Ribosomal DNA (rDNA) identification of the culturable bacterial flora on monetary coinage from 17 currencies.

Introduction

Currency in the form of banknotes and metal coinage represents a universal medium for the transmission of bacteria in the environment and among humans. Although there have been numerous reports on the contamination of currency with illicit drugs including cocaine, heroin, and amphetamines (Jenkins, 2001; Oyler, Darwin, & Cone, 1996), there have been relatively few studies detailing the microbiological status of currency. Therefore, the aim of the investigation reported here was to identify the bacterial microflora on monetary coinage from 17 countries by employment of polymerase chain reaction (PCR) sequenced-based molecular identification of rDNA from bacterial cultures.

Materials and Methods

Monetary coinage of 17 nationalities, which had been in general circulation in the country of origin, was examined as detailed (Table 1). Coins were collected at random by several laboratory personnel visiting each country. On return home to the United Kingdom, researchers placed the foreign coinage in separate, clean plastic bags until analysis. A mixture of bronze and silver/other alloy coins (approximately 300 grams) was randomly selected and was cultured aerobically in tryptone soya broth (TSB) (Oxoid CM0129, Oxoid Ltd., Basingstoke, England) for 72 hours at 30[degrees]C. Following this step, 20 [micro]l of broth was inoculated onto Columbia blood agar (BA) (Oxoid CM331) supplemented with 5 percent volume-per-volume (v/v) defibrinated horse blood for 72 hours at 30[degrees]C, and the resulting colonies were purified by further subculture, as detailed above, for a further 72 hours.

All molecular manipulations were carried out in accordance with good molecular diagnostic practices (GMDPs) as detailed in the guidelines of Millar, Xu, and Moore (2002). Bacterial DNA was extracted from purified colonies demonstrating differing morphologies; the Roche High Purity PCR Template Kit (Roche Diagnostics Ltd., United Kingdom) was employed

in accordance with the manufacturer's instructions. Extracted DNA was stored at -20[degrees]C prior to PCR amplification. The researchers analyzed the extracts for the presence of bacterial DNA by targeting two regions of the 165 rDNA gene loci as detailed in Figure 1. PCR reaction mixes (50 [micro]L) had the following characteristics: 10 millimolars (mM) Tris-HCl; pH 8.3; 50 mM KCl; 2.5 mM Mg[Cl.sub.2]; 200 [micro]M (each) dATP, dCTP, dGTP, and dTTP; 1.25 units of Taq DNA polymerase (Amplitaq; Perkin Elmer), 0.2 [micro]M (each) of the 16S rRNA primers, as detailed (Figure 1) and 4 [micro]L of DNA template containing approximately 50 ng DNA per milliliter extract. Following a "hot start," the reaction mixtures were subjected to the following thermal cycling parameters in a Perkin Elmer 2400 thermocycler: 96[degrees]C for 3 minutes followed by 40 cycles of 96[degrees]C for 1 minute, 55[degrees]C for 1 minute, and 72[degrees]C for 1 minute, followed by a final extension at 72[degrees]C for 10 minutes. During each run, molecular-grade water (Biowhittaker Inc., United States) instead of DNA was included randomly as a negative control, and Staphylococcus aureus DNA was included as a positive control.

PCR products were analyzed by electrophoresis through 2 percent weight-per-volume (w/v) agarose gels (Gibco, United Kingdom) containing ethidium bromide (5 [micro]g per 100 mL) and were visualized with a UV transilluminator using a gel image analysis system (UVP Products, England). All images were archived as digital graphic (*bmp) files. PCR products were sequenced with the Thermo Sequenase fluorescent-labeled primer Cycle Sequencing Kit with 7-deaza-dGTP (Amersham Phamacia Biotech) and analyzed on the ALF II sequencer (Amersham Phamacia Biotech) as previously described (Moore, Xu, & Millar, 2002). The authors compared the resulting sequences (approximately 1,000 base pairs [bp] obtained) (Figure 1) with those stored in the Genbank Data Base using BLASTn alignment software (http://www.blast.genome.ad.jp). For identification to the species or genus level, the criteria previously published by Goldenberger, Kunzli, Vogt, Zbinden, and Altwegg (1997) were employed.

[FIGURE 1 OMITTED]

Results and Discussion

Twenty-five isolates were obtained from the coinage, as described in Table 1. Of these, 25 (100 percent) were Gram positive, and the most prevalent genus observed was Bacillus, which accounted for 10 of 25 isolates (40 percent) and was isolated from 10 of 17 countries (58.8 percent). The next most prevalent was Staphylococcus spp., which accounted for 7 of 25 isolates (28 percent) and was isolated from 7 of 17 countries (41.2 percent). Within the Bacillus isolates, the authors identified six species and four species within Staphylococcus isolates (Table 1). All resulting 16S rDNA sequences have been submitted to GenBank, as detailed in Table 1.

The aim of this study was to identify the endogenous culturable bacterial flora on monetary coinage from 17 countries. Given that microbial identification of such environmental isolates is problematic with rapid-identification techniques based on biochemical gallery strips--mainly because of a lack of biochemical profiles of these organisms--it was decided that all identifications should be performed with broad-range or universal 16S rDNA, as previously done in the identification of environmental organisms (Moore et al., 2002).

Previous studies on the cultivation of bacteria from money have included studies involving notes, coins, and both (Havas, 2000; Pachter, Kozer, Pachter, & Weiner, 1997; Shukla, 1980). A recent Hungarian study (Havas, 2000) of the bacteriological state of 115 notes and 71 coins demonstrated that there were significantly higher numbers of bacteria on the notes than on the coins and that members of the Entero-bacteriaceae family, enterococci, and Bacillus cereus were found only on the notes. That study concluded that the sale of food items and the collection of money should be separated, and that frequent handwashing should be promoted, as control measures to reduce contamination. A further study of 100 notes and 102 coins collected from staff in one hospital found that 3 percent of coins and 11 percent of notes were contaminated with opportunistic pathogens including Staphylococcus spp., Bacillus spp., and Corynebacterium spp. (Pachter et al., 1997). This study contrasts with an earlier study in 1972 (Abrams & Waterman, 1972), which demonstrated that 70 percent of 150 coins and 70 percent of 50 notes were contaminated with bacteria. Of these, 20 of the 105 contaminated coins (19 percent), and 21 of the 35 contaminated notes (60 percent) contained opportunistic pathogens, including Staph, aureus., E. coli, and Ps. aeruginosa (Abrams & Waterman, 1972).

In the study reported here, 25 organisms were identified, and the majority of them have been identified in clinical infection. Table 2 details the previous role of these organisms in cutaneous-associated infection. The presence of these organisms on the coins may be attributed to 1) environmental contamination, in the case of Bacillus spp. and 2) Staphylococcus spp. arising from the normal commensal skin flora. Consequently, given the organisms identified in this study, it is not believed that monetary coinage presents any particular risk to public health. The authors support, however, the principles of basic hygiene in terms of proper handwashing and the avoidance of handling money when working with food or when dressing wounds and other infected skin lesions.

In conclusion, this study demonstrated that money from 17 countries was contaminated by environmental Gram-positive flora, in particular Bacillus spp., and that the universal 16S rDNA-PCR approach coupled with automated direct sequencing provides a rapid means of identifying the contaminated organisms present.

TABLE 1 Distribution and Identification of Culturable Bacterial Flora on
Monetary Coinage from 17 Countries

Country      Identification     Number of  Percentage  GenBank Accession
             (Closest           Bases      Homology    Number of Isolate
             Phylogenetic       Analyzed               Sequenced
             Match)

Australia    Bacillus           1,015      100         AF540983
             thuringiensis/
             cereus
Austria      Bacillus           1,015       99         AY144451
             megaterium
Belgium      Staphylococcus       982      100         --
             epidermidis
             Bacillus           1,014       99         AF540987
             litoralis
Canada       Staphylococcus     1,000      100         AY144447
             aureus
France       Streptococcus sp.    972       99         AY144448
             Staphylococcus       976      100         --
             aureus
             Microbacterium       999       99         AY144450
             sp.
Hong Kong    Bacillus subtilis    970      100         AY144452
             Microbacterium       949      100         AF540988
             sp./
             Curtobacterium
             sp.
             Micrococcus        1,003      100         AY144446
             luteus
Israel       Staphylococcus       995      100         AF540985
             epidermidis
             Micrococcus          906      100         --
             luteus
Italy        Kocaria palustris    961      100         AY144445
Japan        Staphylococcus       972      100         --
             hominis
             Bacillus             970      100         --
             thuringiensis/
             cereus
Republic     Bacillus lentus    1,015       97         --
of Ireland
             Staphylococcus       981      100         AY144443
             schleiferi
South        Kocuria              984      100         --
Africa       palustris/
             Micrococcus sp.
Spain        Bacillus subtilis    970      100         --
Switzerland  Bacillus           1,002       99         AF540984
             circulans
The          Paenibacillus sp.  1,001       99         AF540982
Netherlands
The          Bacillus           1,001      100         --
People's     thuringiensis/
Republic of  cereus
China
United       Staphylococcus       988      100         AY144444
Kingdom      hominis
United       Bacillus             972      100         --
States       thuringiensis/
             cereus

TABLE 2 Previous Reports of Human Clinical Cutaneous Infection
Associated with Microflora Found on Coins in the Current Study

Organism               Evidence as Pathogen in    Reference
                       Cutaneous or Cutaneous-
                       Related Infection

Bacillus spp.          10/29 Japanese patients    Matsumoto et al., 2000
                       developed septicaemia
                       from Bacillus
                       contamination of puncture
                       site
                       filarial lymphedema
Bacillus megaterium    Uncommon
Bacillus litoralis     Uncommon
Bacillus lentus        Uncommon
Bacillus subtilis      Acute                      Oyler, Darwin, & Cone,
                       dermatolymphangioadenitis  1996
Bacillus circulans     Endophthalmitis            Tandon, Tay-Kearney,
                                                  Metcalf, & McAllister,
                                                  2001
                       Cutaneous infection of a   Logan, Old, & Dick,
                       wound                      1985
                       Catheter-related           Castagnola et al.,
                       infection                  1997
Kocuria spp.           Advanced noma lesions      Paster et al., 2002
Microbacterium spp.    Well-documented            Alonso-Echanove et
                       opportunistic pathogen,    al., 2001; Funke,
                       particularly involving     Haase, Schnitzler,
                       sepsis in immuno-          Schrage, & Reinert,
                       compromised patients, as   1997
                       well as endophthalmitis
Micrococcus luteus     Septic shock               Albertson, Natsios,
                                                  & Gleckman, 1978
                       Contamination of           Mino de Kaspar,
                       automated surgical         Grasbon, & Kampik,
                       equipment                  2000
Paenibacillus spp.     Uncommon
Staphylococcus aureus  Common causal agent of     Hanakawa et al., 2002
                       bullous impetigo           Sharma & Verma, 2001;
                       Staphylococcus scalded-    Stulberg et al., 2002;
                       skin syndrome
                       Furunculosis               Sharma & Verma, 2001;
                                                  Stulberg et al., 2002;
                       Folliculitis
                       Cellulitis                 Garcia Fernandez,
                                                  Pedragosa Jove, &
                                                  Castells Rodellas,
                                                  1999
                       Common agent of            Balaban & Rasooly,
                       food poisoning             2000
Staphylococcus         Skin lesions               Akiyama, Kanzaki,
epidermidis                                       Tada, & Arata, 1998
Staphylococcus         Purulent skin lesions      Stulberg, Penrod, &
hominis                                           Blatney, 2002
                       Hidradenitis suppurativa   Jemec, Faber,
                                                  Gutschik, & Wendelboe,
                                                  1996
                       Contaminants of catheters  Wilkins & Patzakis,
                                                  1990
Staphylococcus         Wound infections           Hernandez et al.,
schleiferi                                        2001; Kluytmans et
                                                  al., 1998
                       Osteomyelitis              Calvo, Hernandez,
                                                  Farinas, Garcia-
                                                  Palomo, & Aguero, 2000
                       Exudative scalp lesion     Aspiroz, Agustin,
                                                  Navarro, Concellon, &
                                                  Boned, 2001
Streptococcus spp.     Cellulitis, erysipelas,    Chiller, Selkin, &
                       impetigo, folliculitis,    Murakawa, 2001
                       furuncles and carbuncles
                       Necrotizing fasciitis      Swoboda-Kopec et al.,
                                                  1999

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Jiru Xu, Ph.D.

John E. Moore, Ph.D.

B. Cherie Millar, Ph.D.

Corresponding Author: Jiru Xu, Professor, Department of Pathogenic Biology, School of Medicine, Xian-Jiaotong University, Xian, Shannxi Province, The People's Republic of China, 760001. E-mail: [e-mail].