Universal 16S rRNA Primers Research Articles

Development of a panel for detection of pathogens in xenotransplantation donor pigs.

There have been high expectations in recent years of using xenotransplantation and regenerative medicine to treat humans, and pigs have been utilized as the donor model. Pigs used for these clinical applications must be microbiologically safe, that is, free of infectious pathogens, to prevent infections not only in livestock, but also in humans. Currently, however, the full spectrum of pathogens that can infect to the human host or cause disease in transplanted porcine organs/cells has not been fully defined. In the present study, we thus aimed to develop a larger panel for the detection of pathogens that could potentially infect xenotransplantation donor pigs. Our newly developed panel, which consisted of 76 highly sensitive PCR detection assays, was able to detect 41 viruses, 1 protozoa, and a broad range of bacteria (by use of universal 16S rRNA primers). The applicability of this panel was validated using blood samples from uterectomy-born piglets, and pathogens suspected to be vertically transmitted from sows to piglets were successfully detected. We estimate that, at least for viruses and bacteria, the number of target pathogens detected by the developed screening panel should suffice to meet the microbiological safety levels required worldwide for xenotransplantation and/or regenerative therapy. This panel provides greater diagnosis options to produce donor pigs so that it would render unnecessary to screen for all pathogens listed. Instead, the new panel could be utilized to detect only required pathogens within a given geographic range where the donor pigs for xenotransplantation have been and/or are being developed.

High Salt Concentration Affects the Microbial Diversity of Cassava during Fermentation, as Revealed by 16S rRNA Gene Sequencing

Cassava plays an important role in the life and diet of several communities worldwide. It is used in various processed forms and has become the focus of research and innovation in recent years. Bioprocessing techniques, such as fermentation, have been utilized to create new avenues for its application in food and functional products. The fermentation of cassava can enhance its nutritional value, improve its shelf life, and increase its digestibility. However, the effect of salt-mediated fermentation on microbial diversity and potential benefits has not been widely reported. In this study, the effect of six levels of salt (NaCl), ranging from 0 to 25% after 50 days of spontaneous cassava fermentation at 25–30 °C, was investigated. A total of 30 bacterial isolates were selected for molecular characterization. A proprietary pool of universal 16S rRNA primers and species-specific primers were used to amplify a wide variety of species in clonal samples. The bacteria observed include Lactiplantibacillus, Limosilactobacillus, and Weissella. The population of Lactiplantibacillus in the cassava microflora increased with and without salt treatment, while Weissella, a new genus, was detected in 20 and 25% of salt-treated samples. Lactiplantibacillus and Weissella accounted for 32 and 68% and 60 and 40% of the bacterial populations detected at 20 and 25% salt treatment, respectively. A total of 17 strains and sequences were identified from the 30 isolates screened. Sequencing results classified the 30 isolates into four groups, of which 76.67% were Limosilactobacillus. Phylogenetic analysis showed that all 17 strains were divided into three clusters. These results show that high salt-mediated fermentation of cassava can trigger a shift in dominance in the bacterial community, changing the diversity of the microbial community.

Opposite selection effects of nZVI and PAHs on bacterial community composition revealed by universal and sphingomonads-specific 16S rRNA primers

Next-generation sequencing method employing the sphingomonads-specific and universal 16s rRNA primers were applied to analyze the response of soil bacterial community to continuous stress of nanoscale zero-valent iron (nZVI) and/or polycyclic aromatic hydrocarbons (PAHs) under suspended conditions. The group specific primer greatly improves taxonomic resolution, the overall OTU numbers increased from 12 by universal primer to 42 by sphingomonads-specific primer. It was found that nZVI and PAHs had opposite selection effects on bacterial community composition, furthermore, the abundance of Sphingopyxis was positively related to nZVI while negatively related to PAHs, whereas abundances of Sphingobium and Sphingosinicella were positively related to PAHs (p < 0.01). The improved taxonomic resolution helps identify the potential keystone taxa, which provides valuable information for future bioremediation technology such as synthetic microbiome.

Rhizospheric PGPR Strains of Wheat, Barley and Trefoil Grown in Ağrı Province

The present study aimed to isolate potential plant growth rhizobacteria (PGPR) from various crops in agricultural areas in Ağrı-Turkey and their characterization. For this goal, rhizospheric soil samples of wheat, barley and trefoil were collected from 12 different locations in Ağrı province. Bacteria isolation studies were carried out with these rhizospheric samples. Then, to determine the PGPR properties of each isolate; nitrogen fixation, phosphate dissolution, siderophore, ammonia and HCN production tests were performed. Molecular identification of active isolates determined as suitable for development of biofertilizers, biostimulants and/or bioprotectants was done by PCR and sequencing applications performed with universal 16S rRNA primers. According to the results, 29 potential PGPR isolates were determined and their molecular characterization was done. These isolates were distributed in Arthrobacter, Bacillus, Brevibacterium, Enterobacter, Pantoea, Pseudomonas, Microbacterium and Stenotrophomonas according to their 16S rRNA sequence similarities.

Incidence of Bacterial Grain Rot Disease, Identification, and Diversity of Burkolderia glumae in Some Rice Varieties in West Java

Penyakit busuk bulir padi oleh bakteri Burkholderia glumae perlu diwaspadai termasuk di Jawa Barat sebagai salah satu sentra produksi padi nasional. Penelitian ini bertujuan untuk menentukan insidensi penyakit, identitas dan keragaman bakteri B. glumae pada beberapa varietas padi di Provinsi Jawa Barat. Pengamatan insidensi penyakit dan pengambilan sampel dilakukan di 9 kabupaten. Bakteri diisolasi dari bulir padi yang bergejala busuk bulir kemudian dilakukan uji biokimia dan fisiologi yang meliputi uji Gram serta uji pertumbuhan pada pH 4.8, dan NaCl 2%. Variasi fenotipik diamati dari warna koloni pada medium S-PG, produksi toksofalvin, respons hipersensitivitas pada daun tembakau, dan uji patogenisitas pada tanaman padi. Primer spesifik JLBgF/JLBgR dan primer universal 16S rRNA, yaitu 27F/1492R digunakan untuk menentukan identitas bakteri secara molekuler. Insidensi penyakit di lapangan berkisar antara 0–73.3%, tertinggi di Kecamatan Dawuan (Karawang) pada var. Mekongga. Berdasarkan uji biokimia dan fisiologi diperoleh 29 isolat terkonfirmasi sebagai B. glumae. Hasil pengamatan fenotipik menunjukkan 10 isolat tergolong koloni tipe A, 19 isolat tipe B; 25 isolat menghasilkan toksoflavin; 29 isolat menimbulkan respons hipersensitivitas pada daun tembakau dan gejala hawar pada tanaman padi. Identifikasi menggunakan primer spesifik dan universal membuktikan 29 isolat adalah B. glumae. Analisis keragaman genotip menunjukkan bahwa isolat KRCH-2 (Karawang) dan INCH-6 (Indramayu) memiliki hubungan kekerabatan yang dekat dengan B. glumae asal Cina dan Amerika

The Dynamic Changes of Gut Microbiota in Muc2 Deficient Mice.

Gut dysbiosis is associated with colitis-associated colorectal carcinogenesis, and the genetic deficiency of the Muc2 gene causes spontaneous development of colitis and colorectal cancer. Whether there are changes of gut microbiota and a linkage between the changes of microbiota and intestinal pathology in Muc2−/− mice are unclear. Muc2−/− and Muc2+/+ mice were generated by backcrossing from Muc2+/− mice, and the fecal samples were collected at different dates (48th, 98th, 118th, 138th, and 178th day). Gut microbiota were analyzed by high-throughput sequencing with the universal 16S rRNA primers (V3–V5 region). All mice were sacrificed at day 178 to collect colonic tissue and epithelial cells for the analysis of histopathology and inflammatory cytokines. On the 178th day, Muc2−/− mice developed colorectal chronic colitis, hyperplasia, adenomas and adenocarcinomas, and inflammatory cytokines (e.g., cyclooxygenase 2 (COX-2), interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin 1 β (IL-1β), i-kappa-B-kinase β (IKKβ)) were significantly increased in colonic epithelial cells of Muc2−/− mice. In general, structural segregation of gut microbiota was observed throughout the experimental time points between the Muc2−/− and Muc2+/+ mice. Impressively, in Muc2−/− mice, Alpha diversities reflected by Shannon and Chao indexes were higher, the phylum of Firmicutes was enriched and Bacteroidetes was decreased, and Desulfovibrio, Escherichia, Akkermansia, Turicibacter, and Erysipelotrichaceae were significantly increased, but Lactobacilli and Lachnospiraceae were significantly decreased. Moreover, the abundance of Ruminococcaceae and butyrate-producing bacteria was significantly higher in the Muc2−/− mice. There were significant differences of gut microbiota between Muc2−/− and Muc2+/+ mice. The dynamic changes of microbiota might contribute to the development of colitis and colitis-associated colorectal carcinogenesis. Therefore, this study revealed specific functional bacteria in the development of colitis and colitis-associated colorectal carcinogenesis, which will benefit the development of preventive and therapeutic strategies for chronic inflammation and its malignant transformation.

Molecular detection and characterization of phytoplasma in association with begomovirus in eggplant.

The samples from eggplants showing mixed symptoms of little leaf and mosaic were collected from two districts (Mirzapur and Varanasi) of Uttar Pradesh, India. The total nucleic acid extracted from these samples was amplified by PCR using universal 16S rRNA primers specific to phytoplasma and primers specific to DNA-A-like sequence of begomovirus. A total of eighteen eggplant samples showing the symptoms of little leaf and mosaic tested positive for the presence of both begomovirus and phytoplasma. The phytoplasma associated with the mixed symptoms of mosaic and little leaf in the eggplant samples was identified as a member belonging to Clover proliferation group (16SrVI) (nucleotide sequence identity of 97.5-97.8%). The characterized begomovirus from the eggplant samples was identified as a strain of previously described bipartite begomovirus tomato leaf curl Palampur virus (ToLCPalV) (92.5-94.1% nucleotide sequence identity), which is known to infect cucurbits and solanaceous crops in India and Ireland. Further, putative recombination events were detected within the 16S rRNA gene F2n/R2 fragment of phytoplasma and DNA-A of strain of ToLCPalV. Most of the sequence variations observed within the phytoplasma were due to intra and interspecific recombination events between eggplant little leaf-16SrVI-D, Ca. P. asteris-16SrI and Ca. P. pruni-16SrIII. Similarly, most of the DNA fragments of newly characterized strain of ToLCPalV appear to have been derived from tomato leaf curl New Delhi virus (ToLCNDV), squash leaf curl China virus (SLCCNV) and ToLCPalV like ancestors. This perhaps is the first evidence of mixed infection of both phytoplasma-begomovirus in eggplant in India.

Bacterial community dynamics in a cooling tower with emphasis on pathogenic bacteria and Legionella species using universal and genus-specific deep sequencing

Cooling towers are the major source of outbreaks of legionellosis in Europe and worldwide. These outbreaks are mostly associated with Legionella species, primarily L. pneumophila, and its surveillance in cooling tower environments is of high relevance to public health. In this study, a combined NGS-based approach was used to study the whole bacterial community, specific waterborne and water-based bacterial pathogens, especially Legionella species, targeting the 16S rRNA gene. This approach was applied to water from a cooling tower obtained by monthly sampling during two years. The studied cooling tower was an open circuit cooling tower with lamellar cooling situated in Braunschweig, Germany. A highly diverse bacterial community was observed with 808 genera including 25 potentially pathogenic taxa using universal 16S rRNA primers. Sphingomonas and Legionella were the most abundant pathogenic genera. By applying genus-specific primers for Legionella, a diverse community with 85 phylotypes, and a representative core community with substantial temporal heterogeneity was observed. A high percentage of sequences (65%) could not be affiliated to an acknowledged species. L. pneumophila was part of the core community and the most abundant Legionella species reinforcing the importance of cooling towers as its environmental reservoir. Major temperature shifts (>10 °C) were the key environmental factor triggering the reduction or dominance of the Legionella species in the Legionella community dynamics. In addition, interventions by chlorine dioxide had a strong impact on the Legionella community composition but not on the whole bacterial community. Overall, the presented results demonstrated the value of a combined NGS approach for the molecular monitoring and surveillance of health related pathogens in man-made freshwater systems.

Does infection play a role in the pathogenesis of granuloma annulare?

Does infection play a role in the pathogenesis of granuloma annulare?

Characterization of the Relative Abundance of the Citrus Pathogen Ca. Liberibacter Asiaticus in the Microbiome of Its Insect Vector, Diaphorina citri, using High Throughput 16S rRNA Sequencing

The relationship between the causal agent of Huanglongbing (HLB), Ca. Liberibacter asiaticus(Las), and the naturally occurring endosymbiotic community of its insect vector, the Asian citrus psyllid (ACP), Diaphorina citri, was studied. Variation was observed in the titer of Las within an ACP population feeding on the same material. The cause of this disparity is unknown, and has implications for Las transmission and the spread of HLB. This study utilizes culture independent methods to establish the relationship between the ACP’s microbial community and Las acquisition. DNA from 21 psyllids was amplified using universal 16S rRNA primers with Illumina adaptor regions and a sample-specific 7- base identifier. These amplicons were then batch-sequenced on the Illumina platform. The resulting sequences were separated by the identifier, and compared to known sequences in a 16S rRNA database. The microbial communities of each psyllid were compared to determine whether a correlation exists between the ACP’s endosymbionts and level of Las acquisition.ACPs were dominated by the same four bacterialgenera regardless of the abundance of Ca.Liberibacter. A combination of qPCR and Illumina sequencing was used to establish an infection gradient among the sampled ACPs. The Ca. Liberibacter titer within the insect was found to have a strong negative relationship with an endosymbiont residing in the syncytium of the mycetocyte and a positive relationship with Wolbachia. These correlations have implications in the acquisition of Las by the ACP as well as the activities of Las within this vector.

Molecular Study of Free-ranging Mule Deer and White-tailed Deer from British Columbia, Canada, for Evidence of Anaplasma spp. and Ehrlichia spp.

Twenty-three free-ranging white-tailed deer (WTD; Odocoileus virginianus) and six mule deer (MD; Odocoileus hemionus) from south-central British Columbia, Canada, were tested for Anaplasma marginale by msp5 gene-specific PCR and Ehrlichia spp. by 16S rRNA or citrate synthase (gltA) gene-specific PCR, as well as by PCR with universal 16S rRNA primers detecting a wide range of bacteria. No deer tested positive for A.marginale. Amplification with universal 16S rRNA primers followed by sequencing of cloned fragments detected an Anaplasma sp. in one of 23 (4.3%) WTD and six of six (100%) MD and Bartonella sp. in four of 23 (17.4%) WTD. The Anaplasma sp. was genetically distinct from A.marginale and all other recognized members of the genus. Four of six (66.7%) MD and 0 of 23 (0%) WTD were Ehrlichia positive by PCR with primers for 16S rRNA and gltA genes. The sequences of gltA PCR fragments were identical to each other and to the respective region of the gltA gene of an Ehrlichia sp. which we detected previously in naturally infected cattle from the same area, suggesting the possibility of biological transmission of this rickettsia between cattle and wild cervids. Antibodies reactive with the MSP5 protein of A.marginale were detected using a competitive enzyme-linked immunosorbent assay in two of six (33.3%) MD, but not in WTD. The two seropositive MD were PCR positive for both the Anaplasma sp. and Ehrlichia sp. detected in this study, suggesting a reaction of antibodies against one or both of these rickettsias with the MSP5 antigen.

Occurrence of ‘Candidatus Phytoplasma aurantifolia’ (16SrII group) in cassava and four other species in Uganda

Cassava (Manihot esculenta) is amongst the most important crops for Uganda, being grown on more than 400 000 ha (FAOSTAT, 2005). Symptoms of leaf yellowing, chlorosis, shortening of internodes, and slight stunting were recently observed in cassava fields in Kawanda. Interestingly, some plants of four other species (sticky mallow, Malvaviscus arborus; croton, Codiaeum variegatum.; Chinese hibiscus, Hibiscus rosa-sinensis.; and passion fruit, Passiflora edulis) growing nearby showed little leaf, chlorosis, and leaf yellowing and deformation. Two samples of leaves with symptoms were collected from each species and total DNA extracted and used as template in a nested PCR with universal 16S rRNA primers R16mF2/R1 and fU5/rU3. PCR amplicons (880 bp) were produced in 8/10 samples, purified, and directly sequenced in both directions. Sequences were manually edited and compared with those of reference material from GenBank using blast. Sequences were aligned using Clustal W (Thompson et al., 1994), and a phylogenetic tree constructed using the neighbour-joining method and 1000 replicates for bootstraps analysis with mega 3·1 (Kumar et al., 2004). Restriction profiles obtained after RFLP assays of PCR amplicons with Sau3AI, HpaII and HaeIII enzymes were all similar to those of ‘Candidatus Phytoplasma aurantifolia’(16SrII group). The 16S rRNA sequences of phytoplasmas detected in cassava (EU315317) and M. arboreus (EU315318), showed 98% of identity with that of the cactus witches’ broom phytoplasma (AJ293216), whilst sequences from C. variegatum (EU315315), H. rosa-sinensis (EU315314), and P. edulis (EU315319) exhibited 99% of identity with that of Catharantus roseus phytoplasma (EU096500). The phylogeny results suggest that two distinct sub-types have been identified in (i) cassava and M. arboreus and in (ii) C. variegatum, H. rosa-sinensis, and P. edulis, in plants growing geographically close to each other. This is the first record of phytoplasma group 16SrII, ‘Ca. Phytoplasma aurantifolia’ in Uganda, extending previous reports of its presence in Africa (Arocha et al., 2007). Further studies are needed of the biological aspects of the relationships between closely related phytoplasma isolates affecting nearby plant hosts. Work in the UK was done under Defra licence No. PHF 174D/5185(08/2005). Sequencing was done by the Sequencing Service (School of Life Sciences, University of Dundee, Scotland, http://www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3·1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer.

A New 'Candidatus Liberibacter' Species Associated with Diseases of Solanaceous Crops.

A new disease of glasshouse-grown tomato and pepper in New Zealand has resulted in plant decline and yield loss. Affected plants are characterized by spiky, chlorotic apical growth, curling or cupping of the leaves, and overall stunting. Transmission electron microscopy revealed the presence of phloem-limited bacterium-like organisms in symptomatic plants. The strategy used to identify the bacterium involved using specific prokaryote polymerase chain reaction (PCR) primers in combination with universal 16S rRNA primers. Sequence analysis of the 16S rRNA gene, the 16S/23S rRNA spacer region, and the rplKAJL-rpoBC operon revealed that the bacterium shared high identity with 'Candidatus Liberibacter' species. Phylogenetic analysis showed that the bacterium is distinct from the three citrus liberibacter species previously described and has been named 'Candidatus Liberibacter solanacearum'. This is the first report of a liberibacter naturally infecting a host outside the Rutaceae family. A specific PCR primer pair was developed for its detection.

Universal 16s rRNA Primers BD1 For Soil Microbial Community Analysis

New universal 16S rRNa primers were constructed and tested. These primers allow identifying correct taxonomic position of bacterial isolates and were shown to be useful in microbial community studies. The primers enable to detect the vast majority of unique 16S rRNa gene sequences. In the study 160 restriction types were found in 16S rRNa clone library (190 clones).

Retrieval of nearly complete 16S rRNA gene sequences from environmental DNA following 16S rRNA‐based community fingerprinting

16S rRNA-based fingerprinting techniques allow rapid analyses of overall bacterial community structure but suffer from a lack of phylogenetic information hitherto retrievable from the short 16S rRNA gene sequences obtained from excised bands. An approach is presented that allows nearly complete 16S rRNA gene sequences to be retrieved for abundant components of the bacterial community as obtained by the community fingerprint, i.e. those reflected by major fingerprint bands. This was achieved by designing a pair of highly specific primers derived from the sequence of an excised band. Combined with universal 16S rRNA primers, these specific primers were applied directly to environmental DNA serving as template. This procedure allowed the generation of a nearly complete 16S rRNA gene sequence of the target taxon by specific polymerase chain reaction (PCR) followed by cycle sequencing down to a relative abundance of at least 1.5% of the environmental DNA. The procedure was exemplified for an epsilonproteobacterium related to Thiomicrospira denitrificans occurring in the central Baltic Sea. This approach is based only on PCR without any cloning step involved. It allows focussing on specific target taxa and is thus rather efficient. This approach should be applicable in general to 16S rRNA or 16S rRNA gene-based fingerprinting techniques and their respective environmental DNA.

Detection of bacterial DNA in joint samples from patients with undifferentiated arthritis and reactive arthritis, using polymerase chain reaction with universal 16S ribosomal RNA primers.

Bacteria are considered to be important in the pathogenesis of several forms of arthritis. The goal of this study was to apply the 16S ribosomal RNA (rRNA)-polymerase chain reaction method for the detection of bacterial DNA in synovial fluid (SF) and synovial tissue (ST) from inflamed joints. Samples from 5 patients with septic arthritis and from 7 with osteoarthritis or arthritis secondary to joint trauma were used as controls. Samples from 6 patients with spondylarthropathy (SpA) and from 20 with undifferentiated arthritis (UA) were analyzed for the presence of bacterial DNA using universal 16S rRNA primers. Automated sequencing and comparative data analysis were performed to identify the species. In the positive control group, the bacterial species cultured from the synovium could be identified in all cases. No bacterial DNA was detected in the SF and ST from patients in the negative control group. In 4 of 6 patients with SpA and 7 of 20 with UA, the analysis of joint samples revealed the presence of bacterial DNA. Sequence analysis indicated the presence of multiple species, which was confirmed by sequencing of cloned products. When the the above techniques were used with a stringent regimen, we were able to demonstrate that it is possible to collect and analyze joint samples without contaminating bacterial DNA. The accumulation of phagocytic cells that contain bacterial DNA of various species could play a role in the pathogenesis of both SpA and UA.

Detection of bacterial dna in joint samples from patients with undifferentiated arthritis and reactive arthritis, using polymerase chain reaction with universal 16S ribosomal RNA primers

Objective Bacteria are considered to be important in the pathogenesis of several forms of arthritis. The goal of this study was to apply the 16S ribosomal RNA (rRNA)-polymerase chain reaction method for the detection of bacterial DNA in synovial fluid (SF) and synovial tissue (ST) from inflamed joints. Methods Samples from 5 patients with septic arthritis and from 7 with osteoarthritis or arthritis secondary to joint trauma were used as controls. Samples from 6 patients with spondylarthropathy (SpA) and from 20 with undifferentiated arthritis (UA) were analyzed for the presence of bacterial DNA using universal 16S rRNA primers. Automated sequencing and comparative data analysis were performed to identify the species. Results In the positive control group, the bacterial species cultured from the synovium could be identified in all cases. No bacterial DNA was detected in the SF and ST from patients in the negative control group. In 4 of 6 patients with SpA and 7 of 20 with UA, the analysis of joint samples revealed the presence of bacterial DNA. Sequence analysis indicated the presence of multiple species, which was confirmed by sequencing of cloned products. Conclusion When the the above techniques were used with a stringent regimen, we were able to demonstrate that it is possible to collect and analyze joint samples without contaminating bacterial DNA. The accumulation of phagocytic cells that contain bacterial DNA of various species could play a role in the pathogenesis of both SpA and UA.