16s rrna sequencing
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Animals ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 118
Author(s):  
Zhengwen Wang ◽  
Xiongxiong Li ◽  
Lingyun Zhang ◽  
Jianping Wu ◽  
Shengguo Zhao ◽  
...  

The objective of this experiment was to evaluate the effect of different EOC (0.1425% cobalt lactate + 1.13% oregano essential oil + 98.7275% carrier) levels on in vitro rumen fermentation and microbial changes. Six EOC levels (treatments: 0 mg·L−1, CON; 50 mg·L−1, EOC1; 100 mg·L−1, EOC2; 400 mg·L−1, EOC3; 800 mg·L−1, EOC4 and 1500 mg·L−1, EOC5) were selected to be used to in vitro incubation. The in vitro dry matter digestibility (IVDMD), in vitro neutral detergent fiber digestibility (IVNDFD), in vitro acid detergent fiber digestibility (IVNDFD), pH, ammonia-nitrogen (NH3-N) concentration, total volatile fatty acid (TVFA) concentration and microbial protein (MCP) concentration were measured after 48 h incubation, after which the groups with significant nutrient digestibility and fermentation parameters were subjected to 16S rRNA sequencing. The results showed that the total gas production (GP) of the EOC5 group was higher than that of the other groups after 12 h of in vitro incubation. TVFA, NH3-N and MCP concentrations were also shown to be higher in group EOC5 than those in other groups (p < 0.05), while NH3-N and MCP concentrations in the EOC2 group were lower than those in other groups significantly (p < 0.05). The molar ratio of acetic acid decreased while the molar ratio of propionic acid increased after the addition of EOC. 16S rRNA sequencing revealed that the rumen microbiota was altered in response to adding EOC, especially for the EOC5 treatment, with firmicutes shown to be the most abundant (43.1%). The relative abundance of Rikenellaceae_RC9_gut_group was significantly lower, while the relative abundance of uncultured_bacterium_f_Muribaculaceae and Succiniclasticum was significantly higher in the EOC5 group than those in other groups (p < 0.05). Comprehensive analysis showed that EOC (1500 mg·L−1) could significantly increase gas production, alter sheep rumen fermentation parameters and microbiota composition.


Cancers ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 7
Author(s):  
Patrycja Proc ◽  
Joanna Szczepańska ◽  
Beata Zarzycka ◽  
Małgorzata Szybka ◽  
Maciej Borowiec ◽  
...  

A child’s mouth is the gateway to many species of bacteria. Changes in the oral microbiome may affect the health of the entire body. The aim of the study was to evaluate the changes in the oral microbiome of childhood cancer survivors. Saliva samples before and after anti-cancer treatment were collected from 20 patients aged 6–18 years, diagnosed de novo with cancer in 2018–2019 (7 girls and 13 boys, 7.5–19 years old at the second time point). Bacterial DNA was extracted, and the microbial community profiles were assessed by 16S rRNA sequencing. The relative abundances of Cellulosilyticum and Tannerella genera were found to significantly change throughout therapy (p = 0.043 and p = 0.036, respectively). However, no differences in the alpha-diversity were observed (p = 0.817). The unsupervised classification revealed two clusters of patients: the first with significant changes in Campylobacter and Fusobacterium abundance, and the other with change in Neisseria. These two groups of patients differed in median age (10.25 vs. 16.16 years; p = 0.004) and the length of anti-cancer therapy (19 vs. 4 months; p = 0.003), but not cancer type or antibiotic treatment.


Author(s):  
Noel Gahamanyi ◽  
Leonard E.G. Mboera ◽  
Mecky I. Matee ◽  
Dieudonné Mutangana ◽  
Raghavendra G. Amachawadi ◽  
...  

A growing number of Campylobacter species other than C. jejuni and C. coli have been considered as emerging human and animal pathogens but their contribution to human gastroenteritis is poorly documented. This study aimed at detecting Campylobacter species from human and cattle faecal samples in Kilosa District, Tanzania using molecular techniques without culture. Seventy (70) faecal samples were collected from five diarrheic and 65 non-diarrheic human patients attending Kilosa District Hospital in Tanzania from July to October 2019. During the same period, 30 faecal samples were also collected from healthy cattle in the same district. Genus and species identification of Campylobacter was conducted on the samples using molecular techniques [the polymerase chain reaction (PCR) and 16S rRNA sequencing]. Phylogenetic analysis was carried out by comparison of the 16S rRNA gene sequences to reference strains by the Neighbor-Joining method in MEGA X. Campylobacter species detection rate by PCR was 65.7% (46/70) and 20% (6/30) in humans and cattle, respectively.  There were five human diarrheic cases, four of which were positive for Campylobacter and of these, two were children ≤15 years of age. In humans, 16S rRNA sequencing revealed that C. concisus was the most predominant species occurring at a frequency of 37.8% (14/37), followed by uncultured Campylobacter spp. 24.3% (9/37) and C. hominis 21.6% (8/37). The least represented species were C. jejuni and C. lanienae, all occurring at 2.7% (1/37). In cattle, five (100%) sequenced PCR products matched with C. lanienae. Phylogenetic analysis revealed that with the exception of C. lanienae, 16S rRNA sequences of Campylobacter species were closely related to the reference strains used (Percent identity: 90.51-96.56%). Based on our findings, we recommend that molecular techniques, mainly PCR be adopted for the direct detection of Campylobacter species during laboratory screening and surveillance studies.


Author(s):  
Qian Jiang ◽  
Xing Liu ◽  
Qifen Yang ◽  
Liang Chen ◽  
Deqin Yang

Microorganisms are confirmed to be closely related to the occurrence and development of cancers in human beings. However, there has been no published report detailing relationships between the oral microbiota and salivary adenoid cystic carcinoma (SACC). In this study, unstimulated saliva was collected from 13 SACC patients and 10 healthy controls. The microbial diversities, compositions and functions were comprehensively analyzed after 16S rRNA sequencing and whole-genome shotgun metagenomic sequencing. The alpha diversity showed no significant difference between SACC patients and healthy controls, while beta diversity showed a separation trend. The SACC patients showed higher abundances of Streptococcus and Rothia, while Prevotella and Alloprevotella were more abundant in healthy controls. The prevalent KEGG pathways, carbohydrate-active enzymes, antibiotic resistances and virulence factors as well as the biomarkers in SACC were determined by functional gene analysis. Our study preliminarily investigated the salivary microbiome of SACC patients compared with healthy controls and might be the basis for further studies on novel diagnostic and treatment strategies.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4005-4005
Author(s):  
Maren Schmiester ◽  
René Maier ◽  
René Riedel ◽  
Marco Frentsch ◽  
Robert R. Jenq ◽  
...  

Abstract Introduction Microbial dysbiosis is associated with increased infectious complications and poorer treatment outcomes in patients with hematologic malignancies (Galloway-Pena et al., 2016, Peled et al., 2020). While the effective reconstitution of microbial diversity by autologous fecal microbiota transfer appears within clinical reach (Malard et al, 2021), diagnosis of dysbiosis typically relies on comparatively slow and laborious molecular techniques. We validated a fast and reliable method to assess and track microbial diversity based on flow cytometric analysis (FCM) of single cell phenotypical traits in stool samples of patients undergoing therapy for aggressive lymphoma disease. Methods Stool samples of 12 patients with B-cell neoplasms and 1 patient with a T-cell neoplasm were collected at the time of diagnosis, directly before each cycle of chemoimmunotherapy administration (CHOP backbone +/- anti-CD20 therapy) and every 3 months during follow-up (2-11 samples per patient totaling n = 77 samples). Microbiome analyses were performed by sequencing the V3/V4 16S rRNA region and by FCM of light scatter properties and DNA content (DAPI staining). Phenotypic and taxonomic alpha diversity (inverse Simpson index, D2) was determined from the flow cytometric profiles and sequencing data, respectively, using the PhenoFlow R package (Props et al., 2016). Longitudinal trends in alpha diversity were evaluated with the SplinectomeR R package (Shields-Cutler et al., 2018). Results Correlation analysis and ordinary least squares regression confirmed a statistically significant association between FCM-based phenotypic and sequencing-based taxonomic alpha diversity in "real-world" patient fecal samples (Pearson's correlation r p = 0.56, r 2 = 0.32, p &lt; 0.001) (Figure 1). Despite the distinct and complex data types generated by the two methods, these results demonstrate that phenotypic diversity measurements obtained from FCM can serve as proxies for taxonomic diversity measurements obtained from 16S rRNA sequencing. Next, we analyzed the dynamics of microbial alpha diversity in our cohort over the course of therapy and follow-up using a spline-based approach optimized for longitudinal data. We demonstrated a statistically significant non-linear trend of alpha diversity with a decrease during the period of chemotherapy administration followed by reconstitution during the immunotherapy and follow-up period (Figure 2) using both FCM and 16S rRNA sequencing (p = 0.001). Notably, the samples collected during the treatment phase were obtained 2-3 weeks after the last administration of chemoimmunotherapy, depicting a progressive and sustained dysbiosis in patients during the months of chemotherapy. Conclusions We demonstrated a clear correlation between microbial alpha diversity as determined by 16S rRNA sequencing and by our flow cytometric-based approach. Using FCM analysis, we found a marked decline in diversity during chemotherapy administration. These results are in line with the dysbiosis pattern observed by 16S rRNA sequencing and with previous studies (Rashidi et al., 2019). Our findings thus demonstrate that FCM could provide a fast, first-line point-of-care monitoring of microbial diversity dynamics. Figure 1 Figure 1. Disclosures Jenq: Merck: Consultancy; Karius: Consultancy; Microbiome DX: Consultancy; Seres: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Kaleido: Membership on an entity's Board of Directors or advisory committees; MaaT Pharma: Membership on an entity's Board of Directors or advisory committees; Prolacta: Membership on an entity's Board of Directors or advisory committees; LISCure: Membership on an entity's Board of Directors or advisory committees. Bullinger: Daiichi Sankyo: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Sanofi: Honoraria; Amgen: Honoraria; Gilead: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Hexal: Consultancy; Novartis: Consultancy, Honoraria; Bayer: Research Funding; Pfizer: Consultancy, Honoraria; Seattle Genetics: Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Menarini: Consultancy; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Astellas: Honoraria. Na: Octapharma: Honoraria, Research Funding; Shire/Takeda: Honoraria, Research Funding; Bristol Myers Squibb: Research Funding.


2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S599-S600
Author(s):  
Hadar Mudrik-Zohar ◽  
Shaqed Carasso ◽  
Tal Gefen ◽  
Anat Zalmanovich ◽  
Michal Katzir ◽  
...  

Abstract Background Infected diabetic foot ulcers (IDFU) are a major complication of diabetes mellitus. These potentially limb-threatening ulcers are challenging to treat due to the impairment of wound healing in diabetic patients and the complex microbial environment characterizing these ulcers. Our aim was to analyze the microbiome of IDFU in association with clinical outcomes. Methods Wound biopsies from IDFU were obtained from hospitalized patients and were analyzed using traditional microbiology cultures, 16S rRNA sequencing and shotgun metagenomic sequencing. Patients’ characteristics, culture-based results and sequencing data were analyzed in association with clinical outcomes. Study Design Results 31 patients were enrolled. Significantly more anaerobic and Gram-negative bacteria were detected with sequencing methods compared to conventional cultures (59% and 76% were anaerobes according to 16SrRNA and metagenomic respectively vs. 26% in cultures, p=0.001, and 79%, 59% and 54% were Gram negative bacteria respectively, p&lt; 0.001). Culture-based results showed that Staphylococcus aureus was more prevalent among patients who were conservatively treated (p=0.048). In metagenomic analysis the Bacteroides genus was more prevalent among patients who underwent toe amputation (p&lt; 0.001). Analysis of metagenomic-based functional data showed that antibiotic resistance genes and genes related to biofilm production and to bacterial virulent factors were more prevalent in IDFU that resulted in toe amputation (p&lt; 0.001). Occurrences and mean relative abundances of the most prevalent bacteria of IDFU Comparison between [A] traditional cultures, 16S rRNA sequencing and metagenomic sequencing results (genera level - 12 samples) [B] traditional cultures and metagenomic sequencing results (species level – 30 samples) [C] traditional cultures and 16S rRNA sequencing results (genera level - 30 samples) CUL – cultures; 16S - 16S rRNA sequencing; MTG – metagenomic sequencing Bacteroides genus association with toe amputation Bacteroides genera was more common among samples of patients who underwent toe amputation compared with samples of patients who were conservatively treated (p &lt; 0.001). Species level analysis showed that Bacteroides fragilis and Bacteroides xylanisolvens predominated IDFU of patients who underwent toe amputation (p=0.04, p=0.002 respectively). No – conservative treatment; Yes – toe amputation. Functional genes differentiating patients who underwent toe amputation from conservatively treated Yellow stars – indicate genes that were associated with bacterial virulent factors, biofilm formation and resistant mechanisms – all were more prevalent in patients who underwent toe amputation (with p values&lt;&gt; Conclusion Molecular sequencing tools uncover the complex biodiversity of IDFU and emphasize the high prevalence of anaerobes and Gram-negative bacteria in these ulcers. Furthermore, sequencing results highlighted the possible association between certain genera, species, and bacterial functional genes to clinical outcomes Disclosures Yossi Paitan, PhD, Ilex Medical Ltd (Employee, Other Financial or Material Support, As of 01.01.2021 I am the Laboratories Manager of Ilex Labs)


Author(s):  
Shweta Singh ◽  
Saswati Chakraborty

Abstract This study demonstrated the successful utilization of a lab-scale baffled horizontal constructed wetland substituted with mixed organic media for zinc removal from high acidity (∼610 mg L−1 as CaCO3) sulfate-rich (∼1,300 mg L−1) wastewater. The wetland was planted with Typha latifolia. The mean zinc concentration in the influent was gradually increased from 0.56 ± 0.02 mg L−1 to 5.3 ± 0.42 mg L−1. The mean zinc concentration in the outflow was 0.22 ± 0.19 mg L−1, accounting 95% zinc removal throughout the study. However, total zinc uptake by plants was 533 mg kg−1, accounting for only 1.2% of total zinc removal and therefore, major zinc retention occurred within wetland media (83%). The overall activity and specific sulfidogenic activity decreased at the end of the study to 1.43 mg COD removed mg TVS−1 d−1 and 0.60 mg sulfate reduced mg TVS−1 d−1, respectively. Additionally, 16S rRNA sequencing revealed major dominant phyla present: Firmicutes (36%), Proteobacteria (16%), Actinobacteria (8.8%), Planctomycetes (7.8%), Chloroflexi (3.5%), Acidobacteria (1.9%) and Fibrobacteres (1.5%).


2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Anirudha R. Dixit ◽  
Christina L. M. Khodadad ◽  
Mary E. Hummerick ◽  
Cory J. Spern ◽  
LaShelle E. Spencer ◽  
...  

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.


2021 ◽  
Vol 12 ◽  
Author(s):  
Younghwan Kwak ◽  
Penglin Sun ◽  
Venkata RamaSravani Meduri ◽  
Diana M. Percy ◽  
Kerry E. Mauck ◽  
...  

Sap-feeding insects in the order Hemiptera associate with obligate endosymbionts that are required for survival and facultative endosymbionts that can potentially modify resistance to stress, enemies, development, and reproduction. In the superfamily Psylloidea, the jumping plant lice (psyllids), less is known about the diversity and prevalence of their endosymbionts compared to other sap-feeding pests such as aphids (Aphididae). To address this knowledge gap, using 16S rRNA sequencing we identify symbionts across divergent psyllid host lineages from around the world. Taking advantage of a new comprehensive phylogenomic analyses of Psylloidea, we included psyllid samples from 44 species of 35 genera of five families, collected from 11 international locations for this study. Across psyllid lineages, a total of 91 OTUs were recovered, predominantly of the Enterobacteriaceae (68%). The diversity of endosymbionts harbored by each psyllid species was low with an average of approximately 3 OTUs. Two clades of endosymbionts (clade 1 and 2), belonging to Enterobacteriaceae, were identified that appear to be long term endosymbionts of the psyllid families Triozidae and Psyllidae, respectively. We also conducted high throughput metagenomic sequencing on three Ca. Liberibacter infected psyllid species (Russelliana capsici, Trichochermes walkeri, and Macrohomotoma gladiata), initially identified from 16S rRNA sequencing, to obtain more genomic information on these putative Liberibacter plant pathogens. The phylogenomic analyses from these data identified a new Ca. Liberibacter species, Candidatus Liberibacter capsica, that is a potential pathogen of solanaceous crops. This new species shares a distant ancestor with Ca. L. americanus, which occurs in the same range as R. capsici in South America. We also detected the first association between a psyllid specializing on woody hosts and the Liberibacter species Ca. L. psyllaurous, which is a globally distributed pathogen of herbaceous crop hosts in the Solanaceae. Finally, we detected a potential association between a psyllid pest of figs (M. gladiata) and a Ca. Liberibacter related to Ca. L. asiaticus, which causes severe disease in citrus. Our findings reveal a wider diversity of associations between facultative symbionts and psyllids than previously reported and suggest numerous avenues for future work to clarify novel associations of ecological, evolutionary, and pathogenic interest.


2021 ◽  
Author(s):  
Anicet Ebou ◽  
Dominique Koua ◽  
Adolphe Zeze

The 16S ribosomal RNA gene is one of the most studied genes in biology. This 16S ribosomal RNA importance is due to its wide application in phylogenetics and taxonomic elucidation of bacteria and archaea. Indeed, 16S ribosomal RNA is present in almost all bacteria and archaea and has, among many other useful characteristics, a low mutation rate. The 16S ribosomal RNA is composed of nine hypervariable regions which are commonly targeted by high throughput sequencing technologies in identification or community studies like metabarcoding studies. Unfortunately, the hypervariable regions do not have the same taxonomic resolution among all bacteria taxa. This requires a preliminary in silico analysis to determine the best hypervariable regions to target in a particular study. Nevertheless, to the best of our knowledge, no automated primer-based open-source tool exists to extract hypervariable regions from complete or near-complete 16S rRNA sequencing data. Here we present HyperEx which efficiently extracts the hypervariable region of interest based on embedded primers or user-given primers. HyperEx implements the Myers algorithm for the exact pairwise sequence alignment. HyperEx is freely available under the MIT license as an operating system independent Rust command-line tool at https://github.com/Ebedthan/hyperex and https://crates.io.


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