Detecting Bacterial Species from Ancient Human Skeletal Samples

This paleopathological study aims to identify Mycobacterium tuberculosis complex (MTBC), Mycobacterium avium complex (MAC) and other Mycobacterium species in silico from skeletal samples that belonged to 28 Polish individuals in the Neolithic period under PRJNA422903 from the Sequence Read Archive (SRA). After next-generation sequencing (NGS), bioinformatics methods are heavily relied upon for identification of pathogens from complex samples. We implemented a bioinformatics pipeline, with custom-built databases, utilizing the following software tools: Trim Galore! and Kraken2. After adapter trimming, Kraken2 was used for taxonomic classifications. We have found that Mycobacterium is present in all 28 individuals. The average percentage of MAC present in the genus Mycobacterium, in all 28 individuals, is 6%. Reads from MTBC makes up an average of 7% of the Mycobacterium genus. We have identified previously unreported strains of MTBC and MAC such as Mycobacterium tuberculosis XDR1219, which is an extensively drug-resistance strain. Our analysis also revealed 14.8% of reads from MTBC belong to Mycobacterium avium hominissuis, which was commonly found in humans and pigs. Additionally, strains of Mycobacterium simiae complex were also discovered. Mycobacterium simiae has been commonly found among immunocompromised individuals. In conclusion, our bioinformatics pipeline has been more effective than other published approaches. This approach broadens the potential scope of paleoepidemiology both to older, sub-optimally preserved samples and to pathogens with difficult intrageneric taxonomy. It is therefore suitable for other studies in paleopathology using NGS technologies

Detecting bacterial species from ancient human skeletal samples

Diagnosis of tuberculosis (TB) via morphological analysis is difficult and often inconsistent. With next-generation sequencing (NGS), ancient host microbiomes can be subjected to metagenomic analyses for the detection of TB in silico. Suitable bioinformatic workflows are needed for reliable ancient DNA (aDNA) analysis of causative agents. This study aims to enhance available bioinformatic screening methods to create more suitable bioinformatic processes and generate insights in relation to TB. This research utilizes publicly available NGS data accessed through the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI). Initial quality control steps included adapter trimming with Trim Galore!. Kraken2 was then used for taxonomic classification with a custom-built database comprised of Mycobacterial genomes from the NCBI. Quantitation and visualization were carried out with Bracken and Krona, respectively. Our workflow was first applied to 28 Neolithic skeletons (SRA number PRJNA422903) representing the Middle Neolithic Brześć Kujawski Group of the Lengyel culture (∼4400–4000 BC, 26 individuals), and the Late Neolithic Globular Amphora culture (∼3100–2900 BC, 2 individuals). Three additional datasets have since been utilized in this research: mummified remains of 265 individuals from Hungary (1731–1838 CE; PRJNA795622), one calcified lung nodule from Lund, Sweden (17th century; PRJNA517266); and dental calculus of four individuals from the Iberian Peninsula (4500-5000 BP; PRJEB46022). Preliminary results for the 28 Neolithic skeletons revealed an average of 7% of the Mycobacterium genus sequencing reads mapping to Mycobacterium tuberculosis complex (MTBC) among all individuals. This work also revealed additional species of MTBC and Mycobacterium avium complex (MAC) that were previously unreported by the originator of datasets, including the extensively drug-resistant (XDR) Mycobacterium tuberculosis XDR1219 and Mycobacterium avium hominissuis. Our bioinformatic workflow has therefore been more effective than previously published approaches and is suitable for future paleopathological studies

Human papillomavirus in amniotic fluid

BACKGROUND: There is evidence to suggest that human papillomavirus (HPV) can cross the placenta resulting in in-utero transmission. The goal of this study was to determine if HPV can be detected in amniotic fluid from women with intact amniotic membranes. METHODS: Residual amniotic fluid and cultured cell pellets from amniocentesis performed for prenatal diagnosis were used. PGMY09/11 L1 consensus primers and GP5+/GP6+ primers were used in a nested polymerase chain reaction assay for HPV. RESULTS: There were 146 paired samples from 142 women representing 139 singleton pregnancies, 2 twin pregnancies, and 1 triplet pregnancy. The women were 78% Caucasian, 5% African American, 14% Asian, and 2% Hispanic. The average age was 35.2 years with a range of 23–55 years. All samples were β-globin positive. HPV was not detected in any of the paired samples. CONCLUSION: Given the age range, race, and ethnicity of the study population, one would anticipate some evidence of HPV if it could easily cross the placenta, but there was none

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

Phylogeny of the Resistance-Nodulation-Cell Division (RND) Superfamily

Our contemporary, comprehensive phylogenetic study spans all domains to understand the Resistance-Nodulation-Cell Division (RND) superfamily’s evolution and points of divergence. Several members of the superfamily are involved in both the acquisition and intrinsic resistance to antibiotics despite origins that are ubiquitous and ancient compared to modern-day antibiotic use (Nikaido 2018). Exhaustive searches of selected representative sequences through BLAST (Mistry et al. 2013) and HMMSearch (Camacho et al. 2009) created the most comprehensive and up-to-date dataset to establish all possible RND homologs. CD-HIT, a clustering software, was used to refine our dataset (Pearson 2013). Multiple sequence alignments like CLUSTAL (Sievers et al. 2011) and MUSCLE (Edgar 2004) were utilized. Each alignment had differing computational methods used by these programs to offer significant information about the structure and evolution of RNDs that gave collective insight. Phylogenetic trees produced from PhyML, a program based on maximum-likelihood algorithm, were our final products to characterize novel members and sub-families of the RND superfamily (Letunic & Bork, 2021). Evolutionary pathways were elucidated through highly likely clustering and branch lengths. Overall, the study provides a significant update to our understanding of the RND superfamily giving rise to previously uncharacterized members and families

Metagenome Assembled Genome of a Novel Phage from the Nunu Microbiome

The application of next-generation sequencing (NGS) has provided new opportunities in expanding our understanding towards microbiomes in homemade fermented foods. This study explores microbiomes from a homemade dairy product called nunu, which is a West African fermented dairy beverage sold in open-air markets without a standardized starter culture or pasteurization. Raw reads with NCBI accession number of PRJEB20873 were first subjected to quality control and adapter trimming with cutadapt before assembly with MetaSPAdes to generate metagenome assembled genomes (MAGs). One MAG consisted of 43,090 bp with a G + C content of 54.82% and 56 predicted open-reading frames (ORFs). Structural and functional annotations revealed shared synteny with phage vB_EcoM_ECOO78 that has been classified as a Jilinvirus. As members of the Myoviridaefamily, Jilinviruses are known to infect the genera of Escherichia and Enterobacter. Functional annotation with BLAST searches revealed eight ORFs that shared no sequence similarities with genes from other Jilinviruses. Seven ORFs have been annotated to function in nucleotide metabolism and replication. Twelve ORFs have been predicted for functions in morphogenesis, including several tail and capsid proteins. Putative small and large terminase subunits were assigned for ORF55 and ORF56. While other Jilinviruses lack integrases, ORF26 was predicted to function as an integrase with sequence similarity of 85.0% when compared against Pseudomonas phage PPpW-3. This putative integrase from our current study also shares a sequence similarity of 88% with a tyrosine-type recombinase/integrase from E. coli. ORF42 shares sequence similarity at N-terminus with Dpo42 from phage vB_EcoM_ECOO78, that prevents E. colibiofilm formation. In conclusion, this novel phage appears to represent the fifth member of the Jilinvirus and further our understanding towards the nunu microbiome

Detecting Bacterial Species from Next Generation Sequencing Data Derived from Ancient Human Skeletal Samples

This study aims to isolate and identify bacteria and single nucleotide polymorphisms (SNPs) found alongside Mycobacterium tuberculosis complex (MTCB) in silico. MTCB is a causative agent of tuberculosis (TB). Our secondary objective is to examine variations of TB, study its paleoepidemiology, and apply this information to present-day public health issues. This research utilized data from the Sequence Read Archive (SRA), number PRJNA422903. This dataset is comprised of DNA obtained from the remains of 28 individuals belonging to Neolithic-period populations. This DNA is being studied in silico through next generation sequencing (NGS), utilizing the following bioinformatics software tools with customized setting: Trim Galore! and Kraken2. We plan to implement a bioinformatics pipeline to pass further fastQ files through trimming with deprivation of the adapter sequence, and Kraken2 will act as a filter allowing us to find unknown pieces of DNA sequences. Using NGS, we will be able to isolate and identify bacteria found alongside MTCB in silico to study the paleoepidemiology of TB prior to the usage of antibiotics. Due to advancements in technology, we will be able to identify more bacterial species and SNPs alongside MTBC than previous scholars in the field of paleoepidemiology. In conclusion, this approach broadens the potential scope of paleoepidemiology both to older, sub optimally preserved samples and to pathogens with difficult intrageneric taxonomy. These approaches could also be utilized in future disease diagnosis and control

Exploring the Microbiome of Korean Industrial Kimchi Fermentation Products

Despite existing over millenniums, Kimchi, a historic side dish of Korean culture, has a cultivation process that remains poorly defined and difficult to control along industrial production lines. Traditionally, Kimchi is made through the process of fermentation, a chemical mechanism in which microorganisms convert sugars to alcohol or an acid. The variation in taste existing across different batches of identically prepared kimchi products supports the idea that the constituents of the kimchi microbiome are generally unknown. The primary objective of this research study is to identify novel phages from fermented microbiomes, specifically within the kimchi microbiome. Furthermore, novel phages could have the capacity to serve as biocontrol agents. The secondary objective of this research study is to identify all microorganisms on a species level to allow the public to fully appreciate the diversity of the kimchi microbiome. Next-generation sequencing (NGS), a tool used to sequence the genetic material of organisms, will be used to identify the kimchi microbiome and continue downstream bioinformatics. Our preliminary analysis on an NGS data set from Korean industrial kimchi products with NCBI accession number of SRX2725663 will be used extensively throughout our research. We utilized six software packages with customized settings in order to analyze the datasets: Kraken2, Trimmonmatic, Velvet, Edena, BLAST, and Seaview. Kraken2 was used to distinguish novel sequences from previously characterized phages and bacterial hosts. After the identification of potential uncharacterized phages, researchers on the project may seek to develop the complete genomes of such phages. Additionally, researchers could use the generated databases from their bioinformatics pipeline to serve as a universally adopted protocol in the detection of novel phages. The utilization of this detection apparatus could reduce the pathogenic bacteria in food microbiomes

Nunu Dairy Microbiome Phage DM-1

With the advent of next-generation sequencing (NGS) on metagenomes, the elucidation of all genetic material from microbiomes has prompted a renewed interest towards uncultivated members of the virosphere. We describe the discovery of a novel phage from a metagenomic dataset on the West African fermented dairy product, nunu, with a custom bioinformatics workflow to potentially serve as a biocontrol agent against pathogenic E. coli. Initial dataset of ERR2014814 from NCBI was first subjected to Kraken2 to extract novel sequencing reads for further de novo assembly into contigs.</p