Investigation into the prevalence, persistence and antibiotic resistance profiles of staphylococci isolated from euro currency



Jim M. O'Mahony, Cork Institute of Technology, Bishopstown, Cork, Ireland. E-mail:



The study set out to sample €10 banknotes for the presence of coagulase-positive staphylococci (CPS) such as Staphylococcus aureus (S. aureus) and coagulase-negative staphylococci (CoNS) in Southern Ireland, to assess the levels of antibiotic resistance among those isolated, and determine the persistence of S. aureus on €10 banknotes and €2 coins.

Methods and Results

We report that 97% of €10 banknotes screened (n = 155) harboured multiple species of staphylococci. From the generated bank of strains, a total of 150 representative staphylococci isolates were used for further study, 71 were CPS and 79 were CoNS. Of these, we found that 62% of the staphylococci demonstrated resistance to at least one of the first-line antibiotics (52·11% of CPS isolates and 76·71% of the CoNS isolates). Resistance to multiple antibiotics was seen in 31·18% of the resistant isolates. In relation to persistence studies, S. aureus was shown to remain viable on euro banknotes and coins for significant periods (on average, 19·33 days on €10 banknotes and 16·67 days on €2 coins) as determined using bioluminescence.


We advocate the expansion of antibiotic surveillance programs, with a view to tracking/monitoring antibiotic resistance dissemination among environmental contaminants. Additionally, we propose that ‘cashless transactions’ should be encouraged in high-risk environments such as hospitals and healthcare settings, as well as stricter infection controls.

Significance and Impact of the Study

Although it is accepted that circulating currency has the potential to harbour disease-causing pathogens, studies investigating prevalence and persistence of such pathogens on euro currency are virtually nonexistent. In an attempt to rectify this, we examined the prevalence of staphylococci on €10 banknotes in Ireland and reported relatively high levels of antibiotic resistance among the isolates. Furthermore, we have established the persistence of S. aureus on euro currency for the first time.


Money has historically been suspected of being a means for disease transmission. Even during the ‘Black Death’ or bubonic and pneumonic plague pandemics in England, historical reports show that money was strongly believed to carry fatal infections. According to records, affected villages would leave money in water troughs filled with vinegar, or under running well water in attempts to decontaminate the money in exchange for food and other supplies from the surrounding uninfected villages (Tolba et al. 2007).

Contamination from the skin, wounds, anal region, nasal secretions and aerosols generated by sneezing and coughing are all potential sources of bacteria on currency (Tagoe et al. 2010). A study in the U.S. reported that a mere 6% of banknotes examined were found to be free from bacterial contamination (Aidoo 2011). Various other international studies have shown similar results reporting that the most prevalent, recurring pathogenic bacteria isolated from circulating banknotes are Staphylococcus species (spp.), Bacillus spp. and Escherichia spp. (Feglo and Nkansah 2010; Tagoe et al. 2010; Ayandele and Adeniyi 2011).

Additionally, according to the CDC, antibiotic resistance in the U.S. costs an estimated $20 billion each year in excess healthcare costs, $35 million in other societal costs and more than 8 million additional days in hospital (CDC 2011). Alarmingly, multidrug-resistant (MDR) bacterial strains whereby the bacteria demonstrate resistance to more than one class of antibiotic are increasingly being reported (Kaur et al. 2012). While examining the available literature, we found that even though a small number of groups while investigating the prevalence of micro-organisms on banknotes and coinage had analysed antibiotic resistance among bacteria recovered from banknotes, (Bhalakia 2005; Tolba et al. 2007; Kumar et al. 2009; Espírito Santo et al. 2010; Ayandele and Adeniyi 2011; Ehwarieme 2011), very few had focused on antibiotic resistance. The antibiotics examined in this study with the exception of cefoxitin (fusidic acid, erythromycin, mupirocin (MUP) and E resistant) and gentamicin) were chosen because of their utility as first- and second-line antibiotics typically used in the treatment of staphylococcal infections.

As well as prevalence and antibiotic resistance profiles, persistence and viability of pathogenic staphylococci on banknotes are largely unstudied. Vriesekoop et al. (2010) reported that bacterial growth and persistence on paper currency is influenced by two factors; the type of material the note is made from and the age of the banknote. Pockets, wallets, purses, cash registers and other ‘closed’ environments provide favourable conditions for bacterial growth and proliferation (Tagoe et al. 2010). Persistence studies proved scarce, and only two such studies were found to have examined persistence on banknotes (Thomas et al. 2008; Kumar et al. 2009). This study therefore set out to examine both the prevalence and persistence of potentially pathogenic staphylococci on European currency.

Materials and methods

Prevalence of staphylococci on circulating banknotes

Sampling and recovery of Staphylococci spp

In total, 155 €10 banknotes from various different commercial retail outlets in Southern Ireland were tested for the presence of coagulase-positive staphylococci (CPS) and coagulase-negative staphylococci (CoNS) over a 6-month period from November through April 2011. All notes were collected using a stratified random sampling approach (Aschengrau and Seage 2003).

Microbial analysis

The entire surface area of the each banknote (back and front) was swabbed using a sterile swab soaked in sterile PBS which was subsequently streaked onto mannitol salt agar (MSA; Sigma) and incubated overnight at 37°C in order to select for staphylococci. Having recorded the number of staphylococci isolates from each note present on MSA, 150 representative isolates were selected, plated to purity and stocked in 80% glycerol for phenotypic confirmation (MSA, Gram stain analysis, coagulase testing and catalase testing).

Antibiotic resistance profiles for each of the 150 isolates were established against fusidic acid (10 μg), erythromycin (15 μg), cefoxitin (30 μg) and gentamicin (10 μg) using the disc diffusion method in accordance with ‘Performance standards for antimicrobial susceptibility testing M100-S20 Guidelines for Staphylococcus species’ CLSI and EUCAST guidelines (CLSI 2010; EUCAST, 2012). As interpretation, criteria have not been outlined by CLSI in relation to fusidic acid, and those described by Jones et al. (2010) were used. For the purpose of this study, the distinction between high-level and low-level MUP resistance was not made, and instead, the demonstration of either level of resistance by staphylococci from circulating euro banknotes was considered significant. Therefore, the method described by Creagh and Lucey (2007), which differentiates between resistant and susceptible staphylococci isolates using the 5-μg disc diffusion method was used.

Persistence of Staphylococcus aureus on euro currency

Bacterial test strain and IVIS (in vivo imaging system)

Staphylococcus aureus Xen29 (Caliper Lifesciences, Hopkinton, MA, USA) was used exclusively as a representative staphylococcal strain for this part of the study. It was derived from ATCC 12600, the parental methicillin-susceptible S. aureus strain. The IVIS (Xenogen Corp., Alameda, CA, USA) consists of a highly sensitive charge-coupled device digital camera with accompanying computer software for image data acquisition and analysis. The system captures photons of light emitted by actively growing bacteria engineered to produce bioluminescence such as Xen29 (Xiong et al. 2005).

€10 banknote study design

Four €10 banknotes were divided evenly into five vertical segments, each representing the five sampling days per week. The notes were then divided horizontally in halves, which specified the areas to be tested each day (Fig. 1). The first banknote was called ‘Week 1’, the second ‘Week 2’ and so on. All notes used in the study were sterilized via exposure to ultraviolet light (CL-1000 ultraviolet cross-linker, UVP Inc., Upland, CA, USA) at 254 nm for 15 min. An overnight S. aureus Xen29 culture was diluted in fresh brain heart infusion broth (BHI; Fluka, Sigma-Aldrich, St Louis, MO, USA) to a 0·5 McFarland standard (Kumar et al. 2009). Across each note, 1·5 ml of the suspension was uniformly sprayed so as to generate uniform coverage and mimic aerosolized transmission. The spray bottle containing the inoculum was held 25 cm from each note at an approximate angle of 45°. Once complete, the notes were permitted to air dry. A fifth banknote was sectioned and sterilized as outlined previously which remained uninoculated and therefore acted as a negative control. All five notes were subsequently incubated at room temperature to mimic ‘real life’ conditions for the appropriate time period (up to 26 days). On each day of each week in the study, the appropriate segment of banknote was examined as follows.

Figure 1.

Illustrative design of experimental protocol. ‘Count I’ and ‘Count II’ specifies the area swabbed during each day of the experiment. Numbers ‘1, 2, 3, 4 and 5’ represent the sampling days 1–5 respectively on week 1 of the study. On each sampling day, the requisite areas were swabbed, reconstituted, diluted and plated, viable bioluminescent colonies were subsequently counted.

Twenty hours postinoculation, segment 1 of the ‘Week 1’ banknote was analysed. The first square in this section called ‘Count I’ (Fig. 1) was swabbed with a sterile cotton swab which was then soaked in 1 ml of sterile phosphate-buffered saline (PBS). A serial dilution down to 10−3 was performed, and 100 μl of each dilution was plated in duplicate onto BHI agar containing 200 μg ml−1 kanamycin (Sigma-Aldrich) with subsequent overnight incubation at 37°C. (The insertion of kanamycin resistance genes into the bacterial chromosome guarantees the selective growth of the bioluminescent S. aureus Xen29 strain on media containing kanamycin 200 μg ml−1, (Hoerr et al. 2012). This process was then repeated on the second square called ‘Count II’ for the same day (Fig. 1).

After overnight incubation, each plate was examined for the appearance of bioluminescent colonies using the IVIS under the following parameters – exposure time: 1 s, binning: medium 4, filter: open, f/stop: 1, field of view: 12·5 cm. As a control, on the first day of each week in the study, two segments from the uninoculated note were processed exactly as outlined above. The full experiment was executed on three separate occasions, and in all cases continued up to and including day 26 (the end of the Week 4 banknote).

€2 coin study design

Sixty €2 coins were sterilized by boiling and swabbing with alcohol (using sterile swabs). An overnight culture of S. aureus Xen29 (grown up in BHI broth supplemented with 200 μg ml−1 kanamycin) was diluted to a 0·5 McFarland standard. One hundred millilitre of the bioluminescent culture was aseptically transferred to a sterile cup (Sarstedt Ltd., Leicester, UK). Thirty €2 coins were dropped sequentially into the vessel which was shaken for 30 s to guarantee thorough exposure. Thereafter, the coins were aseptically removed with forceps, permitted to air dry and incubated at room temperature until the appropriate sampling day. The first coin was designated ‘Week 1, day 1’, the second ‘Week 1, day 2’ and so on up to and including ‘Week 6, day 5’.

On Week 1 day 1 the first coin was placed into a falcon tube containing 10 ml of BHI broth and 200 μg ml−1 kanamycin. This was incubated overnight at 37°C, shaking at 200 rpm after which time, a loopful of this suspension was spread onto BHI agar supplemented with 200 μg ml−1 kanamycin and incubated overnight at 37°C. The plate was then examined under the IVIS for the presence of bioluminescence under the following parameters – exposure time: 1 s, binning: medium 4, filter: open, f/stop: 1, field of view: 12·5 cm. On each day an uninoculated €2 coin was also examined (using the same procedure) which acted as the negative control.


Prevalence of staphylococci on circulating banknotes

We found that 97% of banknotes tested were positive for staphylococci growth. From a total of 155 × €10 notes collected and analysed, 150 were contaminated by CPS and/or CoNS. Overall, the average number of staphylococcal colonies recovered per note was 22·19. Isolate numbers recorded ranged from 1 to 79 colonies per banknote. In total, 150 representative isolates were chosen, and of these, 71 were CPS and 79 were CoNS as determined by MSA plating, Gram staining, catalase and coagulase tests.

A definitive relationship between bacterial contamination and the condition of banknote existed which was found to be inversely proportional (Fig. 2). It was evident that as the condition of the note deteriorated, the numbers of bacteria recovered increased. The average number of colonies recovered from the different banknote grades ranged from 3·4 on AU notes to 52·24 on P grade notes (Table S1).

Figure 2.

Relationship between banknote grade and number of colonies recovered. A definite relationship between bacterial contamination and the condition/grade of the banknote existed which was found to be inversely proportional. The date of sampling (between November and April) did not seem to have an effect on bacterial numbers recovered. The abbreviations used were outlines by the International Bank Note Society (IBNS) which are used to categorize banknote grades. AU, about uncirculated; EF, extremely fine; G, good; F, fair; P, poor. (For further detail on banknote grades refer to Table S1).

Antibiotic resistance profiles

Overall, 93/150 isolates (62%) demonstrated resistance to one or more of the antibiotics (cefoxitin, fusidic acid, erythromycin and/or MUP). Specifically, 37 of 71 CPS isolates (52·11%) and 56 of 79 CoNS isolates (76·71%) demonstrated antibiotic resistance.

CPS and CoNS isolates demonstrated greatest resistance to erythromycin (48·4% overall) at similar levels of 48·6 and 48·2%, respectively. Levels of resistance against fusidic acid recorded for both bacteria were also high at levels of 40·5% for CPS and 33·9% for CoNS. In relation to MUP, 29·7% of CPS isolates proved resistant, whereas 48·2% resistance was observed among the CoNS isolates (Table 1). With regard to cefoxitin resistance (which indicates methicillin resistance and consequently flucloxacillin resistance), very low levels were observed (5·4% for CPS and 1·79% for the CoNS isolates). EUCAST and the British society for antimicrobial chemotherapy (BSAC) guidelines recommend a cefoxitin disc concentration of 10 μg, in this study, however, CLSI guidelines were followed which recommend 30-μg discs the higher concentration that may have contributed to the low levels of resistance we observed (EUCAST, 2012, BSAC 2012). One hundred percent sensitivity to gentamicin was recorded for CPS and CoNS (Table 1).

Table 1. Summary of antibiotic resistance profiles
StrainNo. of resistant isolates (%)FOX 30 μg (%)FD 10 μg (%)E 15 μg (%)MUP 5 μg (%)
  1. CoNS, coagulase-negative staphylococci; CPS, coagulase-positive staphylococci.

  2. The incidence of bacterial resistance to the following antibiotics was examined; cefoxitin (FOX) 30 μg, mupirocin (MUP) 5 μg, erythromycin (E) 15 μg, fusidic acid (FD) 10 μg. Gentamicin (CN) 10 μg was not included in the table as no resistance was recorded. ‘0’ indicates complete sensitivity to the antibiotic.

Overall93/150 (62)3 (3·2)34 (36·6)45 (48·4)38 (40·9)
CPS37/71 (52·11)2 (5·4)15 (40·5)18 (48·6)11 (29·7)
CoNS56/79 (76·71)1 (1·79)19 (33·9)27 (48·2)27 (48·2)

Multiple drug resistance, whereby the isolate was resistant to more than one antibiotic was observed in 29/93 (31·18%) resistant isolates recovered from the €10 banknotes. Of this figure, 27·59% CPS (eight isolates) and 72·41% CoNS isolates (21 isolates) were found to be resistant to two or more of the antibiotics in our study. Based on the multiple resistance profile of the isolates, an arbitrary profile number (I–VII) was assigned to each new profile type to facilitate comparative analysis across the isolates. From this data, a summary table was produced (Table 2) which demonstrates that the most common profile was I (E and FD resistant), closely followed by profile type II (MUP and E resistant). The least common profiles were types III, V, VI and VII. Only one single isolate was classified as profile type III by demonstrating resistance to all four of the first-line antibiotics examined. Gentamicin (CN) was not included in this table as no resistance was recorded among any of the isolates in our study.

Table 2. Multiple antibiotic resistance profile types and number of isolates demonstrating each
Profile typeResistance toNo. of isolatesNo. of CPSNo. of CoNS
  1. CoNS, coagulase-negative staphylococci; CPS, coagulase-positive staphylococci.

  2. Cefoxitin (FOX) 30 μg, mupirocin (MUP) 5 μg, erythromycin (E) 15 μg and fusidic acid (FD) 10 μg.

IE, FD1147
IIMUP, E1019

Persistence of Staphylococcus aureus on euro currency

Having established that euro banknotes are abundantly contaminated with Staphylococcus spp. the next stage of our research was to investigate the persistence of staphylococci on both euro coinage and banknotes using a representative strain of S. aureus (Xen29) and bioluminescence as a means of monitoring viability.

Our in vitro study revealed the ability of the bacteria to survive for an average of at least 19·33 days at room temperature after which point bioluminescent colonies were nondetectable. S. aureus persisted on €2 coins for an average of 16·67 days postinoculation, after which point an absence of bioluminescence was noted. The study was carried out on three occasions. Study 1 resulted in Xen29 persistence for 15 days, study 2 for 18 days and study 3 for 17 days postinoculation.


The concept that coinage and banknotes can act as vehicles for microbial transmission has been well recognized; however, studies incorporating both the prevalence and persistence of micro-organisms on euro currency are virtually nonexistent. We found that 97% of banknotes examined in our study were contaminated by staphylococci. Of the banknotes we studied, only five did not harbour staphylococci. Interestingly, all five of these notes were considered to be in mint condition. The number of staphylococci colonies recovered ranged between 1 and 79 CFU per banknote, which was in line with Kumar et al. (2009) who reported the total number of staphylococci recovered on Indian notes as ranging from 18 to 69 CFU per banknote. The average number of staphylococci recovered in our study was 22·19 colonies per note.

Antibiotic resistance (and increasingly multidrug resistance), which was once confined to hospitals, has evolved to include communities all across the globe and, according to the CDC, virtually all types of pathogenic bacteria are becoming resistant to antibiotic treatments (Marshall et al. 2009). From international tourists to war-displaced refugees, international human movement is more prevalent than ever before. This provides the opportunity for a variety of antimicrobial-resistant bacteria to be carried from one geographic location to another (Van der Bij and Pitout 2012). Consequently, antibiotic resistance is an escalating problem worldwide, and its emergence has been mainly attributed to the excessive and inappropriate usage of therapeutic drugs. Paper currency provides a large surface area as a reservoir for potentially pathogenic micro-organisms, and antibiotic profile analysis from our study clearly indicates a marked resistance among the bacteria isolated from Irish banknotes, especially against commonly used antibiotics (Tagoe et al. 2010). Bhalakia concluded that S. aureus along with other resistant organisms can be transmitted from hospitals into the community with the exchange of paper currency (Kumar et al. 2009).

With regard to the isolates recovered during our study, worryingly, we found that 62% of our staphylococcal isolates demonstrated resistance to at least one antibiotic (specifically, 52·11% of CPS isolates, and a substantial 76·71% among the CoNS isolates examined showed resistance; Table 1). The recent global increase in the emergence of MDR clinical strains of bacteria has presented us with the frightening scenario of entering a ‘post-antibiotic era’ of untreatable infections and epidemics (Kaur et al. 2012). According to the World Health Organisation, MDR infections are responsible for approximately 25 000 patient deaths in the E.U and additional healthcare costs and productivity losses of 1·5 million euro annually (World Health Organisation 2011).

In our study, resistance to multiple antibiotics was exhibited by eight CPS isolates (27·59%) and by 21 CoNS isolates (72·41%) findings similar to those reported by Neel, whereby 25·85% of S. aureus isolated from paper currency were MDR (Neel 2012). In general, resistance to multiple antibiotics was much greater among CoNS than recorded among CPS (Table 2). These findings are significant, given that the most frequently isolated member of CoNS, Staphylococcus epidermidis has been heavily implicated in the spread of MDR. The ubiquitous nature of S. epidermidis as a commensal organism makes it an ideal carrier and reservoir for antibiotic resistance genes, in particular, those which do not impose a major fitness cost to the bacteria (such as SCCmec in MRSA; Otto 2009).

Having established that staphylococci are abundant on €10 banknotes, we chose to examine the persistence of S. aureus under simulated conditions. To the best of our knowledge, this is the first study of its kind and the selection of the €2 coin and €10 note was due to their high frequency of handling. Our in vitro analysis demonstrated the pathogen's ability to persist for on average at least 19·33 days and 16·67 days €10 notes and €2 coins, respectively. With regard to €10 banknotes, the only similar study available to use as a comparison was by Kumar et al. (2009) However, they found that S. aureus persisted on Indian paper currency for only 8 days, nearly 2 weeks less than our study. As comparable inoculum levels were used, the discrepancy in results may be attributed to differing compositions of banknotes between that of euro notes and Indian rupees or differences between the bacterial strains used. Additionally, Thomas et al. (2008)showed that human Influenza A virus can survive and maintain infectivity on Swiss paper banknotes for up to 17 days. Despite the numbers of bacteria that persisted on the notes being relatively low, numerous infectious intestinal diseases are capable of causing illnesses even at low doses and therefore, particularly at risk are the immunocompromised in society (Tagoe et al. 2010).

In high-risk settings such as hospitals, the constant flow of human traffic means money is being exchanged and handled by an enormous variety of people both healthy and sick. Studies have revealed women doctors’ purses, hospital hand sanitizers and textiles such as bed linens (even after laundering) as potential fomites involved in the spread of infection within healthcare settings (Eiref et al. 2012; Feldman and Feldman 2012; Fijan and Šostar Turk 2012; Sigler and Hensley 2012). We propose money circulating between staff, patients and visitors for use in canteens, gift shops and for pay-parking, etc. exposes already ill and immunocompromised patients to opportunistic or even pathogenic bacteria. Even at low levels, exposure to bacteria from banknotes could have devastating consequences and may also play a role in the spread of nosocomial infections and bacterial resistance in the community. We recommend that antibiotic surveillance programs should be expanded, with a view to tracking/monitoring antibiotic resistance dissemination among environmental fomites including currency.

It has been reported that bacterial growth on paper currency is influenced by two main factors; the type of material the note is made from and the age of the banknote, that is to say older cotton-based notes especially those that are particularly soiled or creased facilitate greater numbers of bacterial growth (Vriesekoop et al. 2010). We propose an investigation into the introduction of an ‘alternative to money’ into hospitals and healthcare settings, in an attempt to protect the already vulnerable from potential infection by the contaminating micro-organisms. In recent years, the world has witnessed the introduction of mobile phone payment options and Near Field Communication (NFC) systems which offer ‘contact-less payment’ services such as Google wallet and Pingit by Barclays Bank. Such secure means of payment offers practical alternatives to cash transactions and has facilitated a societal shift towards a ‘cashless’ society which could aid in reducing exposure to some extent. Furthermore, along with stricter infection control policies in hospitals, governments (particularly in underdeveloped countries) should do more to improve ‘hand hygiene’ awareness such as the introduction of campaigns and seminars in attempts to curb the spread of bacteria and resulting antibiotic resistance associated with contaminated currency.