Combining Comprehensive Analysis of Off-Site Lambda Phage Integration with a CRISPR-Based Means of Characterizing Downstream Physiology

ABSTRACT During its lysogenic life cycle, the phage genome is integrated into the host chromosome by site-specific recombination. In this report, we analyze lambda phage integration into noncanonical sites using next-generation sequencing and show that it generates significant genetic diversity by targeting over 300 unique sites in the host Escherichia coli genome. Moreover, these integration events can have important phenotypic consequences for the host, including changes in cell motility and increased antibiotic resistance. Importantly, the new technologies that we developed to enable this study—sequencing secondary sites using next-generation sequencing and then selecting relevant lysogens using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based selection—are broadly applicable to other phage-bacterium systems. IMPORTANCE Bacteriophages play an important role in bacterial evolution through lysogeny, where the phage genome is integrated into the host chromosome. While phage integration generally occurs at a specific site in the host chromosome, it is also known to occur at other, so-called secondary sites. In this study, we developed a new experimental technology to comprehensively study secondary integration sites and discovered that phage can integrate into over 300 unique sites in the host genome, resulting in significant genetic diversity in bacteria. We further developed an assay to examine the phenotypic consequence of such diverse integration events and found that phage integration can cause changes in evolutionarily relevant traits such as bacterial motility and increases in antibiotic resistance. Importantly, our method is readily applicable to other phage-bacterium systems. IMPORTANCE Bacteriophages play an important role in bacterial evolution through lysogeny, where the phage genome is integrated into the host chromosome. While phage integration generally occurs at a specific site in the host chromosome, it is also known to occur at other, so-called secondary sites. In this study, we developed a new experimental technology to comprehensively study secondary integration sites and discovered that phage can integrate into over 300 unique sites in the host genome, resulting in significant genetic diversity in bacteria. We further developed an assay to examine the phenotypic consequence of such diverse integration events and found that phage integration can cause changes in evolutionarily relevant traits such as bacterial motility and increases in antibiotic resistance. Importantly, our method is readily applicable to other phage-bacterium systems.

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Next Generation Sequencing for the Prediction of the Antibiotic Resistance in Helicobacter pylori: A Literature Review

Antibiotics ◽

10.3390/antibiotics10040437 ◽

2021 ◽

Vol 10 (4) ◽

pp. 437

Author(s):

Ilaria Maria Saracino ◽

Matteo Pavoni ◽

Angelo Zullo ◽

Giulia Fiorini ◽

Tiziana Lazzarotto ◽

...

Keyword(s):

Antibiotic Resistance ◽

Next Generation Sequencing ◽

Literature Review ◽

Molecular Mechanisms ◽

World Health ◽

Next Generation ◽

Culture Methods ◽

Phenotypic Resistance ◽

H Pylori ◽

Generation Sequencing

Background and aims: Only a few antimicrobials are effective against H. pylori, and antibiotic resistance is an increasing problem for eradication therapies. In 2017, the World Health Organization categorized clarithromycin resistant H. pylori as a “high-priority” bacterium. Standard antimicrobial susceptibility testing can be used to prescribe appropriate therapies but is currently recommended only after the second therapeutic failure. H. pylori is, in fact, a “fastidious” microorganism; culture methods are time-consuming and technically challenging. The advent of molecular biology techniques has enabled the identification of molecular mechanisms underlying the observed phenotypic resistance to antibiotics in H. pylori. The aim of this literature review is to summarize the results of original articles published in the last ten years, regarding the use of Next Generation Sequencing, in particular of the whole genome, to predict the antibiotic resistance in H. pylori.Methods: a literature research was made on PubMed. The research was focused on II and III generation sequencing of the whole H. pylori genome. Results: Next Generation Sequencing enabled the detection of novel, rare and complex resistance mechanisms. The prediction of resistance to clarithromycin, levofloxacin and amoxicillin is accurate; for other antimicrobials, such as metronidazole, rifabutin and tetracycline, potential genetic determinants of the resistant status need further investigation.

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Next generation sequencing reveals limitation of qPCR methods in quantifying emerging antibiotic resistance genes (ARGs) in the environment

Applied Microbiology and Biotechnology ◽

10.1007/s00253-021-11202-4 ◽

2021 ◽

Author(s):

Bo Li ◽

Tao Yan

Keyword(s):

Antibiotic Resistance ◽

Next Generation Sequencing ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Next Generation ◽

Generation Sequencing

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Molecular evolution and genetic diversity analysis of SFTS virus based on next-generation sequencing

Biosafety and Health ◽

10.1016/j.bsheal.2021.02.002 ◽

2021 ◽

Author(s):

Aqian Li ◽

Lin Liu ◽

Wei Wu ◽

Yang Liu ◽

Xiaoxia Huang ◽

...

Keyword(s):

Genetic Diversity ◽

Next Generation Sequencing ◽

Molecular Evolution ◽

Diversity Analysis ◽

Next Generation ◽

Genetic Diversity Analysis ◽

Generation Sequencing

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Profile of HBV Integration in the Plasma DNA of Hepatocellular Carcinoma Patients

Current Genomics ◽

10.2174/1389202919666181002144336 ◽

2019 ◽

Vol 20 (1) ◽

pp. 61-68 ◽

Cited By ~ 1

Author(s):

Weiyang Li ◽

Xiaofang Cui ◽

Qing Huo ◽

Yanwei Qi ◽

Yuhui Sun ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Next Generation Sequencing ◽

Hepatitis B ◽

Direct Evidence ◽

Plasma Samples ◽

Plasma Dna ◽

Initial Experiment ◽

Integration Sites ◽

Significant Enrichment ◽

Generation Sequencing

Background: Hepatitis B Viral (HBV) infection is one of the major causes of Hepatocellular Carcinoma (HCC). Mounting evidence had provided that the HBV integration might be a critical contributor of HCC carcinogenesis. </P><P> Objective and Methods: To explore the profile of HBV integration in the plasma DNA, the method of next-generation sequencing, HBV capture and bioinformatics had been employed to screen for HBV integration sites in the plasma samples. Results: In the initial experiment, a total of 87 breakpoints were detected in the 20 plasma samples. The distribution of breakpoints showed that there was significant enrichment of breakpoints in the region of intron. Furthermore, the HBV breakpoints were prone to occur in the region of X protein (1,700-2,000bp) in the plasma samples. The pathway analysis had revealed that the HBV integrations sites were specifically enriched in the cancer pathway. Conclusion: Altogether, our results had provided direct evidence for the HBV integration in plasma DNA, and they might be potentially useful for future HCC prognosis and diagnosis.

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Genome-wide analysis of ivermectin response by Onchocerca volvulus reveals that genetic drift and soft selective sweeps contribute to loss of drug sensitivity

10.1101/094540 ◽

2016 ◽

Cited By ~ 2

Author(s):

Stephen R. Doyle ◽

Catherine Bourguinat ◽

Hugues C. Nana-Djeunga ◽

Jonas A. Kengne-Ouafo ◽

Sébastien D.S. Pion ◽

...

Keyword(s):

Genetic Diversity ◽

Next Generation Sequencing ◽

Genetic Drift ◽

Onchocerca Volvulus ◽

Molecular Pathways ◽

Ivermectin Treatment ◽

Genetic Changes ◽

Genome Wide ◽

Parasite Populations ◽

Generation Sequencing

ABSTRACTBackgroundTreatment of onchocerciasis using mass ivermectin administration has reduced morbidity and transmission throughout Africa and Central/South America. Mass drug administration is likely to exert selection pressure on parasites, and phenotypic and genetic changes in several Onchocerca volvulus populations from Cameroon and Ghana - exposed to more than a decade of regular ivermectin treatment - have raised concern that sub-optimal responses to ivermectin’s anti-fecundity effect are becoming more frequent and may spread.Methodology/Principal FindingsPooled next generation sequencing (Pool-seq) was used to characterise genetic diversity within and between 108 adult female worms differing in ivermectin treatment history and response. Genome-wide analyses revealed genetic variation that significantly differentiated good responder (GR) and sub-optimal responder (SOR) parasites. These variants were not randomly distributed but clustered in ~31 quantitative trait loci (QTLs), with little overlap in putative QTL position and gene content between countries. Published candidate ivermectin SOR genes were largely absent in these regions; QTLs differentiating GR and SOR worms were enriched for genes in molecular pathways associated with neurotransmission, development, and stress responses. Finally, single worm genotyping demonstrated that geographic isolation and genetic change over time (in the presence of drug exposure) had a significantly greater role in shaping genetic diversity than the evolution of SOR.Conclusions/SignificanceThis study is one of the first genome-wide association analyses in a parasitic nematode, and provides insight into the genomics of ivermectin response and population structure of O. volvulus. We argue that ivermectin response is a polygenically-determined quantitative trait in which identical or related molecular pathways but not necessarily individual genes likely determine the extent of ivermectin response in different parasite populations. Furthermore, we propose that genetic drift rather than genetic selection of SOR is the underlying driver of population differentiation, which has significant implications for the emergence and potential spread of SOR within and between these parasite populations.Author summaryOnchocerciasis is a human parasitic disease endemic across large areas of Sub-Saharan Africa, where more that 99% of the estimated 100 million people globally at-risk live. The microfilarial stage of Onchocerca volvulus causes pathologies ranging from mild itching to visual impairment and ultimately, irreversible blindness. Mass administration of ivermectin kills microfilariae and has an anti-fecundity effect on adult worms by temporarily inhibiting the development in utero and/or release into the skin of new microfilariae, thereby reducing morbidity and transmission. Phenotypic and genetic changes in some parasite populations that have undergone multiple ivermectin treatments in Cameroon and Ghana have raised concern that sub-optimal response to ivermectin’s anti-fecundity effect may increase in frequency, reducing the impact of ivermectin-based control measures. We used next generation sequencing of small pools of parasites to define genome-wide genetic differences between phenotypically characterised good and sub-optimal responder parasites from Cameroon and Ghana, and identified multiple genomic regions differentiating the response types. These regions were largely different between parasites from both countries but revealed common molecular pathways that might be involved in determining the extent of response to ivermectin’s anti-fecundity effect. These data reveal a more complex than previously described pattern of genetic diversity among O. volvulus populations that differ in their geography and response to ivermectin treatment.

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