus aureus 29,56% 36,64% 81,03% of corridor Coliforms spp 72,38% 46,62% 79,72% Pseudomonas spp 93,09% 64,49% 88,44% Candida spp. 68,88% 56,21% 68,47% Acinetobacter 44,74% Toilet floor Staphylococcus aureus 58,75% 51,33% 85,88% Coliforms spp 89,15% 78,13% 78,31% Pseudomonas spp 55,28% 75,94% 78,57% Candida spp. 82,90% 67,80% 71,78% Acinetobacter spp. 74,25% Toilet sinks Staphylococcus aureus 55,74% 52,50% 95,59% Coliforms spp 81,56% 75,83% 85,12% Pseudomonas spp 67,53% 50,41% 95,16% Candida spp. 50,38% 27,93% 94,86% Acinetobacter 16,39% 31,25% 75,99% It was found that after two months of use, the prolonged action of the Chrisal probiotics caused a substantial decrease in the pathogenic microbial load compared to the areas that were treated with traditional products. In many cases the populations of the microorganisms of interest were A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 10

reduced by close to 90%. One example was from the sink in the toilets, which is a critical surface for patients. The second phase of the study was undertaken at the Rehap Centre San Giogio which is a 90 bed hospital with a constructed area of 30,000 square meters. It involved two types of research. The first was to confirm at a different location the pathogen reduction data from the preliminary study, and the second was to determine if the reduction in pathogens translated to a reduction in nosocomial infections. The results of the research demonstrated that the use of the probiotic cleaning products displayed the following advantages: Figure 1. a. The pathogen load in the hospital was demonstrated to decrease over the course of time through both in vitro and su campo testing. This was verified by 14,400 samples that were taken and analyzed. These samples were done in real, operational conditions and on different types of surfaces in the presence of continuous recontamination. b. After comparing the cleaning procedures that were used for both the chemical products and the probiotic cleaning products they saw that the probiotic products reduced pathogens by a factor of 70-80% better than the chemical cleaners. c. They also demonstrated that when using the chemical cleaning, the action of the disinfection of S. aureus and E. coli was relatively short-lived. S. aureus values increased by 80% from the first testing to re-testing six and one-half hours later. E- coli values increased by 145% during that time span. (Figure 1.) d. In contrast, the probiotic cleaning prolonged the effect of the cleaning. The values of S. aureus seven hours after cleaning were 48% what they were 13 hours earlier and values of E. coli were 40% of the values from 13 hours later. (Figure 1.) Chemical cleaning 30 minutes after disinfection 7 hours after disinfection S. aureus 54.50 cfu/100 cm² 97.95 cfu/100 cm² E. coli 9.41 cfu/100 cm² 23.01 cfu/100 cm² Chrisal cleaning 6 hours before cleaning 7 hours after cleaning S. aureus 30.50 cfu/100 cm² 14.70 cfu/100 cm² E. coli 11.16 cfu/100 cm² 4.60 cfu/100 cm² A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 11

The reduction in the hospital environment of potential pathogens was significant and the load of potential pathogens was reduced dramatically (Figure 2.). Figure 2. Overall Reduction of pathogens from probiotic cleaning compared to chemical cleaning S. aureus 90% Pseudomonas 82% Candida spp. 90% Coliforms 92% Acinetobacter spp 78% From January 2012 to October 2012 there was a reduction was 34.3% in Hospital Acquired Infections at the Rehab Center S. Giorgio. The conclusion was that the use of probiotic enhanced cleaners significantly reduces pathogens in the hospital environment for greater durations than disinfectants, and that along with good hospital practices and good hand hygiene there can be significant reductions in Hospital Acquired Infections. Over the period to from October 2012 to September 2014 the reduction in HAI s increased to 69% and the hospital estimated that they had saved in excess of 500,000 per year. Two papers have been published in peer-reviewed journals. They are: 1. Vandini A, Temmerman R, Frabetti A, Caselli E, Antonioli P, et al. (2014) Hard Surface Biocontrol in Hospitals Using Microbial-Based Cleaning Products. PLoS ONE 9(9): e108598. doi:10.1371/journal.pone.0108598 2. Vandini A, Frabetti A, Antonioli P, Platano D, Branchini A, et al. (2014) Reduction of the Microbiological Load on Hospital Surfaces Through Probiotic-Based Cleaning Procedures: A New Strategy to Control Nosocomial Infections. J Microbiol Exp 1(5): 00027. DOI: 10.15406/ jmen.2014.01.00027 4. Shriners Hospital for Children, Tampa, Florida The Shriners Hospitals for Children has the Mission to: Provide the highest quality care to children with neuromusculoskeletal conditions, burn injuries and other special healthcare needs within a compassionate, familycentered and collaborative care environment. Provide for the education of physicians and other healthcare professionals. Conduct research to discover new knowledge that improves the quality of care and quality of life of children and families. This mission is carried out without regard to race, color, creed, sex or sect, disability, national origin or ability of a patient or family to pay. A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 12

Their vision is: Shriners Hospitals for Children will be the unquestioned leader, nationally and internationally, in caring for children and advancing the field in its specialty areas. Rationale for the Evaluation of Chrisal Products at the Shriners Hospital in Tampa The Shriners Hospitals for Children has the long-term goal of: Providing better infection control Reduce respiratory problems among patients and staff. Improve worker safety Accomplish better cleaning in the hospital Reduce material and labor costs Protect the environment. Important additional factors include problems with infection control and cleaning that have come to light. These include: The major impact biofilm has in the propagation of a wide variety of pathogens. Disinfectants are known to play a role in the development of resistant pathogenic bacteria Recognition that existing cleaners and disinfectants exacerbate the respiratory problems experienced by children. Cleaners and disinfectants can also engender allergic reactions in both the staff and the patients, creating a search for less toxic alternatives. The Shriners hospital study was designed to investigate a new paradigm in cleaning that met their long term goals. The study was not designed to consider a substitution of products; rather it was part of an in-depth strategy to find solutions that would solve current concerns while anticipating future requirements. Experimental Design Twenty-five locations within the hospital were monitored including public areas and patient treatment areas. Examples are shown below. Each area was first cleaned with the hospital s normal cleaning products then: ATP (Adenosine Triphosphate) testing was done using a Hygiena ATP Meter and swabs. A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 13

Cultures were taken of the same area using 3-M culture swabs that were then delivered to the hospital laboratory and poured onto 3-M culture media. The media was incubated for 24 hours before being checked and frozen for photographs. Subsequent testing for all areas followed this protocol. Results Changes in the Presence of Bacterial contamination through the testing period. The results below are typical of what was seen at all of the test areas. Any contamination that was present was eliminated by the second or third week of cleaning. Conclusions The final result of the Shriners Hospital study demonstrated that the Chrisal probiotic cleaners exceeded the criteria set forth in the goal to find new methods and solutions that: (1) Improved cleaning, (2) Reduced infection, (3) Eliminated adverse respiratory and allergic response in patients and staff. (4) Reduced both material and labor costs, (5) Reduced the negative impact on the environment with toxic chemicals. A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 14

5. Midland Medical Clinic Chrisal has been used for four years at a medical Clinic in Fort Lauderdale, FL. This clinic has a high AIDS patient population, and many are immuno-compromised. Following is a reference letter from the Medical Director of the Clinic written in January 2010. The Clinic has continued to use Chrisal products since then. A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 15

6. The University of Liverpool Testing the effect of Chrisal cleaning products on viability of Staphylococcus aureus and Clostridium difficile. Dr Malcolm Horsburgh, 28th April 12th May 2009 Bacterial Pathogenesis Group, School of Biological Sciences, University of Liverpool. Conclusion: Chrisal 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. 7. The University of Ulster MRSA Study of Healthcare Worker Infection & Domestic Environment Contamination Report by Prof. D. McDowell, University of Ulster Test Period from January 2009 May 2009 Results: Preliminary results have clearly demonstrated that, when the Chrisal Product was correctly and consistently applied, (i.e. initial specialist treatment, followed by ongoing regular non-specialist/consumer maintenance treatments) the product[s] were able to: Initially eliminate (or reduce to undetectably low numbers) all of the MRSA from these complex contaminated domestic environments (when the colonised humans and animals were temporarily excluded). Significantly limit the numbers and incidence/dissemination of MRSA in this domestic environment, during the course of the study (more than 100 days) despite very significant pressures towards heavy and widespread re-contamination, associated with the re-entry of persistently MRSA positive (colonised) individuals into the tested environment. 8. The Medical Institute of Ostrava Independent effectiveness trial of probiotic cleaning products Kateřina Matějová, RNDr. Erich Pazdziora, CSc., RNDr. Věra Toršová, CSc., Medical Institute of Ostrava, Czech Republic Survivability testing was done on 5 species of bacteria: Staphylococcus aureus CCM 3953 Enterococcus faecalis CCM 4224 Klebsiella pneumoniae CCM 4415 Pseudomonas aeruginosa CCM 1960 Staphylococcus aureus - Methicillin resistant (MRSA) isolated from a nosocomial infection Results: After 5 days of cleaning with the Chrisal product, none of the test strains survived. All surfaces were colonised by Bacillus. Test results on MRSA show that after 5 days of cleaning also MRSA was completely removed from the various surfaces, except for textile. However, we need to draw the attention to the fact that the textile was constantly wet, so we can conclude that A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 16

the textile needs to be dry in order to achieve a decent beneficial cleaning. (Hence the reason for the development of the Chrisal Allergy Free (BioMist in Canada) spray that applies the beneficial bacteria to textile surfaces without making them wet; remark added by Chrisal) The best result was measured against gram negative rod shaped bacteria, whereas Enterococcus faecalis demonstrated to be the most resistant bacteria from all test strains. This resistance of Enterococcus faecalis is also know from test results with disinfectants. Test results on MRSA indicated that even a very high MRSA load can be completely removed after 3 days of Chrisal cleaning. Chrisal Cleaning Products are Used Around the Globe Chrisal cleaning products are being used at these and many more healthcare facilities around the globe. There are also on-going and new studies that are proving that even after extended use, Chrisal cleaning products continue to deliver consistent results in terms of: Improving cleanliness Reducing the presence of pathogenic bacteria Reducing nosocomial infections Protecting worker safety. Saving money, both in terms of cleaning costs and the reduced expense for antibiotics and shorter hospital stays due to reduced nosocomial infections. As of December 2014 there are now over 50 hospitals in Italy using Chrisal products as well as in Belgium, Germany, Switzerland Russia, the US, Canada, and other countries. Please refer any questions to: John Edwards Source Probiotics Ltd. edwardsj@sourceprobiotics.com (250) 519-0244 A New Paradigm of Cleaning for Healthcare- The Chrisal Probiotic Cleaning System Page 17