16S Ribosomal RNA Sequencing Research Articles

Diagnostic difficulties of Lactobacillus casei bacteraemia in immunocompetent patients: A case report

IntroductionLactobacilli are currently proposed as probiotic agents in several dietary products. In blood cultures, they are usually considered as contaminants, but in recent years they have been recognized as causal infectious agents of endocarditis, urinary tract infections, meningitis, intra-abdominal infections and bacteraemia.Case presentationWe report a case of Lactobacillus casei bacteraemia in a 66-year-old immunocompetent man with a history of fever of unknown origin. Leuconostoc bacteraemia was demonstrated by blood culture, but a later polymerase chain reaction analysis with sequencing of 16S ribosomal RNA identified Lactobacillus casei and a successful antibiotic therapy was performed.ConclusionBacteraemia caused by probiotic organisms is rare but underestimated, since they are normally regarded as contaminants and their role as primary invaders is not always easily established. Although the consumption of probiotic products cannot be considered a risk factor in the development of diseases caused by usually non-pathogenic bacteria, specific individual clinical histories should be taken into account. This report should alert both clinicians and microbiologists to the possibility of unusual pathogens causing serious illnesses and to the use of 16S ribosomal RNA sequencing for molecular identification as a powerful tool in confirming the diagnosis of infrequent pathogens.

Phylogenetic analysis of Spiroplasmas from three freshwater crustaceans ( Eriocheir sinensis, Procambarus clarkia and Penaeus vannamei) in China

Disease epizootics in freshwater culture crustaceans (crab, crayfish and shrimp) gained high attention recently in China, due to intensive developments of freshwater aquacultures. Spiroplasma was identified as a lethal pathogen of the above three freshwater crustaceans in previous studies. Further characterization of these freshwater crustacean Spiroplasma strains were analyzed in the current study. Phylogenetic position was investigated by analysis of partial nucleotide sequences of 16S ribosomal RNA (rRNA), gyrB and rpoB genes, together with complete sequencing of 23S rRNA gene and 16S–23S rRNA intergenetic spacer regions (ISRs). Phylogenetic analysis of these sequences showed that the above-mentioned three freshwater crustacean Spiroplasma strains were identical and had a close relationship with Spiroplasma mirum. Furthermore, the genomic size, serological studies and experimental infection characteristics confirmed that three freshwater crustacean Spiroplasma strains are a single species other than traditional S. mirum. Therefore, these data suggest that a single species of Spiroplasma infects all three investigated freshwater crustaceans in China, and is a potential candidate for a new species within the Spiroplasma genus. These results provide critical information for the further investigations in fresh aquaculture epizootics related to tremor diseases, caused by this infectious agent.

Gut Microbiota and Its Possible Relationship With Obesity

Obesity results from alterations in the body's regulation of energy intake, expenditure, and storage. Recent evidence, primarily from investigations in animal models, suggests that the gut microbiota affects nutrient acquisition and energy regulation. Its composition has also been shown to differ in lean vs obese animals and humans. In this article, we review the published evidence supporting the potential role of the gut microbiota in the development of obesity and explore the role that modifying the gut microbiota may play in its future treatment. Evidence suggests that the metabolic activities of the gut microbiota facilitate the extraction of calories from ingested dietary substances and help to store these calories in host adipose tissue for later use. Furthermore, the gut bacterial flora of obese mice and humans include fewer Bacteroidetes and correspondingly more Firmicutes than that of their lean counterparts, suggesting that differences in caloric extraction of ingested food substances may be due to the composition of the gut microbiota. Bacterial lipopolysaccharide derived from the intestinal microbiota may act as a triggering factor linking inflammation to high-fat diet-induced metabolic syndrome. Interactions among microorganisms in the gut appear to have an important role in host energy homeostasis, with hydrogen-oxidizing methanogens enhancing the metabolism of fermentative bacteria. Existing evidence warrants further investigation of the microbial ecology of the human gut and points to modification of the gut microbiota as one means to treat people who are over-weight or obese.

Infected abdominal aortic aneurysm caused by Brucella abortus: A case report

This case report describes an infected aneurysm caused by Brucella abortus. To the best of our knowledge, this case represents the first reported occurrence of an infected abdominal aortic aneurysm caused by this pathogen. The clinical findings included mild fever, fatigue, cold sweats, and abdominal pain. The diagnosis was reached by computed tomography scanning. The patient was treated by urgent surgery, which consisted of resection of the aneurysmal sac, prosthetic graft replacement, and omental wrapping. Antibiotics were administrated for 3 months. A bacteriologic specimen was isolated from the blood culture. At 1 year 10 months after surgery, the patient remains in good health.

Biodiversity among luminescent symbionts from squid of the genera Uroteuthis, Loliolus and Euprymna (Mollusca: Cephalopoda).

Luminescent bacteria in the family Vibrionaceae (Bacteria: γ-Proteobacteria) are commonly found in complex, bilobed light organs of sepiolid and loliginid squids. Although morphology of these organs in both families of squid is similar, the species of bacteria that inhabit each host has yet to be verified. We utilized sequences of 16S ribosomal RNA, luciferase α-subunit (luxA) and the glyceraldehyde-3-phosphate dehydrogenase (gapA) genes to determine phylogenetic relationships between 63 strains of Vibrio bacteria, which included representatives from different environments as well as unidentified luminescent isolates from loliginid and sepiolid squid from Thailand. A combined phylogenetic analysis was used including biochemical data such as carbon use, growth and luminescence. Results demonstrated that certain symbiotic Thai isolates found in the same geographic area were included in a clade containing bacterial species phenotypically suitable to colonize light organs. Moreover, multiple strains isolated from a single squid host were identified as more than one bacteria species in our phylogeny. This research presents evidence of species of luminescent bacteria that have not been previously described as symbiotic strains colonizing light organs of Indo-West Pacific loliginid and sepiolid squids, and supports the hypothesis of a non-species-specific association between certain sepiolid and loliginid squids and marine luminescent bacteria.

INVESTIGATION OF THE PHYLOGENETIC RELATIONSHIPS OF SARCOCYSTIS SPP. FROM GREYLAG (ANSER ANSER) AND WHITE-FRONTED (ANSER ALBIFRONS) GEESE TO OTHER CYST FORMING COCCIDIA USING 18S AND 28S rRNA GENE SEQUENCES

Based on cyst morphology, Sarcocystis cysts type I were found in one White-fronted goose (Anser albifrons) and cysts type III in one Greylag goose (Anser anser) and two White-fronted geese. Sarcocysts isolated from infected birds as intermediate host have not been previously described and are unnamed. Type III sarcocysts detected in White-fronted and Greylag geese may illustrate the case of polyhostal nature of sarcocysts when same-species parasites infest intermediate hosts of different species. The obtained partial sequences of 18S and 28S ribosomal RNA showed the highest homology for the genera Sarcocystis and Frenkelia. In the tree of phylogenetic relationships, the species involved in this study were grouped with Frenkelia microti, Frenkelia glareoli, Sarcocystis muris and Sarcocystis neurona. Analysis of the partial sequences of 18S and 28S ribosomal RNA revealed the phylogenetic and taxonomic status of the investigated Sarcocystis spp.

A Bioinformatics Approach to Ribosomal Antibiotics Research Using Peptide-Peptide Nucleic Acid (PNA) Conjugates

1* Abstract Peptide - Peptide Nucleic Acid (PNA) conjugates are known for their antibacterial effects on several pathogens. Here we designed five PNA-peptide conjugates that consist of sequences originating from conservative sequences of 16S ribosomal RNA (rRNA) from hundreds of different bacterial species. Their antibacterial effects were tested on E. coli K12 and Klebsiella pneumoniae. The result shows the sequence-dependent antibacterial or bactericidal effects of the designed peptide-PNA conjugates. We also developed a bioinformatics tool and examined any possible off-target effects of the peptide-PNA we designed. Using this tool, we also predicted the least off-target sequences for further studies. Designing peptide-PNAs utilizing conservative 16S rRNA sequences may pave a way to developing broad-spectrum ribosomal antibiotics fighting life-threatening, drug-resistant infections.

The Bacteriology of Pleural Infection by Genetic and Standard Methods and Its Mortality Significance

Antibiotic choices for pleural infection are uncertain as its bacteriology is poorly described. Pleural fluid from 434 pleural infections underwent standard culture and a screen for bacteria by amplification and sequencing of bacterial 16S ribosomal RNA gene. Approximately 50% of community-acquired infections were streptococcal, and 20% included anaerobic bacteria. Approximately 60% of hospital-acquired infections included bacteria frequently resistant to antibiotics (methicillin-resistant Staphylococcus aureus, 25%; Enterobacteriaceae, 18%; Pseudomonas spp., 5%, enterococci, 12%). Mortality was increased in hospital-acquired infection (hospital, 17/36 [47%]; community, 53/304 [17%]; relative risk, 4.24; 95% confidence interval, 2.07-8.69; p < 0.00001; chi(2), 1 df = 17.47) and in gram-negative (10/22 [45%]), S. aureus (15/34 [44%]), or mixed aerobic infections (13/28 [46%]), compared with streptococcal infection (23/137 [17%]) and infection including anaerobic bacteria (10/49 [20%]; p < 0.00001, chi(2), 4 df = 23.35). Pleural infection differs bacteriologically from pneumonia and requires different treatment. Antibiotics for community-acquired infection should treat aerobic and anaerobic bacteria. Hospital-acquired, gram-negative S. aureus and mixed aerobic infections have a high mortality rate.

Genotyping of Giardia in Dutch patients and animals: A phylogenetic analysis of human and animal isolates

Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis) is a protozoan organism that can infect the intestinal tract of many animal species including mammals. Genetic heterogeneity of G. duodenalis is well described but the zoonotic potential is still not clear. In this study, we analysed 100 Giardia DNA samples directly isolated from human stool specimens, to get more insight in the different G. duodenalis assemblages present in the Dutch human population. Results showed that these human isolates could be divided into two main Assemblages A and B within the G. duodenalis group on the basis of PCR assays specific for the Assemblages A and B and the DNA sequences of 18S ribosomal RNA and the glutamate dehydrogenase ( gdh) genes. Genotyping results showed that G. duodenalis isolates originating from Dutch human patients belonged in 35% of the cases to Assemblage A (34/98) and in 65% of the cases to Assemblage B (64/98) whereas two human cases remained negative in all assays tested. In addition, we compared these human samples with animal samples from the Netherlands and human and animal samples from other countries. A phylogenetic analysis was carried out on the DNA sequences obtained from these Giardia and those available in GenBank. Using gdh DNA sequence analysis, human and animal Assemblage A and B Giardia isolates could be identified. However, phylogenetic analysis revealed different sub-clustering for human and animal isolates where host–species-specific assemblages (C, D, E, F and G) could be identified. The geographic origin of the human and animal samples was not a discriminating factor.

The use of stable isotope probing techniques in bioreactor and field studies on bioremediation

Stable isotope probing (SIP) is a molecular technique that allows investigators to follow the flow of atoms in isotopically enriched molecules through complex microbial communities into metabolically active microorganisms. Thus, SIP has immense promise for discovering microorganisms responsible for ecologically important biogeochemical reactions in nature. Applications of SIP to biodegradation and bioremediation processes are still in their infancy. In the past few years, approximately a dozen biodegradation studies using SIP based on the analysis of labeled DNA, RNA or phospholipid fatty acids have been completed. Results have begun to link biomarkers (especially sequences of 16S ribosomal RNA and functional genes) to biodegradation reactions in naturally occurring microbial communities. As extensive compilations of ecologically important genotypes and phenotypes accrue, predictive abilities for contaminant metabolism in particular habitats may be achieved.

Phylogenetic analysis of six species of pacific abalone (Haliotidae) based on DNA sequences of 16s rRNA and cytochrome c oxidase subunit I mitochondrial genes

Six species of abalones (Haliotidae) are found on the Korean coasts. Identification and characterization of these abalones are usually based on morphologic characters. In this research we compared the partial sequences of the mitochondrial 16S ribosomal RNA and cytochrome c oxidase subunit I genes to identify species using molecular data and to determine their phylogenetic relationships. Sequence alignments and phylogenetic analysis revealed that the 6 species fell into 2 distinct groups which were genetically distant from each other and exhibited little internal phylogenetic resolution. One group included Haliotis discus hannai, H. discus discus, H. madaka, and H. gigantea, while the other group contained H. diversicolor supertexta and H. diversicolor diversicolor. The 16S rRNA sequences were relatively more conserved than to the COI sequences, but both gene sequences provided sufficient phylogenetic information to distinguish among the 6 species of Pacific abalone, and thus could be valuable molecular characters for species identification.

Phylogeny and Evolution of Butterflies of the Genus Parnassius: Inferences from Mitochondrial 16S and ND1 Sequences

Phylogenetic relationships among species of the genus Parnassius and its related taxa were analyzed by comparing nucleotide sequences of mitochondrial 16S ribosomal RNA (504 sites) and NADH-dehydrogenase subunit 1 (469 sites). In the phylogenetic trees, Parnassius was found to be most closely related to Hypermnestra helios, whereas Archon apollinus, which has been classified in the tribe Parnassiini together with Parnassius and Hypermnestra, was more closely related to members of the tribe Zerynthiini. Within the Parnassius clade, six major clades corresponding to species groups were well supported, although the phylogenetic relationships among them were not clear. Although the results of the present study were in agreement with those of a previous phylogenetic study based on mitochondrial NADH-dehydrogenase subunit 5 sequences, our study strongly supported a close relationship between Parnassius and Hypermnestra, which was not well supported in the previous study.

A Case of Melioidosis Presenting as Migrating Pulmonary Infiltration: The First Case in Korea

Melioidosis is an infection of the Gram-negative bacterium Burkholderia pseudomallei. While it is known as an important cause of sepsis or chronic abscess-forming disease in Southeast Asia and northern Australia, no case has yet been reported in Korea. A 50-yr-old man visited our hospital for intermittent fever associated with dry cough and sputum. Roentgenographic examination showed migrating pulmonary infiltration. Symptoms and chest radiograph and computed tomography (CT) image findings did not improve despite use of fluoroquinolone antibiotics. Gram-negative bacteria were isolated on bronchoscopic washing culture and were identified as B. pseudomallei on DNA sequencing of 16S ribosomal RNA with 100% homology. Treatment for melioidosis was commenced with high dose ceftazidime, and the patient's fever, cough, and sputum were improved and the lesion on chest radiograph and CT almost disappeared.

Genetic identity of JapaneseSympetrum frequensand KoreanS. depressiusculuminferred from mitochondrial 16S rRNA sequences (Odonata: Libellulidae)

Abstract The Japanese endemic Sympetrum frequens is considered as the insular vicariant of S. depressiusculum, widely distributed in the Eurasian Continent. In Korea, morphologically intermediate specimens have been collected, mixed with typical S. depressiusculum. The taxonomical status of these two species is thus questionable. To clarify their status, sequencing of mitochondrial 16S ribosomal RNA (rRNA) was performed on 77 specimens of Sympetrum species collected from Korea and Japan. The pairwise differences between 378 nucleotides of S. frequens and S. depressiusculum, including the intermediate type, revealed only minor differences (< 0.5%, average 0.48%). The neighbor-joining phylogenetic tree indicated that all S. frequens and S. depressiusculum form one clade, suggesting that they pertain to a single species. The tree also suggests that the S. frequens population from Hokkaido is different from all other populations.

Phylogenetic Analysis and Rapid Detection of Genus Phellinus using the Nucleotide Sequences of 18S Ribosomal RNA

Analysis of phylogenetic relationship was performed among Phellinus species based on 18S ribosomal subunit sequence data. Twenty-five strains of 19 Phellinus species including P. linteus were examined in this study. Regions of 18S ribosomal subunit were very conserved, but some variable regions between Phellinus species were observed. The species-specific detection primers, modified by 2 or 3 nucleotides in sense primer were designed based on 18S ribosomal DNA (rDNA) sequence data. The 210 bp PCR bands were detected with annealing temperature 48°C. The 18S 2 F-18S 4 R detection primer set distinguished P linteus from various Phellinus species but some species like P baumii, P weirianius, P rhabarberinus and P pomaceus also had weak reactivity on this primer set. The 18S 3 F-18S 4 R primer set distinguished only P. linteus from various Phellinus species, although sensitivity with this primer set was lower than that of 18S 2 F-18 4 R primer set. These primer sets would be useful for the detection of only P. linteus among unknown Phellinus species rapidly.

Identification of characteristic oligonucleotides in the bacterial 16S ribosomal RNA sequence dataset.

The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S rRNA is characteristic of any particular bacterial grouping was developed. A measure of signature quality, Q(s), was formulated and subsequently calculated for every individual oligonucleotide sequence in the size range of 5-11 nucleotides and for 15mers with reference to each cluster and subcluster in a 929 organism representative phylogenetic tree. Subsequently, the perfect signature sequences were compared to the full set of 7322 sequences to see how common false positives were. The work completed here establishes beyond any doubt that highly characteristic oligonucleotides exist in the bacterial 16S rRNA sequence dataset in large numbers. Over 16,000 15mers were identified that might be useful as signatures. Signature oligonucleotides are available for over 80% of the nodes in the representative tree.

Genetic Approaches to Studying Protein Synthesis: Effects of Mutations at Ψ516 and A535 in Escherichia coli 16S rRNA

A genetic system for the study of ribosomal RNA function and structure was developed. First, the ribosome binding sequence of the chloramphenicol acetyltransferase gene and the message binding sequence of 16S ribosomal RNA were randomly mutated and alternative highly functional sequences were selected and characterized. From this set of mutants, a single clone was chosen and subjected to a second round of mutagenesis to optimize the specificity of the system. In the resulting system, plasmid-encoded ribosomes efficiently and exclusively translate specific mRNA containing the appropriate ribosome binding sequences. This system allows facile isolation and analysis of mutations that would normally be lethal and allows direct selection of rRNA mutants with predetermined levels of ribosome function. The system was used to examine the effects of mutations at the sole pseudouridine (Psi) in Escherichia coli 16S rRNA which is located at position 516 of the conserved 530 loop. The nucleotide opposite Psi516 in the hairpin, A535, was also mutated. The data show that a pyrimidine (Psi or C) is required at position 516, while substitutions at position 535 reduce ribosome function by < 50%. A requirement for base pair formation between Psi516 and A535 was not indicated.

Phylogeographic analysis of the threatened and endangered superconglutinate-producing mussels of the genus Lampsilis (Bivalvia: Unionidae).

Several species of freshwater unionid mussels in the genus Lampsilis exhibit a remarkable reproductive strategy. Female mussels of these species enclose their larvae in a minnow-like lure, called a 'superconglutinate', to attract piscivorous fishes. When a fish attempts to ingest the superconglutinate the lure ruptures and the larvae are released to parasitize the fish. Of the four species of mussel which exhibit this strategy and are endemic to the Gulf Coast drainages of the southeastern United States, three are protected under the Endangered Species Act, and one is recognized as imperiled. Phylogenetic analysis of nucleotide sequences of the mitochondrial 16S ribosomal RNA and the first subunit of the cytochrome oxidase c genes was conducted on 18 individual specimens representing these four species and six outgroup taxa. Phylogenetic analyses of these data support the monophyly of the superconglutinate-producing mussels, and indicates a strong geographical component to the data. The zoogeographic patterns of the four taxa included in the study are congruent with those seen in freshwater vertebrates, and are consistent with a vicariant pattern resulting from fluctuations in sea level during the Pleistocene. Despite the strong geographical structuring of the data, only one species, Lampsilis subangulata, was recovered as monophyletic. The authors attribute the lack of support for the monophyly of the remaining species to insufficient sequence variation and the recent origin of the ancestor of these taxa. Based on these data, any future captive breeding projects aimed at augmenting or re-establishing populations should do so only from the appropriate source populations so as to maintain the genetic integrity of these nascent species.

Extensive complementarity of the Shine-Dalgarno region and 3'-terminal sequence of 16S ribosomal RNA is inefficient for translation in vivo

Translation initiation in Escherichia coli involves as a rule complementary interactions between a Shine-Dalgarno (SD) sequence upstream of the initiation codon and a highly conserved 3'-end sequence of 16S rRNA (anti-SD). The translation efficiency is believed to be directly affected by the affinity of the ribosome to the mRNA initiation region. Earlier, high-affinity RNA ligands to E. coli ribosomes were selected by the SELEX approach, with the ligands containing an extended SD-sequence well represented. In this work, we examined the ability of artificial ribosome binding sites (RBSs) containing such an extended (10-nt) SD-sequence (superSD) to drive translation in vivo, as well as its ability to form the translation initiation complex in vitro. Toe print experiments showed the formation of a ternary initiation complex on mRNA comprising superSD. Moreover, they proved the formation of an extended SD-duplex in the binary 30S-mRNA complex. Nevertheless, the superSD appeared to be inefficient in translation in vivo. We believe that the initiation complex involving a superSD-element is too stable to be functional; it may impede the transition from initiation to elongation, thus disrupting the transcription-translation coupling and inhibiting the formation of polysomes.

Phylogenetic Comparison of Endosymbionts with Their Host Ants Based on Molecular Evidence

Some Formicid ants have symbiotic intracellular bacteria in the epithelial cells of their midgut. These endosymbionts are believed to be derived from a common ancestor. A recent study revealed that endosymbionts of the ant genus Camponotus are closely related to Enterobacteriaceae, but their relationship to endosymbionts of other genera of ants is unknown. In this study, the nucleotide sequences of 16S ribosomal RNA (rRNA) of endosymbionts and mitochondrial cytochrome oxidase subunit I (COI) of their host were determined in five genera of the subfamily Formicinae (Hymenoptera: Formicidae). Based on these molecular data, we constructed phylogenetic trees in order to characterize the systematic position of the symbionts and to estimate the relationship of symbionts and hosts. The analysis showed that the endosymbionts were all connected with the Enterobacteriaceae but did not constitute a monophylitic group, while the three genera belonging to the tribe Camponotini, the endosymbionts and their hosts made a clade. The topologies of these trees were identical for the most part. These results suggest that the endosymbionts of ants have plural origins, and that in the Camponotini, ancestral symbionts have coevolved with their host ants, which are so divergent to several genera as to construct one tribe.