Universal 16S rRNA Gene Primers Research Articles

Isolation and Identification of Actinomycetes-A Potential Bioprospective Candidates

Actinomycetes were isolated from the vegetable decomposing soil sample by serial dilution agar plating method. Five actinomycetes (A1 to A5) were obtained and the antimicrobial activity was carried out by Kirby Bauer agar diffusion method against selected eleven bacterial and eight fungal pathogens. Actinomycete strain A4 demonstrated the most activity. The most active actinomycete strain (A4) was identified phenotypically as Streptomyces spp. using macroscopic and microscopic examination. Following that, bioactive substances were purified using silica gel column chromatography and identified using Gas Chromatography-Mass Spectrometry. The major compounds identified were canadaline (35.79%), 2’4-diphromophenoxyphenol(31.07%). The ethyl acetate crude extract was assayed for anticancer activity by MTT method on HepG2cells with 10µl of extract showed 48.93% viability of HepG2 cells and significantly (p-value 0.000151 < 0.05 α-values) active among different concentration of crude A4 extract. The crude extract of A4 actinomyces exhibited the maximum inhibition of 47.55% at 10 10µl concentration and the activity were significant (p-value 0.01485) <0.05 α-values. The DNA was isolated from actinomycete strain (A4) and the 16SrRNA gene was amplified using universal eubacterial 16SrRNA gene primers and it was genotypically identified as Streptomyces coelicolor.

First Report of Pectobacterium parvum Causing Diseases on Potato in Inner Mongolia, China.

Soft rot enterobacterial plant pathogens Pectobacterium spp. and Dickeya spp. have caused devastating blackleg, aerial stem rot, and soft rot of potato tubers (Charkowski 2018). In August 2021, potato plants (cv. Xisen6# or Maiken1) with blackleg or aerial stem rot symptoms were observed in two commercial fields in Xinghe County, Ulanqab City, Inner Mongolia Autonomous Region, China. The plants were wilted, and the crown stem showed gradual degradation and browning. The disease incidence was around 3 to 7% and 20 to 25% in Xinghe Zhangyou Village (33 ha) and Bianjia Village (7 ha), respectively. Pathogens were isolated on crystal violet pectate agar (CVP) plates (Ge et al. 2018). Briefly, symptomatic stem tissues were surface sterilized in 75% ethanol, ground, then serial dilutions were cultured on CVP plates (Handique et al. 2022). The plates were incubated at 28oC for 2 days. Pure colonies of Pectobacterium spp. were obtained from the pits of CVP plates and sequenced for identification using the universal 16S rRNA gene primers 27F/1492R (Monciardini et al. 2002). Results of the comparison of 16S sequences against NCBI GenBank showed 100% sequence identity to P. parvum FN20211T (CP087392.1) type strain for the three colonies designated as ZRIMU1006 (1542/1542 bp), ZRIMU1019 (1542/1542 bp), and ZRIMU1020 (1542/1542 bp). Sequences were deposited under accession numbers OP941529, OP941525, OP941526, respectively. Additionally, six housekeeping gene sequences were used to confirm identification at the species level and were uploaded to GenBank: fusA (OP793177, OP793171, OP793172), gapA (OP793221, OP793216, OP793217), gyrB (OP793265, OP793259, OP793260), infB (OP793310, OP793304, OP793305), pgi (OP793355, OP793349, OP793350), and rplB (OP793400, OP793394, OP793395). Phylogenetic trees constructed using the MEGA X program of concatenated sequences of the housekeeping genes sequences show that the three isolates grouped with P. parvam FN20211T, confirming that they are P. parvam. Pathogenicity tests for stem rot were done by injecting a bacterial suspension into potato seedlings (cv. Favorita) grown from seed tubers. The tubers were planted in perlite potting mix and 3 weeks after emergence, a 100 μl bacterial suspension (105 CFU/ml) or sterile phosphate-buffered solution was injected at the stem base. The bacterial injection experiment was performed twice in a greenhouse with five plants inoculated per bacterial strain. Plants were covered with plastic bags to maintain 100% humidity at 25°C for 2 days. Seven days after inoculation, the inoculated area of the seedlings had rotted or turned black, while the controls remained symptomless. Symptomatic tissues from each strain were processed as above and placed on CVP plates to reisolate the Pectobacterium spp. 16S rRNA sequence analysis confirmed the bacteria to be similar in sequence to the inoculated strains, thus completing Koch's postulates. Soft rot development was performed by adding bacterial suspension (100 μl, 105 CFU/ml) on tuber slices. The infected tubers rotted, while the controls were symptomless. The vacuum infiltration method on tuber that is used to test pathogens for blackleg did not result in the development of blackleg symptoms. Briefly, five tubers were inoculated with the pathogen by vacuum infiltration and planted in potting mix. The plants showed wilting after emergence, but no blackleg symptoms were observed. Recently, multiple new Pectobacterium species including P. parmentieri, P. polaris, and P. punjabense, were identified to cause potato disease in different provinces of China (Cao et al. 2021; Handique et al. 2022). In China, P. parvum was first isolated from Brassica, and in the year 2022, it was reported to cause aerial stem-rot on potato in Hebei province in China (Wang et al. 2022). Inner Mongolia is a major potato-seed-producing province and the incidence of new strains of Pectobacterium in the province causing aerial stem rot and soft rot of tuber might cause a reduction in seed production. This report will draw attention to the management of P. parvum by the seed-producing companies in Inner Mongolia which distributes the seed throughout China.

First Report of Streptomyces niveiscabiei Causing Potato Common Scab in Shanxi and Gansu, China.

Multiple species of Streptomyces cause common scab disease in potato (Solanum tuberosum) (Kämpfer et al. 1991). Potato tubers (cv. Jinshu1 #5 and Longshu #6) with severe pitted common scab symptoms were observed from two farms in Chaozhou in Shanxi Province and in Tianzhu in Gansu Province during the national disease survey of bacterial diseases of potatoes in 2021. The disease incidence was around 30% on the 6.7 ha of the Chaozhou farm and 10% on the 0.7 ha on the Tianzhu farm. Three tubers with scab symptoms were surface disinfested with 3% sodium hypochlorite for 1 min. The symptomatic tissue was then ground in sterile water. Serially diluted ground samples were cultured on Streptomyces ISP Medium 5 agar plates (Shirling and Gottlieb 1966) and incubated at 280C for 5 days. Eight pure Streptomyces colonies were obtained and sequenced for identification using the universal 16S rRNA gene primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3') (Monciardini et al. 2002) by colony PCR. Blast results of the sequences against the NCBI GenBank for the eight isolates, ZRIMU1508, ZRIMU1510, ZRIMU1511, ZRIMU1512, ZRIMU1514, ZRIMU1515, ZRIMU1516 and ZRIMU1530 (Accession numbers: OP941573 - OP941580), showed more than 99% sequence identity to S. niveiscabiei NRRLB-24457T type strain. Additionally, 12 housekeeping gene sequences, acnA (OP997624 - OP997625), atpD (OP997622 - OP997623), dnaN (OP997620 - OP997621), gap (OP997618 - OP997619), gyrA (OP997614 - OP997615), gyrB (OP997612 - OP997613), infB (OP997610- OP997611), mdh (OP997608 - OP997609), recA (OP997602 - OP997603), rplB (OP997600 - OP997601), rpoB (OP997598- OP997599), and trpB (OP997594 - OP997595), were extracted from the genome sequences of two strains, ZRIMU1510 and ZRIMU1530, and uploaded to GenBank. Genes for pathogenicity, txtA (OP997593 - OP997594), tomA (OP997596 - OP997597) and Nec1(OP997606 - OP997607), were also identified from the genome sequence and uploaded to GenBank. The housekeeping genes and the pathogenicity genes showed over 98% identity with S. niveiscabiei. Phylogenetic trees were constructed using concatenated housekeeping gene sequences (Kumar et al. 1994) and the cladogram showed that the isolates ZRIMU1510 and ZRIMU1530 grouped with the type strain NRRLB-24457T. Pathogenicity tests were done by drench application of 100 ml spore suspensions (104 CFU/ml) of ZRIMU1530, ZRIMU1510, or phosphate buffer into pots with potato plants (cv. Favorita) grown in potting mix. Five tubers were planted and inoculated with each pathogen or phosphate buffer as the negative control. The plants were then placed in a greenhouse with 12 h of light per day, irrigated regularly, and harvested after 3 months. The newly formed tubers were checked for disease symptoms. Tubers from pots inoculated with ZRIMU1530 and ZRIMU1510 exhibited typical symptoms of common scab with raised corky lesions with deep pits, but the negative controls remained asymptomatic. The pathogens were reisolated from the lesions and confirmed to be identical to the original isolates by 16S rRNA gene sequences, thus completing Koch's postulates. The pot experiment was conducted twice: first in May 2022 and second in February 2023. To our knowledge, this is the first report of S. niveiscabiei causing common scab of potato in Shanxi and Gansu, China. S. niveiscabiei was first reported in Korea (Park et al. 2003) and this report will draw attention to the study and management of scab pathogens in China.

Combined Culture and DNA Metabarcoding Analysis of Cyanobacterial Community Structure in Response to Coral Reef Health Status in the South China Sea

Cyanobacteria occupy an extraordinarily diverse array of ecological niches in coral reefs because they play multifaceted roles, including primary carbon and nitrogen fixation, calcification, nutrient cycling, and oxygen production, as well as coral reef degradation through skeletal biocorrosion and polymicrobial diseases. In this study, cyanobacterial diversity in sediment, water, and coral tissues were explored in relation to coral health status (slightly, moderately, and severely damaged) of coral reefs at Weizhou Island, South China Sea. Microscopy of taxa morphological characteristics was combined with 16S rRNA gene metabarcoding. Fifteen and forty-three cyanobacterial genera were identified based on universal prokaryotic 16S rRNA gene primers and cyanobacteria-specific 16S rRNA gene primers metabarcoding, respectively, indicating a more sophisticated efficiency of the latter. In addition, three out of seven cyanobacterial strains that were isolated and identified based on morphology and phylogeny could not be detected using either molecular method. Therefore, culture-based combined cyanobacteria-specific 16S rRNA gene metabarcoding are highly recommended in future routine surveys. There was a clear distinction in cyanobacterial assemblage composition among locations with different coral health statuses, with degraded reefs exhibiting approximately a 1.25-fold increase in species compared to healthy habitats. In addition, the spreading of potentially toxic cyanobacteria, such as Nostoc and Lyngbya, in the degraded reef implies putative links to reef degradation. This study provides novel insights into the taxonomical diversity of cyanobacteria in tropical coral reefs. Metabarcoding is recommended as an effective tool for revealing cyanobacterial diversity patterns and thereby providing critical information for the effective management of coral reef ecosystems.

Characterization of ‘Candidatus Phytoplasma asteris’-related strain association with leaf yellowing of Wrightia antidysenterica (Arctic Snow) in Tripura, India

Wrightia antidysenterica is a small compact and evergreen ornamental shrub. Little leaf and yellowing symptoms were recorded on W. antidysenterica plants in June 2018 at College of Agriculture campus, Lembucherra, Tripura. The expected amplicons of ~1.25 kb and ~480 bp were obtained in symptomatic leaf samples with the phytoplasma specific universal 16S rRNA and secA gene primers. Sequence comparison analysis of 16S rRNA and secA gene confirmed the association of ‘Ca. P. asteris’-related strain. However, comparing the restriction profiles of 16S rRNA gene in virtual RFLP analysis revealed that the phytoplasma was a 16SrI group related strain. To our knowledge, this is the first report of phytoplasma association with W. antidysenterica little leaf and yellowing in world.

Reducing host DNA contamination in 16S rRNA gene surveys of anthozoan microbiomes using PNA clamps

Efforts to study the microbial communities associated with corals can be limited by inefficiencies in the sequencing process due to high levels of host amplification by universal bacterial 16S rRNA gene primers. Here, we develop an inexpensive peptide nucleic acid (PNA) clamp that binds to a target sequence of host DNA during PCR and blocks amplification. We then test the ability of this PNA clamp to mitigate host contamination and increase overall microbial sequence coverage on samples from three coral species: the gorgonians Eunicea flexuosa and Gorgonia ventalina, and the scleractinian Porites panamensis. The 20-bp PNA clamp was designed using DNA from E. flexuosa. Adding the PNA clamp during PCR increased the percentage of microbial reads in E. flexuosa samples more than 11-fold. Microbial community diversity was similar without- and with-PNA clamps, as were the relative frequencies of the ten most abundant ASVs (amplicon sequence variants), indicating that the clamps successfully blocked host DNA amplification while simultaneously increasing microbial DNA amplification proportionally across the most abundant taxa. The reduction of E. flexuosa DNA correlated with an increase in the abundance of rarer taxa. The clamp also increased the average percentage of microbial reads in another gorgonian, G. ventalina, by 8.6-fold without altering the microbial community beta diversity, and in a distantly related scleractinian coral, P. panamensis, by nearly double. The reduction of host contamination correlated with the number of nucleotide mismatches between the host amplicon and the PNA clamp. The PNA clamp costs as little as $0.48 per sample, making it an efficient and cost-effective solution to increase microbial sequence coverage for high-throughput sequencing of coral microbial communities.

Diversity of Betaproteobacteria revealed by novel primers suggests their role in arsenic cycling

High arsenic concentration in groundwater is a severe environmental problem affecting human health, particularly in countries of South and South-East Asia. The Bengal Delta Plain (BDP) distributed within India and Bangladesh is a major arsenic-affected region where groundwater is the primary source of drinking water. Previous studies have indicated that members of the bacterial class Betaproteobacteria constitute a major fraction of the microbial community in many of the aquifers within this region. Bacteria belonging to this class are known to be involved in redox cycling of arsenic as well as other metals such iron and manganese, thereby impacting arsenic mobilization and immobilization. While microbial diversity in arsenic-contaminated environments is generally assessed using universal 16S rRNA gene primers, targeted evaluation of Betaproteobacteria diversity remains poorly constrained. In this study, bacterial diversity was investigated in the groundwater from two shallow aquifers (West Bengal, India) based on 16S rRNA gene clone libraries and sequencing using a custom-designed pair of primers specific to Betaproteobacteria. Specificity of the primers was confirmed in silico as well as by the absence of PCR amplification of other bacterial classes. Four major families (Burkholderiaceae, Comamonadaceae, Gallionellaceae and Rhodocyclaceae) were detected among which members of Burkholderiaceae represented 59% and 71% of the total community in each aquifer. The four OTUs (operational taxonomic units; 97% sequence identity) within Burkholderiaceae were close phylogenetic relatives of bacteria within the genus Burkholderia known to solubilize phosphate minerals. Additionally, the OTUs belonging to Gallionellaceae were closely related to the members of the genera Gallionella and Sideroxydans, known to oxidize iron under microaerophilic conditions. These results suggest that members of Betaproteobacteria can potentially influence iron and phosphorus cycling which can influence biogeochemistry in arsenic-contaminated aquifers of the BDP.

First report of 16Sr II‐C subgroup phytoplasma association with Acacia mangium in Tripura, India

AbstractLeaf yellowing symptoms were observed on Acacia mangium in the Sipahijala district of Tripura, India, during June 2017. Symptomatic and asymptomatic leaf samples (three of each) were collected from roadside trees of A. mangium for DNA extraction using the CTAB method. Amplicons of ~1.25 kb and ~480 bp were detected in all the symptomatic samples using the phytoplasma‐specific universal 16S rRNA and secA gene primers. Pair wise sequence analysis of 16S rRNA gene sequences, virtual RFLP and phylogenetic analysis revealed that the phytoplasma strain associated with A. mangium belonged to phytoplasma subgroup 16SrII‐C. This is the first report of an association between the 16SrII‐C subgroup and A. mangium leaf yellowing.

Rapid PCR identification of Prevotella copri in an Australian cohort of pregnant women.

Gut bacteria from the genus Prevotella are found in high abundance in faeces of non-industrialised communities but low abundance in industrialised, Westernised communities. Prevotella copri is one of the principal Prevotella species within the human gut. As it has been associated with developmental health and disease states, we sought to (i) develop a real-time polymerase chain reaction (PCR) to rapidly determine P. copri abundance and (ii) investigate its abundance in a large group of Australian pregnant mothers.The Barwon Infant Study is a pre-birth cohort study (n = 1074). Faecal samples were collected from mothers at 36 weeks gestation. Primers with a probe specific to the V3 region of P. copri 16S rRNA gene were designed and optimised for real-time PCR. Universal 16S rRNA gene primers amplified pan-bacterial DNA in parallel. Relative abundance of P. copri was calculated using a 2-ΔCt method.Relative abundance of P. copri by PCR was observed in 165/605 (27.3%) women. The distribution was distinctly bimodal, defining women with substantial (n = 115/165, 69.7%) versus very low P. copri expression (n = 50/165, 30.3%). In addition, abundance of P. copri by PCR correlated with 16S rRNA gene MiSeq sequencing data (r2 = 0.67, P < 0.0001, n = 61).We have developed a rapid and cost-effective technique for identifying the relative abundance of P. copri using real-time PCR. The expression of P. copri was evident in only a quarter of the mothers, and either at substantial or very low levels. PCR detection of P. copri may facilitate assessment of this species in large, longitudinal studies across multiple populations and in various clinical settings.

Elevated CO2 has a significant impact on denitrifying bacterial community in wheat roots

Elevated CO2 (eCO2) stimulates plant growth and photosynthesis, which affect root deposition, leading to altered structure and function of the root microbiome. We studied the effect of eCO2 on wheat-root microbiome composition and plant development, with an emphasis on denitrifying communities. Wheat plants were grown in a greenhouse with continuous fertigation for 6 weeks under ambient CO2 (400 ppm) or eCO2 (850 ppm). The total bacterial community was quantified using qPCR with universal 16S rRNA gene primers, and denitrifying genes (nirK, nirS, nosZ) were measured. In addition, total (16S-based) and N2O-reducing (nosZ-based) bacterial community compositions in the soil and roots were analyzed by amplicon sequencing during plant growth. eCO2 had a significant impact on abundance of the studied denitrifying genes, particularly during the late stages of wheat growth before spike formation. Moreover, eCO2 had a significant impact on N2O-reducing community structure in roots. This effect was more pronounced on Burkholderiales and Rhizobiales, with a minor effect on Pseudomonadales. In addition, as expected, bacterial community structure (total and N2O-reducing bacteria), and denitrifying gene abundance, were primary influenced by habitat (soil vs. roots), and secondarily by plant developmental stage. In summary, it is suggested that eCO2 may change root microbiome, enhance wheat development and N demand without an increase in N2O emission.

Improved quadruplex real-time PCR assay for the diagnosis of diphtheria.

Introduction. Diphtheria is caused by toxigenic strains of Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis. For diagnostic purposes, species identification and detection of toxigenic strains (diphtheria toxin (tox)-positive strains) is typically performed using end-point PCR. A faster quadruplex real-time PCR (qPCR) was recently developed (De Zoysa et al. J . Med . Microbiol. 2016;65(12):1521-1527).Aims. We aimed to improve the quadruplex method by adding a 16S rRNA gene target as an internal processing control, providing confirmation of the presence of bacterial DNA in the assays, thus avoiding the possibility of false-negative reporting.Methodology. Universal 16S rRNA gene primers and a probe were defined. The novel method was tested using 36 bacterial isolates and 17 clinical samples. Experimental robustness to temperature and reagent concentration variations was assessed.Results. The method allows detection of the tox gene and distinguishing C. diphtheriae (including the newly described species Corynebacterium belfantii) from C. ulcerans and C. pseudotuberculosis. Complete diagnostic specificity, sensitivity and experimental robustness were demonstrated. The lower limit of detection for C. diphtheriae, C. ulcerans and tox targets was 1.86 genome copies per 5 µl reaction volume. The method was successfully used on two distinct qPCR technologies (LightCycler 480, Roche Diagnostics and Rotor-Gene Q, Qiagen) and in two laboratories (Institut Pasteur, Paris, France and Public Health England - National Infection Service, London, UK).Conclusion. This work describes validation of the improved qPCR quadruplex method and supports its implementation for the biological diagnosis of diphtheria.

Isolation and characterization of heterotrophic nitrification–aerobic denitrification and sulphur-oxidizing bacterium Paracoccus saliphilus strain SPUM from coastal shrimp ponds

The Paracoccus sp. is one of the best-characterized prokaryote that has served as a model organism to study the dentrification and sulphur oxidation processes. However, its ability of dentrification and sulphur oxidation in coastal shrimp ponds is not reported much. In the present work, the Gram-negative, neutrophilic, facultatively lithoautotrophic bacterium Paracoccus saliphilus strain SPUM isolated from coastal shrimp ponds of Urran, Maharashtra, was studied for its efficiency of simultaneous heterotrophic nitrification–aerobic denitrification and sulphur oxidation processes. The maximum removal rate of nitrite and nitrate was 11.22 ± 0.31 and 14.17 ± 0.31 mg of NO3-N/l respectively after 24 h of incubation, while the sulphate-sulphur production observed was 190 ± 4.3 mg l−1 with a change in pH from 8.0 to 7.4 ± 0.08 after 12 days of incubation. The strain was characterized using universal 16S rRNA gene primers revealing high similarity (> 99%) with Paracoccus saliphilus belonging to α-proteobacteria. The isolate could express sulphate thiolesterase/sulphate thiohydrolase, soxB gene which is essential for sulphur oxidation. From all the results, it has been found that the strain SPUM could play a major role in simultaneous aerobic nitrification/denitrification and sulphur oxidation processes to overcome the toxicity of nitrogenous and sulphur-reducing compounds respectively in coastal aquaculture and wastewater systems.

Primer Design for an Accurate View of Picocyanobacterial Community Structure by Using High-Throughput Sequencing.

High-throughput sequencing (HTS) of the 16S rRNA gene has been used successfully to describe the structure and dynamics of microbial communities. Picocyanobacteria are important members of bacterioplankton communities, and, so far, they have predominantly been targeted using universal bacterial primers, providing a limited resolution of the picocyanobacterial community structure and dynamics. To increase such resolution, the study of a particular target group is best approached with the use of specific primers. Here, we aimed to design and evaluate specific primers for aquatic picocyanobacterial genera to be used with high-throughput sequencing. Since the various regions of the 16S rRNA gene have different degrees of conservation in different bacterial groups, we therefore first determined which hypervariable region of the 16S rRNA gene provides the highest taxonomic and phylogenetic resolution for the genera Synechococcus, Prochlorococcus, and Cyanobium An in silico analysis showed that the V5, V6, and V7 hypervariable regions appear to be the most informative for this group. We then designed primers flanking these hypervariable regions and tested them in natural marine and freshwater communities. We successfully detected that most (97%) of the obtained reads could be assigned to picocyanobacterial genera. We defined operational taxonomic units as exact sequence variants (zero-radius operational taxonomic units [zOTUs]), which allowed us to detect higher genetic diversity and infer ecologically relevant information about picocyanobacterial community composition and dynamics in different aquatic systems. Our results open the door to future studies investigating picocyanobacterial diversity in aquatic systems.IMPORTANCE The molecular diversity of the aquatic picocyanobacterial community cannot be accurately described using only the available universal 16S rRNA gene primers that target the whole bacterial and archaeal community. We show that the hypervariable regions V5, V6, and V7 of the 16S rRNA gene are better suited to study the diversity, community structure, and dynamics of picocyanobacterial communities at a fine scale using Illumina MiSeq sequencing. Due to its variability, it allows reconstructing phylogenies featuring topologies comparable to those generated when using the complete 16S rRNA gene sequence. Further, we successfully designed a new set of primers flanking the V5 to V7 region whose specificity for picocyanobacterial genera was tested in silico and validated in several freshwater and marine aquatic communities. This work represents a step forward for understanding the diversity and ecology of aquatic picocyanobacteria and sets the path for future studies on picocyanobacterial diversity.

Sulfur oxidation by Achromobacter xylosoxidans strain wsp05 reveals ecological widening over which thiotrophs are distributed.

Achromobacter xylosoxidans is a versatile bacterium known for its ability to degrade aromatic compounds. However, its ability to oxidize sulfur compounds for electron and energy source is not reported much. In the present work, the Gram-negative bacterium Achromobacter xylosoxidans strain wsp05 isolated from a waste stabilization ponds (WSPs) system was studied for its ability to oxidize reduced sulfur compounds. The strain was able to oxidize thiosulfate and sodium sulfite. To observe the effect of physicochemical parameters on the rate of sulfur oxidation, strain wsp05 was grown in thiosulfate (20mM) containing minimal salt medium at varied pH, temperature and ammonium and phosphate ions concentration. Maximum thiosulfate oxidation was observed at 30°C with initial pH of 7-7.2. The strain was characterized using universal 16S rRNA gene primers revealing high similarity (> 99%) with Achromobacter xylosoxidans NBRC 15126T belonging to β-proteobacteria. In the present study, we investigated the sulfur oxidation properties of the Achromobacter xylosoxidans strain wsp05, which revealed an ecological and phylogenetic widening over which the thiotrophs are distributed.

Design and evaluation of universal 16S rRNA gene primers for high-throughput sequencing to simultaneously detect DAMO microbes and anammox bacteria

To develop universal 16S rRNA gene primers for high-throughput sequencing for the simultaneous detection of denitrifying anaerobic methane oxidation (DAMO) archaea, DAMO bacteria, and anaerobic ammonium oxidation (anammox) bacteria, four published primer sets (PS2–PS5) were modified. The overall coverage of the four primer pairs was evaluated in silico with the Silva SSU r119 dataset. Based on the virtual evaluation, the two best primer pairs (PS4 and PS5) were selected for further verification. Illumina MiSeq sequencing of a freshwater sediment and a culture from a DAMO–anammox reactor using these two primer pairs revealed that PS5 (341b4F–806R) was the most promising universal primer pair. This pair of primers detected both archaea and bacteria with less bias than PS4. Furthermore, an anaerobic fermentation culture and a wastewater treatment plant culture were used to verify the accuracy of PS5. More importantly, it detected DAMO archaea, DAMO bacteria, and anammox bacteria simultaneously with no false positives appeared. This universal 16S rRNA gene primer pair extends the existing molecular tools for studying the community structures and distributions of DAMO microbes and their potential interactions with anammox bacteria in different environments.

Relations of microbiome characteristics to edaphic properties of tropical soils from Trinidad.

Understanding how community structure of Bacteria, Archaea, and Fungi varies as a function of edaphic characteristics is key to elucidating associations between soil ecosystem function and the microbiome that sustains it. In this study, non-managed tropical soils were examined that represented a range of edaphic characteristics, and a comprehensive soil microbiome analysis was done by Illumina sequencing of amplicon libraries that targeted Bacteria (universal prokaryotic 16S rRNA gene primers), Archaea (primers selective for archaeal 16S rRNA genes), or Fungi (internal transcribed spacer region). Microbiome diversity decreased in the order: Bacteria > Archaea > Fungi. Bacterial community composition had a strong relationship to edaphic factors while that of Archaea and Fungi was comparatively weak. Bacterial communities were 70–80% alike, while communities of Fungi and Archaea had 40–50% similarity. While each of the three component communities differed in species turnover patterns, soils having relatively similar bacterial communities also housed similar archaeal communities. In contrast, the composition of fungal communities had no correlation to bacterial or archaeal communities. Bacterial and archaeal diversity had significant (negative) correlations to pH, whereas fungal diversity was not correlated to pH. Edaphic characteristics that best explained variation between soils in bacterial community structure were: total carbon, sodium, magnesium, and zinc. For fungi, the best variables were: sodium, magnesium, phosphorus, boron, and C/N. Archaeal communities had two sets of edaphic factors of equal strength, one contained sulfur, sodium, and ammonium-N and the other was composed of clay, potassium, ammonium-N, and nitrate-N. Collectively, the data indicate that Bacteria, Archaea, and Fungi did not closely parallel one another in community structure development, and thus microbiomes in each soil acquired unique identities. This divergence could in part reflect the finding that unknown factor(s) were stronger than edaphic characteristics in shaping fungal and archaeal communities.

Prevalence and phylogenetic analysis of haemoplasmas from cats infected with multiple species

Mycoplasma haemofelis (Mhf), ‘Candidatus Mycoplasma haemominutum’ (CMhm) and ‘Candidatus Mycoplasma turicensis’ (CMt) are agents of feline haemoplasmosis and can induce anaemia in cats. This study aimed to determine the prevalence and phylogeny of haemoplasma species in cats from Brazil's capital and surrounding areas, and whether correlation with haematological abnormalities existed. Feline haemoplasmas were found in 13.8% of 432 cats. CMhm was the most prevalent species (in 13.8% of cats), followed by Mhf (11.1%) and CMt (4.4%). Over 80% of haemoplasma-infected cats harboured two or more feline haemoplasma species: 7.1% of cats were co-infected with Mhf/CMhm, 0.4% with CMhm/CMt and 3.9% with Mhf/CMhm/CMt. Male gender was significantly associated with haemoplasma infections. No association was found between qPCR haemoplasma status and haematological variables, however CMhm relative copy numbers were correlated with red blood cell (RBC) numbers and packed cell volume (PCV). Haemoplasma 16S rRNA gene sequences (>1Kb) were derived from co-infected cats using novel haemoplasma species-specific primers. This allowed 16S rRNA gene sequences to be obtained despite the high level of co-infection, which precluded the use of universal 16S rRNA gene primers. Within each species, the Mhf, CMhm and CMt sequences showed >99.8%, >98.5% and >98.8% identity, respectively. The Mhf, CMhm and CMt sequences showed >99.2%, >98.4% and >97.8% identity, respectively, with GenBank sequences. Phylogenetic analysis showed all Mhf sequences to reside in a single clade, whereas the CMhm and CMt sequences each grouped into three distinct subclades. These phylogeny findings suggest the existence of different CMhm and CMt strains.

Diagnosis of Carrion’s Disease by Direct Blood PCR in Thin Blood Smear Negative Samples

Bartonella bacilliformis is the etiologic agent of Carrion's disease. This disease has two well established phases, the most relevant being the so called Oroya Fever, in which B. bacilliformis infect the erythrocytes resulting in severe anemia and transient immunosuppression, with a high lethality in the absence of adequate antibiotic treatment. The presence of B. bacilliformis was studied in 113 blood samples suspected of Carrion’s disease based on clinical criteria, despite the absence of a positive thin blood smear, by two different PCR techniques (using Bartonella-specific and universal 16S rRNA gene primers), and by bacterial culture. The specific 16S rRNA gene primers revealed the presence of 21 B. bacilliformis and 1 Bartonella elizabethae, while universal primers showed both the presence of 3 coinfections in which a concomitant pathogen was detected plus Bartonella, in addition to the presence of infections by other microorganisms such as Agrobacterium or Bacillus firmus. These data support the need to implement molecular tools to diagnose Carrion’s disease.

Diversity Analysis of Bacterial Community from Permafrost Soil of Mo-he in China

The permafrost soil of Mo-he in Northeast China presents a typical cold environment colonized by psychrophilic microorganisms. This study is aimed at assessing the bacterial communities of permafrost soil of Mo-he in China by sequencing the 16S rRNA genes and Mothur analysis. PCR products with universal 16S rRNA gene primers were cloned and partially sequenced, and bacterial identification at the species was performed by comparative analysis with the GenBank/EMBL/DDBJ database. A total of 266 clones were obtained with the average length of 1,050bp. Mothur analysis showed that the coverage value of clone library was 53.78%, Shannon diversity (H) was 4.03, Simpson diversity value was 0.018, and 74 operational taxonomic units were generated. Through phylogenetic assignment using BLASTN by more than 97% similarity, a total of 87 tentative taxa were identified. The majority of bacterial sequences recovered in this study belonged to the Acidobacteria, Proteobacteria, Verrucomicrobia, Bacteroidetes, Chloroflexi and Chlorobi. Among them, Acidobacteria are dominant community, accounting for 30.1% of total bacteria, followed by Proteobacteria which accounted for 22.2%. This result reflected the acidic characteristics of the permafrost soil of which pH value was 6.0. Our study indicated that the permafrost soil of Mo-he in China has a high diversity of bacteria and represents a vast potential resource of novel bacteria. As far as we knew, this is the first report on bacterial diversity of permafrost soil of Mo-he in China.

Microflora profiling of infected root canal before and after treatment using culture-independent methods

This study aimed to profile the microflora in infected root canals before and after root canal treatment using culture-independent methods. Six infected root canals in single-rooted teeth with periapical lesions from five subjects were included. Quantification of total bacteria was performed by real-time PCR with primers targeting 16S rRNA genes. PCR products with universal 16S rRNA gene primers were cloned and partially sequenced, and bacterial identification at the species level was performed by comparative analysis with the GenBank database. The concentration of extracted DNA before treatment was higher than that after root canal treatment, although the difference was not statistically significant. Sequence analysis revealed that oral bacteria such as Fusobacterium, Streptococcus, Olsenella, and Pseudoramibacter detected in cases before root canal treatment disappeared after treatment. These results suggest that the root canal microflora are distinct before and after root canal treatment, and that treatment changes the microflora in both quantity and quality.