scholarly journals Managing Contamination and Diverse Bacterial Loads in 16S rRNA Deep Sequencing of Clinical Samples: Implications of the Law of Small Numbers

mBio ◽  
2021 ◽  
Author(s):  
Ruben Dyrhovden ◽  
Martin Rippin ◽  
Kjell Kåre Øvrebø ◽  
Randi M. Nygaard ◽  
Elling Ulvestad ◽  
...  
Keyword(s):  
Infectious Disease ◽  
16S Rrna ◽  
Deep Sequencing ◽  
Gradual Increase ◽  
Clinical Samples ◽  
Materials Management ◽  
Bacterial Dna ◽  
Dna Contamination ◽  
Sequencing Studies ◽  
The Law

There has been a gradual increase in 16S deep sequencing studies on infectious disease materials. Management of bacterial DNA contamination is a major challenge in such diagnostics, particularly in low biomass samples.

Sterile paper points as a bacterial DNA-contamination source in microbiome profiles of clinical samples

2013 ◽  
Vol 41 (12) ◽  
pp. 1297-1301 ◽  
Author(s):  
Joyce van der Horst ◽  
Mark J. Buijs ◽  
Marja L. Laine ◽  
Daniël Wismeijer ◽  
Bruno G. Loos ◽  
...  
Keyword(s):  
Clinical Samples ◽  
Contamination Source ◽  
Bacterial Dna ◽  
Dna Contamination

scholarly journals Aerococcus suis sp. nov., isolated from clinical specimens from swine

2007 ◽  
Vol 57 (6) ◽  
pp. 1291-1294 ◽  
Author(s):  
A. I. Vela ◽  
N. García ◽  
M. V. Latre ◽  
A. Casamayor ◽  
C. Sánchez-Porro ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Molecular Genetic ◽  
16S Rrna Gene Sequencing ◽  
Clinical Samples ◽  
Rrna Gene ◽  
Biochemical Tests ◽  
Sequencing Studies ◽  
Micro Organisms

Biochemical and molecular genetic studies were performed for five isolates of unknown Gram-positive, catalase-negative, cocci-shaped micro-organisms obtained from clinical samples from pigs. The micro-organisms were tentatively identified as Aerococcus species on the basis of the results from cellular morphological and biochemical tests. 16S rRNA gene sequencing studies confirmed the provisional identification of the isolates as members of the genus Aerococcus, but the micro-organism did not correspond to any recognized species of this genus. The nearest phylogenetic relatives of these unknown cocci isolated from pigs were Aerococcus viridans (95.9 % 16S rRNA gene sequence similarity) and Aerococcus urinaeequi (95.8 %). The unknown bacterium, however, was distinguishable from these two species and from other animal aerococci by using biochemical tests. On the basis of both phenotypic and phylogenetic findings, the isolates represent a novel species of the genus Aerococcus, for which the name Aerococcus suis sp. nov. is proposed. The type strain is 1821/02T (=CECT 7139T=CCUG 52530T).

scholarly journals 16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles

Microbiome ◽  
2014 ◽  
Vol 2 (1) ◽  
pp. 31 ◽  
Author(s):  
Jun Hang ◽  
Valmik Desai ◽  
Nela Zavaljevski ◽  
Yu Yang ◽  
Xiaoxu Lin ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Temperature Stability ◽  
Clinical Samples ◽  
Rrna Gene ◽  
16S Rrna Gene Pyrosequencing

scholarly journals Whole-Genome Relationships among Francisella Bacteria of Diverse Origins Define New Species and Provide Specific Regions for Detection

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Jean F. Challacombe ◽  
Jeannine M. Petersen ◽  
La Verne Gallegos-Graves ◽  
David Hodge ◽  
Segaran Pillai ◽  
...  
Keyword(s):  
New Species ◽  
16S Rrna ◽  
Response Times ◽  
Amino Acid Sequences ◽  
Clinical Samples ◽  
Whole Genome ◽  
Multiple Gene ◽  
Content Type ◽  
Environmental Surveys ◽  
Genome Comparisons

ABSTRACT Francisella tularensis is a highly virulent zoonotic pathogen that causes tularemia and, because of weaponization efforts in past world wars, is considered a tier 1 biothreat agent. Detection and surveillance of F. tularensis may be confounded by the presence of uncharacterized, closely related organisms. Through DNA-based diagnostics and environmental surveys, novel clinical and environmental Francisella isolates have been obtained in recent years. Here we present 7 new Francisella genomes and a comparison of their characteristics to each other and to 24 publicly available genomes as well as a comparative analysis of 16S rRNA and sdhA genes from over 90 Francisella strains. Delineation of new species in bacteria is challenging, especially when isolates having very close genomic characteristics exhibit different physiological features—for example, when some are virulent pathogens in humans and animals while others are nonpathogenic or are opportunistic pathogens. Species resolution within Francisella varies with analyses of single genes, multiple gene or protein sets, or whole-genome comparisons of nucleic acid and amino acid sequences. Analyses focusing on single genes (16S rRNA, sdhA), multiple gene sets (virulence genes, lipopolysaccharide [LPS] biosynthesis genes, pathogenicity island), and whole-genome comparisons (nucleotide and protein) gave congruent results, but with different levels of discrimination confidence. We designate four new species within the genus; Francisella opportunistica sp. nov. (MA06-7296), Francisella salina sp. nov. (TX07-7308), Francisella uliginis sp. nov. (TX07-7310), and Francisella frigiditurris sp. nov. (CA97-1460). This study provides a robust comparative framework to discern species and virulence features of newly detected Francisella bacteria. IMPORTANCE DNA-based detection and sequencing methods have identified thousands of new bacteria in the human body and the environment. In most cases, there are no cultured isolates that correspond to these sequences. While DNA-based approaches are highly sensitive, accurately assigning species is difficult without known near relatives for comparison. This ambiguity poses challenges for clinical cases, disease epidemics, and environmental surveillance, for which response times must be short. Many new Francisella isolates have been identified globally. However, their species designations and potential for causing human disease remain ambiguous. Through detailed genome comparisons, we identified features that differentiate F. tularensis from clinical and environmental Francisella isolates and provide a knowledge base for future comparison of Francisella organisms identified in clinical samples or environmental surveys.

scholarly journals Alteration of Bacterial Communities in Anterior Nares and Skin Sites of Patients Undergoing Arthroplasty Surgery: Analysis by 16S rRNA and Staphylococcal-Specific tuf Gene Sequencing

2020 ◽  
Vol 8 (12) ◽  
pp. 1977
Author(s):  
Søren Iversen ◽  
Thor Bech Johannesen ◽  
Anna Cäcilia Ingham ◽  
Sofie Marie Edslev ◽  
Staffan Tevell ◽  
...  
Keyword(s):  
16S Rrna ◽  
Bacterial Communities ◽  
Gene Sequencing ◽  
Alpha Diversity ◽  
Clinical Samples ◽  
Staphylococcal Species ◽  
Tuf Gene ◽  
Staphylococcus Hominis ◽  
The Impact ◽  
Arthroplasty Surgery

The aim was to study alterations of bacterial communities in patients undergoing hip or knee arthroplasty to assess the impact of chlorhexidine gluconate soap decolonisation and systemic antibiotic prophylaxis. A Swedish multicentre, prospective collection of samples obtained from elective arthroplasty patients (n = 83) by swabbing anterior nares, skin sites in the groin and the site of planned surgery, before and after arthroplasty surgery, was analysed by 16S rRNA (V3-V4) gene sequencing and a complementary targeted tuf gene sequencing approach to comprehensively characterise alterations in staphylococcal communities. Significant reductions in alpha diversity was detected for both bacterial (p = 0.04) and staphylococcal (p = 0.03) groin communities after arthroplasty surgery with significant reductions in relative Corynebacterium (p = 0.001) abundance and Staphylococcus hominis (p = 0.01) relative staphylococcal abundance. In nares, significant reductions occurred for Staphylococcus hominis (p = 0.02), Staphylococcus haemolyticus (p = 0.02), and Staphylococcus pasteuri (p = 0.003) relative to other staphylococci. Staphylococcus aureus colonised 35% of anterior nares before and 26% after arthroplasty surgery. Staphylococcus epidermidis was the most abundant staphylococcal species at all sampling sites. No bacterial genus or staphylococcal species increased significantly after arthroplasty surgery. Application of a targeted tuf gene sequencing approach provided auxiliary staphylococcal community profiles and allowed species-level characterisation directly from low biomass clinical samples.

scholarly journals Molecular diagnosis of orthopaedic device infection direct from sonication fluid by metagenomic sequencing

2017 ◽  
Author(s):  
Teresa L. Street ◽  
Nicholas D. Sanderson ◽  
Bridget L. Atkins ◽  
Andrew J. Brent ◽  
Kevin Cole ◽  
...  
Keyword(s):  
Diagnostic Information ◽  
Species Level ◽  
Metagenomic Sequencing ◽  
Tissue Samples ◽  
Bacterial Dna ◽  
Dna Contamination ◽  
Prosthetic Joint ◽  
Device Infection ◽  
Fluid Culture ◽  
Orthopaedic Device

AbstractCulture of multiple periprosthetic tissue samples is the current gold-standard for microbiological diagnosis of prosthetic joint infections (PJI). Additional diagnostic information may be obtained through sonication fluid culture of explants. However, current techniques can have relatively low sensitivity, with prior antimicrobial therapy and infection by fastidious organisms influencing results. We assessed if metagenomic sequencing of complete bacterial DNA extracts obtained direct from sonication fluid can provide an alternative rapid and sensitive tool for diagnosis of PJI.We compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other orthopaedic device infections. Reads from Illumina MiSeq sequencing were taxonomically classified using Kraken. Using 50 samples (derivation set), we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and validated our findings in 47 independent samples.Compared to sonication fluid culture, the species-level sensitivity of metagenomic sequencing was 61/69(88%,95%CI 77-94%) (derivation samples 35/38[92%,79-98%]; validation 26/31[84%,66-95%]), and genus-level sensitivity was 64/69(93%,84-98%). Species-level specificity, adjusting for plausible fastidious causes of infection, species found in concurrently obtained tissue samples, and prior antibiotics, was 85/97(88%,79-93%) (derivation 43/50[86%,73-94%], validation 42/47[89%,77-96%]). High levels of human DNA contamination were seen despite use of laboratory methods to remove it. Rigorous laboratory good practice was required to prevent bacterial DNA contamination.We demonstrate metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings combined with increasing availability of portable, random-access sequencing technology offers the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI.

scholarly journals Control of artefactual variation in reported inter-sample relatedness during clinical use of a Mycobacterium tuberculosis sequencing pipeline

2018 ◽  
Author(s):  
David H Wyllie ◽  
Nicholas Sanderson ◽  
Richard Myers ◽  
Tim Peto ◽  
Esther Robinson ◽  
...  
Keyword(s):  
Consensus Sequence ◽  
Read Depth ◽  
Pairwise Distance ◽  
Contact Tracing ◽  
Clinical Samples ◽  
Bacterial Dna ◽  
Consensus Sequences ◽  
Minor Variant ◽  
Validation Set ◽  
Genomic Regions

ABSTRACTContact tracing requires reliable identification of closely related bacterial isolates. When we noticed the reporting of artefactual variation between M. tuberculosis isolates during routine next generation sequencing of Mycobacterium spp, we investigated its basis in 2,018 consecutive M. tuberculosis isolates. In the routine process used, clinical samples were decontaminated and inoculated into broth cultures; from positive broth cultures DNA was extracted, sequenced, reads mapped, and consensus sequences determined. We investigated the process of consensus sequence determination, which selects the most common nucleotide at each position. Having determined the high-quality read depth and depth of minor variants across 8,006 M. tuberculosis genomic regions, we quantified the relationship between the minor variant depth and the amount of non-Mycobacterial bacterial DNA, which originates from commensal microbes killed during sample decontamination. In the presence of non-Mycobacterial bacterial DNA, we found significant increases in minor variant frequencies of more than 1.5 fold in 242 regions covering 5.1% of the M. tuberculosis genome. Included within these were four high variation regions strongly influenced by the amount of non-Mycobacterial bacterial DNA. Excluding these four regions from pairwise distance comparisons reduced biologically implausible variation from 5.2% to 0% in an independent validation set derived from 226 individuals. Thus, we have demonstrated an approach identifying critical genomic regions contributing to clinically relevant artefactual variation in bacterial similarity searches. The approach described monitors the outputs of the complex multi-step laboratory and bioinformatics process, allows periodic process adjustments, and will have application to quality control of routine bacterial genomics.

scholarly journals Detection of Nocardia by 16S Ribosomal RNA Gene PCR and Metagenomic Next-Generation Sequencing (mNGS)

2022 ◽  
Vol 11 ◽  
Author(s):  
Juanjuan Ding ◽  
Bing Ma ◽  
Xupeng Wei ◽  
Ying Li
Keyword(s):  
16S Rrna ◽  
Ribosomal Rna ◽  
Human Herpesvirus ◽  
Antibiotic Sensitivity ◽  
Common Species ◽  
16S Ribosomal Rna ◽  
Clinical Samples ◽  
Klebsiella Pneumonia ◽  
Antimicrobial Drugs ◽  
Detection And Identification

In this study, the aim was to investigate the discriminatory power of molecular diagnostics based on mNGS and traditional 16S ribosomal RNA PCR among Nocardia species. A total of fourteen clinical isolates from patients with positive Nocardia cultures and clinical evidence were included between January 2017 and June 2020 in HeNan Provincial People’s Hospital. DNA extraction and 16S rRNA PCR were performed on positive cultures, and pathogens were detected by mNGS in these same samples directly. Among the 14 Nocardia isolates, four species were identified, and N. cyriacigeorgica (8 cases) is the most common species. Twelve of the 14 Nocardia spp. isolates were identified by the two methods, while two strains of N. cyriacigeorgica were not identified by mNGS. All tested isolates showed susceptibility to trimethoprim-sulfamethoxazole (SXT), amikacin and linezolid. Apart from Nocardia species, other pathogens such as Acinetobacter baumannii, Klebsiella pneumonia, Aspergillus, Enterococcus faecalis, Human herpesvirus, etc., were detected from the same clinical samples by mNGS. However, these different pathogens were considered as colonization or contamination. We found that it is essential to accurately identify species for determining antibiotic sensitivity and, consequently, choosing antibiotic treatment. 16S rRNA PCR was useful for identification of nocardial infection among species, while this technique needs the clinicians to make the pre-considerations of nocardiosis. However, mNGS may be a putative tool for rapid and accurate detection and identification of Nocardia, beneficial for applications of antimicrobial drugs and timely adjustments of medication.

scholarly journals Probability of infectious disease in humans during epidemic

2021 ◽  
Author(s):  
AM Karmishin ◽  
IV Borisevich ◽  
VI Skvortsova ◽  
AA Goryaev ◽  
SM Yudin
Keyword(s):  
Infectious Disease ◽  
Biological Object ◽  
Epidemiological Modeling ◽  
Pathogen Virulence ◽  
The Core ◽  
Special Cases ◽  
Quantitative Characteristics ◽  
Infective Dose ◽  
The Law ◽  
Generalized Time

Popular SIR models and their modifications used to generate predictions about epidemics and, specifically, the COVID-19 pandemic, are inadequate. The aim of this study was to find the laws describing the probability of infection in a biological object. Using theoretical methods of research based on the probability theory, we constructed the laws describing the probability of infection in a human depending on the infective dose and considering the temporal characteristics of a given infection. The so-called generalized time-factor law, which factors in the time of onset and the duration of an infectious disease, was found to be the most general. Among its special cases are the law describing the probability of infection developing by some point in time t, depending on the infective dose, and the law that does not factor in the time of onset. The study produced a full list of quantitative characteristics of pathogen virulence. The laws described in the study help to solve practical tasks and should lie at the core of mathematical epidemiological modeling.

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