Total DNA Extraction Research Articles

Investigation of Upper Respiratory Carriage of Bacterial Pathogens among University Students in Kampar, Malaysia.

The carriage of bacterial pathogens in the human upper respiratory tract (URT) is associated with a risk of invasive respiratory tract infections, but the related epidemiological information on this at the population level is scarce in Malaysia. This study aimed to investigate the URT carriage of Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa among 100 university students by nasal and oropharyngeal swabbing. The presence of S. aureus, K. pneumoniae and P. aeruginosa was assessed via swab culture on selective media and PCR on the resulting isolates. For S. pneumoniae, H. influenzae and N. meningitidis, their presence was assessed via multiplex PCR on the total DNA extracts from chocolate agar cultures. The carriage prevalence of H. influenzae, S. aureus, S. pneumoniae, K. pneumoniae, N. meningitidis and P. aeruginosa among the subjects was 36%, 27%, 15%, 11%, 5% and 1%, respectively, by these approaches. Their carriage was significantly higher in males compared to females overall. The S. aureus, K. pneumoniae and P. aeruginosa isolates were also screened by the Kirby-Bauer assay, in which 51.6% of S. aureus were penicillin-resistant. The outcomes from carriage studies are expected to contribute to informing infectious disease control policies and guidelines.

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants.

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

Electrochemical Detection of ompA Gene of C. sakazakii Based on Glucose-Oxidase-Mimicking Nanotags of Gold-Nanoparticles-Doped Copper Metal-organic Frameworks.

The present work developed an electrochemical genosensor for the detection of virulence outer membrane protein A (ompA, tDNA) gene of Cronobacter sakazakii (C. sakazakii) by exploiting the excellent glucose-oxidase-mimicking activity of copper Metal-organic frameworks (Cu-MOF) doped with gold nanoparticle (AuNPs). The signal nanotags of signal probes (sDNA) that biofunctionalized AuNPs@Cu-MOF (sDNA-AuNPs@Cu-MOF) were designed using an Au-S bond. The biosensor was prepared by immobilization capture probes (cDNA) onto an electrodeposited AuNPs-modified glassy carbon electrode (GCE). AuNPs@Cu-MOF was introduced onto the surface of the GCE via a hybridization reaction between cDNA and tDNA, as well as tDNA and sDNA. Due to the enhanced oxidase-mimicking activity of AuNPs@Cu-MOF to glucose, the biosensor gave a linear range of 1.0 × 10-15 to 1.0 × 10-9 mol L-1 to tDNA with a detection limit (LOD) of 0.42 fmol L-1 under optimized conditions using differential pulse voltammetry measurement (DPV). It can be applied in the direct detection of ompA gene segments in total DNA extracts from C. sakazakii with a broad linear range of 5.4-5.4 × 105 CFU mL-1 and a LOD of 0.35 CFU mL-1. The biosensor showed good selectivity, fabricating reproducibility and storage stability, and can be used for the detection of ompA gene segments in real samples with recovery between 87.5% and 107.3%.

MP15-04 PRESENCE OF HPV IN PROSTATE TISSUE FROM PATIENTS SUBMITTED TO PROSTATE BIOPSY

You have accessJournal of UrologyCME1 Apr 2023MP15-04 PRESENCE OF HPV IN PROSTATE TISSUE FROM PATIENTS SUBMITTED TO PROSTATE BIOPSY Nalisson Pereira, Emerson Martins, Mateus Quintela, Arthur Cunha, and Jaques Waisberg Nalisson PereiraNalisson Pereira More articles by this author , Emerson MartinsEmerson Martins More articles by this author , Mateus QuintelaMateus Quintela More articles by this author , Arthur CunhaArthur Cunha More articles by this author , and Jaques WaisbergJaques Waisberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003235.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostate cancer (PCa) is the second most common cancer among men. Environmental and hereditary factors are assumed to play crucial roles in prostate carcinogenesis. In addition, increasing epidemiological evidence, along with molecular research, suggests that viral infection would be a potential cofactor for the development of PCa. Recently, it has been estimated that virus infection represents approximately 10% of the worldwide cancer burden. HPV, one of the most important oncogenic viruses. Based on the genomic sequence of L1, the gene encoding the main capsid protein, more than 200 types of HPV have been identified and characterized, with at least 14 high-risk types which may cause cancer. The aim of this study was to evaluate the presence of HPV in prostate tissue in patients undergoing transrectal ultrasound-guided prostate biopsy and its possible relationship with prostate cancer. METHODS: 157 prostate tissue samples were obtained from patients undergoing screening for PCa by transrectal ultrasound guided biopsy, collected at IOM (Instituto de Oncologia e Mastologia de Macapá, Amapá, Brazil) from December 2018 to January 2021. One fragment was placed in a microcentrifuge tube with 1mL of RNAlater. After collection, the vials were stored at -20C until being processed. Total DNA extraction was performed in the Microbiology and Immunology Laboratory of the Federal University of Amapá (UNIFAP). The Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) method was used for HPV detection. The analysis to investigate the relationship between the presence of HPV and cancer was the extension of Fisher's exact test and the alpha significance level equal to 5% was applied. RESULTS: 157 patients, 95 (60.5%) with PCa, 55 (35.0%) without cancer, and 7 (4.5%) as inconclusive (ASAP). The presence of HPV was 7/95 =(7.4%) in patients with cancer, and 2/55 =(3.6%) in those without evidence of cancer or inconclusive diagnosis ASAP. The inferential results revealed that the presence of HPV was not related to the diagnosis of cancer when the ASAP diagnosis was considered in the analysis (p=0.663) and, also when the ASAP diagnosis was not included (p=0.487). CONCLUSIONS: There was no L1 gene expression and significant levels of HPV L1 in prostate tissue. These findings suggest no oncogenic activity of HPV in PCa. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e192 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nalisson Pereira More articles by this author Emerson Martins More articles by this author Mateus Quintela More articles by this author Arthur Cunha More articles by this author Jaques Waisberg More articles by this author Expand All Advertisement PDF downloadLoading ...

Multigene typing of Giardia Duodenalis isolated from tuberculosis and non-tuberculosis subjects.

Giardia duodenalis is a cryptic protozoan, which has eight assemblages (A-H). Assemblages A and B are the main genotypes reported from humans with probable anthroponotic and zoonotic transmission. The current study aimed to characterize G. duodenalis assemblages in tuberculosis (TB) patients and healthy subjects using multilocus genotyping (MLG). Thirty Giardia-positive stool samples, which were obtained from TB patients and healthy subjects were included in the study. After total DNA extraction, three β-giardin (bg), triosephosphate isomerase (tpi), glutamate dehydrogenase (gdh) genes were amplified and sequenced. Obtained sequences were compared to the GenBank database to characterize assemblages. Phylogenetic analysis using Maximum Likelihood (ML) and Tamura 3-parameter was performed for each gene. From 30 Giardia-positive subjects, 17 (57%) and 13 (43%) were from healthy and TB-infected subjects, respectively. There was no significant co-existence of Giardia and tuberculosis (P-value = 0.051). In addition, 14 (46.7%) and 16 (53.3%) of Giardia isolates were from asymptomatic and symptomatic subjects, respectively. PCR amplification was successful in 25 single samples (83.3%) consisted of 20 for tpi, 15 for bg, and 13 for gdh genes. Accordingly, 13/25 (52%) and 8/25 (32%) belonged to assemblage A and assemblages B, respectively, whereas 4/25 (16%) were either assemblage A or B with different genes at the same time. Significant correlation between assemblages and TB, age, and symptoms was not seen. The phylogenetic analyses represented no separation based on TB and gastrointestinal symptoms. Assemblage A was the predominant genotype in samples. The high frequency of assemblage AII indicated importance of anthroponotic transmission of Giardia in both healthy and TB patients. In addition, considering the exclusive reports of sub-assemblage AIII in wild ruminants, the presence of AIII in the current study have to be carefully interpreted. The inconsistency between the assemblage results of either bg or gdh loci with tpi gene signifies the insufficiency of single gene analysis and the necessity for MLG in molecular epidemiology of G. duodenalis.

Polymorphism of mitochondrial DNA genes involved in asthenozoospermia in infertile patients of Côte d’Ivoire

Objective: The relationship between mitochondrial DNA (mtDNA) polymorphisms and abnormalities in sperm quality has been the subject of several studies, with the objective of improving the treatment of male infertility. This study, which contributes to the identification of genetic markers of sperm abnormalities, was conducted to study mtDNA mutations in the asthenozoospermia profile. Methods: This case-control study included 30 patients with asthenozoospermia and 28 with normospermia after spermogram and spermocytogram analyses. After the extraction of total DNA from the spermatozoa of 58 ejaculates from these individuals using the phenol-chloroform method, the amplification of genes of interest in mtDNA using specific primers was performed by conventional polymerase chain reaction, and sequencing was used to detect mutations. Results: Male patients with asthenozoospermia in the tertiary sector had significantly more mutant- than wild-type (P = 0.0005) MT-CO II genes. Similarly, for the same gene, males with asthenozoospermia and primary infertility had significantly more mutants than the wild-type (P = 0.001). Sequencing revealed 29 mutations that were observed only with asthenozoospermia, which could be the basis for low sperm mobility. Conclusion: This study identified several mutations in mtDNA genes that could be considered genetic markers of asthenozoospermia if confirmed in a deeper study.

THE ABILITY OF matK AND trnL-trnL-trnF INTERGENIC SPACER TO DISCERN CERTAIN SPECIES ACCESSIONS OF THE FAMILIES SOLANACEAE AND FABACEAE

Indonesia, as a mega-biodiversity country, has many kinds of tropical fruits. Solanaceae and Fabaceae families contain plant species that produce fruits and vegetables for human food, such as ‘rimbang’ (Solanum torvum), ‘terung asam’ (S. ferox), ‘kabau’ (Archidendron microcarpum), ‘jengkol’ (A. pauciflorum), and ‘petai’ (Parkia speciosa). This study aimed to determine the ability of DNA barcodes (matK and trnL-trnL-trnF intergenic spacer [IGS]) in distinguishing some of those tropical fruit plants. Plant materials came from Riau Province, Indonesia. Methods developed include total DNA extraction from fresh leaves and PCR using the primer pairs to amplify matK and trnL-trnL-trnF IGS. The PCR products underwent sequencing, with the DNA sequences analyzed using MEGA6. In this study, the obtained matK and trnL-trnL-trnF IGS sequences showed lengths ranging from 800 bp to 892 bp and 872 bp to 1113 bp, respectively. The matK and trnL-trnL-trnF IGS sequences of S. ferox, A. microcarpum, and A. pauciflorum provided the first sequences reported in those species. Several nucleotide variations in matK sequences emerged among the studied accessions caused by substitution mutation, including transversion and transition. No indel (insertion-deletion) mutation in the studied matK sequences occurred. Indel can also cause variations in trnL-trnL-trnF IGS sequences among the studied accessions aside from substitution mutation. For species S. torvum, S. ferox, and P. speciosa, there came about some decisive nucleotides based on the matK sequences. Moreover, for species S. torvum, S. ferox, A. microcarpum, A. pauciflorum, and P. speciose, there occurred few critical nucleotides based on trnL-trnL-trnF IGS. Also, there was a critical nucleotide for large-fruited ‘jengkol’ (one of the populations in A. pauciflorum) based on trnL-trnL-trnF IGS. However, both DNA barcodes could not differentiate the two types of S. ferox, i.e., ‘terung asam’ and ‘terung bulu.’ Both DNA barcodes showed to differentiate some species members of both families Solanaceae and Fabaceae into separate clusters. Moreover, compared with matK, the trnL-trnL-trnF IGS indicated uniqueness in discriminating populations within species.

Genetic Variants of ISL1 Gene Promoter Identified from Congenital Tetralogy of Fallot Patients Alter Cellular Function Forming Disease Basis.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease in newborns. ISL1 is a master transcription factor in second heart field development, whereas the roles of ISL1 gene promoter variants in TOF patients have not been genetically investigated. Total DNA extraction from 601 human subjects, including 308 TOF patients and 293 healthy controls, and Sanger sequencing were performed. Four variants (including one novel heterozygous variant) within the ISL1 gene promoter were only found in TOF patients. Functional analysis of DNA sequence variants was performed by using the dual-luciferase reporter assay and demonstrated that three of the four variants significantly decreased the transcriptional activity of ISL1 gene promoter in HL-1 cells (p < 0.05). Further, the online JASPAR database and electrophoretic mobility shift assay showed that the three variants affected the binding of transcription factors and altered ISL1 expression levels. In conclusion, the current study for the first time demonstrated that the variants identified from the ISL1 gene promoter region are likely involved in the development of TOF by affecting the transcriptional activity and altering the ISL1 expression level. Therefore, these findings may provide new insights into the molecular etiology and potential therapeutic strategy of TOF.

Quantification of the hepatitis B virus cccDNA: evidence-based guidelines for monitoring the key obstacle of HBV cure

ObjectivesA major goal of curative hepatitis B virus (HBV) treatments is the reduction or inactivation of intrahepatic viral covalently closed circular DNA (cccDNA). Hence, precise cccDNA quantification is essential in...

Influence of DNA from non-viable sources on the riverine water and biofilm microbiome, resistome, mobilome, and resistance gene host assignments

Shotgun metagenomic studies have revealed the diversity, relative abundance, and hosts of antibiotic resistance genes (ARGs) across environmental matrices. There is motivation to combine this method with viability-based techniques to better define ARG hazard. The objectives of this study were to evaluate the performance of different methods for extracellular DNA (eDNA) and putative “non-viable” cell DNA separation to understand the influence on ARG-host assignments. Paired water and biofilm samples were collected along a land use gradient. To study putative “viable-cell” DNA, samples were treated with propidium monoazide (i.e., PMA-DNA). To study eDNA, intracellular and extracellular DNA were separated. qPCR revealed differences in total 16S rRNA gene copies in water for filter vs. centrifuge-concentrated samples, but otherwise there were no differences in gene copy concentrations between DNA fractions. Next, metagenomic sequencing was performed on PMA-DNA and total DNA extracts revealing significant differences between the two for bacterial community structure and ARG profiles. Putative viable taxa containing pathogenic ARG hosts were identified in biofilm and water. Removing PMA-bound DNA improved N50 and assembly mapping compared to total DNA extracts. This study demonstrates the impact of different sample preparation methods on informing the hazard potential associated with riverine ARGs in water and biofilm.

The impact of Ceftriaxone on Relative Copy Numbers of Mitochondrial and Chloroplast DNA in Grapevine Leaves

The cultivation of micropropagated grapevine plants in vitro is currently undergoing improvements in existing methods and the development of new cultivation techniques due to the problem of bacterial contamination caused by multidrug-resistant microorganisms. Explants (Vitis vinifera L., ‘Chardonnay’ variety) were cultured on Murashige-Skoog basal medium supplemented with different concentrations of ceftriaxone: 0 mg/L, 250 mg/L, and 1000 mg/L. After 30 days, morphometric characteristics of the micropropagated plants and relative copy numbers of mitochondrial and chloroplast DNA were evaluated. Leaf samples (5-10 mg) were randomly selected from each plant group for subsequent total DNA extraction. Quantitative RT-PCR was performed using LightCycler 480 SYBR Green I Master Mix (LifeScience, Roche) and analyzed with a LightCycler 96 automated analyzer (Roche Life Science). The relative copy numbers of NAD1 (mitochondrial DNA) and rps16 (chloroplast DNA) genes were determined using the GAPDH gene (chromosomal DNA) as the reference. The 2-DCt and 2-DDCt algorithms were used for quantitative assessment. Ceftriaxone at concentrations of 250 and 1000 mg/l reduces the relative number of copies of chloroplast and mitochondrial DNA, which indicates the suppression of photosynthesis and oxidative phosphorylation in grape microplants. The experimental scheme developed by us can be successfully used as a test system for assessing the degree of influence of various biogenic and abiogenic factors on plant objects in order to optimize their cultivation.

Skim-Nanopore sequencing for routine genomic evaluation and bacterial pathogen detection in cattle

Context Genotyping-by-sequencing, the use of sequence reads to genotype single-nucleotide polymorphisms (SNPs), has seen an increase in popularity as a tool for genomic prediction. Oxford Nanopore Technologies (Nanopore) sequencing is an emerging technology that produces long sequence reads in real-time. Recent studies have established the ability for low-coverage Nanopore sequence data to be used for genomic prediction. However, the value proposition of Nanopore sequencing for individuals could be improved if both genotyping and disease diagnosis are achieved from a single sample. Aims This study aimed to demonstrate that Nanopore sequencing can be used for both rapid genotyping and as a disease diagnostic tool using the same sample in livestock. Methods Total DNA extracts from nasal swabs collected from 48 feedlot cattle presenting with clinical signs of bovine respiratory disease (BRD) were sequenced using the Nanopore PromethION sequencer. After 24 h of sequencing, genotypes were imputed and genomic estimated breeding values (GEBVs) for four traits were derived using 641 163 SNPs and corresponding SNP effects. These GEBVs were compared with GEBVs derived from SNP array genotypes and calculated using the same SNP effects. Unmapped sequence reads were classified into taxa using Kraken2 and compared with quantitative real-time polymerase chain reaction (qPCR) results for five BRD-associated pathogens of interest. Key results Sequence-derived genotypes for 46 of the 48 animals were produced in 24 h and GEBV correlations ranged between 0.92 and 0.94 for the four traits. Eleven different BRD-associated pathogens (two viruses and nine bacterial species) were detected in the samples using Nanopore sequence data. A significant (P &lt; 0.001) relationship between Nanopore and qPCR results was observed for five overlapping species when a maximum threshold cycle was used. Conclusions The results of this study indicated that 46 cattle genomes can be multiplexed and accurately genotyped for downstream genomic prediction by using a single PromethION flow cell (ver. R9.4) in 24 h. This equates to a cost of AUD35.82 per sample for consumables. The concordance between qPCR results and pathogen proportion estimates also indicated that some pathogenic species, in particular bacterial species, can be accurately identified from the same test. Implications Using Nanopore sequencing, routine genotyping and disease detection in livestock could be combined into one cost-competitive test with a rapid turnaround time.

The influence of storage conditions and total DNA extraction protocol on the results of molecular analysis of the European spruce bark beetle (Ips typographus L.)

One of the key steps of the molecular identification of bark beetles is obtaining a sufficient quantity of high-quality DNA extract. In this study, we investigated the influence of different storage procedures for Ips typographus (L.) specimens and various DNA extraction protocols on the quantity and quality of DNA intended for use in molecular diagnostics. Adult beetles were frozen at -20 °C, either dry or in ethanol. We tested four different protocols for DNA extraction. We compared the quantity of extracted DNA and assessed its quality with PCR and Sanger sequencing. Different storage protocols had no significant effect on the quantity of DNA extracted. However, freezing specimens in ethanol provided higher-quality DNA for molecular applications. Only two of the extraction protocols produced sequenceable amplicons, and the difference in the amount of extracted DNA between them was not significant. We propose the optimal combination of storing specimens in ethanol at -20°C and using the Nucleospin Insect DNA extraction kit from Macherey Nagel, enabling a timeefficient identification process.

The relative copy number of mitochondrial and chloroplast DNA in young and mature leaves of different grape varieties

The copy number of DNA matrices of subcellular organelles (plants) can serve as an indicator of the intensity of photosynthesis and oxidative phosphorylation processes. We assessed the relative copy numbers (RCN) of mitochondrial and chloroplast DNA in young and mature leaves of three grape varieties: ‘Traminer Pink’, ‘Chardonnay’, and ‘Syrah’, grown under field conditions. Leaf samples (5–10 mg) were randomly selected from each group of plants for subsequent total DNA extraction. The qRTPCR reaction was performed using LightCycler 480 SYBR Green I Master Mix (LifeScience, Roche) and a LightCycler 96 Automatic Analyzer (Roche Life Science). The relative copy numbers of the NAD1 gene (mitochondrial DNA) and rps16 gene (chloroplast DNA) were determined using the GAPDH gene (chromosomal DNA) as a reference. Quantitative assessment was conducted using the 2--Ct и 2---Ct algorithms. It has been established that the relative copy number (RCN) values of chloroplast and mitochondrial DNA vary and depend on the grape variety and leaf maturity. RCN of chloroplast and mitochondrial DNA is significantly higher in mature grape leaves of all studied varieties, indicating a higher intensity of photosynthesis and oxidative phosphorylation in mature grape leaves compared to young leaves. When assessing the MacroErgic Balance (MEB) indicator, it can be concluded that from 2 to 4% of the energy obtained in chloroplasts through photosynthetic processes is used for the production of macroergic compounds in the mitochondria of various grape varieties in both young and mature leaves. The experimental scheme we have developed can be successfully used as a testing system to assess the potential yield of various grape varieties.

Conventional tobacco products harbor unique and heterogenous microbiomes

While an increasing number of studies have evaluated tobacco microbiomes, comparative microbiome analyses across diverse tobacco products are non-existent. Moreover, to our knowledge, no previous studies have characterized the metabolically-active (live) fraction of tobacco bacterial communities and compared them across products. To address these knowledge gaps, we compared bacterial communities across four commercial products (cigarettes, little cigars, cigarillos and hookah) and one research cigarette product. After total DNA extraction (n = 414) from all samples, the V3V4 region of the 16S rRNA gene was sequenced on the Illumina HiSeq platform. To identify metabolically-active bacterial communities within these products, we applied a coupled 5-bromo-2′-deoxyuridine labeling and sequencing approach to a subset of samples (n = 56). Each tobacco product was characterized by its signature microbiome, along with a shared microbiome across all tobacco products consisting of Pseudomonas aeruginosa, P. putida, P. alcaligenes, Bacillus subtilis, and Klebsiella pneumoniae. Comparing across products (using Linear discriminant analysis Effect Size (LEfSe)), a significantly higher (p < 0.05) relative abundance of Klebsiella and Acinetobacter was observed in commercial cigarettes, while a higher relative abundance of Pseudomonas and Pantoea was observed in research cigarettes. Methylorubrum and Paenibacillus were higher in hookah, and Brevibacillus, Lactobacillus, Bacillus, Lysinibacillus, and Staphylococcus were higher in little cigars and cigarillos. Across all products, the majority of the metabolically-active bacterial communities belonged to the genus Pseudomonas, followed by several genera within the Firmicutes phylum (Bacillus, Terribacillus, and Oceanobacillus). Identification of some metabolically-active pathogens such as Bacillus cereus and Haemophilus parainfluenzae in commercial products is of concern because of the potential for these microorganisms to be transferred to users' respiratory tracts via mainstream smoke. Future work is warranted to evaluate the potential impact of these tobacco bacterial communities on users' oral and lung microbiomes, which play such an important role on the spectrum from health to disease.

Identification of Leishmania species and frequency distribution of LRV1 and LRV2 viruses on cutaneous leishmaniasis patients in Isfahan Province, Iran

PurposeLeishmaniasis is one of the most serious health problems in developing countries. Iran is one of the endemic regions of cutaneous leishmaniasis. Leishmania RNA virus (LRV) is a dsRNA virus member of the Totiviridae family, which was first detected in the promastigotes of Leishmania braziliensis guyanensis. Our study aimed to investigate possible changes in the predominant and causative strains of CL and screening the LRV1 and LRV2 species genome from Leishmania species isolated from the lesions of patients. Materials and methodsDirect smear samples obtained from 62 patients with leishmaniasis referring to the Skin Diseases and Leishmaniasis Research Center in Isfahan province during 2021–2022 were examined. Total DNA extraction procedures and conservation of site-specific multiplex PCR and nested PCR were performed for detecting Leishmania species. The molecular identification of LRV1 and LRV2 viruses, samples were used for total RNA extraction and real-time (RT)-PCR analysis, followed by conducting a restriction enzyme assay to confirm the PCR products. ResultsOf the total Leishmania isolates, 54 and 8 isolates were identified as L. major and L. tropica, respectively. LRV2 was identified in 18 samples affected by L. major, while LRV1 was only detected in one of the samples with L. tropica. No LRV2 was found in any samples with L. tropica. The results showed that there was a significant relationship between LRV1 and the type of leishmaniasis (Sig. ​= ​0.009, P ​≤ ​0.05), while this relationship was not observed between LRV2 and the type of leishmaniasis. ConclusionsThe presence of a significant number of LRV2 in isolated samples, as well as the recognition of LRV1 in one of the Old World leishmaniasis species, which is a new result, could pave the way for investigating further aspects of this disease and successful treatment strategies in future studies.

Effect of feeding raw potato starch on the composition dynamics of the piglet intestinal microbiome.

Raw potato starch (RPS) is resistant to digestion, escapes absorption, and is metabolized by intestinal microflora in the large intestine and acts as their energy source. In this study, we compared the effect of different concentrations of RPS on the intestinal bacterial community of weaned piglets. Male weaned piglets (25-days-old, 7.03±0.49 kg) were either fed a corn/soybean-based control diet (CON, n = 6) or two treatment diets supplemented with 5% RPS (RPS5, n = 4) or 10% RPS (RPS10, n = 4) for 20 days and their fecal samples were collected. The day 0 and 20 samples were analyzed using a 16S rRNA gene sequencing technology, followed by total genomic DNA extraction, library construction, and high-throughput sequencing. After statistical analysis, five phyla and 45 genera accounting for over 0.5% of the reads in any of the three groups were further analyzed. Furthermore, short-chain fatty acids (SCFAs) in the day 20 fecal samples were analyzed using gas chromatography. Significant changes were not observed in the bacterial composition at the phylum level even after 20 d post feeding (dpf); however, the abundance of Intestinimonas and Barnesiella decreased in both RPS treatment groups compared to the CON group. Consumption of 5% RPS increased the abundance of Roseburia (p<0.05) and decreased the abundance of Clostridium (p<0.01) and Mediterraneibacter (p< 0.05). In contrast, consumption of 10% RPS increased the abundance of Olsenella (p<0.05) and decreased the abundance of Campylobacter (p<0.05), Kineothrix (p<0.05), Paraprevotella (p<0.05), and Vallitalea (p<0.05). Additionally, acetate (p<0.01), butyrate (p<0.05), valerate (p = 0.01), and total SCFAs (p = 0.01) were upregulated in the RPS5 treatment group. Feeding 5% RPS altered bacterial community composition and promoted gut health in weaned piglets. Thus, resistant starch as a feed additive may prevent diarrhea in piglets during weaning.

Extraction of Total DNA and RNA from Marine Filter Samples and Generation of a Universal cDNA as Universal Template for Marker Gene Studies.

Microbial communities play an important role in marine ecosystem processes. Although the number of studies targeting marker genes such as the 16S rRNA gene has increased during the last years, the vast majority of marine diversity are rather unexplored. Moreover, most studies focused on the entire microbial community and thus do not assess the active fraction of the microbial community. Here, we describe a detailed protocol for the simultaneous extraction of DNA and RNA from marine water samples and the generation of cDNA from the isolated RNA that can be used as a universal template in various marker gene studies.

Three Distinct Annotation Platforms Differ in Detection of Antimicrobial Resistance Genes in Long-Read, Short-Read, and Hybrid Sequences Derived from Total Genomic DNA or from Purified Plasmid DNA.

Recent advances and lower costs in rapid high-throughput sequencing have engendered hope that whole genome sequencing (WGS) might afford complete resistome characterization in bacterial isolates. WGS is particularly useful for the clinical characterization of fastidious and slow-growing bacteria. Despite its potential, several challenges should be addressed before adopting WGS to detect antimicrobial resistance (AMR) genes in the clinical laboratory. Here, with three distinct ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), different approaches were compared to identify best practices for detecting AMR genes, including: total genomic DNA and plasmid DNA extractions, the solo assembly of Illumina short-reads and of Oxford Nanopore Technologies (ONT) long-reads, two hybrid assembly pipelines, and three in silico AMR databases. We also determined the susceptibility of each strain to 21 antimicrobials. We found that all AMR genes detected in pure plasmid DNA were also detectable in total genomic DNA, indicating that, at least in these three enterobacterial genera, the purification of plasmid DNA was not necessary to detect plasmid-borne AMR genes. Illumina short-reads used with ONT long-reads in either hybrid or polished assemblies of total genomic DNA enhanced the sensitivity and accuracy of AMR gene detection. Phenotypic susceptibility closely corresponded with genotypes identified by sequencing; however, the three AMR databases differed significantly in distinguishing mobile dedicated AMR genes from non-mobile chromosomal housekeeping genes in which rare spontaneous resistance mutations might occur. This study indicates that each method employed in a WGS workflow has an impact on the detection of AMR genes. A combination of short- and long-reads, followed by at least three different AMR databases, should be used for the consistent detection of such genes. Further, an additional step for plasmid DNA purification and sequencing may not be necessary. This study reveals the need for standardized biochemical and informatic procedures and database resources for consistent, reliable AMR genotyping to take full advantage of WGS in order to expedite patient treatment and track AMR genes within the hospital and community.

Deep kinetoplast genome analyses result in a novel molecular assay for detecting Trypanosoma brucei gambiense-specific minicircles.

The World Health Organization targeted Trypanosoma brucei gambiense (Tbg) human African trypanosomiasis for elimination of transmission by 2030. Sensitive molecular markers that specifically detect Tbg type 1 (Tbg1) parasites will be important tools to assist in reaching this goal. We aim at improving molecular diagnosis of Tbg1 infections by targeting the abundant mitochondrial minicircles within the kinetoplast of these parasites. Using Next-Generation Sequencing of total cellular DNA extracts, we assembled and annotated the kinetoplast genome and investigated minicircle sequence diversity in 38 animal- and human-infective trypanosome strains. Computational analyses recognized a total of 241 Minicircle Sequence Classes as Tbg1-specific, of which three were shared by the 18 studied Tbg1 strains. We developed a minicircle-based assay that is applicable on animals and as specific as the TgsGP-based assay, the current golden standard for molecular detection of Tbg1. The median copy number of the targeted minicircle was equal to eight, suggesting our minicircle-based assay may be used for the sensitive detection of Tbg1 parasites. Annotation of the targeted minicircle sequence indicated that it encodes genes essential for the survival of the parasite and will thus likely be preserved in natural Tbg1 populations, the latter ensuring the reliability of our novel diagnostic assay.