Dr.Leyla Sağlam

1. YILIN MAKALELERİ Dr.Leyla Sağlam TTD 12.yıllık Kongresi 2009 Antalya Atatürk Üniversitesi Tıp fakültesi Göğüs Hastalıkları Anabilim Dalı Erzurum

3. Tobacco flakes on cigarette filters grow bacteria: A potential health risk to the smoker? J L Pauly, J D Waight, G M Paszkiewicz Tob.Control 2008;17;i49-i52 Department of Immunology, Roswell Park Cancer Institute Buffalo, NY, USA Correspondence to: Dr John L Pauly, USA; john.pauly@roswellpark.org Received 16 July 2007 Accepted 18 October 2007

4. ABSTRACT (1) • Bacterial growth from a single flake of tobacco wasdocumented for cigarettes that had been urchased recently from local vendors and from cigarettes that had been stored for more than six years in a warehouse. • In a novel tobacco flake assay, a pack of cigarettes was opened within the sterile environment of a laminar flow hood. • A single flake of tobacco was collected randomly and aseptically from the middle of the cigarette column and placed onto the surface of a blood agar plate. • The test cigarettes included eight different popular US brands, and these were from three different tobacco companies.

5. ABSTRACT (2) • After 24 hours of incubation at 37uC, theplates showed bacterial growth for tobacco from all brands of cigarettes. Further, more than 90% of the individual tobacco flakes of a given brand grew bacteria. • Likewise, bacteria grew from microparticulate tobacco that had been sieved from cigarettes. Tobacco flakes were observed lying loosely on the cut surface of the filter of cigarettes in newly opened packs, and bacteria grew from cigarette filters that had been touched to the surface of a blood agar plate. • In conclusion, the results of these studies predict that diverse microbes and microbial toxins are carried by tobacco microparticulates that are released from the cigarette during smoking, and carried into mainstream smoke that is sucked deep into the lung.

6. introduction (1) Investigators have expressed concern as to the health risks associated with diverse microbes and microbial toxins in smoking and smokeless tobacco products. A partial listing of the microbial agents that have been identified includes bacteria,1 bacterial toxins,2–4 fungal spores,5 various mycotoxins, including aflatoxin 6 as well as zymosan (b-1,3-glucan) of mould cell walls.7 The health concern of these agents is not restricted to smoking tobacco but includes the association of microbes and microbial toxins of smokeless tobacco products with oral disease.8 9 Bacteria have been identified on cigarette tobacco flake. One study of cigarettes collected from different countries, assayed with state-of-the art molecular probe technology, identified 23 different species of bacteria in cigarette tobacco.1 In addition to bacteria, cigarette tobacco is known to contain mould. In one investigation, all 14 brands of cigarettes that were tested were shown to have tobacco that was heavily contaminated with a spectrum of fungal spores.

7. introduction (2) The most prevalent fungal spore was Aspergillus fumigatus. The authors note that invasive aspergillosis is a significant cause of morbidity and mortality in immunocompromised patients.5 It is widely recognised that chronic inflammation is associated with cancer.10–12 Moreover, studies of human beings13–15 and laboratory animals 15–17 have shown that tobacco-associated inflammation is associated with lung cancer and non-neoplastic pulmonary diseases. In the context of chronic inflammation, it is significant that the tobacco in cigarettes has bacterial-derived endotoxins (LPS, lipopolysaccharides). 2–4 LPS, derived from Gram-negative bacteria (for example, Escherichia coli) is a potent inducer of inflammation.18–20 Very small amounts of LPS (,50 mg) delivered into the lungs of healthy subjects have been shown to induce pulmonary inflammation.21 22 Studies reported here are the first to address the health risks that may be associated with microbes and toxins on tobacco flakes on cigarette filters.

8. MATERIALS AND METHODS Test and control cigarettes Test cigarettes used in these studies were different popular brand US filter cigarettes that had been purchased recently from local vendors. In some experiments, test cigarettes were obtained also from a warehouse where they had been stored for more than six years. In addition to these test cigarettes, all experiments included procedurecontrol cigarettes that had been autoclaved to kill the bacteria on the tobacco (121uC, 15 minutes).

9. Bacterial assays of cigarette tobacco flake A series of exploratory experiments for assessing the growth of bacteria from cigarette tobacco using conventional microbiological procedures that employ nutrient broth tube culture gave rise to a novel blood agar plate assay that permitted the detection of bacteria on a single tobacco flake. This method is described below. Cigarettes were opened in a sterile fibre-free and article-free environment provided by a laminar flow hood and by research personnel experienced in working aseptically. With a gloved hand and sterile forceps, a cigarette was removed from a new pack of cigarettes. Then, the cigarette was placed onto a platform and a longitudinal incision into the cigarette barrel was made with a scalpel. A randomly selected tobacco flake was then obtained with forceps. The flakes differed in length, colour and other features. The flake was placed onto the surface of a blood agar plate (catalogue No 221239;Becton, Dickinson and Company, Sparks, MD, USA). A grid of eight pie-shaped sections had been previously drawn on the bottom surface of the agar dish with a felt-tipped black marker (see fig 1).

10. The tobacco flake assay plates were placed in an inverted position in a 37uC incubator having a humidified atmosphere of 5% CO2 in air. After 24 hours and 48 hours of incubation, the plates were examined for microbial growth. A digital camera was used to collect an image of the plate. Tobacco powder was obtained septically by sieving cigarette tobacco flakes using a cup-shaped sterile metal screen (,1 mm cut-off). The particles were shaken from the sieve directly onto blood agar plates. Following incubation, the plates were examined with the naked eye and a tereo-zoom microscope (magnification, 68–40). Bacteria associated with tobacco flakes on the cut surface of a cigarette filter (fig 1A) were assessed in the following manner. The cut surface of a newly opened pack of cigarettes wasexamined for the presence of tobacco flakes and particles. Filters having tobacco particles visible to the naked eye were scored aspositive; filters that were tobacco-free were scored as negative. The filter of the cigarettes, regardless of the filter score was then tested for bacteria. In this assay, a randomly selected cigarette was removed from the pack, then held in a vertical position to the agar plate, and the cut surface of the filter touched to the agar surface for three seconds before removing. The cigarette filter assay plates were incubated and read as described above

11. RESULTSBacterial growth from a single tobacco flake The results of a representative study are as follows. Cigarettes tested were from eight different brands, and these included different styles (for example, full flavour, ‘‘light’’ and menthol) and packaging (for example, box and soft pack). From each cigarette brand, 12 flakes were assayed. The flakes were distributed as four flakes to each of three blood agar plates. Examination of the blood agar plates after 24 hours of incubation showed that microbial colonies grew from all cigarette brands. Expressed on a per flake basis, microbial growth at 24 hours was recorded for 78 of the 84 tobacco flakes (92.9%). Of the 84 test flakes 68 (81.0%) bacterial colonies were associated with destruction of the red blood cells (for example, haemolysis; fig 1B). A subsequent reading of the same plates at 48 hours revealed that all of the flakes had microbial growth, and that all tobacco flakes were associated with haemolysis (100%, n=84/84). In many instances, a single flake produced bacterial colonies that differed with respect to size, shape, morphology, colour and margin (fig 1B).

12. In contrast to the observations of test cigarettes, no microbial growth was detected with same-brand control cigarettes that had been autoclaved before testing. Further, no bacterial growth was observed on the plate that was not associated with a tobacco flake. Thus, the observed microbial growth could not be ascribed to improper handling of the test cigarettes, airborne contaminants, improper assay conditions or technical error. A high frequency of bacterial growth (.50% of the flakes) was also observed in assays of tobacco flake from vintage cigarettes (three brands).The question was raised as to whether bacterial growth would be observed for small pieces of tobacco leaf (for example, tobacco dust, fines and microparticulates) that could be inhaled deep into the lung. The tobacco powder, prepared as described in Materials and methods, was cast onto blood agar plates. Examination of the powder tobacco assay plates at 24 hours

13. showed that microbial growth occurred for 90% of tobacco particulates (fig 1C). Positive results for bacteria were obtained for tobacco powder derived from both new and vintage cigarettes. During this investigation, the question arose as to whether the tobacco flake lying loosely on the cut surface of the filter of cigarettes in newly opened packs would also contain bacteria (fig 1A). In one representative study, the filters of the cigarettes from 11 different brands of five different tobacco companies were assayed. For all brands (n=11/11), cigarettes were observed with tobacco flake on the filter. An inspection of the cut surface of the cigarette filter with the naked eye revealed that 127 of 208 (61.1%) contained tobacco. Numerous small tobacco particles were observed in the field of cellulose acetate filter fibres with a stereo-zoom microscope. The filters of cigarettes from this collection were assayed. For all brands (n=11/11), the filters showed positive assay results for bacteria. At a 24 hour reading (fig 1D), bacterial growth was positive for 39 of 88 (44.3%) of the filters tested. At a 48-hour reading of the same blood agar plates, bacterial growth was recorded for two more filters (41/88; 46.6%). For 24-hour and 48-hour readings in no instance (n=8/8 filters) was there bacterial growth for control cigarettes that had been autoclaved

14. (B) Petri dish (diameter, 100 mm) of blood agar that had been marked with a felt-tipped pen into eight pie-shaped sections (a-h), each of which contained a single flake of tobacco from different brands of cigarettes. All sections display microbial growth that arose during 24 hours of incubation of the blood agar plate. The dotted circle in section "a" illustrates an area of bacterial-associated haemolysis (yellow field), and section "h" illustrates a single flake of tobacco.

15. (D) View of a cigarette filter assay for bacteria in which the cut surface of a cigarette filter had been touched to the surface of a blood agar plate. Microbial growth (sections "a", "d", "e" and "f") was observed at 48 hours for four of the eight filters tested. Note the haemolysis in sections "a", "d" and "f".

16. (C) Shown are numerous and diverse microbial colonies that were generated during 24 hours of incubation of a blood agar plate that contained tobacco microparticulates that had been sieved from cigarette tobacco.

17. Figure 1 (A) View of tobacco flakes and tobacco fines on the cut surface of the filter of several cigarettes in a newly opened box. The diversity of the size and shape of the tobacco particles is shown in the enlarged view of the site denoted with the small box.

18. DISCUSSION (1) The blood agar assay scheme described here is a technically simple and reproducible method of growing bacteria from tobacco flake and microparticulates. Bacteria have been grown successfully from the flakes of all tobacco products tested thus far. These include diverse smoking (for example, cigarettes and cigars) and smokeless products (that is, chewing tobacco; for example, long-cut/pouch, dry snus and moist snus). The most salient feature of our work is the observation that bacterial growth was documented from minute pieces of tobacco on the cut surface of the cigarette filter. Concern is expressed that the tobacco microparticles will be carried in the bolus of mainstream smoke and deposited deep into the smoker’s lung. The inhaled tobacco flake may be envisioned as a foreign body that delivers diverse bacteria, fungi, spores and microbial toxins.

19. DISCUSSION (2) Activation of the lung macrophages can be induced by any one of many agents associated with viable or dead microbes, and endotoxin.2–5,8,9,18,19,21,22 Tobacco used in smoking and smokeless products is not washed; thus, the flakes may contain soil as well as residues of agents used in plant propagation (for example, insecticides and fungicides). The flake will also contain diverse additives—tobacco casings (for example, flavourings), humectants and other substances. The potential health risk is further established with the knowledge that the long-term smoker, who does not clear deposited tobacco smoke particulates (‘‘tar’’) from the lung may have: (a) impaired mucociliary clearance mechanisms, nonproductive cough and reduction of other host-defence mechanical forces for eliminating the inhaled tobacco microparticulates, 23 (b) a compromised immune response,20 (c) chronic pulmonary inflammation1–3,7,10–14,18–22 and (d) recurring respiratory infections.24 US cigarettes are made with tobacco from different countries of the world.

20. DISCUSSION (3) Notable is that today more than 125 countries grow tobacco, many of which are developing countries.25 Thus, cigarettes may contain bacteria and mould imported from around the world. The question arises as to whether there is adequate regulatory control for cultivating, insect-abating and mould-abating, curing, exporting, storage and blending tobacco of different types, including plants from Third World countries. 26 The reason why the issue of microbes and toxins in cigarette smoke has received so little attention among those assessing mainstream cigarettes may be due to a misperception. Some have argued that the bacteria and mould pose no risk to the smoker because the tobacco flake in the cigarette column is prevented from entering mainstream smoke by the filter.2 It has also been opined that the high levels of microbial toxins and viable fungal spores that have been found in the tobacco column of cigarettes would be destroyed or greatly reduced in the cigarette

21. DISCUSSION (4) column during the burning of the tobacco.1–3, 5 These views are challenged by the fact that tobacco flake is often seen lying loosely on the filter of cigarettes of newly pened packs. Most probably, the tobacco on the filter is the result of highspeed manufacturing procedures and transportation of the cigarettes to vendors worldwide. Also, as the cigarettes are consumed, the remaining cigarettes in the pack are jostled, and this is likely to increase tobacco particulates that are released from the tip of the cigarette to translocate to the face of the filter. Thus, the tobacco flake observed on the mouth end of the filter is as diverse as the blend of tobaccos in the cigarette column. During normal smoking behaviour, tobacco flakes are discharged from the filter. More than 50 years ago it was reported that tobacco flakes were observed routinely in the ainstream smoke captured in the first three puffs of all six filter cigarettes studied (Tareyton, Winston, Kent, L&M, Marlboro and Viceroy).27

22. DISCUSSION (5) The critic may argue that many common foods contain viable bacteria. It is to be noted that, in contrast to tobacco, most vegetables and perishable foods are: (a) often washed before consuming; (b) refrigerated or frozen; (c) sometimes chemically treated (for example, spinach; washed with ‘‘chlorinated water’’ to destroy E coli); or (d) preserved (for example, dried, salted or smoked). We have included in our studies tests of other flakelike substances that included different spices (for example, thyme, garlic, oregano and rosemary) as well as ground coffee and tea. Almost all of the spices analysed showed little or no bacterial growth. Likewise, coffee and tea displayed very little or no bacterial growth. Most important, however, is that a distinction must be made between the propensity of a health risk from inhaling tobacco flake and ingesting tobacco or foodstuffs. Unlike the gastrointestinal tract, the deep lung of a healthy subject is sterile—free of bacteria and mould.

23. DISCUSSION (6) Accordingly, it is reasonably anticipated that inhaling moist tobacco flake with bacteria, spore or toxins into the lung would provoke chronic macrophage-mediated inflammation and, thereby, be a heretofore undefined health risk associated with cigarette smoking. As is generally known, tobacco must be cured before it is used to manufacture cigarettes or other tobacco products. Although different curing processes are used, most methods subject tobacco to conditions that favour the growth of bacteria and mould (for example, high temperature, elevated humidity, sustained darkness and poor ventilation). For more than two decades, the tobacco companies have studied microbes on tobacco. For example, RJ Reynolds28 and Philip Morris29 have analysed the growth of aerobic bacteria, anaerobic bacteria and mould on different types of tobacco, under different curing conditions and during storage for more than a year. Also notable is that Imperial Tobacco has developed an easy-to-search database on the micro-organisms associated with tobacco.30 The database is intended to provide information

24. DISCUSSION (7) on the microscopic appearance of the colonies and their biochemical capabilities (for example, enzymes, specific biochemical reactions and growth at various temperatures).28 29 Today, there exists a need to catalogue the microbial analytes (for example, bacteria, mould, spores, endotoxins, mycotoxins) in different smoking and smokeless tobacco products. In addition to bacteria and spores, concern is also expressed as to the toxins produced by bacteria and mould.1–6 17 19–21 A potent bacterial endotoxin, LPS, has been found in raw tobacco,3 mainstream smoke1–3 and secondhand smoke.3 LPS has been studied extensively and is widely recognised as a potent inducer of inflammation, particularly pulmonary inflammation.18–22 Different methods have been proposed, some of which are currently in use to treat raw tobacco for bacteria and mould.

25. DISCUSSION (8) By way of example, Philip Morris has crafted different washing methods: ‘‘…for treating tobacco before or during curing to lower or eliminate bacterial populations and/or activity, fungal growth, and/or tobacco-specific nitrosamines or bacterial endotoxin levels…’’. The bacterial population of tobacco leaves is about 105–106 bacteria/gram and increases 10-fold to 20-fold during curing. The authors note that the bacterial populations found on the bright or Virginia tobacco leaves are primarily Gram-negative bacteria, including pseudomonads and enterobacters. These bacteria form LPS, which can remain as a residue even after the bacteria have been destroyed.31 Other examples are the US patents awarded for microwave radiation, heat or gasses, applied during appropriate times in the curing cycle, to reduce the content and prevent the formation of carcinogenic nitrosamines in tobacco and marijuana.9 32 33

26. DISCUSSION (9) Considerable effort has been devoted to assessing the carcinogenicity and toxicity of the thousands of different chemicals that are in mainstream smoke which arises from the burning of tobacco.3435 In various comprehensive reports of tobacco-smoke toxicology, the subject of microbes and microbial toxins and their association with oral and pulmonary diseases is absent. The studies reported here highlight the need to research the effect of inhaling flakes harbouring diverse microbes and microbial toxins.

27. What this paper adds Cigarette tobacco contains bacteria, fungal spores and microbial toxins. We report here the results of studies that document the growth of bacteria from cigarette tobacco microparticulates that were assayed with blood agar plates. Our findings are the first to raise the question as to the potential health risks associated with microbes and microbial toxins of raw tobacco flakes that are known to be released from the cut surface of the cigarette filter into mainstream smoke that is drawn into the lung.

28. Acknowledgements: The authors wish to express their appreciation for the assistance of Linda Charles-Steele, laboratory supervisor, Department of Clinical Microbiology. • Funding: This work was supported by NIH CCSG Sub Award 5P30CA01605631 and a • Clinical Innovator Award of the Flight Attendant Medical Research Institute. • Competing interests: JLP has served as an expert witness in court cases against the tobacco industry for which he received monetary compensation. JDW and GMP have no competing interests to declare.

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