ATM - Journal of Modern Biotechnology

JOURNAL OF MODERN BIOTECHNOLOGY, VOL. 2, NO. 1, pp 14–22, January 2013 Copyright © 2013, by Madras Institute of Biotechnology. All Right Reserved. www.thebiotech.org

Research Article

Enumeration and Characterisation of Coliforms from Automated Teller Machine (ATM) Centers in Urban Areas Veerappan Saroja, Senthilkumar Kamatchiammal*, Karthikeyan Brinda and Sakkaravarthy Anbazhagi National Environmental Engineering Research Institute, Chennai Zonal Laboratory, CSIR Madras complex, Taramani, Chennai -600113, Tamil Nadu, India. *Correspondence Author e-mail: [email protected] Received 17 December 2012; Revised 28 December 2012; Accepted 31 December 2012

Abstract An Automated Teller Machine (ATM) is a computerized telecommunications device that provides the customers of a financial institution with access to financial transactions in a public space without the need for a human clerk or bank teller. Diversity of people using the ATM centers is increasing day by day. As ATM centers are usually air conditioned, the cold and damp environment may favour the growth of micro organisms. This study involves the collection of samples by using cotton swabs from various ATM centers in Chennai. Sixty five samples were analyzed for the presence of predominant pathogenic bacteria. Among the 65 samples 27 (41.54%) of the samples were positive for the presence of E.coli, 25 (38.46%) for Klebsiella sp, 8 (12.3%) for Shigella sp. and 6 (9.23%) for Vibrio sp and the total microbial load in nutrient agar ranged from 40 to 1.9 x 105 CFU. The obtained results were confirmed by using Amplification of target genes, Lac z and Lam b (commonly present in lactose fermenting microbes) was performed by using Lac z and Lam b primers. Also, BLAST-n analysis of the sequenced PCR product served as additional confirmation for these studies. As an overview this study proposes the banks to take preventive and corrective actions against spread of infectious diseases through ATMs and further alert the users of ATM that it is not only money you draw but also, microbes. Keywords: Automated Teller Machine (ATM); Coliforms; PCR; BLAST ___________________________________________________________________________________________________ INTRODUCTON Microbes are the oldest forms of life on earth. Some types have existed for billions of years. Bacteria are one such type of microorganisms that are ubiquitous in the environment, capable of growing on any surface including liquids when the nutritional and environmental factors are favourable. Although the vast majority of bacteria are harmless, a few pathogenic strains of bacteria are found to be causing infectious diseases. The delicate natural balance of ecological systems is often modified through human activities that result in organic pollutants which bring about either abrupt or gradual changes in the conditions of the natural environment. With

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the continuing expansion and proliferation of urban areas, and the growing threat of population explosion people become busy in their day to day life having no time to withdraw money from bank using the traditional means. Hence they move with the new advancement called the Automatic Teller Machine (ATM). Today, the widespread use of electronic technology in healthcare is another source of contamination. In recent years, more people are now moving towards using the ATMs for their banking needs. According to a survey by Bank net India, 95% people now prefer this modern channel to traditional mode of banking, almost 60% people use an ATM at least once a week. As we all know microbes can survive on many surfaces ranging from VOLUME 2· NUMBER 1· JANUARY 2013

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those in domestic kitchens and hospitals, to commonly used office equipment such as computer keyboards, telephones, mobile phones, it is not a surprise that the Automated teller machine are also one among them. An ATM is a computerized telecommunications device. ATMs are known by various other names including automated transaction machine, automated banking machine, money machine, bank machine, cash machine, hole-in-the-wall and Any Time Money (in India) but there is one more name which people are not aware of “ANY TIME MICROBES”, though this name sounds interesting it’s a bitter truth that we all need to accept. As ATM centers are usually air conditioned, the cold and damp environment favours the growth of a wide range of microorganisms from pathogens to harmless microbes. Healthcare-associated infections are a major concern to clinicians and healthcare consumers. The emergence of multidrug-resistant organisms and other pathogenic microorganisms has made treatment of infections from these organisms more costly and complex. Studies have demonstrated a variety of reservoirs in the environment that have served as sources of contamination (Huang et al., 2006; De Gialluly et al., 2006; McFarland et al., 1989; Noskin et al., 1995). Convenient hand-held devices and various other electronic point-of-care systems these are all new found sources for infection transmission. A steady influx of computers in patient rooms or computers on wheels (COWS) that are moved from bedside to bedside for electronic documentation will be touched by many caregivers; and, as a result, harmful bacteria has been found lurking on computer keyboards thus making it easy for germs to spread to patients and among healthcare workers Coliform bacteria are the commonly-used bacterial indicator of sanitary quality of foods and water. Schultz et al., 2003; studied 100 keyboards in 29 clinical areas at an inner-city tertiary-care Veterans Affairs Medical Center. 95 keyboards were positive for microorganisms. Streptococcus, Clostridium perfingens, Enterococcus (including harbour vancomycin-resistant Enlerococcus faecium), Staphylococcus aureus, fungi and gram-negative organisms were isolated. Noskin et al., 2005; studied both Computer keyboards and keyboard covers to determine their ability to harbour vancomycinresistant Enterococcus faecium (VRE), Methicillinresistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (PSEA). 15 VOLUME 2· NUMBER 1· JANUARY 2013

Coliforms from automated teller machines

The keyboard and covers harboured MRSA and VRE Ear longer periods of time than PSEA. With increased contact with keyboard there was increased recovery of bacteria on users' hands. Though the computers could hold these pathogens, the study also found that they could be easily cleaned. Rutala et al., 2006; studied the degree of microbial contamination, the efficacy of different disinfectants, and the cosmetic and functional effects of the disinfectant on computer keyboard. In this study, an attempt has been made to identify the diversity of bacteria, especially Coliforms, present in ATM centers from in and around Chennai, Tamil Nadu, India.

MATERIALS AND METHODS Sample collection Sixty five samples were collected from different ATM centers in Chennai. Sampling was done 3 times from these ATM centers on different days. The samples were collected using sterile cotton swabs. Sterile test tubes with screw caps were used to transport the cotton swabs. Inside the ATM room the swab was taken out and rubbed on the touch screen and buttons. Samples were collected during peak hours (i.e.) when more number of people would be using the ATM centers. Hence sampling was done between 9am to 11am and the samples were brought to the laboratory within 1 hour. In the laboratory, the samples were preserved by adding 2 ml of Phosphate Buffer Solution (PBS) to each test tube under sterile conditions and stored in a refrigerator at 4oC until use. All the samples were inoculated as (triplicates) on Nutrient agar, SS (Salmonella Shigella) agar, TCBS (Thiosulphate Citrate Bile Salt) Agar and M-EC (Media for E.coli) Test Agar by spread plate method. Samples were then incubated at 37oC for overnight. Subsequently 500 µl of sample was processed for DNA isolation. After 24 hrs the incubated plates were observed for growth of bacteria. A single colony was isolated and sub cultured on Luria Bertani (LB) broth for further preliminary and biochemical tests. The colonies isolated from SS Agar, TCBS Agar and MEC Test Agar only were considered for further tests. Gram staining was followed to find out whether the isolated colonies belonged to Gram Positive or Gram Negative bacterium (Pelczar, 1993). Biochemical tests were

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performed to confirm the pathogens using IMViC test, Urease test, TSI test and Carbohydrate fermentation test. Recovery of DNA Total genomic DNA was extracted from the PBS buffer directly by the procedure of Del Sel et al., 1989; using this procedure, 500 µl of samples were used for the isolation of DNA. DNA from bacterial cells was released by alkaline lysis with Sodium Dodecyl Sulfate (SDS) treatment. The Cetyl Trimethyl Ammonium bromide (CTAB) was used to remove proteins and carbohydrates. The DNA was further purified by using chloroform-isoamyl alcohol (24:1) and phenol-chloroform-isoamyl alcohol (24:24:2) extractions followed by precipitation with isopropanol. After centrifugation at 12,000 x g for 15 min, the pelleted DNA was washed once with cold 70% alcohol and dried under vacuum. That pellets were dissolved by 1x TE buffer.This procedure was able to recover 100 to 150µg of purified genomic DNA from each sample. Primers used in this study Common primer for lactose fermenting group of microbes which are intended to be coliforms including E.coli, the marker for faecal contamination, were used for this study. Bacterial pathogens Primers were chosen to amplify a 326base- pair (bp) region of the LacZ gene, based on the sequence reported by Kalnins et al., 1983; And also a 554 bp region of the lam B gene based on the sequence reported by Bedouelle et al., 1980; The primers used in this study are given in (Table 1). Polymerase chain reaction Amplification of a segment of the coding region of Escherichia coli lacZ by using a PCR primer annealing temperature of 50°C detected E. coli and other coliform bacteria (including Shigella sp. but not Salmonella sp.) and non-coliform bacteria. Amplification of a region of E. coli lamB by using a primer annealing temperature of 50°C selectively detected E. coli and Salmonella and Shigella sp. Amplification of the DNA extracted from the ATM samples was carried out as per Asim et al., 1990. PCR amplification of lacZ and lamB provides a basis for a method to detect indicators of fecal contamination, and amplification of lamB in particular permits detection of E. coli and enteric pathogens (Salmonella and Shigella sp).


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DNA amplification was carried out in gene amplification PCR system, using 2 X PCR master mixes (Genei). The PCR master mix normally contained lx PCR amplification buffer (10x buffer contains 50 mM KCl, 100 mM Tris hydrochloride [pH 8.13], 15 mM MgCl2, and 0.1% [wt/vol] gelatin), 200 p.M each of the dNTPs, 0.4 µM of each of the primers and 2.5 U of Taq DNA polymerase were added to the master mix. Oligonucleotide primers were synthesized by Integrated DNA Technologies, USA. After adding the template DNA, the whole mixture was initially denatured at 94°C for 3 minutes. Then a total number of 30 PCR cycles were run under the following conditions. Denaturation at 94°C for 1 min, primer annealing at 50°C for 1 min, DNA extension at 72 °C for 2 min. 10 µl of amplified PCR product was electrophoresed on a 1% agarose gel and photographed under UV light. DNA sequence DNA sequencing was carried out using ABI 310 Genetic Analyser (Shankra Nethralaya, Chennai, India) using both the forward and reverse primers to confirm the results. The multiple sequence alignment was analyzed by using (http://www.align.genome.jp/).

RESULTS Nutrient agar showed growth of microbes in all the 65 samples ranging from 40 CFU/ml to 9.7×104CFU/ml. According to these results, the highest bacterial contamination was reported in sample number 32 and the lowest bacterial contamination was reported in sample number 2 (Table 2). These samples were further tested with MEC agar which revealed that 27 out of 67 samples showed yellow colonies indicating the presence of E.coli. The result for MEC agar ranged from 20 CFU/ml to 7.4×104CFU/ml. The highest number of CFU was reported in sample number 30. In SS agar Klebsiella sp., 25 samples tested positive for Klebsiella sp ranging from 80 CFU/ml to 9.8×104CFU/ml. The highest was reported sample number 26. Then 8 samples indicated the presence of Shigella sp in SS agar Shigella sp ranging from 40CFU/ml to 1.7×104CFU/ml, reporting the highest in sample number 28. Finally, 6 samples showed positive results in TCBS agar ranging from 1.2 x 102 CFU to 4.4×102 CFU indicating presence of yellow colour colonies that shows

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the existence of Vibrio sp with highest record on sample number 16. Sample number 30 and sample number 31 showed the presence of all four enteric pathogens. And, three enteric pathogens namely E.coli, Klebsiella sp, Shigella sp was found in sample numbers 48 and 50 except for Vibrio sp. (Table 2). Thus as an overview amongst 65 samples 27 (41.54%), samples were positive for the presence of E.coli, 25 (38.46%) for Klebsiella sp, 8 (12.3%) for Shigella sp. and 6 (9.23%) for Vibrio sp. (Figure 1). All samples which showed bacterial growth on selective media were then confirmed for the presence of

enteric pathogens like E.coli, Klebsiella sp, Shigella sp and Vibrio sp using cultural, staining and biochemical examinations. Of the 65 samples analysed, 24 samples showed growth only on nutrient agar while there was no growth on the other specific media (i.e.) nearly 36.92% of the ATM centres are free from enteric pathogens. About 41 samples had one or more enteric pathogens present (i.e.) nearly 63.08% of the ATM centres possess potentially enteric pathogens. All the above values determined are on an average of triplicates.

Table 1: Target Gene and their respective sequences and positions. Target gene Primers

Sequence (5’–3’)

Sense primer

Position

ATG AAA GCT GGC TAC AGG AAG GCC 1675 and 1698

LacZ Antisense primer GGT TTA TGC AGC AAC GAG ACG TCA 2001 and 2025 Sense primer

GGA TAT TTC TGG TCC TGG TGC CGG

4899 and 4922

Antisense primer ACT TGG TGC CGT TGT CGT TAT CCC

5429 and 5452

LamB

Table2: Expression of bacterial counts in nutrient agar and specific media, MEC agar- E. coli, SS agar –Klebsiella sp., SS agar – Shigella sp. and TCBS agar – Vibrio sp. in terms of CFU.

Sample No.

Nutrient agar

Mec agar – E.coli

SS agar-Klebsiella sp.

SS agar-Shigella sp.

TCBS agar-Vibrio sp.

1 2 3 4 5 6 7 8 9 10 11 12

4×104 40 1.2×104 7.2×103 5.6×103 3.6×104 7.2×104 1.8×104 2.3×104 2.6×102 5.2×104 7×104

7.5×103 NIL 5.4×102 NIL NIL NIL 20 NIL 7.6×103 NIL 3.2×104 40

NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL 7.2×103

NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL


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13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

2.7×104 1.3×104 1.6×104 6.4×103 8.1×103 6.5×103 8.4×104 8.8×103 1.1×104 1.8×105 1.3×104 7.8×104 8.4×104 1.9×105 5.7×104 4.8×104 1.8×105 7.8×104 1.9×104 9.7×104 5.2×102 2.5×103 7.6×102 8.4×102 3×104

1.5×103 NIL 1.4×104 NIL NIL 7×102 NIL NIL 60 NIL 2.8×103 NIL NIL NIL NIL 3.6×104 NIL 3.2×104 40 6.4×104 NIL 1×103 NIL

NIL 7.9×103 NIL 4.9×104 7.9×103 NIL 6.2×104 NIL 9.4×102 7×104 NIL NIL 8.8×103 9.8×104 6.5×103 NIL NIL 7.4×104 1.5×104 NIL NIL NIL NIL

NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL 1.7×104 1.1×103 3.2×102 2.7×104 NIL NIL 3.1×103 NIL

NIL NIL NIL 4.4×102 3.8×102

NIL NIL

NIL NIL

NIL NIL

NIL NIL

38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53

2×102 1.4×103 3.4×104 4.7×103 1.5×104 1.4×102 2.6×102 5.8×104 8×103 1.1×104 4.8×104 1.2×104 1.6×104 7.1×104 1.2×104 1.9×104

NIL NIL NIL NIL 80 NIL NIL NIL 1×102 8.6×102 1.3×103 60 4.8×104 NIL NIL NIL

NIL NIL 1.2×104 NIL 2.9×103 NIL NIL 4.2×104 NIL NIL 5.7×104 NIL 5.6×103 1.2×103 NIL NIL

NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL 1.4×104 NIL 2.9×103 NIL NIL NIL

NIL NIL 1.4×102 NIL NIL NIL NIL 3.6×102 NIL NIL NIL NIL NIL NIL NIL NIL


NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL NIL 2.9×104 60 1.2×102 NIL NIL NIL NIL


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2.7×103 1.3×104 7.8×103 7.1×104 5.2×104 6×102 2.8×102 2.6×104 1.6×102 8.6×102 3.9×103 5.4×103

ATM Samples

54 55 56 57 58 59 60 61 62 63 64 65

70 60 50 40 30 20 10 0

1.2×102 NIL NIL 7.8×102 NIL NIL 1.4×102 NIL 60 NIL 8×102 NIL

7.2×102 NIL NIL 80 5.1×104 NIL 2.4×103 1.1×104 2.8×102 NIL NIL NIL

NIL NIL NIL NIL 2.8×102 NIL NIL NIL NIL NIL NIL NIL


Nutirent Agar

MEC Agar

Un known Microbes

E.coli

SS Agar

Klebsiella sp

SS Agar

TCBS Agar

Shigella sp

vibrio sp

Bacterial Colonies Figure 1:

Graphical representation on number of coliforms present in ATM sample.

PCR was evaluated for detection of coliforms from 65 samples collected at various ATM centres in Chennai. In all the PCR positive samples, a distinct 326 bp (lac Z), 554 bp (lam B) products were visible on the agarose gel when stained with ethidium bromide (Figures 2 & 3). The identities of the bands were further confirmed by sequencing. BLAST comparison of the nucleotide sequence with other sequences available at the GenBank showed that the PCR amplified sequence comprised of the LacZ and LamB gene. The accession numbers for the submitted sequence is HM449754 and HM449759 respectively.


DISCUSSION In this study an attempt was made to identify probable diversity of Enteric pathogens that are present in ATM centers in Chennai, and the results thus identified provides supporting evidences for pathogenic microbes like E.coli, Klebsiella spp, Shigella spp, Vibrio spp, to be present at the ATM centers. The moisture content inside the ATM centers facilitates the survival of pathogens as the ATM centers are air-conditioned. This study was conducted to create awareness to public and scientific community on the possible diseases that may spread due to the presence of



these microbes at the ATM centers. Human occupational activities, without hygienic intervention, could introduce

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the risk of infection through ATM machines.

Figure 2: Electrophoresis gel results confirming the presence of Lac z regions (326bp) for the samples obtained from ATMs. Lane 1: 100 bp Marker Lane 2: Negative control Lane 3: Positive control and Lane 4, 5, 6, 8, 10 ,11, 12, 13, 14, 16, 17, 18 confirming the positive results. Lane 7, 9 and 15 confirming the negative results.

Figure 3: Electrophoresis gel results confirming the presence of Lam B regions (554bp) for the samples obtained from ATMs. Lane 1: 100 bp Marker Lane 2: Negative control Lane 3: Positive control and



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Lanes 4, 5, 6, 8, 10 ,11, 12, 13, 14, 16, 17, 18 confirming the positive results. Lanes 7, 9 and 15 confirming the negative results. In this study more focus was given to enteric pathogens, as coliform bacteria’s in water using polymerase chain they are the common ones that spread diseases through reaction (PCR) amplification and gene probe detection touch, improper sanitary activities of the individual. provided as a cutting edge to this research in identifying Transient pathogens are excreted in faecal, through various the existence of coliforms in ATM centers. Use of nucleic body fluids or tissues by persons infected or colonized by acid techniques provides a rapid identification and these pathogens (Barker et al., 2005). When diarrhoea confirmation. Further confirmation with the sequences results in many liquid stools per day, hands easily become showed the identities with the LacZ genome in blastn contaminated because billions of pathogen cells are analysis. present. Previous studies also report the presence of This research is considered to be reliable as the presence of coliforms in coins, paper currencies, and public telephones coliforms was detected using consistent methods used by and the introduction and spread of contamination in those researchers. This also further provides evidence that even cases also through handling like in our study (Ferdinandus ATM machines are risk to handle with, as diseases may et al., 2001). This work is considered to be important as spread through the use of these unclean machines. The risk virulent strains of pathogenic microbes like E.coli, of food-borne infection associated with crossKlebsiella, Shigella, and Vibrio can cause diseases like contamination depends on two factors: the level of gastroenteritis, urinary tract infections, septicemia, contamination on the surfaces and the probability of its dysentery, vomiting, stomach cramps, flatulence etc., transfer to the foods being consumed (Bloomfield et al., Irrespective of all enteric pathogens positive in special 1997). Hand washing before and after use of ATM centers media were positive for E.coli, the indicator for faecal may be impractical and imperfect, but it might be desirable contamination, further confirms the source of instead to clean the keys and screens with alcohol or other contamination is due to the insanitary human activities. In disinfectants on a regular basis. Similar recommendations this study two ATM centers one from a highly populated have been made by previous researchers (Neeley et al., area and the other near sewage outfalls tested positive for 2005, Rutala et al., 2006 ) and may be pertinent to other three pathogens, excluding Salmonella. And, four out of settings, such as the one investigated in this study. This six samples that reported positive for Vibrio were sampled research shall serves as a base for researches to identify from ATM centers nearer to fishermen colonies, specific strains of species causing disease and effective supporting the spread of contamination fishes and through disinfecting procedures that reduce the risk of ATM handling of marine water. This is supported by the work of acquired infection. Khin et al., 1989; where currency notes handled by fish mongers had contaminations. Even though 22 samples failed to have E.coli, Salmonella, Shigella and Vibrio they CONCLUSION tested positive on nutrient agar indicating the possibilities Gone are those days where people need to worry while of microbial contamination other than feacal carrying cash while travelling to places. Now days in this contamination. Earlier researches on electronic devices speedy world ATMs have become a necessary evil. People like keyboards provide evidences for existence of often tend to realise that the ATMs not only dispense cash Streptococcus, Clostridium perfingens, Enterococcus but also microbes. As the number of ATMs increase day supports the contamination of ATM surfaces. Also we by day the increase of pathogens and spread of diseases observed some rhizoidal colonies on nutrient agar which tend to increase. Banks have taken high security measures represents the member of Actinomycetes group indicate to prevent theft of cash at the ATMs, however they have the contamination from the soil as they were the common failed to take preventive and security initiatives on residents of soil microflora. This research is an extension maintaining safe and clean ATM machines. Currently, the to identify pathogenic microbes at ATM machines that is cleaning of ATMs has reduced drastically and thus more widely used among all ages of human race (Bej et al., contaminated ATMs are present in and around the city. In 1990) on identification of LacZ and lamB regions for




this high populated world care should be taken to protect the human race from spread of infectious diseases for the betterment of human life.

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