Ribosomal Deoxyribonucleic Acid Sequence Research Articles

Nonalcoholic Fatty Liver Disease: Changes in Gut Microbiota and Blood Lipids.

The global prevalence of nonalcoholic fatty liver disease (NAFLD) is 25%. This study aimed to explore differences in the gut microbial community and blood lipids between normal livers and those affected by NAFLD using 16S ribosomal deoxyribonucleic acid sequencing. Gut microbiome profiles of 40 NAFLD and 20 non-NAFLD controls were analyzed. Information about four blood lipids and 13 other clinical features was collected. Patients were divided into three groups by ultrasound and FibroScan, those with a normal liver, mild FL (FL1), and moderate-to-severe FL (FL2). FL1 and FL2 patients were divided into two groups, those with either hyperlipidemia or non-hyperlipidemia based on their blood lipids. Potential keystone species within the groups were identified using univariate analysis and a specificity-occupancy plot. Significant difference in biochemical parameters ion NAFLD patients and healthy individuals were identified by detrended correspondence analysis and canonical correspondence analysis. Decreased gut bacterial diversity was found in patients with NAFLD. Firmicutes/Bacteroidetes decreased as NAFLD progressed. Faecalibacterium and Ruminococcus 2 were the most representative fatty-related bacteria. Glutamate pyruvic transaminase, aspartate aminotransferase, and white blood cell count were selected as the most significant biochemical indexes. Calculation of areas under the curve identified two microbiomes combined with the three biochemical indexes that identified normal liver and FL2 very well but performed poorly in diagnosing FL1. Faecalibacterium and Ruminococcus 2, combined with glutamate pyruvic transaminase, aspartate aminotransferase, and white blood cell count distinguished NAFLD. We speculate that regulating the health of gut microbiota may release NAFLD, in addition to providing new targets for clinicians to treat NAFLD.

Acartia arbruta (previously A. tonsa) in British Columbia: a bioindicator of climate-driven ecosystem variability in the northeast Pacific Ocean

Abstract Climate change is driving biogeographic shifts in marine zooplankton. In the northeast (NE) Pacific, the distribution of the estuarine copepod, Acartia tonsa ( Dana, 1849), is generally limited to California except during marine warming events where it is found as far north as British Columbia (BC). Documentation of such events relies on accurate species identification. Phylogenetic reconstruction of the genus Acartia using 768 mitochondrial cytochrome oxidase subunit I Deoxyribonucleic Acid (DNA) sequences collected worldwide demonstrates that NE Pacific A. tonsa is distinct from all conspecifics and congeners. Males of NE Pacific A. tonsa are larger with slight differences in the fifth pair of legs as compared to geographically isolated conspecifics. Accordingly, we propose NE Pacific A. tonsa be reclassified to Acartia arbruta. Analysis of mitochondrial 16S ribosomal RNA DNA sequences from 154 specimens of A. arbruta collected from California, Oregon and BC suggests that A. arbruta detected in BC is a mixture of southern populations. Southern populations are likely driven north during “warm” phases of the oceanographic processes indexed by the Pacific Decadal Oscillation, which is positively correlated with A. arbruta biomass anomalies on the continental shelf of Vancouver Island, BC. The presence of A. arbruta in BC waters is a useful bioindicator of marine warming events.

Morphological and molecular identification of Pfenderius heterocaeca (Trematode: Paramphistomoidea) from Sumatran elephant (Elephas maximus sumatranus).

Background and Aim:Paramphistomiasis is common in tropical countries such as Indonesia and affects livestock and various endemic wild animals such as Sumatran elephants. However, the specific species of paramphistomoid worm that causes paramphistomiasis are rarely reported. The study aims at identifying paramphistomoid worm that infects Sumatran elephants.Materials and Methods:Flukes were collected from the feces of five semi-captive Sumatran elephants that lived at Tegal Yoso Elephant Response Unit in Way Kambas National Park, in 2018, after treatment of oxyclozanide 1 g at the dose of approximately 5-8 mg/kg of body weight. Eight paramphistomoid worms were flattened and stained in Semichon’s carmine for morphological identification, and five other worms were used for molecular identification at second internal transcribed spacer (ITS-2) of ribosomal deoxyribonucleic acid sequence.Results:Forty-five flukes were collected from five Sumatran elephants in Lampung, Indonesia. Eight paramphistomoid worms were morphologically identified as Pfenderius heterocaeca and five isolates did not show any variation in ITS-2. Phylogenetic analysis showed that there was a close genetic relationship between our sample and Chiorchis fabaceus that had a family similar to the samples.Conclusion:Based on the morphological and molecular characteristics, the paramphistomoids found in Sumatran elephant on Way Kambas National Park are P. heterocaeca.

Pseudomonas oryzihabitans sepsis in a 1-year-old child with multiple skin rashes: a case report

BackgroundPseudomonas oryzihabitans is a Pseudomonas bacterial organism rarely implicated in human infections. The bacterium has been isolated in a few reported cases of neurosurgical infections and patients with end-stage cirrhosis, sickle cell disease, and community-acquired urinary tract infections. Limited information exists in developing countries, however, because of the lack of advanced microbiological tools for identification and characterization of this bacterium. This case report describes the isolation of a rare Pseudomonas bacterium in a patient presenting with sepsis and skin infection.Case presentationA 1-year-old girl was presented to a hospital in the northeastern part of Ghana with a 1-week history of pustular rashes on her scalp and neck, which occasionally ruptured, along with discharge of yellowish purulent fluid. The child is of Mole-Dagbon ethnicity and hails from the northern part of Ghana. Pseudomonas oryzihabitans was identified in the patient’s blood culture using the 16S ribosomal deoxyribonucleic acid sequencing technique. The rash on the patient’s scalp and skin resolved after continuous treatment with gentamicin while her condition improved clinically.ConclusionsThis finding suggests the potential of this bacterium to cause disease in unsuspected situations and emphasizes the need to have evidence for the use of the appropriate antibiotic in clinical settings, particularly in rural settings in Africa. It also brings to the fore the unreliability of conventional methods for identification of Pseudomonas bacteria in clinical samples and thus supports the use of 16S ribosomal deoxyribonucleic acid in making the diagnosis.

MICROORGANISMS AS CHEMICAL FACTORIES FOR ISOLATION OF META-BOLOMES FROM MESOPHILIC SOIL

Objective: To investigate the micro-flora of the mesophilic soil of Rajasthan for isolation of novel compounds having antibacterial potentials.Methods: In present experiments, bacterial colonies were isolated from four different regions of Jaipur, Rajasthan and screened for antimicrobial efficacy against five selected pathogens Pseudomonas aeruginosa MTCC 7093, Staphylococcus aureus MTCC 7443, Escherichia coli MTCC 40, Klebsiella pneumoniae MTCC 530, and Bacillus subtillis MTCC 121. Antimicrobial efficacy against the selected strains was performed. The potential efficacy of the extract was also screened for gas chromatography–mass spectroscopy (GC-MS) analysis for novel metabolites screening. Further, potent bacterial strains were identified at the molecular level by 16S ribosomal deoxyribonucleic acid (DNA) sequencing method.Results: After the primary screening, 29 microbial isolates were selected for the screening of bioactivity. Results displayed zones of inhibition ranging from 5 mm till maximum 13 mm. Soil testing indicated survival conditions for microbes isolated, and biochemical tests supported the identification of screened isolates. The potentially isolated strains S-III C, S-III D and S-IV D were identified at the molecular level using 16S ribosomal DNA sequencing as Bacillus shackletonii, Bacillus thuringiensis and Bacillus subtilis subsp. inaquosorum, respectively.Conclusion: Extraction of active metabolites from soil microbiota, against five pathogenic bacteria, is far better, safe and economical method. This study will help in exploring new compounds against increasing number of resistant pathogenic strains with an aim to reduce demand of medicinal plants for extraction of effective antimicrobial compounds. Bacillus strains (S-IIIC, S-IIID and S-IVC) isolated from soil microflora possess antimicrobial activity and can be used for isolation of antibiotics at industrial levels.

Identification of Colletotrichum spp. associated with fruit rot of Capsicum annuum in North Western Himalayan region of India using fungal DNA barcode markers

The DNA barcode approach was used to identify and establish association of Colletotrichum species complex with fruit rot disease of chili (Capsicum annuum L.) in North-Western Himalayan region of India. Twenty isolates of five morphologically identified Colletotrichum species collected from commercial chili growing areas were identified using deoxyribonucleic acid (DNA) barcode marker genes, 5.8S ribosomal ribonucleic acid flanking internal transcribed spacers 1 & 2 and β-tubulin gene. Morpho-cultural identification requires expertise to delineate C. gloeosporioides, C. boninense and C. acutatum complexes from each other, as these species possess minute variation in spore shape and size. Ribosomal DNA and β-tubulin sequence analysis along with species-specific marker amplification established the association of seven Colletorichum spp. viz., C. truncatum (syn. Colletotrichum capsici), C. coccodes, C. karstii, C. kahawae, C. nymphaeae, C. fructicola and C. gloeosporioides complex with fruit rot of chili. Phylogenetic analysis of 35 Colletotrichum sequences including authentic type sequences validated the identified sequences with strong bootstrap support. This approach delineated morphologically identified species with great ease into more reliable genotype based speciation of various Colletorichum complexes. The DNA barcode markers have direct implications for plant pathologists in relation to diagnostics in fields and for the purpose of quarantine and disease management.

Rapid detection of health-care-associated bloodstream infection in critical care using multipathogen real-time polymerase chain reaction technology: a diagnostic accuracy study and systematic review.

There is growing interest in the potential utility of real-time polymerase chain reaction (PCR) in diagnosing bloodstream infection by detecting pathogen deoxyribonucleic acid (DNA) in blood samples within a few hours. SeptiFast (Roche Diagnostics GmBH, Mannheim, Germany) is a multipathogen probe-based system targeting ribosomal DNA sequences of bacteria and fungi. It detects and identifies the commonest pathogens causing bloodstream infection. As background to this study, we report a systematic review of Phase III diagnostic accuracy studies of SeptiFast, which reveals uncertainty about its likely clinical utility based on widespread evidence of deficiencies in study design and reporting with a high risk of bias. Determine the accuracy of SeptiFast real-time PCR for the detection of health-care-associated bloodstream infection, against standard microbiological culture. Prospective multicentre Phase III clinical diagnostic accuracy study using the standards for the reporting of diagnostic accuracy studies criteria. Critical care departments within NHS hospitals in the north-west of England. Adult patients requiring blood culture (BC) when developing new signs of systemic inflammation. SeptiFast real-time PCR results at species/genus level compared with microbiological culture in association with independent adjudication of infection. Metrics of diagnostic accuracy were derived including sensitivity, specificity, likelihood ratios and predictive values, with their 95% confidence intervals (CIs). Latent class analysis was used to explore the diagnostic performance of culture as a reference standard. Of 1006 new patient episodes of systemic inflammation in 853 patients, 922 (92%) met the inclusion criteria and provided sufficient information for analysis. Index test assay failure occurred on 69 (7%) occasions. Adult patients had been exposed to a median of 8 days (interquartile range 4-16 days) of hospital care, had high levels of organ support activities and recent antibiotic exposure. SeptiFast real-time PCR, when compared with culture-proven bloodstream infection at species/genus level, had better specificity (85.8%, 95% CI 83.3% to 88.1%) than sensitivity (50%, 95% CI 39.1% to 60.8%). When compared with pooled diagnostic metrics derived from our systematic review, our clinical study revealed lower test accuracy of SeptiFast real-time PCR, mainly as a result of low diagnostic sensitivity. There was a low prevalence of BC-proven pathogens in these patients (9.2%, 95% CI 7.4% to 11.2%) such that the post-test probabilities of both a positive (26.3%, 95% CI 19.8% to 33.7%) and a negative SeptiFast test (5.6%, 95% CI 4.1% to 7.4%) indicate the potential limitations of this technology in the diagnosis of bloodstream infection. However, latent class analysis indicates that BC has a low sensitivity, questioning its relevance as a reference test in this setting. Using this analysis approach, the sensitivity of the SeptiFast test was low but also appeared significantly better than BC. Blood samples identified as positive by either culture or SeptiFast real-time PCR were associated with a high probability (> 95%) of infection, indicating higher diagnostic rule-in utility than was apparent using conventional analyses of diagnostic accuracy. SeptiFast real-time PCR on blood samples may have rapid rule-in utility for the diagnosis of health-care-associated bloodstream infection but the lack of sensitivity is a significant limiting factor. Innovations aimed at improved diagnostic sensitivity of real-time PCR in this setting are urgently required. Future work recommendations include technology developments to improve the efficiency of pathogen DNA extraction and the capacity to detect a much broader range of pathogens and drug resistance genes and the application of new statistical approaches able to more reliably assess test performance in situation where the reference standard (e.g. blood culture in the setting of high antimicrobial use) is prone to error. The systematic review is registered as PROSPERO CRD42011001289. The National Institute for Health Research Health Technology Assessment programme. Professor Daniel McAuley and Professor Gavin D Perkins contributed to the systematic review through their funded roles as codirectors of the Intensive Care Foundation (UK).

Isolation and identification of cadmium- and lead-resistant lactic acid bacteria for application as metal removing probiotic

The purpose of the present study was to isolate and identify the metal-resistant lactic acid bacteria from sediments of coastal aquaculture habitats for removal of cadmium and lead from ambience. Collected sediment samples were used to isolate the cadmium- and lead-resistant bacterial colonies by spread plate technique using agar media (De Man, Rogosa and Sharpe) supplemented with cadmium or lead at 50 mg/l. Isolates were identified by bacterial colony polymerase chain reaction and sequencing of 16S ribosomal deoxyribonucleic acid. Metal removing probiotic was determined by characterizing the lactic acid yield in culture media, viability in fish intestine, metal-resistant and metal-removal efficiencies. 16S ribosomal deoxyribonucleic acid sequencing data of five (Cd10, Cd11, Pb9, Pb12 and Pb18) and other all isolates clearly showed 99 % similarities to Enterococcus faecium and Bacillus cereus, respectively. The Pb12 exhibited higher lactic acid yield (180 mmol) than that of the remaining E. faecium strains and excellent viability without pathogenicity; therefore, further study was carried out using Pb12 strain. The selected Pb12 strain showed elevated metal resistant (minimum inhibitory concentrations 120 and 800 mg/l for cadmium and lead, respectively) and removal efficiencies [Cadmium 0.0377 mg/h/g and lead 0.0460 mg/h/g of cells (wet weight)]. From the viability and metal removal points of view, it can be concluded that isolated metal-resistant E. faecium Pb12 strains might be used as potential probiotic strains for removing heavy metals from fish intestinal milieu to control the progressive bioaccumulation of heavy metals in the fish.

Degradation of benzyldimethyl hexadecylammonium chloride by Bacillus niabensis and Thalassospira sp. isolated from marine sediments

Following enrichment in its presence, two strains of bacteria, isolated from marine sediments, were shown to degrade the quaternary ammonium surfactant benzyldimethyl hexadecylammonium chloride (BDHAC) in a minimal salts medium. The bacteria identified by 16S ribosomal deoxyribonucleic acid sequencing were shown to belong to several genera and determined to be Bacillus niabensis and Thalassospira sp. Initial investigations demonstrated that the bacteria were capable of degrading BDHAC when it was present at concentrations in the range 2–4 mg mL−1. In media containing BDHAC, up to 90% was degraded within 7 days, but limited growth of the strain was observed at 2 and 4 mg mL−1 BDHAC. Preliminary analysis of samples after degradation experiment by electrospray ionization mass spectrometry/mass spectrometry produced a peak with a parent–daughter ion transition of 136 → 91, corresponding to N,N-dimethylbenzylamine. The presence of this potential metabolite suggests the cleavage of the C-alkyl-N bond as a step in BDHAC catabolism.

Diversity and structure of ectomycorrhizal and co-associated fungal communities in a serpentine soil

The community of ectomycorrhizal (ECM) and co-associated fungi from a serpentine site forested with Pinus sylvestris and Quercus petraea was explored, to improve the understanding of ECM diversity in naturally metalliferous soils. ECM fungi were identified by a combination of morphotyping and direct sequencing of the nuclear ribosomal internal transcribed spacer region 2 and of a part of the large-subunit region. Co-associated fungi from selected ECM were identified by restriction fragment length polymorphism and sequencing of representative clones from libraries. Polymerase chain reaction with species-specific primers was applied to assess patterns of association of ECM and co-associated fungi. Twenty ECM species were differentiated. Aphyllophoralean fungi representing several basidiomycete orders and Russulaceae were dominant. Phialocephala fortinii was the most frequently encountered taxon from the diverse assemblage of ECM co-associated fungi. A ribotype representing a deeply branching ascomycete lineage known from ribosomal deoxyribonucleic acid sequences only was detected in some ECM samples. A broad taxonomic range of fungi have the potential to successfully colonise tree roots under the extreme edaphic conditions of serpentine soils. Distribution patterns of ECM-co-associated fungi hint at the importance of specific inter-fungal interactions, which are hypothesised to be a relevant factor for the maintenance of ECM diversity.

The radiation of Thaparocleidus (Monogenoidea: Dactylogyridae: Ancylodiscoidinae): phylogenetic analyses and taxonomic implications inferred from ribosomal DNA sequences

The monophyly of Thaparocleidus Jain 1952 and its phylogenetic relationship with Pseudancylodiscoides were assessed using the D1-D2 domains of the large-subunit ribosomal deoxyribonucleic acid sequences, and taxonomic implications were discussed concerning the relative taxonomic importance of the characters of the reproductive complex and those of the haptoral scleties for future genus erection and combination within the Ancylodiscoidinae. Thaparocleidus contained divergent genetic lineages and was not resolved as a monophyletic group. The first lineage was represented by two species found on Pangasium sutchi (Pangasidae), and the second one contained 12 species all from Silurus astus (Siluridae). Three clades were observed for species from S. astus, consistent with results of morphological analyses, indicating that 12 Thaparocleidus species could be divided into three groups by morphological examinations of the male copulatory organ (MCO). Pseudancylodiscoides spp. was more closely related to Thaparocleidus spp. from S. astus, and morphologically, it was found that shapes of MCOs of these species were the same type but different from that of Thaparocleidus spp. from P. sutchi. Therefore, based on results of molecular phylogenetic analyses and morphological examinations, we propose to abolish Pseudancylodiscoides and to erect a new genus to accommodate Thaparocleidus species from S. astus and Pseudancylodiscoides spp. It is also suggested to erect another new genus to accommodate two Thaparocleidus species from the P. sutchi, whose MCO shapes are different from other Thaparocleidus species.

Chronic Polymyositis Associated with Disseminated Sarcocystosis in a Captive-born Rhesus Macaque

A 2-year-old, captive-born, clinically healthy male, rhesus macaque, was euthanatized as part of an experimental study. At necropsy, diffuse pale streaking of the trunk, lumbar, and limb muscles were noted macroscopically. On histology, numerous elongated cysts that contained crescent-shaped basophilic spores were found in the fibers of skeletal muscles. Scattered affected myofibers were degenerate and accompanied by eosinophilic-to-granulomatous inflammation. Sarcocysts had prominent villus-like projections with the morphology of a type 11 sarcocyst wall similar to Sarcocystis neurona but possessing many more villus microtubules than is reported for S. neurona. In addition, bradyzoites were very long, up to approximately 12 microm in length. The protozoa were consistent with a Sarcocystis sp., based on histology and ultrastructure, however, a definitive identification of the species was not possible. Nonspecific immunohistochemical crossreaction with Sarcocystis cruzi antisera was observed. The 18S ribosomal deoxyribonucleic acid sequence showed 91% similarity to Sarcocystis hominis, 90% similarity to Sarcocystis buffalonis, and 89% similarity to Sarcocystis hirsuta. Interestingly, the ITS1 sequence showed very little homology to any sequence in GenBank, suggesting that this is possibly a unique Sarcocystis sp. Sarcocystosis is often considered an incidental finding, particularly in wild-caught animals, with little clinical significance. However, as demonstrated in this report and others, disseminated sarcocystosis can occur in captive-born rhesus macaques with or without clinical signs. In some cases interference with research results can occur; including death in fulminant cases.

Diverse Tetracycline Resistant Bacteria and Resistance Genes from Coastal Waters of Jiaozhou Bay

Environmental microbiology investigation was carried out in Jiaozhou Bay to determine the source and distribution of tetracycline-resistant bacteria and their resistance mechanisms. At least 25 species or the equivalent molecular phylogenetic taxa in 16 genera of resistant bacteria could be identified based on 16S ribosomal deoxyribonucleic acid sequence analysis. Enterobacteriaceae, Pseudomonadaceae, and Vibrionaceae constituted the majority of the typical resistant isolates. Indigenous estuarine and marine Halomonadaceae, Pseudoalteromonadaceae, Rhodobacteraceae, and Shewanellaceae bacteria also harbored tetracycline resistance. All the six resistance determinants screened, tet(A)-(E) and tet(G), could be detected, and the predominant genes were tet(A), tet(B), and tet(G). Both anthropogenic activity-related and indigenous estuarine or coastal bacteria might contribute to the tet gene reservoir, and resistant bacteria and their molecular determinants may serve as bioindicators of coastal environmental quality. Our work probably is the first identification of tet(E) in Proteus, tet(G) in Acinetobacter, tet(C) and tet(D) in Halomonas, tet(D) and tet(G) in Shewanella, and tet(B), tet(C), tet(E), and tet(G) in Roseobacter.

Dilution-to-Extinction Culturing of Psychrotolerant Planktonic Bacteria from Permanently Ice-covered Lakes in the McMurdo Dry Valleys, Antarctica

Lakes in the McMurdo Dry Valleys of Antarctica are characterized by a permanent ice cover and little or no anthropogenic influence. Although bacterial cultures have been obtained from these habitats, recent culture-independent studies indicate that the most abundant microbes in these systems are not yet cultivated. By using dilution-to-extinction cultivation methods with sterilized and nutrient-amended lake water as media, we isolated 148 chemotrophic psychrotolerant bacterial cultures from fresh surface water of Lake Fryxell and the east lobe of Lake Bonney and the hypersaline, suboxic bottom water from the west lobes of Lake Bonney. Screening of the 16S ribosomal ribonucleic acid (rRNA) genes of the cultures by restriction fragment length polymorphism (RFLP) yielded 57 putatively pure psychrotolerant, slow growing cultures grouped into 18 clusters. The sequencing of 16S rRNA genes of randomly selected representatives of each RFLP cluster revealed that the corresponding isolates belong to the Alphaproteobacteria (six RFLP patterns), Betaproteobacteria (six RFLP patterns), Bacteroidetes (four RFLP patterns), and Actinobacteria (two RFLP patterns). Phylogenetic analysis of the sequences showed that the vast majority of the isolates were not closely related to previously described species. Thirteen of 18 RFLP patterns shared a 16S ribosomal deoxyribonucleic acid sequence similarity of 97% or less with the closest described species, and four isolates had a sequence similarity of 93% or less with the nearest described species. Phylogenetic analysis showed that these sequences were representatives of deeply branching organisms in the respective phylum. A comparison of the isolates with 16S rRNA clone libraries prepared from the same environments showed substantial overlap, indicating that dilution-to-extinction culturing in natural lake water media can help isolate some of the most abundant organisms in these perennially ice-covered lakes.

Ribosomal DNA sequences indicate isolated populations of Ichthyophonus hoferi in geographic sympatry in the north‐eastern Pacific Ocean

AbstractInfections of Ichthyophonus hoferi, a cosmopolitan parasite of marine fish, have recently been reported in rockfish, Sebastes spp., from the north‐eastern Pacific. Because I. hoferi also infects Pacific herring, Clupea pallasi Valenciennes, and salmonids in this region, we wanted to determine if Ichthyophonus parasites from rockfishes, Pacific herring and chinook salmon, Oncorhynchus tshawytscha (Walbaum), were the same. Small subunit ribosomal deoxyribonucleic acid sequence data revealed two haplotypes that were fixed among host species in geographic sympatry, one from rockfish and the other from both Pacific herring and salmon. These isolated populations of Ichthyophonus could be part of the same species that are ecologically separated because of host behaviours, or they could be distinct species that are host specific. Dietary patterns of the hosts indicate that ecological separation among hosts is possible, but the presence of distinct species may better explain the observed Ichthyophonus haplotype association with host species.

Initiation of transcription of ribosomal deoxyribonucleic acid sequences in isolated nuclei of Physarum polycephalum: studies using nucleoside 5'-[gamma-S]triphosphates and labeled precursors.

Initiation of transcription of ribosomal deoxyribonucleic acid sequences in isolated nuclei of Physarum polycephalum: studies using nucleoside 5'-[gamma-S]triphosphates and labeled precursors.