Tag Archives: Whole Genome Sequencing

Identifying what makes some gut bacteria strains life-threatening in pre-term babies

Researchers from the Quadram Institute and University of East Anglia have identified what makes some strains of gut bacteria life-threatening in pre-term babies.

The findings will help identify and track dangerous strains and protect vulnerable neonatal babies.

A major threat to neonatal babies with extremely low birth weight is necrotizing enterocolitis (NEC).

Rare in full-term babies, this microbial infection exploits vulnerabilities destroying gut tissue leading to severe complications. Two out of five cases are fatal.

One bacterial species that causes especially sudden and severe disease is Clostridium perfringens. These are common in the environment and non-disease-causing strains live in healthy human guts.

So what makes certain strains so dangerous in preterm babies?

Prof Lindsay Hall and Dr Raymond Kiu from the Quadram Institute and UEA led the first major study on C. perfringens genomes from preterm babies, including some babies with necrotizing enterocolitis.

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The research team analyzed C. perfringens genomes from the faecal samples of 70 babies admitted to five UK Neonatal Intensive Care Units (NICUs).

Based on genomic similarities, they found one set had a lower capacity to cause disease. This allowed a comparison with the more virulent strains.

The less virulent group lacked genes responsible for production of a toxin called PFO and other factors needed for colonization and survival.

This study has begun to construct genomic signatures for C. perfringens associated with healthy preterm babies and those with necrotizing enterocolitis.

Exploring genomic signatures from hundreds of Clostridium perfringens genomes has allowed us potentially to discriminate between ‘good’ bacterial strains that live harmlessly in the preterm gut, and ‘bad’ ones associated with the devastating and deadly disease necrotizing enterocolitis.

We hope the findings will help with ‘tracking’ deadly C. perfringens strains in a very vulnerable group of patients – preterm babies.”

Prof Lindsay Hall, UEA’s Norwich Medical School and the Quadram Institute

Larger studies, across more sites and with more samples may be needed but this research could help identify better ways to control necrotizing enterocolitis.

The team previously worked alongside Prof Paul Clarke and clinical colleagues at the Norfolk and Norwich University Hospital NICU. And they demonstrated the benefits of providing neonatal babies with probiotic supplements.

The enterocolitis gut microbiome of neonatal infants is significantly disrupted, making it susceptible to C. perfringens overgrowth.

Prof Hall said: “Our genomic study gives us more data that we can use in the fight against bacteria that cause disease in babies – where we are harnessing the benefits of another microbial resident, Bifidobacterium, to provide at-risk babies with the best possible start in life.”

Dr Raymond Kiu, from the Quadram Institute, said: “Importantly, this study highlights Whole Genome Sequencing as a powerful tool for identifying new bacterial lineages and determining bacterial virulence factors at strain level which enables us to better understand disease.”

This research was supported by the Biotechnology and Biological Sciences Research Council, part of UKRI, and the Wellcome Trust.

The study was led by researchers at Quadram Institute and the University of East Anglia, in collaboration with colleagues at Imperial College, London, the University of Glasgow, the University of Cambridge, Newcastle University and Northumbria University.

‘Particular genomic and virulence traits associated with preterm infant-derived toxigenic Clostridium perfringens strains’ is published in Nature Microbiology.

Source:
Journal reference:

Kiu, R., et al. (2023). Particular genomic and virulence traits associated with preterm infant-derived toxigenic Clostridium perfringens strains. Nature Microbiology. doi.org/10.1038/s41564-023-01385-z.

A novel approach to quantify personal information contained within gut metagenome data

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In a recent study published in Nature Microbiology, researchers used shotgun sequencing to extract human reads from deoxyribonucleic acid (DNA) in fecal samples of 343 Japanese individuals comprising the main dataset of this study.

They used this gut metagenome data to reconstruct personal information. Some study participants also provided whole genome sequencing (WGS) data for ultra-deep metagenome shotgun sequencing analysis.

Study: Reconstruction of the personal information from human genome reads in gut metagenome sequencing data. Image Credit: KaterynaKon/Shutterstock.comStudy: Reconstruction of the personal information from human genome reads in gut metagenome sequencing data. Image Credit: KaterynaKon/Shutterstock.com

Background

The knowledge regarding the human microbiome, microorganisms inhabiting the human body, has expanded considerably in the last ten years, thanks to rapid advancements in technologies like metagenome shotgun sequencing.

This technology allows the sequencing of the non-bacterial component of the microbiome samples, including host DNA. For instance, in fecal samples, the amount of host DNA is less than 10% but is removed to protect the privacy of donors.

Human germline genotype in metagenome data is substantial to enable the re-identification of individuals. However, researchers and donors should recognize that it is highly confidential, so sharing it with the community requires careful consideration.

Apart from ethical concerns related to sharing this data, it is necessary to understand that if human reads in metagenome data are not removed before deposition, what kind of personal information (e.g., sex and ancestry) could this data help recover?

In addition, human reads in gut metagenome data could be a good resource for stool-based forensics, robust variant calling, and polygenic risk scores based estimates of disease risks (e.g., type 2 diabetes).

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Since this data could help quantitatively and precisely reconstruct genotype information, it could complement human WGS data.

About the study

In the present study, researchers applied a few humans reads in the gut metagenome data of the main study dataset to reconstruct personal information, including genetic sex and ancestry. For predicting genetic sex and the ancestries of these 343 individuals, they used sequencing depth of the sex chromosomes and modified likelihood score-based method, respectively.

In addition, the researchers developed methods to re-identify a person from a genotype dataset. Furthermore, they combined two harmonized genotype-calling approaches, the direct calling of rare variants and the two-step imputation of common variants, to reconstruct genotypes.

The main dataset of the study included 343 Japanese participants, whereas the validation dataset for the genetic sex prediction analysis comprised 113 Japanese individuals.

The multi-ancestry dataset, which helped the researchers validate ancestry prediction analysis, comprised 73 individuals of various nationalities, including samples from individuals in New Delhi, India.

The female and male participants in each dataset were 196 & 147, 65 & 48, and 25 & 48, respectively. Likewise, the age range for these three datasets was 20 to 88, 20 to 81, and 20 to 61 years, respectively.

Results and conclusion

Given that human reads in the gut metagenome data were derived consistently from all chromosomes, the read depth of the X chromosome was nearly double in females and that of the Y chromosome in males.

So, in a logistic regression analysis, when the researchers applied a 0.43 Y:X chromosome read-depth ratio to the validation dataset, which correctly predicted the genetic sex of 97.3% of the study samples.

In human microbiome and genetic research, the feasibility of sex prediction using human gut metagenome data could help remove mislabelled samples.

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The study analysis also helped researchers remarkably predict ancestry in 98.3% of individuals using 1,000 Genomes Project (1KG) data as a reference.

However, the likelihood score-based method often misclassified South Asian (SAS) samples as American (AMR) and European (EUR), especially when the number of human reads was small. It is understandable because the genetic diversity of the SAS population is complex.

The likelihood score-based method also efficiently utilized the data from genomic areas with low coverage demonstrating the quantitative power of gut metagenome data to re-identify individuals and successfully re-identified 93.3% of individuals.

Despite ethical concerns, the re-identification method used in this study could help in the quality control of multi-omics datasets comprising gut metagenome and human germline genotype data.

In addition, the authors successfully reconstructed genome-wide common variants using genomic approaches. Historically researchers used stool samples as a source of germline genomes for wild and domestic animals but not humans.

Thus, further development of suitable methodologies could help efficiently utilize the human genome in gut metagenome data and benefit animal research.

Nonetheless, the study remarkably demonstrated that optimized methods could help reconstruct personal information from the human reads in gut metagenome data.

Moreover, the findings of this study could serve as a guiding resource to devise best practices for using the already accumulated gut metagenome data of humans.

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Journal reference:

Denmark experienced a surge in invasive Strep A Infections during the 2022-2023 winter season

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During the 2022-2023 winter season Denmark experienced a surge in infections caused by group A streptococci (GAS), including the more dangerous, invasive types of infections (iGAS). Incidence of iGAS is highest among the elderly, but the largest relative increase from previous seasons was seen among children. The study is being presented to the European Congress of Clinical Microbiology & Infectious Diseases (ECCMID 2023, Copenhagen, 15-18 April), by Thor Bech Johannesen and Steen Hoffmann, Statens Serum Institut, Copenhagen, Denmark, and colleagues.

Following the implementation of lockdown measures to prevent spread of COVID-19 in March 2020, the number of invasive infections caused by GAS, including more dangerous invasive types (iGAS), decreased. However, during November 2022, an increasing number of these infections occurred in all regions of Denmark, with incidence rates reaching three times the pre-lockdown levels in January-March 2023. While there is no policy on mandatory reporting of GAS infection in Denmark, clinical microbiology laboratories nationwide submit isolates of iGAS to Statens Serum Institut (SSI) for further characterization on a voluntary basis.

Since 2018 approximately 90% of all iGAS cases in Denmark have been submitted to SSI for whole genome sequencing (WGS). For the period 2018 through March 2023, the authors extracted these WGS data and all records from the Danish Microbiology Database (MiBa) with culture-proven GAS and iGAS (invasive GAS being defined as GAS isolated from an anatomical region that should be sterile). Repeated specimens from the same patient of either GAS or iGAS within a 30-day-period were excluded. Potential date of death was collected from the Danish Civil Registration System.

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Incidence of GAS as well as iGAS decreased notably following the restrictions in March 2020. The incidence of both remained at low levels until October 2022 and then dramatically increased in December 2022, peaking between January and March 2023 (Figure 1). The incidence of iGAS was highest in the age groups 65-84 years (4.0/100,000) and 85+ years (5.2/100,000) (Figure 2). Fatalities from iGAS in absolute numbers have also increased, but the case fatality rates for all age groups were similar to previous seasons (approximately 15% overall, and 30% in those aged 85 years and older – rates in children are low and vary due to low absolute numbers).

The strains ST28 emm1 (also known as M1) and ST36 emm12, which have both been virtually absent since April 2020, accounted for 53% and 28%, respectively, of iGAS infections in 2023. A new subvariant of M1 emerged in 2022 and has become the dominant subvariant in 2023, accounting for 30% of all iGAS cases (Figure 3). In addition to a distinct core genome, this variant is characterised by its acquisition of a bacteriophage carrying the virulence factor SpeC, a known key exotoxin. From initial analyses, the novel M1 subvariant does not appear to be significantly more virulent than other M1 variants circulating in Denmark, however, M1 variants in general are more likely to cause invasive disease, and iGAS patients infected with M1 variants are more often in need of intensive care. No significant difference was found in mortality rates for individual variants.

The authors conclude: “Since December 2022, the incidence of iGAS-cases in Denmark has been unusually high, partly driven by the emergence of a new M1 subvariant, which has been responsible for 30% of iGAS cases in 2023. Although a large proportion of the variants currently circulating in Denmark have a high capacity for virulence, we estimate that the current surge is largely due to extensive community spread, possibly combined with a low level of immunity in the general population following two years of extraordinarily low incidence rates.

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Genes encoding T cell receptors vary greatly between persons and populations, study reveals

Researchers from Karolinska Institutet have discovered that the genes encoding our T cell receptors vary greatly between persons and populations, which may explain why we respond differently to for example infections. The findings, presented in the journal Immunity, also demonstrate that some gene variants are inherited from Neanderthals.

T-cells that are part of our immune system are central in the protection against infections and cancer. With the help of TCRs, the cells recognize foreign invaders and tumor cells.

“It was previously unknown how variable human TCR genes are”, says Gunilla Karlsson Hedestam, professor at the department of microbiology, tumor and cell biology at Karolinska Institutet and the study’s lead author.

Using deep sequencing of blood samples, the researchers examined TCR genes in 45 people originating from sub-Saharan Africa, East Asia, South Asia and Europe. The researchers showed that these genes vary greatly between different persons and population groups. The results were confirmed by analyses of several thousand additional cases from the 1000 Genomes project.

We found that every individual, other than identical twins, has a unique set of TCR gene variants. These differences reveal possible mechanisms underlying the wide range of responses to infections and vaccines that we observe at the population level.”

Martin Corcoran, first author of the study

“We discovered 175 new gene variants, which doubles the number of known TCR gene variants. An unexpected and surprising finding is that certain gene variants originate from Neanderthals and one of these is present in up to 20% of modern humans in Europe and Asia.”

Gunilla Karlsson Hedestam explains that the variation in these genes cannot be detected with the standard methods used in whole genome sequencing, but with the development of specialized deep sequencing methods and analysis software that allow highly precise definition of B- and T-cell receptor genes, this is now possible.

“As these genes are among the most variable in our genome, the results also provide new information about how our immune system has developed over the course of history, says Martin Corcoran. We are particularly interested in uncovering the function of the TCR variants we have inherited from Neanderthal ancestors. The frequency of these variants in modern humans suggests an advantageous function in our biology and we are keen to understand this”, adds Martin Corcoran.

The findings and the new TCR gene database the researchers now publish can be of great importance in the development of new therapeutic approaches in the future.

“Understanding human genetics is fundamental for the development of targeted treatments. The methods described in the study provide new opportunities, not the least in the cancer field where T-cells are central to several promising forms of immunotherapy”, says Gunilla Karlsson Hedestam.

The results can also shed light on other areas of research.

“The findings can lead to the development of new diagnostics and therapies in a range of medical disciplines, including precision medicine”, says Gunilla Karlsson Hedestam.

What is the next step in your research?

“We are now investigating the functional significance of several of the newly discovered gene variants and how this variation impacts our T-cell responses. We are also planning extended studies involving large groups of individuals to examine the role of TCR gene variation in diseases we know involve T cells, such as infectious diseases, cancer, and autoimmune disorders”, says Gunilla Karlsson Hedestam.

Main funding for the study comes from an ERC Advanced Grant and the Swedish Research Council.

Source:
Journal reference:

Corcoran, M., et al. (2023) Archaic humans have contributed to large-scale variation in modern human T cell receptor genes. Immunity. doi.org/10.1016/j.immuni.2023.01.026.

Study confirms the growing resistance in non-pathogenic Listeria species

In the food processing industry, the deadly bacteria Listeria monocytogenes is monitored closely. Not only can the bacteria make people extremely ill, it is known to be developing resistance to various food safety measures across the world.

However, two ‘harmless’ species of Listeria are also developing a surprising number of characteristics potentially harmful to humans.

A Whole Genome Sequencing study in South Africa, from a team of researchers with first author Dr Thendo Mafuna at the University of Johannesburg, shows some of the changing characteristics of Listeria found in the country.

The study shows that Listeria innocua strains are developing resistance to temperature, pH, dehydration and other stresses; as well as hypervirulence genetically identical to that of Listeria monocytogenes.

Some strains of L. innocua and L. welshimeri in the study show all three genes for resistance to a widely-used disinfectant, from the quaternary ammonium compound (QAC or QUAT) group of chemicals.

Two strains of L. innocua they analyzed have developed three or more concerning pathogenic characteristics, including CRISPR CAS-type adaptive immune systems.

The two non-pathogenic strains of Listeria were sampled in raw, dried and processed meats at commercial food processing facilities in the country.

The study confirms other research showing growing resistance in non-pathogenic Listeria species in other parts of the world.

Shared genes with pathogenic species

The Listeria innocua that we tested has some of the genes that are also found in pathogenic Listeria monocytogenes.

Dr Thendo Mafuna, First Author, University of Johannesburg

Mafuna is from the Department of Biochemistry at the University of Johannesburg.

These shared genes between L. innocua and L. monocytogenes are also responsible for disease in humans; and stress tolerance such as resistance to the disinfectant Benzalkonium chloride (BC or BAC).

Research from others has shown that though Listeriosis is rarely caused by L. innocua, it does happen more often in people with compromised immune systems, he adds.

Benzalkonium chloride (BAC) is a member of a group of chemicals called Quaternary ammonium compounds, or QUATs. Quats are found in many common disinfectant products. They have been shown to be highly effective at killing bacteria, fungi and viruses.

All the L. innocua strains they tested also had the complete LIPI-4 hypervirulence gene sequence, which can cause disease in humans, he says. The LIPI-4 sequence they found in L. innocua is identical to that found in pathogenic L. monocytogenes, as recorded by the Pasteur Institute in Paris, France.

From raw, dried and processed meats

The samples and isolates analyzed in this study were collected between 2014 and 2019 by the South African Government’s Department of Agriculture, Land Reform and Rural Development (DALRRD). These were submitted to the Agricultural Research Council (ARC) at Onderstepoort Veterinary Research SA for analysis.

In total, 258 isolates from butcheries, abbatoirs, retail outlets, cold stores and processing facilities all over the country were studied. Of these, 38 were found to be nonpathogenic L. Innocua; and another three isolates found to be nonpathogenic L. welshimeri.

The isolates came from raw whole, raw processed, dried, and processed cooked, beef, chicken and pork meats. Dr. Itumeleng Matle at the Bacteriology Division, ARC in Onderstepoort did the microbiological analysis of Listeria isolation and identification.

The Whole Genome Sequencing (WGS) was done by Dr. Rian E. Pierneef at the ARC’s Biotechnology Platform at Onderstepoort.

Mafuna then compared the genome sequences with those recorded by the Pasteur Institute, in Paris, France; and performed the analysis for the study.

On the lookout

“We need to look at our own facilities in South Africa to really see what is happening. Our analyses of these bacteria can help us predict which sequence types to be on the look out for,” says Mafuna.

It is the number of harmful characteristics that the L. innocua strains share with L. monocytogenes that is concerning, he adds.

Food processors need to look out for Listeria innocua because it is becoming resistant to disinfectants that are used in industry to get rid of them. It would also be helpful to try different types of disinfectants to surfaces, he says. Switching from one type to another may prevent or delay the bacteria developing resistance to one type of disinfectant.

“Big industrial food processors may want to investigate how efficient BC or quat disinfectants are in their facilities. This can be done by taking swabs before cleaning and again after cleaning, culturing those, to see how well the disinfectant regimes are working,” says Mafuna.

Source:
Journal reference:

Mafuna, T., et al. (2022) Comparative Genomics of Listeria Species Recovered from Meat and Food Processing Facilities. Microbiology Spectrum. doi.org/10.1128/spectrum.01189-22.

Study investigates spread of Klebsiella superbug between humans and the environment

An international team of scientists investigating transmission of a deadly drug-resistant bacteria that rivals MRSA, has found that whilst the bugs are found in livestock, pets and the wider environment, they are rarely transmitted to humans through this route.

The researchers, led by Professor Ed Feil from the Milner Centre for Evolution at the University of Bath, investigated the prevalence of Klebsiella, a family of bacterial species that lives harmlessly in the intestines, but can be dangerous if it spreads to other parts of the body.

Klebsiella pneumoniae is the most well-known species in this family, which can cause pneumonia, meningitis, urinary tract infections and infections in the bloodstream.

These bacteria are now highly resistant to antibiotics, with some strains even resistant to carbapenems, one of the so-called “last resort” class of antibiotics which is only used when no other antibiotic treatment works.

Klebsiella has overtaken MRSA as a health problem in the UK, with rates steadily increasing. The WHO has recognised the bacteria as a critical priority healthcare associated pathogen.

As well as being found in hospitals, the microbe has also previously been detected in the environment, including livestock and wastewater, but until now it wasn’t clear whether the bacteria were passed between clinical and non-clinical environments.

In the largest scale study ever conducted, the team collected 6,548 samples over a 15 month period from different locations in and around the Italian city of Pavia, where this pathogen is a major problem in hospitals, and analyzed them using whole genome sequencing techniques to detect and identify any Klebsiella bacteria present.

The team swabbed patients in hospitals and healthy ‘carriers’ in the community, took samples from farms, puddles, domestic animals and even house flies and other insects to detect where the bacteria was present.

From this, they found 3,482 isolates including 15 different species of Klebsiella, with half of the positive samples containing K. pneumoniae.

When the team genetically sequenced the bacteria to find which strains were present, they found that there was very little overlap between those bugs found in the hospitals and those found in the environment.

Professor Ed Feil, who led the study, said: “Klebsiella infections are becoming increasingly resistant to antibiotics, so whereas you used to be able to treat most urinary tract infections easily, now it is more common that patients are getting infections that keep coming back and causing problems.

Klebsiella can also cause pneumonia, which kills around half of patients. These bacteria are a bigger problem in the UK than MRSA.

“Our researchers wanted to find out whether resistant bacterias are now spreading into pets, farms, livestock, plants, and water, and so we wanted to investigate where Klebsiella is found and monitor how it spreads, to inform how best to prevent and control outbreaks.

“We found it was present everywhere, but were surprised that the strains found in hospital were different to those found in the environment, indicating there’s very little transfer between the two habitats: humans nearly always catch this from other humans.

“This confirms the best way to control infection for these bacteria remains stringent hospital hygiene, and that there is less chance that outbreaks may be caused by contact with animals or the environment than previously feared, at least in a high-resource country like Italy.”

The fear was that farmers might get these bacteria from their livestock or soil, that we could get infected by contaminated salad or get sick if we swim in lakes that are infected.

Our research did not give any evidence of this, however, we did find resistant klebsiella in pets, such as cats and dogs. Vets and owners should be aware of this, as these animals could pose a risk for spreading the bacteria.”

Dr Harry Thorpe, first author of the paper, University of Oslo (Norway)

The project consortium, called SpARK, was led by Bath but included researchers from the UK (Wellcome Sanger Institute, Universities of Bristol and Glasgow), Norway, France, Finland and Italy. The work was funded by the Joint Programming Initiative on Antimicrobial Resistance (JPI-AMR), and the MRC, and published in Nature Microbiology.

Professor Feil said: “This is the biggest, most systematic study that has been conducted at the same time in a single geographical location.

“We looked at transmission of strains, however antibiotic resistance can be conferred to other strains very easily when they swap and pick up circular pieces of DNA called plasmids.

“Next we want to track how plasmids are transferred between strains, using a technique called long read sequencing.”

The team has recently been awarded a network grant by the JPIAMR to do this, which builds on a GW4 research community and was supported by the GW4 AMR Alliance.

Source:
Journal reference:

Thorpe, H.A., et al. (2022) A large-scale genomic snapshot of Klebsiella spp. isolates in Northern Italy reveals limited transmission between clinical and non-clinical settings. Nature Microbiology. doi.org/10.1038/s41564-022-01263-0.