Persistence of Escherichia coli in the microbiomes of red Romaine lettuce (Lactuca sativa cv. ‘Outredgeous’) and mizuna mustard (Brassica rapa var. japonica) - does seed sanitization matter?

Abstract Background Seed sanitization via chemical processes removes/reduces microbes from the external surfaces of the seed and thereby could have an impact on the plants’ health or productivity. To determine the impact of seed sanitization on the plants’ microbiome and pathogen persistence, sanitized and unsanitized seeds from two leafy green crops, red Romaine lettuce (Lactuca sativa cv. ‘Outredgeous’) and mizuna mustard (Brassica rapa var. japonica) were exposed to Escherichia coli and grown in controlled environment growth chambers simulating environmental conditions aboard the International Space Station. Plants were harvested at four intervals from 7 days post-germination to maturity. The bacterial communities of leaf and root were investigated using the 16S rRNA sequencing while quantitative polymerase chain reaction (qPCR) and heterotrophic plate counts were used to reveal the persistence of E. coli. Result E. coli was detectable for longer periods of time in plants from sanitized versus unsanitized seeds and was identified in root tissue more frequently than in leaf tissue. 16S rRNA sequencing showed dynamic changes in the abundance of members of the phylum Proteobacteria, Firmicutes, and Bacteroidetes in leaf and root samples of both leafy crops. We observed minimal changes in the microbial diversity of lettuce or mizuna leaf tissue with time or between sanitized and unsanitized seeds. Beta-diversity showed that time had more of an influence on all samples versus the E. coli treatment. Conclusion Our results indicated that the seed surface sanitization, a current requirement for sending seeds to space, could influence the microbiome. Insight into the changes in the crop microbiomes could lead to healthier plants and safer food supplementation.

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Comparison of MI, Chromocult® coliform, and Compass CC chromogenic culture-based methods to detect Escherichia coli and total coliforms in water using 16S rRNA sequencing for colony identification

Journal of Water and Health ◽

10.2166/wh.2017.174 ◽

2017 ◽

Vol 15 (3) ◽

pp. 353-359

Author(s):

Andrée F. Maheux ◽

Sébastien Bouchard ◽

Ève Bérubé ◽

Michel G. Bergeron

Keyword(s):

Public Health ◽

Escherichia Coli ◽

16S Rrna ◽

Distribution System ◽

Water Distribution ◽

Total Coliform ◽

16S Rrna Sequencing ◽

Total Coliforms ◽

Specificity And Sensitivity ◽

Rrna Sequencing

The MI, Chromocult® coliform, and Compass CC chromogenic culture-based methods used to assess water quality by the detection of Escherichia coli and total coliforms were compared in terms of their specificity and sensitivity, using 16S rRNA sequencing for colony identification. A sewage water sample was divided in 2-μL subsamples for testing by all three culture-based methods. All growing colonies were harvested and subjected to 16S rRNA sequencing. Test results showed that all E. coli colonies were correctly identified by all three methods, for a specificity and a sensitivity of 100%. However, for the total coliform detection, the MI agar, Chromocult® coliform agar, and Compass CC agar were specific for only 69.2% (9/13), 47.2% (25/53), and 40.5% (17/42), whereas sensitive for 97.8% (45/46), 97.5% (39/40), and 85.7% (24/28), respectively. Thus, given the low level of specificity of these methods for the detection of total coliforms, confirming the identity of total coliform colonies could help to take public health decisions, in particular for cities connected to a public drinking water distribution system since the growth of few putative total coliform colonies on chromogenic agar is problematic and can lead to unnecessary and costly boiling notices from public health authorities.

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The Limits and Avoidance of Biases in Metagenomic Analyses of Human Fecal Microbiota

Microorganisms ◽

10.3390/microorganisms8121954 ◽

2020 ◽

Vol 8 (12) ◽

pp. 1954

Author(s):

Emma Bergsten ◽

Denis Mestivier ◽

Iradj Sobhani

Keyword(s):

16S Rrna ◽

Dna Extraction ◽

Fecal Microbiota ◽

16S Rrna Sequencing ◽

Marker Genes ◽

Bacteria Identification ◽

16S Rna ◽

Rrna Sequencing ◽

The Impact ◽

Identification And Quantification

An increasing body of evidence highlights the role of fecal microbiota in various human diseases. However, more than two-thirds of fecal bacteria cannot be cultivated by routine laboratory techniques. Thus, physicians and scientists use DNA sequencing and statistical tools to identify associations between bacterial subgroup abundances and disease. However, discrepancies between studies weaken these results. In the present study, we focus on biases that might account for these discrepancies. First, three different DNA extraction methods (G’NOME, QIAGEN, and PROMEGA) were compared with regard to their efficiency, i.e., the quality and quantity of DNA recovered from feces of 10 healthy volunteers. Then, the impact of the DNA extraction method on the bacteria identification and quantification was evaluated using our published cohort of sample subjected to both 16S rRNA sequencing and whole metagenome sequencing (WMS). WMS taxonomical assignation employed the universal marker genes profiler mOTU-v2, which is considered the gold standard. The three standard pipelines for 16S RNA analysis (MALT and MEGAN6, QIIME1, and DADA2) were applied for comparison. Taken together, our results indicate that the G’NOME-based method was optimal in terms of quantity and quality of DNA extracts. 16S rRNA sequence-based identification of abundant bacteria genera showed acceptable congruence with WMS sequencing, with the DADA2 pipeline yielding the highest congruent levels. However, for low abundance genera (<0.5% of the total abundance) two pipelines and/or validation by quantitative polymerase chain reaction (qPCR) or WMS are required. Hence, 16S rRNA sequencing for bacteria identification and quantification in clinical and translational studies should be limited to diagnostic purposes in well-characterized and abundant genera. Additional techniques are warranted for low abundant genera, such as WMS, qPCR, or the use of two bio-informatics pipelines.

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Transient Effect of Infant Formula Supplementation on the Intestinal Microbiota

Nutrients ◽

10.3390/nu13030807 ◽

2021 ◽

Vol 13 (3) ◽

pp. 807

Author(s):

Ning Chin ◽

Gema Méndez-Lagares ◽

Diana H. Taft ◽

Victoria Laleau ◽

Hung Kieu ◽

...

Keyword(s):

16S Rrna ◽

Gut Microbiome ◽

Bacterial Species ◽

Bifidobacterium Longum ◽

The United States ◽

16S Rrna Sequencing ◽

T Cell Subsets ◽

Rrna Sequencing ◽

Stool Samples ◽

The Impact

Breastfeeding is the gold standard for feeding infants because of its long-term benefits to health and development, but most infants in the United States are not exclusively breastfed in the first six months. We enrolled 24 infants who were either exclusively breastfed or supplemented with formula by the age of one month. We collected diet information, stool samples for evaluation of microbiotas by 16S rRNA sequencing, and blood samples for assessment of immune development by flow cytometry from birth to 6 months of age. We further typed the Bifidobacterium strains in stool samples whose 16S rRNA sequencing showed the presence of Bifidobacteriaceae. Supplementation with formula during breastfeeding transiently changed the composition of the gut microbiome, but the impact dissipated by six months of age. For example, Bifidobacterium longum, a bacterial species highly correlated with human milk consumption, was found to be significantly different only at 1 month of age but not at later time points. No immunologic differences were found to be associated with supplementation, including the development of T-cell subsets, B cells, or monocytes. These data suggest that early formula supplementation, given in addition to breast milk, has minimal lasting impact on the gut microbiome or immunity.

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Haemophilus parainfluenzaeInfective Endocarditis Diagnosed by Direct 16S rRNA Sequencing of Vegetation

Laboratory Medicine Online ◽

10.3343/lmo.2012.2.2.111 ◽

2012 ◽

Vol 2 (2) ◽

pp. 111

Author(s):

Sung-Hee Oh ◽

Min-Chul Cho ◽

Jae-Wook Kim ◽

Dongheui An ◽

Mun-Hui Jeong ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Sequencing ◽

Rrna Sequencing

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Full-length versus short amplicon 16S rRNA sequencing: benefits, drawbacks and risks

10.26226/morressier.5f3288739a6b12016a74ff57 ◽

2020 ◽

Author(s):

Isabel Abellan-Schneyder ◽

Andrea Janina Bayer ◽

Sandra Reitmeier ◽

Klaus Neuhaus

Keyword(s):

16S Rrna ◽

Full Length ◽

16S Rrna Sequencing ◽

Rrna Sequencing

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Full-length versus short amplicon 16S rRNA sequencing: benefits, drawbacks and risks

10.26226/morressier.5f3392ca9d1718ca4c8b2f06 ◽

2020 ◽

Author(s):

Andrea Janina Bayer ◽

Sandra Reitmeier ◽

Klaus Neuhaus ◽

Isabel Abellan-Schneyder

Keyword(s):

16S Rrna ◽

Full Length ◽

16S Rrna Sequencing ◽

Rrna Sequencing

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Phenotypic and phylogenetic characterization of Lactobacillus species isolated from traditional Lighvan cheese

Food Production, Processing and Nutrition ◽

10.1186/s43014-021-00065-x ◽

2021 ◽

Vol 3 (1) ◽

Author(s):

Haleh Forouhandeh ◽

Sepideh Zununi Vahed ◽

Hossein Ahangari ◽

Vahideh Tarhriz ◽

Mohammad Saeid Hejazi

Keyword(s):

16S Rrna ◽

Random Amplified Polymorphic Dna ◽

Bacterial Species ◽

16S Rrna Sequencing ◽

Lactobacillus Species ◽

Culture Methods ◽

Phylogenetic Characterization ◽

Rrna Sequencing ◽

Specific Culture ◽

Lighvan Cheese

Abstract Lighvan cheese (Lighvan panir) is among the most famous traditional cheese in Iran for its desired aroma and flavor. Undoubtedly, the lactic acid bacteria especially the genus Lactobacillus are the critical factors in developing the aroma, flavor, and texture in Lighvan cheese. In this study, the Lactobacillus population of the main Lighvan cheese was investigated. The Lactobacillus of the main Lighvan cheese was isolated using specific culture methods according to previously published Guidelines. Then, the phylogenetic features were investigated and the phenotypic characteristics were examined using specific culture methods. Twenty-eight Gram-positive bacterial species were identified belonged to the genus Lactobacillus. According to the same sequences as each other, three groups (A, B, and C) of isolates were categorized with a high degree of similarity to L. fermentum (100%) and L. casei group (L. casei, L. paracasei, and L. rhamnosus) (99.0 to 100%). Random amplified polymorphic DNA (RAPD) fingerprint analysis manifested the presence of three clusters that were dominant in traditional Lighvan cheese. Cluster І was divided into 4 sub-clusters. By the result of carbohydrate fermentation pattern and 16S rRNA sequencing, isolates were identified as L. rhamnosus. The isolates in clusters II and III represented L. paracasei and L. fermentum, respectively as they were identified by 16S rRNA sequencing and fermented carbohydrate patterns. Our result indicated that the specific aroma and flavor of traditional Lighvan cheese can be related to its Lactobacillus population including L. fermentum, L. casei, L. paracasei, and L. rhamnosus. Graphical abstract

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Coupled DNA-labeling and sequencing approach enables the detection of viable-but-non-culturable Vibrio spp. in irrigation water sources in the Chesapeake Bay watershed

Environmental Microbiome ◽

10.1186/s40793-021-00382-1 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

Leena Malayil ◽

Suhana Chattopadhyay ◽

Emmanuel F. Mongodin ◽

Amy R. Sapkota

Keyword(s):

16S Rrna ◽

Irrigation Water ◽

Brackish Water ◽

Water Sources ◽

16S Rrna Sequencing ◽

Vbnc State ◽

Recycled Water ◽

Dna Labeling ◽

Rrna Sequencing ◽

Brdu Labeling

AbstractNontraditional irrigation water sources (e.g., recycled water, brackish water) may harbor human pathogens, including Vibrio spp., that could be present in a viable-but-nonculturable (VBNC) state, stymieing current culture-based detection methods. To overcome this challenge, we coupled 5-bromo-2′-deoxyuridine (BrdU) labeling, enrichment techniques, and 16S rRNA sequencing to identify metabolically-active Vibrio spp. in nontraditional irrigation water (recycled water, pond water, non-tidal freshwater, and tidal brackish water). Our coupled BrdU-labeling and sequencing approach revealed the presence of metabolically-active Vibrio spp. at all sampling sites. Whereas, the culture-based method only detected vibrios at three of the four sites. We observed the presence of V. cholerae, V. vulnificus, and V. parahaemolyticus using both methods, while V. aesturianus and V. shilonii were detected only through our labeling/sequencing approach. Multiple other pathogens of concern to human health were also identified through our labeling/sequencing approach including P. shigelloides, B. cereus and E. cloacae. Most importantly, 16S rRNA sequencing of BrdU-labeled samples resulted in Vibrio spp. detection even when our culture-based methods resulted in negative detection. This suggests that our novel approach can effectively detect metabolically-active Vibrio spp. that may have been present in a VBNC state, refining our understanding of the prevalence of vibrios in nontraditional irrigation waters.

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Antibiotic-Induced Dysbiosis of Microbiota Promotes Chicken Lipogenesis by Altering Metabolomics in the Cecum

Metabolites ◽

10.3390/metabo11080487 ◽

2021 ◽

Vol 11 (8) ◽

pp. 487

Author(s):

Tao Zhang ◽

Hao Ding ◽

Lan Chen ◽

Yueyue Lin ◽

Yongshuang Gong ◽

...

Keyword(s):

16S Rrna ◽

Gut Microbiota ◽

High Performance ◽

Fat Deposition ◽

16S Rrna Sequencing ◽

Oral Antibiotics ◽

Ms Analysis ◽

Rrna Sequencing ◽

Cecal Microbiota ◽

Metabolite Network

Elucidation of the mechanism of lipogenesis and fat deposition is essential for controlling excessive fat deposition in chicken. Studies have shown that gut microbiota plays an important role in regulating host lipogenesis and lipid metabolism. However, the function of gut microbiota in the lipogenesis of chicken and their relevant mechanisms are poorly understood. In the present study, the gut microbiota of chicken was depleted by oral antibiotics. Changes in cecal microbiota and metabolomics were detected by 16S rRNA sequencing and ultra-high performance liquid chromatography coupled with MS/MS (UHPLC–MS/MS) analysis. The correlation between antibiotic-induced dysbiosis of gut microbiota and metabolites and lipogenesis were analysed. We found that oral antibiotics significantly promoted the lipogenesis of chicken. 16S rRNA sequencing indicated that oral antibiotics significantly reduced the diversity and richness and caused dysbiosis of gut microbiota. Specifically, the abundance of Proteobacteria was increased considerably while the abundances of Bacteroidetes and Firmicutes were significantly decreased. At the genus level, the abundances of genera Escherichia-Shigella and Klebsiella were significantly increased while the abundances of 12 genera were significantly decreased, including Bacteroides. UHPLC-MS/MS analysis showed that antibiotic-induced dysbiosis of gut microbiota significantly altered cecal metabolomics and caused declines in abundance of 799 metabolites and increases in abundance of 945 metabolites. Microbiota-metabolite network revealed significant correlations between 4 differential phyla and 244 differential metabolites as well as 15 differential genera and 304 differential metabolites. Three metabolites of l-glutamic acid, pantothenate acid and N-acetyl-l-aspartic acid were identified as potential metabolites that link gut microbiota and lipogenesis in chicken. In conclusion, our results showed that antibiotic-induced dysbiosis of gut microbiota promotes lipogenesis of chicken by altering relevant metabolomics. The efforts in this study laid a basis for further study of the mechanisms that gut microbiota regulates lipogenesis and fat deposition of chicken.

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