16S rDNA Copy Research Articles

Quality and composition of archived nucleic acids after use in SARS-CoV-2 molecular testing

BackgroundReverse transcription real-time PCR (rRT-PCR) has been a gold-standard method to detect SARS-CoV-2, for which quality assessment of nucleic acids (NAs) is not needed. In order to prepare for future use, we evaluated NA quality from archived SARS-CoV-2 rRT-PCR samples. MethodsNA samples were collected in February 2021 and extracted using the QIAamp DSP Virus Spin Kit, (53 SARS-CoV-2-positive and 100 SARS-CoV-2-negative). Quality, quantity, and purity of NA were measured spectrophotometrically or fluorescently. Droplet digital PCR was used to characterize the double strand DNA (dsDNA) origin and composition by quantifying 16S rDNA and RPP30. ResultsThe RIN and purity were not significantly different between groups (p = 0.3828). RNA quantity was significantly higher than dsDNA in both groups (p < 0.0001); both dsDNA and RNA quantity were significantly higher in positive samples (dsDNA, RNA p = 0.021). For dsDNA, 16S rDNA copies were significantly greater than RPP30 in both groups (p < 0.0001), and RPP30 were significantly higher in positive samples (p < 0.0001). ConclusionsArchived NA quality after SARS-CoV-2 rRT-PCR was guaranteed for subsequent molecular research using human or bacterial DNA, especially for short targets.

Melting temperature mapping method in children: Rapid identification of pathogenic microbes

IntroductionThe melting temperature (Tm) mapping method (TM) identifies bacterial species by intrinsic patterns of Tm values in the 16S ribosomal RNA gene (16S rDNA) extracted directly from whole blood. We examined potential clinical application of TM in children with bloodstream infection (BSI). MethodsThis was a prospective observational study at a children's hospital in Japan from 2018 to 2021. In patients with diagnosed or suspected BSI, we investigated the match rates of pathogenic bacteria identified by TM and blood culture (BC), the inspection time to identification of TM, and the amount of bacterial DNA in blood samples. ResultsThe median age of 81 patients (93 samples) was 3.6 years. Of 23 samples identified by TM, 11 samples matched the bacterial species with BC (positive-match rate, 48 %). Of 64 TM-negative samples, 62 samples were negative for BC (negative-match rate, 97 %). Six samples, including one containing two pathogenic bacterial species, were not suitable for TM identification. In total, the matched samples were 73 of 93 samples (match rate, 78 %). There were seven samples identified by TM in BC-negative samples from blood collected after antibiotic therapy. Interestingly, the bacteria were matched with BC before antibiotic administration. These TM samples contained as many 16S rDNA copies as the BC-positive samples. The median inspection time to identification using TM was 4.7 h. ConclusionsIn children with BSI, TM had high negative-match rates with BC, the potential to identify the pathogenic bacteria even in patients on antibiotic therapy, and more rapid identification compared to BC. Registering clinical trialsUMIN000041359https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000047220.

RAD51 and RAD51B Play Diverse Roles in the Repair of DNA Double Strand Breaks in Physcomitrium patens.

RAD51 is involved in finding and invading homologous DNA sequences for accurate homologous recombination (HR). Its paralogs have evolved to regulate and promote RAD51 functions. The efficient gene targeting and high HR rates are unique in plants only in the moss Physcomitrium patens (P. patens). In addition to two functionally equivalent RAD51 genes (RAD1-1 and RAD51-2), other RAD51 paralogues were also identified in P. patens. For elucidation of RAD51's involvement during DSB repair, two knockout lines were constructed, one mutated in both RAD51 genes (Pprad51-1-2) and the second with mutated RAD51B gene (Pprad51B). Both lines are equally hypersensitive to bleomycin, in contrast to their very different DSB repair efficiency. Whereas DSB repair in Pprad51-1-2 is even faster than in WT, in Pprad51B, it is slow, particularly during the second phase of repair kinetic. We interpret these results as PpRAD51-1 and -2 being true functional homologs of ancestral RAD51 involved in the homology search during HR. Absence of RAD51 redirects DSB repair to the fast NHEJ pathway and leads to a reduced 5S and 18S rDNA copy number. The exact role of the RAD51B paralog remains unclear, though it is important in damage recognition and orchestrating HR response.

FISHing for ciliates: Catalyzed reporter deposition fluorescence in situ hybridization for the detection of planktonic freshwater ciliates.

Planktonic ciliate species form multiple trophic guilds and are central components of freshwater food webs. Progress in molecular analytical tools has opened new insight into ciliate assemblages. However, high and variable 18S rDNA copy numbers, typical for ciliates, make reliable quantification by amplicon sequencing extremely difficult. For an exact determination of abundances, the classical morphology-based quantitative protargol staining is still the method of choice. Morphotype analyses, however, are time consuming and need specific taxonomic expertise. Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) may represent a promising tool for the analysis of planktonic ciliates by combining molecular identification with microscopic quantification. We tested the applicability of CARD-FISH using nine cultured ciliate species. Eight species- and three genus-specific oligonucleotide probes were designed based on their 18S rRNA genes. The CARD-FISH protocol was adapted and the specificity of probes was established. We subsequently examined the precision of quantitation by CARD-FISH on single cultures and mock assemblages. Successful tests on lake water samples proved that planktonic ciliates could be identified and quantified in field samples by CARD-FISH. Double hybridizations allowed studying interspecific predator prey interactions between two ciliate species. In summary, we demonstrate that CARD-FISH with species-specific probes can facilitate studies on the population dynamics of closely related, small sized or cryptic species at high sampling frequencies.

Local Environmental Conditions Promote High Turnover Diversity of Benthic Deep-Sea Fungi in the Ross Sea (Antarctica).

Fungi are a ubiquitous component of marine systems, but their quantitative relevance, biodiversity and ecological role in benthic deep-sea ecosystems remain largely unexplored. In this study, we investigated fungal abundance, diversity and assemblage composition in two benthic deep-sea sites of the Ross Sea (Southern Ocean, Antarctica), characterized by different environmental conditions (i.e., temperature, salinity, trophic availability). Our results indicate that fungal abundance (estimated as the number of 18S rDNA copies g−1) varied by almost one order of magnitude between the two benthic sites, consistently with changes in sediment characteristics and trophic availability. The highest fungal richness (in terms of Amplicon Sequence Variants−ASVs) was encountered in the sediments characterized by the highest organic matter content, indicating potential control of trophic availability on fungal diversity. The composition of fungal assemblages was highly diverse between sites and within each site (similarity less than 10%), suggesting that differences in environmental and ecological characteristics occurring even at a small spatial scale can promote high turnover diversity. Overall, this study provides new insights on the factors influencing the abundance and diversity of benthic deep-sea fungi inhabiting the Ross Sea, and also paves the way for a better understanding of the potential responses of benthic deep-sea fungi inhabiting Antarctic ecosystems in light of current and future climate changes.

Diagnostic predictability of miR-4535 and miR-1915-5p expression in amniotic fluid for foetal morbidity of infection.

Clinical prediction of foetal inflammatory response syndrome (FIRS) is highly necessary. We have previously reported that miR-4535 and miR-1915-5p are potential biomarkers for severe chorioamnionitis based on the results of microRNA array analysis. Therefore, we evaluated the relationship between foetal morbidity of infection and miR-4535, miR-1915-5p, interleukin (IL)-6, or 16S rDNA copy number levels in amniotic fluid from pregnant women with chorioamnionitis. Amniotic fluid from 57 pregnant women with preterm premature membrane rupture or threatened premature labour were collected. Infants with WBC counts <5000/μL or >20,000/μL, CRP >0.5mg/mL, or IgM >20mg/mL at birth received a diagnosis of suspicious foetal infection, and those requiring antibiotic administration for >5 days were considered infected newborns. miR-4535, miR-1915-5p, and IL-6 levels and 16S rDNA copy number were evaluated. Mann-Whitney U test and Dunn's test were used for comparison. The area under the curve (AUC) and Youden index were calculated to examine the diagnostic accuracy of foetal morbidity of infection. miR-4535, miR-1915-5p, 16S rDNA, and IL-6 were significantly higher in patients with severe chorioamnionitis than in patients with chorionitis or sub-chorionitis (P<0.05). miR-4535 and miR-1915-5p levels were significantly associated with WBC counts <5000/μL or >20,000/μL, CRP >0.5mg/mL, or IgM >20mg/mL (P<0.05). AUC values of miR-4535 and miR-1915-5p indicated moderate or low accuracy for foetal morbidity of infection, while those of IL-6 and 16S rDNA seemed unreliable. MiR-4535 and miR-1915-5p levels in amniotic fluid may be considered clinically predictive for foetal morbidity of infection.

Determination of bacterial abundance and communities in the nipple drinking system of cascading cage layer houses

Water quality is critical for egg production and animal health in commercial layer housing systems. To investigate microbial contamination in nipple drinking system in layer houses, the bacterial abundance and communities in water pipes and V-troughs on different tiers (e.g., 1st, 3rd, 5th, and 7th tiers) of a layer house with 8 overlapping cage tiers were determined using qRT-PCR and 16S rRNA sequencing. The water bacterial abundance (i.e., genome 16S rDNA copy number, WBCN) in water pipes and V-troughs did not significantly differ among tiers, but they were 46.77 to 1905.46 times higher in V-troughs than that in water pipes (P < 0.05) for each tier. Illumina sequencing obtained 1,746,303 effective reads from 24 water samples in V-troughs of 4 tiers (six samples from each tier). Taxonomic analysis indicated that the 1st and 5th tiers were predominated by Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes, while the 3rd and 7th tiers were predominated by Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria. The top four genera were Acinetobacter, Streptococcus, Rothia and Comamonas among measured tiers. The high bacterial abundance and bacterial OTUs of water in the V-troughs reflect poor water quality, which may adversely affect growth and health of laying hens. Therefore, it is suggested that water quality in the V-tough should be checked more frequently in commercial layer houses.

A potential probiotic Leuconostoc mesenteroides TBE-8 for honey bee

An isolated bacterium TBE-8, was identified as Leuconostoc mesenteroides according to the sequences of 16S rDNA and the 16S–23S rDNA intergenic spacer region. The probiotic properties of the L. mesenteroides TBE-8 strain were characterized and revealed that TBE-8 could utilize various carbohydrates, exhibited high tolerance to sucrose’s osmotic pressure and acidic conditions, and could mitigate the impact of the bee pathogen Paenibacillus larvae. In addition, we found that the TBE-8 broth increased the expression of the nutrition-related genes major royal jelly protein 1 and vitellogenin in bees by approximately 1400- and 20-fold, respectively. The expression of genes encoding two antibacterial peptides, hymenoptaecin and apidaecin, in the bee abdomen was significantly increased by 17- and 7-fold in bees fed with the TBE-8 fermented broth. Furthermore, we fed four-frame bee colonies with 50% sucrose syrup containing TBE-8 and can detect the presence of approximately 2 × 106 16S rDNA copies of TBE-8 in the guts of all bees in 24 h, and the retention of TBE-8 in the bee gut for at least 5 days. These findings indicate that the L. mesenteroides TBE-8 has high potential as a bee probiotic and could enhance the health of bee colonies.

Soil type influence nutrient availability, microbial metabolic diversity, eubacterial and diazotroph abundance in chickpea rhizosphere.

Rhizosphere microbial communities are dynamic and play a crucial role in diverse biochemical processes and nutrient cycling. Soil type and cultivar modulate the composition of rhizosphere microbial communities. Changes in the community composition significantly alter microbial function and ecological process. We examined the influence of soil type on eubacterial and diazotrophic community abundance and microbial metabolic potential in chickpea (cv. BG 372 and cv. BG 256) rhizosphere. The total eubacterial and diazotrophic community as estimated through 16S rDNA and nifH gene copy numbers using qPCR showed the soil type influence with clear rhizosphere effect on gene abundance. PLFA study has shown the variation in microbial community structure with different soil types. Differential influence of soil types and cultivar on the ratio of Gram positive to Gram negative bacteria was observed with most rhizosphere soils corresponding to higher ratios than bulk soil. The rhizosphere microbial activities (urease, dehydrogenase, alkaline phosphatase and beta-glucosidase) were also assessed as an indicator of microbial metabolic diversity. Principal component analysis and K-means non-hierarchical cluster mapping grouped soils into three categories, each having different soil enzyme activity or edaphic drivers. Soil type and cultivar influence on average substrate utilization pattern analyzed through community level physiological profiling (CLPP) was higher for rhizosphere soils than bulk soils. The soil nutrient studies revealed that both soil type and cultivar influenced the available N, P, K and organic carbon content of rhizosphere soil. Our study signifies that soil type and cultivar jointly influenced soil microbial community abundance and their metabolic potential in chickpea rhizosphere.

Degradation of antimicrobial resistance genes within stockpiled beef cattle feedlot manure

Degradation of antimicrobial resistance genes (ARG) in manure from beef cattle administered (kg−1 feed) 44 mg of chlortetracycline (CTC), 44 mg of chlortetracycline plus sulfamethazine (CTCSMZ), 11 mg of tylosin (TYL), or no antimicrobials (Control) was examined. Manure was stockpiled and quantitative PCR (qPCR) was used to assess tetracycline [tet(C), (L), (M), (W)], erythromycin [erm(A), (B), (F), (X)], and sulfamethazine [sul(1), (2)] ARG and 16S rDNA. After 102 d, copies of all ARG decreased by 0.3 to 1.5 log10 copies (g dry matter)−1. Temperature in the interior of piles averaged ≥ 55 °C for 10 d, except for CTCSMZ, but did not reach 55 °C at pile exteriors. Compared to Control, CTCSMZ increased (P < 0.05) tet(C), tet(M), tet(W), sul(1), and sul(2) in stockpiled manure. Copies of 16S rDNA remained higher (P < 0.05) in CTCSMZ than Control for the first 26 d. Levels of most ARG did not differ between the interior and exterior of stockpiles. Our results suggest that stockpiled manure would still introduce ARG to land upon manure application, but at levels lower than if manure was applied fresh.

Phylogenetic diversity contributes more to sediment magnetism than abundance during incubation of iron-reducing sediment from a non-active volcanic lake in Northeast China.

This study aimed to analyse bacterial community and biomineralization products from Wudalianchi non-active volcanic field and the relationship between magnetization and bacterial community. Eighteen sediment samples obtained from Wenbo Lake, high-throughput sequencing and quantitative PCR (qPCR) were separately employed to investigate the bacterial community composition dynamics and abundance variation of the sediment sample with the highest iron-reducing capacity during incubation. The mineralization products were characterized by transmission electron microscopy, scanning electron microscopy, X-ray diffraction (XRD), Raman spectroscopy, vibrating sample magnetometer (VSM) and variable-temperature magnetism analyses. The results showed that the highest iron reduction rate was 98·06%. Seven phyla were identified as dominant bacterial phyla during the incubation process. Iron-reducing bacteria (FeRB) including Geobacter, Desulfosporosinus and Clostridium were involved in the iron mineralization process. The 16S rDNA copy numbers of sediment decreased quickly and then stayed steady during the incubation. Bacteria with rod-shaped and spheroid species were involved in extracellular iron reduction to produce magnetic particles with massive aggregation and columnar structures on the mineral surface morphologies. The materials produced by the microbial community over the incubation period were sequentially identified as siderite, magnetite and maghemite. The magnetism of the mineral samples gradually increased from 0·31748 to 33·58423emug-1 with increased incubation time. The final products showed relatively stable magnetism under 0-400K. Meanwhile, the saturation magnetization (MS ) of the mineralized substance was tightly associated with bacterial diversity (P<0·05). Bacterial community varied during incubation of iron-reducing sediment of volcanic lake. Various iron mineral crystals were in turn formed extracellularly by FeRB. The magnetism of mineralized products was tightly associated with bacterial community. These results not only help us to better understand the iron mineralization of FeRB in the volcanic lake sediments but also provide basic information for the future application of FeRB in environmental bioremediation.

Caffeic acid modulates methane production and rumen fermentation in an opposite way with high-forage or high-concentrate substrate in vitro.

Plant secondary metabolites, including tannins, saponins and phenolic acids, possess potential methane (CH4 ) inhibition bioactivity. Caffeic acid (CA), as one of the typical phenolic acids, serves as a promising rumen CH4 inhibitor, but the underlying mechanisms and investigations with typical formulated rations are still not well documented. Therefore, a batch culture study was conducted to investigate the effects of CA on methanogenesis, rumen fermentation and growth of ruminal microorganisms when high-forage or high-concentrate substrates are fermented. After 48 h incubations, adding CA up to 40 g kg-1 dry matter linearly reduced (P < 0.05) the disappearance of dry matter, neutral detergent fiber (NDFD), total gas, methanogenesis, total volatile fatty acid and 16S rDNA copy numbers of Ruminococcus albus and Butyrivibrio fibrisolvens, and increased 16S rDNA copy numbers of methanogens for the high-forage treatment. For the high-concentrate treatment, CA exerted opposite effects (P < 0.05) on the above variables, except that CA did not affect (P > 0.05)16S rDNA copy numbers of methanogens or R. albus. Caffeic acid inhibited in vitro methanogenesis and rumen fermentation with high-forage substrate incubation. Contrarily, CA benefited in vitro fermentation and enhanced methanogenesis with high-concentrate substrate incubation. It suggests that CA modulates methanogenesis and rumen fermentation mainly by affecting the growth of cellulolytic bacteria in vitro. © 2020 Society of Chemical Industry.

Non-Specific Antibodies Induce Lysosomal Activation in Atlantic Salmon Macrophages Infected by Piscirickettsia salmonis.

Piscirickettsia salmonis, an aggressive intracellular pathogen, is the etiological agent of salmonid rickettsial septicemia (SRS). This is a chronic multisystemic disease that generates high mortalities and large losses in Chilean salmon farming, threatening the sustainability of the salmon industry. Previous reports suggest that P. salmonis is able to survive and replicate in salmonid macrophages, inducing an anti-inflammatory environment and a limited lysosomal response that may be associated with host immune evasion mechanisms favoring bacterial survival. Current control and prophylaxis strategies against P. salmonis (based on the use of antibiotics and vaccines) have not had the expected success against infection. This makes it urgent to unravel the host-pathogen interaction to develop more effective therapeutic strategies. In this study, we evaluated the effect of treatment with IgM-beads on lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The impact of IgM-beads on cytotoxicity induced by P. salmonis in infected cells was evaluated by quantification of cell lysis through release of Lactate Dehydrogenase (LDH) activity. Bacterial load was determined by quantification of 16S rDNA copy number by qPCR, and counting of colony-forming units (CFU) present in the extracellular and intracellular environment. Our results suggest that stimulation with antibodies promotes lysosomal activity by lowering lysosomal pH and increasing the proteolytic activity within this organelle. Additionally, incubation with IgM-beads elicits a decrease in bacterial-induced cytotoxicity in infected Atlantic salmon macrophages and reduces the bacterial load. Overall, our results suggest that stimulation of cells infected by P. salmonis with IgM-beads reverses the modulation of the lysosomal activity induced by bacterial infection, promoting macrophage survival and bacterial elimination. This work represents a new important evidence to understand the bacterial evasion mechanisms established by P. salmonis and contribute to the development of new effective therapeutic strategies against SRS.

Bentonite-Based Organic Amendment Enriches Microbial Activity in Agricultural Soils

Bentonite-based organic amendments may have the potential to enhance soil microbial properties. The experiment was carried out from 2014 to 2017 comprising four treatments: NPK fertilizer (nitrogen, phosphorus and potassium mineral fertilizer as a control), NPK + cattle manure, NPK + bentonite, and NPK + combination of manure with bentonite (MB) to verify this hypothesis. The effect of treatments on seven different soil microbial properties was measured: dehydrogenase activity (DHA), bacterial phospholipid fatty acid content, fungal phospholipid fatty acid content, microbial biomass carbon (Cmic), 16S rDNA, 18S rDNA, and ammonia-oxidizing bacteria in soil. The results showed that solely bentonite treatment increases the bacterial and fungal biomass, which was further confirmed by the increased 16S rDNA and 18s rDNA gene copy numbers. The only significantly decreased values upon treatment with solely bentonite were recorded for DHA and Cmic. The ammonia-oxidizing bacteria population increased with the sole application of bentonite and reached its maximum value when bentonite was applied with manure. The MB treatment showed the highest value for all seven measured properties. In summary, the application of bentonite solely might increase or decrease the soil activity, but its addition, along with manure, always promotes an abundance of soil microorganisms and their activity. The co-application of bentonite with manure altered the soil microbial properties in a 3-year field experiment in favor of increased microbial biomass, which is beneficial for agriculture and environment and reveals the potential for the restoration of polluted lands.

Exclusive use of digital PCR allows an absolute assay of heat-killed Lactobacilli in foods targeting multiple copies of 16S rDNA

The real-time PCR (qPCR) and digital PCR (dPCR) to amplify a single-copy of house-keeping genes (i.e., hsp60, pheS or tuf) are used for the assay of limited microbial species. In general, with a single-copy gene, there are obviously varied DNA sequences for even the same microbial species, which could cause difficulties with design of primers and probes for PCR when targeting various single copy genes. In general, for identification by dPCR (as a representative case: Lactobacillus paracasei), accumulated DNA sequence information of 16S rDNA, which is much more frequently used, should be targeted. In contrast, next-generation sequencing revealed that there are five copies of 16S rDNA in a live L. paracasei MCC1849. Therefore, we aimed to reveal, if heat-killed L. paracasei supplemented in nutritional foods that aid the host immune system have the relevant five copies per chromosomal DNA, and if the relevant copies remain unchanged on the same chromosomal DNA or remain to be different chromosomal DNA fragments. So, we revealed the actual distribution of the potential original five copies of 16S rDNA using our innovative dPCR, in which both 16S rDNA and hsp60 genes were simultaneously elongated. The molecular ratios of 16S rDNA/hsp60 dispersed in the dPCR chip were then estimated. The 16S rDNA/hsp60 molecular ratios of the heat-killed L. paracasei in foods, resultantly ranged from 5.0 to 7.2, being the same or higher than that of the five copies determined by next-generation sequencing. The 16S rDNA copy number/ratio indicated the chromosomal DNA molecular number and the associated cell number. As significance, different nutritional foods could potentially cause the loss of chromosomal DNA of supplemented beneficial microbes to a much greater degree. Our absolute dPCR does not require standard correlative samples for the estimation of final products. The estimation principle of the ratio of 16S rDNA/a house-keeping single-copy gene by our absolute dPCR could lead to a useful and accurate assay for various nutritional foods.

Survival of staphylococci and transmissibility of their antimicrobial resistance genes in milk after heat treatments

There is growing concern that the milk heat treatments, which are the main antibacterial safeguard in the dairy industry, are not sufficient in inactivating the antimicrobial resistance (AMR) genes of staphylococci and may induce a viable but non-culturable (VBNC) state of these microorganisms. This study investigated the persistence and quantification of blaZ, mecC and tetK plasmid-mediated AMR genes copy numbers of two staphylococcal strains in both milk and Tris-EDTA (TE) buffer. During subsequent storage after pasteurization, all tested genes showed increased copy numbers. By electroporation of these genes to the Staphylococcus aureus RN42200 electro-competent strain, both mecC and tetK genes were still expressive and transferable. The formation of VBNC cells was estimated with viability staining and quantitative PCR of 16S rDNA copy numbers of both staphylococcal strains. The copy numbers increased over the storage period, which suggests that the pasteurization process was a stress factor and likely induces VBNC state in tested staphylococci. This explains the continual increase in the copy numbers of AMR genes. In contrast, after the sterilization treatment all the genes showed decreases in copy numbers. Further studies are required to evaluate the efficiency of currently practiced pasteurization and to assess the hazard level of VBNC staphylococci.

Poor Unstable Midgut Microbiome of Hard Ticks Contrasts With Abundant and Stable Monospecific Microbiome in Ovaries.

Culture-independent metagenomic methodologies have enabled detection and identification of microorganisms in various biological systems and often revealed complex and unknown microbiomes. In many organisms, the microbiome outnumbers the host cells and greatly affects the host biology and fitness. Ticks are hematophagous ectoparasites with a wide host range. They vector a number of human and animal pathogens and also directly cause major economic losses in livestock. Although several reports on a tick midgut microbiota show a diverse bacterial community, in most cases the size of the bacterial population has not been determined. In this study, the microbiome was quantified in the midgut and ovaries of the ticks Ixodes ricinus and Rhipicephalus microplus before, during, and after blood feeding. Although the size of bacterial community in the midgut fluctuated with blood feeding, it was overall extremely low in comparison to that of other hematophagous arthropods. In addition, the tick ovarian microbiome of both tick species exceeded the midgut 16S rDNA copy numbers by several orders of magnitude. This indicates that the ratio of a tick midgut/ovary microbiome represents an exception to the general biology of other metazoans. In addition to the very low abundance, the tick midgut diversity in I. ricinus was variable and that is in contrast to that found in the tick ovary. The ovary of I. ricinus had a very low bacterial diversity and a very high and stable bacterial abundance with the dominant endosymbiont, Midichloria sp. The elucidation of this aspect of tick biology highlights a unique tissue-specific microbial-invertebrate host interaction.

Amplicon-Based Illumina Sequencing and Quantitative PCR Reveals Nanoplankton Diversity and Biomass in Surface Water of Qinhuangdao Coastal Area, China

Aureococcus anophagefferens caused brown tides for three consecutive years from2009 to2011 in the coastal waters of Qinhuangdao, China, with numerous, widespread ecological and economic impact on ecosystems. To understand the population dynamics of nanoplankton during the brown tides, sequences of the V9 region of the 18S rDNA gene, used as a marker, were analyzed by Illumina sequencing to assess nanoplankton biomass, and real-time fluorescence quantitative PCR was performed to analyze spatial variation in the 18S rDNA copy concentrations of nanoplankton off the Qinhuangdao coast in July,2011. The results showed that A. anophagefferens and Minutocellus polymorphus were the dominant species in the local phytoplankton community during the brown tide in July2011. The highest 18S rDNA copy concentrations of A. anophagefferens and M. polymorphus were detected at stations SHG and FN, respectively. The central area most strongly affected by the brown tide migrated southward from2011 to2013. Redundancy analysis (RDA) showed that the decreasing NOx concentration might provide suitable nutrient conditions for the A. anophagefferens outbreak. During the brown tide caused by A. anophagefferens, other phytoplankton, such as diatoms, cryptophytes, chlorophytes, dinoflagellates and other flagellates, could co-occur with it. For zooplankton, due to less selective feeding behavior, Amoebozoa was the most abundant zooplankton at station SHG, while Ciliophora was the most abundant zooplankton at other stations for its more selective feeding.

Genetic diversity of diazotrophs and total bacteria in the phyllosphere of Pyrus serotina, Prunus armeniaca, Prunus avium, and Vitis vinifera.

The phyllosphere, which supports a large number of microorganisms, represents the interface between the aboveground parts of plants and air. In this study, four nifH clone libraries were constructed from the phyllosphere of Pyrus serotina (L), Vitis vinifera (P), Prunus armeniaca (X), and Prunus avium (Y). Clones related to Skermanella (L, 12.1%; X, 15.6%; Y, 62.5%; P 70.8%), Bradyrhizobium (X, 2.1%; P, 15.1%; L, 63.7%), Erwinia (X, 68.8%), Pseudomonas (L, 3.3%; P, 7.6%), and Chroococcidiopsis (P, 0.9%; L, 4.4%, X; 5.2%, Y; 19.6%) were present at high percentages, highlighting their critical role in contributing nitrogen to the phyllosphere ecosystem. The 16S rDNA sequence analysis suggested that phyllosphere-associated bacteria were affiliated with a wide range of taxa, encompassing members from Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Cyanobacteria, Tenericutes, and Deinococcus-Thermus. Additionally, the abundance of the nifH gene and 16S rDNA was assessed with quantitative PCR. The number of copies of nifH and 16S rDNA ranged from 1.14 × 103 to 1.49 × 104 and from 3.72 × 106 to 7.02 × 107 copies/g fresh leaf sample, respectively. In conclusion, our work sheds light on the microbial communities of the phyllosphere that are important for plant growth. Moreover, we observed a unique composition of nitrogen-fixing bacteria in each phyllosphere sample, suggesting the existence of specific interactions between these functional microorganism and plants, which may provide information or be a reference for the development of bacterial fertilizers.

Quantification of a cell culture contaminant using 16S rDNA.

In this study, we identified a "black dot"-like cell culture contaminant as a species belonging to the genus of Pusillimonas using 16S rDNA sequencing. Among all antibiotics tested, a combinatorial treatment of ampicillin and gentamicin both at 100 µg/mL was able to eliminate this contaminant. The contaminant was then visualized by fluorescence microscopy using propidium iodide staining and was found inside the cytosol of contaminated A549 cells. To characterize the efficacy of antibiotics for contaminant removal, we devised a quantitative method to determine the average number of 16S rDNA copies associated with a single A549 cell, which is directly proportional to the average number of contaminant per A549 cell. By using primers specific to the 16S rDNA sequence of the contaminant, we were able to estimate contaminants per single contaminated cell using both qPCR-based relative and absolute quantification.