Comparison of Aerobic and E. Coli Colony-Forming Units Isolated From Circulating Paper and Plastic $20 Canadian Banknotes Submitted by: Ryan Olivier Submitted to: British Columbia Institute of Technology Environmental Health 2013 Copyrighted material. All rights reserved. This work may not be reproduced in any way, or by any means, without expressed permission of the author. Abstract Introduction Paper currency serves as an ideal breeding ground for microorganisms. Furthermore, the combination of its widespread use and its constant exchange make paper currency a likely agent for disease transmission. Recently the Bank of Canada has begun issuing plastic banknotes; theoretically, these plastic bills are less prone to contamination due to the inherent properties of plastic and specifically added antibacterial agents. Purpose The purpose of this study was to determine if the new plastic banknotes harbor fewer bacterium than comparable (in age and denomination) paper bills. The objective was to sample both paper and plastic bills, and statistically verify whether there is (or is not) a difference in microbial load. Methods Standard microbiological methods were followed to test paper and plastic bills. The sample bills were tested using 3M aerobic colony Petrifilm plates and E. coli/coliform Petrifilm plates. The number of colonies counted on the Petrifilm plates (both types) was used to indicate associated contamination levels. Results On average, the plastic bills had lower counts of aerobic bacteria and E. coli coliforms. However, there was not a statistically significant difference of contamination rates between plastic and paper bills (p-value: 0.090332). A low power (0.380125) indicates a beta error may have occurred and that a larger sample size is required to provide more accurate results. Conclusions The main conclusions resulting from this study include the following: 1. Contamination rates for plastic bills are statistically similar to those of paper bills. 2. On average (mean and median data), plastic bills had fewer aerobic bacteria and E. coli coliforms than paper bills. 3. Canadian bills have a similar contamination rate as US bills. This study showed a 6.6% rate of heavily contaminated Canadian bills, compared to 7% in the United States (Lamichhane, Adhikary, Guatam, & Maharjan, 2009). 4. Contamination rates varied greatly. While the majority of bills had relatively low contamination rates, a select few had extremely high rates.   ii Table of Contents Abstract………………………………………………………………………………………...ii 1.0 Introduction………………………………………………………………………………...1 2.0 Literature Review…………………………………………………………………………..2 2.1 Significance to Public Health……………………………………………………....2 2.2 Prevalence……………………….………………………………………………….2 2.2.1 Worldwide………………………………………………………………..3 2.2.2 Developing Nations…………………………………..…………………..3 2.2.3 Developed Nations.……………………………………………………....4 2.2.4 United States and Canadian Currency………………………………...….4 2.3 Pathogens of Concern………………………………………………………….…...5 2.3.1 Common Pathogens Found on Currency………………………………....5 2.3.2 Vulnerable Populations…………………………………………..….........6 2.4 Implications for Food Premises………………………………………………….....6 2.4.1 Foodborne Illness and Currency……………………………………….....6 2.4.2 Mobile Food Vending Operations…………………………………..........7 2.5 Canadian Guidelines and Regulations……………………………….......……........7 2.5.1 Bank of Canada…………………………………………....…………......7 2.5.2 Food Premises Regulation……………………………………………......8 2.6 Introduction of New Canadian Bills………………………………………….….....8 2.6.1 New Canadian Notes Enter Circulation…………………………….….....8 2.6.2 Public Health Features………………………………………………........8 3.0 Purpose of Study………………………………………………………………………........9 4.0 Methods and Materials...………………………………………………………….…...........9 4.1 Materials Used………………………………………………………………….…...9 Table 4.1-1 Scientific Materials Required for This Experiment…………….......9 Table 4.1-2 Statistical Materials Required for This Experiment…………….......9 4.2 Standard Methods……………………………………………………......................10 4.2.1 Sampling Media…………………………………………………..............10 4.2.2 Description of Media……………………………………………………..10   iii 4.3 Alternative Methods……………………………………………………………...11 4.4 Justification of Methods……………………………………………………….....12 4.5 Reliability and Validity of Measures…………………………………………......12 4.6 Inclusion and Exclusion Criteria………………………………………………....13 4.7 Pilot Study………………………………………………………………………..13 5.0 Statistical Analysis……………………………………………………………………….14 5.1 Description of Data……………………………………………………………....14 5.2 Statistical Package Used………………………………………………………....14 5.3 Descriptive Statistics……………………………………………………………..14 5.4 Inferential Statistics……………………………………………………………....14 6.0 Results......................………………………………………………………………..…....15 Table 6.0-1 Descriptive Statistical Results Using NCSS 8.....................…….15 Table 6.0-2 Statistical Results of the Paired t-Test Using NCSS 2012…...….16 6.1 Interpretation of Results.........................................................................................16 6.1.1 Descriptive Statistics Result....................................................................16 6.1.2 Inferential Statistics Results....................................................................16 6.2 Alpha and Beta Error Discussion……………...............…………………….…...17 7.0 Discussion..........................................................................................................................17 8.0 Limitations.........................................................................................................................19 9.0 Conclusions........................................................................................................................19 10.0 Recommendations............................................................................................................19 11.0 Future Research Recommendations.................................................................................20 References…………………………………………………………………………………......v Appendix A: – 3M Petrifilm Aerobic and E. coli Plate Count Procedures.………………...viii Appendix B: – Sampling Techniques Tested…….….…………………………………….......x Appendix C: – Total Bacterial Count on Paper Bills ………………………………………...xi Appendix D: – Total Bacterial Count on Plastic Bills............................................................xiii Appendix E: – Descriptive Statistics Report for Paper Bills………………………………....xv Appendix F: – Descriptive Statistics Report for Plastic Bills……………………………......xvi Appendix G: – Paired t-Test Report for Paper vs. Plastic Bills……………………………..xvii   iv 1.0 Introduction Money is the most widely used and sought after service on the planet. The transfer of paper currency has been the model of economic exchange since its introduction in China circa 1000 AD (Bernholtz, 2003). In the late 1800s and early 1900s, scientists began to theorize that the transmission of money was associated with the transmission of disease (Schaarschmidt, 1884) (Hilditch, 1908) (Morrison, 1910) (Boyer, 1921). Modern scientific techniques have confirmed these theories and have shown that viable pathogenic organisms (viruses, bacteria, and fungi) can be isolated on the surfaces of both paper and coin currency (Kuria, Wahome, Jobalamin, & Kariuki, 2009) (Lamichhane, Adhikary, Guatam, & Maharjan, 2009) (Thomas, Vogel, Wunderli, Suter, & Witschi, 2008). Research has shown that paper currency serves as an ideal breeding ground for microorganisms for several reasons. First, the paper bills offer a large surface area for organisms and organic debris to collect (Ayandele & Adeniyi, 2011). Secondly, folds and/or deliberate depressions or projections specifically engineered into the bills’ design as anti-counterfeiting methods serve as settling sites for both organisms and debris, which allow the microorganisms to live longer (Lamichhane, Adhikary, Guatam, & Maharjan, 2009). Lastly, banknotes weave their way through the population for many years before they come to rest. Studies indicate that the age and denomination of a bill have a direct correlation with the contamination observed (e.g., older bills had the most contamination while newer bills had the least) (Pradeep, Marulasiddaiah, & Chetana, 2012). Physical transfer of material from hands, surfaces, and the environment can contaminate paper currency (Ahmed, Parveen, Nasreen, & Feroza, 2010) (Kuria, Wahome, Jobalamin, & Kariuki, 2009). Individuals from almost every socio-economic background routinely hold and   1 transfer paper currency. Any object that can spread communicable diseases throughout a diverse population should be considered a risk to public health. Therefore, paper currency has an important role in the transmission of pathogenic microorganisms and presents a moderate risk to public health. The Bank of Canada has ceased making paper currency. Beginning in 2011, the bank began issuing new polypropylene (plastic) currency notes for $100 and $50 bills, with all other denominations scheduled for transition by 2013. The new bills have four advertised features to lessen the microbial load on circulating notes: less pore space for colonization, easier to physically clean (waterproof), absorb little to no moisture, and are impregnated by an antibacterial agent (Bank of Canada, 2012b). This study aims to test these claims by examining the microbial load on circulating paper and plastic Canadian currency. 2.0 Literature Review 2.1 Significance to Public Health Paper currency and coins can serve as agents for transmission of microorganisms (fomites) and are frequently and freely passed from person to person. This section will limit its focus to the prevalence of contamination, common isolated pathogens from banknotes, risks associated with food establishments, and the introduction of the new plastic Canadian banknotes. 2.2 Prevalence Two constant aspects of the studies researched show that denomination and age of a bill directly correlate with contamination. Currency notes of lower denominations were the most contaminated, presumably because lower denomination notes pass through more hands in their lifetime than the higher denomination notes (Lamichhane, Adhikary, Guatam, & Maharjan, 2009) (Ayandele & Adeniyi, 2011). Other studies demonstrated that the age of the currency note   2 had a positive correlation with microbial contamination. Increased contact time is presumed to escalate contamination (Barro, Bello, & Savadogo, 2006) (Igumbor, Obl, & Bessong, 2007). 2.2.1 Worldwide Studies from around the world have reported high rates of microbial contamination of currency notes in circulation (Pradeep, Marulasiddaiah, & Chetana, 2012) (Barro, Bello, & Savadogo, 2006) (Ayandele & Adeniyi, 2011). Although every location contained endemic bacterium, the microorganisms most commonly isolated on paper money included members of the family Enterobacteriacea, Mycobacterium tuberculosis, Vibrio cholerae, Bacillus sp., Staphylococcus sp., Micrococcus sp., and Corynebacterium sp. (Ahmed, Parveen, Nasreen, & Feroza, 2010). Common background contaminants of paper money were environmental organisms such as gram-positive flora (especially Bacillus sp.) and those arising from human normal skin flora such as Staphylococcus aureus (Ahmed, Parveen, Nasreen, & Feroza, 2010) 2.2.2 Developing Nations Developing nations have the highest rates of currency contamination. After researching studies conducted around the world, it became clear that poor nations with large, impoverished populations were funding these studies (India, Nepal, Myanmar, Vietnam, several parts of Africa, and others) (Pradeep, Marulasiddaiah, & Chetana, 2012) (Igumbor, Obl, & Bessong, 2007) (Barro, Bello, & Savadogo, 2006). One particular study conducted in the Venda region of South Africa showed that bacteria and fungi were isolated from 96% of the used banknotes collected in the study (Barro, Bello, & Savadogo, 2006). No microorganisms were isolated from new banknotes received directly from the bank. The source of contamination on the used notes must be from handling and use. Of particular concern was the isolation of Shigella and Salmonella from the currency, which   3 indicated fecal contamination. This finding supports the theory that individuals who prepare food after handling contaminated currency notes have a higher risk of infecting themselves and others with foodborne pathogens. 2.2.3 Developed Nations Microbial contamination of paper money is not only confined to developing nations. Several studies from the United States reported contamination of coins and paper bills and revealed the presence of pathogenic microbes like Staphylococcus aureus, Escherichia coli, and Klebsiella enterobacter (Vriesekoop, Russell, & Alvarez-Mayorga, 2010). One such study of US currency isolated 93 types of bacteria (belonging to the species Staphylococcus, Streptococcus, Enterobacter, Acinetobacter, Pseudomonas, Bacillus, Diptheroids, Klebsiella pneumoniae, and Escherichia vulneris) (Ahmed, Parveen, Nasreen, & Feroza, 2010). 2.2.4 United States and Canadian Currency The United States and Canadian type of banking systems revolve around a continuous cycle of printing new bills while destroying old “contaminated and mutilated” banknotes. This system is, to date, the most effective method of getting contaminated banknotes out of circulation before they can cause health problems to vulnerable individuals (Bank of Canada, 2012a) (Federal Reserve Bank, 2012). A study of American coins and currency revealed the presence of pathogenic bacteria on 18% of the coins and 7% of the bills (Raloff, 2010). The study stated that the “cleanest” banknotes contained 20 CFUs (colony-forming units) and the “dirtiest” banknotes contained more than 25,000 CFUs (Raloff, 2010). No published studies of Canadian currency contamination were found at the time of this writing. However, in 2007 a former British Columbia Institute of Technology (BCIT) student   4 (Tony Gill) conducted a research paper titled, “Is the Amount of Contamination on Money a Significant Threat to Public Health?” Although his research provided data regarding paper $20 bills, the data will not be used in this study because it met the definition of exclusion data listed in Section 4.6. Gill’s study did, however spark my interest in the association between contaminated banknotes and foodborne illness. 2.3. Pathogens of Concern 2.3.1 Common Pathogens Found on Currency Potentially dangerous bacterial agents that have been isolated on paper currency include the following: 1. Streptococcus and Staphylococcus that have developed resistance to conventional antibiotics (Lamichhane, Adhikary, Guatam, & Maharjan, 2009). 2. E. coli is usually nonpathogenic, but some strains can cause serious (potentially fatal) food-poisoning infections (Lamichhane, Adhikary, Guatam, & Maharjan, 2009). 3. Enterobacter cloacae is associated with urinary tract and respiratory tract diseases (Lamichhane, Adhikary, Guatam, & Maharjan, 2009). 4. Staphylococcus epidermidis is usually nonpathogenic but can cause infection in patients whose immune system is compromised (Lamichhane, Adhikary, Guatam, & Maharjan, 2009). 5. K. pneumoniae is a virulent organism that can cause pneumonia, typically along with urinary tract and wound infections, particularly in immunocompromised individuals (Lamichhane, Adhikary, Guatam, & Maharjan, 2009).   5 6. Enterobacter aerogenes is a nosocomial and pathogenic bacterium that causes opportunistic infections in skin and other tissues (Lamichhane, Adhikary, Guatam, & Maharjan, 2009). 7. Salmonella choleraesuis can cause salmonellosis, an acute gastroenteritis with sudden onset of headache, abdominal pain, diarrhea, nausea, and sometimes vomiting (Lamichhane, Adhikary, Guatam, & Maharjan, 2009). 8. S. aureus can cause a range of illnesses, from minor skin infections such as pimples, impetigo boils, and abscesses, to life-threatening diseases such as pneumonia, meningitis, osteomyelitis endocarditis, toxic shock syndrome (TSS), and septicemia (Lamichhane, Adhikary, Guatam, & Maharjan, 2009). 2.3.2 Vulnerable Populations Most of the bacteria mentioned in Section 2.3.1 do not typically cause infections in healthy people. Rather they have been known to cause infections in young children and those with a suppressed immune system (including those with HIV, undergoing chemotherapy, or taking other medications that suppress the immune system). 2.4 Implications for Food Premises 2.4.1 Foodborne Illness and Currency Data accumulated during the last 20 years indicate that pathogens on currency notes could represent a potential cause of foodborne illness (Micheals, 2002). Many food outlets rely heavily on the exchange of paper currency for their products. If the same person is handling both money and food products (especially ready-to-eat products), the risk of cross-contamination increases (Green, Selman, & Radke, 2006). These findings have resulted in several changes regarding how food is prepared and handled in the food service industry. In some instances, the   6