Amplification Of Ribosomal RNA Genes Research Articles

O-281 Characterization of Vaginal microbiota and reproductive outcomes associated with assisted reproductive technologies through next-generation sequencing, from Indian population

Abstract Study question Is there any association between the composition of the vaginal microbiota and the reproductive outcomes in infertile women’s undergoing for assisted reproductive treatments (ART)? Summary answer Higher microbial abundance in non-pregnant patients compared to pregnant patients. These pathogens could be used as a possible marker for a higher IVF failure rate. What is known already Various next- generation sequencing methods have been used to study bacterial populations in infertile Women. Vaginal microbiota could have an influence on success rates of fertility treatments. The bacterial microbiota varies by the population. There is no direct evidence on this subject to guide clinical decision making or its influence on the hierarchy of fertility treatments. While the presence of pathogenic bacteria non-Lactobacilli dominated microbiota is associated with poor reproductive outcomes in assisted reproductive treatments (ART). Additional studies, particularly those using metagenomics approaches, may be useful. Study design, size, duration A total of 240 infertile women’s were included in the study. The study population consists of patients attending to the ART unit of a tertiary medical center Udaipur, India, between November 2021 to October 2023. Depending on the biochemical pregnancy results, they were divided into two groups. Group 1 (n = 128) was pregnancy (P), and Group 2 (n = 112) non- pregnancy (NP) group, Patients were aged between (20-45 years). The ethical approval was obtained from the institute. Participants/materials, setting, methods DNA was extracted from vaginal swab samples collected at the time of oocyte pick up using the commercially available ( DNeasy Power Soil kit). The Vaginal microbiota of samples were analyzed using 16S ribosomal RNA gene amplification (MinION) Oxford Nanopore Ltd. Bioinformatics analysis was performed using QIIME2 and Microbiome Analyst packages. Alpha, beta diversity and taxonomic characterization were compared for microbial composition. The findings of the principle coordinate analysis were shown using R version 4.0. Main results and the role of chance Our results show that higher Shannon and Simpson index were obtained in the non- pregnancy (NP) group compared to the pregnancy (P) group. In addition, the partial least squares discriminant analysis (PLS-DA) revealed a difference in microbial composition between pregnancies (P) and non- pregnancy (NP) groups, as well as a higher microbial abundance in non-pregnant patients compared to pregnant patients. At the phylum level Firmicutes, Actinobacteria, Proteobacteria, Fusobacteria, and Bacteroidetes are the most common predominant in the pregnancy (P) group (80.77, 9.52, 9.18, 0.04, and 0.40%, respectively) and the non- pregnancy (NP) group (72, 61, 12.41, 8.01, and 3.75%). A higher abundance of Firmicutes (80.77 vs. 72.61%) and Proteobacteria (9.18 vs. 8.01%), and a lower abundance of Actinobacteria (9.52 vs. 12.41%, p = 0.03), Fusobacteria (0.04 vs. 8.01%, p = 0.02), and Bacteroidetes (0.40 vs. 3.75%, p = 0.03) were observed in pregnancy (P) group. At the genus level, pregnancy group (P) increased the abundance of probiotics lactic acid bacteria (74.61 vs 63.09%) and decreased the abundance of the pathogen Gardnerella (6.03 vs 7.24%, p = 0.03), Atopobium (0.84 vs 4.14%, p = 0.02), Sneathia (0.03 vs. 3.75%, p = 0.02), and Prevotella (0.38 vs. 3, 02%, p = 0.04). Limitations, reasons for caution The limited sample size is the primary constraint of this study. Larger studies with a larger sample size are needed to confirm the distinct microbiome patterns identified in female reproductive tracts samples in connection to infertility and live birth rate (LBR) outcomes. Wider implications of the findings Vaginal microbiota composition associated to different clinical outcomes in patients undergoing ART might have profound implications. This may lead to failed fertility treatments and reduced live birth rate (LBR). More studies are needed to clarify the relative effectiveness of treatments for infertility. Trial registration number NA

Multi-omics data elucidate parasite-host-microbiota interactions and resistance to Haemonchus contortus in sheep

BackgroundThe integration of molecular data from hosts, parasites, and microbiota can enhance our understanding of the complex biological interactions underlying the resistance of hosts to parasites. Haemonchus contortus, the predominant sheep gastrointestinal parasite species in the tropics, causes significant production and economic losses, which are further compounded by the diminishing efficiency of chemical control owing to anthelmintic resistance. Knowledge of how the host responds to infection and how the parasite, in combination with microbiota, modulates host immunity can guide selection decisions to breed animals with improved parasite resistance. This understanding will help refine management practices and advance the development of new therapeutics for long-term helminth control.MethodsEggs per gram (EPG) of feces were obtained from Morada Nova sheep subjected to two artificial infections with H. contortus and used as a proxy to select animals with high resistance or susceptibility for transcriptome sequencing (RNA-seq) of the abomasum and 50 K single-nucleotide genotyping. Additionally, RNA-seq data for H. contortus were generated, and amplicon sequence variants (ASV) were obtained using polymerase chain reaction amplification and sequencing of bacterial and archaeal 16S ribosomal RNA genes from sheep feces and rumen content.ResultsThe heritability estimate for EPG was 0.12. GAST, GNLY, IL13, MGRN1, FGF14, and RORC genes and transcripts were differentially expressed between resistant and susceptible animals. A genome-wide association study identified regions on chromosomes 2 and 11 that harbor candidate genes for resistance, immune response, body weight, and adaptation. Trans-expression quantitative trait loci were found between significant variants and differentially expressed transcripts. Functional co-expression modules based on sheep genes and ASVs correlated with resistance to H. contortus, showing enrichment in pathways of response to bacteria, immune and inflammatory responses, and hub features of the Christensenellaceae, Bacteroides, and Methanobrevibacter genera; Prevotellaceae family; and Verrucomicrobiota phylum. In H. contortus, some mitochondrial, collagen-, and cuticle-related genes were expressed only in parasites isolated from susceptible sheep.ConclusionsThe present study identified chromosome regions, genes, transcripts, and pathways involved in the elaborate interactions between the sheep host, its gastrointestinal microbiota, and the H. contortus parasite. These findings will assist in the development of animal selection strategies for parasite resistance and interdisciplinary approaches to control H. contortus infection in sheep.Graphical

16S rRNA Next-Generation Sequencing May Not Be Useful for Examining Suspected Cases of Spontaneous Bacterial Peritonitis.

Background and Objectives: Ascites, often associated with liver cirrhosis, poses diagnostic challenges, particularly in detecting bacterial infections. Traditional methods have limitations, prompting the exploration of advanced techniques such as 16S rDNA next-generation sequencing (NGS) for improved diagnostics in such low-biomass fluids. The aim of this study was to investigate whether the NGS method enhances detection sensitivity compared to a conventional ascites culture. Additionally, we aimed to explore the presence of a microbiome in the abdominal cavity and determine whether it has a sterile condition. Materials and Methods: Ten patients with clinically suspected spontaneous bacterial peritonitis (SBP) were included in this study. A traditional ascites culture was performed, and all ascites samples were subjected to 16S ribosomal RNA gene amplification and sequencing. 16S rRNA gene sequencing results were interpreted by comparing them to positive and negative controls for each sample. Results: Differential centrifugation was applied to all ascites samples, resulting in very small or no bacterial pellets being harvested. The examination of the 16S amplicon sequencing libraries indicated that the target amplicon products were either minimally visible or exhibited lower intensity than their corresponding negative controls. Contaminants present in the reagents were also identified in the ascites samples. Sequence analysis of the 16S rRNA gene of all samples showed microbial compositions that were akin to those found in the negative controls, without any bacteria isolated that were unique to the samples. Conclusions: The peritoneal cavity and ascites exhibit low bacterial biomass even in the presence of SBP, resulting in a very low positivity rate in 16S rRNA gene sequencing. Hence, the 16S RNA sequencing method does little to enhance the rate of positive samples compared to traditional culture methods, including in SBP cases.

Detection of Multidrug resistant cariogenic bacteria among Diabetic Patients attending a Tertiary Hospital in Ado Ekiti, South-Western, Nigeria

Microorganisms colonizing the oral cavity have a significant probability of spreading on epithelial surfaces to neighboring sites. Many reports have associated oral bacteria to oral infectious diseases as well as systemic diseases such as cardiovascular disease and diabetes. Oral care is usually neglected in the control of health problems linked with diabetes which may contribute to the increased morbidity and complications from oral health problems. The study aimed to identify the resident microbial flora and assess the possible carriage of cariogenic bacteria among people with diabetes in Ado- Ekiti, Nigeria. Oral swabs were collected from 306 consenting patients. Culture-based isolation was done using standard techniques on Mitis salivarius agar supplemented with 1% Potassium Tellurite, 10% chocolate agar and MacConkey agar at 37 oC for 18-48 hours. The susceptibility patterns of the isolates were detected using standard techniques as recommended by CLSI. Selected bacteria isolates were genomically identified using the bacterial universal primer 16S rRNA and STR-1 and STR 2 primers (partial tuf genes). Amplified genes were sequenced and phylogenetic analysis was carried out using MEGA 7 software. A total of 447 microbial isolates were identified among which were Staphylococcus spp (14.5%), Klebsiella pneumoniae (3.4%), Escherichia coli (2.5%), Candida spp (8%) while the streptococcal organisms such as Streptococcus pyogenes (35.2%), Streptococcus mitis (14%), Streptococcus mutans (7.3%) and Streptococcus salivarius (15.1%). Streptococcus mutans was susceptible to meropenem (71.4%) but highly resistant to Penicillin (100%), cefuroxime (71.4%) cefotaxime (68.6%). Streptococcus mitis was highly resistant to Amoxicillin/Clavulanate (100%), Cloxacillin (100%) and Cefuroxime (70.1%) but was found susceptible to Meropenem (77.3%). 16S ribosomal RNA gene amplification and subsequent sequencing of the isolates revealed twenty-six (26) de-novo bacterial strains. In conclusion, the study showed that diabetic individuals carry multidrug-resistant cariogenic bacteria that may predispose them to other oral diseases. There is a need to incorporate routine oral microbiological screening into routine care for diabetic patients in Nigeria.

P-067 Characterization of seminal microbiota in patients with asthenozoospermic and normozoospermic using next-generation sequencing

Abstract Study question How are the semen microbiota compositions of patients with asthenozoospermic and normozoospermic different, According to data from 16S rRNA gene-specific Next-generation sequencing (NGS)? Summary answer Large numbers of microbes can be present in seminal fluid, and there are differences in the semen microbiota between asthenozoospermic and normozoospermic semen samples. What is known already Male factor is attributable in up to 50% of cases of infertility. Most studies on normal and abnormal sperm microbiota are based on 16S rRNA gene-specific next-generation sequencing (NGS). Microbiome analysis based on subunit 16S rRNA sequencing is a fast tool that can enable the identification of all the pathogenic microorganisms associated with semen in clinical pathology. Studies on the impact of semen micro-biomes in asthenozoospermic and normozoospermic samples could improve the results of assisted reproductive technologies. The major bacterial diversity in asthenozoospermic and normozoospermic samples belongs to the genera Lactobacillus, Prevotella, Staphylococcus, and Anaerococcus. Study design, size, duration Two eighty patients with their own semen samples were included in the study. The study population consists of patients attending to the “Pacific IVF Center” Pacific Medical University and Hospital (Udaipur, India) from November 2020 to October 2022. Depending on the spermiogram results, they were divided into two groups. Group 1 (n = 127) was asthenozoospermic, and Group 2 (n = 153) had normozoospermic semen samples, Patients were aged between (20-45 years). Ethical approval was obtained from the institute. Participants/materials, setting, methods Genomic DNA was extracted from samples using commercially available (Qiagen, DNeasy Power Soil kit). The amount of extracted DNA was measured using a Nanodrop spectrophotometer (Thermo Scientific). The microbiota of semen was analyzed using 16S ribosomal RNA (rRNA) gene amplification (MinION) Oxford Nanopore Ltd. Bioinformatics analysis was performed using QIIME2 and Microbiome Analyst packages. Alpha, beta diversity, and taxonomic characterization were compared for the seminal microbiome in asthenozoospermic and normozoospermic semen samples. Main results and the role of chance Different bacterial communities were detected when asthenozoospermic and normozoospermic semen samples were analyzed. In patients with asthenozoospermic parameters, a higher alpha diversity index tendency was found. In normozoospermic semen samples, a p-value of bacterial diversity was non-significant (p = 0.14 for the Shannon index), and significant differences in asthenozoospermic semen samples (p = 0.01 for the Shannon index). In the beta diversity analysis, no significant differences were observed between both groups Relative abundance analysis identified the bacterial communities, Lactobacillus, Streptococcus, and Ureaplasma in normozoospermic semen samples, and Anaerococcus and Gardnerella in asthenozoospermic samples, Comparison of asthenozoospermic and normozoospermic samples with alpha diversity (p = 0.06 for the Shannon index and p = 0.08 for the Simpson index) and beta diversity (p < 0.001) showed significant differences. Relative abundance in normozoospermic semen samples identified the bacterial communities, Lactobacillus (p = 3.70E-4), Prevotella (p = 8.11E-4), and in asthenozoospermic semen samples Anaerococcus (p = 0.004) and Gardnerella (p = 0.004). Limitations, reasons for caution The limited sample size is the primary constraint of this study. Larger studies with a larger sample size are needed to confirm the distinct microbiome patterns identified in asthenozoospermic and normozoospermic samples in connection to male infertility. Wider implications of the findings Further research could determine the detection rate of the described bacterial diversity in semen with other pathologies. Determining the relationship between sperm microbiota characteristics and infertility could enable the development of new algorithms for treating patients with reproductive disorders, An abnormal seminal microbiome appears to be strongly correlated with infertility. Trial registration number N/A

Intestinal fatty acid binding protein is associated with cardiac function and gut dysbiosis in chronic heart failure.

The gut microbiota in patients with chronic heart failure (HF) is characterized by low bacterial diversity and reduced ability to synthesize beneficial metabolites. These changes may facilitate leakage of whole bacteria or bacterial products from the gut into the bloodstream, which may activate the innate immune system and contribute to the low-grade inflammation seen in HF. In this exploratory cross-sectional study, we aimed to investigate relationships between gut microbiota diversity, markers of gut barrier dysfunction, inflammatory markers, and cardiac function in chronic HF patients. In total, 151 adult patients with stable HF and left ventricular ejection fraction (LVEF) < 40% were enrolled. We measured lipopolysaccharide (LPS), LPS-binding protein (LBP), intestinal fatty acid binding protein (I-FABP), and soluble cluster of differentiation 14 (sCD14) as markers of gut barrier dysfunction. N-terminal pro-B-type natriuretic peptide (NT-proBNP) level above median was used as a marker of severe HF. LVEF was measured by 2D-echocardiography. Stool samples were sequenced using 16S ribosomal RNA gene amplification. Shannon diversity index was used as a measure of microbiota diversity. Patients with severe HF (NT-proBNP > 895 pg/ml) had increased I-FABP (p < 0.001) and LBP (p = 0.03) levels. ROC analysis for I-FABP yielded an AUC of 0.70 (95% CI 0.61-0.79, p < 0.001) for predicting severe HF. A multivariate logistic regression model showed increasing I-FABP levels across quartiles of NT-proBNP (OR 2.09, 95% CI 1.28-3.41, p = 0.003). I-FABP was negatively correlated with Shannon diversity index (rho = -0.30, p = <0.001), and the bacterial genera Ruminococcus gauvreauii group, Bifidobacterium, Clostridium sensu stricto, and Parasutterella, which were depleted in patients with severe HF. In patients with HF, I-FABP, a marker of enterocyte damage, is associated with HF severity and low microbial diversity as part of an altered gut microbiota composition. I-FABP may reflect dysbiosis and may be a marker of gut involvement in patients with HF.

Assessment of bacterial diversity associated with assisted reproductive technologies through next-generation sequencing

BackgroundTo assess bacterial diversity in infertile couples with their biochemical pregnancy outcomes. Using a retrospective case-control study design, participants were recruited for collection of vaginal swab, follicular fluid, endometrial fluid, and semen samples. The microbial composition was analyzed by 16S ribosomal RNA (rRNA) gene amplification with (MinION) Oxford Nanopore Ltd.ResultsOur findings revealed that age and endometrial thickness had a significant impact on the pregnancy success rate of pregnant (P) and non-pregnant (NP) patients receiving IVF, with high levels of luteinizing hormone, estrogen, and progesterone in the P group. In addition, the partial least squares discriminant analysis (PLS-DA) revealed a difference in microbial composition between the P and NP groups, as well as a higher microbial abundance in non-pregnant patients compared to pregnant patients. After comparison between pregnant patients and non-pregnant patients, pregnant patients had a higher abundance of Firmicutes and Proteobacteria and a lower abundance of Actinobacteria, Fusobacterium, and Bacteroidetes at the phylum level. Non-pregnant patients had a lower abundance of the probiotics lactobacillus and a higher abundance of infections Prevotella and Gardnerella at the genus level. As a result, a disordered microbiota in non-pregnant patients, characterized by a decrease in probiotics and an increase in pathogens, could be used as a possible marker for a higher IVF failure rate.ConclusionAlteration of the microbiota of the reproductive tract or the presence of certain microbes, regardless of the degree of pathogenicity that can affect fertilization, as well as implantation and subsequent embryonic development. This could result in failed fertility treatments and a lower live birth (LBR) rate.

Intestinal microflora provides biomarkers for infertile women with endometrial polyps

Background Endometrial polyps (EPs) are related to infertility; however, there are no biomarkers for identification. Objective We evaluated changes in the intestinal microflora to identify microflora-based biomarkers that may be useful for detecting EPs. Material and methods Intestinal specimens were prospectively collected from 100 women: 25 infertile women with EPs (InfEP + group), 25 infertile women without EPs (InfEP- group), and 50 healthy women (Fertile group). The microbiota composition was analyzed using 16S ribosomal RNA gene amplification and the intestinal expression of selected human genes using quantitative reverse transcription polymerase chain reaction. Results The InfEP + group had higher proportions of Prevotella, Streptococcus, Fusobacterium, Fenollaria, and Porphyromonas than the InfEP- and Fertile groups, while the Fertile group had higher proportions of Faecalibacterium, Bacteroides, and Blautia. We constructed a microbial dysbiosis index based on the intestinal microbiota at the genus level as a predictive model. The most accurate model to predict the presence of EPs was that including the Fertile and InfEP + groups (area under the curve: 0.89, 95% confidence interval: 0.79–0.96). The InfEP- and Fertile groups had significant differences in microflora composition compared with the InfEP + group. Conclusion The intestinal microflora may be a useful biomarker for identifying EPs in infertile women.

First report of Fusarium oxysporum and Globisporangium ultimum as causal agents of pomegranate fruit rot in Tunisia

First report of <i>Fusarium oxysporum</i> and <i>Globisporangium ultimum</i> as causal agents of pomegranate fruit rot in Tunisia

A Capillary Electrophoresis Method for Analyzing the Diversity of Vaginal Microbiome

16S ribosomal RNA (rRNA) gene sequence analysis is the common method to identify the bacteria in human vaginal flora. While specific DNA primers were designed to target 16S rRNA gene sequences, DNA fragment analysis using capillary electrophoresis can obtain more accurate qualitative and quantitative information on the microbiome. This study aimed to assess the ability of capillary electrophoresis method to analyze the diversity of vaginal microbiome and provide a theoretical basis for the accurate gene detection of vaginal flora. We collected 75 vaginal secretion samples from female outpatients aged 25 - 50, who had undergone rou-tine gynecologic examinations in Fujian provincial hospital from March 2021 to April 2021. Clinical diagnosis was based on the results of microscopic examination of Gram-stained specimens and biochemical tests of bacteria (pH value, catalase, leukocyte esterase, sialidases, β-glucuronidase, and acetylglucosaminidase). Vaginal secretion samples were collected and then total bacterial DNA was extracted. We used six pairs of fluorescent dye tagged specific primers that were designed based on the 16S rRNA genes of four Lactobacillus species (L. iners, L. crispatus, L. jensenii, L. gasseri), Gardnerella vaginalis, and Atopobium vaginae. PCR products of six species of bacteria were detected and analyzed by a 3130 Genetic Analyzer. Seventy-five samples were divided into two groups according to the vaginal microbiome evaluation, including 50 cases which had vaginal bacteria balance and 25 cases which had vaginal bacteria disorder. PCR amplification of 16S ribosomal RNA genes of L. iners, L. crispatus, L. jensenii, L. gasseri, Gardnerella vaginalis, and Atopobium vaginae was successfully performed on the DNA extracted from vaginal secretion samples. Four Lac-tobacillus species were detected in 4 - 33 cases of "Balance" group, and Gardnerella vaginalis was detected in 23 cases of "Disorder" group and, simultaneously, Atopobium vaginae was detected in 20 cases. Based on the technique of DNA fragment analysis using capillary electrophoresis method, the most common vaginal bacteria in Chinese healthy women are L. iners and L. crispatus. Gardnerella vaginalis and Atopobium vaginae are the most common pathogenic bacteria detected in the patients who had vaginal bacteria disorder. Using capillary electrophoresis method to detect the vaginal bacteria will be useful for accurate identifica-tion of vaginal microbiome. There will be an application value to find out the composition of the vaginal microbiome rapidly and detect specific gene markers to identify potential pathogenic bacteria when women are at risk of serious illness before they develop obvious symptoms.

First Report of Moldy Core of Sweet Tango Apples from New Zealand Caused by Alternaria arborescens.

First Report of Moldy Core of Sweet Tango Apples from New Zealand Caused by Alternaria arborescens.

Multiplexed Signal Ion Emission Reactive Release Amplification (SIERRA) Assay for the Culture-Free Detection of Gram-Negative and Gram-Positive Bacteria and Antimicrobial Resistance Genes.

The global prevalence of antibiotic-resistant bacteria has increased the risk of dangerous infections, requiring rapid diagnosis and treatment. The standard method for diagnosis of bacterial infections remains dependent on slow culture-based methods, carried out in central laboratories, not easily extensible to rapid identification of organisms, and thus not optimal for timely treatments at the point-of-care (POC). Here, we demonstrate rapid detection of bacteria by combining electrochemical immunoassays (EC-IA) for pathogen identification with confirmatory quantitative mass spectral immunoassays (MS-IA) based on signal ion emission reactive release amplification (SIERRA) nanoparticles with unique mass labels. This diagnostic method uses compatible reagents for all involved assays and standard fluidics for automatic sample preparation at POC. EC-IA, based on alkaline phosphatase-conjugated pathogen-specific antibodies, quantified down to 104 bacteria per sample when testing Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa lysates. EC-IA quantitation was also obtained for wound samples. The MS-IA using nanoparticles against S. aureus, E. coli, Klebsiella pneumoniae, and P. aeruginosa allowed selective quantitation of ∼105 bacteria per sample. This method preserves bacterial cells allowing extraction and amplification of 16S ribosomal RNA genes and antibiotic resistance genes, as was demonstrated through identification and quantitation of two strains of E. coli, resistant and nonresistant due to β-lactamase cefotaximase genes. Finally, the combined immunoassays were compared against culture using remnant deidentified patient urine samples. The sensitivities for these immunoassays were 83, 95, and 92% for the prediction of S. aureus, P. aeruginosa, and E. coli or K. pneumoniae positive culture, respectively, while specificities were 85, 92, and 97%. The diagnostic platform presented here with fluidics and combined immunoassays allows for pathogen isolation within 5 min and identification in as little as 15 min to 1 h, to help guide the decision for additional testing, optimally only on positive samples, such as multiplexed or resistance gene assays (6 h).

Bile Microbiota in Liver Transplantation: Proof of Concept Using Gene Amplification in a Heterogeneous Clinical Scenario.

Objective: Historically, bile in the biliary tract has been considered sterile. Most of the series are based on patients with biliary tract diseases or the bile has been obtained with procedures susceptible to contamination.Methods: We evaluated the bile in a heterogeneous cohort of liver donors and recipient patients, with samples obtained in a sterile way, directly from the gallbladder and the common bile duct.Results: We assessed the bile microbiota in six liver donors and in six liver recipients after whole or split liver procedures in adult or pediatric recipients. Bile samples were studied using PCR sequencing of the 16S ribosomal RNA gene amplification (rDNA).Conclusions: We demonstrated that the bile is sterile, thereby ruling this out as a source of contamination following transplant.

Diagnosis of pleural empyema/parapneumonic effusion by next-generation sequencing

Background Although a microbiological diagnosis of pleural infection is clinically important, it is often complicated by prior antibiotic treatment and/or difficulties with culturing some bacterial species. Therefore, we aimed to identify probable causative bacteria in pleural empyema/parapneumonic effusions by combining 16S ribosomal RNA (rRNA) gene amplification and next-generation sequencing (NGS). Methods Pleural fluids were collected from 19 patients with infectious effusions and nine patients with non-infectious malignant effusions. We analysed DNA extracted from the pleural fluid supernatant by NGS using the Genome Search Toolkit and GenomeSync database, either directly or after PCR amplification of the 16S rRNA gene. Infectious and non-infectious effusions were distinguished by semi-quantitative PCR of the 16S rRNA gene. Results Only 8 (42%) effusions were culture-positive, however, NGS of the 16S rRNA gene amplicon identified 14 anaerobes and 7 aerobes/facultative anaerobes in all patients, including Streptococcus sp. (n = 6), Fusobacterium sp. (n = 5), Porphyromonas sp. (n = 5), and Prevotella sp. (n = 4), accounting for >10% of the total genomes. The culture and NGS results were discordant for 3 out of 8 patients, all of whom had previously been treated with antibiotics. Total (2ΔCT value in semi-quantitative PCR of the 16S rRNA gene) and specific (total bacterial load multiplied by the proportion of primary bacteria in NGS) bacterial loads could efficiently distinguish empyema/parapneumonic effusion from non-infectious effusion. Conclusion Combining NGS with semi-quantitative PCR can facilitate the diagnosis of pleural empyema/parapneumonic effusion and its causal bacteria.

Urinary Microbiome Characteristics in Female Patients with Acute Uncomplicated Cystitis and Recurrent Cystitis.

Traditionally, the diagnostic mainstay of recurrent urinary tract infection has been urinary culture. However, the causative uropathogen of recurrent cystitis has not been well established. Urine DNA next-generation sequencing (NGS) can provide additional information on these infections. Herein, we compared urine NGS results and urine cultures in patients with acute uncomplicated cystitis (AUC) and recurrent cystitis (RC), and evaluated the difference in microbiome patterns in the NGS results. Patients who underwent urine culture and NGS due to AUC or RC were retrospectively reviewed. All urine samples were collected via a transurethral catheter and studied utilizing a type of NGS called 16S ribosomal RNA gene amplification and sequencing. The sensitivity of urine NGS was significantly higher than that of conventional urine culture (69.0% vs. 16.7%, p < 0.05). The detection rate of urine NGS was slightly lower in the RC group than in the AUC group (67.7% vs. 72.7%). Microbiome diversity was significantly higher in the RC group compared to the AUC group (p = 0.007), and the microbiome composition was significantly different between the AUC and RC groups. In the urine NGS results, Pseudomonas, Acinetobacter, and Enterobacteriaceae were found in the AUC group, and Sphingomonas, Staphylococcus, Streptococcus, and Rothia spp. were detected in the RC group. Urine NGS can significantly increase the diagnostic sensitivity compared to traditional urine culture methods, especially in RC patients. AUC and RC patients had significant differences in bacterial diversity and patterns. Therefore, recurrent cystitis might be approached from a different perspective.

High Culturable Bacterial Diversity From a European Desert: The Tabernas Desert.

One of the most diverse ecological niches for microbial bioprospecting is soil, including that of drylands. Drylands are one of the most abundant biomes on Earth, but extreme cases, such as deserts, are considered very rare in Europe. The so-called Tabernas Desert is one of the few examples of a desert area in continental Europe, and although some microbial studies have been performed on this region, a comprehensive strategy to maximize the isolation of environmental bacteria has not been conducted to date. We report here a culturomics approach to study the bacterial diversity of this dryland by using a simple strategy consisting of combining different media, using serial dilutions of the nutrients, and using extended incubation times. With this strategy, we were able to set a large (254 strains) collection of bacteria, the majority of which (93%) were identified through 16S ribosomal RNA (rRNA) gene amplification and sequencing. A significant fraction of the collection consisted of Actinobacteria and Proteobacteria, as well as Firmicutes strains. Among the 254 isolates, 37 different genera were represented, and a high number of possible new taxa were identified (31%), of which, three new Kineococcus species. Moreover, 5 out of the 13 genera represented by one isolate were also possible new species. Specifically, the sequences of 80 isolates held a percentage of identity below the 98.7% threshold considered for potentially new species. These strains belonged to 20 genera. Our results reveal a clear link between medium dilution and isolation of new species, highlight the unexploited bacterial biodiversity of the Tabernas Desert, and evidence the potential of simple strategies to yield surprisingly large numbers of diverse, previously unreported, bacterial strains and species.

Molecular detection and genetic characterization of Anaplasma marginale and Anaplasma platys-like (Rickettsiales: Anaplasmataceae) in water buffalo from eight provinces of Thailand

BackgroundAnaplasmosis, an animal disease caused by rickettsial bacteria in the genus Anaplasma, is of considerable economic importance in livestock animals in many countries worldwide. The objectives of this study were to determine the identity, prevalence, and geographic distribution of Ehrlichia and Anaplasma in naturally infected water buffalo in Thailand using PCR amplification and sequencing of the 16S ribosomal RNA and heat shock protein groEL genes. A total of 456 buffalo blood samples from Thailand were investigated. Species identification and genetic differentiation of intra-population and inter-population with the global isolates were conducted based on nucleotide sequences. Interplay between the infection and host factors was also assessed.ResultsOverall, 41% of water buffalo were found to be infected with rickettsial organisms in the family Anaplasmataceae, but Ehrlichia spp., Neorickettsia spp., and Wolbachia spp. were not found in any of the sequenced samples in this study. Female buffalo were more frequently infected with bacteria in the family Anaplasmataceae than males [71 out of 176 females (40.3%) versus 11 out of 47 males (23.4%)]. The Odds Ratio value indicated that the risk of infection for female buffalo was 2.2-fold higher than that for males (p < 0.05). We detected three haplotypes of A. marginale 16S rRNA gene and they were placed in a clade that was closely related to the A. marginale in buffalo in China; and cattle in Thailand, Uganda, and China. Homology searching of groEL sequences against the GenBank™ database using the BLASTn algorithm revealed that the obtained sequences had a high percentage similarity (98.36–99.62%) to A. platys sequences. The groEL sequences of three A. platys-like isolates were clustered in the same clade as the A. platys from the tick Rhipicephalus microplus in China.ConclusionsOur data showed that the apparently healthy buffalo were naturally infected by bacteria in the family Anaplasmataceae at a relatively high prevalence. We also report the finding of A. platys-like infections in water buffalo in Thailand for the first time. Water buffalo serving as the reservoir host of anaplasmosis is of concern for managing the disease control and prevention in ruminants.

Rhizopus arrhizus (syn. R. oryzae) Causing Sunflower Head Rot in Hebei Province, China

<i>Rhizopus arrhizus</i> (syn. <i>R. oryzae</i>) Causing Sunflower Head Rot in Hebei Province, China

Immune-mediated haemolytic anaemia secondary to haemotrophic mycoplasma infection in a pet ewe

Haemotrophic Mycosplasma species (haemoplasmas) are adapted to adhere to erythrocyte cell membranes of hosts,1 resulting in a range of clinical presentations. Clinical signs of Mycoplasma ovis (previously known as Eperythrozoon...

Practical considerations for the study of the oral microbiome

In the oral cavity, complex microbial community is shaped by various host and environmental factors. Extensive literature describing the oral microbiome in the context of oral health and disease is available. Advances in DNA sequencing technologies and data analysis have drastically improved the analysis of the oral microbiome. For microbiome study, bacterial 16S ribosomal RNA gene amplification and sequencing is often employed owing to the cost-effective and fast nature of the method. In this review, practical considerations for performing a microbiome study, including experimental design, molecular analysis technology, and general data analysis, will be discussed.