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Excised hypocotyls from developing soybean (Glycine max (L.) merr. cv. Jack) were cultivated on agar-solidified medium until callus formed. The calli were then propagated in liquid medium until stable, relatively uniform, finely-divided suspension cultures were obtained. Cells were typically transferred to fresh medium at 7-day intervals. Cultures were harvested by filtration five days (early log phase) or eight days (late log phase) after transfer. In order to evaluate dynamic changes, both intracellular and extracellular proteins were analyzed by 2-dimensional difference gel electrophoresis. Selected spots were subjected to in-gel tryptic-digestion and the resultant peptides were analyzed by nLC-MS/MS. In follow-up studies gel-free shot-gun analyses led to identification of 367 intracellular proteins and 188 extracellular proteins.
This article describes the Pulsed-field gel electrophoresis clustering of the predominant Salmonella strains (Salmonella ser. Albany, Salmonella ser. Brancaster, and Salmonella ser. Corvallis) isolated from poultry and processing environment in wet market and small-scale processing plant in Penang and Perlis, the northern states of Malaysia. Agar disk diffusion assay was performed to determine the phenotypic antibiotic resistance of these Salmonella strains. The most common antibiograms among the three predominant Salmonella serovars were reported. The presence of integrase genes and antibiotic resistance genes conferring to resistance against β-lactams, aminoglycosides, tetracyclines, quinolones, sulphonamides and chloramphenicol, was detected via PCR amplification.
Effluents discharged by poultry meat industries are heavily polluted with raw materials, such as fat, blood residues, and proteins. Thus, untreated effluents directly discharged into the environment may constitute a public health threat. This study aims to evaluate the bacterial diversity of three water qualities: industrial poultry wastewater (PWW), tap water (TW), and PWW diluted with TW (50 : 50) (V/V) (TWPWW) by the combination of culture-independent and culture-dependent approaches. The total bacterial DNA was extracted using phenol/chloroform method. The hypervariable 16S rRNA region V3-V5 was amplified by PCR using universal primers. The amplicons were separated by vertical electrophoresis on a polyacrylamide gel of increasing denaturing gradient according to their richness in GC bases. Selected bands were reamplified and sequenced. Pure isolated bacteria from nutrient agar medium were characterized according to their morphological and biochemical characteristics. Genomic DNA from pure strains was extracted by boiling method, and a molecular amplification of the 16S-23S ITS region of the 16S rRNA gene was performed using the universal primers. Selected isolates were identified by sequencing. Results showed a high bacterial load and diversity in PWW in comparison with TW and TWPWW. A collection of 44 strains was obtained, and 25 of them were identified by sequencing. Proteobacteria represented 76% of isolated bacteria Gamma-Proteobacteria was the predominate isolate (68%). Other isolates were Firmicutes (8%), Bacteroidetes (12%), and Actinobacteria (8%). These isolates belong to different genera, namely, Pseudomonas, Acinetobacter, Proteus, Empedobacter, Corynebacterium, Enterobacter, Comamonas, Frondibacter, Leclercia, Staphylococcus, Atlantibacter, Klebsiella, and Microbacterium.
Accurate identification of slowly growing nontuberculous mycobacteria (SG-NTM) of clinical significance remains problematic. This study evaluated a novel method of SG-NTM identification by amplification of the mycobacterial 16S-23S rRNA internal transcribed spacer (ITS) region followed by resolution of amplified fragments by sequencer-based capillary gel electrophoresis (SCGE). Fourteen American Type Culture Collection (ATCC) strains and 103 clinical/environmental isolates (total n = 24 species) of SG-NTM were included. Identification was compared with that achieved by high performance liquid chromatography (HPLC), in-house PCR and 16S/ITS sequencing. Isolates of all species yielded a SCGE profile comprising a single fragment length (or peak) except for M. scrofulaceum (two peaks). SCGE peaks of ATCC strains were distinct except for peak overlap between Mycobacterium kansasii and M. marinum. Of clinical/environmental strains, unique peaks were seen for 7/17 (41%) species (M. haemophilum, M. kubicae, M. lentiflavum, M. terrae, M. kansasii, M. asiaticum and M. triplex); 3/17 (18%) species were identified by HPLC. There were five SCGE fragment length types (I-V) each of M. avium, M. intracellulare and M. gordonae. Overlap of fragment lengths was seen between M. marinum and M. ulcerans; for M. gordonae SCGE type III and M. paragordonae; M. avium SCGE types III and IV, and M. intracellulare SCGE type I; M. chimaera, M. parascrofulaceum and M. intracellulare SCGE types III and IV; M. branderi and M. avium type V; and M. vulneris and M. intracellulare type V. The ITS-SCGE method was able to provide the first line rapid and reproducible species identification/screening of SG-NTM and was more discriminatory than HPLC.
The dataset described in this paper provides information on the morphological features of 24 different species of the genera Bacillus, Paenibacillus, Brevibacillus, Lysinibacillus, and Rummeliibacilluswhen growing in HiCrome Bacillus agar. The species studied are common contaminants of honey. In support to the recent publication entitled "HiCrome Bacillus agar for presumptive identification of Bacillus and related species isolated from honey samples" (2), a collection of 197 bacterial isolates belonging to 24 different species of aerobic spore-forming bacteria have been screened for their colony appearance and color and any substrate color change of HiCrome Bacillus agar at 24 and 48 h of incubation. Two simple flowcharts utilizing a combination of colony and media characteristics in the chromogenic medium and a set of simple biochemical and morphological tests were developed for quick presumptive identification.
We recently established a method for isolating functional single cells from environmental samples using a micromanipulator (Functional single-cell (FSC) isolation), and applied it to the study of denitrifying bacteria in rice paddy soil (Ashida et al. 2010. Appl Microbiol Biotechnol 85:1211-1217). To further examine the advantages and possible disadvantages of the FSC method, we isolated denitrifying bacteria from the same rice paddy soil sample using both FSC and standard agar plate dilution (APD) methods and compared in this study. The proportion of denitrifying bacteria in the total isolates was more than 6-fold larger with FSC isolation (57.1%) compared with the APD method (9.2%). Denitrifying bacteria belonging to Alphaproteobacteria and Bacilli were commonly isolated using both methods, whereas those belonging to Betaproteobacteria, which had been found to be active in the denitrification-inductive paddy soil, were isolated only with the FSC method. On the other hand, Actinobacteria were only isolated using the APD method. The mean potential denitrification activity of the FSC isolates was higher than that of the APD isolates. Overall, FSC isolation was confirmed to be an excellent method for studying denitrifying bacteria compared with the standard agar plate dilution method.
The success of invertebrates throughout evolution is an excellent illustration of the efficiency of their defence strategies. Caenorhabditis elegans has proven to be an appropriate model for transcriptome studies of host-pathogen interactions. The aim of this paper is to complement this knowledge by investigating the worm's response to a Staphylococcus aureus infection through a 2-dimensional differential proteomics approach.
Chicken is a leading source of thermotolerant Campylobacter, which triggers human foodborne enteritis. This study evaluated thermotolerant Campylobacter contamination of retail chicken in southern Brazil, using qualitative and quantitative analyses. Selective enrichment in Bolton broth for 24 and 48 h after plating onto modified charcoal-cefoperazone-deoxycholate (mCCD) agar and Preston agar was assessed. The combined results of the detection and enumeration methods revealed a frequency of 70% occurrence of thermotolerant Campylobacter in chicken samples. Campylobacter was enumerated in 60% of the samples, whereas 46% of the samples were positive in the qualitative analysis. Quantitative analysis showed average counts of 3.10 ± 0.15 log10 CFU/sample. Higher numbers of Campylobacter-positive samples were found using 24-h enrichment before plating onto Preston agar (46%) than onto mCCD agar (2%). The majority of isolated strains were identified as Campylobacter jejuni, and Campylobacter coli was also found but to a lesser extent. Subtyping revealed a clear distinction between strains isolated from different chicken sources. The enriched samples plated onto mCCD agar showed extensive spreading of nonproducing extended-spectrum β-lactamases Proteus mirabilis that hampered the identification of Campylobacter colonies. P. mirabilis strains showed resistance to cefoperazone, trimethoprim, and polymyxin B present in broth and plate media used and were inhibited by rifampicin present in Preston agar. The results underline the effect of the spread of contaminant strains on Campylobacter cultures, which might be prevented using a recently revised International Organization for Standardization method for qualitative analysis of chicken.
The acquisition of iron is important for the pathogenicity of bacteria and blood. Three different culture environments (Fe stimulation, blood agar plate and normal plate) were used to stimulate Enterobacter cloacae, and their respective pathogenicities were compared at the proteomic, mRNA and metabolomic levels.
To evaluate the water compartment antibiotic-resistance contamination rates, 11 wells, five streams, and four treatment plants located in the Oltrepò Pavese area were screened for the presence of third generation cephalosporins resistant Gram-negative bacteria. Enterobacteriaceae were also characterized for the Extended-Spectrum-β-Lactamases (ESBLs), carbapenemases, and mcr-1 genes presence. From December 2014 to November 2015, 246 water samples were filtered, plated on Plate Count Agar, MacConkey Agar, and MacConkey Agar with cefotaxime. Isolates were species identified using AutoSCAN-4-System and ESBLs, carbapenemases, and colistin resistance determinants were characterized by PCR, sequencing, and microarray. Plasmid conjugative transfer experiments, PCR-based Replicon typing, Pulsed-Field Gel Electrophoresis, Multi-Locus-Sequence-Typing, and in-silico plasmid characterization were performed. A total of 132 enterobacteria isolates grew on MacConkey agar with cefotaxime: 82 (62.1%) were obtained from streams, 41 (31.1%) from treatment plants, and 9 (6.8%) from wells. Thirty out of 132 (22.7%) isolates, mainly belonging to Escherichia coli (n = 15) species, showed a synergic effect with piperacillin-tazobactam. A single ESBL gene of blaCTX-M-type was identified in 19/30 isolates. In further two E. coli strains, a blaCTX-M-1 gene co-existed with a blaSHV-type ESBL determinant. A blaSHV-12 gene was detected in two isolates of E. coli (n = 1) and Klebsiella oxytoca (n = 1), while any ESBL determinant was ascertained in seven Yersinia enterocolitica strains. A blaDHA-type gene was detected in a cefoxitin resistant Y. enterocolitica from a stream. Interestingly, two Klebsiella pneumoniae strains of ST307 and ST258, collected from a well and a wastewater treatment plant, resulted KPC-2, and KPC-3 producers, respectively. Moreover, we report the first detection of mcr-1.2 ST10 E. coli on a conjugative IncX4 plasmid (33.303 bp in size) from a stream of Oltrepò Pavese (Northern Italy). Both ESBLs E. coli and ESBLs/carbapenemase-producing K. pneumoniae strains showed clonal heterogeneity by Pulsed-Field Gel Electrophoresis and Multi-Locus-Sequence-Typing. During one-year study and taking in account the whole Gram-negative bacterial population, an average percentage of cefotaxime resistance of 69, 32, and 10.3% has been obtained for the wastewater treatment plants, streams, and wells, respectively. These results, of concern for public health, highlight the need to improve hygienic measures to reduce the load of discharged bacteria with emerging resistance mechanisms.
Molecular characteristics of vancomycin resistant enterococci isolates from Bermuda Island is currently unknown. This study was conducted to investigate phenotypic and genotypic characteristics of VRE isolates from Bermuda Island using the chromogenic agar, E-tests, polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Eighteen E. faecium isolates were completely analyzed and were all resistant to vancomycin, susceptible to linezolid and quinupristin/dalfopristin, positive for vanA and esp genes. The MLST analysis confirmed most isolates were of the sequence types linked to clonal complex 17 (CC17) that is widely associated with outbreaks in hospitals. Infection control measures, antibiotic stewardship, and surveillance activities will continue to be a priority in hospital on the Island.
A total of sixty raw milk samples were collected from (street vendors and shops) from Baghdad city, Iraq. The samples were inoculated into peptone water and, then, subcultured onto MacConkey agar and Blood agar. Identification of isolates was confirmed by microscopic examination, cultural characteristic, biochemical tests, Vitek (VITEK®2 system), and Biolog GN substrate reactions followed by 16S rRNA and specific genes sequencing. Of 60 raw cow's milk samples, Providencia spp. were identified only in 4 samples (6.67%) and P. rettgeri was the most common, 2/4 (50%), followed by P. stuartii and P. vermicola, 1/4 (25%). Antimicrobial susceptibility tests were conducted against ten antibiotics by the disc diffusion method. All Providencia isolates showed multidrug resistance (MDR), and the absolute resistant was 100% to tetracycline, erythromycin, and doxycycline and 50% against ampicillin\sulbactam and amoxicillin/clavulanic acid. They were highly susceptible (100%) to trimethoprim, imipenem, and chloramphenicol. These findings indicate that milk might be contaminated with Providencia spp. leading to transmission to humans causing poisoning, diarrhea, and other infections. This is the first study of isolated Providencia spp. from raw cow's milk.
The plasmid-borne fosfomycin resistance gene fosA3 has been identified in Escherichia coli (E. coli) from various animals but has rarely been reported in ducks. In this study, we investigated the fosA3 prevalence and molecular characteristics of fosA3-harboring E. coli strains from ducks in Shandong province of China. In 416 E. coli isolates, 91 (21.88%) were identified as fosA3-bearing strains, and the fosfomycin-resistant phenotype of 88 of the 91 fosA3-harboring strains was successfully transferred to the recipient strains. Seven different genetic structures surrounding the fosA3 gene were detected and 2 new contexts were discovered among the fosA3-carrying E. coli. Twenty fosA3-harboring isolates and their trans-conjugants were randomly selected for pulsed-field gel electrophoresis (PFGE) typing and S1-nuclease PFGE, respectively. The PFGE patterns revealed that the 20 randomly selected fosA3-bearing isolates were not a result of clonal dissemination. S1-PFGE showed that 15 of the 20 randomly selected trans-conjugants carried a single plasmid, and these 15 plasmids that harbored fosA3 (55-190 kb) were distributed into the following replicon types: IncF (n = 11), IncI1 (n = 1), IncN (n = 1), untypable (n = 1), and W-FIC (n = 1). Additionally, as vectors for fosA3 in E. coli, F-:A1:B6, N/ST1, IncI1/ST2, W-FIC, and one untypable plasmid had never been reported before. These observations highlighted the importance of ducks as a reservoir for multidrug-resistant fosA3-carrying E. coli.
Agrobacterium-mediated plant galls are often misdiagnosed as nematode-mediated knots, even by experts, because the gall symptoms in both conditions are very similar. In the present study, we developed biosensor strains based on agrobacterial opine metabolism that easily and simply diagnoses Agrobacterium-induced root galls. Our biosensor consists of Agrobacterium mannitol (ABM) agar medium, X-gal, and a biosensor. The working principle of the biosensor is that exogenous nopaline produced by plant root galls binds to NocR, resulting in NocR/nopaline complexes that bind to the promoter of the nopaline oxidase gene (nox) operon and activate the transcription of noxB-lacZY, resulting in readily visualized blue pigmentation on ABM agar medium supplemented with X-gal (ABMX-gal). Similarly, exogenous octopine binds to OccR, resulting in OoxR/octopine complexes that bind to the promoter of the octopine oxidase gene (oox) operon and activate the transcription of ooxB-lacZY, resulting in blue pigmentation in the presence of X-gal. Our biosensor is successfully senses opines produced by Agrobacterium-infected plant galls, and can be applied to easily distinguish Agrobacterium crown gall disease from nematode disease.
Root metabolites and soil microbial community structure in the rhizosphere play critical roles in crop growth. Here, we assessed the efficiency of conventional and tissue culture propagation methods in modulating the soil health and microbiota in the rhizosphere of sugarcane (Saccharum officinarum L.) plants. The seeding canes were obtained using newly planted and two-year ratooned canes propagated by conventional (CSN and CSR) or tissue culture (TCN and TCR) methods. Changes in soil fertility, root metabolites and soil microbial community structure in the rhizosphere of sugarcane plants obtained using these canes were assessed. The activities of soil β-glucosidase and aminopeptidase, soil microbial biomass nitrogen, and abundances of soil beneficial microbes, both at phyla and genera levels, were significantly higher in the rhizosphere of sugarcane plants in TCN and TCR treatments than those in that of plants in CSN and CSR treatments. Furthermore, flavonoid and flavonol biosynthesis and alanine, aspartate and glutamate metabolism were significantly upregulated in the roots of TCR and TCN plants compared with those in the roots of CSN and CSR plants. These results suggest that the tissue culture propagation method is a sustainable method for sugarcane cultivation to improve soil fertility and health in sugarcane rhizosphere.
Although many protocols have been previously developed for genomic DNA (gDNA) extraction from S. cerevisiae, to take advantage of recent advances in laboratory automation and DNA-barcode sequencing, there is a need for automated methods that can provide high-quality gDNA at high efficiency. Here, we describe and demonstrate a fully automated protocol that includes five basic steps: cell wall and RNA digestion, cell lysis, DNA binding to magnetic beads, washing with ethanol, and elution. Our protocol avoids the use of hazardous reagents (e.g., phenol, chloroform), glass beads for mechanical cell disruption, or incubation of samples at 100°C (i.e., boiling). We show that our protocol can extract gDNA with high efficiency both from cells grown in liquid culture and from colonies grown on agar plates. We also show results from gel electrophoresis that demonstrate that the resulting gDNA is of high quality.
In an effort to axenically culture the previously uncultivable populations of the rhizobacteria of Lucerne (Medicago sativa L.), we propose plant-only teabags culture media to mimic the nutritional matrix available in the rhizosphere. Here, we show that culture media prepared from Lucerne powder teabags substantially increased the cultivability of Lucerne rhizobacteria compared with a standard nutrient agar, where we found that the cultivable populations significantly increased by up to 60% of the total bacterial numbers as estimated by Quantitative Real-time Polymerase Chain Reaction (qRT-PCR). Cluster analysis of 16S rDNA Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) of cultivable Colony-Forming Units (CFUs) revealed a more distinct composition and separation of bacterial populations recovered on the plant-only teabags culture media than those developed on a standard nutrient agar. Further, the new plant medium gave preference to the micro-symbiont Sinorhizobium meliloti, and succeeded in isolating a number of not-yet-cultured bacteria, most closely matched to Novosphingobium sp., Lysobacter sp. and Pedobacter sp. The present study may encourage other researchers to consider moving from the well-established standard culture media to the challenging new plant-only culture media. Such a move may reveal previously hidden members of rhizobacteria, and help to further explore their potential environmental impacts.
To simplify the methodology for the isolation of Campylobacter spp. from retail broiler meat, we evaluated 108 samples (breasts and thighs) using an unpaired sample design. The enrichment broths were incubated under aerobic conditions (subsamples A) and for comparison under microaerobic conditions (subsamples M) as recommended by current reference protocols. Sensors were used to measure the dissolved oxygen (DO) in the broth and the percentage of oxygen (O2) in the head space of the bags used for enrichment. Campylobacter isolates were identified with multiplex PCR assays and typed using pulsed-field gel electrophoresis (PFGE). Ribosomal intergenic spacer analyses (RISA) and denaturing gradient gel electrophoresis (DGGE) were used to study the bacterial communities of subsamples M and A after 48 h enrichment.
Lignocellulosic biomass such as barley straw is a renewable and sustainable alternative to traditional feeds and could be used as bioenergy sources; however, low hydrolysis rate reduces the fermentation efficiency. Understanding the degradation and colonization of barley straw by rumen bacteria is the key step to improve the utilization of barley straw in animal feeding or biofuel production. This study evaluated the hydrolysis of barley straw as a result of the inoculation by rumen fluid of camel and sheep. Ground barley straw was incubated anaerobically with rumen inocula from three fistulated camels (FC) and three fistulated sheep (FR) for a period of 72 h. The source of rumen inoculum did not affect the disappearance of dry matter (DMD), neutral detergent fiber (NDFD). Group FR showed higher production of glucose, xylose, and gas; while higher ethanol production was associated with cellulosic hydrolysates obtained from FC group. The diversity and structure of bacterial communities attached to barley straw was investigated by Illumina Mi-Seq sequencing of V4-V5 region of 16S rRNA genes. The bacterial community was dominated by phylum Firmicutes and Bacteroidetes. The dominant genera were RC9_gut_group, Ruminococcus, Saccharofermentans, Butyrivibrio, Succiniclasticum, Selenomonas, and Streptococcus, indicating the important role of these genera in lignocellulose fermentation in the rumen. Group FR showed higher RC9_gut_group and group FC revealed higher Ruminococcus, Saccharofermentans, and Butyrivibrio. Higher enzymes activities (cellulase and xylanase) were associated with group FC. Thus, bacterial communities in camel and sheep have a great potential to improve the utilization lignocellulosic material in animal feeding and the production of biofuel and enzymes.
Rumen bacteria make the greatest contribution to rumen fermentation that enables the host animal to utilize the ingested feeds. Agro-industrial byproducts (AIP) such as olive cake (OC) and date palm byproducts (discarded dates (DD), and date palm fronds (DPF)) represent a practical solution to the deficiency in common feed resources. In this study, thirty-six growing Barki lambs were divided into three groups to evaluate the effect of untraditional diets including the AIP on the growth performance. Subsequently, nine adult Barki rams were used to evaluate the effect of experimental diets on rumen fermentation and rumen bacteria. Three rations were used: common concentrate mixture (S1), common untraditional concentrate mixture including OC and DD (S2), and the same concentrate mixture in S2 supplemented with roughage as DPF enriched with 15% molasses (S3). The animals in S2 group showed higher dry matter intake (DMI) and lower relative growth rate (RGR) as compared to the animals in S1 group. However, the animals in S3 group were the lowest in DMI but achieved RGR by about 87.6% of that in the S1 group. Rumen pH, acetic and butyric acids were more prevalent in animals of S3 group and rumen ammonia (NH3-N), total volatile fatty acids (TVFA), propionic acid were higher in S1. Rumen enzymes activities were higher in S1 group followed by S3 and S2. The bacterial population was more prevalent in S1 and microbial diversity was higher in the S3 group. Principal coordinate analysis revealed clusters associated with diet type and the relative abundance of bacteria varied between sheep groups. The bacterial community was dominated by phylum Bacteroidetes and Firmicutes; whereas, Prevotella, Ruminococcus, and Butyrivibrio were the dominant genera. Results indicate that diet S3 supplemented by OC, DD, and DPF could replace the conventional feed mixture.
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