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ORIGINAL RESEARCH article

Front. Public Health

Sec. Environmental Health and Exposome

Volume 13 - 2025 | doi: 10.3389/fpubh.2025.1693267

Bacterial Community Structure Associated with Smokeless Tobacco Reference Products under Different Storage Conditions and Durations

Provisionally accepted
Shuang  LiuShuang LiuIsaac  GreenhutIsaac GreenhutLuke  MoeLuke Moe*
  • University of Kentucky Department of Plant and Soil Sciences, Lexington, United States

The final, formatted version of the article will be published soon.

The microbiology of smokeless tobacco products (STPs), such as moist snuff, snus, and loose-leaf chewing tobacco, has recently received significant interest owing to the impact of microbes on product storage and safety. Tobacco leaf-associated microbes, as well as microbes introduced during product manufacturing, may play a role in formation of carcinogenic nitrosamine compounds during manufacturing and product spoilage upon storage. The Center for Tobacco Reference Products at the University of Kentucky has, since 1968, provided tobacco reference products for non-clinical research purposes. These products, including cigarettes, cigars, and STPs, are commercially produced and meant to be representative of off-the-shelf products. Reference products provide the opportunity to enhance reproducibility and reduce batch-to-batch variability. In this study, the microbial communities of smokeless tobacco reference products 3S1 (loose-leaf chewing tobacco), 3S3 (moist snuff), 1S4 (Swedish-style snus) and 1S5 (American snus) were analyzed using culture-based and culture-independent analysis. Bacterial and fungal loads were assessed on three media types, and 16S rDNA amplicon sequencing was used to track the bacterial community structure as a function of time and product storage temperature. Culturable loads were consistently highest with moist snuff (~106-107 CFU/g) and lowest with the snus products (~102-103 CFU/g). Bacterial community structure varied according to product, with Firmicutes and Proteobacteria the primary phyla observed. At the This is a provisional file, not the final typeset article genus level, the most commonly observed operational taxonomic units (OTUs) belonged to Tetragenococcus and Staphylococcus, but their relative abundances differed according to product. The moist snuff product showed the most significant shift in microbial community structure according to storage temperature, with an increase in Atopostipes, Staphylococcus, and Carnobacteriacea OTUs at room temperature and an increase in Lentibacillus at 37ºC. From these studies, we conclude that elevated storage temperatures will alter STP microbial communities but that storage at -20ºC is sufficient for long-term storage of the reference products.

Keywords: Tobacco, microbiome, smokeless tobacco, Reference Product, Tobacco storage

Received: 27 Aug 2025; Accepted: 01 Oct 2025.

Copyright: © 2025 Liu, Greenhut and Moe. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Luke Moe, luke.moe@uky.edu

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