mBiosphere

Hot new manufacturing tool: a temperature-controlled microbe

Many manufacturing processes rely on microorganisms to perform tricky chemical transformations or make substances from simple starting materials. A study published in mBio today describes a way to control a heat-loving archaeon with a temperature switch: it makes a product at low temperatures but not at high temperatures. The authors say the innovation could make it easier to use microorganisms as miniature factories for the production of needed materials like biofuels.

Originally isolated from hot marine sediments, the hyperthermophile Pyrococcus furiosus grows best at temperatures around 100ºC (212ºF). Like other hyperthermophiles, P. furiosus' enzymes are stable at the high temperatures that facilitate many industrial processes, making it a well-used tool in biotechnology and manufacturing. But not all products can be made at high heat. Some enzymes will only work at lower temperatures.

The authors inserted a gene from another organism into P. furiosus and coaxed it to use that gene to make a new product by simply lowering the temperature. The donor organism, Caldicellulosiruptor bescii, prefers to grow at a relatively cool 78ºC, so the protein product of its gene, lactate dehydrogenase, is most stable at that comparatively low temperature.

The lactate dehyrogenase gene was inserted into a strategic spot, right next to a cold shock promoter that "turns on" the genes around it when P. furiosus is out in the cold at 72ºC. This essentially gives manufacturers a switch for controlling lactate production: put the organism at 72ºC to turn on lactate production, restore it to 100ºC to turn it off, thus preventing the need for chemical inducers. What's more, since P. furiosus is mostly shut down at these lower temperatures, making the new product doesn't interfere with its metabolism, or vice-versa.

The lead author on the study, Michael Adams of the University of Georgia, explains that this is the key benefit of this system: although P. furiosus now makes the enzyme that carries out the process, at these lower temperatures the organism's other metabolic processes don't get in the way.

"The hyperthermophile is essentially the bioreactor that contains the foreign enzymes," says Adams. P. furiosus just supplies cofactors and a cytoplasmic environment for the highly active foreign enzymes, according to Adams. This makes for a cleaner, more controllable reaction.

This is the first time a targeted modification of a hyperthermophile has been accomplished, say the authors, providing a new perspective on engineering microorganisms for product and biofuel formation.

E. coli: can subtractive reverse vaccinology help design a vaccine?

Escherichia coli is no stranger to the human body. In around 20% of us, E. coli is the predominant species in our gastrointestinal tract, where it lives as a commensal. But when E. coli gets out of hand it can cause anything from gastroenteritis to sepsis to urinary tract infections. It's those problematic aspects that Nesta et al. target in their study in mBio this week. Using a subtractive reverse vaccinology approach, the co-authors uncovered an E. coli protein specific to pathogenesis and designed a vaccine to target it. They argue that a broadly cross-protective E. coli vaccine would yield significant public health benefits.

Subtractive reverse vaccinology is a bioinformatics method in which scientists compare the genome sequences of pathogens and related non-pathogens to identify genes specific to disease for the purpose of developing a vaccine. Nesta et al. identified an E. coli gene they called FdeC this way and showed that it is involved in adhesion and the formation of macrocolonies in disease outside the gastrointestinal tract. They had some measure of success with a FdeC-based vaccine - in a mouse model it proved protective for some infection sites but not others. According to the authors, this evidence of protection argues that functionally characterizing potential vaccine candidates and determining the role of these candidates in pathogenesis could support the development of more effective vaccines - for E. coli and other causes of infection.

Commentary: using phages for treating cystic fibrosis promising, builds on decades of research

Can phages eventually help cystic fibrosis patients live longer? In a Commentary appearing in mBio this week, Harald Brüssow explains the significance of another recent mBio paper that tested the ability of bacteriophages to clear infections by Pseudomonas aeruginosa.

Worldwide, an estimated 700,000 - 1,000,000 people are living with cystic fibrosis, a genetic disorder that leads to the buildup of thick, mucous secretions in the lungs that lead to chronic infections and death at a young age. Among the bacteria that take advantage of the lungs of CF patients, P. aeruginosa is particularly problematic because it forms thick biofilm fortifications that protect it from percussive treatment and antibiotics.

Brüssow lists the reasons that phage therapy is an attractive choice for treating lung infections in cystic fibrosis patients and puts the positive findings in the context of other successful phage applications. Phage therapy isn't as far-fetched as it may sound, and the progress documented in the Alemayehu et al. paper provides an encouraging outlook, according to Brüssow.

The Black Queen Hypothesis: how microbes lose a necessary function and survive to tell the tale

Pared down genomes are the norm in symbiotic microbes, but how do non-symbionts get away with cutting out functions it would appear that they need? The authors of an Opinion piece this week explain their ideas about the matter. They say microbes that shed necessary functions may well be getting others to do the hard work for them, an adaptation that can encourage microorganisms to live in cooperative communities.

The Black Queen Hypothesis, as they call it, puts forth the idea that eliminating one function and getting another organism to carry out that process instead confers a selective advantage. In these cases, it would make evolutionary sense for a microbe to lose a burdensome gene for a function it doesn't have to perform for itself. The authors, Richard Lenski and J. Jeffrey Morris of Michigan State University, and Erik Zinser of the University of Tennessee, named the hypothesis for the queen of spades in the game Hearts, in which the usual strategy is to avoid taking this card. The organism stuck with the job of carrying out the process for another organism is, according to their theory, holding the queen of spades, a card that is a burden for the holder, but necessary for the cardgame.

"It's a sweeping hypothesis for how free-living microorganisms evolve to become dependent on each other," says Richard Losick of Harvard University, who edited the paper. "The heart of the hypothesis is that many genetic functions provide products that leak in and out of cells and hence become public goods," he says.

To illustrate their hypothesis, the authors apply it to one particular microbial system that has been a source of some confusion: one of the most common plankton species in the open ocean, Prochlorococcus, which has a much smaller genome than you might expect from a free-living bacterium. Scientists have wondered how Prochlorococcus has managed to be extremely successful while shedding important genes, including the gene for catalase-peroxidase, which allows it to neutralize hydrogen peroxide, a compound that can damage or even kill cells. Prochlorococcus relies on the other microorganisms around it to remove hydrogen peroxide from the environment, say the authors, allowing it to fob off its responsibilities on the unlucky card holders around it. This is an instance of how one species can profit from paring down while relying on other members of the community to help out.

Losick says the Black Queen Hypothesis offers a new way of looking at complicated, inter-dependent communities of microorganisms. "I have a special interest in how bacteria form biofilms, complex natural communities that often consist of many different kinds of bacteria. The Black Queen Hypothesis provides a valuable new way to think about how the members of these biofilm communities coevolved.”

Meningitis: A protein in spinal fluid is associated with survival

Bacterial meningitis strikes around 1,000 people in the U.S. every year, according to the CDC, and 10-14% of those cases are fatal. Researchers are drilling down into what happens in the spinal fluid to make a bad case of meningitis and deadly one, but they still know little about the role of one protein involved in the process: a component of the immune system called complement C3. This week, mBio features a study that shows the presence of complement C3 in spinal fluid correlates with survival, a fact that indicates it plays a key role in the pathogenesis of bacterial meningitis.

Looking for proteins that indicate survival, the team used samples of cerebrospinal fluid (CSF) from 80 patients with bacterial meningitis and 10 non-infected controls. The fluid samples were a useful resource for the team: they represented 40 meningitis patients who survived and 40 who did not. They measured the levels of several central nervous system and serum proteins that had been identified as possible indicators in earlier proteomics work. Goonetilleke et al. found that complement C3 was present at significantly higher concentration in the spinal fluid of patients who later pulled through than in patients who later died.

What does this mean? Complement C3 aids innate immunity by coating pathogens with fragments of itself, which stimulates destruction of the pathogen by other components of the immune system or the release of proinflammatory mediators. It's easy to see that if a person's spinal fluid is depleted in C3, this cascade of immune reactions wouldn't be effective and the pathogen might run rampant, endangering the patient's life. Knowing that C3 is present at greater concentrations in patients who survive, it may well serve as a prognostic marker - a sort of oracle that can indicate just how severe a case of bacterial meningitis is bound to become.

Another interesting aspect of the study is what it says about apoptosis, a process that has been the target of experimental therapies for bacterial meningitis. In mice, apoptosis (programmed cell death) is an important mechanism for neuronal cell death, which can cause neurological problems in survivors. If apoptosis were also a factor in human neuronal damage from bacterial meningitis, then treatments that inhibit apoptosis could prevent some of the neurological damage wrought by the pathogen. But in their study, Goonetilleke et al. found that standard apoptosis markers were absent in patients' spinal fluid, suggesting that apoptosis is not a factor in human cases of meningitis and that treatment with citicoline and caspase inhibitors to prevent apoptosis is unlikely to have an effect.

Microneedles could be a shot in the arm for public health

Seasonal flu is serious. Every year, flu kills around 40,000 Americans, but only 30 to 40% of the population gets a flu shot. But what if the annual flu shot wasn't a shot at all? If you could be immunized with a scratchy little pad pressed to your skin, would you be more likely to get that vaccine? We are one step closer to that option thanks to the results of a study in mBio this week, which looked at the immunological reaction to immunization with metal microneedle patches coated with influenza antigens.

Metal microneedle patches look kind of like little squares of rough velcro. They're designed to deliver antigen to the surface of the skin and the cells just below the surface to the epidermis. The skin is an important actor in the immune system, so delivering a vaccine to skin cells provides access to macrophages, Langerhans cells, and dermal dendritic cells, all of which are crucial for initiating adaptive immune responses. The conventional mode of vaccination - intramuscular - is (need I remind you?) painful, but it has another drawback as well: when using intramuscular injections, full immunization for influenza is often only achieved with high doses or multiple doses (ouch). The other option, the intranasal flu vaccine, is only approved for people between the ages of 2 and 49, a range that excludes those most vulnerable to flu-related complications: the elderly.

Using mice, Martin et al. looked at the immune response to influenza antigens administered with metal microneedle patches. They showed that immunization elicited an increase in cytokines at the immunization site that are important for recruitment of neutrophils, monocytes and dendritic cells. They also observed dendritic cells loaded with influenza viruses migrating from the place where they were deposited in the skin, which suggests they can travel to the lymph nodes - something desirable in the case of a vaccine.

These results, combined with the group's earlier work, which showed flu vaccine delivered via microneedle patches offers the same or better protection as injections, paints a rosy picture for the future of these little devices. Maybe microneedles can eventually make a dent in the annual number of flu deaths?

Commentaries: how to balance risk of H5N1 escape with benefits of research?

Where can you safely work with avian H5N1 influenza? The debate over H5N1 continues*, and this  week we present two Commentaries (by Imperiale and Hanna and García-Sastre) that can help stimulate an honest discussion about the most appropriate place to carry out research on newly developed strains. As Arturo Casadevall and Tom Shenk point out in an accompanying editorial, the decision about where to house H5N1 research has profound implications for society and poses an explicit trade-off between safety and preparedness for future outbreaks.

The Commentaries offer opposing views of the appropriate level of security for dealing with H5N1 viruses. The authors agree that, with a case fatality rate as high as 50% or more, H5N1 could create a pandemic of disastrous proportions, but they differ in their opinions of how to strike a balance between biosecurity and effective research.

BSL-4 is too much, enhanced BSL-3 is just right

Adolfo García-Sastre of the Mount Sinai School of Medicine argues that the H5N1 viruses in question may well be less pathogenic than they were before passage through ferrets, but they could still be quite dangerous, so preventing human exposure is crucial. However, he says, the ultimate level of biosecurity, BSL-4, is excessive in this case and would stifle the pace of discovery. There are both therapeutics and vaccines available for H5N1, says García-Sastre, so he advocates for conducting the research in enhanced BSL-3 facilities, which he says offer the necessary security measures, including interlocked rooms with negative pressure, Hepa-filtered air circulation, and appropriate decontamination and/or sterilization practices for material leaving the facility.

Bad virus, top security

Michael Imperiale and Michael Hanna, on the other hand, make their case that the H5N1 viruses merit BSL-4 containment. Although H5N1 that cannot transmit from human to human would normally be handled in a BSL-3 facility, researchers changed the virus' biosafety profile when they enhanced its ability to transmit between mammals. According to Imperiale and Hanna, the vaccine for H5N1 is not widely available, and drug resistance and a slow distribution system for antiviral drugs mean a small outbreak could never be contained.

Read the Commentaries and make up your own mind about the issue. Where do you stand?

*In case you've been under a rock these past few months, here's the back story: this fall, the U.S. National Science Advisory Board for Biosecurity recommended that the authors of two avian H5N1 investigations and the scientific journals that planned to publish the studies to withhold crucial details of the research in the interest of biosecurity. The researchers had used ferrets to develop strains of influenza that can transmit from mammal-to-mammal, something the virus did not readily do before. The concern is that the viruses are now honed to kill. If they've maintained their case fatality rate and if humans are anything like ferrets (and they are), these viruses could cause a pandemic of biblical proportions.

This week: "Livestock MRSA" in hospitals

After the study linking MRSA in livestock to antibiotic use from last week, MRSA makes another appearance in mBio, but this time the focus is on hospital-acquired infections. A human-specific, tetracycline-sensitive strain of Staphylococcus aureus (methicillin-sensitive S. aureus, or MSSA) is becoming an increasing problem in health care settings, and it is closely related to the MRSA strains called ST398, which favor livestock and farm workers.

Uhlemann et al. founded their study on one troubling observation: the emergence of this ST398 MSSA strain in households in Northern Manhattan, a region well-known for its low density of livestock farms. Unlike their livestock-associated relatives, however, MSSA isolates of the ST398 lineage are easily transmissible between humans, a fact that is making them problematic in hospitals. ST398 MSSA is also well-adapted to the human host, deploying a bevy of mobile genetic elements, surface adhesins, and the ability to adhere to cells in human skin called keratinocytes. The human ST398 MSSA is now apparently spreading independent of animal contact, causing conditions that range from soft tissue infections to invasive bloodstream infections, but it is also a benign resident of nasal tissues and household surfaces.

Opinion: H5N1 just as dangerous as feared, now requires action

The debate continues over H5N1 influenza, but this week we have a Commentary from Michael Osterholm and Nicholas Kelley, both of the Center for Infectious Disease Research and Policy at the University of Minnesota. Osterholm has been in the news recently discussing H5N1 and just how deadly it might actually be. According to Osterholm and Kelley, certain reviews that play down the fatality case rates of H5N1 (like the review in Science this week ) are off the mark in their summation of newly developed H5N1 virus strains. They say H5N1 viruses are just as scary as we always thought they were, and it is time to move the discussion forward and figure out how to cope with that fact.

In case you've been under a rock these past few months, here's the story: this fall, the NSABB recommended that the authors of two H5N1 investigations and the scientific journals that planned to publish the studies to withhold crucial details of the research in the interest of biosecurity. The researchers had used ferrets to develop strains of influenza that can transmit from mammal-to-mammal, something the virus did not readily do before.

The concern is that the viruses are now honed to kill: if they've maintained their case fatality rate and if humans are anything like ferrets (and they are), these viruses could cause a pandemic of biblical proportions. However, some authors (including authors of previous Commentaries in mBio and the authors of the piece in Science) have argued that the virus must be attenuated after passage through ferrets and that the case fatality rate for H5N1 is not nearly as high as we'd been led to believe.

In the Commentary, Osterholm and Kelley present their case that H5N1 is a very dangerous virus based on their analysis of published studies of the seroepidemiology of H5N1 in humans. H5N1 flu infections have exceedingly high mortality, they say, and current vaccines and antiviral drugs will not pull us out of a global H5N1 pandemic. "We believe that the assertion that the case-fatality rate of H5N1 influenza in humans may be overestimated is based on a flawed data analysis," says Osterholm.

Analysis of reports of H5N1 seroprevalence that include data from the 1997 Hong Kong outbreak as well as data from 2004 to date will give a misleading impression because the 1997 outbreak was a very different "biologic event" that is recognized as such by the WHO, because the 1997 H5N1 virus has a significantly different genotype from that of later H5N1 viruses. This is why the WHO does not include the Hong Kong H5N1 virus data in any analysis of H5N1 transmission, and the 1997 Hong Kong virus is not recommended for inclusion in H5N1 vaccines, Osterholm explained.

Seroepidemiologic studies that have examined the exposure of various groups of people to H5N1 viruses from 2004 onward indicate that only a small segment of the population has ever been exposed to H5N1, and that among those that have been exposed, many become seriously ill or die.

"The available seroepidemiologic data for human H5N1 infection support the current WHO reported case-fatality rates of 30% to 80%," the authors write. In the event of an H5N1 pandemic, they point out, if the virus is even one tenth or one twentieth as virulent as has been documented in these smaller outbreaks, the resulting fatality rate would be worse than in the 1918 pandemic, in which 2% of infected individuals died.

Vaccines will not head off an H5N1 pandemic either, the authors say, since the time required to develop and manufacture an influenza vaccine specific to new outbreak strain has resulted in "too little, too late" vaccine responses for the 1957, 1968, and 2009 influenza pandemics, and not much in the process has changed since 2009.

"The technology behind our current influenza vaccines is simply not sufficient to address the complex challenges associated with an influenza pandemic in the 21st century," Osterholm and Kelley say.

This is the heart of the matter, they say: there has been enough discussion about how severe an H5N1 pandemic might be. Moving forward, the current controversy has provided a valuable opportunity for scientists and public policy experts to discuss influenza research and preparedness and create "a roadmap for the future." The discussion among scientists and policy makers needs to move on from whether H5N1 poses a serious international threat - as it clearly does - and begin discussing how we can prevent these viruses from escaping labs and how scientists can share their flu-related results with those who have a need to know.

There are critical questions that need to be answered, the authors say. For instance, how can scientists conduct virus-transmission studies in mammals safely and how can scientists share research methods and results with those who have a need to know? We also need to come to agreement on how to ensure that strains of H5N1 viruses created in the lab don't escape those controlled environments, the authors say. And new, more effective vaccine technologies are needed that can enable substantially faster production. Resolving these issues could allow H5N1 research and preparedness to serve as a springboard for solving similar problems with existing or emerging pathogens.

MRSA in livestock acquired drug resistance on the farm, now infects humans

A strain of MRSA that humans can contract from livestock most likely became drug resistant due to the use of antibiotics on the farm. That's according to the authors of a study in mBio this week, who looked closely at the genetic relationships among strains of the antibiotic resistant bacterium MRSA (methicillin-resistant Staphylococcus aureus). They discovered that ST398, a type of MRSA found in livestock that can also be passed to humans was originally a human strain, and it developed resistance to antibiotics once it was picked up by farm animals. The finding illustrates a very close link between antibiotic use on the farm and potentially lethal human infections.

MRSA is the well-known cause of a variety of invasive skin infections that can quickly turn life-threatening, but in 2003, a novel form of MRSA called ST398 emerged in livestock. Today, ST398 regularly infects farm workers and others who come into contact with infected livestock with any of several types of acute infections, including skin and soft tissue infections, respiratory infections, and bacteremia (also called sepsis). The strain can now be found in pigs, turkeys, cattle, and other livestock and has been detected in 47% of meat samples in the U.S..

It has been thought that overuse of antibiotics in livestock production could be fueling antibiotic resistance in bacteria, including S. aureus. In 2001, the Union of Concerned Scientists estimated that livestock producers in the U.S. used 24.6 million pounds of antibiotics per year for non-therapeutic purposes, a controversial practice that has now been banned in the European Union. The mBio study draws a line between the exposure of S. aureus to antibiotics on farms and the development of a form of MRSA that can threaten human lives, a correlation that has long been suspected but has been difficult to study directly.

A team of researchers from the Translational Genomics Research Institute (TGen) in Flagstaff, Arizona, the Statens Serum Institut in Denmark, and several other institutions sequenced the genomes of 88 different S. aureus isolates that are all closely related to ST398 to determine the family relationships among antibiotic sensitive strains and antibiotic resistant strains from both humans and animals.

An analysis of the various genomes revealed that ST398 most likely evolved from an antibiotic-sensitive strain of S. aureus that came from humans. Once it found a home in livestock, the genome sequences indicate this strain changed rapidly, acquired some new genes, and differentiated into many different types, including ST398, which is resistant to a few different antibiotics.

"Most of the ancestral human strains were sensitive to antibiotics, whereas the livestock strains had acquired resistance on several independent occasions," says Ross Fitzgerald of the University of Edinburgh in Scotland, who reviewed the paper for mBio. This implies that the bacterium picked up the ability to fend off antibiotics after it migrated into livestock, says Fitzgerald.

The fact that ST398 originally came from a human is significant, says Fitzgerald, because it shows that infection is a two-way street. "Intensive farming practices could promote the transfer of bacteria between different host species including humans to animals," says Fitzgerald. ST398's family tree shows that sharing bacteria with livestock could well mean that those bacteria come back to us with the ability to defeat antibiotics.