Tag: Antimicrobial Resistance

Choose This Year’s Metagenomics Award Winner

Posted on September 3, 2019 by - ProxiMeta

Congratulations to Dr. Ben Tully on winning this year’s Project ProxiMeta: 2019 Metagenomics Award! Read more about his project, 4. The Complete Hydrothermal Microbial Metal Metabolism

This summer, researchers from across the U.S. sent in short proposals for a chance to win a full-service ProxiMeta™ microbiome workup for a sample of their choice. ProxiMeta combines shotgun metagenomics with in vivo proximity ligation (Hi-C) and necessary bioinformatic tools to help researchers assemble high-quality microbial genomes directly from complex microbiome samples.

 

 

HOW TO VOTE

Each project was assessed by a panel of scientists for scientific merit, novelty, impact, and feasibility, and four finalists were selected. Cast your vote on Twitter for your favorite project.

 

 

THE FINALISTS

1. The Gut Microbiome as a Risk Factor for Arsenic-Induced Cancer

Twitter Name: Gut & As-Induced Cancer

It is estimated that ~200 million people worldwide are exposed to arsenic concentrations exceeding current safety standards. Our collaborators have recently demonstrated that mice and human microbiomes can protect mice from arsenic toxicity. While human stool supplementation fully restores protection to arsenic in germ-free mice, researchers were only able to isolate one microbe, Faecalibacterium prausnitzii, that successfully conferred protection to both parent and infant mice. These results are huge because arsenic poses the highest lifetime risk for developing cancer in humans.We will investigate the role of arsenic-transforming bacteria within the gastrointestinal (GI) microbiome as another possible risk factor.

In nature, arsenic-reducing microorganisms are well known for their ability to generate more toxic arsenic products called arsenites, which are typically formed in anaerobic environments like the gut. Past research indicates that ingested arsenic may also be transformed into the toxic product arsenite by gut microbes thus increasing the risk for the host. On the other hand, arsenite-oxidizing microbes may also provide a benefit to the host by lowering arsenite concentrations. The ability of the microbiome to transform arsenic is determined by its genetic composition, therefore ProxiMeta sequencing technology will allow us to immediately analyze our collaborators rodent stool samples for genetic clues regarding this mysterious protection. Our project goals are to expand on this knowledge by: (1) characterizing the genetic basis for protection to arsenic provided by the microbiome (2) identifying, and then isolating, the bacteria-harboring arsenic transforming genes involved in protection.

We predict that differences in the gut metagenome composition will explain the incidences in arsenic susceptibility within a population or even at the family level. This project will provide important insight regarding how gut microbes contribute to cancer and may lead to novel therapies and probiotics that could target the microbiome of arsenic-exposed individuals.

2. Evaluating Antimicrobial Resistance in Backyard Poultry Environments

Twitter: AMR in Backyard Poultry

Approximately 13 million rural, urban, and suburban US residents reported owning backyard poultry (BYP) in 2014, and interest in BYP ownership is nearly four times that amount. BYP ownership has risen recently due to product quality, public health, ethical, and animal welfare concerns of commercial operations. However, BYP ownership and disease treatment is largely under-regulated, unlike commercial poultry production. Lack of regulation poses public health concerns of transmission of antimicrobial resistant (AMR) bacteria, such as AMR strains of Salmonella, Mycoplasma gallisepticum, and Escherichia coli commonly associated with BYP. BYP owners (2014 survey) were largely uninformed about poultry diseases and treatments but were interested in learning more on disease management.

The combination of a lack of regulation and public information warrants further research into the bacterial communities of BYP and their environments. Cloacal and environmental swabs were collected as part of a 2018 citizen science study where BYP owners reported current and historical poultry antibiotic usage. We propose to conduct shotgun metagenomic sequencing and proximity ligation using the ProxiMeta platform, allowing for increased detection of full-length AMR gene alleles compared to that revealed by short-read sequencing. The combination of PacBio reads with HiC intercontig ligation analysis allows for identification of potential gene transfer events of AMR genes within communities and potential dissemination throughout the environment.

This analysis is especially important considering the public health concerns of AMR persistence in backyard environments. Additionally, investigation of lytic and prophage presence would allow investigation of phage-mediated bacterial regulation that would not be possible with short-read sequencing alone. ProxiMeta analysis of these samples would provide the most comprehensive insight of AMR presences and persistence in BYP environments to date. These findings will be critical for new regulation and disease management for the increasing number of BYP flocks, which currently pose a potential health risk.

3. Unraveling the Metagenomics of Contamination

Twitter: Steel Site Contamination

We propose a metagenome characterization of contaminated Munger Landing sediment located in the St. Louis River, Duluth, MN USA. Seasonal samples are already collected and stored; of which one will be sequenced. Munger landing, is located downstream from the U.S. Steel Superfund site and contaminants include PAHs, dioxins, PCBs, and heavy metals.

Soil condition is integral to high productivity and ecosystem balance at all trophic levels. Human activities erode soil condition through agriculture, mining, sewage outflows and/or chemical/waste disposal into waterways. These practices alter the chemical structure of the soil and break down the microbial community processes responsible for ensuring the balance of biogeochemical cycling patterns in the soil. We hypothesize the activity of these pathways involved in cycling of nitrogen, phosphorus and carbon are altered in contaminated soil systems.

Metagenomic profiling of Munger Landing will provide data to examine microbes, metabolic pathways, and contaminant-processing genes present in the community that can be characterized further using qRTPCR. This project will be presented within a community college microbiology course module. Curriculum utilizing real-world data and the sequencing technology from Phase Genomics will teach students experimental design, troubleshooting, hypothesis testing, data analysis and how to communicate the broader impacts of a study to society, the field of environmental microbiology or conservation.

In the future, this data will assist in designing a longitudinal metagenomic and metatranscriptomic study to assess the ability of remediation to ‘recover’ bacterial community function at the Munger Landing site; slated to start in 2020-2021 as compared to two uncontaminated control sites. Ten sites, slated for remediation, have been identified as having high chemical and heavy metal contamination for the St. Louis River Estuary. The Munger Landing project will establish a workflow that can be applied to other contaminated sites.

4. The Complete Hydrothermal Microbial Metal Metabolism

Twitter: Hydrothermal Microbiome

Hydrothermal vents replenish the oceans with much-needed micronutrients, spewing iron, magnesium, nickel, and other metals from the earth’s crust. These metal micronutrients are used as biological cofactors for organisms throughout the marine food chain. Boiling, sterile hydrothermal fluids quickly cool and are colonized by highly specialized microorganisms that begin to cycle the metal species mixing with the seawater. Though regularly sampled, rarely have hydrothermal plumes been tracked through the water column to establish how microbial colonization occurs through time and space. We lack understanding regarding the replicability of colonization to what extent stochastic processes shape microbial community structure.

While on station at the East Pacific Rise hydrothermal vent field, size-fractionated samples (0.2, 3.0 and 5.0-μm) were collected in the hydrothermal plume emanating from Bio Vent. Samples fluids were collected from the source through the first 1-km of dispersal – the key distance for colonization – and this effort was repeated over the course of 10-days – to determine the replicability of natural colonization events. The application of standard metagenomics sequencing and microbial genome reconstruction through binning would provide novel insight into the cycling of metals within the plume but the use of cross-linked DNA techniques would deliver an unprecedented understanding of how strain diversity impacts colonization and how microbes interact with extrachromosomal elements in the environment.

While some microbes are poised to take advantage of reduced metal species for lithotrophic growth, microbes from the water column that become entrained in the plume will need metal-resistance adaptations to alleviate stress from the elevated metal concentrations present. Metal-resistance genes dispersed through the viral and plasmid pools are essential elements for understanding the functioning of the microbial community in this globally important source of metals to the oceans and effective interpretation of the community can only be achieved through cross-linked DNA metagenomic techniques.

*All finalists projects are owned by verified researchers at U.S. academic institutions.

 

RESOURCES

 

Hi-C Technology Links Antimicrobial Resistance Genes to the Microbiome

Posted on December 4, 2018 by - Hi-C, Metagenomics, ProxiMeta, Uncategorized

 

Antibiotic resistance is a rapidly growing global health threat as bacteria share and spread resistance genes via plasmids and other mobile genetic elements. Several teams of researchers applied a new method to understand which microorganisms house genes for antibiotic resistance within complex microbiome communities.
Read the paper, Linking the Resistome and Plasmidome to the Microbiome.

 

ANTIMICROBIAL RESISTANCE ON THE RISE

 

According to the World Health Organization, antimicrobial resistance (AMR) in microbial pathogens is expected to take 10 million lives by 2050 if there are no new pharmaceutical or technological advancements dedicated to combating this pressing problem. For almost a century, medicine has made remarkable impact on human life by using antibiotics to treat infections, but this has led to a very concerning overuse problem, stoking an arms race between antibiotics and the pathogens they target. The CDC points out that at least 30% of antibiotic prescriptions are unnecessary and there is a massive contribution to antibiotic overuse in the food and agriculture industry where each year 130,000 tons of antibiotics are given to food animal livestock. Both of these problems correlate with the rise of AMR.

 

Though there are naturally occurring antibiotic-resistant bacteria, there are two mechanisms by which bacteria can acquire antimicrobial resistance genes (ARGs) and become resistant: 1) through spontaneous genetic mutations and/or 2) by acquiring genetic material from other microbes via plasmids, viruses, or other means of horizontal gene transfer. Due to the evolutionary pressure exerted on microbes by antibiotic overuse, pathogens resistant to these antibiotics within our body, hospitals, and the environment become reservoirs of transmittable AMR genes that can rapidly spread and accumulate within a single microbe contributing to the emergence of multidrug-resistant microbes commonly known as superbugs.

 

PROXIMITY-LIGATION (HI-C) LINKS ARG AND PLASMIDS TO THEIR HOSTS

 

One of the biggest obstacles faced by scientists when studying AMR is the inability to determine which microbes are carrying and spreading specific ARGs. Because these genes often travel on mobile elements, they can move dynamically between different species and can therefore be found in numerous organisms without one clear parental host. When attempting to sequence the DNA of a mixed microbial sample, all the DNA is purified from all the cells at the same time and the host-plasmid connection is severed, making it nearly impossible to determine where each mobile element came from or if they were shared among several species. In this newly published paper, researchers highlight a novel method for linking ARGs and other mobile genetic elements to their hosts directly from microbiome samples using the latest version of the proximity-ligation (Hi-C) data analysis tool, ProxiMeta Hi-C.

 

Phase Genomics CEO, Dr. Ivan Liachko, describes how our Hi-C platform solves one of microbiologists’ greatest problems pertaining to the linking of plasmids with their hosts.

 

Hi-C utilizes in vivo proximity-ligation which can assemble complete genomes down to the strain-level directly from mixed-population samples as well as physically links plasmids/ARGs to their host. This method is particularly useful for researchers studying the “dark-matter” of the microbiome because the method does not require culturing nor a priori information about a sample.

 

USING HI-C TO TRACK ARGs IN THE MICROBIOME

 

Lead author Thibault Stalder from the University of Idaho used the ProxiMeta Hi-C kit on a complex microbiome wastewater community, a suspected AMR reservoir, to learn more about which bacteria carry ARGs. After the Hi-C library was sequenced, Phase Genomics used the data to inform contig clustering of hundreds of genomes, most of which are novel, with our cloud-based software – ProxiMeta. Using the genome clusters found by ProxiMeta, the Hi-C linkages of each ARG-, plasmid-, and integron-bearing contigs to each genome were measured to determine which species physically hosted the relevant mobile elements.

 

ProxiMeta was able to cluster contigs into >1000 genome clusters and search for over 30 groups of ARGs, plasmids, and integrons which speed up the adaptive process of newly integrated ARGs (Figure 1, circle plot). For each of these genes, we inferred hosts (Figure 2). Moreover, these organisms generally belonged to families known to host each known gene (marked with an “X” in Figure 2), supporting the accuracy of the analysis. In the future, this information will allow us to track the spread of AMR in complex communities consisting of many diverse organisms.

 

Microbiome Antibiotic Resistance Genes and Plasmids

Figure 1: Hi-C linkage between ARGs, plasmid markers, and integrons among clusters belonging to Alpha, Beta, Gamma and Delta Proteobacteria.

 

Over 200 genome clusters had strong Hi-C links to ARGs, of which 12 had high-quality assemblies. These resultant genomes include both gram positive and gram-negative bacteria and most belonged to species that were previously unsequenced. ARGs were mostly linked to genome clusters belonging to the Gammaproteobacteria, Betaproteobacteria and Bacteroidetes (Figure 2, below).

 

Microbiome Antibiotic Resistance Genes AMR and Plasmids

Figure 2: Normalized Hi-C links between ARGs, plasmids, and families of bacteria.

 

 

FUTURE DIRECTIONS

 

This method can be useful for researchers not only studying the microbiome, but the virome as well. Phages, or viruses, also distribute genetic information amongst bacteria to influence host biology, much like plasmids. Several previous studies showed that in vivo proximity-ligation can be used to link phages with their hosts directly from mixed complex samples, much like was done with plasmids and AMR genes in this study. This information could be crucial to labs and companies that are now engineering phages that could replace the widespread use of antibiotics and combat AMR.

 

This year, antibiotic resistant bugs have infected more than 2 million people globally; 23,000 of those individuals will die because of our inability to fight these superbugs. By using ProxiMeta Hi-C to better understand the genomics of microbial communities suspected to be AMR reservoirs, researchers can identify ARG carriers down to the strain-level and quantify how prevalent these genes are. With further exploration, this tool could one day offer a new solution to limit the spread of these genes and reverse the trend of increasing antibiotic resistance and save lives.

 

BRING A HI-C KIT INTO YOUR LAB TODAY

 

Phase Genomics offers a wide variety of proximity-ligation products and services including Hi-C preparation kits and a range of different cloud-based bioinformatic analysis platforms. Power your microbiome research with ProxiMeta Hi-C and our easy Hi-C kits; assemble hundreds of complete genomes for novel, unculturable microbes, and associate plasmids with hosts directly from raw microbiome samples using ProxiMeta Hi-C.