Tag Archives: microbiology

Pseudomonas: Even More Bad-Assed Than We Thought

The first time I met the genus Pseudomonas, I was a brand-new graduate student doing a rotation in David Figurski’s lab at Columbia University. Dave works on “promiscuous” plasmids that can move to many different species of bacteria. These plasmids pay their rent by providing multiple antibiotic resistance genes and other goodies to the bugs that host them. In that lab, we commonly cultured new strains with three or four different antibiotics in order to select for particular plasmids. Novice that I was, it seemed to me that such cultures would be pretty foolproof in terms of contamination – I mean, how many “wild” bacteria could live in such a drug-laden soup?

Pseudomonas aeruginosa, for one. It wasn’t long before I managed to contaminate a plate with something that grew bright green colonies and had a weird grape-like odor. A senior grad student in the lab diagnosed it immediately. “But how can it survive on there?” I asked. “It’s Pseudomonas,” he laughed.

Pseudomonas killing another bacterium with secreted toxins.

The last thing you see: P. aeruginosa (red) secretes toxins into another bacterium (green/blue), causing its cell wall to fall apart. Image by James Easter.

While many bacteria pick up drug resistance genes here and there through mobile genetic elements such as plasmids and transposons, P. aeruginosa has resistances baked right into its genome. It carries multiple drug-specific resistance genes as well as general-purpose pumps that throw antibiotics out of the cell before they can even act.

One Pseudomonas cousin, now known as Burkholderia cepacia, takes this tough-guy act a step further; it can actually use penicillin as a food source. Imagine hornets passing around a can of Raid so they can take long pulls from it while commenting on its pleasant flavor, and you’ve got the general idea. These are some seriously bad-assed bugs.

Given all that, perhaps it shouldn’t come as a surprise that P. aeruginosa is also covered in venomous spines. That was the conclusion of a paper published last summer by researchers at the University of Washington, and discussed in an accompanying News & Views article in Nature.

The authors start off the paper with an excellent description of the microbial world:

Competition for niches among bacteria is widespread, fierce and deliberate. These organisms produce factors ranging in complexity from small diffusible molecules to multicomponent machines, in order to inhibit the proliferation of rival cells.

It’s a jungle down there at the micrometer scale, and P. aeruginosa is the honey badger.

This is a heavily armed conflict on all sides. While we often think of antibiotics as brilliant products of medical research, microbes invented them. Penicillin is just a fungus’s way to get ahead of its bacterial competitors; penicillin resistance is the bacterial side’s countermeasure. We’re naive newcomers to a battle that’s been raging for billions of years.

One of the more sophisticated siege machines in play is called the Type VI secretion system, a set of proteins found in many Gram-negative bacteria. The Type VI system injects proteins from one cell directly into another when they touch, in a manner similar to the way some bacteriophages transfer their genetic material into their hosts. That similarity could be a case of convergent evolution, in which form followed function, or the bacteria might have ripped off the mechanism directly from an ancient phage infection that went awry. I favor the latter explanation, largely because it fits so well with the general Pseudomonas attitude. Scrapping an infecting pathogen to build a weapon just sounds like something it would do.

In P. aeruginosa, the Type VI system exports at least three proteins, called Tse1, Tse2, and Tse3. All three, the new paper explains, are poisonous to other Gram-negative bacteria. Tse1 and Tse3 attack the peptidoglycan structure that lies in the periplasmic space between the outer and inner membranes of Gram-negative cells, causing the cell to fall apart or lyse. Earlier work had shown that Tse2 is toxic when it enters the cytoplasm, but it’s not clear exactly how it kills. When P. aeruginosa contacts another Gram-negative bacterium, it can use its Type VI secretion system to inject these toxins into the periplasmic space, killing off its competition so it can colonize a new food source.

That’s all well and good, but there’s a problem: the Tse proteins can also kill P. aeruginosa. To solve that, the bacteria carry antidotes, called Tsi1, Tsi2, and Tsi3. Each poison/antidote pair is expressed from a bicistronic operon (two genes transcribed from a single promoter), so whenever a cell manufactures the poison, it simultaneously produces the antidote for itself.

If Pseudomonas is producing both poison and antidote, though, why not just secrete the poison into the environment, antibiotic-style, rather than inject it through direct contact? It could be that these particular toxins don’t work well when diluted in the surrounding fluid, or that the whole toxin-plus-injection system evolved as a complete setup. Evolution doesn’t analyze a problem and calculate the most efficient way of doing things, it just tries out solutions until something works, and this works. It’s also possible that Pseudomonas is playing a more sophisticated game that we still haven’t fully uncovered. Transfering a toxin through direct contact could allow the bacterium to perform some kind of recognition step so that it won’t inject its brethren. Bayonets are less likely to cause collateral damage than bombs.

Whatever Pseudomonas is up to, it’s certainly a good system to keep studying. Besides being a pathogen in its own right, especially in patients with cystic fibrosis or immune deficiencies, this genus provides a nice sampling of the kinds of adaptations bacteria have evolved to survive in ferociously competitive environments. If it shows up as a contaminant in your lab, though, just throw those plates into the autoclave. That’s one thing even Pseudomonas can’t survive – yet.

1. Alistair B. Russell, Rachel D. Hood, Nhat Khai Bui, Michele LeRoux, Waldemar Vollmer & Joseph D. Mougous (2011). Type VI secretion delivers bacteriolytic
effectors to target cells, Nature, DOI: 10.1038/nature10244

Exploring the Sourdoughome

I love it when my interests intersect, so this new report from researchers in Italy and Belgium, on the microbiota of sourdough breads, definitely caught my attention. As the authors explain:

This study aimed at the identification of the [lactic acid bacteria] (LAB) and yeast microbiotas of 19 Italian sourdoughs used for the manufacture of traditional/typical Italian breads. The dominating LAB and yeasts were monitored by culture-dependent methods. Multivariate statistical analyses were performed in order to find the correlation between ingredients and the composition of the sourdough microbiotas, as well as the effects of the latter on the biochemical characteristics of sourdoughs.


My own applied microbiology project.

It seems that Italian sourdough is a particularly good subject for this, as the country is home to about 200 different types of bread, many of them leavened with regionally unique sourdough starters. These distinct bread recipes also use different types of flour and employ different procedures for “back-slopping,” or propagating the mixed bacterial-yeast culture. If there’s anything Italians love more than bread, it’s disagreeing about how to do things.

To see what the different starters look like microbially and chemically, the team took samples from 19 different sourdoughs, then cultured and identified their bacterial and fungal constituents to the level of species and strains. They also analyzed such parameters as pH, lactic acid concentration, and levels of free amino acids (FAA), gamma amino butyric acid (GABA), and other byproducts of fermentation.

Sure enough, the diverse baking techniques have led to diverse microbial and biochemical traits in the sourdough starters. There are a few dominant species of lactic acid bacteria and yeasts – Lactobacillus sanfranciscencis is the top bacterial species and the venerable Saccharomyces cerevisiae dominates the yeast communities – but they’re joined by a large supporting cast of related microbes, and each region seems to have its own specific combination of sub-strains. The resulting breads are equally varied, from Pane di Altamura (pH 4.03, 82mM lactic acid) to Pane Casareccio di Genazo (pH 4.14, 63.7mM lactic acid) to Pagnotta del Dittaino (pH 3.70, 83mM lactic acid).

The differences might affect more than just the flavor of the bread. As the researchers comment in the paper:

In addition, FAA and GABA produced by LAB may increase the nutritional value of the breads. For instance, the amount of GABA in 150 g of Pane di Matera PGI represents the minimum effective daily dose to get positive effects in humans.

The “positive effects” they’re talking about include lowering blood pressure in people with mild hypertension. Perhaps that’s another way the famous “Mediterranean diet” offsets the effects of that region’s delicious meats and cheeses.

One potential limitation of the study was that it relied on culturing the sourdough microbes in order to identify them. As metagenomic studies have recently revealed, the culturable part of the microbial world is just the tip of the iceberg. There’s a whole universe of bacterial, fungal, protozoan, and viral life out there that just can’t survive in any of the relatively small number of culture media available in the lab. That said, sourdough starters have been selected for a certain type of culturability, so there probably aren’t too many unculturable organisms in these samples. If it grows in a deliberately maintained culture in the kitchen, it will probably do so in the lab, too.

I hope the authors are planning to do follow-up studies on other sourdoughs, and perhaps on some beers. Belgian beers should be particularly interesting, as that country’s brewers have pursued their art in as many ways as Italian bakers have theirs.

Meanwhile, I’ll keep doing my own work in this field, which consists of baking a loaf of sourdough every few weeks. My starter allegedly originated in 1847 along the Oregon Trail, though it’s been passaged by many people in different parts of the US since then. The really charming thing about it is that a dedicated group of volunteers still distributes this starter to anyone who asks, for the cost of a self-addressed stamped envelope. I don’t know whether it produces enough GABA to lower anyone’s blood pressure, but it sure does taste good.

ResearchBlogging.orgMinervini, F., Di Cagno, R., Lattanzi, A., De Angelis, M., Antonielli, L., Cardinali, G., Cappelle, S., & Gobbetti, M. (2011). Lactic Acid Bacterium and Yeast Microbiotas of 19 Sourdoughs Used for Traditional/Typical Italian Breads: Interactions between Ingredients and Microbial Species Diversity Applied and Environmental Microbiology, 78 (4), 1251-1264 DOI: 10.1128/AEM.07721-11

Sewage Treatment, Coral Disease, and Koch’s Postulates

Coral reefs are in a tight spot these days. Increasing CO2 levels and rising ocean temperatures aren’t doing them much good, but their biggest problems are more direct. Overfishing is wiping out important predators, the aquarium trade picks off whatever looks pretty, agricultural and other runoff is clogging the filter-feeders, and some folks are even blowing them apart with dynamite.

Places with strict environmental regulations and protected marine preserves are generally doing a better job protecting their reefs, but even there we may be doing damage without realizing it. For example, what if coral reefs are catching human diseases?

That seems to be exactly what’s happening in Caribbean elkhorn coral (Acropora palmata), an iconic and structurally crucial reef species that’s been dying from a mysterious condition called white pox, or acropora serratiosis. The disease has been so deadly that the US EPA declared A. palmata an endangered species in 2006. Now, scientists have fingered human sewage as the source of the pathogen causing white pox.

A diver swims past an elkhorn coral colony. Image courtesy James Porter, University of Georgia.

A diver swims past a healthy elkhorn coral colony on Molasses Reef, near Key Largo, FL. Many other elkhorn colonies are dying from an infection that may be caused by a human pathogen. Image courtesy James W. Porter, University of Georgia.

Researchers at Rollins College and the University of Georgia described the finding yesterday in PLoS ONE:

Here we hypothesize that [Serratia marcescens] strain PDR60 isolated from two distinct environments, one terrestrial (human wastewater) and one marine (APS-affected A. palmata, apparently healthy Siderastrea siderea and Coralliophila abbreviata) causes APS [acroporid serratiosis] in A. palmata. To examine this hypothesis we conducted challenge experiments by inoculating eight isolates of Serratia marcescens representing three strains onto A. palmata fragments maintained in closed seawater aquaria. Our results confirm strain PDR60 as a coral pathogen through fulfillment of Koch’s postulates. These results are also consistent with the hypothesis that non-host corals and predatory snails may function as interepizootic reservoirs or vectors of the APS pathogen. Furthermore, we show that S. marcescens isolated from human wastewater causes APS in as little as four days, unequivocally verifying humans as a source of a marine invertebrate disease.

S. marcescens is a ubiquitous gram-negative bacterium. It’s in dirt, sewage, and probably your shower. If you haven’t noticed it, it’s because you have a working immune system. People who aren’t so fortunate – especially in hospitals – can get serious opportunistic S. marcescens infections. When this bug first turned up as a possible culprit in white pox, I figured it was probably an opportunist in the elkhorn corals as well. Perhaps the coral somehow got the anthozoan equivalent of a suppressed immune system, and Serratia took advantage of the situation.

The new work suggests otherwise, though I’m not sure it quite seals the case. The investigators experimentally infected elkhorn corals growing in tanks of purified saltwater, and found that a single inoculation with a pure S. marcescens strain cultured from sewage effluent was enough to give the corals a virulent case of white pox. This is called fulfilling Koch’s postulates, and it’s the Holy Grail of epidemiology. We can now say for sure that S. marcescens from human sewage causes white pox.

Or can we? I’m certainly convinced that the bacterial strain in sewage is capable of causing the distinctive pathogenesis of this disease, consisting of bleached white zones that spread across the coral colony. But we need to read the fine print.

The coral colonies these researchers used were harvested from “healthy” wild corals. That’s the only practical way to get experimentally useful amounts of this slow-growing creature. Unlike mice or guinea pigs, we can’t just breed up a bunch of stock from a well-characterized lab strain. However, picking apparently healthy corals from the wild doesn’t prove that they really are healthy. They’re presumably exposed to the same stresses and insults that afflict their white pox-infected neighbors, and could very well be on the brink of contracting the disease themselves. Maybe they’re already infected with the real underlying cause of the disease, and are just one wound or stressor away from getting the S. marcescens component that will finish them off. Because it’s currently impossible to characterize all aspects of the immunological and infectious status of a coral sample, we can’t know whether the bacterium alone is enough to cause disease.

Worse, we can state with certainty that the corals in these experiments were exposed to unusual stress. The scientists chipped off a piece of the colony (traumatic injury), sampled its mucoid coating (open wound), then carried it by boat to a laboratory tank (physiological stress).

That’s not to say I don’t believe the conclusions or the authors’ recommendations. Indeed, the measures they suggest include improved sewage treatment plants throughout the Caribbean, a step that’s clearly a good idea for a long list of reasons, whether or not it will save the elkhorn coral. Humans already get well-documented cases of sewage-borne diseases, and many Caribbean towns use inadequate treatment systems that raise the risk of these infections. That’s why Florida is already in the process of upgrading the treatment plants throughout the Keys. Of course, someone should also take a long, hard look at the waste treatment (or lack thereof) on cruise ships in international waters.

In the meantime, I hope researchers will continue studying white pox, with an eye toward preventing and perhaps treating it. As one of the authors points out in an accompanying press release, the stakes are high, even if the metaphors are a bit mixed:

“These bacteria do not come from the ocean, they come from us,” said Porter. Water-related activities in the Florida Keys generate more than $3 billion a year for Florida and the local economy. “We are killing the goose that lays the golden egg, and we’ve got the smoking gun to prove it,” [University of Georgia Ecology Professor James] Porter said.

1. Sutherland, K., Shaban, S., Joyner, J., Porter, J., & Lipp, E. (2011). Human Pathogen Shown to Cause Disease in the Threatened Eklhorn Coral Acropora palmata PLoS ONE, 6 (8) DOI: 10.1371/journal.pone.0023468

High-Definition Microscopy Movies – Now in 3-D

In a paper appearing right now in Nature Methods, researchers at the Howard Hughes Medical Institute’s Janelia Farm campus describe a new type of microscopy that’s just chock full of awesomeness. By shooting a special pulsating light source into the side of a sample and imaging the photons coming off at a right angle to the light, they can construct highly detailed 3-dimensional views of individual cells. That’s cool on its own, but what takes it over the top is that they can also zoom the view very rapidly through individual cells. The result is true three-dimensional movies of living cells.

Here, for example, is their movie of African green monkey cells carrying out endocytosis:

Vesicles forming. Movie courtesy Eric Betzig/Janelia Farm*

The waves crashing across the surface of the cell in the top panel are called membrane ruffles, and they’re forming the vacuoles (big globular structures) you can see in the bottom panels. Having learned the textbook version of this process, which is inevitably accompanied by a drawing showing a dimple forming in the cell surface, I find this fascinating to watch. Instead of dimples, we have something that looks more like a cat’s tongue drinking water, which in retrospect makes a lot of sense. The cell isn’t passively letting its food push into it, it’s actively eating.

The other thing that occurs to me as I watch the movie is: HOLY CRAP WE CAN ACTUALLY SEE THIS! Other microscopy techniques offer high resolution, or the ability to see subcellular structures in three dimensions, or time-lapse moviemaking capabilities in live cells, but not all three simultaneously. I think this technique is going to become extremely popular in cell biology labs.

Now if you’ll excuse me, I have to go spend another hour looping this movie – and the others in the paper’s supplementary data – and staring at them with my jaw on the floor.

* If anyone knows a straightforward way to embed a Quicktime movie in a WordPress post, please explain in the comments. Otherwise, just click the link and watch it in the subsequent window. Sorry for the inconvenience. David kindly sent instructions for embedding the video in the post. Thanks!

Microbes Munched Macondo’s Methane

Back in May, I blogged about a proposal to monitor dissolved methane levels in the Gulf of Mexico as a surrogate marker for oil. The idea was that the Deepwater Horizon blowout was spewing a mixture of oil and methane into the water, and while measuring oil levels is relatively complicated, dissolved methane is pretty easy to track. Measuring methane would have provided a good estimate of the actual volume of the oil spill.

I don’t know how far that effort got, but if researchers didn’t measure the gas promptly, it’s too late now. As John Kessler and his colleagues report in the latest online issue of Science, a massive bacterial bloom quickly ate the excess methane.

Sampling the Gulf of Mexico with a rosette.

Sampling the water column in the Gulf of Mexico with a rosette.

According to data from the NOAA research vessel Pisces, dissolved methane levels dropped rapidly in the Gulf once BP capped the Macondo well. Just four months after the initial blowout, methane levels were nearly normal again.

That’s good, because methane in the water column alters the water chemistry in potentially harmful ways, and methane released into the atmosphere is a potent greenhouse gas. Instead, the methane from the well seems to have fed a bacterial bloom, which will presumably enter the food web and the normal oceanic carbon cycle. It could still have downstream impacts on the ecosystem, but they should be less severe than researchers had initially feared.

It also illuminates an interesting aspect of normal ocean microbiology. Deep methane releases are a regular phenomenon on the seabed, stemming from hydrothermal vent eruptions and the breakdown of solid methane hydrates. Based on the rapid bacterial response to the Deepwater Horizon blowout, the researchers propose that methanotrophic bacteria react similarly to these natural methane releases, quickly recycling the gas back into the marine ecosystem.

Too bad we can’t get them to eat the oil off the beaches that fast.

Happy Hand Washing Day

Today is the United Nations’ first worldwide Hand Washing Day. Yes, it means exactly what it sounds like. As the BBC reports:

“The message we are really trying to get out is the importance of correctly washing your hands with soap and water at the critical times,” Unicef’s senior Sanitation and Hygiene programme adviser, Therese Dooley, said.

“And those critical times are before you cook or prepare food, before you eat and after using the toilet and after cleaning a baby.”

Public health experts estimate that regular hand washing can reduce the risk of diarrheal diseases, currently one of the world’s leading causes of death, by more than 40%. Now if only we could ensure that everyone in the world had enough clean water to wash with …

Bringing Back an Oldie to Fight TB

In a new paper in PLoS Medicine, researchers have stumbled onto a promising “new” treatment for that resurgent scourge, tuberculosis:

“Rifapentine is back,” says Johns Hopkins infectious disease specialist Eric Nuermberger, M.D., whose studies in mice, to be published in the Public Library of Science journal PLoS Medicine online Dec. 17, have found it so promising as an initial treatment for active TB that clinical trials are scheduled to begin next year in at least eight countries.

The mouse studies showed that substituting higher and daily doses of rifapentine for another antibiotic, rifampin, cured mice two to three times faster than the much older, standard regimen of drugs that includes rifampin. Researchers say if tests in people confirm the findings in mice, the average time to clear the potentially fatal bacterial infection could be reduced from six months to three or less.

Of course, that last “if” is a mighty big one. A longstanding saying in biomedical research is that mice lie and monkeys exaggerate. Still, this marks one of the few pieces of potentially good news in the ongoing fight against TB. Interestingly, rifapentine is a very old antibiotic that fell out of favor (and out of production) years ago, so the new work resurrects a forgotten antibiotic to treat a resurrected classical disease. If the strategy does pan out, we’ll just have to hope that rifapentine won’t fall into the same cycle of lax control, overprescription, and general misuse that’s made so many other antibiotics fail.

An Antibacterial … What?

Among the deluge of press releases I see each day, there are inevitably a few odd ones. This news from computer technology maker ATEN, however, was exceptionally odd. It seems they’ve developed a new line of antibacterially-coated KVM switches. A KVM switch, as if you didn’t know, is the switch the über-geeks who administer server farms use to switch a single keyboard, video monitor, and mouse between multiple computers. And why does the world need antibacterial KVM switches? The PR flack at ATEN is glad you asked:

The average desk harbors 400 times more bacteria than the average toilet seat, and some of the most germ-contaminated items include the keyboard and mouse, according to a study conducted by the University of Arizona. Despite this fact, network administrators rarely have time to clean their desktops which can lead to the spread of bacteria in the data center. As a result, the presence of microbes contributes to the spread of pneumonia, the flu, pink eye and strep throat, among other extremely contagious viruses.

“We have designed these groundbreaking and nanocoated enterprise KVM switches to serve the needs of network administrators who operate in ‘clean room’ environments such as hospitals, laboratories, manufacturing facilities and others,” said Sampson Yang, CEO, ATEN Technology, Inc. “Beyond these specific environments, product protective antimicrobial nanocoating can benefit data centers and multi-user environments, as well as server rooms within libraries, schools or government facilities where protection is critical.”

Can you count the factual errors and logical fallacies in the above two paragraphs? I see at least five, maybe six, but I’ll close with a nod to one of my biggest pet peeves. All together now, chant it with me: Bacteria Are Not Viruses.