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Bacterial Contamination of Packaged Smokeless Tobacco Sold in India

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Bacterial Contamination of Packaged Smokeless Tobacco Sold in India Research Paper Tobacco Prevention & Cessation Bacterial contamination of packaged smokeless tobacco sold in India Rashmi Mehra1, Vikrant Mohanty1, Aswini Y. Balappanavar1, Shivam Kapoor1 ABSTRACT INTRODUCTION About 21.4% of India’s population uses smokeless tobacco products (SLT), yet limited data are available on their microbial contamination. To AFFILIATION understand the potential microbiological risks associated with SLT use, the 1 Department of Public Health present study aims to investigate bacterial contamination of tobacco and the Dentistry, Maulana Azad Institute of Dental Sciences, New Delhi, types of microbes that could be cultured from SLT products. India METHODS Twenty-two brands of SLT products, including paan masala, khaini, gutka and tobacco-containing dentifrices were examined and cultured by using CORRESPONDENCE TO Rashmi Mehra. Department of appropriate selective and differential media including MacConkey agar and CLED Public Health Dentistry, Maulana agar. This was followed by a sequence of further identification by biochemical Azad Institute of Dental Sciences, New Delhi, India. tests. E-mail: [email protected] RESULTS All 22 types of SLT products showed growth of aerobic bacteria. The most KEYWORDS common bacteria isolated were Pseudomonas aeruginosa followed by Streptococcus contamination, bacteria, faecalis. Other bacteria that were isolated from products, in traces, included toxicology, smokeless tobacco, Klebsiella spp., E. coli, and Bacillus subtilus. tobacco constituents CONCLUSIONS This study raises and addresses the issue of bacterial contamination Received: 27 March 2019 of packaged SLT products. SLT users might be subjected to a significant health Revised: 28 September 2019 Accepted: 4 December 2019 hazard, especially those who are immunocompromised. Tob. Prev. Cessation 2020;6(February):11 https://doi.org/10.18332/tpc/115064 INTRODUCTION thought to be less toxic than smoked products even According to the Global Adult Tobacco Survey though they are associated with many adverse health (GATS) 2016–2017, 21.4% of India’s population uses effects including nicotine addiction, oral lesions, oral smokeless tobacco products (SLT)1. Smokeless tobacco and pancreatic cancer, and cardiovascular disease6. use consists of chewing paan (mixture of lime, pieces Many of these adverse health outcomes are attributed of areca nut, tobacco and spices wrapped in betel leaf), to chemical carcinogens present in smokeless tobacco chewing gutka or paan masala (scented tobacco mixed products including tobacco-specific nitrosamines with lime and areca nut, in powder form), and mishri (TSNAs) and polycyclic aromatic hydrocarbons6. (a kind of toothpaste used for rubbing on gums). India TSNAs are thought to be among the most important has one of the largest number and relative share of tobacco-associated carcinogens due to their toxicity tobacco users in the world2. It has been estimated that and high abundance in SLT products7. Different 90% of oral cancer cases in India are attributable to TSNAs are formed from the reaction of alkaloids with tobacco use of any kind3. A review by Boffeta et al.4 nitrite8, and levels of available nitrite are influenced estimated that more than half of all oral cancers in by nitrite-reducing bacteria that are known to be part India are caused by SLT use. of a variety of bacteria, or microbiota, associated with Unlike smoked forms of tobacco, SLT relies on tobacco products9. absorption of nicotine through the oral mucosa5. SLT manufacturing in India falls into two In terms of toxicity, SLT products are commonly categories: registered/organised and unregistered/ Published by European Publishing on behalf of the European Network for Smoking and Tobacco Prevention (ENSP). © 2020 Mehra R. et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution NonCommercial 4.0 International License. 1 (http://creativecommons.org/licenses/by-nc/4.0) Research Paper Tobacco Prevention & Cessation unorganised. The estimated share of SLT production products followed by a second phase that included by the unorganised sector was 11% in 201110. microbiological analysis of the collected products. Further, different SLT products manufactured Ethical clearance was obtained from the Institutional in India undergo various processes of curing and Ethical Review Board before product analysis. mixing. Plain chewing tobacco is sold in sachets In our study, we have included only the SLT along with a pack of slaked lime which the user can products manufactured by the organised sector. mix. Gul (tobacco containing dentifrice) is a dry The city of Delhi is geographically divided into 5 powder-like tobacco preparation made using fine zones, namely North, South, East, West and Central. tobacco leaf dust, molasses, lime, and red soil. It is The Central zone of Delhi was chosen based on then packaged into plastic sachets or tin boxes. convenience for collection of samples. A single Gutka is a wet non-perishable paan substitute, investigator collected commercially available SLT commercially manufactured containing areca nut, products from tobacco shops located in the Central slaked lime, catechu, sun dried tobacco leaves, zone till no new products were found. The study was flavourings, and sweeteners. Loose locally sold conducted in October 2018. tobacco leaves are manufactured using an air-curing The current study included a total of 22 products process. Most products are sold in plastic packaging including 7 brands of paan masala, 7 gutka, 6 khaini, to ensure longer shelf life. Each step tends to be a and 2 tobacco containing dentifrices. Following potential inception ground for contamination as collection, the products were identified, noting the there are no stipulated guidelines for quality control batch number and date of manufacture if mentioned. and avoidance of microbiological contamination of tobacco products. Sample analysis In 2010, to curtail environmental effects of For the second part of the study, all samples were tobacco product packages, directions were issued to transported and stored at room temperature prior ban the sale of tobacco products like gutka, tobacco to examination. The purchased products were then and paan masala in plastic pouches. The Ministry placed in a UV chamber for 30 minutes to ensure of Environment and Forests finalised an enforced decontamination of the outer surface of the packets. this regulation by March 2011. They are packed in In addition, all laboratory tools that were used during various sizes and materials including plastics and experiments were sterilized by autoclaving and flame paper packaging, stored in differing climatic and sterilized immediately prior to use. geographic conditions at various tobacco kiosks One gram of each product was dissolved in 10 mL across the country2. of normal saline solution. After appropriate mixing, Microbiological contamination of SLT along samples were centrifuged and cultures prepared with the impact of varying climatic and geographic by spreading 0.1 mL of each sample on different conditions might pose an additional risk by altering media and incubated for 48–72 hours at 37oC. The the nature of the intended constituents of the number of estimated colony-forming units (CFU) packaged SLT product. Further, microbiological for each sample was counted. Discrete colonies of contamination might contribute to the conversion aerobic bacteria were sub-cultured for purification of nitrites and alkaloids into TSNAs, which are by streaking in fresh solid media11. major carcinogenic agents. Hence, the present The following media were used as per the American study was conducted with the aim to investigate Public Health Association (APHA 1985)12 and World bacterial contamination of commercially available Health Organization (WHO 1997)13 guidelines. SLT products and the types of bacteria that could be Further identification of isolated bacteria was cultured from them. made by gram staining and biochemical tests (oxidase test, urease test, coagulase test, Indole METHODS production test, and citrate utilisation test). These Product sampling tests were performed according to Finegold The study was conducted in two phases. The first and Baron14. A modified summary of the testing phase was the collection of commercially available SLT performed is provided in Figure 1. Tob. Prev. Cessation 2020;6(February):11 https://doi.org/10.18332/tpc/115064 2 Research Paper Tobacco Prevention & Cessation Figure 1. Modified summary ofFigure biochemical 1: Modified tests Summaryperformed of biochemical tests performed Gram Staining Gram Negative Gram Positive Cocci Bacilli Cocci Bacilli Oxidase Catalase Negative Positive Negative Positive Coagulase Enterobacteriaecae 1,2,3 Negative Positive Pseudomonas aeruginosa Staphylococcus Triple Sugar Iron -ve Saprophyticus Indole -ve Citrate -ve 1. Escherichia coli 2. Klebsiella, Enterobacter, 3. Proteus, Providencia, Triple Sugar Iron Acid Slant Acid Serratia Morganella Deep Triple Sugar Iron Acid Slant Acid Triple Sugar Iron Acid Alkaline No H2S Deep Slant Acid Deep Indole +ve Indole -ve Phenylalanine deaminase +ve Methyl Red +ve Methyl Red -ve (only this group is positive) Citrate -ve Citrate +ve Data analysis Figure 2. Bacterial contamination of SLT product Data obtained were entered on a digital spreadsheet using Microsoft Excel 2016. Descriptive inferences were derived and plotted in the form of bar diagrams and charts. RESULTS A total of 22 brands of SLT products were analysed for bacterial
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