In world, people exchange money by different mechanisms. Citizens of most developed countries use credit or debit card system, whereas the majority of developing countries are still depend on hand-to-hand cash turnover for marketing. From its nature, cash-money is fragile and easy to be lost in addition to its exposure to contamination by different microorganisms. As a result, cash-money exchanges are considered as one of the potential vehicle for the spread of pathogenic microorganisms. Communities have different habits of storing, counting, using and managing cash-money that could be conducive for the growth and transmission of pathogenic microorganisms. As a result, both bacterial and fungal pathogens of human are reported from different countries [1-3].

The abundance and type of pathogenic microorganisms existing on cash-money varies based of the livelihood of the bearer [3,4]. For instance, in India, the cash-money obtained from bank, hospitals and municipals are highly contaminated with various pathogenic bacteria as compared to those obtained from butchers and food sellers [5]. A study conducted at some parts of Ethiopia showed the high prevalence of E. coli that ranges from 67.2% at open market milk sellers to 87.2% at dairy station milk sellers [6]. Cash-money collected from different classes of Libyans also showed variation in load of pathogenic microorganisms in which the abundance of Enterobacter agglomerans>Pseudomonas spp.>Staphylococcus aureus>Enterobacter cloacae>Klebsiella pneumonia>E. coli [7]. Similarly, in Nepal abundance of heterotrophic aerobic bacteria>coliform bacteria>Staphylococci are found on cash-money [1].

Contamination level of cash-money varies based on its type and value of the money [1,6]. A study showed the negative correlation between value of the money and microbial load [2]. For instance, prevalence of E. coli on Ethiopian Birr (ETB) of hundred notes is less than on ETB of one note [6]. Pathogenic microorganisms from cash-money vary in their resistance to the minimum inhibitory concentration of antibiotics. E. coli isolated from Ethiopian cash-money circulating in the western part of the country were resistant to neomycin than other antibiotics tested [6].

Ethiopia is known by enormous culture, customs and classes that vary among regions and localities. Contamination level of Ethiopian cash-money could be the reflection of the community lifestyle. Prevalence and drug susceptibility study of E. coli on Ethiopian birr was studied at the Eastern part of the country where majority of the communities’ livelihood depends on business but not studied in the western part of the country where the main livelihood is farming. Therefore, this study was to investigate abundance of pathogenic microorganisms on Ethiopian cash-money of different birr notes and coins collected from Nekemte town communities having different livelihood and to test their antibiotics susceptibility.

Description of the study sites

The study was conducted at Nekemte town, 331 km from Addis Ababa to the western part of the country. Nekemte is a market town; and according to 2012 national census, its total population is estimated to 110,688. Majority of the community lifestyles include businesspersons, smallholder farmer, and government employers. As the town is connecting many town of the country, money exchange rate is expected to be high.

Sampling and its preparation

Cash-money samples were purposively collected from butchers, beggars, gamblers, street food sellers, restaurant workers, bank workers and taxi drivers. Totally, 120 different ETB samples holding 50 cents, one birr (coin), one birr (note), five birr note and ten birr note were included in this experiment. Sampling was conducted in triplicates including a control of new ETB obtained from Commercial Bank of Ethiopia Nekemte district. Each collected cash-money was aseptically transferred into sterilized separate bottles containing 10 ml of sterile buffered peptone water solution. Then, the bottles were shaken vigorously for about 2 minutes to dislodge the microorganisms into the medium. The samples were removed aseptically from the bottle with a sterile forceps, rinsed with water and dried to recover.

Estimation of cultivatable bacterial load

From appropriate dilutions, 0.1 ml of the aliquot was spread plated on Plate Count Agar (PCA) and the plates were incubated at 35°C for 24 h. After incubation, plates with colonies between 30 and 300 were counted to estimate the abundances of aerobic mesophilic bacteria (AMB) [8]. Similarly, the diluents were spread-plate on Salmonella ShigellaAgar (SSA), Xylose Lysine Deoxycholate (XLD) Agar, Eosin Methylene Blue (EMB) Agar, MacConkey (MAC) agar and incubated at 37°C for 24 h. After which, different types of colonies of bacteria with different colors like pink to red purple colonies on EMBA, Red to  Black centered colonies on SSA and XLD Agar were counted as member of a family of Enterobacteriaceae [9]. A purplish colony surrounded by reddish zone on MAC agar and Metallic green sheen on EMB Agar were counted as coliforms [8]. Spore forming bacteria (Bacillus) were differentiated by spore staining from PAC and the colonies were counted [10]. Typical golden yellow colour colonies and red colonies were counted as Staphylococcus and Streptococcus from Mannitol Salt Agar (MSA).

Estimation of fungal load

Colonies of yeast and mold were determined by direct plate count from Potato Dextrose Agar (PDA) supplemented with 0.06 g/L chloramphenicol. Smooth (non-hairy) colonies without extension at periphery were counted as yeasts whereas hairy colonies were counted as mould after 48 h of incubation at 29°C [9].

Antimicrobial susceptibility of cultivatable bacteria

The bacterial isolates were tested for sensitivity to different generations of antibiotics by disc diffusion method on Mueller-Hinton agar using the commercial antibiotics such Ampicillin (AMP), Cephalexin (LEX), Cefuroxime (CXM) and Cefixime (CFM). Concentration (µg/ml) of the antibiotics used during the experiment was 10, 16, 5, and 8 for AMP, LEX, CFM, and CXM, respectively [11]. The disc note was impregnated with appropriate amounts of antibiotics, and put on the surface of solid Mueller Hinton agar which was pre-seeded with a pure culture of the test organism and incubated for 24 h at 37°C. Sensitivity of the test organisms was verified with a clear zone and the level of sensitivity was estimated by measuring a clear zone using ruler and is expressed in mm. Based on the measurement of the inhibition zones (IZ in mm), effectiveness of the antibiotics against the inoculated bacteria was expressed as ‘Sensitive’, ‘Moderately sensitive’ and ‘Resistance’ for IZ>14, 712].

Abundance of cultivatable bacterial groups

Abundance of different bacterial groups such as aerobic mesophilic bacteria (AMB), coliforms, enterobacteriaceae, staphylococcus, and Bacillus were significantly varied (P<0.01) based on sources of samples and type of money. The study showed that sample from “1ETB note” and “50 cents” were the highest and the lowest in count of cultivable microorganisms, respectively. Amongst the sample sites, the maximum and minimum mean count of cultivatable microorganisms was from “Beggars” (255.8 ± 1.0 CFU/ml) and “Bank workers” (0.00 CFU/ml), respectively (Table 1).

Table 1: Abundance of bacterial groups isolated from Ethiopian cash-money of different types with respect to their sources of sampling.

The mean count of aerobic mesophilic bacteria (AMB) isolated from “beggars” was the highest on samples of “1ETB notes” (255.8 ± 1.0 CFU/ml) and there was no AMB on “50 cents” from “butchers” and “restaurant workers”. Similarly, the highest mean counts of Enterobacteriaceae were on “1ETB note” sampled from “beggars” (138.1 ± 1.0 CFU/ml); whereas “50 cents” collected from “butchers” and “restaurant workers” did not contain Enterobacteriaceae (Table 1). The number of coliform bacteria and Staphylococcus bacterial groups from “street food sellers” were 44.0 ± 1.0 CFU/ml and 103.0 ± 1.0 CFU/ml, respectively on “1ETB note”.

Likewise, the mean counts of Bacillus bacteria counted from “1ETB notes” were 33.8 ± 1.0 CFU/ml from “beggars” (Table 1). In general, AMB, Enterobacteriaceae, Staphylococcus, coliform and Bacillus were observed on all samples isolated from the different bearer except from “restaurant worker” and “butcher”. All types of money from taxi driver did not contain Bacillus except “1ETB note” (Table 1).

Abundance of yeasts and molds

The mean counts of fungal load isolated from the sampling sources were higher for yeasts than molds. The highest load of yeasts was 125.75 ± 1.42 CFU/ml on samples of “1ETB” from “street food sellers”. Similarly, the maximum fungal molds count was 20.25 ± 0.52 CFU/ml on “1ETB” isolated from “beggars”. Yeasts and molds were not isolated from “50 cents” collected from “gamblers”, “restaurant workers” and “butcher” (Table 2).

Table 2: Abundance of fungal groups isolated from Ethiopian cash-money of different types with respect to their sources of sampling.

Abundance of pathogenic bacteria

The result in Table 3 showed that the type of money collected from different sites had bacterial pathogens with different degree of contamination except “50 cents” from “butchers” and “restaurant workers” from which no cultivable type of pathogenic bacteria was isolated. Load of some specific pathogenic microorganisms on ETB from different type and different bearer showed significant variation (p<0.01) (Table 3). The bacterial contamination of note-money was exceeding the contamination of coins. Among note-money, 1ETB was more contaminated with bacteria pathogens compared to the coins with six folds differences (Table 3). The highest load was of pathogenic bacteria was Staphylococcus (91.9 ± 1.0 CFU/ml) on “1ETB note” from “street food sellers” followed by Salmonella (35.1 ± 1.0 CFU/ml) on “1ETB note” and Klebsiella (34.8 ± 1.0 CFU/ml) on “10ETB” from “gamblers” and “beggars”, respectively (Table 3).

Table 3: Abundance of bacterial pathogens isolated from Ethiopian cash-money of different denomination from different sites.

Samples from “street food sellers” and “taxi drivers” showed the dominancy of Staphylococcus with 91.9 CFU/ml but Bacillus was the least from the sources (Table 3). Similarly, samples collected from the “gamblers” were majorly loaded with Salmonella but lowest mean count was Bacillus from the source. Samples from “restaurant workers” wereheavily loaded with Klebsiella as compared to the other pathogens. Samples from “beggars”, “butchers” and “bank workers” were dominantly contaminated by E. coli, Klebsiella and Bacillus with load of 35.8, 26.1 and 18.0 CFU/ml, respectively (Table 3).

Drug susceptibility of pathogenic bacteria

As shown in Table 4, Bacillus was resistant to the antibiotics tested in this study. Besides, Shigella was resistant to Ampicillin and Cefalexine, and E. coli was resistant to Cefuroxime (Table 4). Staphylococcus, Enterobacter and Pseudomonas were “highly sensitive” to “moderately sensitive” to the antibiotics tested in this experiment [12].

Table 4: Intrinsic antibiotics resistance of bacterial pathogens isolated from Ethiopian cash-moneys

Cefuroxime was about four times effective of Ampicillin and Cefalexine in inhibiting growth of Salmonella. Besides, Shigella, Klebsiella and E. coli were “highly sensitive” to Cefixine. In general, Cefixine and Cefuroxime were highly effective and broad in inhibition of the pathogens except Bacillus and E. coli (Table 4).

Money acts as the reservoir of different groups of microorganisms for the transmission among community. In this study of bacterial pathogens such as Staphylococcus, Streptococcus, Escherichia coli, Salmonella, Shigella, Enterobacter, Klebsiella, Bacillus and Pseudomonas were detected on ETB samples of “50 cents”, 1ETB coin”, “1ETB note”, “5ETB” and “10ETB”. In addition, yeasts and molds also detected on the Ethiopian money samples. Similarly, different kinds of pathogenic bacteria and fungi were reported from currencies of Ghana [13], Saudi Arabia [14], and Bangladesh [15] although the load and survival rate of microorganisms on the currencies of different countries varies [16]. Ethiopian cash-money collected from different bearer at Easter and Southwestern part of the country were heavily loaded with microbes [6,17]. However, the mean microbial load in the present sample was up to 10-200 folds higher as compared to the one reported from the Southwestern part of the country [17]. The big difference in microbial load on the cash-money collected from different areas could depend of lifestyle of the community, and duration of the samples in market and its transfer rate in the area. Particularly, poor handling practices and personal hygiene of the bearers could contribute to the observed differences on microbial counts. For instance, cash-money collected from community working in hospitals, municipals and banks are more contaminated than those sampled from butchers and food sellers in India [5].

In this study, money notes were more contaminated than the coins. Particularly, “1ETB note notes” were more contaminated by bacteria and fungi followed by 10ETB. This could be also associated with the long duration of “1ETB note” in market as it was on the process of replacing with 1ETB coin”. Besides, “1ETB note” could be in high rate of transfer because it is small in value for change. Similarly, the inverse correlation between money value and microbial load on Sudanese currencies [2,18], Libyan note currencies [7] and Ethiopian currencies [19] was previously reported. The less contamination of “ETB coins” as compared to “ETB notes” could be associated with the nature of coins as it is not comfortable for the existence and reproduction of microbial pathogens. However, report from Nepal showed that coins are more contaminated with bacteria than note or polymer notes that are in contrary to the current result [1].

The load of bacterial pathogens and fungus on the samples collected from “beggars” and “street food sellers” were high as compared to samples from other sources. Similarly, in Nepal the cash-money sampled from public transport were found to be extremely contaminated with various pathogenic bacteria followed by the cash-money obtained from “butchers” and “food sellers” [20]. The lifestyle of the bearers could give chance for the reproduction of the pathogens, which make them potentially vehicles for the transmission of these pathogens.

The current global health problem is resistance of pathogens to multiple antibiotics. In this study Bacillus from all samples were found resistant to Ampicillin, Cefalexin, cefixime, and Cefuroxime. E. coli is also resistant to Cefalexin and Cefuroxime although it was highly sensitive to Ampicillin and Cefixime. However, previous study showed the high susceptibility of E. coli from samples of Ethiopian cash-money to different antibiotics [6]. Therefore, variation could depend on target action of the antibiotics.

Ethiopian cash-moneys are loaded with bacteria and fungi. The bacterial group like Aerobic mesophilic bacteria, Coliform, Enterobacteriaceae, Staphylococcus and Bacillus were isolated from the cash-money. The specific pathogens like Salmonella, Shigella, Klebsiella, Bacillus and Pseudomonas were isolated from the cash-money. However, the abundance of these bacteria varied based on bearers and types of money. “1ETB note” was more contaminated by the bacterial and fungal group as compared to other money types, and microbial load on coins were significantly low. The genus Bacillus was very resistant to Ampicillin, Cefalexine, Cefixime and Cefuroxime. Shigella responded only for Cefixine and the antibiotics tested in this experiment were effective against Staphylococcus.

This research was conducted in Wollega University, Microbiology laboratory. The authors like to thank the University for allowing us to conduct the experiment in the lab without bench fee.