scholarly journals Off Earth Identification of Bacterial Populations Using 16S rDNA Nanopore Sequencing

Genes ◽  
2020 ◽  
Vol 11 (1) ◽  
pp. 76 ◽  
Author(s):  
Aaron S. Burton ◽  
Sarah E. Stahl ◽  
Kristen K. John ◽  
Miten Jain ◽  
Sissel Juul ◽  
...  
Keyword(s):  
High Performance ◽  
Space Station ◽  
Bacterial Cells ◽  
Sequencing Data ◽  
Mock Community ◽  
Bacterial Populations ◽  
Microbial Identification ◽  
Staphylococcus Capitis ◽  
Staphylococcus Hominis ◽  
Community Standard

The MinION sequencer has made in situ sequencing feasible in remote locations. Following our initial demonstration of its high performance off planet with Earth-prepared samples, we developed and tested an end-to-end, sample-to-sequencer process that could be conducted entirely aboard the International Space Station (ISS). Initial experiments demonstrated the process with a microbial mock community standard. The DNA was successfully amplified, primers were degraded, and libraries prepared and sequenced. The median percent identities for both datasets were 84%, as assessed from alignment of the mock community. The ability to correctly identify the organisms in the mock community standard was comparable for the sequencing data obtained in flight and on the ground. To validate the process on microbes collected from and cultured aboard the ISS, bacterial cells were selected from a NASA Environmental Health Systems Surface Sample Kit contact slide. The locations of bacterial colonies chosen for identification were labeled, and a small number of cells were directly added as input into the sequencing workflow. Prepared DNA was sequenced, and the data were downlinked to Earth. Return of the contact slide to the ground allowed for standard laboratory processing for bacterial identification. The identifications obtained aboard the ISS, Staphylococcus hominis and Staphylococcus capitis, matched those determined on the ground down to the species level. This marks the first ever identification of microbes entirely off Earth, and this validated process could be used for in-flight microbial identification, diagnosis of infectious disease in a crewmember, and as a research platform for investigators around the world.

scholarly journals Staphylococcal Communities on Skin Are Associated with Atopic Dermatitis and Disease Severity

2021 ◽  
Vol 9 (2) ◽  
pp. 432
Author(s):  
Sofie Marie Edslev ◽  
Caroline Meyer Olesen ◽  
Line Brok Nørreslet ◽  
Anna Cäcilia Ingham ◽  
Søren Iversen ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Alpha Diversity ◽  
Skin Microbiota ◽  
Specific Primers ◽  
Staphylococcus Lugdunensis ◽  
Disease Symptoms ◽  
Lesional Skin ◽  
Staphylococcus Capitis ◽  
Staphylococcus Hominis

The skin microbiota of atopic dermatitis (AD) patients is characterized by increased Staphylococcus aureus colonization, which exacerbates disease symptoms and has been linked to reduced bacterial diversity. Skin bacterial communities in AD patients have mostly been described at family and genus levels, while species-level characterization has been limited. In this study, we investigated the role of the bacteria belonging to the Staphylococcus genus using targeted sequencing of the tuf gene with genus-specific primers. We compared staphylococcal communities on lesional and non-lesional skin of AD patients, as well as AD patients with healthy controls, and determined the absolute abundance of bacteria present at each site. We observed that the staphylococcal community, bacterial alpha diversity, and bacterial densities were similar on lesional and non-lesional skin, whereas AD severity was associated with significant changes in staphylococcal composition. Increased S. aureus, Staphylococcus capitis, and Staphylococcus lugdunensis abundances were correlated with increased severity. Conversely, Staphylococcus hominis abundance was negatively correlated with severity. Furthermore, S. hominis relative abundance was reduced on AD skin compared to healthy skin. In conclusion, various staphylococcal species appear to be important for skin health.

scholarly journals Stochasticity in Colonial Growth Dynamics of Individual Bacterial Cells

2013 ◽  
Vol 79 (7) ◽  
pp. 2294-2301 ◽  
Author(s):  
Konstantinos P. Koutsoumanis ◽  
Alexandra Lianou
Keyword(s):  
Kinetic Parameters ◽  
Single Cells ◽  
Growth Curves ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Initial Population ◽  
Bacterial Cells ◽  
Stochastic Growth ◽  
Bacterial Populations ◽  
Content Type

ABSTRACTConventional bacterial growth studies rely on large bacterial populations without considering the individual cells. Individual cells, however, can exhibit marked behavioral heterogeneity. Here, we present experimental observations on the colonial growth of 220 individual cells ofSalmonella entericaserotype Typhimurium using time-lapse microscopy videos. We found a highly heterogeneous behavior. Some cells did not grow, showing filamentation or lysis before division. Cells that were able to grow and form microcolonies showed highly diverse growth dynamics. The quality of the videos allowed for counting the cells over time and estimating the kinetic parameters lag time (λ) and maximum specific growth rate (μmax) for each microcolony originating from a single cell. To interpret the observations, the variability of the kinetic parameters was characterized using appropriate probability distributions and introduced to a stochastic model that allows for taking into account heterogeneity using Monte Carlo simulation. The model provides stochastic growth curves demonstrating that growth of single cells or small microbial populations is a pool of events each one of which has its own probability to occur. Simulations of the model illustrated how the apparent variability in population growth gradually decreases with increasing initial population size (N0). For bacterial populations withN0of >100 cells, the variability is almost eliminated and the system seems to behave deterministically, even though the underlying law is stochastic. We also used the model to demonstrate the effect of the presence and extent of a nongrowing population fraction on the stochastic growth of bacterial populations.

scholarly journals Prokaryotic Single-Cell RNA Sequencing by In Situ Combinatorial Indexing

2019 ◽  
Author(s):  
Sydney B. Blattman ◽  
Wenyan Jiang ◽  
Panos Oikonomou ◽  
Saeed Tavazoie
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Low Cost ◽  
Bacterial Cells ◽  
Single Experiment ◽  
Bacterial Populations ◽  
Single Cell Rna Sequencing ◽  
Gene Expression Heterogeneity ◽  
Mrna Polyadenylation

AbstractDespite longstanding appreciation of gene expression heterogeneity in isogenic bacterial populations, affordable and scalable technologies for studying single bacterial cells have been limited. While single-cell RNA sequencing (scRNA-seq) has revolutionized studies of transcriptional heterogeneity in diverse eukaryotic systems, application of scRNA-seq to prokaryotes has been hindered by their extremely low mRNA abundance, lack of mRNA polyadenylation, and thick cell walls. Here, we present Prokaryotic Expression-profiling by Tagging RNA In Situ and sequencing (PETRI-seq), a low-cost, high-throughput, prokaryotic scRNA-seq pipeline that overcomes these technical obstacles. PETRI-seq uses in situ combinatorial indexing to barcode transcripts from tens of thousands of cells in a single experiment. PETRI-seq captures single cell transcriptomes of Gram-negative and Gram-positive bacteria with high purity and low bias, with median capture rates >200 mRNAs/cell for exponentially growing E. coli. These characteristics enable robust discrimination of cell-states corresponding to different phases of growth. When applied to wild-type S. aureus, PETRI-seq revealed a rare sub-population of cells undergoing prophage induction. We anticipate broad utility of PETRI-seq in defining single-cell states and their dynamics in complex microbial communities.

scholarly journals A METAGENOMIC ASSESSMENT OF BACTERIAL CONTAMINATION OF DUST EVENTS IN SENEGAL

2021 ◽  
Vol 9 (03) ◽  
pp. 509-526
Author(s):  
Alioune Marone ◽  
◽  
Malick Mbengue ◽  
Gregory Jenkins ◽  
Demba Ndao Niang ◽  
...  
Keyword(s):  
Respiratory Diseases ◽  
Rrna Gene ◽  
Source Population ◽  
Sequencing Analysis ◽  
Human Pathogens ◽  
Sequencing Data ◽  
Bacterial Populations ◽  
African Dust ◽  
Hypervariable Regions ◽  
Dust Events

Previous work in the Caribbean and West Africa have shown that air samples taken during dust events contain microorganisms (bacteria, fungi, viruses), including human pathogens that can cause many respiratory diseases. To better understand the potential downstream effect of bacteria dust on human health and public ecosystems, it is important to characterize the source population. In this study, we aimed to explore the bacterial populations of African dust samples collected between 2013-2017. The dust samples were collected using the spatula method, then the hypervariable regions (V3 and V4) of the 16S rRNA gene were amplified using PCR followed byMiSeq Illumina sequencing. Analysis of the sequencing data were performed using MG-RAST. At the phylum level, the proportions of Actinobacteria (22%), Firmicutes (20%), Proteobacteria (19%), and Bacteroidetes (13%) were respectively predominant in all dust samples. At the genus level, Bacillus(16%), Pseudomonas(10%), Nocardiodes and Exiguobacterium (5%) are the most dominated genera in African dust samples collected in this study.The study showed that molecular characterization of dust microbial population remains a very efficient method, also applicable to the search for viruses and fungi in this type of sample. It is important to note that the majority of microorganisms identified in this study can cause respiratory diseases.

scholarly journals Refinement of metabolite detection in cystic fibrosis sputum reveals heme negatively correlates with lung function

2018 ◽  
Author(s):  
Nathaniel R. Glasser ◽  
Ryan C. Hunter ◽  
Theodore G. Liou ◽  
Dianne K. Newman ◽  
Keyword(s):  
Mass Spectrometry ◽  
Cystic Fibrosis ◽  
Lung Function ◽  
High Performance ◽  
16S Rrna Gene Sequencing ◽  
Initial Study ◽  
Rrna Gene ◽  
Sequencing Data ◽  
Unknown Compound ◽  
Mountain West

SummaryPseudomonas aeruginosalung infections are a leading cause of morbidity and mortality in cystic fibrosis (CF) patients (1, 2). Our laboratory has studied a class of small molecules produced byP. aeruginosaknown as phenazines, including pyocyanin and its biogenic precursor phenazine-1-carboxylic acid (PCA). As phenazines are known virulence factors (3), we and others have explored the possibility of using phenazine concentrations as a marker for disease progression (4–6). Previously, we reported that sputum concentrations of pyocyanin and PCA negatively correlate with lung function in cystic fibrosis patients (6). Our study used high performance liquid chromatography (HPLC) to quantify phenazines by UV–vis absorbance after extraction from lung sputum. Since our initial study, methods for metabolite analysis have advanced considerably, aided in large part by usage of mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS). Because a more recent study employing LC-MS/MS revealed a surprising decoupling ofP. aeruginosametabolites in sputum and the detection ofP. aeruginosathrough culturing or microbiome profiles (4), we decided to check whether we could reproduce our previous findings by analyzing sputum samples from a different patient cohort with a new LC-MS instrument in our laboratory. Our new samples were provided by the Mountain West CF Consortium Sputum Biomarker study (7). In the course of performing our new analyses, comparison of our old HPLC data to our new LC-MS data led us to realize that the peak previously assigned to PCA instead originates from heme, and the peak assigned to pyocyanin originates from an as-yet unknown compound. This correction only affects the measurements of phenazines in sputum, and we are confident in the phenazine measurements from isolated cultures and the 16S rRNA gene sequencing data from that study (6). Here we outline the basis for our correction and present additional data showing that heme concentration negatively correlates with lung function in cystic fibrosis patients.

scholarly journals Occurrence of Bacterial Stem Rot, Caused by Erwinia chrysanthemi, on Field-Grown Tomato in Florida

Plant Disease ◽  
1998 ◽  
Vol 82 (7) ◽  
pp. 831-831 ◽  
Author(s):  
D. O. Chellemi ◽  
H. A. Dankers ◽  
K. Hill ◽  
R. E. Cullen ◽  
G. W. Simone ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Methyl Esters ◽  
Sole Source ◽  
Erwinia Chrysanthemi ◽  
Identification System ◽  
Bacterial Cells ◽  
Sterile Water ◽  
Microbial Identification ◽  
Pure Cultures ◽  
Tomato Isolate

In September 1997, wilted 4-week-old tomato (Lycopersicon esculentum Mill.) plants were observed in a commercial production field in St. Lucie County, FL. Closer inspection of affected plants revealed hollow stems and petioles with dark, water-soaked lesions. Diseased tissue was macerated and streaked onto nutrient agar (NA) and crystal violet pectate (CVP) agar. After incubation for 2 days at 30°C, isolates produced pits on the CVP agar. Isolates were transferred onto NA and the incubation and transfer procedure was performed two additional times to obtain pure cultures. Suspensions of bacterial cells were injected into tomato and tobacco leaves to test for a hypersensitive or pathogenic reaction. Isolates produced collapsed necrotic tissue on tomato while no reaction was observed on tobacco. Tests for differentiating species and subspecies in the ‘carotovora’ group of Erwinia were conducted following the protocol of Dickey and Kelman (1). With known cultures of E. carotovora subsp. carotovora and E. chrysanthemi as controls, the isolate from tomato was determined to function as a facultative anaerobe, utilize asparagine as a sole source of carbon and nitrogen, and give positive reactions for pectate degradation, phosphatase, and growth at 37°C. Known cultures of E. carotovora subsp. carotovora, E. chrysanthemi, and the tomato isolate were grown on trypticase soy broth agar for 24 h at 28°C and their cellular fatty acids derivatized to fatty acid methyl esters (FAMEs). Statistical analyses of FAME profile data (MIDI Microbial Identification System, Newark, DE, version 3.60) identified the tomato isolate as Erwinia chrysanthemi. Pathogenicity was determined by inoculating 50-day-old tomato plants (cv. SunPride) with a suspension of E. chrysanthemi obtained from nutrient broth plates incubated at 24°C for 60 h. Three plants each were inoculated with the E. chrysanthemi identified from tomato, sterile water, and known cultures of E. chrysanthemi and E. carotovora subsp. carotovora by placing a drop at the junction of the petiole and stem and passing a sterile needle through the drop into the stem. Plants were maintained in a greenhouse. Dark, water-soaked cankers were observed on the stems of plants inoculated with E. chrysanthemi, including the tomato isolate and E. carotovora subsp. carotovora, after 7 days. No symptoms were observed on plants inoculated with sterile water. Reisolation of the pathogen and identification was performed with tissue from one of the symptomatic inoculated plants. Analyses of FAMEs confirmed E. chrysanthemi as the causal agent. This is the first report of E. chrysanthemi causing a vascular disease of field-grown tomato in Florida. Reference: (1) R. S. Dickey and A. Kelman. 1988. Pages 44–59 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. N. W. Schaad, ed. American Phytopathological Society, St. Paul, MN.

Characterization of biofilm producing methicillin resistant coagulase negative Staphylococci from India

2021 ◽  
Keyword(s):  
Biofilm Formation ◽  
Resistance Rate ◽  
Coagulase Negative Staphylococci ◽  
Methicillin Resistant ◽  
Antimicrobial Susceptibility Pattern ◽  
Healthcare Settings ◽  
Staphylococcus Capitis ◽  
Staphylococcus Epidermis ◽  
Staphylococcus Hominis

Abstract Methicillin-resistant coagulase-negative staphylococci (MR-CoNS) cause infectious diseases due to their potential to form biofilm and further colonization in hospital materials. This study evaluated the antibiotic susceptible phenotypes, biofilm-producing ability, and biofilm-associated genes (mecA, icaAD, bap, cna, and fnbA). Biofilm formation was detected through Congo red agar (CRA) method and MTP method. The presence of biofilm and associated genes in MR-CoNS were detected by PCR. A total of 310 (55.95%) isolates produced the biofilm. Among these isolates, Staphylococcus haemolyticus (34.83%), Staphylococcus epidermis (31.93%), Staphylococcus capitis (16.77%), Staphylococcus cohnii (10.96%), and Staphylococcus hominis (5.48%) were identified. The antimicrobial susceptibility pattern of CoNS isolates indicated resistance to cefoxitin (100%), erythromycin (94.8%), ciprofloxacin (66.7%), sulfamethoxazole/trimethoprim (66.7%), gentamicin (66.12%), and clindamycin (62.9%). Resistance rate to mupirocin was 48.5% in S. epidermidis and 38.9% in S. haemolyticus isolates. All isolates were sensitive to vancomycin and linezolid. The prevalence rates of icaAD, bap, fnbA, and cna were 18.06%, 12.5%, 47.4%, and 27.4%, respectively. icaAD and bap genes were detected in 18.06% and 12.5% of MR-CoNS isolates. fnbA and cna genes were detected in 47.41% and 27.41% of MRCoNS isolates. icaAD positive strains exhibited a significant increase in the biofilm formation compared with those that lacked icaAD (0.86 (0.42, 1.39) versus 0.36 (0.14, 0.75), respectively; P < 0.001). In conclusion, the majority of MR-CoNS isolates were biofilm producers, and S. capitis, which possessed icaAD genes, ranked as the great biofilm producer than other Staphylococcus. The study’s findings are important to form a strategy to control biofilm formation as an alternative strategy to counter the spread of MR-CoNS in healthcare settings.

scholarly journals A Novel Weissella cibaria Strain UTNGt21O Isolated from Wild Solanum quitoense Fruit: Genome Sequence and Characterization of a Peptide with Highly Inhibitory Potential toward Gram-Negative Bacteria

Foods ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1242
Author(s):  
Gabriela N. Tenea ◽  
Pamela Hurtado ◽  
Clara Ortega
Keyword(s):  
Cell Death ◽  
Ethylenediaminetetraacetic Acid ◽  
Sequence Similarity ◽  
Genome Mining ◽  
Chelating Agent ◽  
Target Cells ◽  
Bacterial Cells ◽  
Sequencing Data ◽  
E Coli ◽  
Weissella Cibaria

A novel Weissella cibaria strain UTNGt21O from the fruit of the Solanum quitoense (naranjilla) shrub produces a peptide that inhibits the growth of both Salmonella enterica subsp. enterica ATCC51741 and Escherichia coli ATCC25922 at different stages. A total of 31 contigs were assembled, with a total length of 1,924,087 bases, 20 contig hits match the core genome of different groups within Weissella, while for 11 contigs no match was found in the database. The GT content was 39.53% and the genome repeats sequences constitute around 186,760 bases of the assembly. The UTNGt21O matches the W. cibaria genome with 83% identity and no gaps (0). The sequencing data were deposited in the NCBI Database (BioProject accessions: PRJNA639289). The antibacterial activity and interaction mechanism of the peptide UTNGt21O on target bacteria were investigated by analyzing the growth, integrity, and morphology of the bacterial cells following treatment with different concentrations (1×, 1.5× and 2× MIC) of the peptide applied alone or in combination with chelating agent ethylenediaminetetraacetic acid (EDTA) at 20 mM. The results indicated a bacteriolytic effect at both early and late target growth at 3 h of incubation and total cell death at 6 h when EDTA was co-inoculated with the peptide. Based on BAGEL 4 (Bacteriocin Genome Mining Tool) a putative bacteriocin having 33.4% sequence similarity to enterolysin A was detected within the contig 12. The interaction between the peptide UTNGt21O and the target strains caused permeability in a dose-, time- response manner, with Salmonella (3200 AU/mL) more susceptible than E. coli (6400 AU/mL). The results indicated that UTNGt21O may damage the integrity of the cell target, leading to release of cytoplasmic components followed by cell death. Differences in membrane shape changes in target cells treated with different doses of peptide were observed by transmission electronic microscopy (TEM). Spheroplasts with spherical shapes were detected in Salmonella while larger shaped spheroplasts with thicker and deformed membranes along with filamentous cells were observed in E. coli upon the treatment with the UTNGt21O peptide. These results indicate the promising potential of the putative bacteriocin released by the novel W. cibaria strain UTNGt21O to be further tested as a new antimicrobial substance.

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