Cornell researchers developed an imaging tool to create intricate spatial maps of the locations and identities of hundreds of different microbial species, such as those that make up the gut microbiome. The tool will help scientists understand how complex communities of microorganisms interact with each other and also their environment, which is to say, us.
WHAT MICROBES ARE IN YOUR GUT AND WHERE?
The team’s paper, “Highly Multiplexed Spatial Mapping of Microbial Communities,” published Dec. 2 in Nature. The paper’s lead author is doctoral student Hao Shi, M.Eng. ’18.
“There are communities of bacteria that live in our bodies and play an important role in human health and biology, and there’s a rich diversity of these microbes. We know this from technologies such as DNA sequencing that create lists of the bacterial species that are present in a community,” said Iwijn De Vlaminck, the Robert N. Noyce Assistant Professor in Life Science and Technology in the Meinig School of Biomedical Engineering, and the paper’s senior author.
“However, there are very limited tools to understand the spatial interactions between these microbes, and those are quite clearly important to understand the metabolism of these communities, and also how these microbes interact with their host,” he said.
De Vlaminck and Shi set out to create their imaging method by using a two-step process called high phylogenetic resolution microbiome mapping by fluorescence in situ hybridization (HiPR-FISH). They collaborated with the labs of co-authors Warren Zipfel, associate professor of biomedical engineering, and Ilana Brito, assistant professor and the Mong Family Sesquicentennial Faculty Fellow in Biomedical Engineering, to incorporate additional imaging and microbiome expertise.
To locate the microbial communities, the researchers designed oligonucleotide probes that target specific bacteria cells based on the presence of a signature gene sequence, 16S ribosomal RNA, and they made another group of probes that label the cells with fluorophores. Then the team used confocal microscopy to light up the fluorescent markers with lasers, and they used machine learning and custom software to decode the fluorescence spectra and interpret the images, resulting in an efficient and cost-effective technology with single-cell resolution.
The researchers created the palette for their spatial maps with a mixture of 10 basic colors that could “paint” a total of 1,023 possible color combinations of E. coli, each fluorescently labeled with a unique binary barcode.
“The imaging itself leads to very beautiful, rich images with all bacterial cells in different colors,” De Vlaminck said. “But to allow the quantitative understanding of microbe interactions, the distances between cells, cluster sizes and so on, you need to be able to interpret these in an automated way by a computer so that you can convert this image into a digitized representation of the community.”
The team applied their technology to two different systems: the gut microbiome in mice and the human oral plaque microbiome. In the case of the gut microbiome, they were able to demonstrate how the spatial associations between different bacteria are disrupted by antibiotic treatment.
Spatial mapping could be an important tool for studying and possibly treating a range of diseases in which bacteria are a major culprit, such as inflammatory bowel disease, colorectal cancer, and infection.
“We’d like to dig deeper into the biology of systems where microbiomes play important roles and try to understand how these kinds of spatial dynamics change when you have a disease in progression,” Shi said. “We want to see if that offers any clues and therapeutic insights that we can harness to help people.”
For example, new data published in The New England Journal of Medicine underscore the shortcomings of advances in testing technology, suggested Sahil Khanna, MBBS, an associate professor of medicine at Mayo Clinic College of Medicine and Science in Rochester, Minn. (2020;382:1320-1330).
At first glance, the study, which used data from 10 sites around the United States to derive a national estimate of the incidence of C. difficile infection (CDI), reported a relatively unchanged rate of the disease over a six-year period: 476,400 cases in 2011 and 462,100 cases in 2017. However, after adjusting for the increasing use of nucleic acid amplification testing (NAAT), the researchers concluded that the incidence of CDI had actually decreased by 24% during the study period, including a 36% drop in healthcare-acquired CDI cases.
The study highlights a problem with NAAT, according to Khanna. “NAAT is approximately 95% sensitive in detecting the C. difficile gene, but it cannot determine if the gene is active and toxin-producing, so it has the potential for overdiagnosis and for producing clinical false positives,” he explained. “Because of this, it’s important that we interpret NAAT results in the context of patient symptoms.”
Clinicians must be selective when deciding which patients should be tested, he said, adding that it only should be used in patients who have acute diarrhea with no obvious alternative explanation and risk factors for CDI such as older age, longer hospitalization, immunosuppression, use of antibiotics, gastric acid-suppressing agents, gastrointestinal surgery, manipulation of the gastrointestinal tract and tube feeding.
“Patients not experiencing an active infection can be colonized with C. difficile, in which case there is a risk of clinical false positives and unnecessary treatment,” Khanna emphasized.
An alternative testing approach recommended by the Infectious Diseases Society of America (IDSA) and the Society for Healthcare Epidemiology of America (SHEA) is the use of a multistep algorithm including glutamate dehydrogenase (GDH) to identify pathogenic bacteria and enzyme immunoassay (EIA) to detect C. difficile toxin (Clin Infect Dis 2018;66:e1-e48). NAAT should be reserved for instances in which results from GDH and EIA are inconclusive, the guidelines recommend.
Laboratories are increasingly adopting a two-step protocol of GDH and EIA, but “NAAT remains the most commonly used test method,” Khanna said.
The treatment landscape for CDI also has changed over the past few years, noted Kim Ly, PharmD, a clinical pharmacy specialist in critical care and infectious diseases at Sunrise Hospital and Medical Center, in Las Vegas. Bezlotoxumab (Zinplava, Merck), a monoclonal antibody, is approved for the combination treatment of toxin B–producing CDI, along with an established antibiotic. Additionally, metronidazole, while still approved for the treatment of CDI, is no longer recommended by IDSA/SHEA as a first-line agent for primary CDI in adults.
“For severe initial episodes of CDI, oral vancomycin and fidaxomicin [Dificid, Merck] are now the preferred agents, and metronidazole is only recommended for nonsevere initial episodes when patients are unable to be treated with oral vancomycin or fidaxomicin,” Ly explained.
For a first recurrence of CDI, the IDSA/SHEA guidelinesrecommend administering oral vancomycin as a tapered and pulsed regimen or fidaxomicin, rather than a standard 10-day course of vancomycin. For subsequent recurrences, clinicians can use the same regimen, with the addition of a standard course of oral vancomycin followed by rifaximin or fecal microbiota transplantation (FMT).
Metronidazole comes into play again in the management of fulminant CDI, Ly noted.“The IDSA/SHEA guidelines recommend treating this with oral or rectal vancomycin 500 mg four times daily along with intravenous metronidazole,” she explained.
Given that antibiotic-induced microbiota disruption “is far and away the number one precipitant for getting recurrent CDI,” selecting the CDI treatment with the least impact on the microbiota is important, said former IDSA president Cynthia Sears, MD, a professor in the Department of Medicine, Division of Infectious Diseases, at the Johns Hopkins University School of Medicine, in Baltimore.
“Vancomycin is the most commonly used therapy for CDI and its recurrences, but it decreases intestinal diversity and so impedes the recovery of the normal microbiota after CDI, setting the stage for CDI recurrence,” Sears said. “We have learned that vancomycin hits the colon with full force when taken orally because it is not absorbed, and it has off-target effects on lots of anaerobic bacteria that are essential to intestinal resistance of CDI.”
Fidaxomicin has less of an effect on the microbiota and has been shown to sometimes decrease the risk for CDI recurrence compared with vancomycin (N Engl J Med 2011;364:422-431), but it can be expensive, she said.
Fecal Microbiota Transplantation
FMT is a less expensive, highly effective treatment that has received increasingly widespread attention, specifically for the management of recurrent CDI. Despite the enthusiasm surrounding the treatment, Sears expressed significant reservations about employing it. “While there’s no question that FMT benefits patients with recurrent CDI, I feel we don’t yet have a quality-controlled product that we know is safe as well as being effective,” she said.
Sears pointed to two recent FDA safety alerts that warned of the harm that FMT can cause. The first, from 2019, reported that stool from a single donor had not been thoroughly screened before FMT and contained extended-spectrum beta-lactamase-producing Escherichia coli. The specimen had been used in separate FMTs for two immunocompromised patients, leading to infection with the pathogen and death in one case (https://bit.ly/2Teockd).
In another FDA safety alert from earlier this year, the organization said a stool bank specimen that had undergone comprehensive screening nevertheless contained enteropathogenic E. coli and Shiga toxin-producing E. coli. Transfer of the stool for the treatment of recurrent CDI resulted in one nonfatal infection and one death (https://bit.ly/31q5LO0).
“Stool banks try very hard to be sure their specimens are free of disease-causing microbes, but if you have very low-level colonization, molecular diagnostics can miss this,” Sears said. More recently, she noted, the FDA has also raised concerns about the possibility of transferring SARS-CoV-2 through FMT, given that the virus can be present in the stool of infected individuals (https://bit.ly/37sMPBX).
What would a safer and equally effective microbiota-based treatment look like?
According to Sears, although microbial diversity seems to be protective against recurrent CDI, there are suggestions that the administration of specific strains may be able to treat CDI and can be produced under the same strict quality control manufacturing processes as other FDA-approved drugs.
One study published in 2015 using human and mouse samples found that colonization with Clostridium scindens, a strain of Firmicutes, increased resistance to CDI (Nature 2015;517:205-208).
Many microbiota-based therapeutics are in the research pipeline as well.
“I am optimistic that we will see something emerge that’s safer and still as effective as FMT for patients,” Dr. Sears said, “whether it’s an orally or rectally administered product.”
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A new study published online in the journal ClinicalInfectious Diseases looked at the use of a non-frozen capsule of microbiome restoration therapy for treating patients with recurrent C. difficile infection.
“Patients with C. difficile are typically managed with antibiotics or fecal transplantation for recurrent C. difficile,” says the study’s author, Sahil Khanna, M.B.B.S., a gastroenterologist at Mayo Clinic. Dr. Khanna says fecal transplantation has been demonstrated to have high success rates by restoring the gut microbiome of patients. However, he says there are several challenges with fecal transplantation including standardization of the product, keeping it frozen, and mitigating the risk of infectious disease transmission during the procedure.
To help reduce the risks, Dr. Khanna and his team studied a transplantation method using a non-frozen capsule instead of whole stool transplantation. An initial dose-finding, the investigator-initiated study looked at the efficacy of different doses of fecal matter and the safety of performing microbiome restoration therapy using an oral product, RBX7455 developed by Rebiotix, Inc. The team found no concerns related to safety.
“Our study has several implications,” says Dr. Khanna. “We think that products like capsules may be able to replace fecal transplantation that is currently done via a colonoscopy. We also think that products that are non-frozen may allow for repeat dosing and for patient-administered self-treatment at home. The good news is that we are moving closer to having safe and effective products to restore the gut microbiome for patients with recurrent C. difficile.”
Dr. Khanna says that larger clinical trials and blinded, placebo-controlled trials are the next step in moving this potential treatment from research into practice.
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Microbiota Restoration in Recurrent C. difficile (C. diff. ) and COVID-19
This activity is intended for gastroenterologists, infectious disease specialists, and other clinicians who care for patients at risk of serious infection.
Activity Purpose: While the incidence of multiply recurrent C. difficile continues to rise, FMT has emerged as a means to break the cycle. Amid the current COVID-19 pandemic, clinicians should be aware of the additional safety concerns that have arisen. Expert faculty will discuss approaches to FMT including sourcing and screening of donor stool, which requires heightened safety measures during the COVID-19 pandemic. This activity is the first in a series of two educational activities that address the latest data on treating patients who experience rCDI.
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