Microbiological Analysis of Bacteria and Fungi from Socks of School Children and Antibacterial Activity of Herbal Foot Disinfectant Spray

INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 9, ISSUE 01, JANUARY 2020 ISSN 2277-8616 Microbiological Analysis Of Bacteria And Fungi From Socks Of School Children And Antibacterial Activity Of Herbal Foot Disinfectant Spray

Summera Rafiq, Mohammed Rafeeq Shaimah Yasmin, Fatima Raja and SK. Jasmine Shahina

Abstract: A sock is an item of clothing worn on the feet and often covering the ankle or some part of the calf. The foot is among the highest producer of sweat in the body as it can produce over 0.12 of perspiration per day. Socks help to absorb this perspiration. However, this is a good environment for the growth of microorganisms including pathogenic bacteria and fungi especially in contact with the human skin which provides them with moisture, warmth and nutrients. A total of 60 samples were collected from the socks of school children and were processed, from which 64 bacteria and 38 fungi were isolated and identified. The prevalence of bacterial isolates were as follows- Staphylococcus aureus, CoNS, Klebsiella pneumoniae, Pseudomonas aeruginosa, Alcaligenes faecalis, Enterobacter aerogenes, Serratia marcescens, E.coli and Citrobacter freundii. Our study also showed the presence of gram positive rods like Bacillus polymyxa, Bacillus stearothermophilus, Bacillus firmus, Bacillus cereus and Bacillus pumilus. Among the fungi our study showed the presence of Yeasts, Rhizopus spp, Mucor, Penicillium spp, Aspergillus flavus, Aspergillus niger, Rhizoctonia solani, Aspergillus fumigatus, Fusarium oxysporum and Lichthemia corymbifera. Antibacterial activity of herbal foot disinfectant spray was performed and it exhibited bactericidal activity by producing a zone of inhibition of 17mm in size.

Keywords: Socks, Bacteria, Fungi, Foot infection, Sweat, Foot disinfectant, Hygiene. ————————————————————

INTRODUCTION: this study was undertaken to detect various fungal and Sock is a Latin word derived from the Latin ―soccus‖, a term bacterial isolates from the socks worn by school children as which is used to describe a "light, low-heeled shoe" [1]. Due this will reflect their personal hygiene and reinforce the to culture and socio-economic advancements, socks have importance of maintaining hygiene in one’s life. become inevitable accessories. Most number of sweat glands is found in foot than any other part of the body. MATERIALS AND METHOD: Wearing shoes and socks can prevent sweat from evaporating or being absorbed and this favours the growth Collection of data: of both bacteria and fungi. The sweat along with the All the school children from whom the samples were bacteria cause bad odour, leading to bromodosis (smelly collected were given a simple questionnaire about their feet). Sweat is colourless and odourless, but it creates a socks hygiene. The questionnaires were collected after suitable environment for certain bacteria to grow and proper consent from their parents and teachers. It had two produce bad odour substances. As part of the human sections which included general information and other flora these bacteria are naturally present on our skin. specific questions about their usage of socks. Closed-toe shoes causes more smell than the open shoes [2]. Odour inside the shoe can increase due to Sample collection and Processing: accumulation of dead skin cells, dirt and oils over the time Sixty samples were collected with the help of sterile swabs [3]. Foot have a rich bacterial flora most of which is not from the socks of school going children belonging to the normally pathogenic if the feet are in good health [4]. age group of 10 to 12 years. The swabs were moistened Lifestyle factors can expose the feet to higher risks of with nutrient broth and normal saline to facilitate proper contamination by certain bacteria. Most of the school swabbing from the surface of the socks. The samples were children do not wash their socks regularly even at an transported to the laboratory for further processing. interval of at least 3-7 days. Some children are forced to wear wet socks due to lack of time to dry them. Wet and Isolation of bacteria: dirty socks are colonized by microorganisms. Nylon which Thirty samples with nutrient broth were incubated at 37oC is a non- for 24 hrs. They were inoculated on to nutrient agar, absorbent causes the perspiration to remain in contact with mannitol salt agar, blood agar and Mac conkey agar. The the feet favouring bacterial growth. Since there was a plates were incubated at 37oC for 24 hrs. Next day the dearth in this area of research, colonies were picked up and gram staining, motility, ______catalase and oxidase were done. The bacterial isolates  Summera Rafiq, Associate Professor, Department of Microbiology, were identified as per the standard protocols. Justice Basheer Ahmed Sayeed College for Women, Chennai-18, Ph: 9840614430. Isolation of fungi:  E-mail : [email protected] The fungal isolation was done with the other thirty samples  Mohammed Rafeeq Shaimah Yasmin, Department of Microbiology, with normal saline. They were inoculated on to Sabouraud Justice Basheer Ahmed Sayeed College for Women, Chennai-18 dextrose agar and the plates were incubated at 25oC to  Fatima Raja,Department of Microbiology, Justice Basheer Ahmed o Sayeed College for Women, Chennai-18 35 C for a week. Identification of the fungus was done as  Dr. SK. Jasmine Shahina, Associate Professor, Department of per standard methods. Antibacterial activity of herbal foot Microbiology, Justice Basheer Ahmed Sayeed College for Women, disinfectant spray by disc diffusion method: Chennai-18. Ph: 9381981919. Antibacterial activity of herbal foot disinfectant  E-mail : [email protected] spray was performed as per the standard protocol of Kirby- 4028 IJSTR©2020 www.ijstr.org INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 9, ISSUE 01, JANUARY 2020 ISSN 2277-8616

Bauer's disc diffusion method[5] Muller-Hinton agar (MHA) was prepared and poured into sterile petriplates aseptically Table 1: Prevalence of gram negative bacteria in socks (n and was allowed to solidify. Standard suspension of the test =22) isolates was made and the turbidity was matched to Mac Farland standard 0.5. The lawn of the test bacterial strain S.No. Name of the bacteria Percentage of was done with the help of sterile cotton swab. Sterile disc the bacteria 1 Klebsiella pneumoniae 6 (27%) was saturated with the foot disinfectant spray. Discs were 2 Pseudomonas aeruginosa 4 (18%) placed onto MHA with sterile forceps. The test plates were 3 Alcaligenes faecalis 3 (14%) incubated aerobically at 37ᵒC for 24 hrs. After incubation, 4 Enterobacter aerogenes 3 (14%) the results were recorded as the presence or absence of 5 Serratia marcescens 3 (14%) inhibition zone. The antibacterial activity was assayed by 6 E.coli 2 (9%) measuring the diameter of the inhibition zone formed 7 Citrobacter freundii 1 (4%) around the disc. Prevalence of gram positive rods: Among 25 gram positive rods, 7 (28%) were found to be RESULTS: Bacillus polymyxa, followed by 11 (44%) Bacillus stearothermophilus, 3 (12%) Bacillus firmus, 3 (12%) Analysis of the questionnaire: Bacillus cereus and 1 (4%) Bacillus pumilus. (Table 2). A total of thirty questionnaires were collected, of which 15 were from girls and the other 15 were from the boys. It was Table 2: Prevalence of gram positive rods in socks (n =25) observed that most of them used cotton socks, which was S.No. Name of the bacteria Percentage of regularly washed. On an average they had at least three the bacteria pairs of socks which were used in turns. Socks of the boys were more contaminated when compared to girls, as they 1 Bacillus polymyxa 7 (28%) had more physical activity. None of them used any kind of antiseptic for washing their socks. It was also found that not 2 Bacillus 11 (44%) much attention was given towards the washing of their stearothermophilus shoes regularly. 3 Bacillus firmus 3 (12%)

Prevalence of bacterial isolates from socks: 4 Bacillus cereus 3 (12%) A total of 30 samples were collected from the socks of school going children and were processed, of which 64 5 Bacillus pumilus 1 (4%) bacteria were isolated and identified. Out the 64 isolates, 17 were found to be gram positive cocci in clusters (27%) followed by 22 were gram negative rods (34%) and 25 were gram positive rods (39%). (Fig 1) Prevalence of fungal isolates from socks: A total of 30 samples were collected from the socks of school children and were processed, of which 38 fungi were isolated and identified. Out of 38 isolates, 13 (34%) were 39% 27% found to be Yeasts followed by 7 (18%) Rhizopus spp, 5 (13%) Mucor, 3 (8%) Penicillium spp, 3 (8%) Aspergillus flavus, 2 (5%) Aspergillus niger, 2 (5%) Rhizoctonia solani, 34% 1(3%) Aspergillus fumigatus, 1(3%) Fusarium oxysporum and 1(3%) Lichtheimia corymbifera. (Table 3)

Table 3 : Prevalence of fungi in socks Fig 1:Bacterial isolates from socks (n =38) Gram positive cocci S.No. Name of the bacteria Percentage of the Gram negative rods bacteria Gram positive rods 1 Yeasts 13 (34%) 2 Rhizopus spp 7 (18%) 3 Mucor 5 (13%) 4 Penicillium spp 3 (8%) 5 Aspergillus flavus 3 (8%) Prevalence of gram positive cocci: 6 Aspergillus niger 2 (5%) Among 17 gram positive cocci in clusters, 7 (41%) were 7 Rhizoctonia solani 2 (5%) found to be Staphylococcus aureus and 10 (59%) were 8 Aspergillus fumigatus 1(3%) found to be Coagulase negative Staphylococci (CoNS). 9 Fusarium oxysporum 1(3%) 10 Lichtheimia corymbifera 1(3%)

Prevalence of gram negative rods: Among 22 gram negative rods, 6 (27%) were found to be Antibacterial activity of herbal foot disinfectant spray Klebsiella pneumoniae, followed by 4 (18%) Pseudomonas by disc diffusion method: aeruginosa, 3 (14%) Alcaligenes faecalis, 3 (14%) Antibacterial activity of herbal foot disinfectant spray was Enterobacter aerogenes, 3 (14%) Serratia marcescens, 2 performed as per the standard procedure of Kirby- Bauer's (9%) E.coli and 1 (4%) Citrobacter freundii. (Table 1)

4029 IJSTR©2020 www.ijstr.org INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 9, ISSUE 01, JANUARY 2020 ISSN 2277-8616 disc diffusion method and it exhibited bactericidal activity by spray was performed as per the standard procedure of producing a zone of inhibition of 17mm in size. Kirby- Bauer's disc diffusion method and it exhibited bactericidal activity by producing a zone of inhibition of DISCUSSION: 17mm in size. The spray was found to be effective due to A critical barrier between the human body and its outer active components like Azadirachta indica, Mentha pipetita environment is human skin. It prevents loss of moisture and oil, citrus oil and ethanol. Presently, there is very little restricts the entry of pathogenic organism [6]. Different information available on the inhibitory effects of herbal foot microbial communities are harboured on skin as it is an disinfectant sprays and our study shows promising excellent ecosystem [7]. Depending on the environmental antimicrobial activity of the spray tested. conditions specific to distinct region of the skin, these microbial communities are distributed across the human CONCLUSION: skin and live in physiologically diverse and topographically Limited research has been done on socks as a possible distinct niches [8], [9]. Generally the normal microflora source of microbial contamination, despite the likelihood coexists asymptomatically with host, but these can cause that socks are rarely completely clean. Contamination is not infection whenever the host immune system gets always sufficient to determine an infection but remains a compromised or skin is damaged and microorganisms possible source, especially if the feet have lesions. Our causing such infection are said to be opportunistic study showed that school children's socks harboured pathogens. The most common colonizers of the skin in microorganisms such as bacteria and fungi. These mammals and birds are the genus Staphylococcus. Sweat microorganisms could have been isolated due to poor glands and mucous membranes surrounding body personal hygiene, like wearing the same socks for over a openings are harboured with large number of week without washing or inappropriate washing or wearing Staphylococci. In our study, 17 gram positive cocci in wet socks. These organisms isolated can be pathogenic, clusters were isolated of which, 7 (41%) were found to be especially to children. Our study showed promising results Staphylococcus aureus and 10 (59%) were found to be of the herbal foot disinfectant spray, which can be used as Coagulase nagative Staphylococci (CoNS). This was found an effective foot sanitizer. Therefore, good hygiene is to be in agreement with Kloos and Schleifer, 1986[10]. Wet certainly the first step towards healthy feet. socks when worn are prone to bacterial and fungal infections [11]. Sweat produced by the body acts as a ACKNOWLEDGMENTS: nutrient which favours bacterial multiplication and gives We thank the Management and Principal for funding this feet, socks, and shoes a strong odour [3]. The infections project under the Scheme of ―Student’s Project sponsored and proliferation of bacteria and fungi are influenced by by Management‖ through Multi-Disciplinary Research and microclimate, temperature, humidity, physical activity, Consultancy Centre (MRCC) of Justice Basheer Ahmed lifestyle [12], [13] and on individual predisposition factors. In Sayeed college for Women (Autonomous). Our thanks are our study 22 gram negative rods and 25 gram positive rods due also Dr.R.Gayathri, P.Priyadarshini and Dr.R.Lakshmi were isolated. Of which 6 (27%) were found to be Klebsiella for their constant support and motivation. pneumoniae, followed by 4 (18%) Pseudomonas aeruginosa, 3 (14%) Alcaligenes faecalis, 3 (14%) Enterobacter aerogenes, 3 (14%) Serratia marcescens, 2 REFERENCES: (9%) E.coli and 1 (4%) Citrobacter freundii, 7 (28%) were [1] "LacusCurtius – Roman Shoes – found to be Bacillus polymyxa, followed by 11 (44%) Soccus" "historyofsocksus"./www.blacksocks.com.. Bacillus stearothermophilus, 3 (12%) Bacillus firmus, 3 Penelope.uchicago.edu. Retrieved 2010-03-19. (12%) Bacillus cereus and 1 (4%) Bacillus pumilus. This [2] Revathi, P., Prabhu, N., Jayaseelan, T.S., Lakshika, S., was found to be in agreement with Li et al., 2011[14] and Manickavasagam, S. & Uma, A.(2015). Interaction of Messina, 2012 [15]. 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