mBiosphere

Tracking Bacterial Imbalance in the ICU

The human microbiome is the diverse population of microorganisms that live on and in the body. Many thrive on the skin and in the mouth, but the majority live in the intestines. Over the last decade or so, microbiologists have become increasingly aware of how a person's microbial mix likely plays a critical role in a variety of medical conditions, as well as how that person responds to treatment.

 

Maintaining a healthy balance of gut bacteria is particularly difficult in a hospital's intensive care unit, or ICU. Every year, more than 5.7 million critically-ill patients are admitted into ICUs in the United States. Treatments like strong antibiotics (to prevent infections), opioids (for pain), or blood pressure-supporting medications can save the sickest patients and prevent death, but they can also wipe out or change colonies of health-promoting microbes. Researchers have long suspected a connection between critical illness and a gut microbiome imbalance, or dysbiosis. This can leave a person vulnerable to the rapid growth and colonization of disease-causing bacteria and hospital-acquired infections – conditions that can lead to fatal conditions like sepsis.

 

“The original disease is bad, but our treatments are also damaging to normal bacteria, and potentially to a patient's long-term outcome,” says Paul Wischmeyer, an anesthesiologist at the University of Colorado-Boulder who also runs a lab dedicated to improving outcomes for critically-ill patients. “Sometimes we have to do these treatments, but what are we going to do to correct the damage our treatments with antibiotics and other drugs do?” 

 

Many ICU patients suffer long-term damage from treatments, he says. “Are we creating survivors or victims in our ICUs today, and can we do better?”

 

Wischmeyer is passionate about looking for nutritional interventions aimed at restoring a critically ill patient's bacterial balance. But developing such interventions will require deep knowledge of how patients' microbiomes respond to the conditions of the ICU – knowledge that's been lacking, mostly. Previous studies have tracked microbial changes in individuals and small numbers of ICU patients, but Wischmeyer had something bigger in mind.

 

He recently led a study to fill some of that knowledge gap. He and his collaborators – including dietitians, pharmacists, statisticians, critical care physicians, and computer scientists – collected fecal, stool and oral samples from 115 patients admitted to the ICUs at four hospitals in the United States and Canada. They analyzed the microbiomes of those samples 48 hours after a patient was admitted, and again when the patient was discharged (or 10 days later, whichever came first). In a new paper in mSphere, they describe what they found.

 

As they suspected, they found that ICU patients had lower abundances of health-promoting bacteria and higher levels of pathogenic species, compared to healthy patients. Wischmeyer says he was astonished at how rapidly pathogenic bacteria flourished.

 

“In some cases, those organisms became 95 percent of the entire gut flora – all made up of one pathogenic taxa – within days of admission to the ICU,” he says. “That was really striking.” Some patients, in fact, were hosting microbiomes that had more in common with corpses than with healthy people.

 

Wischmeyer, who will move his lab to Duke University in the fall, sees a variety of ways to use the study's findings. In addition to designing recovery plans aimed at restoring healthy bacteria in the gut, researchers may use this approach to gauge a patient's response to therapy. “Can we take these data and begin to make predictions about what happens to some of these patients?” he wonders.

 

The roots and implications of the study are personal for Wischmeyer. During high school, more than two decades ago, doctors diagnosed him with inflammatory bowel disease. He has undergone more than 20 surgeries to treat the condition. 

 

“The drugs they treated me with often made me worse, not better,” he says. His symptoms first materialized soon after he'd been treated with antibiotics for strep throat, and he wonders if that treatment treatment triggered the disease by changing the bacterial balance of his gut. The experience drove him to learn more about gut microbes and their role in health.

 

“I became passionate about nutrition and how the gut can be affected by changes in our “health” environment,” he says. He hopes his findings will lead to future studies on new ways to correct dysbiosis. “I think this is a first step of a large road map for improving health by optimizing the makeup of the bacterial “friends” that share our body with us throughout life.”

--Stephen Ornes

Researchers Identify Multidrug-resistant E. coli Bacteria from New Jersey Patient

Antimicrobial resistance has been a growing concern in the health care community. But a publication by Chinese researchers in The Lancet Infectious Diseases last fall kicked things up a notch. The work found the mcr-1 gene, which confers resistance to the antibiotic colistin, in Escherichia coli isolates taken from raw meat, pigs raised as food animals and a small percentage of hospitalized patients. Most concerning, mcr-1 exists on a plasmid, meaning it could potentially spread antibiotic resistance to other bacterial species.

“When this happened, everyone started getting concerned, because if the resistance is on a plasmid and can spread, it’s going to eventually affect the use of colistin --- which is increasingly used as a last resort for multidrug-resistant infections --- as a drug to treat humans,” says Barry N. Kreiswirth, Ph.D., founding director of the Public Health Research Institute (PHRI) Tuberculosis Center at New Jersey Medical School, part of Rutgers University in Newark, N.J.

The paper prompted Kreiswirth and others to re-examine their own collections of bacterial isolates from countries worldwide to look for colistin-resistant strains harboring mcr-1, prompting numerous additional publications. Kreiswirth’s laboratory then decided to look at carbapenem-resistant bacteria from a nearby hospital in Newark. He and his colleagues pulled about 50 strains, finding one E. coli strain from a New Jersey patient with a complicated urinary tract infection that carried not only mcr-1 but also bla_NDM-5, which confers resistance to broad-spectrum carbapenem antibiotics used to treat multidrug-resistant Gram-negative infections.

Around the same time this spring, Walter Reed investigators published their report of mcr-1 found in a Pennsylvania patient in Antimicrobial Agents and Chemotherapy. The Centers for Disease Control and Prevention in June then issued a health alert calling for  Enterobacteriaceae isolates with a minimum inhibitory concentration (MIC) to colistin of 4 µg/ml or higher to be tested for confirmation and the presence of mcr-1. The mcr-1 isolate from Kreiswirth’s group had a colistin MIC of only 3 µg/ml, however. Kreiswirth knew it was time to publish his own findings, which appeared this week in mBio.

“Others in the U.S. had found mcr-1 positives, but the significance of this paper was this was the first example so far in our country of having the colistin resistance associated within the same strain as carbapenem resistance,” says Kreiswirth.

The strain, isolated in 2014 from a 76-year-old man who had emigrated from India a year prior to developing infection, did respond to other antimicrobial agents and was treated successfully. But Kreiswirth says the case presents a sound reminder to monitor and track multidrug-resistant organisms: “The good news is that this did not cause a major outbreak of drug-resistant infection. The bad news is that since this occurred two years ago, there are clearly other strains out there we haven’t detected yet. The carbapenem resistance and the colistin resistance genes are on separate plasmids, which means they can spread to other bacteria.”

Kreiswirth’s lab used various molecular techniques to identify and compare the different bacteria found in the man’s urine samples, finding that the E. coli strain harbored resistance to several classes of antibiotics, including aminoglycosides, beta-lactams, chloramphenicol, fluoroquinolones, rifampin, sulfonamides, and tetracycline. Additional testing found that the plasmids isolated were highly similar to ones previously reported by Kreiswirth’s group from clinical infections in China.

The lab also identified the E. coli strain as a variant of ST405, one of the main disease-causing strains of the bacteria, which has frequently been associated with community-onset urinary tract infections, says lead study author José R. Mediavilla, MBS, MPH, a research teaching specialist at PHRI.

“Most mcr-1 cases appear to be happening in E. coli, the most common cause of urinary tract infections,” Mediavilla says. “These strains are probably already in the community and could spread further, essentially building toward a situation where you’re going to have difficult if not impossible to treat urinary infections. Active surveillance efforts involving all colistin- and carbapenem-resistant organisms are imperative to determine mcr-1 prevalence and prevent further dissemination.”

-- Karen Blum

The inflammatory nature of a bad recycler

Neisseria gonorrhoeae TEM

Being a bad recycler implies creating more waste because items aren’t being reincorporated into the production chain. Plastic water bottles can be broken down and turned into new plastic bottles, gardening gloves, or fleece – any of which means less oil needs to be harvested and refined to the polymers that constitute these different items. Bacteria, in general, also tend to be very good recyclers. The energy it takes to reuse a compound is generally less than to build the molecular structure from scratch.

An example of bacteria recycling efficiency comes with the cell wall, made of peptidoglycan, which must be remodeled for cell growth and division. Rather than cleave and release peptidoglycan polymers, many bacteria recycle the fragments freed during remodeling to incorporate for new wall synthesis. In Neisseria species, the majority of the freed peptidoglycan fragments are transported into the cytoplasm through the AmpG permease. However, not all Neisseria are equally proficient at this peptidoglycan recycling, and new research published in the Journal of Bacteriology demonstrates that N. gonorrhoeae is the least efficient recycler among related Neisseria species, with an inflammatory effect.

Authors Jia Mun (Elizabeth) Chan and Joseph Dillard investigated different human-associated species of Neisseria. Most Neisseria are commensals that very rarely cause disease, while two – N. gonorrhoeae and N. meningiditis – are associated with disease. These species release peptidoglycan fragments that act as pathogen-associated molecular patterns, which are recognized by pattern recognition receptors, such as NOD1. N. gonorrhoeae is especially wasteful, losing 15% of its peptidoglycan fragments to the extracellular environment via inefficient recycling, compared to the 4-5% lost by other Neisseria species.

 

HPLC of radiolabeled peptidoglycan shows more fragments released with gonococcal vs meningococcal ampG gene

The scientists confirmed the peptidoglycan shedding was due to poor recycling, and not an increase in fragment generation, by looking at the ampG genes of the different Neisseria species. By replacing the native ampG gene with the gonococcal version in asymptomatic colonizers N. sicca or N. mucosa, the scientists observed an increase in peptidoglycan shedding. Although a pathogen, N. meningiditis can also colonize without causing disease, which may be in part due to its minimal shedding. Replacing the meningococcal ampG with the gonococcal ampG led to almost 40% more peptidoglycan release from this strain (see right).

The difference appears to be in the sequence itself, since neither mRNA nor protein levels were higher in gonococcal ampG variants (if anything, the gonococcal transcript were lower). The sequences of the meningococcal and gonococcal AmpG proteins vary in only nine amino acids, and by generating chimeric constructs and then using site-directed mutagenesis, the team was able to pinpoint the three amino acids that affect recycling efficiency.

The large amounts of peptidoglycan shed by N. gonorrhoeae are more likely to alert the immune system to an unwelcome microbial stranger, leading to inflammation and immune cell influx. Given the similarity of N. meningitidis peptidoglycan release levels to other, nonpathogenic Neisseria species, the results of this study help explain why N. meningitidis can sometimes colonize without causing overt or immediate symptoms, while N. gonorrhoeae infection is almost always accompanied by a strong inflammatory response.

-- Julie Wolf

Photo credits: N. gonorrhoeae TEM, Figure from JBacteriology article

 

We're moving!

mBiosphere is excited to announce that starting September 1^st, 2016, our blog content will be moving to asm.org.

We’ve enjoyed interacting with readers on this site, and will continue to bring you the latest in ASM member research, conferences, education and more at our new site: asm.org/index.php/mBiosphere. Please update your blogroll and RSS feeds!

We’re pleased to join the increasing index of ASM blogs, including Microbial Sciences, Around the World, Education, Careers, Zika Diaries, Small Things Considered, and bLog Phase. We’re committed to bringing you the best in the microbial sciences and look forward to our new home at asm.org!

Improving the Quality of Dairy Products

Bitter tasting yogurt or cheese may not make it to your refrigerator, but it is produced and the result of pesky bacteria. The microbial composition of raw milk impacts the quality, shelf life, and safety of processed milk and other dairy products. Controlling the quality of these products is tricky—bacteria can enter milk on the farm, during transport, storage, and processing. While pathogens are destroyed by pasteurization, not all bacteria are eliminated and some can cause defects, such as bad tastes or holes in cheese, which can lead to food waste.

“The food that we get in our supermarket is the best of the best. Right now, there is a lot of mystery about why dairy products spoil or have defects. There doesn’t seem to be a logical pattern or some problem in the facility that can explain why certain products are defective and others are not," said Maria Marco, PhD, associate professor, Department of Food Science & Technology, University of California-Davis.

While scientists have intensively studied the microbial ecology of fresh produce and animal products, little is known about the influences of storage, transport, and processing facilities. To shed light on the issue, Dr. Marco and colleagues set out to identify the microbiota of raw milks collected for large-scale product manufacturing in California. California is the largest producer of milk in the United States, producing 20% of the total U.S. milk production. The scientists analyzed the bacteria in raw milk arriving in 899 tanker trucks at two different dairy processors in the California Central Valley in the fall of 2013 and the spring and summer of 2014.

In a study published online in mBio, the researchers report that bacteria varied by season and were highly diverse, with roughly 50% of the taxa present at less than 1% relative abundance. As a comparison, roughly 20% of human fecal communities are composed of taxa below 1% relative abundance. Milk also had a core microbiome composed of 29 different taxa, including Streptococcus, Staphylococcus, and unidentified Clostridiales.

Another important finding was what happened to the milk after it got to the dairy processing plant. “We saw this interesting shift of the types of bacteria that are dominant in the milk when it goes from the truck to the silos where the milk is stored before pasteurization," said Dr. Marco. The conditions or microbial exposures at the processing facility outweighed the raw milk microbiome, and the bacterial composition changed distinctly within some, but not all silos, a short time after transfer.

“This study was an exploratory mission to find out what types of bacteria are in our raw milk and what happens to them when they reach the built environment," said Dr. Marco. "We now need to tackle the bigger problem of how can we control those microbes in an effective way. The ultimate goal in all of this research is to get dairy products with longer shelf life, less spoilage, and less waste."

By knowing the types of microbes present in foods, scientists can devise ways to get rid of them early, so they don’t get into the final product and cause quality problems.

For example, now that researchers know that certain microbes are more prevalent in certain seasons in California, taking additional steps during these seasons may improve dairy food quality.

Dr. Marco said the study findings might also make those who drink raw milk wary. While all milk sold across state lines must be pasteurized, consuming raw milk is legal in all 50 states.

--Kate O'Rourke