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      Human Pathogens Abundant in the Bacterial Metagenome of Cigarettes

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          Abstract

          Background

          Many studies have evaluated chemical, heavy metal, and other abiotic substances present in cigarettes and their roles in the development of lung cancer and other diseases, yet no studies have comprehensively evaluated bacterial diversity of cigarettes and the possible impacts of these microbes on respiratory illnesses in smokers and exposed nonsmokers.

          Objectives

          The goal of this study was to explore the bacterial metagenomes of commercially available cigarettes.

          Methods

          A 16S rRNA-based taxonomic microarray and cloning and sequencing were used to evaluate total bacterial diversity of four brands of cigarettes. Normalized microarray data were compared using principal component analysis and hierarchical cluster analysis to evaluate potential differences in microbial diversity across cigarette brands.

          Results

          Fifteen different classes of bacteria and a broad range of potentially pathogenic organisms were detected in all cigarette samples. Most notably, we detected Acinetobacter, Bacillus, Burkholderia, Clostridium, Klebsiella, Pseudomonas aeruginosa, and Serratia in ≥ 90% of all cigarette samples. Other pathogenic bacteria detected included Campylobacter, Enterococcus, Proteus, and Staphylococcus. No significant variability in bacterial diversity was observed across the four different cigarette brands.

          Conclusions

          Previous studies have shown that smoking is associated with colonization by pathogenic bacteria and an increased risk of lung infections. However, this is the first study to show that cigarettes themselves could be the direct source of exposure to a wide array of potentially pathogenic microbes among smokers and other people exposed to secondhand smoke. The overall public health implications of these findings are unclear at this time, and future studies are necessary to determine whether bacteria in cigarettes could play important roles in the development of both infectious and chronic respiratory diseases.

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          Most cited references39

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          Estimates of global mortality attributable to smoking in 2000.

          Smoking is a risk factor for several diseases and has been increasing in many developing countries. Our aim was to estimate global and regional mortality in 2000 caused by smoking, including an analysis of uncertainty. Following the methods of Peto and colleagues, we used lung-cancer mortality as an indirect marker for accumulated smoking risk. Never-smoker lung-cancer mortality was estimated based on the household use of coal with poor ventilation. Relative risks were taken from the American Cancer Society Cancer Prevention Study, phase II, and the retrospective proportional mortality analysis of Liu and colleagues in China. Relative risks were corrected for confounding and extrapolation to other regions. We estimated that in 2000, 4.83 (uncertainty range 3.94-5.93) million premature deaths in the world were attributable to smoking; 2.41 (1.80-3.15) million in developing countries and 2.43 (2.13-2.78) million in industrialised countries. 3.84 million of these deaths were in men. The leading causes of death from smoking were cardiovascular diseases (1.69 million deaths), chronic obstructive pulmonary disease (0.97 million deaths), and lung cancer (0.85 million deaths). Smoking was an important cause of global mortality in 2000. In view of the expected demographic and epidemiological transitions and current smoking patterns in the developing world, the health loss due to smoking will grow even larger unless effective interventions and policies that reduce smoking among men and prevent increases among women in developing countries are implemented.
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            Gastric cancer.

            The past decade has seen many advances in knowledge about gastric cancer. Notably, tumour biology and lymphatic spread are now better understood, and treatment by surgical and medical oncologists has become more standardised. Since refrigerators have replaced other methods of food conservation, Helicobacter pylori has become a factor in the cause of gastric cancer. Cancers that arise at the oesophagogastric junction might be further examples of wealth-associated disease. To tailor treatment better, the western hemisphere needs to borrow from the East by establishing screening programmes for early diagnosis, through careful surgical resection, and through detailed analysis of tumour spread. In Europe and the USA, most patients reach treatment with cancers already at an advanced stage. For these patients, three important randomised trials are underway that evaluate combined therapy. Cytostatic drugs, especially angiogenesis inhibitors have proved disappointing; however, basic research efforts to detect familial gastric cancers and to assess minimally residual disease look more hopeful.
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              High-density universal 16S rRNA microarray analysis reveals broader diversity than typical clone library when sampling the environment.

              Molecular approaches aimed at detection of a broad-range of prokaryotes in the environment routinely rely on classifying heterogeneous 16S rRNA genes amplified by polymerase chain reaction (PCR) using primers with broad specificity. The general method of sampling and categorizing DNA has been to clone then sequence the PCR products. However, the number of clones required to adequately catalog the majority of taxa in a sample is unwieldy. Alternatively, hybridizing target sequences to a universal 16S rRNA gene microarray may provide a more rapid and comprehensive view of prokaryotic community composition. This study investigated the breadth and accuracy of a microarray in detecting diverse 16S rRNA gene sequence types compared to clone-and-sequencing using three environmental samples: urban aerosol, subsurface soil, and subsurface water. PCR products generated from universal 16S rRNA gene-targeted primers were classified by using either the clone-and-sequence method or by hybridization to a novel high-density microarray of 297,851 probes complementary to 842 prokaryotic subfamilies. The three clone libraries comprised 1391 high-quality sequences. Approximately 8% of the clones could not be placed into a known subfamily and were considered novel. The microarray results confirmed the majority of clone-detected subfamilies and additionally demonstrated greater amplicon diversity extending into phyla not observed by the cloning method. Sequences matching operational taxonomic units within the phyla Nitrospira, Planctomycetes, and TM7, which were uniquely detected by the array, were verified with specific primers and subsequent amplicon sequencing. Subfamily richness detected by the array corresponded well with nonparametric richness predictions extrapolated from clone libraries except in the water community where clone-based richness predictions were greatly exceeded. It was concluded that although the microarray is unreliable in identifying novel prokaryotic taxa, it reveals greater diversity in environmental samples than sequencing a typically sized clone library. Furthermore, the microarray allowed samples to be rapidly evaluated with replication, a significant advantage in studies of microbial ecology.
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                Author and article information

                Journal
                Environ Health Perspect
                Environmental Health Perspectives
                National Institute of Environmental Health Sciences
                0091-6765
                1552-9924
                March 2010
                22 October 2009
                : 118
                : 3
                : 351-356
                Affiliations
                [1 ] Maryland Institute for Applied Environmental Health, University of Maryland College Park School of Public Health, College Park, Maryland, USA
                [2 ] Environmental Microbial Genomics Group, Laboratoire Ampère, Ecole Centrale de Lyon, France
                Author notes
                Address correspondence to A.R. Sapkota, University of Maryland College Park, School of Public Health, Maryland Institute for Applied Environmental Health, Room 2234P, SPH Building, College Park, MD 20742 USA. Telephone: (301) 405-1772. Fax: (301) 405-8397. E-mail: ars@ 123456umd.edu

                The authors declare they have no competing financial interests.

                Article
                ehp-118-351
                10.1289/ehp.0901201
                2854762
                20064769
                75008d8e-71ee-4884-a272-e31ff39867c5
                This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI.
                History
                : 14 July 2009
                : 22 October 2009
                Categories
                Research

                Public health
                bacteria,smoking,tobacco,bacterial metagenome,cigarettes,pathogens
                Public health
                bacteria, smoking, tobacco, bacterial metagenome, cigarettes, pathogens

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