Fluorescence In Situ Hybridization-flow Cytometry Research Articles

Denitrifying phosphorus removal (DPR) performance and metabolic mechanisms of Tetrasphaera at long-term nitrite exposure

Tetrasphaera has been identified as the main polyphosphate-accumulating organism (PAOs) in enhanced biological phosphate removal (EBPR), but the ability of Tetrasphaera to use nitrite as electron acceptor for denitrifying phosphorus removal (DPR) is still unclear. This study investigated the DPR performance and mechanism, microbial structures, and FNA inhibition of Tetrasphaera-dominated culture in the long-term acclimatization at different nitrite concentrations (10, 20, 40 and 50 mgN/L). The results showed that the most optimal influent nitrite concentration was 20 mgN/L (C/N = 6), and the removal efficiency of nitrite and phosphorus reached above 90 % and 95 %, respectively. Fluorescence In Situ Hybridization-Flow Cytometry (FISH-FCM) showed that the population abundance of Tetrasphaera was 80.95 %, in which Tet2-892 clade was the most abundant and mainly responsible for DPR with nitrite as electron acceptor. When the nitrite concentration reached 50 mgN/L (FNA = 12.80 µgHNO2-N/L, pH = 7.0 ± 0.1), electron supply deficiency and FNA inhibition resulted in only 43.0 % and 68.4 % of nitrite and phosphorus removal efficiency, respectively. The intracellular amino acid and glycogen metabolism were inhibited by FNA under this condition. A significant accumulation of N2O was observed, resulting from a combination of the insufficient electron supply, absence of nos-Z gene, and long-term FNA inhibition. DPR dominated by Tetrasphaera with appropriate nitrite as electron acceptor provides a new way to achieve nitrogen and phosphorus simultaneous removal. In addition, this study found that Tet2-892 clade of Tetrasphaera was more tolerant to FNA compared to Accumulibacter and GAOs, which provides a strategy for inhibiting GAOs activity to maintain the stability of the EBPR system.

Utility of Flow Cytometry and Fluorescence in Situ Hybridization in Follow-up Monitoring of Multiple Myeloma

Introduction Diagnosis and follow-up monitoring of Multiple Myeloma (MM) requires documentation of (clonal) plasma cells using morphological bone marrow assessment and other ancillary studies including CD138 immunohistochemistry (IHC), flow cytometry (FC), and myeloma-specific fluorescence in situ hybridization (FISH). While IHC and FC have traditionally been shown to be the more sensitive methods for the assessment of residual disease, the detection rates of FISH have markedly increased with the recent use of magnetic cell sorting for CD138 cell enrichment. We, therefore, aimed to revisit the clinical utility of these ancillary studies in the diagnosis and follow-up of MM. Methods We retrospectively reviewed the reports of bone marrow biopsy specimens received for the initial diagnosis or follow-up assessment of MM from October 2015 to January 2019. All cases that had both FC and FISH performed were included in the analysis. Patient demographics, treatment history and the results of bone marrow evaluation were recorded. Results During the study period, we evaluated a total of 1,712 biopsy specimens from 464 MM patients (mean±SD age: 64.0±9.4 years; females 41.4%). Of these, 87 cases were submitted for initial diagnosis and 1,625 cases had already established MM and were being evaluated for post-treatment residual disease. For both initial diagnosis and follow-up cases, CD138+ IHC demonstrated highest plasma cell burden (mean: 49.1% and 4.6%, respectively) followed by morphological aspirate count (mean: 29.6% and 2.3%, respectively) and FC (mean: 10.9% and 1.8%, respectively). Both FC and FISH had comparable detection rates at the time of initial diagnosis (95.4% versus 97.7%, respectively) and for follow-up cases (28.6% versus 28.2%, respectively). The FC and FISH results were concordant in 97.7% of the initial diagnosis cases and 89.7% of follow-up cases. Discordant cases with positive FISH results and negative FC results included 2 (2.3%) initial diagnosis cases and 81 (5.0%) follow-up cases whereas those with negative FISH and positive FC results included 87 (5.3%) follow-up cases. In comparison with all concordant cases, the FISH positive, FC negative discordant cases were older in age, more likely to have received treatment with Daratumumab, and less likely to have received stem cell transplantation (p <0.05) (Table 1). When compared to the FC and FISH positive concordant cases; both discordant groups had significantly lower plasma cell counts detected by the aspirate smear and CD138+ IHC (Figure 1). Among all FC positive cases, the discordant cases had lower FC detected plasma cell counts and were less likely to have aberrant CD56 expression as compared to the concordant cases (p <0.05) (Table 2). Finally, among all FISH positive cases, the discordant cases were less likely to have 13q lesion and aneuploidy of chromosomes 5, 9, and 15 (p <0.05) as compared to the concordant cases. Also, between these groups, the percentage of plasma cells with individual FISH aberrations was significantly lower among the discordant cases (Table 3). Conclusion Overall, FISH (with magnetic cell sorting) and FC have comparable detection rates at the initial diagnosis and follow-up assessment. Approximately 10% of the follow-up cases have discordant FISH and FC results where residual disease is detected by only one of these modalities. Also, the discordant cases have significantly lower plasma cell counts as compared to the FC and FISH positive concordant cases. Therefore, this study suggests that both FC and FISH are useful for the follow-up monitoring of MM, especially in the cases with low plasma cell burden. Finally, the higher frequency of Daratumumab treatment in FISH positive, FC negative discordant cases might suggest Daratumumab's potential interference with the residual disease detection by FC. Disclosures No relevant conflicts of interest to declare.

Application of Fluorescence in situ hybridization-Flow cytometry (FISH-FCM) technique to detect and quantify Vibrio cholerae population from different geographic regions

Rapid and species-specific detection, and quantification of pathogenic bacteria are fundamental for monitoring and assessment of the risk they pose to any ecosystem. The study employed Vibrio cholerae, a human pathogen responsible for the life-threatening diarrhoeal disease, cholera and one among the most unwanted from marine bioinvasion point of view. The present study coupled fluorescence in situ hybridization (FISH) technique, a powerful tool in molecular phylogenetic discrimination, with flow cytometry (FCM), a technique used for rapid and accurate quantification of both viable but non-cultivable and non-viable microorganisms. The FISH-FCM technique was used for the first time to quantify V. cholerae (includes cultivable and non-cultivable) from different geographic regions of Southeast Asia (Brunei, Indonesia, Lao PDR, Myanmar, Philippines, Singapore, Thailand, Vietnam) and India (Goa, west coast of India). The data acquired from the analyses provides a snap shot view of the total bacterial abundance with special reference to V. cholerae. As the method developed, it was evaluated with bacterial samples collected from different sites in Southeast Asia and India, and the application of this technique to different geographical regions appears feasible. Considering that the continuous growth of the shipping industry and ballast water as one of the primary vectors responsible for the global transport of pathogenic microorganisms, the risk they present needs immediate attention. This technique will be useful in the quick and accurate detection of specific pathogens. It may also provide significant insights to quarantine measures for Ballast Water Management.

Bayesian inference on stochastic gene transcription from flow cytometry data.

MotivationTranscription in single cells is an inherently stochastic process as mRNA levels vary greatly between cells, even for genetically identical cells under the same experimental and environmental conditions. We present a stochastic two-state switch model for the population of mRNA molecules in single cells where genes stochastically alternate between a more active ON state and a less active OFF state. We prove that the stationary solution of such a model can be written as a mixture of a Poisson and a Poisson-beta probability distribution. This finding facilitates inference for single cell expression data, observed at a single time point, from flow cytometry experiments such as FACS or fluorescence in situ hybridization (FISH) as it allows one to sample directly from the equilibrium distribution of the mRNA population. We hence propose a Bayesian inferential methodology using a pseudo-marginal approach and a recent approximation to integrate over unobserved states associated with measurement error.ResultsWe provide a general inferential framework which can be widely used to study transcription in single cells from the kind of data arising in flow cytometry experiments. The approach allows us to separate between the intrinsic stochasticity of the molecular dynamics and the measurement noise. The methodology is tested in simulation studies and results are obtained for experimental multiple single cell expression data from FISH flow cytometry experiments.Availability and implementationAll analyses were implemented in R. Source code and the experimental data are available at https://github.com/SimoneTiberi/Bayesian-inference-on-stochastic-gene-transcription-from-flow-cytometry-data.Supplementary information Supplementary data are available at Bioinformatics online.

Molecular analysis of infant fecal microbiota in an Asian at-risk cohort–correlates with infant and childhood eczema

BackgroundStudies have suggested that selective microbial targets prevail in the fecal microbiota of infants with eczema. This study evaluated the composition of fecal microbiota of infants who developed eczema in the first 5 years of life and compared these with those of healthy controls.FindingsChildren who developed eczema in the first 2 years, those with eczema at 5 years of age and healthy controls were selected from the placebo arm of a birth cohort of at-risk infants participating in a randomized double-blind trial on the protective effects of supplemental probiotics in early life on allergic outcomes. Molecular evaluation of fecal microbiota were conducted using Fluorescence In Situ Hybridization-Flow Cytometry (FISH-FC) for fecal samples collected. Longitudinal analysis of fecal microbiota composition at three days, one and three months and one year of life revealed higher abundance of Enterobacteriaceae [coefficient (B): 1.081, 95% CI: 0.229-1.933, adj p = 0.014] and Clostridium perfringens [coefficient (B): 0.521, 95% CI: 0.556-0.988, adj p = 0.03] in those who developed eczema in the first 2 years life. In those with eczema at 5 years of age, a lower abundance of Bifidobacterium was observed [coefficient (B): -27.635, 95% CI: -50.040 - -5.231, adj p = 0.018].ConclusionsThe differences in infant fecal microbiota observed in eczema subjects in this study support the notion that relative abundance of selective microbial targets may contribute to the subsequent development of eczema in childhood.

Identification and quantification of toxic and nontoxic strains of the harmful dinoflagellate Alexandrium tamarense using fluorescence in situ hybridization and flow cytometry

The co‐occurrence of morphologically identical toxic and nontoxic ribotypes of the biotoxin producing marine dinoflagellate Alexandrium tamarense presents a significant problem for its identification and enumeration, particularly in a regulatory monitoring context. To address this, we have developed a fluorescence in situ hybridization‐flow cytometry (FISH‐FC)—based method of cell identification and enumeration. This employed the taxa specific oligonucleotide probes TamToxC and TamA to fluorescently label (with the fluorochromes CY.3 and FITC) Group I (toxic) and Group III (nontoxic) A. tamarense ribotypes, respectively. Detection was by fluorescence activated flow cytometric analysis. The FISH‐FC method allowed effective discrimination between laboratory cultures of Group I and Group III ribotypes, with toxic and nontoxic cells creating distinct, easily identifiable, clusters in a flow cytometer bi‐plot of side scatter (SSC) versus the green (FL1) fluorescence detection channel. Comparison of estimates of cell abundance obtained by the FISH‐FC technique with those obtained by microscopy (Sedgwick Rafter technique) showed no statistically significant difference across a range of concentrations. Subsequently, the methodology was successfully applied on natural seawater samples, spiked with known concentrations of toxic and nontoxic A. tamarense cells at environmentally relevant concentrations.

Allergic diseases of the skin and drug allergies – 2013. Longitudinal analysis of fecal microbiota of infants followed-up for eczema till 2 years of age

Methods Children with eczema at 2 years old (n=26) and their matched (for gender, mode of delivery and feeding in first 6 months) healthy controls (n=26) were selected from the placebo group of a cohort of at-risk infants participating in an randomized double-blind placebo controlled trial on the protective effects of supplemental probiotics (first 6 months) on eczema and allergies. Children with eczema were subclassified into atopic eczema (n=12) and non-atopic eczema (n=14). Molecular evaluation of fecal microbiota were conducted using Fluorescence In Situ Hybridization-Flow Cytometry (FISH-FC) for fecal samples collected at 3 days, 1, 3, and 12 months. Probes were selected to target Eubacterium rectale-Clostridium coccoides group (Erec482), Clostridium leptum subgroup (Clep866 and the corresponding competitor probes), Bacteroides-Prevotella group (Bac303), Bifidobacterium genus (Bif164), Atopobium group (Ato291), LactobacilliEnterococci group (Lab158), Enterobacteriaceae family (Enter1432) and Clostridium perfringens (Cperf191). Linear mixed model was used to evaluate the longitudinal differences (i.e. 4 time points) of bacterial targets while adjusting for gender, mode of delivery, feeding up to 6 months, and allergic rhinitis and wheezing within the eczema group at 2 years of age. Results Longitudinal analyses over four time points showed that higher relative abundance of Enterobacteriaceae [coefficient (B): 1.104, 95% confidence interval (CI):0.175-2.033, adj p=0.022] in children with eczema by 2 years of age. Similar observations were made when eczema group was subanalyzed into non-atopic and atopic eczema, where higher relative abundance of Enterobacteriaceae [B:1.357, 95%CI; 0.382-2.332, adj p=0.008] and [B:1.165, 95%CI; 0.221-2.109, adj p=0.019] were observed respectively as compared to healthy controls. Relative abundance of Clostridium perfringens were also higher when subanalyzed for non-atopic [B:0.572, 95%CI; 0.0009-1.144, adj p=0.050] and atopic eczema [B:0.000451, 95%CI: 0.0001-0.0007, adj p=0.012] compared to healthy controls.

Improvement of FISH–FCM enumeration performance in filamentous yeast species in activated sludge by snailase partial digestion

This paper developed a novel strategy to improve the fluorescence in situ hybridization-flow cytometry (FISH-FCM) enumeration performance in filamentous yeast species in activated sludge by snailase partial digestion to fully disaggregate filamentous yeast chains into single cells. A 2 h 2% snailase partial digestion liberated more rod-shaped yeast single cells from intertwined filamentous yeast samples than did sonication disaggregation, based on an optical microscopic observation and the forward-light-scatter frequency histogram of FCM analysis. However, adding snailase resulted in a fluorescence-quenching phenomenon of the hybridized filamentous yeast cells, which was minimized by lowering the snailase concentration. An approximately 3 h 0.5% snailase partial digestion conducted between sonication and hybridization significantly improved the FISH-FCM enumeration performance for filamentous yeast species by 37%. The results presented here will facilitate the rapid detection, identification and exact enumeration of specific filamentous fungal species in environmental samples.

Tracking composition and dynamics of nitrification and denitrification microbial community in a biofilm reactor by PCR-DGGE and combining FISH with flow cytometry

A compact suspended carrier biofilm reactor (SCBR) was operated at three different C/N ratios (C/N = 10:1, 5:1 and 3:1) with focus on reactor performance and microbial community composition of nitrifying and denitrifying bacteria. The process was capable of achieving over 90% removal of chemical oxygen demand (COD) and over 83.3% of simultaneous nitrification and denitrification (SND) efficiency. Denaturing gradient gel electrophoresis (DGGE) analysis showed that the diversity of ammonia-oxidizing bacteria (AOB) community decreased with inoculation sludge, C/N ratio 3:1, 10:1 and 5:1 in turn. Phylogenetics analysis indicated that there were three distinct groups of AOB in Betaproteobacteria subdivision, where Nitrosomonas were the dominant members in the biofilm. Fluorescence in situ hybridization-flow cytometry (FISH-FCM) results revealed that Alpha-, Beta- and Gamma-proteobacteria accounted for over 50% of all cells. Additionally, the clusters of Nitrosomonas, Nitrosospira, Nitrobacter, Nitrospira and AOB of Betaproteobacteria yielded a similar distribution pattern about 5–12% of all cells. Nested analysis of variance assay (ANOVA) demonstrated that the C/N ratio did not significantly affect the shift of populations in different groups of nitrifying and denitrifying bacteria detected by FISH-FCM.

Effect of a Milk Formula Containing Probiotics on the Fecal Microbiota of Asian Infants at Risk of Atopic Diseases

The fecal microbiota of 37 infants with (n = 20) or without (n = 17) probiotic administration was evaluated on D 3, and at 1, 3, and 12 mo by fluorescence in situ hybridization-flow cytometry (FISH-FC), PCR, and bacteriological culture methods. They represent consecutive subjects of an ongoing double-blind, placebo-controlled trial on a probiotic formula (LGG and Bifidobacterium longum) administered during the first 6 mo of life. Despite varying composition in each baby, there was a general bacterial colonization pattern in the first year. Bifidobacteria increased markedly (p = 0.0003) with a parallel decrease in Enterobacteriaceae (p < 0.001) and Bacteroides-Prevotella (p = 0.005) populations. Eubacterium rectale-Clostridium coccoides (p < 0.001) and Atopobium (p = 0.039) groups also gradually increased. This overall pattern was unaffected by probiotic administration (p > 0.05). B. longum (p = 0.005) and Lactobacillus rhamnosus (p < 0.001) were detected more frequently in probiotic group during supplementation, but no difference after supplementation had ceased (p > 0.05). Cultured lactic acid bacteria were also more numerous in the probiotic-administered babies during treatment period (log CFU/g 8.4 versus 7.4; p = 0.035). Our results indicate that supplemented strains could be detected but did not persist in the bowel once probiotic administration had ceased.

Fluorescence In Situ Hybridization-Flow Cytometry-Cell Sorting-Based Method for Separation and Enrichment of Type I and Type II Methanotroph Populations

A fluorescence in situ hybridization-flow cytometry (FISH/FC)-based method was optimized using artificial mixtures of pure cultures of methanotrophic bacteria. Traditional oligonucleotide probes targeting 16S rRNAs of type I (MG84/705 probe) and type II (MA450 probe) methanotrophs were labeled with fluorescein or Alexa fluor and used for FISH, followed by fluorescence-activated FC analysis and cell sorting (FACS). The method resulted in efficient separation of target cells (type I or type II methanotrophs) from the artificial mixtures. The method was then applied for detection and enrichment of type I and type II methanotroph populations from a natural sample, Lake Washington sediment. Cells were extracted from the sediment, fixed, and subjected to FISH/FC/FACS. The resulting subpopulations were analyzed by reverse transcriptase PCR surveys of 16S rRNA, pmoA (encoding a subunit of particulate methane monooxygenase), and fae (encoding formaldehyde-activating enzyme) genes. The functional gene analysis indicated specific separation of the type I and type II methanotroph populations. 16S rRNA gene analysis revealed that type I methanotrophs comprised 59% of the subpopulation separated using the type I-specific probe and that type II methanotrophs comprised 47.5% of the subpopulation separated using the type II-specific probe. Our data indicate that the FISH/FC/FACS protocol described can provide significant enrichment of microbial populations of interest from complex natural communities and that these can be used for genetic tests. We further tested the possibility of direct whole-genome amplification (WGA) from limited numbers of sorted cells, using artificial mixtures of microbes whose genome sequences are known. We demonstrated that efficient WGA can be achieved using 10(4) or more cells separated by 16S rRNA-specific FISH/FC/FACS, while fewer cells resulted in less specific WGA.