Strain-specific qPCR Research Articles

Highly accurate and sensitive absolute quantification of bacterial strains in human fecal samples

BackgroundNext-generation sequencing (NGS) approaches have revolutionized gut microbiome research and can provide strain-level resolution, but these techniques have limitations in that they are only semi-quantitative, suffer from high detection limits, and generate data that is compositional. The present study aimed to systematically compare quantitative PCR (qPCR) and droplet digital PCR (ddPCR) for the absolute quantification of Limosilactobacillus reuteri strains in human fecal samples and to develop an optimized protocol for the absolute quantification of bacterial strains in fecal samples.ResultsUsing strain-specific PCR primers for L. reuteri 17938, ddPCR showed slightly better reproducibility, but qPCR was almost as reproducible and showed comparable sensitivity (limit of detection [LOD] around 104 cells/g feces) and linearity (R2 > 0.98) when kit-based DNA isolation methods were used. qPCR further had a wider dynamic range and is cheaper and faster. Based on these findings, we conclude that qPCR has advantages over ddPCR for the absolute quantification of bacterial strains in fecal samples. We provide an optimized and easy-to-follow step-by-step protocol for the design of strain-specific qPCR assays, starting from primer design from genome sequences to the calibration of the PCR system. Validation of this protocol to design PCR assays for two L. reuteri strains, PB-W1 and DSM 20016 T, resulted in a highly accurate qPCR with a detection limit in spiked fecal samples of around 103 cells/g feces. Applying our strain-specific qPCR assays to fecal samples collected from human subjects who received live L. reuteri PB-W1 or DSM 20016 T during a human trial demonstrated a highly accurate quantification and sensitive detection of these two strains, with a much lower LOD and a broader dynamic range compared to NGS approaches (16S rRNA gene sequencing and whole metagenome sequencing).ConclusionsBased on our analyses, we consider qPCR with kit-based DNA extraction approaches the best approach to accurately quantify gut bacteria at the strain level in fecal samples. The provided step-by-step protocol will allow scientists to design highly sensitive strain-specific PCR systems for the accurate quantification of bacterial strains of not only L. reuteri but also other bacterial taxa in a broad range of applications and sample types.-oGjUmj5e8MXZDxyDjzcm-Video

Schauerella fraxinea gen. nov., sp. nov., a bacterial species that colonises ash trees tolerant to dieback caused by Hymenoscyphus fraxineus

The tolerance of ash trees against the pathogen Hymenoscyphus fraxineus seems to be associated with the occurrence of specific microbial taxa on leaves. A group of bacterial isolates, primarily identified on tolerant trees, was investigated with regard to their taxonomic classification and their potential to suppress the ash dieback pathogen. Examination of OGRI values revealed a separate species position. A phylogenomic analysis, based on orthologous and marker genes, indicated a separate genus position along with the species Achromobacter aestuarii. Furthermore, analysis of the ratio of average nucleotide identities and genome alignment fractions demonstrated genomic dissimilarities typically observed for inter-genera comparisons within this family. As a result of these investigations, the strains are considered to represent a separate species within a new genus, for which the name Schauerella fraxinea gen. nov., sp. nov. is proposed, with the type strain B3P038T (=LMG 33092 T = DSM 115926 T). Additionally, a reclassification of the species Achromobacter aestuarii as Schauerella aestuarii comb. nov. is proposed.In a co-cultivation assay, the strains were able to inhibit the growth of a H. fraxineus strain. Accordingly, a functional analysis of the genome of S. fraxinea B3P038T revealed genes mediating the production of antifungal substances. This potential, combined with the prevalent presence in the phyllosphere of tolerant ash trees, makes this group interesting for an inoculation experiment with the aim of controlling the pathogen in an integrative approach. For future field trials, a strain-specific qPCR system was developed to establish an efficient method for monitoring the inoculation success.

Development and evaluation of genomics informed real-time PCR assays for the detection and strain typing of Mycobacterium avium subsp. paratuberculosis.

This study aimed to identify specific genomic targets for the detection and strain typing of Map and analyse their sensitivity and specificity, and detect Map directly from faeces. A comparative genomics approach was used to identify specific genomic targets for the detection and strain typing of Map. A Map specific qPCR using the primer pair 7132 that targets a DNA segregation ATPase protein was able to detect all strains of Map and is more sensitive than the current Johne's disease PCR assays with a sensitivity of 0.0002 fg µl-1. A strain specific qPCR using the Atsa primer pair that targets the arylsulfase gene was able to differentiate between Type S and Type C strains of Map and was more sensitive than the IS1311 PCR and REA with a sensitivity of 40 fg µl-1 and was specific for Type S Map. Both assays successfully detected Map directly from faeces. This study developed and validated two genomics informed qPCR assays, 7132B Map and Atsa Type S and found both assays to be highly specific and sensitive for the detection of Map from culture and directly from faeces. This is the first time that a probe-based qPCR has been designed and developed for Map strain typing, which will greatly improve the response time during outbreak investigations.

Strain-specific quantitative detection of two putative biocontrol strains for suppression of ash dieback

Plant diseases caused by microorganisms are a major threat to agricultural and forest ecosystems and production. The application of microorganisms as biocontrol agents can be a more sustainable way to suppress pathogens and/or strengthen plant health. In this study, we wanted to demonstrate an efficient way to monitor the inoculation success of promising bacterial strains on plants by qPCR, using the example of common ash and the bacterial strains Luteimonas fraxinea D4P002 and Aureimonas altamirensis C2P003. For the design of the strain-specific qPCR systems, we used a workflow mainly based on the comparison of a target genome with a reference genome database by the software Neptune. We were able to find specific sequences for both strains D4P002 and C2P003, and the designed qPCR systems were found to be highly sensitive, strain specific and the applied approach showed a high recovery rate from plant material. Strain-specific qPCR assays were applied to monitor the colonization and persistence of the two bacterial strains on ash seedlings eight weeks after inoculation. Both inoculated strains were found on different organs of the ash seedlings, with the highest abundance on the older inoculated leaves. On freshly grown plant parts, the inoculated strains could also be identified. The results of this study gave us the opportunity to establish an efficient way to trace inoculation success by specific quantification of the inoculated bacterial strains. Based on the described workflow, strain-specific detection systems can be easily designed for other biocontrol agents to observe their persistence after inoculation. The suggested procedure might be helpful in the evaluation of the biocontrol of plant diseases in horticulture, forestry or agriculture.

Observations supporting hypothetical commensalism and competition between two Campylobacter jejuni strains colonizing the broiler chicken gut.

Campylobacter jejuni is the most prevalent bacterial foodborne pathogen in humans. Given the wide genetic diversity of C. jejuni strains found in poultry production, a better understanding of the relationships between these strains within chickens could lead to better control of this pathogen on farms. In this study, 14-day old broiler chickens were inoculated with two C. jejuni strains (103 or 107 CFU of D2008b and 103 CFU of G2008b, alone or together) that were previously characterized in vitro and that showed an opposite potential to compete for gut colonization in broilers. Liver samples and ileal and cecal contents were collected and used to count total C. jejuni and to quantify the presence of each strain using a strain specific qPCR or PCR approach. Ileal tissue samples were also collected to analyze the relative expression level of tight junction proteins. While a 103 CFU inoculum of D2008b alone was not sufficient to induce intestinal colonization, this strain benefited from the G2008b colonization for its establishment in the gut and its extraintestinal spread. When the inoculum of D2008b was increased to 107 CFU - leading to its intestinal and hepatic colonization - a dominance of G2008b was measured in the gut and D2008b was found earlier in the liver for birds inoculated by both strains. In addition, a transcript level decrease of JAM2, CLDN5 and CLDN10 at 7 dpi and a transcript level increase of ZO1, JAM2, OCLN, CLDN10 were observed at 21 dpi for groups of birds having livers contaminated by C. jejuni. These discoveries suggest that C. jejuni would alter the intestinal barrier function probably to facilitate the hepatic dissemination. By in vitro co-culture assay, a growth arrest of D2008b was observed in the presence of G2008b after 48 h of culture. Based on these results, commensalism and competition seem to occur between both C. jejuni strains, and the dynamics of C. jejuni intestinal colonization and liver spread in broilers appear to be strain dependent. Further in vivo experimentations should be conducted to elucidate the mechanisms of commensalism and competition between strains in order to develop adequate on-farm control strategies.

Synergistic interactions prevail in multispecies biofilms formed by the human gut microbiota on mucin.

Bacterial species in the human gut predominantly exist in the form of mixed-species biofilms on mucosal surfaces. In this study, the biofilm-forming ability of many human gut bacterial strains (133 strains recovered from human faeces) on mucin-coated and non-coated polystyrene surfaces was determined. A significant variation (P< 0.05) in the biofilm-forming ability of many bacterial species on both surfaces was noticed. Based on some preliminary trials, four bacterial species were selected (Bifidobacterium bifidum, Bifidobacterium longum subsp. infantis, Parabacteroides distasonis and Bacteroides ovatus), which could not form any abundant biofilm individually under the invitro conditions investigated, but produced abundant biofilms when co-cultured in different combinations of two, three and four species, giving an evidence of synergistic interactions in multispecies biofilm formation. There was a 4.74-fold increase in the biofilm mass when all strains developed a biofilm together. Strain-specific qPCR analysis showed that B.bifidum was the most dominant species (56%) in the four-species biofilm after 24 h, followed by B.longum subsp. infantis (36.2%). Study involving cell free supernatant of the cooperating strains showed that cell viability as well as physical presence of cooperating cells were prerequisites for the observed synergy in biofilms. The molecular mechanism behind these interactions and subsequent effects on the functionality of the strains involved were not determined in our study but merit further work.

Wolbachia Inter-Strain Competition and Inhibition of Expression of Cytoplasmic Incompatibility in Mosquito

Successful field trials have been reported as part of the effort to develop the maternally transmitted endosymbiontic bacteria Wolbachia as an intervention agent for controlling mosquito vectors and their transmitted diseases. In order to further improve this novel intervention, artificially transinfected mosquitoes must be optimized to display maximum pathogen blocking, the desired cytoplasmic incompatibility (CI) pattern, and the lowest possible fitness cost. Achieving such optimization, however, requires a better understanding of the interactions between the host and various Wolbabachia strains and their combinations. Here, we transferred the Wolbachia wMel strain by embryonic microinjection into Aedes albopictus, resulting in the successful establishment of a transinfected line, HM (wAlbAwAlbBwMel), with a triple-strain infection comprising wMel, wAlbA, and wAlbB. Surprisingly, no CI was induced when the triply infected males were crossed with the wild-type GUA females or with another triply infected HC females carrying wPip, wAlbA, and wAlbB, but specific removal of wAlbA from the HM (wAlbAwAlbBwMel) line resulted in the expression of CI after crosses with lines infected by either one, two, or three strains of Wolbachia. The transinfected line showed perfect maternal transmission of the triple infection, with fluctuating egg hatch rates that improved to normal levels after repeated outcrosses with GUA line. Strain-specific qPCR assays showed that wMel and wAlbB were present at the highest densities in the ovaries and midguts, respectively, of the HM (wAlbAwAlbBwMel) mosquitoes. These finding suggest that introducing a novel strain of Wolbachia into a Wolbachia-infected host may result in complicated interactions between Wolbachia and the host and between the various Wolbachia strains, with competition likely to occur between strains in the same supergroup.

Safety and Effect of a Low- and High-Dose Multi-Strain Probiotic Supplement on Microbiota in a General Adult Population: A Randomized, Double-Blind, Placebo-Controlled Study

Few studies have focused on dose-response analyses of multi-strain probiotics in the general adult population. This study aimed at comparing how a low- and high-dose of a multi-strain probiotic supplement (containing Lactobacillus helveticus R0052, Lactobacillus rhamnosus R0011, Lactobacillus casei R0215, Pediococcus acidilactici R1001, Bifidobacterium breve R0070, Bifidobacterium longum ssp. longum BB536, Lactobacillus plantarum R1012, Lactococcus lactis ssp. lactis R1058) affected microbiota composition, transit persistence and safety in adults. After a 7-d baseline, participants were randomized to receive capsules containing 5 or 25 billion CFU, or placebo daily for 28 days, followed by a 7-d washout. Digestive health and general wellness were assessed. Fecal microbiota composition was analyzed using 16S rRNA gene amplicon sequencing and strain persistence, by qPCR. Participants’ gastrointestinal and general wellbeing were unaffected. No adverse events were associated with either dose. Supplemented strains contributed to the Lactobacillus and Bifidobacterium genera detected in stool, with 0.40 ± 0.11% and 0.51 ± 0.26%, respectively, in the high-dose group. Strain-specific qPCR assays revealed variable levels of post-intervention persistence between strains. Sequencing and composition analyses using the 16S V4 region revealed a decrease in Holdemania and increase in Bacteroidales. The formulation was well tolerated in this sample of the general adult population, even at the higher dose. The strains appear to have influenced microbiota composition minimally, as expected in the absence of dysbiosis, and consistently with the dose administered. Overall, the results provide a rationale to study the effects this formulation on microbiota composition in individuals exhibiting dysbiosis associated with metabolic disorders or obesity.

Design of Bacterial Strain-Specific qPCR Assays Using NGS Data and Publicly Available Resources and Its Application to Track Biocontrol Strains.

Biological control is emerging as a feasible alternative to chemical pesticides in agriculture. Measuring the microbial biocontrol agent (mBCA) populations in the environment is essential for an accurate environmental and health risk assessment and for optimizing the usage of an mBCA-based plant protection product. We hereby show a workflow to obtain a large number of qPCR markers suitable for robust strain-specific quantification. The workflow starts from whole genome sequencing data and consists of four stages: (i) identifying the strain-specific sequences, (ii) designing specific primer/probe sets for qPCR, and (iii) empirically verifying the performance of the assays. The first two stages involve exclusively computer work, but they are intended for researchers with little or no bioinformatic background: Only a knowledge of the BLAST suite tools and work with spreadsheets are required; a familiarity with the Galaxy environment and next-generation sequencing concepts are strongly advised. All bioinformatic work can be implemented using publicly available resources and a regular desktop computer (no matter the operating system) connected to the Internet. The workflow was tested with five bacterial strains from four different genera under development as mBCAs and yielded thousands of candidate markers and a triplex qPCR assay for each candidate mBCA. The qPCR assays were successfully tested in soils of different natures, water from different sources, and with samples from different plant tissues. The mBCA detection limits and population dynamics in the different matrices are similar to those in qPCR assays designed by other means. In summary, a new accessible, cost-effective, and robust workflow to obtain a large number of strain-specific qPCR markers is presented.

Establishment success and crop growth effects of an arbuscular mycorrhizal fungus inoculated into Swiss corn fields

A major strategy to increase the sustainability of agricultural systems consists of enhancing internal ecosystem processes that support crop production and reduce external resource inputs. However, specific approaches to achieve this goal still need to be identified. Here, we investigated whether inoculation with a high dose of a well-characterized strain of a plant symbiotic arbuscular mycorrhizal (AM) fungus into Swiss corn fields leads to successful establishment of the fungus in plant roots and can generate agronomic benefits for maize production.We used single-molecule real-time (SMRT) DNA sequencing to assess community composition of native AM fungi and identified environmental management and biological factors affecting AM fungal abundance, establishment success of the introduced fungus and effects of AMF inoculation on corn yield.While native AM fungal abundance was negatively related to soil P contents, we found significantly positive relationships between soil P contents and establishment success of the inoculated fungus. There was a significantly negative relationship between inoculum establishment and abundance of native AM fungi. Although molecular quantification using strain-specific qPCR indicated that the inoculated strain strongly increased in abundance in roots from most soils investigated, total AM fungal root colonization was only significantly increased in one soil, indicating successful competition of the inoculant for root niche space against native AM fungi. Positive effects on corn yield were only observed when inoculation increased root colonization and were negatively correlated to P fertilization levels.The results imply that phosphorus plays a major role in defining the abundance of native AM fungi and the composition of their communities and that these effects can determine establishment success of the inoculant. The results further indicate that positive effects on crop yield may only be expected when potentially achievable root colonization levels are not yet reached and AMF communities are not well developed.

Strain-Specific Quantification of Native Probiotic Bacillus spp. and Their Effect on Liver Function and Gut Microflora of Experimental Rats.

Safety and efficacy was investigated for two candidate probiotic B. flexus MCC2427 and B. licheniformis MCC2512 via in vivo studies on albino Wistar rats. In acute toxicity assay, rats were fed with single dose of 1010cfumL-1 of probiotics. The follow-up studies for next 14days did not reveal any toxicity-related criteria indicating the non-toxicity nature of probiotics. In 90-day repeated dosage studies, the cultures were administered in three doses (106, 107, 108cfumL-1). Results showed no overt toxic effect and no drastic treatment-related changes pertaining to histopathology of vital organs. DNA fingerprinting indicated the lack of bacterial translocation. Superoxide dismutase and catalase activity indicated their antioxidant potential. Reduced serum cholesterol with improved HDL-cholesterol specified the cholesterol-reducing ability of the cultures, which was also apparent with increased excretion of cholic acid in feces. Both probiotic cultures positively altered the gut microbial environment, retained lactic acid bacterial effect, and simultaneously reduced pathogenic strains. A sensitive and rapid tool was developed using strain-specific qPCR primers, which facilitated appropriate estimation of test culture in feces. The data strongly advocate the safety of tested probiotics at levels used in the study.

Suppression subtractive hybridisation and real-time PCR for strain-specific quantification of the probiotic Bifidobacterium animalis BAN in broiler feed

To ensure quality management during the production processes of probiotics and for efficacy testing in vivo, accurate tools are needed for the identification and quantification of probiotic strains. In this study, a strain-specific qPCR assay based on Suppression Subtractive Hybridisation (SSH) for identifying unique sequences, was developed to quantify the strain Bifidobacterium animalis BAN in broiler feed. Seventy potential BAN specific sequences were obtained after SSH of the BAN genome, with a pool of closely related strain genomes and subsequent differential screening by dot blot hybridisation. Primers were designed for 30 sequences which showed no match with any sequence database entry, using BLAST and FASTA. Primer specificity was assessed by qPCR using 45 non-target strains and species in a stepwise approach. Primer T39_S2 was the only primer pair without any unspecific binding properties and it showed a PCR efficiency of 80% with a Cq value of 17.32 for 20ng BAN DNA. Optimised feed-matrix dependent calibration curve for the quantification of BAN was generated, ranging from 6.28×103cfug−1 to 1.61×106cfug−1. Limit of detection of the qPCR assay was 2×101cfug−1 BAN. Applicability of the strain-specific qPCR assay was confirmed in a spiking experiment which added BAN to the feed in two concentrations, 2×106cfug−1 and 2×104cfug−1. Results showed BAN mean recovery rates in feed of 1.44×106±4.39×105cfug−1 and 1.59×104±1.69×104cfug−1, respectively. The presented BAN-specific qPCR assay can be applied in animal feeding trials, in order to control the correct inclusion rates of the probiotic to the feed, and it could further be adapted, to monitor the uptake of the probiotic into the gastrointestinal tract of broiler chickens.

Monitoring Lysobacter capsici AZ78 using strain specific qPCR reveals the importance of the formulation for its survival in vineyards.

Survival in the phyllosphere is a critical feature for biofungicides based on non-spore forming bacteria. Moreover, knowledge of their persistence on plants is important to design effective formulations and application techniques. With this scope, the aim of this work was to develop a specific method to monitor the fate in the environment of Lysobacter capsici AZ78, a biocontrol agent of Plasmopara viticola, and to evaluate the contribution of formulation in its persistence on grapevine leaves. A strain-specific primer pair derived from REP-PCR fingerprinting was used in quantitative PCR experiments to track the evolution of L. capsici AZ78 population in vineyards. The population reached between 5 and 6 log10 cells gram of leaf(-1) after application and decreased by more than 100 times in one week. Multiple regression analysis showed that unfavourable temperature was the main environmental factor correlating with the decrease of L. capsici AZ78 persistence on grapevine leaves. Importantly, the use of formulation additives protected L. capsici AZ78 against environmental factors and improved its persistence on the leaves by more than 10 times compared to nude cells. Formulation and the knowledge about the persistence of L. capsici AZ78 in vineyards will be useful to develop commercial biofungicides for foliar application.

Identification and quantification of viable Bifidobacterium breve strain Yakult in human faeces by using strain-specific primers and propidium monoazide

To develop a quick and accurate PCR-based method to evaluate viable Bifidobacterium breve strain Yakult (BbrY) in human faeces. The number of BbrY in faeces was detected by using strain-specific quantitative real-time PCR (qPCR) derived from a randomly amplified polymorphic DNA analysis. And using propidium monoazide (PMA) treatment, which combined a DNA-intercalating dye for covalently linking DNA in dead cells and photoactivation, only viable BbrY in the faeces highly and significantly correlated with the number of viable BbrY added to faecal samples within the range of 10(5) -10(9) cells per g of faeces was enumerated. After 11 healthy subjects ingested 10·7 log CFU of BbrY daily for 10 days, 6·9 (± 1·5) log CFU g(-1) [mean (± SD)] of BbrY was detected in faeces by using strain-specific transgalactosylated oligosaccharide-carbenicillin (T-CBPC) selective agar medium. Viable BbrY detected by qPCR with PMA treatment was 7·5 (± 1·0) log cells per g and the total number (viable and dead) of BbrY detected by qPCR without PMA treatment was 8·1 (± 0·8) log cells per g. Strain-specific qPCR with PMA treatment evaluated viable BbrY in faeces quickly and accurately. Combination of strain-specific qPCR and PMA treatment is useful for evaluating viable probiotics and its availability in humans.