Viable bacteria abundant in cigarettes are aerosolized in mainstream smoke.

Multiple studies have demonstrated that cigarettes harbor bacterial pathogens. Yet, to our knowledge, there are no published data to date on whether or not these microorganisms can be aerosolized and transmitted to the respiratory tract of users. To address this knowledge gap, we characterized cigarette bacterial communities and evaluated whether or not they could be aerosolized in mainstream smoke. Filtered and unfiltered cigarettes were tested. Non-smoked tobacco leaf, enriched non-smoked tobacco leaf extract and enriched mainstream smoke extract samples (n = 144) were incubated on trypticase soy agar, and resulting bacterial colonies were sequenced. Total DNA was also extracted, followed by PCR amplification of the 16S rRNA gene, sequencing and analysis using UCHIME, QIIME and R packages. The predominant bacterial genera cultured from the mainstream smoke of unfiltered cigarettes were Bacillus, Terribacillus, Paenibacillus and Desulfotomaculum. Culturable bacteria were not recovered from the smoke of filtered products. However, sequencing data demonstrated no significant differences in bacterial community diversity in the smoke of filtered versus unfiltered cigarettes, suggesting that other non-culturable bacteria may be aerosolized in mainstream smoke as well. Our study provides novel evidence that tobacco-associated bacterial communities are viable, can be aerosolized in mainstream smoke, and could potentially be transferred to the oral cavity and respiratory tract of smokers.

Investigating the Distribution of Strains of Erwinia amylovora and Streptomycin Resistance in Apple Orchards in New York Using Clustered Regularly Interspaced Short Palindromic Repeat Profiles: A 6-Year Follow-Up.

Fire blight, caused by the bacterium Erwinia amylovora, is one of the most important diseases of apple. The antibiotic streptomycin is routinely used in the commercial apple industries of New York (NY) and New England to manage the disease. In 2002 and again, from 2011 to 2014, outbreaks of streptomycin resistance (SmR) were reported and investigated in NY. Motivated by new grower reports of control failures, we conducted a follow-up investigation of the distribution of SmR and E. amylovora strains for major apple production regions of NY over the last 6 years (2015 to 2020). Characterization of clustered regularly interspaced short palindromic repeat (CRISPR) profiles revealed that a few "cosmopolitan" strains were widely prevalent across regions, whereas many other "resident" strains were confined to one location. In addition, we uncovered novel CRISPR profile diversity in all investigated regions. SmR E. amylovora was detected only in a small area spanning two counties from 2017 to 2020 and was always associated with one CRISPR profile (41:23:38), which matched the profile of SmR E. amylovora, discovered in 2002. This suggests the original SmR E. amylovora was never fully eradicated and went undetected because of several seasons of low disease pressure in this region. Investigation of several representative isolates under controlled greenhouse conditions indicated significant differences in aggressiveness on 'Gala' apples. Potential implications of strain differences include the propensity of strains to become distributed across wide geographic regions and associated resistance management practices. Results from this work will directly influence sustainable fire blight management recommendations for commercial apple industries in NY state and other regions.

Using full chloroplast genomes of 'red' and 'yellow' Bixa orellana (achiote) for kmer based identification and phylogenetic inference.

BACKGROUND: Full chloroplast genomes provide high resolution taxonomic discrimination between closely related plant species and are quickly replacing single and multi-locus barcoding regions as reference materials of choice for DNA based taxonomic annotation of plants. Bixa orellana, commonly known as "achiote" and "annatto" is a plant used for both human and animal foods and was thus identified for full chloroplast sequencing for the Center for Veterinary Medicine (CVM) Complete Chloroplast Animal Feed database. This work was conducted in collaboration with the Instituto de Medicina Tradicional (IMET) in Iquitos, Peru. There is a wide range of color variation in pods of Bixa orellana for which genetic loci that distinguish phenotypes have not yet been identified. Here we apply whole chloroplast genome sequencing of "red" and "yellow" individuals of Bixa orellana to provide high quality reference genomes to support kmer database development for use identifying this plant from complex mixtures using shotgun data. Additionally, we describe chloroplast gene content, synteny and phylogeny, and identify an indel and snp that may be associated with seed pod color. RESULTS: Fully assembled chloroplast genomes were produced for both red and yellow Bixa orellana accessions (158,918 and 158,823 bp respectively). Synteny and gene content was identical to the only other previously reported full chloroplast genome of Bixa orellana (NC_041550). We observed a 17 base pair deletion at position 58,399-58,415 in both accessions, relative to NC_041550 and a 6 bp deletion at position 75,531-75,526 and a snp at position 86,493 in red Bixa orellana. CONCLUSIONS: Our data provide high quality reference genomes of individuals of red and yellow Bixa orellana to support kmer based identity markers for use with shotgun sequencing approaches for rapid, precise identification of Bixa orellana from complex mixtures. Kmer based phylogeny of full chloroplast genomes supports monophylly of Bixaceae consistent with alignment based approaches. A potentially discriminatory indel and snp were identified that may be correlated with the red phenotype.

Metabolically-active bacteria in reclaimed water and ponds revealed using bromodeoxyuridine DNA labeling coupled with 16S rRNA and shotgun sequencing.

Understanding the complex microbiota of agricultural irrigation water is vital to multiple sectors of sustainable agriculture and public health. To date, microbiome characterization methods have provided comprehensive profiles of aquatic microbiotas, but have not described which taxa are likely metabolically-active. Here, we combined 5­bromo­2'-deoxyuridine (BrdU) labeling with 16S rRNA and shotgun sequencing to identify metabolically-active bacteria in reclaimed and agricultural pond water samples (n = 28) recovered from the Mid-Atlantic United States between March 2017 and January 2018. BrdU-treated samples were significantly less diverse (alpha diversity) compared to non-BrdU-treated samples. The most abundant taxa in the metabolically-active fraction of water samples (BrdU-treated samples) were unclassified Actinobacteria, Flavobacterium spp., Pseudomonas spp. and Aeromonas spp. Interestingly, we also observed that antimicrobial resistance and virulence gene profiles seemed to be more diverse and more abundant in non-BrdU-treated water samples compared to BrdU-treated samples. These findings raise the possibility that these genes may be associated more with relic (inactive) DNA present in the tested water types rather than viable, metabolically-active microorganisms. Our study demonstrates that the coupled use of BrdU labeling and sequencing can enhance understanding of the metabolically-active fraction of bacterial communities in alternative irrigation water sources. Agricultural pond and reclaimed waters are vital to the future of sustainable agriculture, and thus, the full understanding of the pathogenic potential of these waters is important to guide mitigation strategies that ensure appropriate water quality for intended purposes.

Microbiota of the Hickey Run Tributary of the Anacostia River.

Water from the Hickey Run Tributary of the Anacostia River is being collected quarterly (beginning August 2018) and analyzed to create high-resolution baseline taxonomic profiles of microbiota associated with this important aquatic ecosystem, which has a long history of exposure to residential and commercial effluents from Washington, DC. These United States National Arboretum Microbial Observatory data are available under NCBI BioProject number PRJNA498951.