Cigarette Smoke, Bacteria, Mold, Microbial Toxins, and Chronic Lung Inflammation

John L. Pauly; Geraldine Paszkiewicz

doi:10.1155/2011/819129

Cigarette Smoke, Bacteria, Mold, Microbial Toxins, and Chronic Lung Inflammation

Pauly, John L.;Paszkiewicz, Geraldine 2011-07-09 00:00:00 Hindawi Publishing Corporation Journal of Oncology Volume 2011, Article ID 819129, 13 pages doi:10.1155/2011/819129 Review Article Cigarette Smoke, Bacteria, Mold, Microbial Toxins, and Chronic Lung Inﬂammation John L. Pauly and Geraldine Paszkiewicz Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buﬀalo, NY 14263, USA Correspondence should be addressed to John L. Pauly, john.pauly@roswellpark.org Received 16 November 2010; Revised 28 February 2011; Accepted 20 March 2011 Academic Editor: Venkateshwar Keshamouni Copyright © 2011 J. L. Pauly and G. Paszkiewicz. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chronic inﬂammation associated with cigarette smoke fosters malignant transformation and tumor cell proliferation and promotes certain nonneoplastic pulmonary diseases. The question arises as to whether chronic inﬂammation and/or colonization of the airway can be attributed, at least in part, to tobacco-associated microbes (bacteria, fungi, and spores) and/or microbial toxins (endotoxins and mycotoxins) in tobacco. To address this question, a literature search of documents in various databases was performed. The databases included PubMed, Legacy Tobacco Documents Library, and US Patents. This investigation documents that tobacco companies have identiﬁed and quantiﬁed bacteria, fungi, and microbial toxins at harvest, throughout fermentation, and during storage. Also characterized was the microbial ﬂora of diverse smoking and smokeless tobacco articles. Evidence- based health concerns expressed in investigations of microbes and microbial toxins in cigarettes, cigarette smoke, and smokeless tobacco products are reasonable; they warrant review by regulatory authorities and, if necessary, additional investigation to address scientiﬁc gaps. 1. Introduction: Chemical and Biological of references is included [1–4]. Most of the chemicals, toxicants, and carcinogens in tobacco smoke arise from the Components of Tobacco and Smoke burning (pyrolysis) of the tobacco [1, 2, 4]. The potential for For many years, scientists have undertaken studies to deﬁne harm has also been studied for chemicals that do not arise from the burning of tobacco. The chemicals include metallic the chemical composition of green tobacco leaf, cured- fermented-stored tobacco leaf, and tobacco smoke with the and nonmetallic elements, isotopes, and salts [1, 2, 4]. In intent of identifying chemicals that may pose a signiﬁcant addition, pesticides and other intact agrochemicals have been health risk [1–4]. An illustration has been prepared of the identiﬁed in tobacco smoke [1, 2, 4]. Also included in this annual increase, from 1954 to 2005, in the total number tabulation of chemicals in smoke are menthol and ﬂavorants of tobacco smoke chemicals that have been identiﬁed [4]. [4]. Today, there is a consensus of opinion that cigarette smoke In 1985, Hoﬀmann and coworkers, who had studied the consists of at least 5,300 diﬀerent chemicals [4]. These chemical composition of tobacco smoke for many years, be- chemicals are present in the complex aerosol that consists of gan formulating a list of chemicals that were designated as a heterogeneous mixture of gas- (vapor-) phase and particu- biologically active, carcinogenic, cocarcinogenic, or tumor- late- (“tar-”) phase components [1–4]. genic, reviewed previously in [4]. The tabulation was revised Detailed listings of the chemicals in mainstream and side- and became the basis for the list of “Hoﬀmann Analytes” stream tobacco smoke are available, and an assessment of [4]. In 1985, diﬀerent working groups met to identify those their propensity for harm has been presented; a partial listing chemicals in tobacco smoke that are most likely to be 2 Journal of Oncology carcinogenic to humans as deﬁned by criteria of the In- 3. Tobacco and Harm Associated with Microbes ternational Association for Research on Cancer (IARC), an Our review of the aforementioned writings [1–4]and many intergovernmental agency forming part of the World Health other related reports, addressing chemicals in tobacco smoke Organization, and by the US National Toxicology Program of cigarettes have shown that the writings do not address (NTP) [1, 2, 4]. the propensity for harm that may be associated with micro- bial elements of smokeless and smoking tobacco articles. A partial listing of tobacco-associated microbial elements 2. The Changing Cigarette include bacteria (Gram positive and Gram negative), bacte- rial spores, fungi (yeast and mold), fungal spores, cell wall The identiﬁcation, classiﬁcation, and concentration of the components (certain glucans and ﬂagellum), and diverse various chemicals in cigarette smoke have been challenged by microbial toxins that include exotoxins and endotoxins. changes in the design of cigarettes. A comprehensive review Examples of bacterial-derived toxins include endotoxins of “The Changing Cigarette” was published by D. Hoﬀmann (lipopolysaccharide, LPS; inﬂammatory factor) and fungal- and I. Hoﬀmann in 1997 [5]. derived mycotoxins (aﬂatoxins, AF type B1; human carcino- Subsequently, other investigators addressed changes in gen) [1–4]. cigarettes and their potential for risk [6–12]. By way of There exists today a concern of the potential health risks example, a partial tabulation of changes in cigarette includes associated with diverse microbial elements that are known (a) increased cigarette length (85 mm king sized and extra to exist in smoking and smokeless tobacco products that long “120’s”) and, for some brands, reduced circumference are currently being marketed. This subject has not been (23 mm “slim” cigarettes), (b) variation in the blend of nat- addressed in the context of national tobacco control policy ural tobaccos of diverse types, country of origin, and curing or regulatory authorities. processes, relative percent tobacco leaf (lamina) versus tobac- Harm is to be recognized as persistent or chronic inﬂam- co ribs/stems, and tobacco weight per rod, (c) incorporation mation. Inﬂammation is mediated by diﬀerent leukocyte of manmade tobacco, sometimes referred to as reconstituted subsets and diﬀerent secreted factors (Figure 1). Inﬂamma- or “sheet” tobacco, (d) introduction of additives to the to- tion not only establishes a microenvironment that fosters bacco (casings) that include diverse ﬂavorings (licorice and the malignant transformation and tumor growth but also honey), humectants to retain tobacco moisture, and menthol promotes microbial colonization. to ameliorate smoke irritation and promote smoking accep- tance by youngsters and “starters” (e) addition of ammonia, 4. Research Objectives to facilitate “freebasing” the nicotine to enhance the pharma- cological eﬀect (impact), (f) application of diverse glues and The goal of this paper is to proﬁle the scientiﬁc and medical printing ink, (g) conﬁguration of diverse cigarette ﬁlter mate- literature addressing microbes in tobacco with the intent to rials (cellulose acetate, paper, or combination of both), (h) determine whether there is suﬃcient evidence to warrant alteration of ﬁlters with charcoal and schemes whether the additional investigations to assess propensity for human carbon was dispersed throughout the ﬁlter plug or retained harm. The impetus for undertaking this work was derived in a ﬁlter cavity, (i) variation in ﬁlter design (ﬁlter length, in part from the fact that several teams of investigators, ﬁber packing/crimping, ﬁber density, and ﬁlter ventilation) including our own, have published observations during the to eﬀect tar delivery (full ﬂavor cigarettes versus ultralight last few years that suggest microbial elements maybe harmful low-tar cigarettes), (j) paper type, paper porosity, with burn to tobacco users. accelerators to promote burning, or with modiﬁcations to Notable in a ﬁrst analysis of the literature on the micro- reduce the propensity for sustained burning and aﬀect a “ﬁre biology of tobacco we discovered that there were few recent safe” designation, and (k) diverse methodologies to reduce reports (1990 to 2010) in peer-reviewed, mainstream, scien- “tar” and nicotine yields in mainstream smoke of cigarettes tiﬁc and medical journals by scientists of tobacco companies. that have been smoked mechanically [6–12]. By way of example, Philip Morris has contracted the Life The topic of “The Changing Cigarette” has been addres- Science Research Oﬃce, Inc., (LSRO, Bethesda, MD), to sed and summarized in a recent report of the Surgeon Gen- identify methods to evaluate tobacco products and with a eral entitled “How Tobacco Smoke Causes Disease” [13]. particular focus on identifying research schemes and assays A review of the scientiﬁc and medical literature has shown for assessing reduced-risk tobacco articles [14]. Three mono- that (a) changing cigarette designs over the last ﬁve decades, graphs published by LSRO in 2007 detailed the chemicals including the introduction of cigarette ﬁlters and low-tar to be assayed and recommended procedures. The subject cigarettes, have not reduced overall disease risk among smok- of microbial ﬂora and microbial toxins was not addressed, ers and may have hindered prevention and cessation eﬀorts, nor were schemes and methodologies for the assessment of (b) there is insuﬃcient evidence that novel tobacco products tobacco associated bacteria, mold, or microbial toxins [14]. reduce individual and population health risks, and (c) the Therefore, the question arose as to whether the issue of introduction of novel tobacco products that are marketed as health risks associated with microbial elements in smokeless reduced-risk cigarettes may encourage tobacco use among and smoking tobacco was not investigated by laboratory youngsters. These changes have challenged tobacco policy scientists working at the tobacco companies or whether the and regulation [13]. subject was studied and the information withheld as private Journal of Oncology 3 inﬂammation. It is acknowledged that immunological re- sponses and inﬂammation would not be a primary inter- est by other investigators whose primary interests are in the disciplines of microbiology/metagenomics, aerosol- “Tar” associated inhalation toxicology, infectious diseases, and clinical pathology (oral and lung). Also, the work presented herein is limited in scope. The authors retrieved numerous TLR documents from databases, but space restrictions permit cit- ing but a few of the writings. Also, many of the writings were MΦ internal documents and were not subjected to peer-review. Some documents cited are old and are addressed herein to provide a historical perspective. Lastly, the documents are TNFα IL-1β LIF OSM IL-4 fragmented and it is recognized that conﬂicting ﬁndings and interpretations may be presented by competing tobacco Type I companies. DC cell Type II Tcell cell 6. Literature Search A computer-based structured search of the literature was conducted. The study scheme included a search of the lit- PMN Capillary MON erature from PubMed (http://www.ncbi.nlm.nih.gov/pub- med) and Scopus (http://www.scopus.com/home.url). Also, included was a search of Google (http://www.google.com/). Figure 1: A schematic view of an alveolus that depicts the eﬀect of A search was also made of patents in the database inhaled tobacco smoke on the terminal (respiratory) structure of of the US Patent and Trade Oﬃce (http://www.uspto.gov/). the lung. Particulate matter “Tar” in tobacco smoke is inhaled deep In addition, searches were made for documents that were into the lung where it is recognized by macrophages. The macro- phages arise from the blood monocytes that migrate into the lung released by the tobacco companies and made public as a where they undergo diﬀerentiation and maturation. Macrophage consequence of the tobacco Master Settlement Agreement. phagocytosis of the chemical-rich “Tar” evokes the production To this end, we searched database records of over 11 million of diverse proinﬂammatory mediators (for details, see Figure 1). documents in the digital archive that were established and Macrophages have toll-like receptors (TLR) that recognize diverse which are maintained currently at the University of Califor- microbes andtoxins(LPS isrecognized byTLR-4). Shown inthis nia, San Francisco (http://legacy.library.ucsf.edu/). We also illustration is the production of ﬁve proinﬂammatory cytokines: searched the database from Tobacco Documents (http://to- tumor necrosis factor, type alpha (TNFα), interleukin 1-beta (IL- baccodocuments.org/). 1β), leukemia inhibitory factor (LIF), oncostatin M (OSM), and The searches were performed using conventional tele- Interleukin-4 (IL-4). These soluble factors interact with other cells of the lung, and the response of these cells is thought to accelerate, gram-style search short-string text formulations with Bool- amplify, and prolong pulmonary inﬂammation. The target cells may ean operators as described in PubMed. Illustrative key search include T cells. The T cell that is depicted herein is representative of words were bacteria, mold,fungi,yeast,tobacco, smoke, many diﬀerent T cell subsets, including T helper cell subsets Th1, endotoxin, mycotoxin, cured, fermented, lipopolysaccharide, Th2, and Th17. Type I epithelial cells are the major cells lining aﬂatoxin, and microbiology. We also used unique search the alveolar space, and facilitating O /CO . The type I cells are 2 2 words, such as author’s name, project designation, report spread out and cover about 90 to 95% of the alveolar surface. codes, cigarette brands, and Bates number. The references The type II cells form only 5 to 10% of the surface but produce cited in the retrieved literature were reviewed to identify surfactant proteins. Polymorphonuclear leukocytes (PMN) mediate other topic-speciﬁc writings Table 1. inﬂammation in multiple ways, including the production of an oxidative burst. Dendritic cells (DC) are professional antigen- presenting cells; they also mediate inﬂammation. 7. Tobacco-Associated Chronic Inﬂammation Chronic inﬂammation is associated with malignant trans- and conﬁdential. The paucity of the literature on health risks formation, tumor growth, and, possibly, tumor metastasis, associated with microbes in smokeless and smoking tobacco reviewed in [44–52]. Examples of the association of cancer is to be contrasted to the numerous reports by tobacco scien- with chronic inﬂammation include (a) lung cancer and tists researching other health-related issues, such as potential cigarette smoke (aerosol), (b) malignant mesothelioma and reduced-risk exposure tobacco products (PREPS) [15]. asbestos (ﬁbers), (c) stomach cancer and H. Pylori(bacteria), (d) malignant melanoma and ultraviolet sun light (irradia- tion), (e) liver cancer and aﬂatoxin (mycotoxin), and (f) can- 5. Perspective and Limitations cer of the uterine cervix and human papilloma virus. Thus, The authors are immunologists and have an active research malignancy at diverse body sites, and of various tissues, is interest in addressing tobacco-associated chronic pulmonary associated with chronic inﬂammation provoked by assorted 4 Journal of Oncology Table 1: History of investigations of microbes and microbial toxins in tobacco and tobacco products. Results are reported for studies that were undertaken to characterize the microbes of tobacco before and during 1896 [16] tobacco fermentation. German bacteriologist H. E. Suchsland announces that the delicate aroma and subtle shades of ﬂavor which aﬀect the palate of the smoker are not due to the tobacco but are attributed to the microbes which aid in the process of tobacco 1899 [17] fermentation. A patent based upon this observation was submitted, presumably to improve the poor quality of German tobacco by adding to the harvested tobacco leaves bacteria that he had isolated and grown in his laboratory from high-quality West Indian tobacco. The microbial degradation of nicotine and nicotinic acid was reported. The morphological and physiological 1954 [18] properties of the nicotine-decomposing bacteria were also described. W. C. Flanders of R. J. Reynolds Tobacco Company issues a 70-page report of a three-year study to determine if the number of microorganisms (bacteria and mold) changed appreciably during aging. Experiments were also 1955 [19] conducted to determine if the recorded changes in the microbes follow the changes in the chemical components of tobaccos. These studies were continued and extended for several years. Pseudomonas aeruginosa and other potentially pathogenic fungi and bacteria were identiﬁed in snuﬀ. Similar 1957 [20] microbial isolates from a patient was the basis for the physician to theorize that some of the snuﬀ-derived microbes may be responsible in part for chronic bronchitis. The results of studies were reported that had been undertaken to characterize the deposition of cigarette smoke particles and debris released from the cigarette ﬁlter into the human respiratory tract. Popular brand cigarettes were smoked mechanically and in a manner to reﬂect normal smoking behavior. The studies documented that tobacco 1958 [21, 22] ﬂakes and ﬁne tobacco leaf debris were released into mainstream smoke from the cigarette ﬁlter of all brands that were tested (Tareyton, Winston, Kent, L&M, Marlboro, and Viceroy). The tobacco ﬂakes and other particulates (ﬁlter ﬁbers and carbon from charcoal ﬁlters) were studied by light and electron microscopy. 1966 [23] Toxic fungi were identiﬁed in tobaccos. Comparative studies were preformed for microbiological activity in the smoke of popular brand nonﬁltered and 1967 [24, 25] ﬁltered cigarettes that had been “cold smoked” or lit. Viable bacteria were found in the smoke of all cigarettes tested. The tobacco from diﬀerent popular brands of cigarettes was analyzed for bacteria. The number of bacteria was determined on “our own” (Philip Morris) and competitive cigarette ﬁllers. This test was run for several months and 1972 [26] each month Viceroy, Brown & Williamson’s product, always showed the lowest degree of “contaminant.” The diﬀerence between the brands was statistically signiﬁcant. Brands tested included Salem, Pall Mall, Chesterﬁeld, Kool, Kent, Viceroy, Winston, and Marlboro. The number of bacteria on Marlboro were “too numerous to count.” A 189-page report was prepared by investigators at the Brown & Williamson Tobacco Company that presents methods for the microbiological examination of tobacco and tobacco products. The writings include the description 1972 [27] of techniques for the quantitative determination of bacteria and fungi and methods for the isolation of potentially human pathogenic microorganisms including Coliform bacteria. Also identiﬁed were Staphylococcus aureus, Enterococci, Pseudomonas, Clostridium,and Aspergillus. A 52-page report that describes a “contact plate method” in which a whole cigarette is rolled over the surface of the nutrient agar dish. Viable microbes that are transferred from the cigarette to the plate are illustrated. Presumably, the 1972 [28] intent of the assay was to measure the growth of microbes that would be transferred from the cigarette paper to the hand of the smoker. Other studies showed the growth of microbes from a natural wrapper of a cigar. Also, culture methods were established for testing for coliform bacteria and for counting viable fungi in tobacco. A 346-page in-house document is produced by the British-American Tobacco Company entitled “Methods for the Microbiological Examination of Tobacco and Tobacco Products.” The authors describe the “Public Health Aspects” of smoking and smokeless tobacco products. They note that “[T]he detection of micro-organisms of health signiﬁcance in tobacco products must be expected to be regarded as undesirable or even unacceptable by public 1972 [29] agencies, regardless of whether there is proof of the signiﬁcance in initiating or spreading infection in man. Therefore, it is suggested that tobacco products should be substantially free, or contain only minimal numbers, of micro-organisms of potential health signiﬁcance to man which could conceivably occur on tobacco...” Suggested standards are presented for tobacco products for various bacteria and fungi, and standards that had been established for food products (ﬁsh, sausage, meat pies, cream yogurt, soft cheese, and pasteurized milk). Philip Morris characterizes the microbial population on Marlboro tobaccos throughout the processing line. Five diﬀerent Marlboro Make-Your-Own tobaccos with various anti-microbial preservatives were evaluated 1991 [30] microbiologically for mold and bacteria over time. The microﬂora of Marlboro raw and tobacco blends were deﬁned for burley, oriental, ﬂue-cured, and other tobacco types. Journal of Oncology 5 Table 1: Continued. Bacillus spores were identiﬁed in chewing tobacco sold in the USA. Broth of the culture microbes evoked plasma 1992 [31] exudation from the oral mucosa when tested using a hamster cheek pouch assay. 1995 [32] In an oral presentation, Hasday describes for the ﬁrst time the presence of endotoxin in cigarette smoke. Scientist from Imperial tobacco (Canada) report the development of an easy-to-search database on the microbes 1990 [33] associated with tobacco. 1999 [34] Bacterial endotoxin was identiﬁed as an active component of cigarette smoke. A US Patent was awarded for a method and system for assay and removal of harmful toxins during the processing of 2004 [35] tobacco products. Microbiologists in Sweden used a mass-spectrophotometry-based assay to document that tobacco smoking increased 2004 [36] dramatically the air concentrations of endotoxin (LPS). The authors note that smoke-derived LPS may be a health risk factor associated with environmental tobacco smoke. A US Patent was assigned to Philip Morris for an “antibacterial lavage” method to treat tobacco leaves so as to eliminate or reduce bacterial endotoxins (LPS) and tobacco-speciﬁc nitrosamines that are formed during the curing 2004 [37] process. Bacteria found on tobacco leaves were reported to be primarily Gram-negative bacteria, including pseudomonades and enterobacters. In the awarded patent, Hempling notes that bacterial endotoxins can remain as a residue on the tobacco even after the bacteria have been destroyed. The microbiological composition of tobacco products was deﬁned using culture and chemical analysis. Tobacco 2004 [36] smoke was analyzed chemically, and LPS was measured for tobacco leaves and cigarette tobacco. US Military publishes a report of an investigation that documents bacterial species diversity of varying brands of 2005 [38] cigarettes made in the Middle East that were thought to be associated with illnesses of American soldiers deployed in Operation Iraqi Freedom. 2006 [39] Cigarette smoke was identiﬁed as the source of elevated levels of endotoxin (LPS) found in indoor air. Identiﬁcation of microﬂora on tobacco using culture-independent methods based on the ampliﬁcation of microbial 2007 [40] 16S rDNA sequences directly from the leaf surfaces. The investigators discovered also that three of ﬁve dominant bacterial species on the tobacco could not be cultivated. The microbiological composition of tobacco products was deﬁned using culture and chemical analysis (of tobacco leaves) or chemical analysis only (tobacco and tobacco smoke). Mesophilic bacteria dominated among the bacteria in 2008 [41] both fresh and cured tobacco leaves; however, a wide range of other bacteria, including Gram-negative bacteria, and fungi were delineated. Microbial ﬂora was compared in studies of tobacco from cigarettes from diﬀerent countries. LPS was also measured. Bacteria grown from a single ﬂake of tobacco from all brands of smoking (cigarette, cigar, and pipe) and smokeless 2008 [42] (snus, snuﬀ, and long cut) tobacco products. In many instances, the bacteria from the tobacco caused hemolysis of blood in blood agar and liquid broth cultures. Twenty-seven species of bacteria were identiﬁed in an analysis of both unaged tobacco and ﬂue-cured tobacco by 2010 [43] 16S rRNA sequence analysis. More species (N = 23) were identiﬁed from the unaged ﬂue-cured tobacco leaves than in the aging leaves (N = 15 species). Fifteen classes of bacteria and a broad range of potentially pathogenic organisms were detected in all cigarette samples studied. In greater than 90% of the tobacco samples, the investigators identiﬁed Acinetobacter, Bacillus, Burkholderia, Clostridium, Klebsiella, Pseudomonas aeruginosa,and Serratia. The bacteria were identiﬁed using a 2010 [43] 16S rRNA-based taxonomic microarray. Cloning and sequencing were used to evaluate total bacterial diversity of four brands of cigarettes. Previous studies have shown that smoking was associated with colonization by pathogenic bacteria and an increased risk of lung infection. This study, however, was the ﬁrst to show that cigarettes themselves could be the source of exposure to a wide array of potentially pathogenic microbes. items that include smoke, bacteria, ﬁbers, irradiation, toxins, smoking may not only have an adverse eﬀect of systemic im- and viruses. munity but also skews both innate and adaptive immune responses [61–65]. 8. Cigarette Smoke, Chronic Inﬂammation, 9. Study Rationale: Evidence-Based Health and Impaired Immunity Risks of Tobacco-Associated Microbes Cigarette smoke is known to induce chronic inﬂammation of the lung [53–60]. More recently, a substantial body of infor- Concern has been expressed by many investigators that mi- mation has been obtained to suggest that long-term cigarette croorganisms on cured tobacco might represent a health risk. 6 Journal of Oncology By way of example, in 1968, Wood [66], a scientist at the reproducibility and smoke toxicity. The results were incon- British American Tobacco Company, wrote a 37-page report clusive. Our search for subsequent studies by Wood address- addressing the possible transfer of viable microorganisms ing this subject failed to identify subsequent experiments or into mainstream smoke. In this internal document, he notes published reports. Studies by Slutzker et al. were negative that cured tobacco, of various types, has long been known [69]. In 1967, Curby reported to The Council for Tobacco to contain bacterial spores. Likewise, Wood [66]and others Research the results of comparative studies that he had [23] have addressed the possibility that tobacco-associated undertaken to determine the microbiological activity in the mold may also represent a health hazard to smokers. Support smoke from ﬁlter and nonﬁlter cigarettes. Diﬀerent popular for this concern was derived in part from a paper published brands of cigarettes were obtained from local vendors in in Science by Forgacs and Carll two years previously in Brookline, Mass, USA. Comparative analyses were made which they reported the identiﬁcation of toxic fungi in of bacteria released from cigarettes that had been “cold tobacco [23]. In the Science paper, the investigators exposed smoked” (not lit) or smoked in the usual manner (lit). The mice to smoke from fungally contaminated hay. The mice tobacco smoke collection system was tested for sterility by developed pulmonary emphysema and other pathological means of conventional microbiology culture procedure and conditions; in contrast, mice exposed to smoke from sterile, by means of electronic analyses of particle size and number. uninoculated hay remained normal clinically. In a letter to Viable bacteria were identiﬁed in the smoke from all ciga- the Associate Scientiﬁc Director of the Council for Tobacco rettes tested. The number of liberated organisms was much Research, dated 1964, Forgacs, with more than 16 years greater when the cigarette was burning [24, 25]. of research experience as a mycologist, states that he had Before proﬁling more recent studies, a brief overview is examined mycologically a number of tobacco products, warranted of what many internal documents of the tobacco including cigarettes that had been purchased on the open industry have entitled the “Microbiology of Tobacco.” market [67]. Forgacs observed that the tobacco of all cigarettes contained fungal mycelia and spores [67]. In part, 10. Microbiology of Tobacco the origin of his health concern is based upon the knowl- edge of (a) widespread fungal contamination of tobacco The “Microbiology of Tobacco” has been the focus of many products, (b) heat stability of the mycotoxins; (c) known studies. It was not surprising to learn from our paper that animal toxicity, (d) reasonable assumption that some of the most of all the major tobacco companies have studied this fungi are carcinogenic, and (e) potency at low doses, see also issue for many years. Listed below are varying topics ad- [68]. dressing bacteria, mold, and mycotoxins in tobacco and ref- Wood argues that erences (a) chemical and microbiological changes during curing “[W]hile it is quite impossible to deduce, from [16, 19, 70–75], this (mouse) experiment, the likely eﬀect of smoke from a cigarette containing fungally (b) bacteria in cigarettes; product comparison (also, see contaminated tobacco, the implications are suf- below) [17, 76–79], ﬁciently important to warrant some considera- (c) databases of tobacco microbes [33, 40, 80], tion of the role which micro-organisms may play (d) tobacco microbe control [81], with regard to smoke toxicity. For instance, it is possible that viable spores might be transferred (e) microﬂora community of tobacco [82–88], to mainstream smoke and thus enter the lungs; (f) quantitative studies of tobacco microﬂora [89–91], pathogenic species, even in small numbers, (g) growth of mold in stored tobacco [26, 92], could clearly have harmful eﬀects, while very large number of otherwise harmless micro- (h) growth of Aspergillus from tobacco [93–95], organisms might lead to a signiﬁcant concen- (i) microbial degradation of nicotine [18, 96], tration of genetic material. Alternatively, during (j) examination of cigarettes from mold-damaged and the vegetative stage of their residence on tobacco nondamaged tobacco [97], the micro-organisms might produce toxins which could transfer direct to smoke or metabo- (k) isolation of viable fungi from snuﬀ [98], lites which on burning could give toxic smoke (l) sterilization/treatment to remove NNK [37, 99–105], constituents.” (m) removal of harmful toxins on tobacco [35, 95], The report by Wood also describes some preliminary (n) inhibiting mycotoxin production [106], experiments which were undertaken to show whether bacte- (o) microbiology of cigarettes, pipes, cigars, and snuﬀ rial or fungal spores could transfer into tobacco smoke. Two [27–30, 107–111]. schemes were used to trap the cigarette smoke; these were a test tube bubbler and a micropore ﬁlter. These samples From about the early 1970s, extensive research was con- from the bubbler and the ﬁlter were tested for the growth of ducted on the Microbiology of Tobacco. Many reports reﬂected microorganisms. Growth of microbes was observed; how- the interest of the major tobacco companies. These stud- ever, technical problems were encountered including poor ies sought to identify diﬀerent bacteria and molds and to Journal of Oncology 7 count the number of colony-forming units (CFU) during Warke [103] studied the microbiological quality of chew- processing. The number of bacteria and molds present in able, often sweet, tobacco mixes known as “Gutka” used by green, freshly harvested tobacco was compared to that of var- millions of children and adults in India where it is made ious stages of curing, fermentation, and long-term storage. and often exported. Of the 15 samples studied, all contained In many cases, more than one million bacteria were found in aﬂatoxins B ,B ,and G . Samples exposed to Co radiation 1 2 2 a gram of tobacco (a 100 mm cigarette has about 0.9 grams displayed a marked reduction of viable CFU. Sterilization of tobacco). Comparative studies included various types of of tobacco in the manufacturing has been described in US tobacco (Bright and Burley) and diﬀerent curing methods Patents [105]. (ﬁeld versus ﬂue cured). In these studies, proﬁles were In 1992, Rubenstein reported the identiﬁcation of large established for leaves of the diﬀerent types of tobacco that number (>10 CFU) of a Bacillus species in chewing tobacco had been picked from various positions of the plant. Diverse sold in the USA [31]. Supernatants of the cultured bacteria environmental conditions were evaluated, and these included evoked a plasma exudate in studies in which the supernatant variations in temperature and moisture. Analyses were made was instilled into an intact hamster cheek pouch. of the number of bacteria in popular brand cigarettes. In many instances, the number of bacteria of a particular 12. Pathogenic Bacteria of Cigarettes company’sbrand wascompared to brands marketed by com- petitors. In addition to cigarettes, studies were performed Some bacteria grow in unique microenvironments, and some for cigars and snuﬀ.Considerable eﬀort was devoted to are diﬃcult to grow using traditional broth- and agar-based deﬁning procedures for the sterilization of tobacco to reduce methods. This technical diﬃcultymay also applytogrowing or prevent the growth of mold. The methods used included bacteria that have adapted to unique conditions that develop (a) washing methods using various solutions (bleach), (b) during the curing and fermentation of tobacco. Accordingly, irradiation with microwave, ultraviolet light, and gamma it is believed that conventional methods may not accurately radiation, (c) exposure to various gases, and (d) treatment deﬁne the microﬂora of diverse tobacco products [43, 113]. with diﬀerent antibacterial and antifungal agents (antibi- Consequently, there may be an incomplete understanding of otics). One scheme was to destroy all of the bacteria on the bacterial diversity in the tobacco of cigarettes and also the freshly harvested green tobacco leaves and then seed the impact these microbes and microbial toxins may impose on leaves for fermentation using selected colonies from in-house the smoker [113]. batch-scale production. Quality control of the tobacco was Recently, the bacterial metagenomic of cigarettes were important as high levels of mold produced an unacceptable characterized using a 16S rRNA-based taxonomic microassay “oﬀ-taste.” as well as traditional cloning and sequencing methods. The brands included Camel, Marlboro, Kool, and Lucky 11. Pathogenic Bacteria of Chewing Tobacco Strike. The results of this study showed that the number of microorganisms in cigarettes may be as vast as the number of Studies have been conducted by investigators of the tobacco chemicals in these products. Fifteen classes of bacteria were industry (see above) and health community to address the identiﬁed [113]. Particularly noteworthy was the identiﬁca- potential of bacteria, molds, yeast, and microbial toxins tion of a broad range of potentially pathogenic microorgan- found in diﬀerent types of smokeless tobacco (snuﬀ,snus, isms detected. More than 90% of the tobacco samples from and long cut) [20, 26, 31, 43, 112, 113]. the cigarettes contained Actinetobacter, Bacillus, Burkholde- ria, Closteridium, Klebsiella, Pseudomonas aerogenosa,and In 1951, Dynert published in The New England Journal of Serratia. Other bacteria that are known to be potentially Medicine a case report of a patient with chronic bronchitis. pathogenic to humans and detected using the metagenomic Pseudomonas aeruginosa, often colonized in COPD patients, technology were Campylobacter, Enterococcus, Proteus,and and a few colonies of Staphylococcus aureus were identiﬁed Staphylococcus [113]. in bacteriological examinations of the subject’s sputum [20]. The patient used snuﬀ, and it was theorized that the snuﬀ Reported also in 2010 were the results of an investigation may have been the source of the pathogens. A study was of the diversities of unaged and ﬂue-cured tobacco leaves then undertaken of 22 samples of previously unopened packs using a 16S rRNA sequence analysis scheme [43]. of snuﬀ. The following microorganisms were grown from Others have reported the identiﬁcation of potentially more than 50% of the snuﬀ samples: Bacillus rubitilles, pathogenic bacteria in commercial cigarettes. One study was Staphylococcus aureus (coagulase positive), Staphylococcus undertaken to assess the bacterial diversity of cigarettes that albus (coagulase positive), Pseudomonas aeruginosa, Staphy- were thought to be linked to severe pneumonitis in US mil- lococcus aureus (coagulase negative), and Staphylococcus albus itary personnel deployed in Operation Iraqi Freedom [38]. (coagulase negative). Eight species of Bacillus, including ﬁve new species, and one In 1991, Varma reported the isolation of nine species of new species of Kurthia were isolated from the cigarettes. Aspergillus in stored leaves of chewing tobacco [112]. Ap- Some of these species have been identiﬁed elsewhere to proximately 18 of the Aspergilli were found to be mycotoxi- cause hypersensitivity pneumonitis and other respiratory genic. All aﬂatoxigenic strains of A. ﬂavus produced aﬂatoxin syndromes [38]. This study was of particular interest to B . Patulin and ochratoxin were produced by A. ochraceus. many because the cigarettes were made in Iraq and not man- Sterigmatocystin was produced by three diﬀerent strains. ufactured by a major tobacco company. Undertaking this 8 Journal of Oncology investigation, the question arose as to whether the cigarettes 14. Transfer of Tobacco Flake to that had been purchased by soldiers from street vendors Mainstream Smoke had been intentionally altered by adding pathogenic bacteria The ﬁlter of a cigarette is often contaminated with loose and/or mold. This theory was disproven. tobacco ﬂakes, tobacco ﬁnes, and tobacco dust. In one exam- Another study was conducted by a group of investigators in Sweden who characterized the bacterial and fungal com- ination, the ﬁlters of 11 brands of cigarettes were examined in freshly opened packs. For all brands, cigarettes were observed munity in warehouse tobacco [41]. with tobacco ﬂakes on the ﬁlter. Examination of the ﬁlters We have reported previously the establishment of a novel bioassay which showed that bacteria were grown routinely with the naked eye showed that 127 of 208 (61.1%) of the ﬁlter had tobacco particles [42]. The release of tobacco ﬂakes from a single ﬂake of tobacco that had been placed on the into mainstream smoke has been described previously [21, surface of a sheep blood agar plate [42]. Of eight popular 22]. brands of cigarettes, bacteria grew from almost all (>90%) The tobacco ﬂakes that contaminate the ﬁlter arise from of the ﬂakes. Similarly, bacteria were grown from a single ﬂake, and also with a high frequency, from tobacco that tobacco that escapes from the nonﬁlter, sometimes called the distal, end of the cigarette. Most probably the ﬂakes are jarred had been retrieved from cigar ﬁller and from smokeless loose during manufacturing, shipment, and daily transporta- tobacco (snus, snuﬀ, and long cut). Some bacteria induced hemolysis of the blood in the agar dishes. The destruction tion, especially in a pack in which more than one-half of the cigarettes have been used [117, 118]. of the red blood cells was readily visible as a yellow The release of ﬂakes from the cut surface can readily be zone surrounding a single tobacco ﬂake. Expanding studies demonstrated by comparing the cut surface of the ﬁlter be- documented the hemolysis of human blood in agar or fore and after smoking the ﬁrst puﬀ.The single ﬂake maybe nutrient broth cultures. Thus, as discussed later, bacteria viewed as a matrix for carrying bacterial and fungal agents in could be carried deep into the respiratory tract by a single mainstream tobacco smoke. Thus, the burning of the tobacco tobacco ﬂake sucked from the cut surface of a cigarette ﬁlter during cigarette smoking does not exclude the exposure to and transported into the bolus of smoke that is inhaled deep into the lung. A single tobacco ﬂake may be envisioned tobacco-associated microbes and microbial toxins. Bacteria are also released from the barrel of the cigarette. as a matrix for delivering diverse bacteria into the respira- This was demonstrated in investigations in which a cigarette tory tract of an immunologically compromised long-term smoker. was rolled over the surface of a nutrient agar dish. 15. Endotoxin (LPS) in Mainstream and 13. Cigarettes with Mold Sidestream Tobacco Smoke Mold has been identiﬁed in the tobacco of popular brand cigarettes, and concern has been raised as to the propensity In 1999, Hasday and his colleagues reported the identiﬁ- of these microbes as a health risk to the smoker. Presented cation of bacterial endotoxin as an active component in cigarette tobacco and cigarette smoke [34]. The authors herein is a partial listing of papers that have identiﬁed mold in cigarettes [78, 114–116]and in marijuana [116]. showed that the dose of LPS delivered from smoking one pack of cigarettes was comparable to that of the LPS that As early as 1971, Papavassiliou and coworkers concluded had been previously shown to be associated with adverse that “[C]igarettes are contaminated with various fungi.” health eﬀects in cotton textile workers. With the knowledge They studied cigarettes that were manufactured in the USA, that LPS is one of the most potent inﬂammation-inducing Canada, England, France, Belgium, Germany, Jordan, and agents, the work by Hasday attracted considerable attention, Egypt. Hundreds of strains of fungi were isolated. The Greek reviewed in [32]. In 2004, Larsson et al. reported that they scientists demonstrate that the most prominent fungi were were able to demonstrate unequivocally that high levels of Aspergillus (28 strains from Greek cigarettes and 35 strains LPS are inhaled during active cigarette smoking and, more from other countries). They raised the question as of the importantly, that environmental tobacco smoke may involve association of the fungi with allergies but commented that inhalation of amounts of endotoxin that are dramatically this issue has not been resolved [114]. greater than those existing in indoor environments free In 1983, Kurup and colleagues reported the identiﬁcation from tobacco smoke [36]. In 2006, these ﬁndings were of allergenic fungi in smoking materials and discussed the conﬁrmed and extended [39]. Particularly notable is that health implications of their ﬁndings [115]. Concern has been studies of Larsson and colleagues used a mass-spectrometry- expressed as to the health risks associated with mold in based assay that circumvents the problems often associated cigarettes. with the biologically based LPS assay. Writing in the Journal of the American Medical Associ- ation, Verweij et al. addressed the propensity of heath risks associated with fungal contaminates of tobacco and mar- 16. Analysis of Findings and Policy ijuana [116]. They concluded that “[A]ll cigarette brands Recommendations tested (N = 14 brands) had some degree of fungal contam- ination, although not every cigarette was found to have a The results of this literature review have documented that the positive culture.” tobacco microﬂora has been the subject of many studies by Journal of Oncology 9 investigators of tobacco industry and academic communities. assessed in the context of other known tobacco- During the last 50 years, there has been an imbalance, associated diseases, including chronic obstruc- however, in the attention devoted to addressing the identiﬁ- tive pulmonary disease, asthma, bronchitis, and cation and propensity of the harm of tobacco- and tobacco- alveolar hypersensitivity. smoke-associated chemicals and in the attention devoted to characterizing microbes and microbial-derived factors. (2) Tobacco-speciﬁc nitrosamines (NNK) are human Ample information has accumulated to suggest that carcinogens that are present in mainstream smoke, microbes and microbial-derived factors may contribute to sidestream smoke, and smokeless products. NNKs the health risks of smoking and smokeless tobacco products. arise primarily from the microbial degradation of Moreover, the microbes may facilitate microbial colonization nicotine in tobacco. Diﬀerent technologies have of the mouth and airway, the induction of chronic inﬂam- proven eﬀective in preventing the formation of mation through the activation of diverse leukocyte subsets, NNKs. It is recommended that these technologies be alteration of the tissue microenvironment, and microbial- implemented and that guidelines for tobacco articles toxin-induced pathologies. The current health concerns be established for reduced NNK-products. recently expressed by investigators of various disciplines, and (3) The criteria, protocols, and procedures used by the with diﬀerent research interests, in peer-reviewed published FDA in the assessment of harm associated with my- research articles are reasonable and validate that additional cotoxins in food products should be applied to investigation of the microbiology of tobacco is warranted. loose leaf tobacco, smoking tobacco products, and The ﬁndings reported herein relate to National Tobacco smokeless tobacco articles. Mycotoxin action levels Control Policy and speciﬁcally FDA Regulation of Tobacco should be established to provide an adequate margin Products [119]. of safety to protect human tobacco users. Based upon the information obtained in this paper, we recommend the following for consideration and possible reg- ulatory action. Abbreviations AFL-B Aﬂatoxin, type B (1) Tobacco products should be assessed with the knowl- 1: 1 edge that they contain bacteria, mold, and microbial CFU: Colony forming unit DC: Dendritic cell toxins. IARC: International Association for Research of Cancer (a) In this context, the designation of tobacco prod- IL-1β: Interleukin-1, beta ucts is to include conventional and novel IL-4: Interleukin-4 products that contain tobacco, including items LIF: Leukemia inhibitory factor which are smoking and smokeless tobacco LPS: Lipopolysaccharide articles. LSRO: Life Science Research Organization (b) National and international registries of known LTDL: Legacy Tobacco Document Library human carcinogens should not be used as the MON: Monocyte sole criteria for assessing tobacco-associated NTP: National Toxicology Program human health risks. Any and all tobacco-asso- OSM: Oncostatin M ciated agents that induce any human pathology PGE Prostaglandin E2 2: should be included in risk assessments. PMN: Polymorphonuclear leukocyte (c) Tobacco in smoking and smokeless tobacco ar- RNS: Reactive nitrogen species ticles should be assessed for their propensity ROS: Reactive oxygen species to induce chronic inﬂammation. Chronic in- TLR: Toll-like receptor ﬂammation is known to be induced by diverse TNFα: Tumor necrosis factor, alpha. bacteria (Gram positive and Gram negative) and fungi, living or dead, whole or fragmented, References and intracellular and membrane components. Chronic inﬂammation is known also to be in- [1] M. Borgerding and H. Klus, “Analysis of complex mixtures— duced by diverse toxins of bacteria and/or fungi cigarette smoke,” Experimental Toxicology and Pathology,vol. including, but not limited to, endotoxins, exo- 57, supplement 1, pp. 43–73, 2005. toxins, and mycotoxins. [2] R. R. Baker, “Smoke chemistry,” in TOBACCO: Production, Chemistry and Technology, D. Layten Davis and M. T. Nielsen, (d) Chronic inﬂammation associated with bacteria, Eds., charter 12, pp. 398–439, Blackwell Science, 2003. fungi, and microbial toxins of tobacco products [3] S. S. Hecht, “Cigarette smoking: cancer risks, carcinogens, should include inﬂammation of any and all tar- and mechanisms,” Langenbeck’s Archives of Surgery, vol. 391, get sites, including tissues of the mouth, naso- no. 6, pp. 603–613, 2006. pharynx, and lung. [4] A. Rodgman and T. A. Perfetti, The Chemical Components of (e) In addition to chronic inﬂammation, harm Tobacco and Tobacco Smoke, CCRC Press, Taylor and Francis of microbial elements of tobacco should be Group, Boca Raton, Fla, USA, 2009. 10 Journal of Oncology [5] D. Hoﬀmann and I. Hoﬀmann, “The changing cigarette, of tobacco,” Recent Advances in Tobacco Science,vol.21, pp. 1950–1995,” Journal of Toxicology and Environmental Health 39–80, 1955. A, vol. 50, no. 4, pp. 307–364, 1997. [20] H. P. Dygert, “Snuﬀ-a source of pathogenic bacteria in [6] G. F.Wayne and G. N.Connolly,“Regulatory assessment of chronic bronchitis,” The New England Journal of Medicine, brand changes in the commercial tobacco product market,” vol. 257, no. 7, pp. 311–313, 1957. Tobacco Control, vol. 18, no. 4, pp. 302–309, 2009. [21] W. K. Farr and A. Revere, Examination of Whole Cigarette [7] D. M. Burns and C. M. Anderson, “Do changes in cigarette Smoke by Light and Electron Microscopy,Life Extension design inﬂuence the rise in adenocarcinoma of the lung?” Foundation, New York, NY, USA, 1958. Cancer Causes Control, vol. 22, pp. 13–22, 2011. [22] W. K. Farr and A. Revere, “Examination of whole cigarette [8] R. J. O’Connor, K. M. Cummings, V. W. Rees et al., smoke by light and electron microscopy,” Journal of the “Surveillance methods for identifying, characterizing, and American Medical Association, vol. 172, no. 4, p. 405, 1960. monitoring tobacco products: potential reduced exposure [23] J. Forgacs and W. T. Carll, “Mycotoxicoses: toxic fungi in products as an example,” Cancer Epidemiology Biomarkers tobaccos,” Science, vol. 152, no. 3729, pp. 1634–1635, 1966. and Prevention, vol. 18, no. 12, pp. 3334–3348, 2009. [24] W. A. Curby, “A preliminary study of the biological activity [9] D. K.Hatsukami, K.A. Perkins,M.G. LeSage et al.,“Nicotine in cigarette smoke,” 1967, Bates Number 11330877-0905. reduction revisited: science and future directions,” Tobacco Retrieved on March 23, 2011 from http://legacy.library.ucsf Control, vol. 19, no. e1, pp. 1–10, 2010. .edu/tid/jtp6aa00. [10] H. Ito, K. Matsuo, H. Tanaka et al., “Nonﬁlter and ﬁlter [25] W. A. Curby, “A preliminary study of the biological activity cigarette consumption and the incidence of lung cancer by in cigarette smoke—part II,” 1967, Bates Number 11330942- histological type in Japan and the United States: analysis 0973. Retrieved on March 23, 2011 from http://legacy.library of 30-year data from population-based cancer registries,” .ucsf.edu/tid/stp6aa00. International Journal of Cancer, vol. 128, no. 8, pp. 1918– [26] R. E. Welty, “Fungi isolated from ﬂue-cured tobacco sold in 1928, 2011. Southeast United States, 1968–1970,” Applied Microbiology, [11] M. Rabinoﬀ, N. Caskey, A. Rissling, and C. Park, “Pharmaco- vol. 24, no. 3, pp. 518–520, 1972. logical and chemical eﬀects of cigarette additives,” American [27] T. G. Mitchell and British-American Tobacco Company, Journal of Public Health, vol. 97, no. 11, pp. 1981–1991, 2007. “Microbiological examination of tobacco products: report [12] Brown & Williamson, “Commonly added ingredients,” 1982, Number RD 969-R,” 1972, Bates number 105501740/1767. Bates Number 521057548/7553. Retrieved on February 22. Retrieved on June 28, 2010 from http://legacy.library.ucsf 2011 from http://legacy.library.ucsf.edu/tid/tmx33f00. .edu/tid/xwp67a99. [13] A Report of the Surgeon General, “How tobacco smoke [28] P. C. Stauber and British American Tobacco Company, “Microbiology of Henri Wintermans cigar production on- causes disease. The biology and behavior basis for smoking- attributable disease,” U.S. Department of Health and Human site studies of the primary process at Eersel: report No. Services, Atlanta, GA; Oﬃce on Smoking and Health, U.S. RD 925R,” 1972, Bates number 107466852/6877. Retrieved Government Printing Oﬃce, Washington, DC 20402, 704 on June 28, 2010 from http://legacy.library.ucsf.edu/tid/ pgs, 2010. http://www.cdc.gov/tobacco/data statistics/sgr/ dpe66a99. 2010/index.htm. [29] T. G. Mitchell and P. C. Stauber, “Methods for the microbio- [14] A. M. Bromnawell, Reports of the Life Science Research logical examination of tobacco and tobacco products, Report Oﬃce (LSRO),Bethesda, MD,Volume I. Biological eﬀects Number 888—R,” 1972, Bates number 105597063/7412. assessment in the evaluation of potential reduced-risk tobacco Retrieved on July 22, 2010 from http://legacy.library.ucsf products, 242 pages; Volume II. Scientiﬁc methods to evaluate .edu/tid/vit379. potential reduced-risk tobacco products, 174 pages; and Vol- [30] Smoke Study Group, “CORESTA—Papers presented at the ume III. Exposure assessment in the evaluation of potential Kallithea Symposium,” 1991, Bates number 2021551986/ reduced-risk tobacco products, 170 pages, 2007. 2194. Retrieved on July 27, 2010 from http://legacy.library [15] L. P. Carter, M.L. Stitzer, J. E.Henningﬁeld,R.J. O’Connor, .ucsf.edu/tid/zhe58e00. K. M. Cummings, and D. K. Hatsukam, “Review: abuse [31] I. Rubinstein and G. W. Pederson, “Bacillus species are liability assessment of tobacco products including potential present in chewing tobacco sold in the United States and reduced exposure products,” Cancer Epidemiology Biomark- evoke plasma exudation from the oral mucosa,” Clinical ers and Prevention, vol. 18, no. 12, pp. 3241–3262, 2009. Diagnostics Laboratory and Immunology, vol. 9, pp. 1057– [16] J. Beherns, “Die beziehungen der microorganisms zum 1060, 1992. tabaksbau and zur tabakferntation,” Zentrabl Bakteriol Par- [32] R. L. Barnes and S. A. Glantz, “Endotoxins in tobacco smoke: asitenk, Abt II, vol. 2, pp. 514–527, 1896. shifting tobacco industry positions,” Nicotine and Tobacco [17] Anonymous, “Tobacco and bacteria,” The London Globe, pp. Research, vol. 9, no. 10, pp. 995–1004, 2007. 7, July 21, 1899. Retrieved on November 11, 2010 from http:// [33] A. Morin, F. Samson, A. Porter, and J. Torrie, “Development query.nytimes.com/gst/abstract.html?res=FA0F14F63F5414- of an easy to-search database on the microbes associated with 728DDDA80A94DF405B8985F0D3. tobacco,” 1990 Bates number 620693477/3480. Retrieved on Nov. 8, 2010 from http://tobaccodocuments.org/rjr/ [18] H. Okino, W. C. Squires, and R. J. Reynolds, “Microbial degradation of nicotine and nicotinic acid. Part I, Isola- 522305212-5212.html. tion of nicotine decomposing bacteria and their morpho- [34] J. D. Hasday, R. Bascom, J. J. Costa, T. Fitzgerald, and logical and physiological properties,” 1954. Bates number W. Dubin, “Bacterial endotoxin is an active component of 508893294/3298. Retrieved on June 24, 2011 from http:// cigarette smoke,” Chest, vol. 115, no. 3, pp. 829–835, 1999. legacy.library.ucsf.edu/tid/xwr83d00. [35] K. S. Lane, “Method and system for assay and removal of [19] A. Wiernik, A. Christakopoulos, L. Johansson, and I. harmful toxins during processing of tobacco products,” US Wahlberg, “Eﬀect of air-curing on the chemical composition patent 6,786,221. September 7, 2004. Journal of Oncology 11 [36] L. Larsson, B. Szponar, and C. Pehrson, “Tobacco smoking [55] H. Van Der Vaart, D. S. Postma, W. Timens, and N. H. T. Ten increases dramatically air concentrations of endotoxin,” Hacken, “Acute eﬀects of cigarette smoke on inﬂammation Indoor Air, vol. 14, no. 6, pp. 421–424, 2004. and oxidative stress: a review,” Thorax, vol. 59, no. 8, pp. 713– [37] W. P. Hempling, G. H. Bokelman, and M. Shulleeta, “Method 721, 2004. [56] M. A. Birrell, S. Wong, M. C. Catley, and M. G. Belvisi, for reduction of tobacco speciﬁc nitrosamines,” US patent 6,755,200, June 29, 2004. “Impact of tobacco-smoke on key signaling pathways in the innate immune response in lung macrophages,” Journal of [38] A. P. Rooney,J. L.Swezey, D. T. Wicklow, and M.J. McAtee, Cellular Physiology, vol. 214, no. 1, pp. 27–37, 2008. “Bacterial species diversity in cigarettes linked to an inves- [57] L. Sorokin, “The impact of the extracellular matrix on in- tigation of severe pneumonitis in U.S. military personnel ﬂammation,” Nature Reviews Immunology, vol. 10, no. 10, pp. deployed in Operation Iraqi Freedom,” Current Microbiology, 712–723, 2010. vol. 51, no. 1, pp. 46–52, 2005. [58] W. Huvenne, C.A.Perez-Novo, L. Derycke et al., “Diﬀerent [39] A. Sebastian, C. Pehrson, and L. Larsson, “Elevated concen- regulation of cigarette smoke induced inﬂammation in upper trations of endotoxin in indoor air due to cigarette smoking,” versus lower airways,” Respiratory Research, vol. 11, no. 110, Journal of Environmental Monitoring, vol. 8, no. 5, pp. 519– pp. 1–9, 2010. 522, 2006. [59] G. S. Kulkarni, P. P. Nadkarni, J. M. Cerreta, S. Ma, and J. [40] M. Zhao, B. Wang, F. Li et al., “Analysis of bacterial commu- O. Cantor, “Short-term cigarette smoke exposure potentiates nities on aging ﬂue-cured tobacco leaves by 16S rDNA PCR- endotoxin-induced pulmonary inﬂammation,” Experimental DGGE technology,” Applied Microbiology and Biotechnology, Lung Research, vol. 33, no. 1, pp. 1–13, 2007. vol. 73, no. 6, pp. 1435–1440, 2007. [60] E. Doz, N. Noulin, E. Boichot et al., “Cigarette smoke- [41] L. Larsson, B. Szponar, B. Ridha et al., “Identiﬁcation of induced pulmonary inﬂammation is TLR4/MyD88 and IL- bacterial and fungal components in tobacco and tobacco 1R1/MyD88 signaling dependent,” Journal of Immunology, smoke,” Tobacco Induced Diseases, vol. 4, no. 4, 2008. vol. 180, no. 2, pp. 1169–1178, 2008. [42] J. L. Pauly, J. D. Waight, and G. M. Paszkiewicz, “Tobacco [61] M. R. Sta ¨mpﬂi and G. P. Anderson, “How cigarette smoke ﬂakes on cigarette ﬁlters grow bacteria: a potential health risk skews immune responses to promote infection, lung disease to the smoker?” Tobacco Control, vol. 17, supplement 1, pp. and cancer,” Nature Reviews Immunology,vol.9, no. 5, pp. i49–i52, 2008. 377–384, 2009. [43] J. Yang, J. Yang, Y. Duan et al., “Bacterial diversities on unaged [62] H. Mehta, K. Nazzal, and R. T. Sadikot, “Cigarette smoking and aging ﬂue-cured tobacco leaves estimated by 16S rRNA and innate immunity,” Inﬂammation Research, vol. 57, no. 11, sequence analysis,” Applied Microbiology and Biotechnology, pp. 497–503, 2008. vol. 88, pp. 553–562, 2010. [63] M. Sopori, “Eﬀects of cigarette smoke on the immune [44] L. M. Coussens and Z. Werb, “Inﬂammation and cancer,” system,” Nature Reviews Immunology, vol. 2, no. 5, pp. 372– Nature, vol. 420, no. 6917, pp. 860–867, 2002. 377, 2002. [45] O. Takeuchi and S. Akira, “Pattern recognition receptors and [64] J. Domagala-Kulawik, “Eﬀects of cigarette smoke on the lung inﬂammation,” Cell, vol. 140, no. 6, pp. 805–820, 2010. and systemic immunity,” Journal of Physiology and Pharma- [46] M. Karin, T. Lawrence, and V. Nizet, “Innate immunity gone cology, vol. 59, no. 6, pp. 19–34, 2008. awry: linking microbial infections to chronic inﬂammation [65] D. G. Yanbaeva,M.A.Dentener, E. C. Creutzberg, G. Wes- and cancer,” Cell, vol. 124, no. 4, pp. 823–835, 2006. seling, and E. F. M. Wouters, “Systemic eﬀects of smoking,” [47] C. Nathan and A. Ding, “Nonresolving inﬂammation,” Cell, Chest, vol. 131, no. 5, pp. 1557–1566, 2007. vol. 140, no. 6, pp. 871–882, 2010. [66] D. Wood, “British-American Tobacco Company,” Prelimi- [48] N. Azad, Y. Rojanasakul, and V. Vallyathan, “Inﬂammation nary observations on the possible transfer of viable micro- and lung cancer: roles of reactive oxygen/nitrogen species,” organisms to mainstream smoke, 1968, Bates number Journal of Toxicology and Environmental Health B,vol.11, no. 570343882/3901. Retrieved on June 24, 2011 from http:// 1, pp. 1–15, 2008. legacy.library.ucsf.edu/tid/jnd51f00. [49] L. Zitvogel, O. Kepp, and G. Kroemer, “Decoding cell death [67] J. Forgacs, Good Samaritan Hospital, Suﬀern, NY to Dr. signals in inﬂammation and immunity,” Cell, vol. 140, no. 6, Robert C. Hockett, Council for Tobacco Research, New pp. 798–804, 2010. York, NY, 2 pages, 2010, http://legacy.library.ucsf.edu/tid/ [50] K. E. de Visser and L. M. Coussens, “The inﬂammatory ppd2aa00. tumor microenvironment and its impact on cancer develop- [68] J. Forgacs, “Mycotoxicoses: the neglected diseases,” Feedstuﬀs, ment,” Contributions to Microbiology, vol. 13, pp. 118–137, vol. 36, no. 18, pp. 124–134, 1966. [69] B. Slutzker, G. Harmon, and P. Edmonds, “Microbiological [51] J. K. Kundu and Y. J. Surh, “Inﬂammation: gearing the jour- content of tobacco smoke,” The American Journal of the ney to cancer,” Mutation Research, vol. 659, no. 1-2, pp. 15– Medical Sciences, vol. 243, pp. 196–201, 1962. 30, 2008. [70] J. M. Greene and S. Caldwell, “Chemical and microbiological [52] E. A. Engles, “Inﬂammation in the development of lung can- changes during ﬂue curing of NK-149 tobacco,” 1989, R. J. cer: epidemiological evidence,” Expert Review of Anticancer Reynolds, Bates number 514848867/8887, retrieved on June Therapy, vol. 8, no. 4, pp. 605–615, 2008. 30, 2010 from http://legacy.library.ucsf.edu/tid/qlm03d00. [53] A. I. D’hulst, K. Y. Vermaelen, G. G. Brusselle, G. F. Joos, [71] Anonymous, Brown & Williamson, “Package 13.0 microbi- and R. A. Pauwels, “Time course of cigarette smoke-induced ology,” No date. Bates number 620648956/9146. Retrieved pulmonary inﬂammation in mice,” European Respiratory on June 28, 2010 from http://legacy.library.ucsf.edu/tid/ Journal, vol. 26, no. 2, pp. 204–213, 2005. key21f00. [54] C. Smith, T. Perfetti, and J. King, “Perspectives on pulmonary [72] Anonymous, British American Tobacco, “Master RD 888-R,” inﬂammation and lung cancer risk in cigarette smokers,” Bates number 105597011/7062. Retrieved on December 11, Inhalation Toxicology, vol. 18, no. 9, pp. 667–677, 2006. 2009 from http://legacy.library.ucsf.edu/tid/unit 37a99. 12 Journal of Oncology [73] C. W. Bacon, R. Wenger, and J. F. Bullock, “Chemical changes on October 27, 2010 from http://tobaccodocuments.org/rjr/ in tobacco during ﬂu-curing,” Industrial & Engineering 500937365-7489.html. Chemistry, vol. 44, no. 2, pp. 292–296, 1952. [90] V. Subbiah, “Sheet2. Tobacco sampling for microﬂora [74] C. O. Jensen, “Uber die natur der tabakfermentation,” Zen- counting,” 1995. Bates number 525450330/0335. Retrieved trabl Bakteriol Parasitenk, Abt II, vol. 21, pp. 469–483, 1908. on October 22, 2010 from http://legacy.library.ucsf.edu/tid/ [75] W. P. Hempling and P. Morris, “Fundamental tobacco micro- ymt60d00. biology,” 1987. Bates number 2022226783/6795. Retrieved [91] W. C. Flanders,R.J.Reynolds, “Quantitative studies of the on March 24, 2010 from http://legacy.library.ucsf.edu/tid/ microbiological ﬂora of tobacco during aging,” 1955. Bates jst58e00. number 501663388/3456. Retrieved on July 1, 2010 from [76] K. J. Brotzge and Brown and Williamson Tobacco Company, http://legacy.library.ucsf.edu/tid/wuk39d00. Quantities of microﬂora recovered from Brown & William- [92] P. C. Stauber, “Investigation of mould growth on stored leaf,” son & competitive cigarette brands, Fall/Winter, 1982, 1983. 1975. Bates number 105425004/5072. Retrieved on June 24, Bates number 598000442/0451. Retrieved on March 16, 2010 2011 from http://legacy.library.ucsf.edu/tid/oit57a99. from http://tobaccodocuments.org/bw/971381.html. [93] R. E. Welty and L. A. Nelson, “Growth of aspergillus repens [77] K. J. Brotzge and Brown & Williamson Tobacco Company, in ﬂue-cured tobacco,” Applied Microbiology, vol. 21, no. 5, “Quantities of microﬂora recovered from Brown & William- pp. 854–859, 1971. son and competitive brands, Spring/Summer 830000,” 1983, [94] T. G. Mitchell, D. A. Johnson, and British-American Tobacco Bates number 657017733/7752. Retrieved on March 16, 2010 Company, “Identiﬁcation of fungi of the Aspergillus ﬂavour from http://legacy.library.ucsf.edu/tid/hnl13f00. group from tobacco,” Report RD 1279. 1975. Bates number [78] L. J. Dewey and G. M. Broaddus, “Bacterial, mold and yeast 105598328/8619. Retrieved on July 1, 2010 from http://li- population counts on RCFS and on RC picked from Pall Mall brary/ucsf/edu/tid/pnp57a99. and Winston cigarettes,” 1970, American Tobacco Company, [95] G. M. Myers, “Aﬂatoxin on tobacco and its removal,” R. J. Bates number 950107079/7080, Retrieved on March 16, 2010 Reynolds, Bates number 519972600/2620. Retrieved on June from http://legacy.library.ucsf.edu/tid/jml11a00. 29, 2010 from http://legacy.library.ucsf.edu/tid/yjk90d00. [79] J. Hill and Brown & Williamson, “Microﬂora standards of [96] J. M. Greene and S. Caldwell, “Chemical and microbiological cocoa casing materials,” 1985. Bates number 62018442/4422. changes during ﬂue curing of NK-149 tobacco,” 1989, R. J. Retrieved on March 17, 2010 from http://legacy.library.ucsf Reynolds, Bates number514848867/8887, Retrieved on June .edu/tid/ski31f00. 30, 2010 from http://legacy.library.ucsf.edu/tid/qlm03d00. [80] M. I. Hofer and Philip Morris, “Research and development— [97] D. G. Vickroy and R. E. Welty, “Evaluations of cigarettes quarterly Report Microbiology 851000/1200,” 1985, Bates made with mold-damaged and nondamaged ﬂue-cured to- bacco,” Beitrag ¨ e zur Tabakforschung, vol. 8, pp. 102–106, number 2028639252/9269. Retrieved on November 8, 2010 from http://legacy.library.ucsf.edu/tid/rew56e00. 1975. [81] Philip Morris, “Biocontrol of tobacco microﬂora,” 1989, [98] M. R. Tansey, “Isolation of thermophilic fungi from snuﬀ,” Bates number 2029139024/9050, retrieved on June 30, 2010 Journal of Applied Microbiology, vol. 29, no. 1, pp. 128–129, from http://legacy.library.ucsf.edu/tid/hoy69e00. 1975. [82] R. E. Welty and American Tobacco, “Plant pathology, 5225, [99] T. Thomas, J. Brandon, W. A. Bailey, and T. A. Kosty, “Me- microﬂora of ﬂue-cured tobacco and their aﬀect on quality,” thod for reducing nitrosamines in tobacco,” US patent 7,757, 1970, Bates number 950251672/1675. Retrieved on June 30, 697. July 20, 2010. 2010 from http://legacy.library.ucsf.edu/tid/ufv31A00. [100] A. Lukic, R. E. Welty, and G. B. Lucas, “Antifungal spectra [83] Anonymous and Philip Morris, “Most populous bacteria: of actinomycetes isolated from tobacco,” Antimicrobial Agents burley tobacco research,” 1999, Bates number 2082730005. and Chemotherapy, vol. 1, no. 4, pp. 363–366, 1972. Retrieved on March 19, 2010 from http://legacy.library.ucsf [101] K. Koga, S. Katsuya, and Japan Tobacco Company, “Method .edu/tid/ddq55c00. of reducing nitrosamines content in tobacco leaves,” US ptent [84] Anonymous, “Further examination of coliform bacteria from 7,556,046. July 7, 2009. cigarettes,” Laboratory report L.337-R. 1970, Bates number [102] M. Cui, M. T. Nielsen, R. R. Hart III, M. L. Overbey, D. 650018029/8046. Retrieved on June 24, 2010 from http:// J. Watson, and J. R. Chipley, “Use of chlorate, sulfur or legacy.library.ucsf.edu/tid/rkl66b00. ozone to reduce tobacco speciﬁc nitrosamine,” U.S. patent 2006/019516 A1. Sept 7, 2006. [85] M. Di Giacomo, M. Paolino, D. Silvestro et al., “Microbial community structure and dynamics of dark ﬁre-cured tobac- [103] R. G. Warke, A. S. Kamat, and M. Y. Kamat, “Irradiation of co fermentation,” Applied and Environmental Microbiology, chewable tobacco mixes for improvement in microbiological vol. 73, no. 3, pp. 825–837, 2007. quality,” Journal of Food Protection, vol. 62, no. 6, pp. 678– [86] T. G. Mitchell and British American Tobacco (BAT), 681, 1999. “Changes in the microﬂora of tobacco leaves during ﬁeld [104] T. G. Mitchell and C. R. Jenkins, “Alternative treatments for growth in England,” 1989, Bates number 400047269/7282, mould control on pipe tobacco,” British American Tobacco, retrieved on June 28, 2010 from http://legacy.library.ucsf Bates number 400661432/1433. Retrieved on June 24, 2011 .edu/tid/num81a99. from http://legacy.library.ucsf.edu/tid/rir56a99. [87] S. A. Ghabrial, “Studies on the microﬂora of air-cured burly [105] D. S. Roth, W. H. Cowart Jr., C. B. Jenkins Jr., and D. M. tobacco,” Tobacco Science, vol. 20, pp. 80–82, 1976. Boyle, “Sterilization process in the manufacturing of snuﬀ,” U.S. patent 5,372,149. December 13, 1994. [88] British American Tobacco, “Film Box Number -1, L1R to L151 R, R&D 1838,” Bates number 402185400/5586, [106] V. Subbiah, “Method of inhibiting mycotoxin production,” US Patent 5,698,599. Dec 16, 1997. Retrieved on June 24, 2010 from http://legacy.library.ucsf .edu/tid/fud91a99. [107] R. P. Newton and Brown & Williamson, “Microbiological [89] W. C. Squires, L. E. Hayes, and R. J. Reynolds, “Tobacco ﬂora: examination of cigarettes,” 1968. Retrieved on June 28, 2010 quantitative studies,” November 9, 1961. 125 pages/Retrieved from http://legacy.library.ucsf.edu/tid/vgj94a99. Journal of Oncology 13 [108] V. C. Johnson, A. M. Palmer, and P. Morris, “Bacteria on cigarette ﬁlters,” Bates number 2000759148. February 12, 1968. Retrieved on November 9, 2010 from http://legacy .library.ucsf.edu/tid/aiw48e00. [109] T. G. Mitchell and British-American Tobacco Company, “Examination of mould—Aﬀected cigarettes from China,” 1989, Bates number 400910779/7783. Retrieved on June 24, 2010 from Legacy at http://legacy.library.ucsf.edu/tid/ qvu0499. [110] J. Hill and Brown & Williamson, “Microbial examination of pipe and smokeless tobacco/541,” 1985, Bates number 620184560/4571. Retrieved on July 22, 2010 from http://leg- acy.library.ucsf.edu/tid/rjq20f00. [111] K. Brotzge and Brown & Williamson, “Microbial exam- ination of pipe, snuﬀ, & chewing tobacco products— Fall/Winter, 83000,” 1984, Bates number 598002147/2156. Retrieved on June 24, 2011 from http://legacy.library.ucsf .edu/tid/zcj41f00. [112] S. K. Varma, A. B. Roy, and A. K. Jha, “Ecotoxicological aspects of Aspergilli present in the phylloplane of shored leaves of chewing tobacco (Nicotiana tobacum),” Myco- pathologia, vol. 113, pp. 19–23, 1991. [113] A. R. Sapkota, S. Berger, and T. M. Vogel, “Human pathogens abundant in the bacterial metagenome of cigarettes,” Envi- ronmental Health Perspectives, vol. 118, no. 3, pp. 351–356, [114] J. Papavassiliou, G. Piperakis, and U. Marcelou-Kinti, “Myco- logical ﬂora of cigarettes,” Mycopathology Mycology Applied, vol. 44, no. 2, pp. 117–120, 1971. [115] V. P. Kurup, A. Resnick, S. L. Kagen, S. H. Cohen, and J. N. Fink, “Allergenic fungi and actinomycetes in smoking materials and their health implications,” Mycopathologica, vol. 82, no. 1, pp. 61–64, 1983. [116] P. E. Verweij, J. J. Kerremans, A. Voss, and J. F. G. M. Meis, “Fungal contamination of tobacco and marijuana,” Journal of the American Medical Association, vol. 284, no. 22, p. 2875, [117] N. B. Rainer, “Cigarettes having minimized loose ends and process for preparing same,” US patent 4,715,388, Dec 29, [118] British American Tobacco Company, “Discussion group on ends quality,” 1985, 161 pages, http://tobaccodocuments.org/ batco/109979765-9924.html. [119] L. Deyton, J. Sharfstein, and M. Hamburg, “Tobacco product regulation—a public health approach,” The New England Journal of Medicine, vol. 362, no. 19, pp. 1753–1756, 2010. 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References (133)

M. Stämpfli, G. Anderson (2009)
How cigarette smoke skews immune responses to promote infection, lung disease and cancer
Nature Reviews Immunology, 9
R. Welty, D. Vickroy (1975)
Evaluations of Cigarettes Made with Mold-Damaged and Nondamaged Flue-cured Tobacco
Beiträge zur Tabakforschung / Contributions to Tobacco Research, 8
Tobacco sampling for microflora counting
Carr Smith, T. Perfetti, J. King (2006)
Perspectives on Pulmonary Inflammation and Lung Cancer Risk in Cigarette Smokers
Inhalation Toxicology, 18
S. Varma, R. Verma, A. Jha (2004)
Ecotoxicological aspects of Aspergilli present in the phylloplane of stored leaves of chewing tobacco (Nicotiana tobaccum)
Mycopathologia, 113
(1987)
Fundamental tobacco microbiology
(1985)
Microflora standards of cocoa casing materials
D. Burns, C. Anderson, N. Gray (2010)
Do changes in cigarette design influence the rise in adenocarcinoma of the lung?
Cancer Causes & Control, 22
(1997)
Method of inhibiting mycotoxin production
Mingqin Zhao, Baoxiang Wang, Fuxin Li, L. Qiu, Fangfang Li, Shumin Wang, J. Cui (2007)
Analysis of bacterial communities on aging flue-cured tobacco leaves by 16S rDNA PCR–DGGE technology
Applied Microbiology and Biotechnology, 73
(2010)
Film Box Number -1, L1R to L151 R, R&D 1838 Bates number 402185400
British American Tobacco
Film Box Number -1, L1R to L151 R, R&D 1838,
T. G. Mitchell and P. C. Stauber
Methods for the microbiological examination of tobacco and tobacco products, Report Number 888—R,
I. Neiman (1974)
[Inflammation and cancer].
Patologicheskaia fiziologiia i eksperimental'naia terapiia, 0 4
(2004)
Method and system for assay and removal of harmful toxins during processing of tobacco products
(1966)
Mycotoxicoses: the neglected diseases
(1955)
Effect of air-curing on the chemical composition of tobacco
V. Kurup, V. Kurup, Abraham Resnick, Abraham Resnick, Steven Kagen, Steven Kagen, Steven Cohen, Steven Cohen, Jordan Fink, Jordan Fink (1983)
Allergenic fungi and actinomycetes in smoking materials and their health implications
Mycopathologia, 82
Anonymous (1899)
Tobacco and bacteria,
V. Subbiah
Sheet2. Tobacco sampling for microflora counting,
Hidemi Ito, K. Matsuo, Hideo Tanaka, D. Koestler, H. Ombao, J. Fulton, A. Shibata, Manabu Fujita, H. Sugiyama, M. Soda, T. Sobue, V. Mor (2011)
Nonfilter and filter cigarette consumption and the incidence of lung cancer by histological type in Japan and the United States: Analysis of 30‐year data from population‐based cancer registries
International Journal of Cancer, 128
Anka Lukic, R. Welty, G. Lucas (1972)
Antifungal Spectra of Actinomycetes Isolated from Tobacco
Antimicrobial Agents and Chemotherapy, 1
(1989)
Examination of mould—Affected cigarettes from China
N. Azad, Y. Rojanasakul, V. Vallyathan (2008)
Inflammation and Lung Cancer: Roles of Reactive Oxygen/Nitrogen Species
Journal of Toxicology and Environmental Health, Part B, 11
C. Bacon, R. Wenger, J. Bullock (1952)
Biochemical Changes in Tobacco During Flue Curing
Industrial & Engineering Chemistry, 44
Jingwen Huang, Jinkui Yang, Y. Duan, W. Gu, Xiaowei Gong, Wei Zhe, Can Su, Ke-Qin Zhang (2010)
Bacterial diversities on unaged and aging flue-cured tobacco leaves estimated by 16S rRNA sequence analysis
Applied Microbiology and Biotechnology, 88
Brown & Williamson Anonymous
Package 13.0 microbiology,
(1999)
Most populous bacteria: burley tobacco research
(1968)
British-American Tobacco Company Preliminary observations on the possible transfer of viable microorganisms to mainstream smoke
(1989)
Chemical and microbiological changes during flue curing of NK149 tobacco
(1972)
Bates number 107466852/6877
A. Sebastian, C. Pehrson, L. Larsson (2006)
Elevated concentrations of endotoxin in indoor air due to cigarette smoking.
Journal of environmental monitoring : JEM, 8 5
Emilie Doz, N. Noulin, E. Boichot, I. Guénon, L. Fick, M. Bert, V. Lagente, B. Ryffel, B. Schnyder, V. Quesniaux, I. Couillin (2008)
Cigarette Smoke-Induced Pulmonary Inflammation Is TLR4/MyD88 and IL-1R1/MyD88 Signaling Dependent1
The Journal of Immunology, 180
Anonymous
Further examination of coliform bacteria from cigarettes,
(2010)
How tobacco smoke causes disease. The biology and behavior basis for smokingattributable disease, " U.S. Department of Health and Human Services
(2010)
Further examination of coliform bacteria from cigarettes Laboratory report L.337-R. 1970, Bates number 650018029/8046
Lennart Larsson, B. Szponar, C. Pehrson (2004)
Tobacco smoking increases dramatically air concentrations of endotoxin.
Indoor air, 14 6
(1972)
Microbiological examination of tobacco products: report Number RD 969-R
(1970)
Bacterial, mold and yeast population counts on RCFS and on RC picked from Pall Mall and Winston cigarettes
(1896)
Die beziehungen der microorganisms zum tabaksbau and zur tabakferntation
M. Karin, T. Lawrence, V. Nizet (2006)
Innate Immunity Gone Awry: Linking Microbial Infections to Chronic Inflammation and Cancer
Cell, 124
J. Hill and Brown & Williamson
Microbial examination of pipe and smokeless tobacco/541,
R. R. Baker (2003)
Smoke chemistry,
TOBACCO: Production, Chemistry and Technology
G. H. Bokelman W. P. Hempling
Method for reduction of tobacco specific nitrosamines,
K. J. Brotzge and Brown & Williamson Tobacco Company
Quantities of microflora recovered from Brown & Williamson and competitive brands, Spring/Summer 830000,
(2000)
Bacteria on cigarette filters
(2010)
Package 13.0 microbiology No date. Bates number 620648956/9146
L. Zitvogel, O. Kepp, G. Kroemer (2010)
Decoding Cell Death Signals in Inflammation and Immunity
Cell, 140
K. Visser, L. Coussens (2006)
The inflammatory tumor microenvironment and its impact on cancer development.
Contributions to microbiology, 13
I. Rubinstein, G. Pedersen (2002)
Bacillus Species Are Present in Chewing Tobacco Sold in the United States and Evoke Plasma Exudation from the Oral Mucosa
Clinical and Vaccine Immunology, 9
(1976)
Studies on the microflora of air-cured burly tobacco
J. Papavassiliou, G. Piperakis, U. Marcelou-Kinti (1971)
Mycological flora of cigarettes
Mycopathologia et mycologia applicata, 44
(2010)
http://legacy.library.ucsf.edu/tid/ ppd2aa00
R. Welty, L. Nelson (1971)
Growth of Aspergillus repens in Flue-Cured Tobacco.
Applied microbiology, 21 5
(1985)
Research and development— quarterly Report Microbiology 851000/1200
A. Rodgman, T. Perfetti (2008)
The Chemical Components of Tobacco and Tobacco Smoke
(2009)
Method of reducing nitrosamines content in tobacco leaves
(1989)
Changes in the microflora of tobacco leaves during field growth in England
J. Kundu, Y. Surh (2008)
Inflammation: gearing the journey to cancer.
Mutation research, 659 1-2
(2011)
Alternative treatments for mould control on pipe tobacco
(2006)
Use of chlorate, sulfur or ozone to reduce tobacco specific nitrosamine
P. Verweij, J. Kerremans, A. Voss, J. Meis (2000)
Fungal contamination of tobacco and marijuana.
JAMA, 284 22
(1982)
Commonly added ingredients
J. Forgacs, W. Carll (1966)
Mycotoxicoses: Toxic Fungi in Tobaccos
Science, 152
G. Wayne, G. Connolly (2009)
Regulatory assessment of brand changes in the commercial tobacco product market
Tobacco Control, 18
O. Takeuchi, S. Akira (2010)
Pattern Recognition Receptors and Inflammation
Cell, 140
(1991)
CORESTA—Papers presented at the Kallithea Symposium
H. Mehta, K. Nazzal, R. Sadikot (2008)
Cigarette smoking and innate immunity
Inflammation Research, 57
W. Huvenne, C. Pérez-Novo, L. Derycke, N. Ruyck, O. Krysko, T. Maes, N. Pauwels, Lander Robays, K. Bracke, G. Joos, G. Brusselle, C. Bachert (2010)
Different regulation of cigarette smoke induced inflammation in upper versus lower airways
Respiratory Research, 11
J. Pauly, J. Waight, G. Paszkiewicz (2008)
Tobacco flakes on cigarette filters grow bacteria: a potential health risk to the smoker?
Tobacco Control, 17
J. Holbrook (1981)
The changing cigarette.
The Western journal of medicine, 134 4
(1984)
Microbial examination of pipe, snuff, & chewing tobacco products— Fall/Winter, 83000
(1975)
Investigation of mould growth on stored leaf
M. Sopori (2002)
Effects of cigarette smoke on the immune system
Nature Reviews Immunology, 2
(1908)
Uber die natur der tabakfermentation
(1975)
Identification of fungi of the Aspergillus flavour group from tobacco
C. Nathan, A. Ding (2010)
Nonresolving Inflammation
Cell, 140
D. Hoffmann, I. Hoffmann (1997)
The changing cigarette, 1950-1995.
Journal of toxicology and environmental health, 50 4
S. Hecht (2006)
Cigarette smoking: cancer risks, carcinogens, and mechanisms
Langenbeck's Archives of Surgery, 391
Amy Sapkota, S. Berger, T. Vogel (2009)
Human Pathogens Abundant in the Bacterial Metagenome of Cigarettes
Environmental Health Perspectives, 118
Kent Sahlin, Eva-karin Sällstedt, David Bishop, M. Tonkonogi (2008)
Turning down lipid oxidation during heavy exercise--what is the mechanism?
Journal of physiology and pharmacology : an official journal of the Polish Physiological Society, 59 Suppl 7
A. Rooney, J. Swezey, D. Wicklow, M. McAtee (2005)
Bacterial Species Diversity in Cigarettes Linked to an Investigation of Severe Pneumonitis in U.S. Military Personnel Deployed in Operation Iraqi Freedom
Current Microbiology, 51
R. Warke, Anu Kamat, M. Kamat (1999)
Irradiation of chewable tobacco mixes for improvement in microbiological quality.
Journal of food protection, 62 6
(1899)
Tobacco and bacteria The London Globe
R. Malone (2010)
China's chances, China's choices in global tobacco control
Tobacco Control, 19
I. Rubinstein and G. W. Pederson (1992)
Bacillus species are present in chewing tobacco sold in the United States and evoke plasma exudation from the oral mucosa,
Clinical Diagnostics Laboratory and Immunology, 9
M. A. Dentener D. G. Yanbaeva (2007)
Systemic effects of smoking,
Chest, 131
Michele Giacomo, Marianna Paolino, D. Silvestro, G. Vigliotta, F. Imperi, P. Visca, P. Alifano, D. Parente (2006)
Microbial Community Structure and Dynamics of Dark Fire-Cured Tobacco Fermentation
Applied and Environmental Microbiology, 73
R. Read (1984)
[Systemic effects of smoking].
Nihon Kokyuki Gakkai zasshi = the journal of the Japanese Respiratory Society, Suppl
D. Wood
British-American Tobacco Company,
L. Sorokin (2010)
The impact of the extracellular matrix on inflammation
Nature Reviews Immunology, 10
R. O’Connor, K. Cummings, V. Rees, G. Connolly, Kaila Norton, D. Sweanor, M. Parascandola, D. Hatsukami, P. Shields (2009)
Surveillance Methods for Identifying, Characterizing, and Monitoring Tobacco Products: Potential Reduced Exposure Products as an Example
Cancer Epidemiology, Biomarkers & Prevention, 18
H. Dygert (1957)
Snuff-a source of pathogenic bacteria in chronic bronchitis.
The New England journal of medicine, 257 7
Girish Kulkarni, P. Nadkarni, J. Cerreta, Shuren Ma, J. Cantor (2007)
SHORT-TERM CIGARETTE SMOKE EXPOSURE POTENTIATES ENDOTOXIN-INDUCED PULMONARY INFLAMMATION
Experimental Lung Research, 33
(1967)
A preliminary study of the biological activity in cigarette smoke
M. Borgerding, H. Klus (2005)
Analysis of complex mixtures--cigarette smoke.
Experimental and toxicologic pathology : official journal of the Gesellschaft fur Toxikologische Pathologie, 57 Suppl 1
L. Deyton, J. Sharfstein, M. Hamburg (2010)
Tobacco product regulation--a public health approach.
The New England journal of medicine, 362 19
(1990)
Development of an easy to-search database on the microbes associated with tobacco
M. Cooke (2010)
The Chemical Components of Tobacco and Tobacco Smoke
Chromatographia, 71
British American Tobacco Anonymous
Master RD 888-R,
(1989)
Biocontrol of tobacco microflora
Microbiology of Henri Wintermans cigar production onsite studies of the primary process at Eersel: report No
M. Rabinoff, N. Caskey, Anthony Rissling, Candice Park (2007)
Pharmacological and chemical effects of cigarette additives.
American journal of public health, 97 11
L. Carter, M. Stitzer, J. Henningfield, R. O’Connor, K. Cummings, D. Hatsukami (2009)
Abuse Liability Assessment of Tobacco Products Including Potential Reduced Exposure Products
Cancer Epidemiology, Biomarkers & Prevention, 18
B. Slutzker, G. Harmon, P. Edmonds (1962)
MICROBIOLOGICAL CONTENT OF TOBACCO SMOKE
The American Journal of the Medical Sciences, 243
J. Hasday, R. Bascom, Joseph Costa, T. Fitzgerald, Wendy Dubin (1999)
Bacterial endotoxin is an active component of cigarette smoke.
Chest, 115 3
(1960)
Examination of whole cigarette smoke by light and electron microscopy
Lennart Larsson, B. Szponar, B. Ridha, C. Pehrson, Jacek Dutkiewicz, E. Krysińska-Traczyk, J. Sitkowska (2008)
Identification of bacterial and fungal components in tobacco and tobacco smoke
Tobacco Induced Diseases, 4
D. Hatsukami, K. Perkins, M. Lesage, D. Ashley, J. Henningfield, N. Benowitz, C. Backinger, M. Zeller (2010)
Nicotine reduction revisited: science and future directions
Tobacco Control, 19
H. Vaart, D. Postma, W. Timens, N. Hacken (2004)
Acute effects of cigarette smoke on inflammation and oxidative stress: a review
Thorax, 59
Richard Barnes, S. Glantz (2007)
Endotoxins in tobacco smoke: shifting tobacco industry positions.
Nicotine & tobacco research : official journal of the Society for Research on Nicotine and Tobacco, 9 10
(1985)
Discussion group on ends quality
T. Tabuchi (1955)
Microbial Degradation of Nicotine and Nicotinic Acid
Bulletin of the Agricultural Chemical Society of Japan, 29
A. Velosa, W. Teodoro, Daniel Anjos, R. Konno, C. Oliveira, M. Katayama, E. Parra, V. Capelozzi, N. Yoshinari (2010)
Collagen V-induced nasal tolerance downregulates pulmonary collagen mRNA gene and TGF-beta expression in experimental systemic sclerosis
Respiratory Research, 11
(1982)
Quantities of microflora recovered from Brown & Williamson & competitive cigarette brands
Ai D'hulst, Ky Vermaelen, G. Brusselle, G. Joos, R. Pauwels (2005)
Time course of cigarette smoke-induced pulmonary inflammation in mice
European Respiratory Journal, 26
J. Brandon T. Thomas
Method for reducing nitrosamines in tobacco,
R. Welty (1972)
Fungi isolated from flue-cured tobacco sold in Southeast United States, 1968-1970.
Applied microbiology, 24 3
(1970)
Plant pathology, 5225, microflora of flue-cured tobacco and their affect on quality
R. P. Newton and Brown & Williamson
Microbiological examination of cigarettes,
M. Birrell, Sissie Wong, M. Catley, M. Belvisi (2008)
Impact of tobacco‐smoke on key signaling pathways in the innate immune response in lung macrophages
Journal of Cellular Physiology, 214
E. Engels (2008)
Inflammation in the development of lung cancer: epidemiological evidence
Expert Review of Anticancer Therapy, 8
Methods for the microbiological examination of tobacco and tobacco products
A Report of the Surgeon General
How tobacco smoke causes disease. The biology and behavior basis for smoking-attributable disease,
(1997)
Aflatoxin on tobacco and its removal
(1994)
Sterilization process in the manufacturing of snuff
J. Domagała-Kulawik (2008)
Effects of cigarette smoke on the lung and systemic immunity.
Journal of physiology and pharmacology : an official journal of the Polish Physiological Society, 59 Suppl 6
M. Tansey (1975)
Isolation of thermophilic fungi from snuff.
Applied microbiology, 29 1
(1987)
Cigarettes having minimized loose ends and process for preparing same
(2009)
Master RD 888-R Bates number 105597011/7062
W. C. Squires H. Okino
Microbial degradation of nicotine and nicotinic acid. Part I, Isolation of nicotine decomposing bacteria and their morphological and physiological properties,
(1961)
Tobacco flora: quantitative studies
(1955)
Quantitative studies of the microbiological flora of tobacco during aging

Publisher: Hindawi Publishing Corporation
Copyright: Copyright © 2011 John L. Pauly and Geraldine Paszkiewicz. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ISSN: 1687-8450
eISSN: 1687-8469
DOI: 10.1155/2011/819129
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Abstract

Hindawi Publishing Corporation Journal of Oncology Volume 2011, Article ID 819129, 13 pages doi:10.1155/2011/819129 Review Article Cigarette Smoke, Bacteria, Mold, Microbial Toxins, and Chronic Lung Inﬂammation John L. Pauly and Geraldine Paszkiewicz Department of Immunology, Roswell Park Cancer Institute, Elm and Carlton Streets, Buﬀalo, NY 14263, USA Correspondence should be addressed to John L. Pauly, john.pauly@roswellpark.org Received 16 November 2010; Revised 28 February 2011; Accepted 20 March 2011 Academic Editor: Venkateshwar Keshamouni Copyright © 2011 J. L. Pauly and G. Paszkiewicz. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chronic inﬂammation associated with cigarette smoke fosters malignant transformation and tumor cell proliferation and promotes certain nonneoplastic pulmonary diseases. The question arises as to whether chronic inﬂammation and/or colonization of the airway can be attributed, at least in part, to tobacco-associated microbes (bacteria, fungi, and spores) and/or microbial toxins (endotoxins and mycotoxins) in tobacco. To address this question, a literature search of documents in various databases was performed. The databases included PubMed, Legacy Tobacco Documents Library, and US Patents. This investigation documents that tobacco companies have identiﬁed and quantiﬁed bacteria, fungi, and microbial toxins at harvest, throughout fermentation, and during storage. Also characterized was the microbial ﬂora of diverse smoking and smokeless tobacco articles. Evidence- based health concerns expressed in investigations of microbes and microbial toxins in cigarettes, cigarette smoke, and smokeless tobacco products are reasonable; they warrant review by regulatory authorities and, if necessary, additional investigation to address scientiﬁc gaps. 1. Introduction: Chemical and Biological of references is included [1–4]. Most of the chemicals, toxicants, and carcinogens in tobacco smoke arise from the Components of Tobacco and Smoke burning (pyrolysis) of the tobacco [1, 2, 4]. The potential for For many years, scientists have undertaken studies to deﬁne harm has also been studied for chemicals that do not arise from the burning of tobacco. The chemicals include metallic the chemical composition of green tobacco leaf, cured- fermented-stored tobacco leaf, and tobacco smoke with the and nonmetallic elements, isotopes, and salts [1, 2, 4]. In intent of identifying chemicals that may pose a signiﬁcant addition, pesticides and other intact agrochemicals have been health risk [1–4]. An illustration has been prepared of the identiﬁed in tobacco smoke [1, 2, 4]. Also included in this annual increase, from 1954 to 2005, in the total number tabulation of chemicals in smoke are menthol and ﬂavorants of tobacco smoke chemicals that have been identiﬁed [4]. [4]. Today, there is a consensus of opinion that cigarette smoke In 1985, Hoﬀmann and coworkers, who had studied the consists of at least 5,300 diﬀerent chemicals [4]. These chemical composition of tobacco smoke for many years, be- chemicals are present in the complex aerosol that consists of gan formulating a list of chemicals that were designated as a heterogeneous mixture of gas- (vapor-) phase and particu- biologically active, carcinogenic, cocarcinogenic, or tumor- late- (“tar-”) phase components [1–4]. genic, reviewed previously in [4]. The tabulation was revised Detailed listings of the chemicals in mainstream and side- and became the basis for the list of “Hoﬀmann Analytes” stream tobacco smoke are available, and an assessment of [4]. In 1985, diﬀerent working groups met to identify those their propensity for harm has been presented; a partial listing chemicals in tobacco smoke that are most likely to be 2 Journal of Oncology carcinogenic to humans as deﬁned by criteria of the In- 3. Tobacco and Harm Associated with Microbes ternational Association for Research on Cancer (IARC), an Our review of the aforementioned writings [1–4]and many intergovernmental agency forming part of the World Health other related reports, addressing chemicals in tobacco smoke Organization, and by the US National Toxicology Program of cigarettes have shown that the writings do not address (NTP) [1, 2, 4]. the propensity for harm that may be associated with micro- bial elements of smokeless and smoking tobacco articles. A partial listing of tobacco-associated microbial elements 2. The Changing Cigarette include bacteria (Gram positive and Gram negative), bacte- rial spores, fungi (yeast and mold), fungal spores, cell wall The identiﬁcation, classiﬁcation, and concentration of the components (certain glucans and ﬂagellum), and diverse various chemicals in cigarette smoke have been challenged by microbial toxins that include exotoxins and endotoxins. changes in the design of cigarettes. A comprehensive review Examples of bacterial-derived toxins include endotoxins of “The Changing Cigarette” was published by D. Hoﬀmann (lipopolysaccharide, LPS; inﬂammatory factor) and fungal- and I. Hoﬀmann in 1997 [5]. derived mycotoxins (aﬂatoxins, AF type B1; human carcino- Subsequently, other investigators addressed changes in gen) [1–4]. cigarettes and their potential for risk [6–12]. By way of There exists today a concern of the potential health risks example, a partial tabulation of changes in cigarette includes associated with diverse microbial elements that are known (a) increased cigarette length (85 mm king sized and extra to exist in smoking and smokeless tobacco products that long “120’s”) and, for some brands, reduced circumference are currently being marketed. This subject has not been (23 mm “slim” cigarettes), (b) variation in the blend of nat- addressed in the context of national tobacco control policy ural tobaccos of diverse types, country of origin, and curing or regulatory authorities. processes, relative percent tobacco leaf (lamina) versus tobac- Harm is to be recognized as persistent or chronic inﬂam- co ribs/stems, and tobacco weight per rod, (c) incorporation mation. Inﬂammation is mediated by diﬀerent leukocyte of manmade tobacco, sometimes referred to as reconstituted subsets and diﬀerent secreted factors (Figure 1). Inﬂamma- or “sheet” tobacco, (d) introduction of additives to the to- tion not only establishes a microenvironment that fosters bacco (casings) that include diverse ﬂavorings (licorice and the malignant transformation and tumor growth but also honey), humectants to retain tobacco moisture, and menthol promotes microbial colonization. to ameliorate smoke irritation and promote smoking accep- tance by youngsters and “starters” (e) addition of ammonia, 4. Research Objectives to facilitate “freebasing” the nicotine to enhance the pharma- cological eﬀect (impact), (f) application of diverse glues and The goal of this paper is to proﬁle the scientiﬁc and medical printing ink, (g) conﬁguration of diverse cigarette ﬁlter mate- literature addressing microbes in tobacco with the intent to rials (cellulose acetate, paper, or combination of both), (h) determine whether there is suﬃcient evidence to warrant alteration of ﬁlters with charcoal and schemes whether the additional investigations to assess propensity for human carbon was dispersed throughout the ﬁlter plug or retained harm. The impetus for undertaking this work was derived in a ﬁlter cavity, (i) variation in ﬁlter design (ﬁlter length, in part from the fact that several teams of investigators, ﬁber packing/crimping, ﬁber density, and ﬁlter ventilation) including our own, have published observations during the to eﬀect tar delivery (full ﬂavor cigarettes versus ultralight last few years that suggest microbial elements maybe harmful low-tar cigarettes), (j) paper type, paper porosity, with burn to tobacco users. accelerators to promote burning, or with modiﬁcations to Notable in a ﬁrst analysis of the literature on the micro- reduce the propensity for sustained burning and aﬀect a “ﬁre biology of tobacco we discovered that there were few recent safe” designation, and (k) diverse methodologies to reduce reports (1990 to 2010) in peer-reviewed, mainstream, scien- “tar” and nicotine yields in mainstream smoke of cigarettes tiﬁc and medical journals by scientists of tobacco companies. that have been smoked mechanically [6–12]. By way of example, Philip Morris has contracted the Life The topic of “The Changing Cigarette” has been addres- Science Research Oﬃce, Inc., (LSRO, Bethesda, MD), to sed and summarized in a recent report of the Surgeon Gen- identify methods to evaluate tobacco products and with a eral entitled “How Tobacco Smoke Causes Disease” [13]. particular focus on identifying research schemes and assays A review of the scientiﬁc and medical literature has shown for assessing reduced-risk tobacco articles [14]. Three mono- that (a) changing cigarette designs over the last ﬁve decades, graphs published by LSRO in 2007 detailed the chemicals including the introduction of cigarette ﬁlters and low-tar to be assayed and recommended procedures. The subject cigarettes, have not reduced overall disease risk among smok- of microbial ﬂora and microbial toxins was not addressed, ers and may have hindered prevention and cessation eﬀorts, nor were schemes and methodologies for the assessment of (b) there is insuﬃcient evidence that novel tobacco products tobacco associated bacteria, mold, or microbial toxins [14]. reduce individual and population health risks, and (c) the Therefore, the question arose as to whether the issue of introduction of novel tobacco products that are marketed as health risks associated with microbial elements in smokeless reduced-risk cigarettes may encourage tobacco use among and smoking tobacco was not investigated by laboratory youngsters. These changes have challenged tobacco policy scientists working at the tobacco companies or whether the and regulation [13]. subject was studied and the information withheld as private Journal of Oncology 3 inﬂammation. It is acknowledged that immunological re- sponses and inﬂammation would not be a primary inter- est by other investigators whose primary interests are in the disciplines of microbiology/metagenomics, aerosol- “Tar” associated inhalation toxicology, infectious diseases, and clinical pathology (oral and lung). Also, the work presented herein is limited in scope. The authors retrieved numerous TLR documents from databases, but space restrictions permit cit- ing but a few of the writings. Also, many of the writings were MΦ internal documents and were not subjected to peer-review. Some documents cited are old and are addressed herein to provide a historical perspective. Lastly, the documents are TNFα IL-1β LIF OSM IL-4 fragmented and it is recognized that conﬂicting ﬁndings and interpretations may be presented by competing tobacco Type I companies. DC cell Type II Tcell cell 6. Literature Search A computer-based structured search of the literature was conducted. The study scheme included a search of the lit- PMN Capillary MON erature from PubMed (http://www.ncbi.nlm.nih.gov/pub- med) and Scopus (http://www.scopus.com/home.url). Also, included was a search of Google (http://www.google.com/). Figure 1: A schematic view of an alveolus that depicts the eﬀect of A search was also made of patents in the database inhaled tobacco smoke on the terminal (respiratory) structure of of the US Patent and Trade Oﬃce (http://www.uspto.gov/). the lung. Particulate matter “Tar” in tobacco smoke is inhaled deep In addition, searches were made for documents that were into the lung where it is recognized by macrophages. The macro- phages arise from the blood monocytes that migrate into the lung released by the tobacco companies and made public as a where they undergo diﬀerentiation and maturation. Macrophage consequence of the tobacco Master Settlement Agreement. phagocytosis of the chemical-rich “Tar” evokes the production To this end, we searched database records of over 11 million of diverse proinﬂammatory mediators (for details, see Figure 1). documents in the digital archive that were established and Macrophages have toll-like receptors (TLR) that recognize diverse which are maintained currently at the University of Califor- microbes andtoxins(LPS isrecognized byTLR-4). Shown inthis nia, San Francisco (http://legacy.library.ucsf.edu/). We also illustration is the production of ﬁve proinﬂammatory cytokines: searched the database from Tobacco Documents (http://to- tumor necrosis factor, type alpha (TNFα), interleukin 1-beta (IL- baccodocuments.org/). 1β), leukemia inhibitory factor (LIF), oncostatin M (OSM), and The searches were performed using conventional tele- Interleukin-4 (IL-4). These soluble factors interact with other cells of the lung, and the response of these cells is thought to accelerate, gram-style search short-string text formulations with Bool- amplify, and prolong pulmonary inﬂammation. The target cells may ean operators as described in PubMed. Illustrative key search include T cells. The T cell that is depicted herein is representative of words were bacteria, mold,fungi,yeast,tobacco, smoke, many diﬀerent T cell subsets, including T helper cell subsets Th1, endotoxin, mycotoxin, cured, fermented, lipopolysaccharide, Th2, and Th17. Type I epithelial cells are the major cells lining aﬂatoxin, and microbiology. We also used unique search the alveolar space, and facilitating O /CO . The type I cells are 2 2 words, such as author’s name, project designation, report spread out and cover about 90 to 95% of the alveolar surface. codes, cigarette brands, and Bates number. The references The type II cells form only 5 to 10% of the surface but produce cited in the retrieved literature were reviewed to identify surfactant proteins. Polymorphonuclear leukocytes (PMN) mediate other topic-speciﬁc writings Table 1. inﬂammation in multiple ways, including the production of an oxidative burst. Dendritic cells (DC) are professional antigen- presenting cells; they also mediate inﬂammation. 7. Tobacco-Associated Chronic Inﬂammation Chronic inﬂammation is associated with malignant trans- and conﬁdential. The paucity of the literature on health risks formation, tumor growth, and, possibly, tumor metastasis, associated with microbes in smokeless and smoking tobacco reviewed in [44–52]. Examples of the association of cancer is to be contrasted to the numerous reports by tobacco scien- with chronic inﬂammation include (a) lung cancer and tists researching other health-related issues, such as potential cigarette smoke (aerosol), (b) malignant mesothelioma and reduced-risk exposure tobacco products (PREPS) [15]. asbestos (ﬁbers), (c) stomach cancer and H. Pylori(bacteria), (d) malignant melanoma and ultraviolet sun light (irradia- tion), (e) liver cancer and aﬂatoxin (mycotoxin), and (f) can- 5. Perspective and Limitations cer of the uterine cervix and human papilloma virus. Thus, The authors are immunologists and have an active research malignancy at diverse body sites, and of various tissues, is interest in addressing tobacco-associated chronic pulmonary associated with chronic inﬂammation provoked by assorted 4 Journal of Oncology Table 1: History of investigations of microbes and microbial toxins in tobacco and tobacco products. Results are reported for studies that were undertaken to characterize the microbes of tobacco before and during 1896 [16] tobacco fermentation. German bacteriologist H. E. Suchsland announces that the delicate aroma and subtle shades of ﬂavor which aﬀect the palate of the smoker are not due to the tobacco but are attributed to the microbes which aid in the process of tobacco 1899 [17] fermentation. A patent based upon this observation was submitted, presumably to improve the poor quality of German tobacco by adding to the harvested tobacco leaves bacteria that he had isolated and grown in his laboratory from high-quality West Indian tobacco. The microbial degradation of nicotine and nicotinic acid was reported. The morphological and physiological 1954 [18] properties of the nicotine-decomposing bacteria were also described. W. C. Flanders of R. J. Reynolds Tobacco Company issues a 70-page report of a three-year study to determine if the number of microorganisms (bacteria and mold) changed appreciably during aging. Experiments were also 1955 [19] conducted to determine if the recorded changes in the microbes follow the changes in the chemical components of tobaccos. These studies were continued and extended for several years. Pseudomonas aeruginosa and other potentially pathogenic fungi and bacteria were identiﬁed in snuﬀ. Similar 1957 [20] microbial isolates from a patient was the basis for the physician to theorize that some of the snuﬀ-derived microbes may be responsible in part for chronic bronchitis. The results of studies were reported that had been undertaken to characterize the deposition of cigarette smoke particles and debris released from the cigarette ﬁlter into the human respiratory tract. Popular brand cigarettes were smoked mechanically and in a manner to reﬂect normal smoking behavior. The studies documented that tobacco 1958 [21, 22] ﬂakes and ﬁne tobacco leaf debris were released into mainstream smoke from the cigarette ﬁlter of all brands that were tested (Tareyton, Winston, Kent, L&M, Marlboro, and Viceroy). The tobacco ﬂakes and other particulates (ﬁlter ﬁbers and carbon from charcoal ﬁlters) were studied by light and electron microscopy. 1966 [23] Toxic fungi were identiﬁed in tobaccos. Comparative studies were preformed for microbiological activity in the smoke of popular brand nonﬁltered and 1967 [24, 25] ﬁltered cigarettes that had been “cold smoked” or lit. Viable bacteria were found in the smoke of all cigarettes tested. The tobacco from diﬀerent popular brands of cigarettes was analyzed for bacteria. The number of bacteria was determined on “our own” (Philip Morris) and competitive cigarette ﬁllers. This test was run for several months and 1972 [26] each month Viceroy, Brown & Williamson’s product, always showed the lowest degree of “contaminant.” The diﬀerence between the brands was statistically signiﬁcant. Brands tested included Salem, Pall Mall, Chesterﬁeld, Kool, Kent, Viceroy, Winston, and Marlboro. The number of bacteria on Marlboro were “too numerous to count.” A 189-page report was prepared by investigators at the Brown & Williamson Tobacco Company that presents methods for the microbiological examination of tobacco and tobacco products. The writings include the description 1972 [27] of techniques for the quantitative determination of bacteria and fungi and methods for the isolation of potentially human pathogenic microorganisms including Coliform bacteria. Also identiﬁed were Staphylococcus aureus, Enterococci, Pseudomonas, Clostridium,and Aspergillus. A 52-page report that describes a “contact plate method” in which a whole cigarette is rolled over the surface of the nutrient agar dish. Viable microbes that are transferred from the cigarette to the plate are illustrated. Presumably, the 1972 [28] intent of the assay was to measure the growth of microbes that would be transferred from the cigarette paper to the hand of the smoker. Other studies showed the growth of microbes from a natural wrapper of a cigar. Also, culture methods were established for testing for coliform bacteria and for counting viable fungi in tobacco. A 346-page in-house document is produced by the British-American Tobacco Company entitled “Methods for the Microbiological Examination of Tobacco and Tobacco Products.” The authors describe the “Public Health Aspects” of smoking and smokeless tobacco products. They note that “[T]he detection of micro-organisms of health signiﬁcance in tobacco products must be expected to be regarded as undesirable or even unacceptable by public 1972 [29] agencies, regardless of whether there is proof of the signiﬁcance in initiating or spreading infection in man. Therefore, it is suggested that tobacco products should be substantially free, or contain only minimal numbers, of micro-organisms of potential health signiﬁcance to man which could conceivably occur on tobacco...” Suggested standards are presented for tobacco products for various bacteria and fungi, and standards that had been established for food products (ﬁsh, sausage, meat pies, cream yogurt, soft cheese, and pasteurized milk). Philip Morris characterizes the microbial population on Marlboro tobaccos throughout the processing line. Five diﬀerent Marlboro Make-Your-Own tobaccos with various anti-microbial preservatives were evaluated 1991 [30] microbiologically for mold and bacteria over time. The microﬂora of Marlboro raw and tobacco blends were deﬁned for burley, oriental, ﬂue-cured, and other tobacco types. Journal of Oncology 5 Table 1: Continued. Bacillus spores were identiﬁed in chewing tobacco sold in the USA. Broth of the culture microbes evoked plasma 1992 [31] exudation from the oral mucosa when tested using a hamster cheek pouch assay. 1995 [32] In an oral presentation, Hasday describes for the ﬁrst time the presence of endotoxin in cigarette smoke. Scientist from Imperial tobacco (Canada) report the development of an easy-to-search database on the microbes 1990 [33] associated with tobacco. 1999 [34] Bacterial endotoxin was identiﬁed as an active component of cigarette smoke. A US Patent was awarded for a method and system for assay and removal of harmful toxins during the processing of 2004 [35] tobacco products. Microbiologists in Sweden used a mass-spectrophotometry-based assay to document that tobacco smoking increased 2004 [36] dramatically the air concentrations of endotoxin (LPS). The authors note that smoke-derived LPS may be a health risk factor associated with environmental tobacco smoke. A US Patent was assigned to Philip Morris for an “antibacterial lavage” method to treat tobacco leaves so as to eliminate or reduce bacterial endotoxins (LPS) and tobacco-speciﬁc nitrosamines that are formed during the curing 2004 [37] process. Bacteria found on tobacco leaves were reported to be primarily Gram-negative bacteria, including pseudomonades and enterobacters. In the awarded patent, Hempling notes that bacterial endotoxins can remain as a residue on the tobacco even after the bacteria have been destroyed. The microbiological composition of tobacco products was deﬁned using culture and chemical analysis. Tobacco 2004 [36] smoke was analyzed chemically, and LPS was measured for tobacco leaves and cigarette tobacco. US Military publishes a report of an investigation that documents bacterial species diversity of varying brands of 2005 [38] cigarettes made in the Middle East that were thought to be associated with illnesses of American soldiers deployed in Operation Iraqi Freedom. 2006 [39] Cigarette smoke was identiﬁed as the source of elevated levels of endotoxin (LPS) found in indoor air. Identiﬁcation of microﬂora on tobacco using culture-independent methods based on the ampliﬁcation of microbial 2007 [40] 16S rDNA sequences directly from the leaf surfaces. The investigators discovered also that three of ﬁve dominant bacterial species on the tobacco could not be cultivated. The microbiological composition of tobacco products was deﬁned using culture and chemical analysis (of tobacco leaves) or chemical analysis only (tobacco and tobacco smoke). Mesophilic bacteria dominated among the bacteria in 2008 [41] both fresh and cured tobacco leaves; however, a wide range of other bacteria, including Gram-negative bacteria, and fungi were delineated. Microbial ﬂora was compared in studies of tobacco from cigarettes from diﬀerent countries. LPS was also measured. Bacteria grown from a single ﬂake of tobacco from all brands of smoking (cigarette, cigar, and pipe) and smokeless 2008 [42] (snus, snuﬀ, and long cut) tobacco products. In many instances, the bacteria from the tobacco caused hemolysis of blood in blood agar and liquid broth cultures. Twenty-seven species of bacteria were identiﬁed in an analysis of both unaged tobacco and ﬂue-cured tobacco by 2010 [43] 16S rRNA sequence analysis. More species (N = 23) were identiﬁed from the unaged ﬂue-cured tobacco leaves than in the aging leaves (N = 15 species). Fifteen classes of bacteria and a broad range of potentially pathogenic organisms were detected in all cigarette samples studied. In greater than 90% of the tobacco samples, the investigators identiﬁed Acinetobacter, Bacillus, Burkholderia, Clostridium, Klebsiella, Pseudomonas aeruginosa,and Serratia. The bacteria were identiﬁed using a 2010 [43] 16S rRNA-based taxonomic microarray. Cloning and sequencing were used to evaluate total bacterial diversity of four brands of cigarettes. Previous studies have shown that smoking was associated with colonization by pathogenic bacteria and an increased risk of lung infection. This study, however, was the ﬁrst to show that cigarettes themselves could be the source of exposure to a wide array of potentially pathogenic microbes. items that include smoke, bacteria, ﬁbers, irradiation, toxins, smoking may not only have an adverse eﬀect of systemic im- and viruses. munity but also skews both innate and adaptive immune responses [61–65]. 8. Cigarette Smoke, Chronic Inﬂammation, 9. Study Rationale: Evidence-Based Health and Impaired Immunity Risks of Tobacco-Associated Microbes Cigarette smoke is known to induce chronic inﬂammation of the lung [53–60]. More recently, a substantial body of infor- Concern has been expressed by many investigators that mi- mation has been obtained to suggest that long-term cigarette croorganisms on cured tobacco might represent a health risk. 6 Journal of Oncology By way of example, in 1968, Wood [66], a scientist at the reproducibility and smoke toxicity. The results were incon- British American Tobacco Company, wrote a 37-page report clusive. Our search for subsequent studies by Wood address- addressing the possible transfer of viable microorganisms ing this subject failed to identify subsequent experiments or into mainstream smoke. In this internal document, he notes published reports. Studies by Slutzker et al. were negative that cured tobacco, of various types, has long been known [69]. In 1967, Curby reported to The Council for Tobacco to contain bacterial spores. Likewise, Wood [66]and others Research the results of comparative studies that he had [23] have addressed the possibility that tobacco-associated undertaken to determine the microbiological activity in the mold may also represent a health hazard to smokers. Support smoke from ﬁlter and nonﬁlter cigarettes. Diﬀerent popular for this concern was derived in part from a paper published brands of cigarettes were obtained from local vendors in in Science by Forgacs and Carll two years previously in Brookline, Mass, USA. Comparative analyses were made which they reported the identiﬁcation of toxic fungi in of bacteria released from cigarettes that had been “cold tobacco [23]. In the Science paper, the investigators exposed smoked” (not lit) or smoked in the usual manner (lit). The mice to smoke from fungally contaminated hay. The mice tobacco smoke collection system was tested for sterility by developed pulmonary emphysema and other pathological means of conventional microbiology culture procedure and conditions; in contrast, mice exposed to smoke from sterile, by means of electronic analyses of particle size and number. uninoculated hay remained normal clinically. In a letter to Viable bacteria were identiﬁed in the smoke from all ciga- the Associate Scientiﬁc Director of the Council for Tobacco rettes tested. The number of liberated organisms was much Research, dated 1964, Forgacs, with more than 16 years greater when the cigarette was burning [24, 25]. of research experience as a mycologist, states that he had Before proﬁling more recent studies, a brief overview is examined mycologically a number of tobacco products, warranted of what many internal documents of the tobacco including cigarettes that had been purchased on the open industry have entitled the “Microbiology of Tobacco.” market [67]. Forgacs observed that the tobacco of all cigarettes contained fungal mycelia and spores [67]. In part, 10. Microbiology of Tobacco the origin of his health concern is based upon the knowl- edge of (a) widespread fungal contamination of tobacco The “Microbiology of Tobacco” has been the focus of many products, (b) heat stability of the mycotoxins; (c) known studies. It was not surprising to learn from our paper that animal toxicity, (d) reasonable assumption that some of the most of all the major tobacco companies have studied this fungi are carcinogenic, and (e) potency at low doses, see also issue for many years. Listed below are varying topics ad- [68]. dressing bacteria, mold, and mycotoxins in tobacco and ref- Wood argues that erences (a) chemical and microbiological changes during curing “[W]hile it is quite impossible to deduce, from [16, 19, 70–75], this (mouse) experiment, the likely eﬀect of smoke from a cigarette containing fungally (b) bacteria in cigarettes; product comparison (also, see contaminated tobacco, the implications are suf- below) [17, 76–79], ﬁciently important to warrant some considera- (c) databases of tobacco microbes [33, 40, 80], tion of the role which micro-organisms may play (d) tobacco microbe control [81], with regard to smoke toxicity. For instance, it is possible that viable spores might be transferred (e) microﬂora community of tobacco [82–88], to mainstream smoke and thus enter the lungs; (f) quantitative studies of tobacco microﬂora [89–91], pathogenic species, even in small numbers, (g) growth of mold in stored tobacco [26, 92], could clearly have harmful eﬀects, while very large number of otherwise harmless micro- (h) growth of Aspergillus from tobacco [93–95], organisms might lead to a signiﬁcant concen- (i) microbial degradation of nicotine [18, 96], tration of genetic material. Alternatively, during (j) examination of cigarettes from mold-damaged and the vegetative stage of their residence on tobacco nondamaged tobacco [97], the micro-organisms might produce toxins which could transfer direct to smoke or metabo- (k) isolation of viable fungi from snuﬀ [98], lites which on burning could give toxic smoke (l) sterilization/treatment to remove NNK [37, 99–105], constituents.” (m) removal of harmful toxins on tobacco [35, 95], The report by Wood also describes some preliminary (n) inhibiting mycotoxin production [106], experiments which were undertaken to show whether bacte- (o) microbiology of cigarettes, pipes, cigars, and snuﬀ rial or fungal spores could transfer into tobacco smoke. Two [27–30, 107–111]. schemes were used to trap the cigarette smoke; these were a test tube bubbler and a micropore ﬁlter. These samples From about the early 1970s, extensive research was con- from the bubbler and the ﬁlter were tested for the growth of ducted on the Microbiology of Tobacco. Many reports reﬂected microorganisms. Growth of microbes was observed; how- the interest of the major tobacco companies. These stud- ever, technical problems were encountered including poor ies sought to identify diﬀerent bacteria and molds and to Journal of Oncology 7 count the number of colony-forming units (CFU) during Warke [103] studied the microbiological quality of chew- processing. The number of bacteria and molds present in able, often sweet, tobacco mixes known as “Gutka” used by green, freshly harvested tobacco was compared to that of var- millions of children and adults in India where it is made ious stages of curing, fermentation, and long-term storage. and often exported. Of the 15 samples studied, all contained In many cases, more than one million bacteria were found in aﬂatoxins B ,B ,and G . Samples exposed to Co radiation 1 2 2 a gram of tobacco (a 100 mm cigarette has about 0.9 grams displayed a marked reduction of viable CFU. Sterilization of tobacco). Comparative studies included various types of of tobacco in the manufacturing has been described in US tobacco (Bright and Burley) and diﬀerent curing methods Patents [105]. (ﬁeld versus ﬂue cured). In these studies, proﬁles were In 1992, Rubenstein reported the identiﬁcation of large established for leaves of the diﬀerent types of tobacco that number (>10 CFU) of a Bacillus species in chewing tobacco had been picked from various positions of the plant. Diverse sold in the USA [31]. Supernatants of the cultured bacteria environmental conditions were evaluated, and these included evoked a plasma exudate in studies in which the supernatant variations in temperature and moisture. Analyses were made was instilled into an intact hamster cheek pouch. of the number of bacteria in popular brand cigarettes. In many instances, the number of bacteria of a particular 12. Pathogenic Bacteria of Cigarettes company’sbrand wascompared to brands marketed by com- petitors. In addition to cigarettes, studies were performed Some bacteria grow in unique microenvironments, and some for cigars and snuﬀ.Considerable eﬀort was devoted to are diﬃcult to grow using traditional broth- and agar-based deﬁning procedures for the sterilization of tobacco to reduce methods. This technical diﬃcultymay also applytogrowing or prevent the growth of mold. The methods used included bacteria that have adapted to unique conditions that develop (a) washing methods using various solutions (bleach), (b) during the curing and fermentation of tobacco. Accordingly, irradiation with microwave, ultraviolet light, and gamma it is believed that conventional methods may not accurately radiation, (c) exposure to various gases, and (d) treatment deﬁne the microﬂora of diverse tobacco products [43, 113]. with diﬀerent antibacterial and antifungal agents (antibi- Consequently, there may be an incomplete understanding of otics). One scheme was to destroy all of the bacteria on the bacterial diversity in the tobacco of cigarettes and also the freshly harvested green tobacco leaves and then seed the impact these microbes and microbial toxins may impose on leaves for fermentation using selected colonies from in-house the smoker [113]. batch-scale production. Quality control of the tobacco was Recently, the bacterial metagenomic of cigarettes were important as high levels of mold produced an unacceptable characterized using a 16S rRNA-based taxonomic microassay “oﬀ-taste.” as well as traditional cloning and sequencing methods. The brands included Camel, Marlboro, Kool, and Lucky 11. Pathogenic Bacteria of Chewing Tobacco Strike. The results of this study showed that the number of microorganisms in cigarettes may be as vast as the number of Studies have been conducted by investigators of the tobacco chemicals in these products. Fifteen classes of bacteria were industry (see above) and health community to address the identiﬁed [113]. Particularly noteworthy was the identiﬁca- potential of bacteria, molds, yeast, and microbial toxins tion of a broad range of potentially pathogenic microorgan- found in diﬀerent types of smokeless tobacco (snuﬀ,snus, isms detected. More than 90% of the tobacco samples from and long cut) [20, 26, 31, 43, 112, 113]. the cigarettes contained Actinetobacter, Bacillus, Burkholde- ria, Closteridium, Klebsiella, Pseudomonas aerogenosa,and In 1951, Dynert published in The New England Journal of Serratia. Other bacteria that are known to be potentially Medicine a case report of a patient with chronic bronchitis. pathogenic to humans and detected using the metagenomic Pseudomonas aeruginosa, often colonized in COPD patients, technology were Campylobacter, Enterococcus, Proteus,and and a few colonies of Staphylococcus aureus were identiﬁed Staphylococcus [113]. in bacteriological examinations of the subject’s sputum [20]. The patient used snuﬀ, and it was theorized that the snuﬀ Reported also in 2010 were the results of an investigation may have been the source of the pathogens. A study was of the diversities of unaged and ﬂue-cured tobacco leaves then undertaken of 22 samples of previously unopened packs using a 16S rRNA sequence analysis scheme [43]. of snuﬀ. The following microorganisms were grown from Others have reported the identiﬁcation of potentially more than 50% of the snuﬀ samples: Bacillus rubitilles, pathogenic bacteria in commercial cigarettes. One study was Staphylococcus aureus (coagulase positive), Staphylococcus undertaken to assess the bacterial diversity of cigarettes that albus (coagulase positive), Pseudomonas aeruginosa, Staphy- were thought to be linked to severe pneumonitis in US mil- lococcus aureus (coagulase negative), and Staphylococcus albus itary personnel deployed in Operation Iraqi Freedom [38]. (coagulase negative). Eight species of Bacillus, including ﬁve new species, and one In 1991, Varma reported the isolation of nine species of new species of Kurthia were isolated from the cigarettes. Aspergillus in stored leaves of chewing tobacco [112]. Ap- Some of these species have been identiﬁed elsewhere to proximately 18 of the Aspergilli were found to be mycotoxi- cause hypersensitivity pneumonitis and other respiratory genic. All aﬂatoxigenic strains of A. ﬂavus produced aﬂatoxin syndromes [38]. This study was of particular interest to B . Patulin and ochratoxin were produced by A. ochraceus. many because the cigarettes were made in Iraq and not man- Sterigmatocystin was produced by three diﬀerent strains. ufactured by a major tobacco company. Undertaking this 8 Journal of Oncology investigation, the question arose as to whether the cigarettes 14. Transfer of Tobacco Flake to that had been purchased by soldiers from street vendors Mainstream Smoke had been intentionally altered by adding pathogenic bacteria The ﬁlter of a cigarette is often contaminated with loose and/or mold. This theory was disproven. tobacco ﬂakes, tobacco ﬁnes, and tobacco dust. In one exam- Another study was conducted by a group of investigators in Sweden who characterized the bacterial and fungal com- ination, the ﬁlters of 11 brands of cigarettes were examined in freshly opened packs. For all brands, cigarettes were observed munity in warehouse tobacco [41]. with tobacco ﬂakes on the ﬁlter. Examination of the ﬁlters We have reported previously the establishment of a novel bioassay which showed that bacteria were grown routinely with the naked eye showed that 127 of 208 (61.1%) of the ﬁlter had tobacco particles [42]. The release of tobacco ﬂakes from a single ﬂake of tobacco that had been placed on the into mainstream smoke has been described previously [21, surface of a sheep blood agar plate [42]. Of eight popular 22]. brands of cigarettes, bacteria grew from almost all (>90%) The tobacco ﬂakes that contaminate the ﬁlter arise from of the ﬂakes. Similarly, bacteria were grown from a single ﬂake, and also with a high frequency, from tobacco that tobacco that escapes from the nonﬁlter, sometimes called the distal, end of the cigarette. Most probably the ﬂakes are jarred had been retrieved from cigar ﬁller and from smokeless loose during manufacturing, shipment, and daily transporta- tobacco (snus, snuﬀ, and long cut). Some bacteria induced hemolysis of the blood in the agar dishes. The destruction tion, especially in a pack in which more than one-half of the cigarettes have been used [117, 118]. of the red blood cells was readily visible as a yellow The release of ﬂakes from the cut surface can readily be zone surrounding a single tobacco ﬂake. Expanding studies demonstrated by comparing the cut surface of the ﬁlter be- documented the hemolysis of human blood in agar or fore and after smoking the ﬁrst puﬀ.The single ﬂake maybe nutrient broth cultures. Thus, as discussed later, bacteria viewed as a matrix for carrying bacterial and fungal agents in could be carried deep into the respiratory tract by a single mainstream tobacco smoke. Thus, the burning of the tobacco tobacco ﬂake sucked from the cut surface of a cigarette ﬁlter during cigarette smoking does not exclude the exposure to and transported into the bolus of smoke that is inhaled deep into the lung. A single tobacco ﬂake may be envisioned tobacco-associated microbes and microbial toxins. Bacteria are also released from the barrel of the cigarette. as a matrix for delivering diverse bacteria into the respira- This was demonstrated in investigations in which a cigarette tory tract of an immunologically compromised long-term smoker. was rolled over the surface of a nutrient agar dish. 15. Endotoxin (LPS) in Mainstream and 13. Cigarettes with Mold Sidestream Tobacco Smoke Mold has been identiﬁed in the tobacco of popular brand cigarettes, and concern has been raised as to the propensity In 1999, Hasday and his colleagues reported the identiﬁ- of these microbes as a health risk to the smoker. Presented cation of bacterial endotoxin as an active component in cigarette tobacco and cigarette smoke [34]. The authors herein is a partial listing of papers that have identiﬁed mold in cigarettes [78, 114–116]and in marijuana [116]. showed that the dose of LPS delivered from smoking one pack of cigarettes was comparable to that of the LPS that As early as 1971, Papavassiliou and coworkers concluded had been previously shown to be associated with adverse that “[C]igarettes are contaminated with various fungi.” health eﬀects in cotton textile workers. With the knowledge They studied cigarettes that were manufactured in the USA, that LPS is one of the most potent inﬂammation-inducing Canada, England, France, Belgium, Germany, Jordan, and agents, the work by Hasday attracted considerable attention, Egypt. Hundreds of strains of fungi were isolated. The Greek reviewed in [32]. In 2004, Larsson et al. reported that they scientists demonstrate that the most prominent fungi were were able to demonstrate unequivocally that high levels of Aspergillus (28 strains from Greek cigarettes and 35 strains LPS are inhaled during active cigarette smoking and, more from other countries). They raised the question as of the importantly, that environmental tobacco smoke may involve association of the fungi with allergies but commented that inhalation of amounts of endotoxin that are dramatically this issue has not been resolved [114]. greater than those existing in indoor environments free In 1983, Kurup and colleagues reported the identiﬁcation from tobacco smoke [36]. In 2006, these ﬁndings were of allergenic fungi in smoking materials and discussed the conﬁrmed and extended [39]. Particularly notable is that health implications of their ﬁndings [115]. Concern has been studies of Larsson and colleagues used a mass-spectrometry- expressed as to the health risks associated with mold in based assay that circumvents the problems often associated cigarettes. with the biologically based LPS assay. Writing in the Journal of the American Medical Associ- ation, Verweij et al. addressed the propensity of heath risks associated with fungal contaminates of tobacco and mar- 16. Analysis of Findings and Policy ijuana [116]. They concluded that “[A]ll cigarette brands Recommendations tested (N = 14 brands) had some degree of fungal contam- ination, although not every cigarette was found to have a The results of this literature review have documented that the positive culture.” tobacco microﬂora has been the subject of many studies by Journal of Oncology 9 investigators of tobacco industry and academic communities. assessed in the context of other known tobacco- During the last 50 years, there has been an imbalance, associated diseases, including chronic obstruc- however, in the attention devoted to addressing the identiﬁ- tive pulmonary disease, asthma, bronchitis, and cation and propensity of the harm of tobacco- and tobacco- alveolar hypersensitivity. smoke-associated chemicals and in the attention devoted to characterizing microbes and microbial-derived factors. (2) Tobacco-speciﬁc nitrosamines (NNK) are human Ample information has accumulated to suggest that carcinogens that are present in mainstream smoke, microbes and microbial-derived factors may contribute to sidestream smoke, and smokeless products. NNKs the health risks of smoking and smokeless tobacco products. arise primarily from the microbial degradation of Moreover, the microbes may facilitate microbial colonization nicotine in tobacco. Diﬀerent technologies have of the mouth and airway, the induction of chronic inﬂam- proven eﬀective in preventing the formation of mation through the activation of diverse leukocyte subsets, NNKs. It is recommended that these technologies be alteration of the tissue microenvironment, and microbial- implemented and that guidelines for tobacco articles toxin-induced pathologies. The current health concerns be established for reduced NNK-products. recently expressed by investigators of various disciplines, and (3) The criteria, protocols, and procedures used by the with diﬀerent research interests, in peer-reviewed published FDA in the assessment of harm associated with my- research articles are reasonable and validate that additional cotoxins in food products should be applied to investigation of the microbiology of tobacco is warranted. loose leaf tobacco, smoking tobacco products, and The ﬁndings reported herein relate to National Tobacco smokeless tobacco articles. Mycotoxin action levels Control Policy and speciﬁcally FDA Regulation of Tobacco should be established to provide an adequate margin Products [119]. of safety to protect human tobacco users. Based upon the information obtained in this paper, we recommend the following for consideration and possible reg- ulatory action. Abbreviations AFL-B Aﬂatoxin, type B (1) Tobacco products should be assessed with the knowl- 1: 1 edge that they contain bacteria, mold, and microbial CFU: Colony forming unit DC: Dendritic cell toxins. IARC: International Association for Research of Cancer (a) In this context, the designation of tobacco prod- IL-1β: Interleukin-1, beta ucts is to include conventional and novel IL-4: Interleukin-4 products that contain tobacco, including items LIF: Leukemia inhibitory factor which are smoking and smokeless tobacco LPS: Lipopolysaccharide articles. LSRO: Life Science Research Organization (b) National and international registries of known LTDL: Legacy Tobacco Document Library human carcinogens should not be used as the MON: Monocyte sole criteria for assessing tobacco-associated NTP: National Toxicology Program human health risks. Any and all tobacco-asso- OSM: Oncostatin M ciated agents that induce any human pathology PGE Prostaglandin E2 2: should be included in risk assessments. PMN: Polymorphonuclear leukocyte (c) Tobacco in smoking and smokeless tobacco ar- RNS: Reactive nitrogen species ticles should be assessed for their propensity ROS: Reactive oxygen species to induce chronic inﬂammation. Chronic in- TLR: Toll-like receptor ﬂammation is known to be induced by diverse TNFα: Tumor necrosis factor, alpha. bacteria (Gram positive and Gram negative) and fungi, living or dead, whole or fragmented, References and intracellular and membrane components. Chronic inﬂammation is known also to be in- [1] M. Borgerding and H. Klus, “Analysis of complex mixtures— duced by diverse toxins of bacteria and/or fungi cigarette smoke,” Experimental Toxicology and Pathology,vol. including, but not limited to, endotoxins, exo- 57, supplement 1, pp. 43–73, 2005. toxins, and mycotoxins. [2] R. R. Baker, “Smoke chemistry,” in TOBACCO: Production, Chemistry and Technology, D. Layten Davis and M. T. Nielsen, (d) Chronic inﬂammation associated with bacteria, Eds., charter 12, pp. 398–439, Blackwell Science, 2003. fungi, and microbial toxins of tobacco products [3] S. S. Hecht, “Cigarette smoking: cancer risks, carcinogens, should include inﬂammation of any and all tar- and mechanisms,” Langenbeck’s Archives of Surgery, vol. 391, get sites, including tissues of the mouth, naso- no. 6, pp. 603–613, 2006. pharynx, and lung. [4] A. Rodgman and T. A. Perfetti, The Chemical Components of (e) In addition to chronic inﬂammation, harm Tobacco and Tobacco Smoke, CCRC Press, Taylor and Francis of microbial elements of tobacco should be Group, Boca Raton, Fla, USA, 2009. 10 Journal of Oncology [5] D. Hoﬀmann and I. Hoﬀmann, “The changing cigarette, of tobacco,” Recent Advances in Tobacco Science,vol.21, pp. 1950–1995,” Journal of Toxicology and Environmental Health 39–80, 1955. A, vol. 50, no. 4, pp. 307–364, 1997. [20] H. P. Dygert, “Snuﬀ-a source of pathogenic bacteria in [6] G. F.Wayne and G. N.Connolly,“Regulatory assessment of chronic bronchitis,” The New England Journal of Medicine, brand changes in the commercial tobacco product market,” vol. 257, no. 7, pp. 311–313, 1957. Tobacco Control, vol. 18, no. 4, pp. 302–309, 2009. [21] W. K. Farr and A. Revere, Examination of Whole Cigarette [7] D. M. Burns and C. M. Anderson, “Do changes in cigarette Smoke by Light and Electron Microscopy,Life Extension design inﬂuence the rise in adenocarcinoma of the lung?” Foundation, New York, NY, USA, 1958. Cancer Causes Control, vol. 22, pp. 13–22, 2011. [22] W. K. Farr and A. Revere, “Examination of whole cigarette [8] R. J. O’Connor, K. M. Cummings, V. W. Rees et al., smoke by light and electron microscopy,” Journal of the “Surveillance methods for identifying, characterizing, and American Medical Association, vol. 172, no. 4, p. 405, 1960. monitoring tobacco products: potential reduced exposure [23] J. Forgacs and W. T. Carll, “Mycotoxicoses: toxic fungi in products as an example,” Cancer Epidemiology Biomarkers tobaccos,” Science, vol. 152, no. 3729, pp. 1634–1635, 1966. and Prevention, vol. 18, no. 12, pp. 3334–3348, 2009. [24] W. A. Curby, “A preliminary study of the biological activity [9] D. K.Hatsukami, K.A. Perkins,M.G. LeSage et al.,“Nicotine in cigarette smoke,” 1967, Bates Number 11330877-0905. reduction revisited: science and future directions,” Tobacco Retrieved on March 23, 2011 from http://legacy.library.ucsf Control, vol. 19, no. e1, pp. 1–10, 2010. .edu/tid/jtp6aa00. [10] H. Ito, K. Matsuo, H. Tanaka et al., “Nonﬁlter and ﬁlter [25] W. A. Curby, “A preliminary study of the biological activity cigarette consumption and the incidence of lung cancer by in cigarette smoke—part II,” 1967, Bates Number 11330942- histological type in Japan and the United States: analysis 0973. Retrieved on March 23, 2011 from http://legacy.library of 30-year data from population-based cancer registries,” .ucsf.edu/tid/stp6aa00. International Journal of Cancer, vol. 128, no. 8, pp. 1918– [26] R. E. Welty, “Fungi isolated from ﬂue-cured tobacco sold in 1928, 2011. Southeast United States, 1968–1970,” Applied Microbiology, [11] M. Rabinoﬀ, N. Caskey, A. Rissling, and C. Park, “Pharmaco- vol. 24, no. 3, pp. 518–520, 1972. logical and chemical eﬀects of cigarette additives,” American [27] T. G. Mitchell and British-American Tobacco Company, Journal of Public Health, vol. 97, no. 11, pp. 1981–1991, 2007. “Microbiological examination of tobacco products: report [12] Brown & Williamson, “Commonly added ingredients,” 1982, Number RD 969-R,” 1972, Bates number 105501740/1767. Bates Number 521057548/7553. Retrieved on February 22. Retrieved on June 28, 2010 from http://legacy.library.ucsf 2011 from http://legacy.library.ucsf.edu/tid/tmx33f00. .edu/tid/xwp67a99. [13] A Report of the Surgeon General, “How tobacco smoke [28] P. C. Stauber and British American Tobacco Company, “Microbiology of Henri Wintermans cigar production on- causes disease. The biology and behavior basis for smoking- attributable disease,” U.S. Department of Health and Human site studies of the primary process at Eersel: report No. Services, Atlanta, GA; Oﬃce on Smoking and Health, U.S. RD 925R,” 1972, Bates number 107466852/6877. Retrieved Government Printing Oﬃce, Washington, DC 20402, 704 on June 28, 2010 from http://legacy.library.ucsf.edu/tid/ pgs, 2010. http://www.cdc.gov/tobacco/data statistics/sgr/ dpe66a99. 2010/index.htm. [29] T. G. Mitchell and P. C. Stauber, “Methods for the microbio- [14] A. M. Bromnawell, Reports of the Life Science Research logical examination of tobacco and tobacco products, Report Oﬃce (LSRO),Bethesda, MD,Volume I. Biological eﬀects Number 888—R,” 1972, Bates number 105597063/7412. assessment in the evaluation of potential reduced-risk tobacco Retrieved on July 22, 2010 from http://legacy.library.ucsf products, 242 pages; Volume II. Scientiﬁc methods to evaluate .edu/tid/vit379. potential reduced-risk tobacco products, 174 pages; and Vol- [30] Smoke Study Group, “CORESTA—Papers presented at the ume III. Exposure assessment in the evaluation of potential Kallithea Symposium,” 1991, Bates number 2021551986/ reduced-risk tobacco products, 170 pages, 2007. 2194. Retrieved on July 27, 2010 from http://legacy.library [15] L. P. Carter, M.L. Stitzer, J. E.Henningﬁeld,R.J. O’Connor, .ucsf.edu/tid/zhe58e00. K. M. Cummings, and D. K. Hatsukam, “Review: abuse [31] I. Rubinstein and G. W. Pederson, “Bacillus species are liability assessment of tobacco products including potential present in chewing tobacco sold in the United States and reduced exposure products,” Cancer Epidemiology Biomark- evoke plasma exudation from the oral mucosa,” Clinical ers and Prevention, vol. 18, no. 12, pp. 3241–3262, 2009. Diagnostics Laboratory and Immunology, vol. 9, pp. 1057– [16] J. Beherns, “Die beziehungen der microorganisms zum 1060, 1992. tabaksbau and zur tabakferntation,” Zentrabl Bakteriol Par- [32] R. L. Barnes and S. A. Glantz, “Endotoxins in tobacco smoke: asitenk, Abt II, vol. 2, pp. 514–527, 1896. shifting tobacco industry positions,” Nicotine and Tobacco [17] Anonymous, “Tobacco and bacteria,” The London Globe, pp. Research, vol. 9, no. 10, pp. 995–1004, 2007. 7, July 21, 1899. Retrieved on November 11, 2010 from http:// [33] A. Morin, F. Samson, A. Porter, and J. Torrie, “Development query.nytimes.com/gst/abstract.html?res=FA0F14F63F5414- of an easy to-search database on the microbes associated with 728DDDA80A94DF405B8985F0D3. tobacco,” 1990 Bates number 620693477/3480. Retrieved on Nov. 8, 2010 from http://tobaccodocuments.org/rjr/ [18] H. Okino, W. C. Squires, and R. J. Reynolds, “Microbial degradation of nicotine and nicotinic acid. Part I, Isola- 522305212-5212.html. tion of nicotine decomposing bacteria and their morpho- [34] J. D. Hasday, R. Bascom, J. J. Costa, T. Fitzgerald, and logical and physiological properties,” 1954. Bates number W. Dubin, “Bacterial endotoxin is an active component of 508893294/3298. Retrieved on June 24, 2011 from http:// cigarette smoke,” Chest, vol. 115, no. 3, pp. 829–835, 1999. legacy.library.ucsf.edu/tid/xwr83d00. [35] K. S. Lane, “Method and system for assay and removal of [19] A. Wiernik, A. Christakopoulos, L. Johansson, and I. harmful toxins during processing of tobacco products,” US Wahlberg, “Eﬀect of air-curing on the chemical composition patent 6,786,221. September 7, 2004. Journal of Oncology 11 [36] L. Larsson, B. Szponar, and C. Pehrson, “Tobacco smoking [55] H. Van Der Vaart, D. S. Postma, W. Timens, and N. H. T. Ten increases dramatically air concentrations of endotoxin,” Hacken, “Acute eﬀects of cigarette smoke on inﬂammation Indoor Air, vol. 14, no. 6, pp. 421–424, 2004. and oxidative stress: a review,” Thorax, vol. 59, no. 8, pp. 713– [37] W. P. Hempling, G. H. Bokelman, and M. Shulleeta, “Method 721, 2004. [56] M. A. Birrell, S. Wong, M. C. Catley, and M. G. Belvisi, for reduction of tobacco speciﬁc nitrosamines,” US patent 6,755,200, June 29, 2004. “Impact of tobacco-smoke on key signaling pathways in the innate immune response in lung macrophages,” Journal of [38] A. P. Rooney,J. L.Swezey, D. T. Wicklow, and M.J. McAtee, Cellular Physiology, vol. 214, no. 1, pp. 27–37, 2008. “Bacterial species diversity in cigarettes linked to an inves- [57] L. Sorokin, “The impact of the extracellular matrix on in- tigation of severe pneumonitis in U.S. military personnel ﬂammation,” Nature Reviews Immunology, vol. 10, no. 10, pp. deployed in Operation Iraqi Freedom,” Current Microbiology, 712–723, 2010. vol. 51, no. 1, pp. 46–52, 2005. [58] W. Huvenne, C.A.Perez-Novo, L. Derycke et al., “Diﬀerent [39] A. Sebastian, C. Pehrson, and L. Larsson, “Elevated concen- regulation of cigarette smoke induced inﬂammation in upper trations of endotoxin in indoor air due to cigarette smoking,” versus lower airways,” Respiratory Research, vol. 11, no. 110, Journal of Environmental Monitoring, vol. 8, no. 5, pp. 519– pp. 1–9, 2010. 522, 2006. [59] G. S. Kulkarni, P. P. Nadkarni, J. M. Cerreta, S. Ma, and J. [40] M. Zhao, B. Wang, F. Li et al., “Analysis of bacterial commu- O. Cantor, “Short-term cigarette smoke exposure potentiates nities on aging ﬂue-cured tobacco leaves by 16S rDNA PCR- endotoxin-induced pulmonary inﬂammation,” Experimental DGGE technology,” Applied Microbiology and Biotechnology, Lung Research, vol. 33, no. 1, pp. 1–13, 2007. vol. 73, no. 6, pp. 1435–1440, 2007. [60] E. Doz, N. Noulin, E. Boichot et al., “Cigarette smoke- [41] L. Larsson, B. Szponar, B. Ridha et al., “Identiﬁcation of induced pulmonary inﬂammation is TLR4/MyD88 and IL- bacterial and fungal components in tobacco and tobacco 1R1/MyD88 signaling dependent,” Journal of Immunology, smoke,” Tobacco Induced Diseases, vol. 4, no. 4, 2008. vol. 180, no. 2, pp. 1169–1178, 2008. [42] J. L. Pauly, J. D. Waight, and G. M. Paszkiewicz, “Tobacco [61] M. R. Sta ¨mpﬂi and G. P. Anderson, “How cigarette smoke ﬂakes on cigarette ﬁlters grow bacteria: a potential health risk skews immune responses to promote infection, lung disease to the smoker?” Tobacco Control, vol. 17, supplement 1, pp. and cancer,” Nature Reviews Immunology,vol.9, no. 5, pp. i49–i52, 2008. 377–384, 2009. [43] J. Yang, J. Yang, Y. Duan et al., “Bacterial diversities on unaged [62] H. Mehta, K. Nazzal, and R. T. Sadikot, “Cigarette smoking and aging ﬂue-cured tobacco leaves estimated by 16S rRNA and innate immunity,” Inﬂammation Research, vol. 57, no. 11, sequence analysis,” Applied Microbiology and Biotechnology, pp. 497–503, 2008. vol. 88, pp. 553–562, 2010. [63] M. Sopori, “Eﬀects of cigarette smoke on the immune [44] L. M. Coussens and Z. Werb, “Inﬂammation and cancer,” system,” Nature Reviews Immunology, vol. 2, no. 5, pp. 372– Nature, vol. 420, no. 6917, pp. 860–867, 2002. 377, 2002. [45] O. Takeuchi and S. Akira, “Pattern recognition receptors and [64] J. Domagala-Kulawik, “Eﬀects of cigarette smoke on the lung inﬂammation,” Cell, vol. 140, no. 6, pp. 805–820, 2010. and systemic immunity,” Journal of Physiology and Pharma- [46] M. Karin, T. Lawrence, and V. Nizet, “Innate immunity gone cology, vol. 59, no. 6, pp. 19–34, 2008. awry: linking microbial infections to chronic inﬂammation [65] D. G. Yanbaeva,M.A.Dentener, E. C. Creutzberg, G. Wes- and cancer,” Cell, vol. 124, no. 4, pp. 823–835, 2006. seling, and E. F. M. Wouters, “Systemic eﬀects of smoking,” [47] C. Nathan and A. Ding, “Nonresolving inﬂammation,” Cell, Chest, vol. 131, no. 5, pp. 1557–1566, 2007. vol. 140, no. 6, pp. 871–882, 2010. [66] D. Wood, “British-American Tobacco Company,” Prelimi- [48] N. Azad, Y. Rojanasakul, and V. Vallyathan, “Inﬂammation nary observations on the possible transfer of viable micro- and lung cancer: roles of reactive oxygen/nitrogen species,” organisms to mainstream smoke, 1968, Bates number Journal of Toxicology and Environmental Health B,vol.11, no. 570343882/3901. Retrieved on June 24, 2011 from http:// 1, pp. 1–15, 2008. legacy.library.ucsf.edu/tid/jnd51f00. [49] L. Zitvogel, O. Kepp, and G. Kroemer, “Decoding cell death [67] J. Forgacs, Good Samaritan Hospital, Suﬀern, NY to Dr. signals in inﬂammation and immunity,” Cell, vol. 140, no. 6, Robert C. Hockett, Council for Tobacco Research, New pp. 798–804, 2010. York, NY, 2 pages, 2010, http://legacy.library.ucsf.edu/tid/ [50] K. E. de Visser and L. M. Coussens, “The inﬂammatory ppd2aa00. tumor microenvironment and its impact on cancer develop- [68] J. Forgacs, “Mycotoxicoses: the neglected diseases,” Feedstuﬀs, ment,” Contributions to Microbiology, vol. 13, pp. 118–137, vol. 36, no. 18, pp. 124–134, 1966. [69] B. Slutzker, G. Harmon, and P. Edmonds, “Microbiological [51] J. K. Kundu and Y. J. Surh, “Inﬂammation: gearing the jour- content of tobacco smoke,” The American Journal of the ney to cancer,” Mutation Research, vol. 659, no. 1-2, pp. 15– Medical Sciences, vol. 243, pp. 196–201, 1962. 30, 2008. [70] J. M. Greene and S. Caldwell, “Chemical and microbiological [52] E. A. Engles, “Inﬂammation in the development of lung can- changes during ﬂue curing of NK-149 tobacco,” 1989, R. J. cer: epidemiological evidence,” Expert Review of Anticancer Reynolds, Bates number 514848867/8887, retrieved on June Therapy, vol. 8, no. 4, pp. 605–615, 2008. 30, 2010 from http://legacy.library.ucsf.edu/tid/qlm03d00. [53] A. I. D’hulst, K. Y. Vermaelen, G. G. Brusselle, G. F. Joos, [71] Anonymous, Brown & Williamson, “Package 13.0 microbi- and R. A. Pauwels, “Time course of cigarette smoke-induced ology,” No date. Bates number 620648956/9146. Retrieved pulmonary inﬂammation in mice,” European Respiratory on June 28, 2010 from http://legacy.library.ucsf.edu/tid/ Journal, vol. 26, no. 2, pp. 204–213, 2005. key21f00. [54] C. Smith, T. Perfetti, and J. King, “Perspectives on pulmonary [72] Anonymous, British American Tobacco, “Master RD 888-R,” inﬂammation and lung cancer risk in cigarette smokers,” Bates number 105597011/7062. Retrieved on December 11, Inhalation Toxicology, vol. 18, no. 9, pp. 667–677, 2006. 2009 from http://legacy.library.ucsf.edu/tid/unit 37a99. 12 Journal of Oncology [73] C. W. Bacon, R. Wenger, and J. F. Bullock, “Chemical changes on October 27, 2010 from http://tobaccodocuments.org/rjr/ in tobacco during ﬂu-curing,” Industrial & Engineering 500937365-7489.html. Chemistry, vol. 44, no. 2, pp. 292–296, 1952. [90] V. Subbiah, “Sheet2. Tobacco sampling for microﬂora [74] C. O. Jensen, “Uber die natur der tabakfermentation,” Zen- counting,” 1995. Bates number 525450330/0335. Retrieved trabl Bakteriol Parasitenk, Abt II, vol. 21, pp. 469–483, 1908. on October 22, 2010 from http://legacy.library.ucsf.edu/tid/ [75] W. P. Hempling and P. Morris, “Fundamental tobacco micro- ymt60d00. biology,” 1987. Bates number 2022226783/6795. Retrieved [91] W. C. Flanders,R.J.Reynolds, “Quantitative studies of the on March 24, 2010 from http://legacy.library.ucsf.edu/tid/ microbiological ﬂora of tobacco during aging,” 1955. Bates jst58e00. number 501663388/3456. Retrieved on July 1, 2010 from [76] K. J. Brotzge and Brown and Williamson Tobacco Company, http://legacy.library.ucsf.edu/tid/wuk39d00. Quantities of microﬂora recovered from Brown & William- [92] P. C. Stauber, “Investigation of mould growth on stored leaf,” son & competitive cigarette brands, Fall/Winter, 1982, 1983. 1975. Bates number 105425004/5072. Retrieved on June 24, Bates number 598000442/0451. Retrieved on March 16, 2010 2011 from http://legacy.library.ucsf.edu/tid/oit57a99. from http://tobaccodocuments.org/bw/971381.html. [93] R. E. Welty and L. A. Nelson, “Growth of aspergillus repens [77] K. J. Brotzge and Brown & Williamson Tobacco Company, in ﬂue-cured tobacco,” Applied Microbiology, vol. 21, no. 5, “Quantities of microﬂora recovered from Brown & William- pp. 854–859, 1971. son and competitive brands, Spring/Summer 830000,” 1983, [94] T. G. Mitchell, D. A. Johnson, and British-American Tobacco Bates number 657017733/7752. Retrieved on March 16, 2010 Company, “Identiﬁcation of fungi of the Aspergillus ﬂavour from http://legacy.library.ucsf.edu/tid/hnl13f00. group from tobacco,” Report RD 1279. 1975. Bates number [78] L. J. Dewey and G. M. Broaddus, “Bacterial, mold and yeast 105598328/8619. Retrieved on July 1, 2010 from http://li- population counts on RCFS and on RC picked from Pall Mall brary/ucsf/edu/tid/pnp57a99. and Winston cigarettes,” 1970, American Tobacco Company, [95] G. M. Myers, “Aﬂatoxin on tobacco and its removal,” R. J. Bates number 950107079/7080, Retrieved on March 16, 2010 Reynolds, Bates number 519972600/2620. Retrieved on June from http://legacy.library.ucsf.edu/tid/jml11a00. 29, 2010 from http://legacy.library.ucsf.edu/tid/yjk90d00. [79] J. Hill and Brown & Williamson, “Microﬂora standards of [96] J. M. Greene and S. Caldwell, “Chemical and microbiological cocoa casing materials,” 1985. Bates number 62018442/4422. changes during ﬂue curing of NK-149 tobacco,” 1989, R. J. Retrieved on March 17, 2010 from http://legacy.library.ucsf Reynolds, Bates number514848867/8887, Retrieved on June .edu/tid/ski31f00. 30, 2010 from http://legacy.library.ucsf.edu/tid/qlm03d00. [80] M. I. Hofer and Philip Morris, “Research and development— [97] D. G. Vickroy and R. E. Welty, “Evaluations of cigarettes quarterly Report Microbiology 851000/1200,” 1985, Bates made with mold-damaged and nondamaged ﬂue-cured to- bacco,” Beitrag ¨ e zur Tabakforschung, vol. 8, pp. 102–106, number 2028639252/9269. Retrieved on November 8, 2010 from http://legacy.library.ucsf.edu/tid/rew56e00. 1975. [81] Philip Morris, “Biocontrol of tobacco microﬂora,” 1989, [98] M. R. Tansey, “Isolation of thermophilic fungi from snuﬀ,” Bates number 2029139024/9050, retrieved on June 30, 2010 Journal of Applied Microbiology, vol. 29, no. 1, pp. 128–129, from http://legacy.library.ucsf.edu/tid/hoy69e00. 1975. [82] R. E. Welty and American Tobacco, “Plant pathology, 5225, [99] T. Thomas, J. Brandon, W. A. Bailey, and T. A. Kosty, “Me- microﬂora of ﬂue-cured tobacco and their aﬀect on quality,” thod for reducing nitrosamines in tobacco,” US patent 7,757, 1970, Bates number 950251672/1675. Retrieved on June 30, 697. July 20, 2010. 2010 from http://legacy.library.ucsf.edu/tid/ufv31A00. [100] A. Lukic, R. E. Welty, and G. B. Lucas, “Antifungal spectra [83] Anonymous and Philip Morris, “Most populous bacteria: of actinomycetes isolated from tobacco,” Antimicrobial Agents burley tobacco research,” 1999, Bates number 2082730005. and Chemotherapy, vol. 1, no. 4, pp. 363–366, 1972. Retrieved on March 19, 2010 from http://legacy.library.ucsf [101] K. Koga, S. Katsuya, and Japan Tobacco Company, “Method .edu/tid/ddq55c00. of reducing nitrosamines content in tobacco leaves,” US ptent [84] Anonymous, “Further examination of coliform bacteria from 7,556,046. July 7, 2009. cigarettes,” Laboratory report L.337-R. 1970, Bates number [102] M. Cui, M. T. Nielsen, R. R. Hart III, M. L. Overbey, D. 650018029/8046. Retrieved on June 24, 2010 from http:// J. Watson, and J. R. Chipley, “Use of chlorate, sulfur or legacy.library.ucsf.edu/tid/rkl66b00. ozone to reduce tobacco speciﬁc nitrosamine,” U.S. patent 2006/019516 A1. Sept 7, 2006. [85] M. Di Giacomo, M. Paolino, D. Silvestro et al., “Microbial community structure and dynamics of dark ﬁre-cured tobac- [103] R. G. Warke, A. S. Kamat, and M. Y. Kamat, “Irradiation of co fermentation,” Applied and Environmental Microbiology, chewable tobacco mixes for improvement in microbiological vol. 73, no. 3, pp. 825–837, 2007. quality,” Journal of Food Protection, vol. 62, no. 6, pp. 678– [86] T. G. Mitchell and British American Tobacco (BAT), 681, 1999. “Changes in the microﬂora of tobacco leaves during ﬁeld [104] T. G. Mitchell and C. R. Jenkins, “Alternative treatments for growth in England,” 1989, Bates number 400047269/7282, mould control on pipe tobacco,” British American Tobacco, retrieved on June 28, 2010 from http://legacy.library.ucsf Bates number 400661432/1433. Retrieved on June 24, 2011 .edu/tid/num81a99. from http://legacy.library.ucsf.edu/tid/rir56a99. [87] S. A. Ghabrial, “Studies on the microﬂora of air-cured burly [105] D. S. Roth, W. H. Cowart Jr., C. B. Jenkins Jr., and D. M. tobacco,” Tobacco Science, vol. 20, pp. 80–82, 1976. Boyle, “Sterilization process in the manufacturing of snuﬀ,” U.S. patent 5,372,149. December 13, 1994. [88] British American Tobacco, “Film Box Number -1, L1R to L151 R, R&D 1838,” Bates number 402185400/5586, [106] V. Subbiah, “Method of inhibiting mycotoxin production,” US Patent 5,698,599. Dec 16, 1997. Retrieved on June 24, 2010 from http://legacy.library.ucsf .edu/tid/fud91a99. [107] R. P. Newton and Brown & Williamson, “Microbiological [89] W. C. Squires, L. E. Hayes, and R. J. Reynolds, “Tobacco ﬂora: examination of cigarettes,” 1968. Retrieved on June 28, 2010 quantitative studies,” November 9, 1961. 125 pages/Retrieved from http://legacy.library.ucsf.edu/tid/vgj94a99. Journal of Oncology 13 [108] V. C. Johnson, A. M. Palmer, and P. Morris, “Bacteria on cigarette ﬁlters,” Bates number 2000759148. February 12, 1968. Retrieved on November 9, 2010 from http://legacy .library.ucsf.edu/tid/aiw48e00. [109] T. G. Mitchell and British-American Tobacco Company, “Examination of mould—Aﬀected cigarettes from China,” 1989, Bates number 400910779/7783. Retrieved on June 24, 2010 from Legacy at http://legacy.library.ucsf.edu/tid/ qvu0499. [110] J. Hill and Brown & Williamson, “Microbial examination of pipe and smokeless tobacco/541,” 1985, Bates number 620184560/4571. Retrieved on July 22, 2010 from http://leg- acy.library.ucsf.edu/tid/rjq20f00. [111] K. Brotzge and Brown & Williamson, “Microbial exam- ination of pipe, snuﬀ, & chewing tobacco products— Fall/Winter, 83000,” 1984, Bates number 598002147/2156. Retrieved on June 24, 2011 from http://legacy.library.ucsf .edu/tid/zcj41f00. [112] S. K. Varma, A. B. Roy, and A. K. Jha, “Ecotoxicological aspects of Aspergilli present in the phylloplane of shored leaves of chewing tobacco (Nicotiana tobacum),” Myco- pathologia, vol. 113, pp. 19–23, 1991. [113] A. R. Sapkota, S. Berger, and T. M. Vogel, “Human pathogens abundant in the bacterial metagenome of cigarettes,” Envi- ronmental Health Perspectives, vol. 118, no. 3, pp. 351–356, [114] J. Papavassiliou, G. Piperakis, and U. Marcelou-Kinti, “Myco- logical ﬂora of cigarettes,” Mycopathology Mycology Applied, vol. 44, no. 2, pp. 117–120, 1971. [115] V. P. Kurup, A. Resnick, S. L. Kagen, S. H. Cohen, and J. N. Fink, “Allergenic fungi and actinomycetes in smoking materials and their health implications,” Mycopathologica, vol. 82, no. 1, pp. 61–64, 1983. [116] P. E. Verweij, J. J. Kerremans, A. Voss, and J. F. G. M. Meis, “Fungal contamination of tobacco and marijuana,” Journal of the American Medical Association, vol. 284, no. 22, p. 2875, [117] N. B. Rainer, “Cigarettes having minimized loose ends and process for preparing same,” US patent 4,715,388, Dec 29, [118] British American Tobacco Company, “Discussion group on ends quality,” 1985, 161 pages, http://tobaccodocuments.org/ batco/109979765-9924.html. [119] L. Deyton, J. Sharfstein, and M. Hamburg, “Tobacco product regulation—a public health approach,” The New England Journal of Medicine, vol. 362, no. 19, pp. 1753–1756, 2010. 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Cigarette Smoke, Bacteria, Mold, Microbial Toxins, and Chronic Lung Inflammation

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Cigarette Smoke, Bacteria, Mold, Microbial Toxins, and Chronic Lung Inflammation

Cigarette Smoke, Bacteria, Mold, Microbial Toxins, and Chronic Lung Inflammation

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