Tag Archives: Global Health

Microbiology

Transforming antibiotic resistance testing: a novel, rapid and affordable technique

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Lily Ramsey, LLMInterview conducted by Lily Ramsey, LLMMay 19 2023
Thought LeadersDr. Sandor KasasResearch LeadEcole Polytechnique Fédérale de Lausanne

News Medical speaks with Dr. Sandor Kasas, a lead researcher at Ecole Polytechnique Fédérale de Lausanne in Switzerland. Here we discuss his recent development of a novel and highly efficient method for rapid antibiotic susceptibility testing using optical microscopy.

The new technique, known as Optical Nanomotion Detection (ONMD), is an extremely rapid, label-free, and single-cell sensitive method to test for antibiotic sensitivity. ONMD requires only a traditional optical microscope equipped with a camera or mobile phone. The simplicity and efficiency of the technique could prove to be a game changer in the field of antibiotic resistance.

Please can you introduce yourself, tell us about your career background, and what inspired your career in biology and medicine?

I graduated in medicine but never practiced in hospitals or medical centers. After my studies, I started working as an assistant in histology at the University of Fribourg in Switzerland. My first research projects included image processing, scanning tunneling, and atomic force microscopy.

Later, and for most of the rest of my scientific carrier, I focused primarily on the biological applications of AFM. For the past ten years, my research interest is about nanomotion, i.e., the study of oscillations at a nanometric scale of living organisms.

Image Credit: dominikazara/Shutterstock.comImage Credit: dominikazara/Shutterstock.com

You started working on biological applications of the atomic force microscope (AFM) in 1992. From your perspective, how has the antibiotic resistance landscape changed over the last two decades? What role has the advancement in technology played in furthering our understanding?

In the early ’90s, the AFM was mainly used for imaging. Later, AFM microscopists noticed that the instrument could also be used to explore the mechanical properties of living organisms. More recently, many “exotic” applications of the AFM have emerged, such as its use to weigh single cells or study their oscillations at the nanometric scale. In the 1990s, antibiotic resistance was not as serious a problem as today, but several teams were already using AFM to assess the effects of antibiotics on bacterial morphology.

The first investigations were limited to structural changes, but later, as the fields of application of AFM expanded, the instrument made it possible to monitor the mechanical properties of the bacterial cell wall upon exposure to antibiotics. In the 2010s, with G. Longo and G. Dietler, we demonstrated that AFM could also track nanoscale oscillations of living organisms. The very first application we had in mind was using the instrument to perform rapid antibiotic susceptibility testing.

We have therefore developed devices based on dedicated AFM technology to perform fast AST (i.e., in 2-4h). AFM-based nanomotion detection instruments are already implemented in medical centers in Switzerland, Spain, and Austria. However, this type of device has some drawbacks, including the need to fix the organism of interest on a cantilever. To overcome this limitation, we have developed with R. Willaert a nanomotion detector based on an optical microscope.

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Your most recent research has led to the development of a novel and highly efficient technique for rapid antibiotic susceptibility testing using optical microscopy. Please could you tell us why the development of rapid, affordable, and efficient testing methods is so important in the world of antimicrobial resistance?

Rapid antibiotic susceptibility testing could reduce the use of broad-spectrum antibiotics. Traditional ASTs based on replication rate require 24 hours (but up to 1 month in the case of tuberculosis) to identify the most effective antibiotic. Doctors prescribe broad-spectrum antibiotics between the patient’s admission to a medical center and the results of the AST.

These drugs quickly improve patients’ conditions but, unfortunately, promote resistance. A rapid AST that could identify the most suitable antibiotic within 2-4 hours would eliminate broad-spectrum antibiotics and increase treatment efficiency and reduce the development of resistant bacterial strains. Since bacterial resistance is a global problem, rapid ASTs should also be implemented in developing countries. Therefore, affordable and simple-to-use tests are needed.

Image Credit: Fahroni/Shutterstock.comImage Credit: Fahroni/Shutterstock.com

Were there any limitations and obstacles you faced in the research process? If so, how did you overcome them?

Antibiotic sensitivity detection with ONMD is very similar to the AFM-based technique. As long as the bacterium is alive, it oscillates; if the antibiotic is effective, it kills the micro-organism, and its oscillations stop. The first limitation we faced when developing the ONMD was our microscopes’ depth of field of view. To prevent the bacteria from leaving the focal plane of the optical microscope during the measurement, we had to constrain the microbes into microfluidic channels a few micrometers high.

Microfabrication of such devices is relatively straightforward in an academic environment, but we were looking for simpler solutions. One option for constructing such a device is to use 10-micron double-sided rubber tape. It allows you to “build” a microfluidic chamber in 5 minutes with two glass coverslips and a puncher.

Another challenge was nanoscale motion detection. For this purpose, we used freely available cross-correlation algorithms that achieve sub-pixel resolution. (i.e., a few nanometers). We first developed the ONMD for larger organisms, such as yeast cells, and expanded the method to bacteria. This further development took us around two years.

You worked alongside Dr. Ronnie Willaert, a professor of structural biology at Vrije Universiteit Brussel, on developing this new rapid AST technique. How did your areas of expertise and research backgrounds complement each other in developing ONMD?

R. Willaert is an expert in yeast microbiology and microfluidics, while our team in Lausanne is primarily involved in AFM-based nanomotion detection and applying AFM to clinically relevant problems. The two teams were supported by a joint grant from the Swiss National Science Foundation and the Research Foundation Flanders (FWO) which enabled the development of the method.

The field of antimicrobial resistance requires a high level of international collaboration, with everyone working together to achieve a common goal. With antimicrobial resistance rising to dangerously high levels in all parts of the world, how important is collaboration in this field?

Our project required expertise in various fields, such as microbiology, microscopy, microfluidics, programming, and data processing. In the development of rapid AST instruments and many others, only a multidisciplinary approach and close collaboration between teams with complementary expertise is today the only path to success.

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You and Dr. Willaert have said, ‘The simplicity and efficiency of the method make it a game-changer in the field of AST.’ Can you please expand on what makes ONMD a game changer in the AST field and what implications this research could have in clinical and research settings?

As mentioned earlier, bacterial resistance is a global health problem. Rapid AST should also be easily implemented in developing countries to limit the spread of resistant strains. The cheaper and simpler the technique, the more likely it is to be used on a large scale. We are convinced that the ONMD approach can meet these requirements. ONMD could also be used for drug discovery or basic research.

While we recognize the importance of rapid AST, what next steps must be taken before this technique can be used globally in research and clinical landscapes?

For fundamental research, there are no other important developments to be made. Any reasonably equipped research center can implement the technique and use it. Regarding implementing the technique in developing countries or extreme environments, stand-alone devices have to be used, which have yet to be manufactured.

There is a rapidly expanding need for efficient AST globally; however, the need for affordable, accessible, and simple techniques are of grave importance in developing countries disproportionately affected by antibiotic resistance due to existing global health disparities. Could this rapid AST technique be utilized in low-middle-income countries to slow the growing spread of multi-resistant bacteria? What would this mean for global health?

We are confident that ONMD-based AST testing can soon be implemented in research centers in both developed and developing countries. However, accreditation by the health authorities is necessary to use it as a standard diagnostic tool; this process can take several years, depending on the government health policy.

What’s next for you and your research? Are you involved in any exciting upcoming projects?

We want to develop a self-contained device for extreme environments. It would consist of a small microscope equipped with a camera and a data processing unit. The microfluidic part of the device could contain different antibiotics ready to be tested.

The ONMD technique could also monitor contamination levels in enclosed environments such as submarines, spacecraft, and space stations. One of our recent projects is funded by the European Space Agency (ESA) to develop a rapid antifungal susceptibility test that could work in microgravity. Additionally, ONMD could be used for even more exciting projects, such as chemistry-independent life detection in the search for extraterrestrial life.

Where can readers find more information?

  • Villalba MI, Rossetti E, Bonvallat A, Yvanoff C, Radonicic V, Willaert RG*, Kasas S.*.Simple optical nanomotion method for single-bacterium viability and antibiotic response testing. PNAS 2023, May 2;120(18):e2221284120. doi: 10.1073/pnas.2221284120. Epub 2023 Apr 24. PMID: 37094120. * Contributed equally. https://doi.org/10.1073/pnas.2221284120
  • Radonicic, V.; Yvanoff, C.; Villalba, M.I.; Devreese, B.; Kasas, S.; Willaert, R.G. Single-Cell Optical Nanomotion of Candida albicans in Microwells for Rapid Antifungal Susceptibility Testing. Fermentation 2023, 9:365. https://doi.org/10.3390/fermentation9040365
  • Parmar P, Villalba MI, Horii Huber AS, Kalauzi A, Bartolić D, Radotić K, Willaert RG, MacFabe DF and Kasas S. Mitochondrial nanomotion measured by optical microscopy. Front. Microbiol. 2023, 14:1133773. https://doi.org/10.3389/fmicb.2023.1133773
  • Starodubtseva MN, Irina A. Chelnokova IA, Shkliarava NM, Villalba MI, Tapalski DV, Kasas S, Willaert RG. Modulation of the nanoscale motion rate of Candida albicans by X-rays. Front. Microbiol. 2023, 14:1133027. https://doi.org/10.3389/fmicb.2023.1133027
  • Radonicic V, Yvanoff C, Villalba MI, Kasas S, Willaert RG. The Dynamics of Single-Cell Nanomotion Behaviour of Saccharomyces cerevisiae in a Microfluidic Chip for Rapid Antifungal Susceptibility Testing. Fermentation. 2022; 8(5):195. https://doi.org/10.3390/fermentation8050195

About Dr. Sandor Kasas

Nanomotion is a fascinating and novel approach to observing living organisms.

Our team focuses almost exclusively on recording the nanomotion of bacterial mitochondria and mammalian cells with optical and AFM-based devices.

Recently, we demonstrated that the technique could be used not only for fast antimicrobial sensitivity testing but also to explore the metabolism of unicellular organisms. We hope our efforts will permit us to expand the application domains of ONMD.

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Microbiology

How the COVID pandemic has improved genomics

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Sponsored Content by MGIMay 4 2023Reviewed by Olivia Frost
insights from industryDavide CacciharelliMolecular Biology and Genomics ProfessorUniversity of Naples

In this interview, Davide Cacchiarelli, Molecular Biology and Genomics Professor at the University of Naples talks to NewsMed about how the COVID pandemic has highlighted the vital role of genomic surveillance and improved genomics.

Please introduce yourself and what inspired your career in molecular biology and genomics?

My name is Davide Cacchiarelli, and I am a molecular biology and genomics professor at the University of Naples. I was inspired by the fact that genomics is classed as an effective tool to improve human health, dissect the molecular events happening in the cell and nucleus, and better understand how cells and organisms work.

Image Credit: ShutterStock/pinkeyes

In The Telethon Institute of Genetics and Medicine, you combine various disciplines with cell biology, molecular biology, and genomics. Why is having a multidisciplinary approach useful when making discoveries, particularly surrounding infectious diseases such as COVID?

The majority of the time, a single omic, measuring only gene expression by RNA sequencing, measuring only epigenetics, or measuring only phenotype, is insufficient to understand how a cell works.

The best solution is to combine all efforts to understand how these events happen, from the nucleus to the cell’s exterior. COVID, in particular, has been a case where acquiring one single omic or a single view of how the system works is ineffective in understanding how COVID behaviors occur in the population or clinically hospitalized patients.

We, therefore, try to combine the general information and patient outcome to get the best result regarding COVID infection.

Davide Cacciarelli at ICG17 – How the COVID pandemic has improved genomics

On what research areas are you and your team at TIGEM currently focusing?

Our group aims to answer various questions, from basic microbiology to developmental biology. Then we can re-engineer it for real regenerative medicine purposes. We also look at how we can effectively use genomics as a medical instrument that can be used to impact the healthcare of patients in our healthcare system.

You have recently co-authored a paper, “Improved SARS-CoV-2 sequencing surveillance allows the identification of new variants and signatures in infected patients.” Can you expand on that?

One of the significant issues in Italy regarding SARS-CoV-2 genome sequencing was the cost. Sequencing the COVID genome was also a tedious and elaborate procedure.

Image Credit: ShutterStock/Kateryna Kon

The main objective was first to make this approach economically affordable and create a proof of printing pulled by which this approach could become a cost-effective method for anyone and any country.

Our second approach, therefore, included integrating the genome information and the transcriptomic profiling of the patient airway epithelia. This helps us to understand how the genome evolves and allows us to track its evolution, in addition to seeing the response of the host respiratory epithelium. Finally, we implemented new ways to classify viral variants based on different characteristics using this approach.

What are the advantages of better identifying new cells, or two variants, for healthcare centers and patients?

The European Center for Disease Control has issued several requirements for next year focused on tracking respiratory viruses. One of these is tracking emerging variants as soon as possible, which we have done with COVID-19. We now know that new, specific variants can emerge in a short timeframe, so immediate tracking is crucial to help contain or at least delay the spreading of possible pathogenic variants.

MGI offers a variety of tools and technology surrounding genomics. Can you tell us more about some of the products used during your research and your experience with them?

At MGI, we have typically applied the COVID and whole genome solutions. We also have the freedom to test the stereo-seq they have in production this month. MGI can offer alternative solutions for various genome sequencing needs.

Image Credit: ShutterStock/peterschreiber.media

At present many sequencing genomic companies are coming up with different solutions. At MGI, we understand that the best genomic solution is the one that better fits your needs. With our experience, for example, with COVID, MGI had the right solution at the right moment.

How important is selecting the right sequencing technology for your research? When undertaking new research, what do you look for in a product/sequencer?

When the primary focus is not on identifying genes or mapping gene expression but on identifying or qualifying gene variants, there must be no issues in the sequencing, as the sequencing issue might be an error in the sequencing and misinterpreted data.

The error rate of MGI technology on DNB sequencing is extremely low, which offers significant benefits. Users can confidently rely on the data at the level of leaders in the field, which is what we look for when we start COVID genome sequencing.

You have often collaborated with other researchers throughout your research projects, especially concerning COVID. How vital have these collaborations been in accelerating your research?

Like many scientists who faced the COVID pandemic, I had much to learn. We used our knowledge in medical genetics and variant interpretation, and the crosstalk we had with virologists, MGI scientists, and genomic specialists was a step towards acquiring the best solution and the best effort to try to get those results as soon as possible, which is crucial for COVID sequencing.

Surprisingly, some scientists who had no interest in healthcare possessed knowledge valuable in tackling COVID issues. The circumstances and contingencies around the event forced them to think outside the box.

Do you believe that if we can understand SARS-CoV-2 better, we could better use this knowledge to prepare ourselves for future pandemics better? What advantages would this have for global health?

COVID did not give us any significant advantages for healthcare, but it may have for science. It highlighted how vital advanced genomics is to track diseases which influenced decisions at the governmental level.

Image Credit: ShutterStock/CKA

Today, several diseases require advanced genome sequencing, such as cancer diagnostics and medical genetics. Given that the issues with this problem affect a small population, you do not feel the urgency to improve specific knowledge or tests.

Therefore, the COVID pandemic has highlighted the vital role of genomic surveillance and improved genomics. Today, we have laboratories that, until two years ago, thought they could never afford to set up a genomic workflow; the pandemic forced them to enter the genomics field. Our mission as genomic scientists is to help them implement this solution in their lab because improving genomics in any lab is the best for healthcare in the future.

There is a saying, “omics for all.” As a scientist, what does that mean to you?

‘Omics for all’ has to be understood in two ways. It is critical to give everybody the chance to have access to omics. However, we need to remember that it is still a medical procedure. Thus, the omics flow offers everybody access to high-quality omics profiling of their genome, but under medical supervision.

Finally, what is the future for you in your research?

I will continue my basic research in my lab: studying how pluripotent cells and stem cells can be manipulated and organized for medical purposes. We also want to use the knowledge accumulated in the COVID pandemic to apply fast, cost-effective, and reliable genome sequencing to other types of screening.

Image Credit: ShutterStock/Anusorn Nakdee

With this in mind, we hope to screen for several hereditary cancers, for example, breast cancer inheritance. Therefore, we can effectively use the COVID strategies we set up for COVID sequencing as proof of principle to apply the sequencing to human and human disease-driving genes.

About MGI

MGI Tech Co., Ltd. (referred to as MGI) is committed to building core tools and technology to lead life science through intelligent innovation. MGI focuses on R&D, production, and sales of DNA sequencing instruments, reagents, and related products to support life science research, agriculture, precision medicine, and healthcare. MGI is a leading producer of clinical high-throughput gene sequencers, and its multi-omics platforms include genetic sequencing, mass spectrometry, medical imaging, and laboratory automation.

Founded in 2016, MGI has more than 1000 employees, nearly half of whom are R&D personnel. MGI operates in 39 countries and regions and has established multiple research and production bases around the world. Providing real-time, comprehensive, life-long solutions, its vision is to enable effective and affordable healthcare solutions for all.

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MGI Tech Co., Ltd. 

Microbiology

Study reveals alarming global burden of antimicrobial resistance in bacterial infections

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Neha MathurBy Neha MathurApr 23 2023Reviewed by Danielle Ellis, B.Sc.

In a recent article published in the Lancet journal, researchers quantified the global bacterial antimicrobial resistance (AMR) burden to present deaths and disability-adjusted life-years (DALYs) attributable to and associated with 23 pathogens, 12 major infectious syndromes, 18 drug categories, and 88 pathogen–drug combinations.

They considered two counterfactual scenarios and used consistent methods to arrive at the study estimates as they had no clue of the extent to which susceptible or no infection would replace drug-resistant infections in a scenario when there was no drug resistance.

Study: Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Image Credit: Tatiana Shepeleva / ShutterstockStudy: Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Image Credit: Tatiana Shepeleva / Shutterstock

Background

Bacterial AMR, an emerging public health threat, is making antibiotic use futile or less effective against many common bacterial diseases affecting animals and humans. A United Kingdom (UK) government-commissioned review of AMR stated that it could claim 10 million lives annually by 2050.

The World Health Organization (WHO) and numerous other researchers have also raised that AMR spread is a pressing issue that needs immediate attention; if left unaddressed, rising AMR will make several bacterial pathogens highly fatal in the near future. The challenge is to gather current data on pathogen–drug combinations contributing to actual bacterial AMR burden for all world regions, even those with minimal surveillance.

According to the authors, studies have only reported AMR-related data for specific regions and a limited number of pathogens and pathogen–drug combinations. For instance, the United States Centers for Disease Control and Prevention (US-CDC) published a report in 2019 on AMR-related deaths for 18 AMR-related threats using surveillance data.

Similarly, Cassini et al. estimated the burden of eight and 16 pathogens and pathogen–drug combinations, respectively, for the European region between 2007 and 2015. Despite the significant contributions made by these studies to the field of AMR, there is a lack of comprehensive global estimates covering all locations, all pathogens, and all pathogen–drug combinations contributing to the rising burden of bacterial AMR.

Key Takeaways

  • First comprehensive assessment of the global burden of antimicrobial resistance (AMR) for bacterial infections.
  • Estimated 4.95 million deaths associated with bacterial AMR in 2019, including 1.27 million deaths attributable to bacterial AMR.
  • Lower respiratory infections accounted for the most deaths associated with resistance in 2019.
  • Six leading pathogens for deaths associated with resistance.
  • One pathogen-drug combination, meticillin-resistant S aureus, caused more than 100,000 deaths attributable to AMR in 2019.
  • Burden of AMR is highest in low-resource settings, particularly in western sub-Saharan Africa.
  • Serious data gaps in many low-income settings

About the study

In the present study, researchers used predictive statistical modeling to generate global estimates of bacterial AMR burden for all world locations, covering 204 countries for which they used all available data from the Global Burden of Diseases (GBD), Injuries, and Risk Factors study. The GBD study collated age- and gender-specific estimates for 369 injuries and illnesses in 204 nations and territories between 1990 and 2019.

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They retrieved data from published scientific literature, multisite research collaborations, clinical trials, research institutes based in low-income and middle-income countries (LMICs), public and private hospital records, diagnostic testing data, surveillance systems of pharmaceutical companies, global, national, and enhanced surveillance systems, and other relevant sources, encompassing 471 million (MN) patient records or isolates and 7,585 study-location years, which they gathered using varied strategies and used for study estimations.

The researchers modeled deaths and DALYs for 204 countries and territories to present cumulative estimates of AMR burden globally and for 21 GBD regions, including seven GBD super-regions.

For the first counterfactual scenario, where susceptible infections substituted all drug-resistant infections, they estimated only deaths and DALYs directly due to AMR. For the second counterfactual scenario, where no infection substituted all drug-resistant infections, they estimated all deaths and DALYs related to resistant infections. Both estimates had different utilities; however, both could inform the development of intermediation strategies to regulate AMR spread.

The study approach comprised ten estimation steps within five all-encompassing modeling components, each with varied data requirements; consequently, input data for each modeling component also varied.

Study findings

Substituting drug-resistant infections by no infections (first counterfactual scenario) and susceptible infections (second counterfactual scenario) would have saved 4.95MN and 1.27MN deaths, respectively, in 2019, implying that in 2019, the global AMR burden related to drug-resistant infections for 88 pathogen–drug combinations was ~4.95MN deaths (95% UI), of which drug resistance alone caused 1.27MN deaths. Moreover, after ischaemic heart disease and stroke, AMR accounted for most deaths in 2019.

Additionally, the study analysis revealed that AMR-related all-age death rates were highest in some LMICs, as opposed to the common notion that the burden of bacterial AMR would be higher in high-resource settings with higher antibiotic consumption. Indeed, AMR is emerging as a more serious problem for some of the world’s poorest countries. The authors noted the highest AMR-related death rates in sub-Saharan Africa and South Asia as a function of the prevalence of resistance and critical lower respiratory, bloodstream, and intra-abdominal infections, in these regions.

The study also highlighted that in LMICs, there are other drivers of the higher AMR burden, like a scarcity of laboratory infrastructure for microbiological testing needed to narrow antibiotic use or make it more targeted. Among other factors, counterfeit antibiotics, poor sanitation and hygiene, poor regulations on antibiotics use, etc., also drive resistance.

Further, the researchers identified six pathogens, E. coli, K. pneumoniae, S. pneumoniae, A. baumannii, S. aureus, and P. aeruginosa, who contributed most to the burden of AMR in 2019; they accounted for 73.4% (95% uncertainty interval) of deaths attributable to bacterial AMR. WHO has recognized all six as priority pathogens; however, except S. pneumoniae, targeted primarily through pneumococcal vaccination, none is the focus of global health intervention programs.

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Seven pathogen–drug combinations caused more than 50000 deaths, highlighting the need for expanding infection prevention and control (IPC) policies targeting the deadliest combinations, bolstering vaccine and antibiotic development, and improving access to essential second-line antibiotics where needed. Furthermore, resistance to β-lactam antibiotics, e.g., penicillins and cephalosporins, and fluoroquinolones accounted for >70% of deaths attributable to AMR across pathogens. These antibiotics are the first line of empirical treatment for severe infections.

In 2017, the WHO published a priority list to inform research priorities related to new antibiotics for pathogens with multidrug resistance that caused deadly infections. However, this list covered only five of the seven pathogen–drug combinations estimated to have caused the most deaths in 2019; for instance, this list did not feature fluoroquinolone-resistant E. coli and meticillin-resistant S. aureus only as a “high” but not a “critical” priority.

Per study estimates, the magnitude of bacterial AMR as a global public health issue is as much as human immunodeficiency virus (HIV) and malaria, perhaps, much higher. Additionally, the AMR pattern varied with geographical location, pathogens, and pathogen–drug combinations. Thus, the regional estimates made in this study could help tailor local responses as the ‘One Size Fits All’ approach might not be appropriate.

Despite concerted data collection efforts, high-quality data on AMR was sparsely available for many LMICs. Nevertheless, an improved scientific understanding of this rapidly emerging health threat should be the highest priority for global health policymakers.

Conclusions

The present study used major methodological innovations, two varying AMR counterfactual scenarios, and comprehensive data to fetch novel insights into the global AMR burden. Most importantly, it incorporated models tested and iterated over years during GBD study analysis. So, when used collectively, these models provided a complete estimate of AMR burden with robust geographical coverage.

Further, the researchers compared findings with other causes of death, offering much-needed context on the scale of the burden of this rapidly growing public health problem. The study analysis confirmed that bacterial AMR posed the biggest threat to human health in sub-Saharan Africa and South Asia, involved a diverse set of pathogens, and is exceptionally high for multiple essential antibiotic classes, including β-lactams and fluoroquinolones.

Furthermore, efforts to build and enhance laboratory infrastructure and bolster national & global AMR plans of action are essential to addressing the universal AMR burden. Future studies should also evaluate the indirect effects of AMR, such as its effect on the prophylaxis of infections in organ transplant recipients.

In the future, the study estimates could inform treatment guidelines against many predominant bacterial pathogens for a given infectious syndrome, which, along with estimates of pathogen–drug burden, could inform their treatment guidelines customized for a specific location.

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Journal reference:
Microbiology

Study expands the knowledge about gut viral diversity in healthy infants

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Reviewed by Emily Henderson, B.Sc.Apr 11 2023

Viruses are usually associated with illness. But our bodies are full of both bacteria and viruses that constantly proliferate and interact with each other in our gastrointestinal tract. While we have known for decades that gut bacteria in young children are vital to protect them from chronic diseases later on in life, our knowledge about the many viruses found there is minimal.

A few years back, this gave University of Copenhagen professor Dennis Sandris Nielsen the idea to delve more deeply into this question. As a result, a team of researchers from COPSAC (Copenhagen Prospective Studies on Asthma in Childhood) and the Department of Food Science at UCPH, among others, spent five years studying and mapping the diaper contents of 647 healthy Danish one-year-olds.

“We found an exceptional number of unknown viruses in the feces of these babies. Not just thousands of new virus species – but to our surprise, the viruses represented more than 200 families of yet to be described viruses. This means that, from early on in life, healthy children are tumbling about with an extreme diversity of gut viruses, which probably have a major impact on whether they develop various diseases later on in life,” says Professor Dennis Sandris Nielsen of the Department of Food Science, senior author of the research paper about the study, now published in Nature Microbiology.

The researchers found and mapped a total of 10,000 viral species in the children’s feces – a number ten times larger than the number of bacterial species in the same children. These viral species are distributed across 248 different viral families, of which only 16 were previously known. The researchers named the remaining 232 unknown viral families after the children whose diapers made the study possible. As a result, new viral families include names like Sylvesterviridae, Rigmorviridae and Tristanviridae.

Bacterial viruses are our allies

This is the first time that such a systematic an overview of gut viral diversity has been compiled. It provides an entirely new basis for discovering the importance of viruses for our microbiome and immune system development. Our hypothesis is that, because the immune system has not yet learned to separate the wheat from the chaff at the age of one, an extraordinarily high species richness of gut viruses emerges, and is likely needed to protect against chronic diseases like asthma and diabetes later on in life.”

Shiraz Shah, first author and senior researcher at COPSAC

Ninety percent of the viruses found by the researchers are bacterial viruses – known as bacteriophages. These viruses have bacteria as their hosts and do not attack the children’s own cells, meaning that they do not cause disease. The hypothesis is that bacteriophages primarily serve as allies:

“We work from the assumption that bacteriophages are largely responsible for shaping bacterial communities and their function in our intestinal system. Some bacteriophages can provide their host bacterium with properties that make it more competitive by integrating its own genome into the genome of the bacterium. When this occurs, a bacteriophage can then increase a bacterium’s ability to absorb e.g. various carbohydrates, thereby allowing the bacterium to metabolize more things,” explains Dennis Sandris Nielsen, who continues:

“It also seems like bacteriophages help keep the gut microbiome balanced by keeping individual bacterial populations in check, which ensures that there are not too many of a single bacterial species in the ecosystem. It’s a bit like lion and gazelle populations on the savannah.”

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Shiraz Shah adds:

“Previously, the research community mostly focused on the role of bacteria in relation to health and disease. But viruses are the third leg of the stool and we need to learn more about them. Viruses, bacteria and the immune system most likely interact and affect each other in some type of balance. Any imbalance in this relationship most likely increases the risk of chronic disease.”

The remaining ten percent of viruses found in the children are eukaryotic – that is, they use human cells as hosts. These can be both friends and foes for us:

“It is thought-provoking that all children run around with 10-20 of these virus types that infect human cells. So, there is a constant viral infection taking place, which apparently doesn’t make them sick. We just know very little about what’s really at play. My guess is that they’re important for training our immune system to recognise infections later. But it may also be that they are a risk factor for diseases that we have yet to discover,” says Dennis Sandris Nielsen.

Could play an important role in inflammatory diseases

The researchers have yet to discover where the many viruses in the one-year-olds come from. Their best answer thus far is the environment:

“Our gut is sterile until we are born. During birth, we are exposed to bacteria from the mother and environment. It is likely that some of the first viruses come along with these initial bacteria, while many others are introduced later via dirty fingers, pets, dirt that kids put in their mouths and other things in the environment,” says Dennis Sandris Nielsen.

As Shiraz Shah points out, the entire field of research speaks to a huge global health problem:

“A lot of research suggests that the majority of chronic diseases that we’re familiar with – from arthritis to depression – have an inflammatory component. That is, the immune system is not working as it ought to – which might be because it wasn’t trained properly. So, if we learn more about the role that bacteria and viruses play in a well-trained immune system, it can hopefully lead us to being able to avoid many of the chronic diseases that afflict so many people today.”

The research groups have begun investigating the role of gut viruses in relation to a number of different diseases that occur in childhood, such as asthma and ADHD.

Source:

University of Copenhagen – Faculty of Science

Journal reference:

Shah, S. A., et al. (2023). Expanding known viral diversity in the healthy infant gut. Nature Microbiology. doi.org/10.1038/s41564-023-01345-7.

Microbiology

Researchers report surprising first steps that promote resistance to commonly prescribed antibiotics

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Reviewed by Emily Henderson, B.Sc.Mar 31 2023

Antibiotic resistance is a global health threat. In 2019 alone, an estimated 1.3 million deaths were attributed to antibiotic resistant bacterial infections worldwide. Looking to contribute a solution to this growing problem, researchers at Baylor College of Medicine have been studying the process that drives antibiotic resistance at the molecular level.

They report in the journal Molecular Cell crucial and surprising first steps that promote resistance to ciprofloxacin, or cipro for short, one of the most commonly prescribed antibiotics. The findings point at potential strategies that could prevent bacteria from developing resistance, extending the effectiveness of new and old antibiotics.

Previous work in our lab has shown that when bacteria are exposed to a stressful environment, such as the presence of cipro, they initiate a series of responses to attempt to survive the toxic effect of the antibiotic.”

Dr. Susan M. Rosenberg, co-corresponding author, Ben F. Love Chair in Cancer Research and professor of molecular and human genetics, biochemistry and molecular biology and molecular virology and microbiology at Baylor

She also is program leader in Baylor’s Dan L Duncan Comprehensive Cancer Center (DLDCCC). “We discovered that cipro triggers cellular stress responses that promote mutations. This phenomenon, known as stress-induced mutagenesis, generates mutant bacteria, some of which are resistant to cipro. Cipro-resistant mutants keep on growing, sustaining an infection that can no longer be eliminated with cipro.”

Cipro induces breaks in the DNA molecule, which accumulate inside bacteria and consequently trigger a DNA damage response to repair the breaks. The Rosenberg lab’s discoveries of the steps involved in stress-induced mutagenesis revealed that two stress responses are essential: the general stress response and the DNA-damage response.

Some of the downstream steps of the process that leads to increased mutagenesis have been revealed previously by the Rosenberg lab and her colleagues. In this study, the researchers discovered the molecular mechanisms of the first steps between the antibiotic causing DNA breaks and the bacteria turning on the DNA damage response.

“We were surprised to find an unexpected molecule involved in modulating DNA repair,” said first author Dr. Yin Zhai, postdoctoral associate in the Rosenberg lab. “Usually, cells regulate their activities by producing specific proteins that mediate the desired function. But in this case, the first step to turn on the DNA repair response was not about activating certain genes that produce certain proteins.”

Instead, the first step consisted of disrupting the activity of a protein already present, RNA polymerase. RNA polymerase is key to protein synthesis. This enzyme binds to DNA and transcribes DNA-encoded instructions into an RNA sequence, which is then translated into a protein.

“We discovered that RNA polymerase also plays a major role in regulating DNA repair,” Zhai said. “A small molecule called nucleotide ppGpp, which is present in bacteria exposed to a stressful environment, binds to RNA polymerase through two separate sites that are essential for turning on the repair response and the general stress response. Interfering with one of these sites turns off DNA repair specifically at the DNA sequences occupied by RNA polymerase.”

“ppGpp binds to DNA-bound RNA polymerase, telling it to stop and backtrack along the DNA to repair it,” said co-corresponding author Dr. Christophe Herman, professor of molecular and human genetics, molecular virology and microbiology and member of the DLDCCCC. The Herman lab found the repair-RNA-polymerase connection previously, reported in Nature.

Rosenberg’s lab discovered that DNA repair can be an error-prone process. As repair of the broken DNA strands progresses, errors occur that alter the original DNA sequence producing mutations. Some of these mutations will confer bacteria resistance to cipro. “Interestingly, the mutations also induce resistance to two other antibiotic drugs the bacteria have not seen before,” Zhai said.

“We are excited about these findings,” Rosenberg said. “They open new opportunities to design strategies that would interfere with the development of antibiotic resistance and help turn the tide on this global health threat. Also, cipro breaks bacterial DNA in the same way that the anti-cancer drug etoposide breaks human DNA in tumors. We hope this may additionally lead to new tools to combat cancer chemotherapy resistance.”

Other contributors to this work include P.J. Minnick, John P. Pribis, Libertad Garcia-Villada and P.J. Hastings, all at Baylor College of Medicine.

Source:

Baylor College of Medicine

Journal reference:

Zhai, Y., Minnick, P. J., Pribis, J. P., Garcia-Villada, L., Hastings, P. J., Herman, C., & Rosenberg, S. M. (2023). ppGpp and RNA-polymerase backtracking guide antibiotic-induced mutable gambler cells. Molecular Cell. doi.org/10.1016/j.molcel.2023.03.003.

Microbiology

Usefulness of dried blood spot samples for monitoring HCV infection in people who inject drugs

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Reviewed by Emily Henderson, B.Sc.Mar 17 2023

A study with people who inject drugs evaluated a minimally invasive test based on dried blood spots (DBS) for the monitoring of hepatitis C virus (HCV) infection. The use of DBS samples for HCV RNA detection and genotyping was shown to effectively assess cure after treatment and to differentiate between reinfection and treatment failure. The results support the viability of decentralizing treatment and post-treatment monitoring for people who inject drugs, who frequently face challenges accessing the healthcare system. The study, which has been published in the Journal of Medical Virology, was carried out as part of a project with support from the “Conquering Hepatitis Via Microelimination” (CHIME) programme and a PFIS grant. Investigators from various research institutions collaborated in the project, including the Clinical Virology and New Diagnostic Tools research group, led by Dr Elisa Martró, at Germans Trias i Pujol Research Institute (IGTP) and Dr Sabela Lens from Hospital Clínic’s Viral Hepatitis Group.

Towards elimination of hepatitis

In line with the strategy proposed by the World Health Organization for the elimination of viral hepatitis as a public health threat by 2030, and the Plan for Prevention and Control of Hepatitis in Catalonia, which Dr Martró actively participates in, her group has been focused for years on simplifying the diagnosis of hepatitis C by developing and validating an assay which can detect the virus RNA using DBS samples. These minimally invasive samples can be collected at harm reduction centres or drug dependence care and follow-up centers (known as CAS in Catalan), improving access to hepatitis C diagnosis for vulnerable populations, such as people who inject drugs. While this new test has demonstrated good clinical performance as a diagnostic tool for detecting HCV RNA before treatment in previous studies by the Clinical Virology and New Diagnostic Tools research group, the use of DBS samples had not been evaluated as a test for cure or for detecting reinfection after treatment.

A multidisciplinary research group has been able to pursue a project with a new model of care for hepatitis C, based on point-of-care diagnosis, treatment, and reinfection follow-up at the REDAN La Mina harm reduction centre. Since 2019, approximately 750 individuals who inject drugs have been tested though this initiative, which was designed by Dr Sabela Lens from Hospital Clínic’s Viral Hepatitis Unit, in collaboration with the Clinical Virology and New Diagnostic Tools Research Group at Germans Trias i Pujol Research Institute (IGTP), led by Dr Martró from the Microbiology Service (LCMN) of the Germans Trias i Pujol Hospital (HUGTiP), as well as CEEISCAT and the Public Health Agency of Catalonia. The project had the support of the “Conquering Hepatitis Via Microelimination” (CHIME) programme from Gilead Sciences awarded to Dr Lens, as well as a PFIS grant of the Instituto de Salud Carlos III and the Fondo Social Europeo awarded to Anna Not, who is a member of Dr Martró’s group, and aligns with the World Health Organization’s global health strategy, which aims to eliminate hepatitis C as a public health problem by 2030.

A model of decentralized care

In this project, Dr Martró’s group aimed to evaluate the clinical performance of a previously developed HCV-RNA assay based on DBS, for the assessment of cure and the detection of recurrent viremia after on-site treatment at the harm reduction center, compared to the commercially available HCV-RNA point-of-care test. Furthermore, they sought to assess the possibility of distinguishing between reinfection and treatment failure through HCV genotyping from baseline and follow-up DBS samples. Typically, these assessments (cure and reinfection) are performed using venipuncture blood samples collected at healthcare centres, which can be difficult for people who inject drugs and have often limited access to the healthcare system. The recently published results demonstrate how the collection of DBS samples before and after treatment can simplify these assessments in decentralized test-and-treat programmes.

“The success of the CHIME project lies in the decentralized diagnosis and treatment provided at REDAN La Mina. A nurse trained in hepatology assessments was included in the study to enrol and visit participants. The hepatologists at Hospital Clínic also reviewed each case and prescribed decentralized treatment. Additionally, Dr Martró’s group carried out HCV detection and sequencing from DBS samples collected before and after treatment. This pilot program involves HCV diagnosis on-site in less than an hour, treatment at the same center, and follow-up to assess reinfection”, states Dr Lens.

Detection made easier

Reinfection is common in people who inject drugs and must be treated to prevent further transmission of the virus. During early reinfection, low levels of the virus may be present, making its detection in DBS samples challenging, as they only contain a small amount of blood. Of the 193 DBS samples tested after treatment, the DBS-based assay showed 100% specificity and sensitivity ranging from 84% to 96% based on different relevant viral load cut-offs, and similar rates as a test of cure (three months after treatment). It must be born in mind that among the patients with recurrent viremia after treatment, one tenth had low viral loads. Moreover, HCV genotyping allowed researchers to classify 73% of viremic cases as either reinfection or treatment failure.

Collection of DBS samples was done before antiviral treatment and after treatment if recurrent viremia was detected by the commercially available point-of-care assay. Anna Not, the first author of the article (which will be part of her PhD), explains that it “the use of DBS allowed us to sequence the virus before and after treatment and compare the sequences to determine if the virus was the same (indicating a treatment failure) or if it was different (indicating reinfection). This information enabled the hepatologist to decide on the most appropriate antiviral combination for the second treatment”.

The research shows the potential of using DBS samples for determining cure and differentiating between reinfection and relapse after antiviral treatment for hepatitis C in people who inject drugs. The use of DBS samples makes it possible to decentralize treatment and follow-up, improving access to care for these people. Even so, Dr Martró points out that “a small number of patients had low viral loads, which can hinder the detection of viremia and genotyping in DBS. As a result, repeat testing (e.g. every six months) is advised for individuals who are at risk of HCV reinfection”.

Source:

Germans Trias i Pujol Research Institute

Journal reference:

Not, A., et al. (2023) Usefulness of dried blood spot samples for monitoring hepatitis C treatment outcome and reinfection among people who inject drugs in a test-and-treat program. Journal of Medical Virology. doi.org/10.1002/jmv.28544.

Microbiology

Research identifies western diet-induced microbial and metabolic contributors to liver disease

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Reviewed by Emily Henderson, B.Sc.Mar 15 2023

New research from the University of Missouri School of Medicine has established a link between western diets high in fat and sugar and the development of non-alcoholic fatty liver disease, the leading cause of chronic liver disease.

The research, based in the Roy Blunt NextGen Precision Health Building at MU, has identified the western diet-induced microbial and metabolic contributors to liver disease, advancing our understanding of the gut-liver axis, and in turn the development of dietary and microbial interventions for this global health threat.

We’re just beginning to understand how food and gut microbiota interact to produce metabolites that contribute to the development of liver disease. However, the specific bacteria and metabolites, as well as the underlying mechanisms were not well understood until now. This research is unlocking the how and why.”

Guangfu Li, PhD, DVM, co-principal investigator, associate professor in the department of surgery and Department of Molecular Microbiology and Immunology

The gut and liver have a close anatomical and functional connection via the portal vein. Unhealthy diets change the gut microbiota, resulting in the production of pathogenic factors that impact the liver. By feeding mice foods high in fat and sugar, the research team discovered that the mice developed a gut bacteria called Blautia producta and a lipid that caused liver inflammation and fibrosis. That, in turn, caused the mice to develop non-alcoholic steatohepatitis or fatty liver disease, with similar features to the human disease.

“Fatty liver disease is a global health epidemic,” said Kevin Staveley-O’Carroll, MD, PhD, professor in the department of surgery, one of the lead researchers. “Not only is it becoming the leading cause of liver cancer and cirrhosis, but many patients I see with other cancers have fatty liver disease and don’t even know it. Often, this makes it impossible for them to undergo potentially curative surgery for their other cancers.”

As part of this study, the researchers tested treating the mice with an antibiotic cocktail administered via drinking water. They found that the antibiotic treatment reduced liver inflammation and lipid accumulation, resulting in a reduction in fatty liver disease. These results suggest that antibiotic-induced changes in the gut microbiota can suppress inflammatory responses and liver fibrosis.

Li, Staveley-O’Carroll and fellow co-principal investigator R. Scott Rector, PhD, Director of NextGen Precision Health Building and Interim Senior Associate Dean for Research -; are part of NextGen Precision Health, an initiative to expand collaboration in personalized health care and the translation of interdisciplinary research for the benefit of society. The team recently received a $1.2 million grant from the National Institutes of Health to fund this ongoing research into the link between gut bacteria and liver disease.

Source:

University of Missouri-Columbia

Journal reference:

Yang, M., et al. (2023). Western diet contributes to the pathogenesis of non-alcoholic steatohepatitis in male mice via remodeling gut microbiota and increasing production of 2-oleoylglycerol. Nature Communications. doi.org/10.1038/s41467-023-35861-1.

Microbiology

Wayne State team discovers a simple technology to detect active TB infection antibodies

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Reviewed by Emily Henderson, B.Sc.Mar 13 2023

A team of faculty from Wayne State University has discovered new technology that will quickly and easily detect active Mycobacterium tuberculosis (TB) infection antibodies. Their work, “Discovery of Novel Transketolase Epitopes and the Development of IgG-Based Tuberculosis Serodiagnostics,” was published in a recent edition of Microbiology Spectrum, a journal published by the American Society for Microbiology. The team is led by Lobelia Samavati, M.D., professor in the Center for Molecular Medicine and Genetics in the School of Medicine. Samavati was joined by Jaya Talreja, Ph.D, and Changya Peng, research scientists in Wayne State’s Department of Internal Medicine.

TB remains a global health threat, with 10 million new cases and 1.7 million deaths annually. According to the latest World Health Organization report, TB is the 13th leading cause of death and the second leading infectious killer after COVID-19. Latent tuberculous infection (LTBI) is considered a reservoir for TB bacteria and subjects can progress to active TB. One-third of the world’s population is infected with TB and, on average, 5 to 10% of those infected with LTBI will develop active TB disease over the course of their lives, usually within the first five years after initial infection.

The gold standard tests to determine whether an infection is active TB are the sputum smear and culture tests. However, these methods require collecting sputum, which is time consuming, expensive, requires trained personnel and lacks sensitivity. The current conventional tests differentiating LTBI from uninfected controls -; such as tuberculin skin tests (TST) and/or interferongamma release assay (IGRA) -; do not differentiate between active TB infection and latent TB. Despite advances in rapid molecular techniques for TB diagnostics, there is an unmet need for a simple inexpensive point-of-care (POC), rapid non-sputum-based test.

Samavati’s research group has worked for more than 15 years to develop technology for detection of antibodies in various respiratory diseases. Her lab has developed a novel non-sputum based technology and has discovered several novel immune-epitopes that differentialy bind to specific immunoglobulin (IgG) in TB-infected subjects. The levels of epitope-specific IgG in seum can differentiate active TB from LTBI subjects, healthy contols and other respiratory diseases. This technology can be used as a simple serum assay non-sputum based serological POC- TB test, which is highly specific and sensitveto diffentiate active TB from LTBI.

“Previously, we developed a T7 phage antigen display platform and after immunoscreening of large sets of serum samples, identified 10 clones that differentially bind to serum antibody (IgG) of active TB patients differentiating TB from other respiratory diseases,” said Samavati. “One of these high-performance clones had homology to the Transketolase (TKT) enzyme of TB bacteria that is an essential enzyme required for the intracellular growth of the bacteria in a host. We hypothesized that abundance of IgG in sera against the identified novel neoantigen that we named as TKTµ may differentiate between active TB, LTBI and other non-TB granulomatous lung diseases such as sarcoidosis. We developed a novel direct Peptide ELISA test to quantify the levels of IgG in serum samples against TKTµ. We designed two additional overlapping M.tb TKT-peptide homologs with potential antigenicity corresponding to M.tb-specific transketolase (M.tb-TKT1 and M.tb-TKT3) and hence standardized three Peptide ELISA (TKTμ, M.tb TKT1 and M.tb TKT3) for the TB serodiagnosis.”

After development and standardization of a direct peptide ELISA for three peptides, the research team tested 292 subjects, and their TKT-peptide ELISA results show that TB patients have significantly higher levels of TKT-specific antibodies compared to patients who were healthy controls and with LTBI. The increased levels of TKT-specific antibodies is presumably associated with growing M.tb bacteria in active TB patients. TKT plays a key role in the switch from the dormancy to proliferative phase and TKT specific IgG may uncover the differences between active TB and LTBI. Thus, IgG-based serodiagnosis of TB with TKT-peptide ELISA is promising.

Currently, commercially available serological TB tests show poor sensitivity and specificity. The ELISA results obtained with the Wayne State team’s discovered TKT peptides yielded high specificity and sensitivity. Their results show that IgG antibodies against transketolase can discriminate active tuberculosis. 

Our TKT peptide ELISA test requires chemically synthesized TKT peptides to coat the wells in the ELISA plate, less than 100µl blood serum sample from patient, detection reagents and an ELISA plate reader. We are extremely enthusiastic about our technology and the fact that with a simple test we can differentiate active TB from LTBI and other respiratory diseases. We believe that our method and TKT peptide ELISA can fit the requirements of the World Health Organization and the Centers for Disease Control and Prevention as a POC screening method.”

Lobelia Samavati, M.D., Professor, Center for Molecular Medicine and Genetics, School of Medicine, Wayne State University

The research team has applied a patent application on its technology and is actively seeking companies interested in investing.

This research was supported by the National Heart, Lung and Blood Institute of the National Institutes of Health, grant numbers 113508 and 148089. The Foundation for Innovative New Diagnostics (FIND, Geneva, Switzerland) provided TB and LTBI samples.

Source:

Wayne State University

Journal reference:

Talreja, J., et al. (2023). Discovery of Novel Transketolase Epitopes and the Development of IgG-Based Tuberculosis Serodiagnostics. Microbiology Spectrum. doi.org/10.1128/spectrum.03377-22.

Microbiology

New study focuses on genetic diversity of E. coli bacteria in hospitalized patients

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Reviewed by Emily Henderson, B.Sc.Mar 13 2023

The human intestine is an environment inhabited by many bacteria and other microorganisms collectively known as the gut microbiome, gut microbiota or intestinal flora. In most people, it contributes to wellness. A healthy gut indicates a stronger immune system, improved metabolism, and a healthy brain and heart, among other functions.

Escherichia coli is one of the bacteria found in practically everyone’s gut microbiota, where it performs important functions, such as producing certain vitamins.

But there’s a vast amount of genetic diversity in the species. Some of its members are pathogenic and can cause diseases such as urinary tract infections. E. coli is the main agent of this type of infection among both healthy people and hospitalized patients or users of healthcare services.”

Tânia Gomes do Amaral, Head of the Experimental Enterobacterial Pathogenicity Laboratory (LEPE), Federal University of São Paulo’s Medical School (EPM-UNIFESP), Brazil

Amaral is first author of an article published in the journal Pathogens on the virulence of these bacteria and their resistance to antibiotics in hospitalized patients.

“Our study focused on hospitalized patients because patients who stay in hospital for a long period are more likely to undergo various procedures, such as urine catheter insertion or venous access. Although these procedures are performed to assure life support, they may facilitate the entry of bacteria into the organism and cause an infection,” Amaral explained.

She earned a PhD in microbiology from EPM-UNIFESP in 1988, conducting part of her research at New York University Medical School and the Center for Vaccine Development at the University of Maryland, Baltimore (UMB) in the United States.

The article reports the findings of a broader study led by Amaral, with 12 co-authors who are researchers and graduate students, on the virulence and drug resistance of E. coli strains associated with urinary tract infections. The study was supported by FAPESP via three projects (18/17353-7, 19/21685-8 and 17/14821-7).

The main aim of this part of the study, described in the master’s dissertation of José Francisco Santos Neto, was to evaluate the diversity and drug resistance of pathogenic E. coli strains isolated from the gut microbiota of inpatients, and to analyze the frequency of endogenous infection (caused by bacteria from the patient’s own microbiota).

The UNIFESP group first investigated the genetic diversity and drug resistance of E. coli strains isolated from the gut microbiota of hospitalized patients, sequencing these strains as well as others isolated from their urine and comparing the results in order to evaluate dissemination of the bacteria in the hospital environment.

“We also compared the genomes of these strains with those of E. coli strains isolated in different parts of the world in order to see if any globally disseminated pathogenic bacteria were present in the study sample,” said Ana Carolina de Mello Santos, a postdoctoral researcher working on the LEPE team.

Urinary tract infections proved to be endogenous for the vast majority of the patients in the study (more than 70%). The results also showed that the patients’ gut microbiota contained at least two genetically different populations of E. coli and that about 30% were colonized by non-lactose-fermenting E. coli strains, which are less common, with some of the patients studied having only such strains in their gut microbiota.

“This finding is most interesting because previous research conducted in other countries to analyze the composition of human gut microbiota didn’t investigate non-lactose-fermenting E. coli,” Santos said.

The authors also note the presence of bacteria with all the genetic markers required for classification as pathogenic and the detection of pathogenic bacteria in the gut microbiota of all patients that had not yet developed an infection. “Hospitalized patients are more susceptible to infection because by definition they are already unwell. Colonization by pathogens is the first step in the spread of hospital-acquired infections now so frequent worldwide,” Santos said.

With regard to antibiotics and other antimicrobials, the authors stress that drug resistance is also a growing global problem, and enterobacterial resistance to third-generation cephalosporins as well as colistin is critical. In all patients whose gut microbiota was colonized by drug-resistant bacteria, the same bacteria also caused endogenous urinary tract infections. In other words, the multidrug-resistant bacteria colonized the gut and traveled to the urinary tract, where they caused an infection.

“In light of these findings, early assessment of gut microbiota in hospitalized patients, at least in cases of E. coli infection, can facilitate and guide their treatment, while also identifying patients who risk progressing to extra-intestinal diseases such as urinary tract infections, which were part of the focus for our study,” Amaral said. “We don’t yet know whether the findings also apply to other bacteria found in gut microbiota, such as the genera Klebsiella, Enterobacter, Pseudomonas and others that can cause infections when they travel to extra-intestinal sites.”

These bacterial genera tend to be even more drug-resistant than E. coli, representing a major public health problem in the hospital environment. As the researchers noted, the World Health Organization (WHO) considers E. coli strains resistant to cephalosporin and colistin to be a critical global health threat. “The presence in human gut microbiota of drug-resistant bacteria associated with severe infectious disease is a matter of great concern, not least because they could spread to people outside the hospital environment,” Amaral said.

Another point raised by the study is the importance of finding out when colonization of the patient’s gut by drug-resistant virulent bacteria occurred. The authors of the article were unable to determine whether the bacteria resistant to cephalosporins and colistin colonized the patients before or after they were hospitalized.

By analyzing the genomes of the strains, however, the researchers were able to identify global risk clones that can cause severe disease and are associated with antimicrobial resistance. “One such clone found in the gut microbiota of two patients was identical to others isolated from urinary tract infections in Londrina, Paraná [a state in South Brazil], and in the United States, as well as European and Asian countries. This shows that some strains found in the study are clones generally associated with infections in all regions of the world,” Amaral said.

This type of information is important when patients are hospitalized. Knowledge of bacterial virulence and drug resistance can be used to prevent infection in parts of the organism outside the intestine and stop the bacteria from spreading to other patients in the same hospital.

Source:

São Paulo Research Foundation (FAPESP)

Journal reference:

Santos-Neto, J.F., et al. (2023) Virulence Profile, Antibiotic Resistance, and Phylogenetic Relationships among Escherichia coli Strains Isolated from the Feces and Urine of Hospitalized Patients. Pathogens. doi.org/10.3390/pathogens11121528.

Microbiology

New analysis shows how convalescent plasma can be used as effective, low-cost COVID-19 treatment

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Reviewed by Emily Henderson, B.Sc.Feb 22 2023

Three years into the COVID-19 pandemic, new variant outbreaks continue to fuel economic disruptions and hospitalizations across the globe. Effective therapies remain unavailable in much of the world, and circulating variants have rendered monoclonal antibody treatments ineffective. But a new analysis shows how convalescent plasma can be used as an effective and low-cost treatment both during the COVID-19 pandemic and in the inevitable pandemics of the future.

In astudy published in Clinical Infectious Diseases, an international team of researchers analyzed clinical data and concluded that among outpatients with COVID-19, antibodies to SARS-CoV-2 given early and in high dose reduced the risk of hospitalization.

If the results of this meta-analysis had somehow been available in March of 2020, then I am certain that millions of lives would have been saved around the world.”

Dr. Adam C. Levine, study author, professor of emergency medicine at Brown University’s Warren Alpert Medical School

While several other early treatments for COVID-19 have had similar results, including antivirals like Paxlovid and monoclonal antibodies, only convalescent plasma, the researchers concluded, is likely to be both available and affordable for the majority of the world’s population both now and early in the next viral pandemic.

“These findings will be helpful for this pandemic, especially in places like China, India and other parts of the world that lack access to antiviral medications like Paxlovid,” Levine said. “And because it provides information on how to more effectively use convalescent plasma as a therapy, this will be even more helpful in the next pandemic. This study is essentially a roadmap for how to do this right the next time.”

Blood plasma from people who have recovered from COVID-19 and contains antibodies against SARS-CoV-2 was used as a treatment early in the pandemic, Levine said -; months before monoclonal antibody treatment or vaccines became available, and more than a year before an effective oral drug therapy was clinically available.

Although convalescent plasma seemed promising, outpatient research was limited, and studies that did exist showed mixed results. One problem was that most studies were conducted in patients already hospitalized with COVID-19, Levine said, largely due to the convenience of conducting research with this population. The objective in the new study was to review all available randomized controlled trials of convalescent plasma in non-hospitalized adults with COVID-19 to determine whether early treatment can reduce the risk of hospitalization.

The analysis included data from five studies conducted in four countries, including Argentina, the Netherlands, Spain, and two in the United States. Levine previously supervised enrollment at Rhode Island Hospital in a clinical trial led by Johns Hopkins Medicine and Johns Hopkins Bloomberg School of Public Health. Across the five studies, a total of 2,620 adult patients had received transfusions of convalescent plasma from January 2020 to September 2022. The researchers conducted an individual participant data meta-analysis to assess how the transfusion timing and dose impacted the patient’s risk of hospitalization during the 28 days after infection.

In their analysis, the researchers found that 160 (12.2%) of 1,315 control patients were hospitalized compared with 111 (8.5%) of 1,305 patients treated with COVID-19 convalescent plasma -; 30% fewer hospitalizations.

Notably, the strongest effects were seen in patients treated both early in the illness and with plasma with high levels of antibodies. In these patients, the reduction in hospitalization was over 50%.

For future pandemics, the goal is to use plasma from donors who have high levels of antibodies, said corresponding study author Dr. David J. Sullivan, a professor of molecular microbiology and immunology at Johns Hopkins Bloomberg School of Public Health and School of Medicine. “This research suggests that we have been underdosing convalescent plasma for many previous pathogens, which impacts effectiveness,” Sullivan said. “It bears repeating: Early and high levels of antibodies increased the beneficial efficacy.”

Levine explained that because convalescent plasma was the only treatment available at the beginning of the pandemic, it was used widely -; and often incorrectly, on hospitalized patients who were already experiencing severe symptoms late in the course of COVID-19. Those symptoms were due to a ramped-up immune response to the virus, not the virus itself, Levine explained.

“By the time the patient was at the point where they’d reached the inflammatory phase that caused severe symptoms, it was too late for treatments like convalescent plasma or even monoclonal antibodies to work,” he said.

What is now known is that convalescent plasma works best when given early in the course of illness. That’s when it can neutralize the virus and get ahead of the body mounting an intense immune response, thereby preventing hospitalization and death, Levine said.

The five drug treatment trials in the analysis took place at a variety of global health care sites, he noted, including nursing homes, outpatient clinics and emergency departments. The diversity across the studies is a sign that the data is likely generalizable to many other types of populations and settings around the world, said Levine, who also directs the Center for Human Rights and Humanitarian Studies at the Watson Institute for International and Public Affairs at Brown.

Levine cited another recently published study in JAMA Network Open that showed that convalescent plasma is effective in reducing mortality in immunocompromised patients. This new meta-analysis provides evidence that convalescent plasma can also be effective in the larger population of adults who are not immunocompromised.

The U.S. Food and Drug Administration allowed early convalescent plasma use in December 2021 for those patients with COVID-19 who were also immunocompromised, but not yet for patients with COVID-19 who are not immunocompromised. The authors said they hope the new study will push the FDA, and other countries around the world, to make early treatment with COVID-19 convalescent plasma available to a much larger group of patients at risk for hospitalization.

A treatment that evolves with the pandemic

The findings come at a time when monoclonal antibodies, the most commonly used treatment for COVID-19, have been shown to be ineffective against new variants of the virus. In November, the FDA revoked emergency authorization of the last monoclonal antibody treatment because it wasn’t expected to have much of an effect against Omicron sub-variants.

In contrast to monoclonal antibody therapies, Levine said, convalescent plasma donated by patients who have recovered from the virus is a treatment that evolves with the pandemic. Because it has antibodies that attach to multiple different parts of the virus, there are still opportunities to attach to a receptor even after the virus mutates and morphs some of its receptors. It’s also less expensive to produce than pharmaceutical antivirals.

In the first year of the pandemic, Levine said, before the development of vaccines and effective treatments, researchers tried many treatment strategies in order to quickly find something that worked to save lives.

“When the next big pandemic hits, we’re going to be in a very similar situation,” Levine said. “Yet at least next time, we’ll have research like this to inform our strategy.”

Source:

Brown University

Journal reference:

Levine, A.C., et al. (2023) COVID-19 Convalescent Plasma Outpatient Therapy to Prevent Outpatient Hospitalization: A Meta-analysis of Individual Participant Data From Five Randomized Trials. Clinical Infectious Diseases. doi.org/10.1093/cid/ciad088.