mBiosphere

Watch Your Step: New Types of Antibiotic Resistance Lurking in Cow Manure

Jo Handelsman likes to follow the bugs—especially those bacteria that might hide new forms of antibiotic resistance within. While the antibiotic resistance that shows up in clinical setting gets scads of attention from researchers, Handelsman’s team at Yale University in New Haven, Connecticut wanted to investigate how new antibiotic resistance (AR) genes might make their way into human pathogens. In particular, in a study published this week in mBio®, they investigated an altogether different form of the farm-to-table movement.

“We were interested in the role of manure,” commonly used as a soil fertilizer in agriculture, says Handelsman, who is also an HHMI Professor. She paints a scenario where manure used on some vegetable crops contains bacteria from the cow’s gut, which could cling to root crops especially, such as carrots. The manure bacteria might then move from the produce to people, or they might transfer AR genes to bacteria that colonize humans. 

“Is this a route for movement of these genes from the barn to the table?” she asks. “This was the first step, looking at the presence of antibiotic resistance genes in the manure.”

Using a powerful functional metagenomics approach, her team surveyed five manure samples from four dairy cows housed at the University of Connecticut. From these samples, the researchers extracted all of the bacterial DNA present, chopped it into bits, and put each bit back into a laboratory strain of Escherichia coli bacteria.

The E. coli were then grown on plates containing four different classes of antibiotics: beta-lactams (like penicillin), aminoglycosides (like kanamycin), tetracycline, and chloramphenicol.  Any E. coli that could grow on the antibiotics likely contained an AR gene isolated from the manure bacteria. The team then used third-generation sequencing to pull out the gene and its flanking DNA to identify both the gene and the likely species of gut bacteria that originally hosted it.

They identified 80 distinct and functional AR genes, 75% of which were novel—genes so divergent in sequence identity from known AR genes that some might not have been found through sequencing efforts alone.

“That was exactly the motivation behind the functional process, to deepen the pool of known genes with AR function,” says Handelsman.  Also, by expressing the genes in E. coli, the team selected for genes that truly confer resistance in a species that can be pathogenic and associated with farm products.

The deep divergence from known AR genes surprised Handelsman. “From an evolutionary perspective, these are distant from the ones we see in the database, which largely represent the AR genes seen in the clinic.”  That signals that cow manure bacteria may not be a major source of resistance that currently causes problems for human patients. However, as lead author Fabienne Wichmann points out, it could also mean that “cow manure harbors an unprecedented reservoir of AR genes that we should know about.” 

In other words, these new AR genes could be the next ones to show up in bacteria infecting humans. There are two ways that could happen—bacteria containing these AR genes colonize humans, or through horizontal gene transfer, the genes jump to other bacteria that already colonize humans. Benign manure bacteria might transfer AR genes to pathogens at any point—in manure, soil, food, or humans.

Expanding our repertoire of known resistance-conferring genes, will help clinicians better predict which kinds of antibiotic resistance they might face in the future, says Wichmann. Studies like this will also give drug developers better information for designing pre-emptive strike inhibitors to AR genes.

This is just the first in a sequence of studies, says Handelsman. “We’re hoping this study will open up a larger field of surveillance, to start looking at new types of resistance before they show up in the clinic.”

 --Kendall Powell

More Evidence Antibacterial Soaps Aren't That Good for You

A study in mBio® this week points to yet another reason to potentially be cautious about the man-made antimicrobial compound triclosan. The agent, commonly found in household soaps, shampoos, toothpastes, medical equipment and other materials, may be finding its way inside human noses, where it can promote the colonization of Staphylococcus aureus bacteria and predispose some people to infection.

The study, from researchers at the University of Michigan, found traces of triclosan in the nasal passages of 41 percent (37 of 90) of healthy adults studied. Among those with traces of triclosan, 64 percent also were colonized with S. aureus. By contrast, only 30 percent of adults without triclosan had S. aureus colonization.

Additional experiments found that S. aureus grown in the presence of triclosan was better able to attach to human proteins, and that rats exposed to triclosan were more susceptible to S. aureus nasal colonization.

Triclosan is “really common in hand soaps, toothpastes and mouthwashes but there’s no evidence it does a better job than regular soap,” says senior study author Blaise Boles, PhD, an assistant professor of molecular, cellular and developmental biology at the university. “This agent may have unintended consequences in our bodies. It could promote S. aureus nasal colonization, putting some people, like those undergoing surgery, at increased risk for infection.”

While triclosan has been around for 40 years, it has been incorporated into many household products within the past decade, says Boles. His is not the first study to note its spread to humans: Other studies have found traces of triclosan in human fluids including serum, urine and milk, and studies in mammals have found that high concentrations of triclosan can disrupt the endocrine system and decrease heart and skeletal muscle function.

“In light of the significant use of triclosan in consumer products and its widespread environmental contamination, our data combined with previous studies showing impacts of triclosan on the endocrine system and muscle function suggest that a reevaluation of triclosan in consumer products is urgently needed,” Boles and colleagues wrote.

With consumer backlash and concerns from the scientific community about triclosan leading to bacterial resistance, triclosan has been banned in Europe and Canada, Boles says. In the U.S., Minnesota could become the first state to ban the agent: the Minneapolis City Council has urged the state to ban triclosan, and Gov. Mark Dayton ordered state agencies to stop using products containing the compound. In addition, the Food and Drug Administration in December issued a rule requiring manufacturers to provide more substantial data to demonstrate the safety and effectiveness of antibacterial soaps. Companies including Johnson & Johnson and Procter & Gamble have announced they are phasing out the use of triclosan in their products.

For now, Boles, who says he would like to conduct a more broad survey to determine if triclosan  influences microbial colonization in additional human body sites, opts not to use products containing triclosan. But surveying products for the chemical is tricky, he notes, as triclosan also is marketed under other names including BioFresh, Microban, Lexol 300, Ster-Zac, Cloxifenolum, and Irgasan DP-300. 

 --Karen Blum

Career Chasing Fungal Pathogens Yields Clues About Susceptible Patients

In the 1970s, June Kwon-Chung was characterizing the life cycle of Cryptococcus neoformans, a fungus that causes a rare disease called cryptococcosis, which results in a fatal brain infection if left untreated. Looking at the fungal sexual spores under the microscope, she noticed two distinct forms—one round, the other shaped as fingerlike projections. She had a hunch she was observing two different species. 

“I thought, this has got to be two different species, genetically, but at the time we didn’t even know how to extract DNA since molecular techniques for Cryptococcus were not yet developed. So I was using classical methods to determine how they differed geographically, epidemiologically, and biologically,” said Kwon-Chung, chief of the Molecular Microbiology Section in the Laboratory of Clinical Infectious Disease at the National Institute of Allergy and Infectious Disease (NIAID) in Bethesda, Maryland.

In a study this week in mBio®, Kwon-Chung’s group reports on a risk factor that appears to make some patients susceptible to that second Cryptococcus species she has described.

The fungal pathogens C. gattii (right) and C. neoformans (left) differ in their sexual spore morphology (top) and their biochemistry, which allows for identification on diagnostic, color-changing agar (bottom). Credit: Kwon-Chung lab at NIAID

Kwon-Chung, a medical mycologist, spent a good chunk of the 1980s and 1990s studying the two forms, which originally had been identified as C. neoformans variety neoformans and a sister variety called C. neoformans variety gattii. Found in the environment, the fungus gets inhaled into the lungs. In immunocompromised patients, it can multiply in the lungs and travel to the brain where it proliferates, causing headaches, memory loss, and swelling. Even with anti-fungal treatment, about one-quarter of patients still die. The advent of AIDS put a spotlight on C. neoformans, because it frequently attacked these immunocompromised patients. Today, it is a major cause of AIDS deaths worldwide, with an estimated 1 million new cases and 625,000 deaths annually.

Kwon-Chung’s research group showed that the C. neoformans variety gattii was biochemically quite different and that it was mostly found in patients from tropical and subtropical regions, including Australia, Brazil, Thailand, Hawaii, Mexico, and central and southern Africa. In 2002, molecular genetics proved what Kwon-Chung already knew—that C. neoformans variety gattii was actually a separate species, which she eventually named Cryptococcus gattii.

Soon after, it became clear that while C. neoformans mostly infected AIDS patients , C. gattii only rarely infected HIV patients and instead struck patients who were apparently  healthy.  At this same time, an epidemic of cryptococcal infections in temperate  Vancouver, British Columbia was strangely found to be caused by C. gattii.

“People began opening their eyes and saying let’s identify whether [a patient has] C. gattii or C. neoformans,” said Kwon-Chung.  The majority of C. gattii patients were so-called immunocompetent, the opposite profile of C. neoformans. “Why is C. gattii going after immunocompetent people?” she asks. “That is the last major question I want to solve before I retire.”

Clearly, it wasn’t attacking every healthy person breathing in the spores, so Kwon-Chung hypothesized that there might be a subtle immunological difference in these patients.  “Since I’m not an immunologist, it was a little bit like shooting in the dark,” she said.

Then, in 2013, a study published by Steven Holland’s group also at NIAID gave her a huge hint. It showed that seven immunocompetent patients with cryptococcal infections had autoantibodies to granulocyte-macrophage colony-stimulating-factor (GM-CSF).  Normally, the GM-CSF cytokine regulates growth and survival of immune cells, particularly macrophages and T cells.

In that study, one patient clearly had a C. gattii infection, three patients had infections by unspecified Cryptococcus and three were identified as having C. neoformans.  Kwon-Chung’s team confirmed that the three unknown strains were C. gattii, and she strongly suspected that the other three were misidentified.

Working with colleagues in China and Australia, her group obtained numerous blood samples from immunocompetent patients with cryptococcosis and found that seven patients who had the GM-CSF antibodies in their blood were all infected by C. gattii. In the mBio® paper, the authors make the case that anti-GM-CSF autoantibodies are a risk factor for C. gattii infection and not necessarily for C. neoformans infection.

“It turns out that patients suffering a C. gattii infection are not exactly immunocompetent,” said Kwon-Chung. The team also found one healthy control patient from China with autoantibodies to GM-CSF in his blood who could be predisposed to C. gattii infection.

Although not proven yet, Kwon-Chung thinks it’s very likely that the autoantibodies make patients susceptible to infections, and not the other way around. If true, testing for anti-GM-CSF antibodies might be used in the future to find patients at risk for C. gattii infections, especially in areas where the fungus is endemic.

With a new hunch to chase, she is not considering retirement anytime soon: “I assume this will open up a huge immunological investigation. We have a lot to do. I can’t just stop here.” 

 

--Kendall Powell

Understanding How Gonorrhea Spreads: It's Seminal

The sexually transmitted infection gonorrhea has been around for centuries, described in early writings from Egypt, China and Japan. Even the bible describes warnings about “unclean discharges from the body.” Yet although the bacteria behind the infection, Neisseria gonorrhoeae, were first identified back in 1879, there are still many questions that remain unanswered about how these organisms spread from person to person.

A study in mBio this week offers clues. Researchers at Northwestern University’s Feinberg School of Medicine in Chicago and the University of Cologne in Germany have found that exposure to seminal plasma – the liquid part of semen containing secretions from the male genital tract – allows N. gonorrhoeae to more easily move and start to colonize in human tissues.

In a series of laboratory experiments, the investigators found that 24 times as much N. gonorrhoeae could pass through a synthetic barrier after being exposed to seminal plasma. In addition, exposure to seminal plasma caused hairlike appendages on the bacteria surface, called pili, to move the cells by a process known as twitching motility. This stimulatory effect could be seen even at low concentrations of seminal plasma and beyond the initial influx of seminal fluid.

Additional tests found that exposure to seminal plasma enhanced the formation of bacterial microcolonies on human epithelial cells (cells that line body cavities), which can also promote the establishment of infection.

“Our study illustrates an aspect of biology that was previously unknown,” said lead author study Mark Anderson of Northwestern. “If seminal fluid facilitates motility, it could help transmit gonorrhea from person to person.”

A photomicrograph of Neisseria gonorrhoeae viewed using a Gram-stain technique. Credit: Centers for Disease Control and Prevention

Today gonorrhea is the second most common sexually transmitted infection in the U.S., according to the Centers for Disease Control and Prevention, with estimates upward of 800,000 new cases each year. Fewer than half are diagnosed and reported. Untreated, gonorrhea can lead to serious and permanent health problems, including pelvic inflammatory disease in women and epididymitis, a painful condition of the testicles that may lead to infertility, in men. Untreated gonorrhea also increases a person’s risk of acquiring or transmitting HIV.

With more than 100 million estimated new cases of gonorrhea annually worldwide, said senior study author H. Steven Seifert of Northwestern, “research characterizing the mechanisms of pathogenesis and transmission of N. gonorrhoeae is important for developing new prevention strategies, since antibiotic resistance of the organism is becoming increasingly prevalent.”

Antibiotics once effective against gonorrhea, including penicillin, tetracycline and fluoroquinolones, are no longer recommended because of high rates of resistance. Only one class of antibiotics, cephalosporins including the oral antibiotic cefixime, has been left to treat the disease. But more recently, evidence from CDC’s Gonococcal Isolate Surveillance Project (GISP) suggests that cefixime, too, is becoming less effective. New gonorrhea treatment guidelines published in 2012 suggest only injectable ceftriaxone be used in combination with one of two oral antibiotics, either azithromycin or doxycycline. 

 

-Karen Blum