Tips from the journals of the American Society for Microbiology

December 16, 2010

Researchers Select Microbes to Improve Chocolate

It may seem hard to imagine improving on the world's best chocolates, but that is the goal of a team of microbiologists from the Free University of Brussels, Belgium. Raw cocoa beans have an astringent, unpleasant taste, and must be cured--which involves fermenting the beans--prior to drying and roasting. The Belgian researchers aim to improve the fermentation process, as well as the taste and health benefits of the resulting chocolate, by optimizing a microbial starter culture. The research is published in the December 2010 issue of the journal Applied and Environmental Microbiology.

Creating chocolate from the beans within the raw cocoa pods, which have roughly the size and shape of rugby balls, is a multistep process. The farmers open the pods manually, depositing beans and pulp in heaps of roughly 150 kg onto banana leaves or into boxes, to ferment in the sun, for up to six days, says corresponding author Luc De Vuyst, of the Free University of Brussels. First, yeast ferment sugars to ethanol. Next, lactic acid bacteria convert citric acid and any remaining sugars to lactic acid, and acetic acid bacteria convert ethanol to acetic acid. Acetic acid then penetrates the cocoa beans, releasing enzymes and substrates within to interact with each other, forming the precursors of the chocolate flavor. Only then are the beans dried, and then transported to chocolate companies, where they are roasted, converting the precursors to flavors

In their quest for a superior starter culture, the researchers are conducting fermentation experiments to investigate growth and metabolite production using strains of lactic and acetic acid bacteria which they isolated from fermentation heaps from different locations. (They showed that the species composition of the fermentation heaps is identical regardless of location of origin, although bacterial strains vary from place to place.) Since the times which fermentation experiments can be carried out onsite are limited to two short growing seasons, and travel from Belgium to Africa or South America is dear, the researchers have developed a cocoa bean pulp simulation medium so that they could conduct their experiments in their laboratory.

"We were able to determine the role of specific bacteria during the fermentation process," says De Vuyst. "In this way, we can select certain strains of the most relevant bacterial communities to use as starter cultures in later experiments in the jungle. For example, one species of acetic acid bacteria was notable for its ability to oxidize ethanol and lactic acid, its general tolerance of acidity, and its moderate heat tolerance, he says. We hope not only to improve the fermentation process, but also to improve the flavor, and health benefits of chocolate."

"Note that the best chocolates of the world are already produced in Belgium," brags De Vuyst. "But we want to produce even better and more diverse chocolates."

(T. Lefeber, M. Janssens, N. Camu, and L. De Vuyst. Kinetic Analysis of Strains of Lactic Acid Bacteria and Acetic Acid Bacteria in Cocoa Pulp Simulation Media toward Development of a Starter Culture for Cocoa Bean Fermentation. Appl. Environ. Microbiol. 76: 7708-7716.)

Synthetic Riboswitches Turn On Bacterial Genes

Scientist show that synthetic riboswitches can control gene expression in a wide variety of bacteria, including some organisms in which such control has been difficult. The research is published in the December 2010 issue of the journal Applied and Environmental Microbiology.

Bacteria regulate their metabolism using riboswitches, sequences of RNA that alter gene expression when they bind a small-molecule metabolite. In earlier work, Justin P. Gallivan's laboratory at Emory University, Atlanta created synthetic riboswitches, which the researchers can use to turn specific genes on or off, to control what the cell does.

In the current study Gallivan shows that can control gene expression in a wide variety of bacteria. More importantly, they have created riboswitches that function in certain pathogens, including Streptococcus pyogenes, the microbe that causes strep throat. That research should lead to a better understand the mechanisms of pathogenicity in these organisms, says Gallivan.

Traditionally, researchers investigate gene function by creating "knock-outs," organisms in which the gene of interest has been removed. The problem with that approach: removing an essential gene kills the cell, says Gallivan. But using riboswitches, the researchers could turn an essential gene off only briefly, to determine its function without killing the cell.

Another potential application of riboswitches is to program bacteria to perform complex tasks, says Gallivan. For example, riboswitches can be used to control bacterial movement. "By developing a riboswitch that responds to the herbicide, atrazine, we reprogrammed cells to follow atrazine," he says. "By then adding a gene that encodes an atrazine-catabolizing enzyme, we created cells that seek and destroy atrazine." Such an organism could be developed for cleaning up pollution."

(S. Topp, C.M.K. Reynoso, J.C. Seeliger, I.S. Goldlust, S.K. Desai, D. Murat, A.W. Puri, A. Komeili, C.R. Bertozzi, J.R. Scott, and J.P. Gallivan, 2010. Appl. Environ. Microbiol. 76:7881-7884.)

Healing Wounds With Silver Preps: Better Preps and Better Assays

Researchers have identified a better method of using silver to treat infected wounds. They report their findings in the December 2010 issue of the journal Antimicrobial Agents and Chemotherapy.

Healing difficult wounds is often a challenge, due, in large part, to the bacterial biofilms that frequently thrive in wounds. Sixty-five percent of hospital-acquired infections involve biofilms, according to an estimate from the Centers for Disease Control and Prevention. Biofilms are far more resistant to antibiotics than bacteria which have not organized themselves in this manner. Certain silver preparations appear to improve antibiotic efficacy against wound infections.

Victoria Kostenko, and John Martinuzzi of the University of Calgary, Alberta, Canada, and their collaborators, concerned that laboratory studies testing the effectiveness of different silver preparations often fail to match observations from clinical studies, provide a new test procedure which corresponds more accurately to observations from clinical and animal studies. They also find that nanocrystaline silver has greater efficacy than conventional silver against biofilms during both one-day and long-term treatment.

The nanocrystaline silver boosts the vulnerability of biofilms to antibiotics as compared to larger silver particles because the small particles expose more surface. The silver atoms on those surfaces interact with macromolecules such as DNA and proteins within the biofilms, disorganizing them, thus degrading their defenses against antibiotics.

One of the advances in the testing methodology involves refreshing the growth media, thereby decreasing the bioavailability of silver particles, as happens in actual wounds as compounds therein bind with the silver particles, says Martinuzzi. That, he says, could be responsible for the discrepancy between laboratory results using standard protocols, and clinical results. "We hope our results will convince others to devise laboratory tests which better account for the wound environment. This will help advance product development."

Interestingly, this investigation grew out of the researchers' efforts to develop bioremediation for oil sands tailing ponds, such as those in northern Alberta. "Biofilms are highly adaptable to hostile environments and great producers of by-products, including surfactants capable of helping in the separation of residual bitumen and consolidation of clay suspensions," says Martinuzzi. "The underlying mechanisms are quite generic and thus we often transfer information between fields. We believe that exploiting biofilms will enable us to reduce the ecological footprint of oil sand operations to acceptable levels."

(V. Kostenko, J. Lyczak, K. Turner, and R.J. Martinuzzi, 2010. Impact of silver-containing wound dressings on bacterial biofilm viability and susceptibility to antibiotics during prolonged treatment. Antim. Agents Chemother. 54:5120-5131.)

Study Suggests Immune System Suppresses HIV-1 To Clinically Undetectable Levels in Long-Term Nonprogressors

Some people infected by HIV-1, known as long-term-nonprogressors, can live free of AIDS for more than 15 years even without medical treatment. A subgroup of these people are characterized by plasma concentrations of virus so low as to defy measurement by standard clinical techniques. A team of researchers now provides strong evidence that these individuals, dubbed HIV-1 controllers, are indeed suppressing the virus rather than simply playing host to a poorly replicating strain of HIV-1. The research is published in the December 2010 issue of the Journal of Virology.

Studies published in 2007 first showed that virus cultured from HIV-1 controllers could replicate just as competently in the laboratory as well-characterized laboratory strains of HIV-1. That suggested that strains infecting HIV-1 controllers were similar to strains in more typical individuals, and that HIV-1 controllers' immune systems were doing an unusually good job against the virus. Nonetheless, extrapolating from laboratory studies, where the environment differs so completely from that of the natural host, is risky, says corresponding author Helene Mens of the National Cancer Institute, National Institutes of Health.

A better way to assay replication competency, Mens posited, would be to study genetic change within the virus over time. The virus mutates fairly consistently with each replication. So Mens obtained samples from HIV-1 controllers, and teamed up with John Coffin of the National Cancer Institute, who specializes in quantifying and amplifying HIV-1 from patients with viral loads below the limit of detection in standard assays. Their research showed a rate of genetic evolution in HIV-1 controllers that was similar to that in patients with more typical progression rates. They also found that most HIV-1 controllers had viremia levels that ranged from below 0.2 to 43 copies per ml plasma, well below the limit of detection using clinical techniques--50-75 copies per ml of plasma--and orders of magnitude below typical levels of viremia, which average around 50,000 copies per ml plasma absent therapy.

The research also indicates that the immune system is mounting a strong cellular immune response, in which certain T cells recognize specific antigens on the surface of infected cells, and cause those cells to commit cellular suicide. This "raises hope that an effective HIV vaccine that can delay disease progression and prevent transmission can be developed," says Mens.

Interestingly, previous studies have suggested that HIV-1 controllers have more immune activation, inflammation, and arteriosclerosis (both of which can be a consequence of ample immune activation) than healthy controls. "It is important to find out of viral replication is driving immune activation and inflammation in HIV-1 controllers, and if they therefore may benefit from antiretroviral therapy," says Mens.

(H. Mens, M. Kearney, A. Wiegand, W. Shao, K. Schonning, J. Gerstoft, N. Obel, F. Maldarelli, J.W. Mellors, T. Benfield, and J.M. Coffin, 2010. HIV-1 continues to replicate and evolve in patients with natural control of HIV infection. J. Virol. 84:12971-12981.)

American Society for Microbiology

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