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Microbial Diversity of Molasses Containing Tobacco (Maassel) Unveils Contamination with Many Human Pathogens

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Microbial Diversity of Molasses Containing Tobacco (Maassel) Unveils Contamination with Many Human Pathogens European Review for Medical and Pharmacological Sciences 2021; 25: 4919-4929 Microbial diversity of molasses containing tobacco (Maassel) unveils contamination with many human pathogens M.A.A. ALQUMBER Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Albaha University, Saudi Arabia Abstract. – OBJECTIVE: Tobacco smoking drugs in today’s modern world. Different meth- remains a worldwide health issue, and the use of ods are currently used to consume tobacco, in- flavored varieties (maassel) embedded in glyc- cluding cigarettes, cigars and waterpipes1. Water- erine, molasses, and fruit essence via shisha pipe (shisha) smoking continues to rise globally2. paraphernalia (waterpipe) is growing globally. Smoking flavored tobacco (maassel), through the 16S rRNA gene pyrosequencing was conduct- shisha, is becoming a global preventable cause of ed on 18 different varieties representing 16 fla- 3,4 vors and three brands in order to study the mi- morbidity and mortality . crobiota of maassel and find whether it contains Scientists studied the chemical composition of pathogenic bacteria. tobacco for many years and illustrated the total MATERIALS AND METHODS: The samples number of chemicals identified in tobacco during were selected randomly from the most utilized the years from 1954 to 20055. In addition, a com- brands within Albaha, Saudi Arabia as deter- prehensive review of these chemicals’ classifica- mined through a questionnaire of 253 smok- ers. In addition, ten-fold serially diluted sam- tion, concentration and changes with time due ples were inoculated on blood agar, MacConkey to changes in the shape, design and composition agar, half-strength trypticase soy agar and malt of cigarettes was reported almost a decade ago6. agar for the enumeration of mesophilic microor- On the other hand, the microbiology of tobacco ganisms, coliforms, Bacillus, thermophilic bac- has not been studied in the same depth, nor have teria, and fungi. RESULTS: methods and standards been available for the mi- A core microbiota was recognized 7 consisting of three phyla (Firmicutes, Proteo- crobiological analyses of tobacco . Nonetheless, bacteria and Actinobacteria) and a total of 571 for more than a century, tobacco was known to different species were identified including many be associated with microbial interactions that pathogens, such as Mycobacterium riyadhense, were seen to be an integral part of the develop- M. chelonae, Shigella sonnei, S. flesneri, Kleb- ment of its aroma and other desirable smoking siella pneumoniae, Salmonella bongori, Coxiel- qualities8-10. Moreover, in 1953, Tamayo and Can- la burnetii, Acinetobacter spp., Staphylococcus cho11 inoculated tobacco leaves with microbes haemolyticus, Streptococcus pseudopneumoni- ae, and Streptococcus sanguinis, showing the to improve the aroma of the end product. Con- contamination of maassel. sequently, proper fermentation with appropriate CONCLUSIONS: The present study suggests microbes is essential for good quality tobacco12-14. that flavored tobaccos are potentially infectious. However, the microbiology of the molasses-con- However, further risk assessment is required to taining tobacco product called maassel was not determine transmission occurrence. studied using culture-independent techniques. Key Words: Difficulty tracing the initial introduction of Microbial diversity, Molasses containing tobacco, maassel –flavored tobacco embedded in glycer- Human pathogens, 16S rRNA. ine, molasses and fruit essence– was previously reported15. Nonetheless, maassel was known to be in use since the 1990s15. Before maassel become Introduction widely used with waterpipes, another fermented type of tobacco was used raw, unflavored and Tobacco is an economically important nonfood produced harsher smoke16. Maassel, on the other product and one of the legal harmful addictive hand, produced smooth aromatic smoke. Thus, Corresponding Author: Mohammed A.A. Alqumber, Ph.D; e-mail: dr.alqumber@gmail.com maali@bu.edu.sa 4919 M.A.A. Alqumber maassel increased the marketability of waterpipe bial colony forming units (cfu) determinations. tobacco smoking by making it appealing to ma- The packets were selected randomly from the ny individuals through its many flavor varieties most utilized brands within Albaha, as deter- and its smooth aromatic smoke2,3. Consequently, mined through a questionnaire of 253 smokers the consumption of maassel through the shisha conducted during the three months preceding paraphernalia (waterpipe) has been recently rec- 2014. The smokers surveyed were recruited by ognized as an increasing worldwide phenome- flyers and posters and through the website: www. non17-19. Ill effects of smoking include significant drqumber.wordpress.com. Surveyed participants increase in heart rate, blood pressure and carbon aged 18-55 years (average age 25 ± 12.6) were all monoxide levels20. Understanding the microbi- maassel waterpipe (shisha) smokers for a mini- al composition of maassel can add information mum of 2 years. The questionnaire was designed about its origins and health effects. to capture anonymous demographic data (sex, Today new molecular technologies can allow age, level of education, address, career) and two for accurate determination of microbial com- questions: (1) what is your favorite maassel? and positions21,22. Determining the microbiology of (2) what is the table of contents of your favorite maassel can provide information about its source masseel? For each tested brand of maassel, the pH and pathogenicity. Thus, this study reports the value was measured, and the water activity (aw) entire microbial composition of maassel as deter- determined (Dacagon Pawkitt, Decagon Devices mined via 16S rDNA pyrosequencing. Eighteen Inc., 2365 NE Hopkins Ct., Pullman, WA 99163, different samples were analyzed. In total, 571 USA). Then samples were subjected to DNA different species were identified, including many extraction and 16S rDNA pyrosequencing and pathogens, plant, and soil microbes. Identified the resulting sequences were analyzed through pathogens include Mycobacterium riyadhense, established bioinformatics pipelines. DNA ex- M. chelonae, Shigella sonnei, S. flesneri, Klebsi- traction utilized the Mobio Powersoil kit per ella pneumoniae, Salmonella bongori, Coxiella manufacturer’s protocol (Mo Bio Laboratories, spp., Acinetobacter spp., Staphylococcus hae- Carlsbad, CA, USA). molyticus, Streptococcus pseudopneumoniae and Streptococcus sanguinis. Determination of Colony Forming Units One gram from each maassel packet was trans- ferred aseptically into 100 ml of normal saline Materials and Methods with 0.05 Tween 80 (Sigma-Aldrich Chemie Gm- bH, Steinheim, Germany) and placed in a shaking Literature Search water bath for 20 minutes at 37°C. After shaking, PubMed (Medline) and EMBASE online data- 10-fold serial dilutions were made and immedi- bases titles and abstracts were searched using the ately spread on duplicate agar media. Blood agar, keywords ‘maassel’, ‘shisha’, ‘waterpipe’, ‘tobac- MacConkey agar (Merck, Darmstadt, Germany), co bacteria’ and ‘tobacco micr*’. The returned half-strength trypticase soy agar (Sigma-Aldrich articles were retrieved and evaluated. Relevant Chemie GmbH, Steinheim, Germany) and malt articles were then discussed as far as applicable. agar (Becton Dickinson and Company, Riyadh, Saudi Arabia) were used for the isolation of Sampling mesophilic (Gram-positive and Gram-negative) Maassel packets (n = 18) representing 16 fla- bacteria, coliforms (e.g., Gram-negative entero- vors (plum, grape, lemon, lemon with mint, mint, bacteria), thermophilic bacteria (e.g., actinomy- orange, watermelon, grapefruit, two apples, cap- cetes), and fungi, respectively. The blood agar puccino, gum, grape with mint and gum mastic) and MacConkey agar plates were incubated for from three different companies: 1) Alfakher To- 24 hours at 37°C, inspected for colony count, and bacco Trading, Ajman, United Arab Emirates; 2) then immediately incubated again at 22°C for 3 Nakhla, Matco, Amman, Jordan and 3) Elbasha, days, inspected for colony count again and lastly Shebin El-Kom, Al-Minufiyah, Egypt were sam- incubated at 4°C for 3 days. The half-strength pled. Samples were collected during the year trypticase soy agar plates were incubated at 55°C 2014. The packets, purchased new and sealed, for 5 days. The malt agar plates were incubated at were delivered to the laboratory unopened. In the 30°C and then 22°C for 4 days at each tempera- laboratory, specimens were collected aseptically ture. The long incubations periods and the use from each packet for DNA extraction and micro- of low temperatures were carried as previously 4920 Microbiology of molasses-containing tobacco reported for the isolation of a wide range of mi- curves were generated; and to know how micro- crobes23. Isolated bacteria were identified using bial community compositions differed between the API 20E (BioMerieux, Marcy, France) while maassel samples, beta (β) diversity, weighted fungi were identified microscopically24. More- UniFrac profiles using principal coordinates anal- over, the BIOLOG (Biolog, Inc., California, CA, ysis (PCoA) were generated, using the workflow USA) was used for identifying the isolated mi- QIIME 1.7.028,29. Here, initially sequences were crobes. Results were reported as cfu/g of maassel. denoised, primers and barcodes trimmed, and resulting sequences truncated to 300 base pairs Sequencing and entered into the standard QIIME pipeline to 16S rRNA gene sequence was targeted
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