INTRODUCTION:

Mobile phones are long range, portable electronic devices for personal telecommunication. Mobile phones have become integral and indispensable accessories of professional and social daily life. They are increasingly becoming an important means of conversation worldwide being easily accessible, economical and user friendly¹. Mobile phones are noncritical environmental surfaces, and research demonstrates the presence of contaminates on these devices^{2, 3, 4}. The constant handling of mobile phones and other electronic gadgets by different users exposes to an array of microorganisms and thus makes a good carrier for microbes⁵. This is especially so with the skin due to the moisture and optimum temperature of human body especially our palms.

Along with these factors, heat generated by mobile phones contributes to harboring bacteria and their transmission on the devices at alarming rate⁶.Hence, it can be envisaged beyond doubt that mobiles and computers act as potential reservoirs of pathogenic microbes and could lead to severe infections. Cell phones can harbor various potential pathogens and serves as an exogenous source of nosocomial infection among hospitalized patients⁷.

With recent advances in the source of information, mobile phone use has become indispensible in hospitals⁸. The use of cell phones often occurs in hospital halls, laboratories, and/or intensive care units when dealing with severe illnesses. The Telecom and regulatory Authority of India (TRAI, 2014-15) reported that the share of mobile phone users in India stood at 996.49 million as compared to 784.32 million a year before. The growth was 75.23 to 79.38% year-on-year. They may also be involved in the transmission of infections in the healthcare systems. Mobile phones are used in close contact with the body and, as for most non-medical electronic equipment, there are no cleaning guidelines available that meet hospital standards, and the hygiene risk involved in using mobile phones or has not yet been determined.

Mobile phones could be a health hazard with many microbes living on the phone. Staphylococci, particularly S. epidermidis are members of the normal flora of the human skin, respiratory and gastrointestinal tracts¹⁰. S. aureus can be also found on the skin and in the noses which can cause illnesses from pimples and boils to pneumonia and meningitis, and is a close relative of Methicillin Resistant Staphylococcusaureus¹¹. Furthermore, the hand serves as a major vehicle of transmission of various microbes. E. coli and related bacteria constitute about 0.1% of gut flora, and fecal-oral transmission is the major route through which pathogenic strains of the bacterium cause disease¹². Proteus mirabilis is one of the most common Gram-negative pathogens. It can cause a variety of community or hospital-acquired infections, including those of the urinary tract, respiratory tract, wounds and burns.

Azadirachta indicabelongs to the botanic family Meliaceae, commonly known as Neem. It is used in traditional medicine as a source of many therapeutic agents. A. indica (leaf, bark and seeds) are known to contain antibacterial and antifungal activities against different pathogenic microorganisms; in addition to antiviral activity¹³. Neem leaves has antibacterial properties and could be used for controlling airborne bacterial contamination in the residential premise^{14, 15}.

Hence mobile phones are likely to get contaminated by various micro-organisms, some of which could very well be pathogenic in nature. Despite being used on a continuous basis, these mobile phones are seldom cleaned. They can also act as fomites for transmission of pathogenic organisms like Staphylococcus aureus, Escherichia coli, Pseudomonas, Acinetobacter, Candida, etc. The CDC¹⁶ recommends a cleaning regimen that’s effective, fast-acting, easy to follow, and economical. Currently, there are no published studies with standards for cleaning mobile phones. Thus, in this study, we investigated bacterial contamination of the mobile phones from ten different areas of Chennai and the efficacy of aqueous neem extract by agar well diffusion method against the isolated bacterial cultures.

MATERIAL AND METHODS:

Bacterial sample collection:

Mobile swab samples were collected from 10 different areas from Chennai. The areas were Guindy, Mambalam, Velachery, Mylapore, Aminjikarai, Tandiarpet, Puraisaivakam, Egmore, Ayanavaram and Perambur. The collected mobile swab samples were separately wrapped in sterile aluminium foil and stored in sterile zip lock cover. Samples were processed within 4 hours¹⁷.

Sample processing and microbial analysis:

The collected swab samples were dipped into 1ml sterile distilled water and spread plate method was performed to isolate the bacteria in nutrient media. Single colonies of bacteria were isolated separately by using streak plate method¹⁸.

Identification of bacterial cultures:

Isolated bacterial cultures were identified by studying colony characteristics, Gram staining and Bio chemical analysis based on Bergey's Manual of Determinative Bacteriology¹⁹.

Colony morphology analysis:

Characterization of colony morphology of the isolated bacterial colonies was done according to Aneja (2012). Size, shape, color, pigmentation, elevation, margin, hemolytic activity etc. were determined under cultural identification. Pure cultures were maintained on nutrient agar slants at 4ºC and used for further analysis.

Gram staining analysis:

Gram’s stain was used to study the morphology and staining properties of the bacterial isolates. The Gram positive bacteria appeared purple color while Gram negative bacteria appeared pink colour²¹.

Bio chemical analysis:

Each bacterial culture was identified according to the standard microbiological methods described by Bergey’s Manual of Systematic Bacteriology¹⁹ to the genus level by different biochemical tests. They are carbohydrate IMViC tests (Indole, Methyl Red, Voges Proskeur and Citrate utilization test), oxidase test and catalase test.

Preparation of aqueous extracts from fresh neem leaves:

The extracts from fresh Neem were prepared immediately after sample collection following different steps. 225 grams of fresh leaves were collected then they were washed with distilled water then they were cut into small pieces. For preparation, 20 g of fresh green neem leaves were soaked into 250 ml of water overnight then next day it was filtered. The filtrate was then evaporated using a rotary evaporator and was stored at 5 °C.

ANTI BACTERIAL ACTIVITY:

Preparation of inoculum:

Stock cultures of isolates were maintained at 4^°C on nutrient agar slant. Active cultures for experiments were prepared by transferring a loop full of bacterial culture from the stock cultures to test tubes of Luria Bertani (LB) broth for bacteria and were incubated for 24 hours at 37^°C. The assay was performed by agar well diffusion method.

Agar Well Diffusion Method:

Antibacterial activity of aqueous extract against isolated bacteria was determined by agar well diffusion method²². The Nutrient agar plates were swabbed with the respective 24 hours broth culture of organisms and kept for 15 minutes in laminar chamber for absorption of cultures. Wells were made in agar plates using a sterile cork borer of 5mm.The aqueous neem extract of various concentrations such as 25µg/ml, 50µg/ml, 100µg/ml were prepared and added to each well. Distilled water was used as negative control and chloramphenicol (100µg/ml) used as positive control. The plates were incubated at 37^°C for 24 hours. The diameters of the zone of inhibition were measured in millimeter by using antibiotic zone measuring scale.

RESULTS:

Collection of Sample:

Ten used mobiles were collected from different areas of Chennai. The mobiles and places were presented in Figure 1 and Table 1. There are a number of procedures available for the isolation of microorganisms from mixed culture. But the initial and the most simpler method of isolation is spread plating on solid agar medium. The purpose of spreading is to isolate individual bacteria. Collected swab samples were brought to the laboratory, processed and spread plate method was performed for isolation of bacterial cultures(Figure 2).

Figure.1Collection of samples using sterile swab sticks

Table 1 :List of the Areas Sample Collected

S.No	Location	Naming
1	Guindy	A
2	Velachery	B
3	Mambalam	C
4	Mylapore	D
5	Tandoirpet	E
6	Aminjakarai	F
7	Puraisaivalkam	G
8	Perambur	H
9	Ayanavaram	I
10	Egmore	J

Fig: 2 Spread plate method for bacterial culture isolation

Con- Control; A- Guindy; B-Velachery; C-Mambalam; D-Mylapore; E-Tandiarpet; F-Aminjikarai; G-Puraisaivakam; H-Perambur; I-Ayanavaram; J-Egmore

Table 2: Physical and Biochemical Characterization of the bacterial isolates

Different characteristics

Physical Characterization

Gram staining

G-ve

G+ve

G-ve

G+ve

G-ve

Biochemical characterization

Catalase test

Oxidase test

Indole production

Voges- Proskauer Test

Methyl Red

Citrate Utilization test

Identified Organisms

Pseudomonas aeruginosa

Klebsiella

pneumoniae

Lactobacillus sp.

Bacillus subtilis

Vibrio

Cholerae

E.coli

Streptococcus sp.

Staphylococcus sp.

Proteus vulgaris

+ Positive, - Negative

MICROORGANISMS

Figure 4: Antibacterial activity of the identified bacterial isolates against the aqueous neem extract

Antibacterial activity of Neem extract on mobile isolates. The data was expressed as mean of triplicates +SD measurements.

EXTRACT AT VARIOUS CONCENTRATIONS:

Antibacterial activity

The identified bacterial species were tested for antibacterial activity of the aqueous neem extract at various concentrations (25µg/ml, 50µg/ml, 100µg/ml).

Overall percent inhibition by active plant extracts against test bacteria is shown in Fig. 4. Anti-Bacterial activity of test strains was, in decreasing order: Pseudomonas aeruginosa> Proteus vulgaris >Lactobacillus sp.> E. coli >Staphylococcus sp.> Streptococcus sp.> Vibrio cholerae> Klabsiella pneumoniae>Bacillus subtilis. In the case of test bacteria, the basis for their differences in susceptibility might be due to the differences in the cell wall composition of Gram +ve and Gram -ve bacteria²³.

DISCUSSION:

Microorganisms can be found almost anywhere in the taxonomic organization of life on the planet. Bacteria are almost always microscopic, while a number of eukaryotes are also microscopic, including mostprotists, some fungi, as well as some animals and plants. Microorganisms are essential tools in biotechnology, biochemistry, genetics, and molecular biology.

Jaya Madhuri et al., 2015 isolated bacterial cultures from various sources like mobile phones, keypads, screens and mouse were used for isolation. The isolated bacterial cultures were identified by gram staining and biochemical test. Among the samples, touchpads were more contaminated with the bacterial population Gram positive rods were observed in most of the samples.

Similarly, Sham et al., 2011²⁴ investigated the bacterial contamination on mobile phones in hospital. A total of 204 mobile phones of Healthcare workers HCWs from medical and dental departments were screened. 99% of the phones demonstrated evidence of bacterial contamination. 64.8% of medical samples showed growth of pathogenic micro-organisms and 37.9% showed growth of Multi drug resistant bacteria. 59.37% of dental samples showed growth of pathogenic micro-organisms and 43.75% showed growth of Multi drug resistant bacteria. Pathogens isolated included Methicillin-resistant Staphylococcus aureus, Methicillin-sensitive Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Acinetobacter, Enterococcus faecalis, and Pseudomonas aeruginosa. According to the questionnaire, 40% admitted to using their phones between examinations of patients. Only 6% used disinfectants to wipe their phones.

Microbial contamination can come from several sources. It can be introduced by the food handlers. It could be introduced directly from the hands, by sneezing or coughing onto the food or utensils and surfaces used around food. It may also be introduced indirectly by transfer from doors, tap or clothes. Microorganisms are the cause of many infectious diseases. The organisms involved include pathogenic bacteria, causing diseases such as plague, tuberculosis and anthrax; protozoa, causing diseases such as malaria, sleeping sickness, dysentery and toxoplasmosis²⁵.

In my present study the cultural, morphological and biochemical properties of the isolates showed that they belonged to the following bacterial genera namely: E.coli, Staphylococcus, Streptococcus, Pseudomonas, Klebsiella, Vibrio cholera, Bacillus, Lactobacilli, Proteus. These microorganisms are may be a disease-causing agent. The neem is an antimicrobial agent, in this study the aqueous neem extract was used to clean the mobile in the way to control the spread of the pathogenic infections; it is recommended that the regular wiping of the neem extract is necessary. From the result of the study, the aqueous neem extract was used as disinfection to control the growth of pathogenic microorganisms and prevention of disease. Further the result of antimicrobial study revealed that the isolated microorganisms should have more resistance, hence strong disinfection is required in future.

CONCLUSION:

In the present study 12 bacterial species from mobile phones of the general public were identified and reported. Organism encountered include Escherichia coli, Pseudomonas sp, Serratia spp, Staphylococcus aureus and Bacillus spp. Gram staining was done to identify and differentiate gram-positive and gram- negative bacteria. The entire biochemical test was performed and Bacillus sp., Staphylococcus aureus, Staphylococcus epidermidis, E coli, Pseudomonas sp. and Serratia sp. were identified. it is suggested that mobile users should follow and adapt cost effective and simple hygienic measures such as this for a safe and healthy living. Most of these bacterial species are harmful and cause infection to humans. . Hence, Neem extracts which are well known antibacterial and antimicrobial agent is used in the as disinfectant. Neem leaves are well known examples for natural antibiotic. Wiping the mobiles with potential extract of neem can protect us from disease causing microbes.

CONFLICT OF INTEREST:

Conflict of interest declared none.

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Received on 05.12.2016 Modified on 14.12.2016

Research J. Pharm. and Tech. 2017; 10(2): 501-507.

DOI: 10.5958/0974-360X.2017.00100.7

Antibacterial Effect of aqueous Neem Extract on Bacterial Isolates from Mobile Phones