Clues about autism may come from the gut

Contact: Joseph Caspermeyer Joseph.Caspermeyer@asu.edu Arizona State University

Bacterial flora inhabiting the human gut have become one of the hottest topics in biological research. Implicated in a range of important activities including digestion, fine-tuning body weight, regulating immune response, and producing neurotransmitters affect that brain and behavior, these tiny workers form diverse communities. Hundreds of species inhabit the gut, and although most are beneficial, some can be very dangerous.

In new research appearing in the journal PLOS ONE, a team led by Rosa Krajmalnik-Brown, a researcher at Arizona State University’s Biodesign Institute, present the first comprehensive bacterial analysis focusing on commensal or beneficial bacteria in children with autism spectrum disorder (ASD).

After publishing earlier research exploring crucial links between intestinal microflora and gastric bypass, Krajmlanik-Brown convinced James Adams— director of the ASU Autism/Asperger’s Research Program—that similar high throughput techniques could be used to mine the microbiome of patients with autism. (Previously, Adams had been studying the relationship between the gut microbiome and autism using traditional culturing techniques.)

“One of the reasons we started addressing this topic is the fact that autistic children  have a lot of GI problems that can last into adulthood,” Krajmalnik-Brown  says. “Studies have shown that when we manage these problems, their behavior improves dramatically.”

Following up on these tantalizing hints, the group hypothesized the existence of distinctive features in the intestinal microflora found in autistic subjects compared to typical children. The current study confirmed these suspicions, and found that children with autism had significantly fewer types of gut bacteria, probably making them more vulnerable to pathogenic bacteria.  Autistic subjects also had significantly lower amounts of three critical bacteria, Prevotella, Coprococcus, and Veillonellaceae.

Krajmalnik-Brown, along with the paper’s lead authors Dae-Wook Kang and Jin Gyoon Park, suggest that knowledge gleaned through such research may ultimately be used both as a quantitative e diagnostic tool to pinpoint autism and as a guide to developing effective treatments for ASD-associated GI problems.  The work also offers hope for new prevention and treatment methods for ASD itself, which has been on a mysterious and rapid ascent around the world.

A disquieting puzzle

Autism is defined as a spectrum disorder, due to the broad range of symptoms involved and the influence of both genetic and environmental factors, features often confounding efforts at accurate diagnosis. The diseases’ prevalence in children exceeds juvenile diabetes, childhood cancer and pediatric AIDS combined.

Controversy surrounds the apparent explosive rise in autism cases. Heightened awareness of autism spectrum disorders and more diligent efforts at diagnosis must account for some of the increase, yet many researchers believe a genuine epidemic is occurring. In addition to hereditary components, Western-style diets and overuse of antibiotics at an early age may be contributing to the problem by lowering the diversity of the gut microflora.

In terms of severe developmental ailments affecting children and young adults, autism is one of the most common, striking about 1 in 50 children. The disorder—often pitiless and perplexing—is characterized by an array of physical and behavioral symptoms including anxiety, depression, extreme rigidity, poor social functioning and an overall lack of independence.

To date, studies of the gut microbiome in autistic subjects have focused primarily on pathogenic bacteria, some of which have been implicated in alterations to brain function.  One example involves gram-negative bacteria containing lipopolysaccharides in their cell walls, which can induce inflammation of the brain and lead to the accumulation of high levels of mercury in the cerebrum.

A new approach

Krajmalnik-Brown and lead author Dae-Wook Kang are researchers in the Biodesign Institute’s Swette Center for Environmental Biotechnology, which is devoted to the use of microbial communities for the benefit of human and environmental health. Their new study is the first to approach autism from a different angle, by examining the possible role of so-called commensal or beneficial bacteria.

Up to a quadrillion (1014) bacteria inhabit the human intestine, contributing to digestion, producing vitamins and promoting GI health. Genes associated with human intestinal flora are 100 times as plentiful as the body’s human genes, forming what some have referred to as a second genome. Various environmental factors can destabilize the natural microbiome of the gut, including antibiotics and specific diets.

In the current study, a cohort of 20 healthy and 20 autistic subjects between 3 and 16 years of age were selected and their gut microflora from fecal samples analyzed by means of a technique known as pyrosequencing. Pyrosequencing is a high-throughput method, allowing many DNA samples to be combined as well as many sequences per sample to be analyzed.

Lower diversity of gut microbes was positively correlated with the presence of autistic symptoms in the study. The authors stress that bacterial richness and diversity are essential for maintaining a robust and adaptable bacterial community capable of fighting off environmental challenges. “We believe that a diverse gut is a healthy gut,” Krajmalnik-Brown says.

The new study detected decreased microbial diversity in the 20 autistic subjects whose fecal samples were analyzed. Specifically, three bacterial genera—Prevotella, Coprococcus and Veillonellaceae—were diminished in subjects with autism, when compared with samples from normal children. (Surprisingly, these microbial changes did not seem directly correlated with the severity of GI symptoms.)

The three genera represent important groups of carbohydrate-degrading and/or fermenting microbes.  Such bacteria could be critical for healthy microbial-gut interactions or play a supportive role for  a wide network of different microorganisms in the gut. The latter would explain the decreased diversity observed in autistic samples.

Bacteria: in sickness and in health

Among the fully classified genera in the study, Prevotella was the most conspicuously reduced in autistic subjects. Prevotella is believed to play a key role in the composition of the human gut microbiome. For this reason, the group undertook a sub-genus investigation of autistic subjects. They found that a species known as Prevotella copri occurred only in very low levels in the autistic samples. The species is a common component in normal children exhibiting more diverse and robust microbial communities.

“We think of Prevotella as a healthy, good thing to have,” Krajmalnik-Brown notes. (Michael Polan’s recent New York Times Magazine story on the microbiome points to the fact that he is proud that his gut microbiome is rich in Prevotella regarding it as a possible sign of a healthy non-Western diet.)

Jin Gyoon Park (the other lead author),  who works in the Virginia G. Piper Center for Personalized Diagnostics, under Joshua LaBaer’s direction, conducted a rigorous bioinformatic and statistical analysis of the intestinal microflora. He believes that the microbiome can be mined in future work to find diagnostic biomarkers for autism and many other diseases. Quantitative diagnoses of this sort have so far been lacking for autism, a disease for which subjective behavior indices are typically used to identify the disorder.

In describing the next steps for the research group, Kang and Park point to more detailed, gene-level analyses aimed at probing bacterial function and further illuminating relationships between human health and the complexities of the microbiome.  Additionally, the group will use the current results as a guide for new treatment studies for autism aimed at modifying bacterial composition in the gut.

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A new, interdisciplinary consortium (Autism Microbiome Consortium) has been formed to investigate the underpinings of autism and the gut microbiome, bringing together the combined skills of neurologists, psychiatrists, neuroimmunologists, epidemiologists, pediatricians, geneticists, biochemists, microbiologists and others. In addition to Rosa Krajmalnik-Brown and James Adams, the group consists of:

Jack Gilbert (University of Chicago), Catherine Lozupone (University of Colorado), Rob Knight (University of Colorado and HHMI). Mady Hornig (Columbia University), Sarkis Mazmanian (California Institute of Technology), Tanya Murphy (University of South Florida), , Paul Patterson (California Institute of Technology), John Alverdy (University of Chicago), Janet Jansson (Lawrence Berkeley Lab), KImberly Johnson (University of Colorado).

Written by: Richard Harth Science Writer: Biodesign Institute richard.harth@asu.edu

New study links fate of personal care products to environmental pollution and human health concerns: Triclosan

Contact: Joe Caspermeyer
joseph.caspermeyer@asu.edu
480-727-0369
Arizona State University

Parental concerns in maintaining germ-free homes for their children have led to an ever-increasing demand and the rapid adoption of anti-bacterial soaps and cleaning agents. But the active ingredients of those antiseptic soaps now have come under scrutiny by the EPA and FDA, due to both environmental and human health concerns.

Two closely related antimicrobials, triclosan and triclocarban, are at the center of the debacle. Whereas triclosan (TCS) has long captured the attention of toxicologists due to its structural resemblance to dioxin (the Times Beach and Love Canal poison), triclocarban (TCC) has ski-rocketed in 2004 from an unknown and presumably harmless consumer product additive to one of today’s top ten pharmaceuticals and personal care products most frequently found in the environment and in U.S. drinking water resources.

Now, Biodesign Institute at Arizona State Univesity researcher Rolf Halden and co-workers, in a feat of environmental detective work, have traced back the active ingredients of soaps – used as long ago as the 1960s – to their current location, the shallow sediments of New York City’s Jamaica Bay and the Chesapeake Bay, the nation’s largest estuary.

“Our group has shown that antimicrobial ingredients used a half a century ago, by our parents and grandparents, are still present today at parts-per-million concentrations in estuarine sediments underlying the brackish waters into which New York City and Baltimore discharge their treated domestic wastewater,” said Halden, a new member of the institute’s Center for Environmental Biotechnology. “This extreme environmental persistence by itself is a concern, and it is only amplified by recent studies that show both triclosan and triclocarban to function as endocrine disruptors in mammalian cell cultures and in animal models.”

Aiding in his team’s research was another type of contamination: the radioactive fallout from nuclear testing conducted in the second half of the last century. Using the known deposition history and half-lives of two radioactive isotopes, cesium-137 and beryllium-7, Halden and his collaborators Steven Chillrud, Jerry Ritchie and Richard Bopp were able to assign the approximate time at which sediments observed to contain antimicrobial residues had been deposited in the two East Coast locations.

By analyzing vertical cores of sediment deposited over time in the two sampling locations on the East Coast, they showed that TCC, and to a lesser extent, TCS, can persist in estuary sediments. TCC was shown to be present at parts per million levels, which could represent unhealthy levels for aquatic life, especially the bottom feeders that are important to commercial fishing industries like shellfish and crabs.

In the Chesapeake Bay samples, the group noticed a significant drop in TCC levels that corresponded to a technology upgrade in the nearby wastewater treatment plant back in 1978. However, earlier work by the team had shown that enhanced removal of TCC and TCS in wastewater treatment plants leads to accumulation of the problematic antimicrobial substances in municipal sludge that often is applied on agricultural land for disposal. Lead author Todd Miller concludes that “little is actually degraded during wastewater treatment and more information is needed regarding the long term consequences these chemicals may have on environmentally beneficial microorganisms.”

Along the way of studying the deposition history of antimicrobials in sediments, the team also discovered a new pathway for the breakdown of antimicrobial additives of consumer products. Deep in the muddy sediments of the Chesapeake Bay, they found evidence for the activity of anaerobic microorganisms that assist in the decontamination of their habitat by pulling chlorine atoms one by one off the carbon backbone of triclocarban, presumably while obtaining energy for their metabolism in the process. “This is good news,” said Halden, “but unfortunately the process does not occur in all locations and furthermore it is quite slow. If we continue to use persistent antimicrobial compounds at the current rate, we are outpacing nature’s ability to decompose these problematic compounds.”

While combining bioenergy production and pollutant destruction has its own appeal, Halden sees a simpler solution to combating the pollution his team discovered: limit the use of antimicrobial personal care products to situations where they improve public health and save lives.

“The irony is that these compounds have no measurable benefit over the use of regular soap and water for hand washing; the contact time simply is too short.” Unfortunately this cannot be said for the bottom-dwelling organisms in the sampling locations on the East Coast. “Here,” Halden concludes, “the affected organisms are experiencing multi-generational, life-time exposures to our chemical follies.”

Halden is planning to continue his research on persistent antimicrobials by studying their body burden and associated health effects in susceptible populations including mothers and their babies.

 

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“Fate of Triclosan and Evidence for Reductive Dechlorination of Triclocarban in Estuarine Sediments” is an original research paper currently in press in the peer-reviewed journal Environmental Science & Technology. The work was funded in part by the Johns Hopkins Center for a Livable Future and the National Institute of Environmental Health Sciences. The study’s authors are T. R. Miller, J. Heidler, S. N. Chillrud, A. DeLaquil, J. C. Ritchie, J. N. Mihalic, R. Bopp, and R. U. Halden.

New salmonella-based ‘clean vaccines’ aid the fight against infectious disease:To accomplish this, a recombinant strain of Salmonella was constructed using genes from another pathogen, Francisella tularensis

* They are using genes from tularensis ” inhaling as few as 10 bacteria could be potentially deadly ” I feel uncomfortable with the Gates foundation funding support utilizing a Bioweapon strain of  Rabbit Fever?

 

New salmonella-based ‘clean vaccines’ aid the fight against infectious disease

A powerful new class of therapeutics, known as recombinant attenuated Salmonella vaccines (RASV), holds great potential in the fight against fatal diseases including hepatitis B, tuberculosis, cholera, typhoid fever, AIDS and pneumonia.

Now, Qingke Kong and his colleagues at the Biodesign Institute at Arizona State University, have developed a technique to make such vaccines safer and more effective. The group, under the direction of Dr. Roy Curtiss, chief scientist at Biodesign’s Center for Infectious Diseases and Vaccinology, demonstrated that a modified strain of Salmonella showed a five-fold reduction in virulence in mice, while preserving strong immunogenic properties.

Their findings appear in the cover story of the current issue of the Journal of Immunology.

Streptococcus pneumoniae, an aerobic bacterium, is the causative agent of diseases including community-acquired pneumonia, otitis media, meningitis, and bacteremia. It remains a leading killer—childhood pneumonia alone causing some 3 million fatalities annually, mostly in poorer countries.

Existing vaccines are inadequate for protecting vulnerable populations for several reasons. Heat stabilization and needle injection are required, which are often impractical for mass inoculation efforts in the developing world. Repeated doses are also needed to induce full immunity. Finally, the prohibitively high costs of existing vaccines often deprive those who need them most.  The problem is exacerbated by the recent emergence of antibiotic-resistant strains of pneumococcus causing the disease, highlighting the urgency of developing safe, effective, and lower-cost antipneumococcal vaccines.

One of the most promising strategies for new vaccine development is to use a given pathogen as a cargo ship to deliver key antigens from the pathogen researchers wish to vaccinate against. Salmonella, the bacterium responsible for food poisoning, has proven particularly attractive for this purpose, as Curtiss explains: “Orally-administered RASVs stimulate all three branches of the immune system stimulating mucosal, humoral, and cellular immunity that will be protective, in this case, against a majority of pneumococcal strains causing disease.”

Recombinant Salmonella is a highly versatile vector—capable of delivering disease-causing antigens originating from viruses, bacteria and parasites.  An attenuated Salmonella vaccine against pneumonia, developed in the Curtiss lab, is currently in FDA phase 1 clinical trials.

In the current research, the team describe a method aimed at retaining the immunogenicity of an anti-pneumonia RASV while reducing or eliminating unwanted side effects sometimes associated with such vaccines, including  fever and intestinal distress. “Many of the symptoms associated with reactogenic Salmonella vaccines are consistent with known reactions to lipid A, the endotoxin component of the Salmonella lipopolysaccharide (LPS),” the the major surface membrane component , Kong explained.   “In this paper, we describe a method for detoxifying the lipid A component of LPS in living cells without compromising the ability of the vaccine to stimulate a desirable immune response.”

To achieve detoxification, Salmonella was induced to produce dephosphoylated lipid A, rendering the vaccine safer, while leaving its ability to generate a profound, system-wide immune response, intact.

To accomplish this, a recombinant strain of Salmonella was constructed using genes from another pathogen, Francisella tularensis, a bacterium associated with tularemia or rabbit fever. Salmonella expressing lipid A 1-phosphatase from tularensis (lpxE) showed less virulence in mice, yet acted to inoculate the mice against subsequent infection by wild-type Salmonella.

In further experiments, the group showed that Salmonella strains could also be constructed to additionally synthesize pneumococcal surface protein A (PspA)—a key antigen responsible for generating antibodies to pneumonia. Again, the candidate RASV displayed nearly complete dephosphorylation of lipid A, thereby reducing toxicity.

Following inoculation with the new Salmonella strain, mice produced a strong antibody response to PspA and showed greatly improved immunity to wild-type Streptococcus pneumoniae, compared with those inoculated with Salmonella lacking  the PspA antigen. Tissue culture studies showing reduction of inflammatory cytokines following application of modified lipid A further buttressed the results.

Francisella LpxE was shown to effectively strip the 1-phosphate group from Salmonella‘s lipid A, without loss of the bacterium’s capacity for colonization. The research holds promise for constructing modified live attenuated Salmonella vaccine strains for humans, with dephosphoylated lipid A providing additional safety benefits.

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The research was supported by grants from the Bill and Melinda Gates Foundation and the National Institute of Health

* Reposted on Request

ASU study finds antimicrobials from personal care products in statewide survey of Minnesota’s rivers and lakes – triclosan

Highlights

  • First statewide U.S. survey finds antimicrobial compounds present in sediments of Minnesota’s rivers, creeks and lakes
  • Personal care product active ingredients triclosan (TCS) and triclocarban (TCC) detected in all samples takenupstreamanddownstreamof wastewater treatment plants
  • Among the two known endocrine disruptors monitored, triclocarban was more abundant than triclosan
  • Study adds tobody of scientific work calling into question the widespread use of antimicrobial compounds that offer nomeasurable benefit for the average consumer

In our zest for cleanliness, have we permanently muddied our nation’s waters?     A science team from Arizona State University, in collaboration with federal partners, has completed the first statewide analysis of freshwater bodies in Minnesota, finding widespread evidence of the presence of active ingredients of personal care products in Minnesota lakes, streams and rivers.   These products are a billion dollar industry and can be found in antimicrobial soaps, disinfectants, and sanitizers to scrub our hands and clean countertops. Hundreds of antimicrobial products are sold in the U.S., many marketed with efficacy claims that remain elusive due to the short duration of the average consumer’s handwashing practices. The fate of these products can be traced from home use to sewers to wastewater treatment plants to eventually, downstream bodies of water.    The research team focused on two active ingredients found prominently in anti-bacterial soaps −triclosan and triclocarban− which have come under scrutiny by the EPA and FDA due to their environmental and human health concerns. These compounds persist for decades in the environment, and both triclocarban and triclosan are among the top ten pharmaceuticals and personal care products most frequently found in the environment and in U.S. drinking water resources.

“This study underscores the extent to which additives of antimicrobial consumer products are polluting freshwater environments in the U.S.; it also shows natural degradation processes to be too slow to counter the continuous environmental release of these endocrine disrupting chemicals,” said Halden, director of Environmental Security at the Biodesign Institute and professor in the Ira A. Fulton School of Sustainable Engineering and the Built Environment. Halden’s research focuses on the interconnectedness of the water cycle and human health, with specialemphasis on the role of manmade products and human lifestyle choices on environmental quality.

In a previous study, Halden’s team found significant concentrations of harmful soap-related chemicals dating back to the 1950’s in sediments of Jamaica Bay and Chesapeake Bay, into which New York City and Baltimore discharge their treated domestic wastewater, respectively.

Upon their use, triclosan and triclocarban are absorbed through the skin and hence contaminate human blood, urine, and even breast milk. Ultimately, these chemicals together with the pharmaceuticals we use end up in our sewage and surface waters. In 2002, the USGS published a landmark study that showed 80 percent of 139 streams sampled from across 30 U.S. states were found to contain measurable levels of organic wastewater contaminants. The human health risks associated with these personal care product chemicals are still not fully understood despite them being used for decades.

In the ASU study, river, creek and lakebed sediment samples from 12 locations upstream and downstream of wastewater treatment plants were analyzed for the presence of antimicrobial compounds.

For Halden’s team, which consisted of postdoctoral researcher Benny Pycke, environmental engineering graduate student and first author Arjun Venkatesan, the results showed that overall concentrations of triclocarban were 3- to 58-times higher than those of the more frequently monitored triclosan.

“We were able to detect these two compounds both upstream and downstream of suspected input sources, and the levels of the antimicrobial soap ingredient triclocarban were usually higher compared to triclosan,” said Venkatesan. “Although triclosan is used in a larger number of formulations and personal care products, we found triclocarban to be more abundant in freshwater environments.” The team also found degradation products of TCC but transformation of this antimicrobial is known to be very slow in natural environments.

“Also, we expected to find these compounds mostly downstream of wastewater treatment plants; but when we consistently found detectable levels upstream and downstream, we realized that there are probably multiple sources contributing to the contamination of these sites, potentially including additional wastewater treatment plants further upstream and runoff from sites where antimicrobial-laden sewage sludge had been applied.”

“Every site is essentially downstream of something,” added Pycke. A site in the immediate vicinity of a wastewater treatment plant near Duluth (St. Louis Bay at Lake Superior) had the greatest concentration of triclocarban and its lower chlorinated derivatives, and the Duluth site and Shagawa Lake site had concentrations three times higher than river and creek sediments. There was a strong correlation between the level of contamination with wastewater treatment plant discharge, stream flow and the population density of the surrounding region.

“As the name suggests, these antimicrobial compounds (triclosan and triclocarban) are incompatible with biological wastewater treatment infrastructure paid for with tax dollars,” said Halden. “Municipalities in Minnesota and across the U.S. work hard using state?of?the?art equipment to keep our freshwater environments clean but they cannot control what consumers, misled by aggressive marketing, discharge into the sewage collection system.”

Wherever antimicrobial personal care products are in use, water and sediment have been contaminated, a situation that certainly is not unique to the state of Minnesota. “Regulatory agencies are aware of the overuse of antimicrobials but no state or federal restrictions have been implemented yet for either triclosan or triclocarban,” said Halden. “Aside from ecological concerns, widespread environmental occurrence of antimicrobials also is a potential public health concern because unwarranted use of antimicrobials can promote drug resistance of human pathogens.”

Halden’s research is developing engineering solutions to clean up environments impacted by antimicrobial compounds. However, he emphasizes that the best solution right now in combating this pollution is for consumers to limit their use of antimicrobial personal care products that, ironically, provide no measurable health benefits to the average consumer, as determined by an expert panel convened by the Food and Drug Administration in 2005.

For this project, ASU was supported by funding from the National Institute of Environmental Health Sciences.

In addition to Halden’s appointment as Director of Environmental Security at ASU’s Biodesign Institute, he holds the title of professor in the School of Sustainable Engineering and the Built Environment, at the Ira. A. Fulton Schools of Engineering, ASU, and adjunct associate professor of Environmental Health Sciences, at the Johns Hopkins Bloomberg School of Public Health.