The Bacteria in the Stone

 Extra-tiny microorganisms may lead to kidney stones and other diseases

 In the fine tradition of transforming lemons into lemonade, here's a story of a laboratory annoyance and how it may lead researchers to a new therapy for one of the most painful maladies that people suffer.

 Almost a decade ago, E. Olavi Kajander faced a problem that has frustrated many a biologist. The biochemist at the University of Kuopio in Finland was trying to culture mammalian cells, but they simply weren't thriving. The cells grew very slowly, if at all, and many contained abnormal bubbles, or vacuoles, within their cytoplasm.

 Scientists usually grow mammalian cells in fetal bovine serum, made from the fluid part of cow blood. The serum is considered sterile, but viruses and mycoplasmas-small bacteria without rigid cell walls-sometimes contaminate it and cause problems. So Kajander and his colleagues examined their sickly cells with an electron microscope.

 The investigators didn't find any viruses or mycoplasmas, but they did capture images of unusually small bacteria inside many of the cells. In the years since then, Kajander and his colleagues have isolated and characterized the tiny microorganisms, dubbing them nanobacteria because they range in diameter from 50 to 500 nanometers (see sidebar).

"They are as small as the largest viruses-and smaller," says Kajander.

 Although his group's work suggested that nanobacteria often contaminate fetal bovine serum, and more rarely human blood, Kajander's research was largely ignored by his colleagues in Finland and the rest of the world.

 Last month, however, nanobacteria stepped into the scientific spotlight. In the July 7 Proceedings of the National Academy of Sciences (PNAS), Kajander and his colleague Neva Ciftcioglu contend that nanobacteria live in urine and, by precipitating calcium and other minerals around themselves, induce the formation of kidney stones.

"We believe that they are the real starting point of most kidney stones," says Kajander.

 That provocative claim, along with the possibility that antibiotics might help people who suffer from chronic stone formation, has caught the attention of some researchers. "It's exciting," says Leroy M. Nyberg, director of urology programs at the National Institute of Diabetes and Digestive and Kidney Diseases in Bethesda, Md. "I think it's the first real theory as to what is the nucleation factor for [kidney stones]."

"I'm sure there will be a lot of controversy around this, but I think controversy is good if it leads to experimentation," adds Dennis A. Carson of the University of California, San Diego, who published a commentary on the research in the same issue of PNAS.

 Why have nanobacteria escaped the attention of microbiologists until recently? First, their small size makes them difficult, if not impossible, to see with traditional light microscopes, says Kajander. Moreover, many of the chemicals used to stain cell walls or other components of traditional bacteria fail to bind to nanobacteria.

 Also, the microbes don't thrive on agar, the jellylike medium used to grow most bacteria. Finally, while many bacteria divide every hour or so, nanobacteria multiply very slowly, doubling in number about every 3 days. This slow growth makes it tough to study their metabolism, explains Kajander.

 Despite such challenges, the Finnish researchers have put together a fair-size dossier on the microbes. When grown in serum, alone or with mammalian cells, they often form a white biofilm. The bacteria also appear able to trick cells that don't normally engulf microbes into incorporating them. In doing so, says Kajander, nanobacteria can trigger the cell suicide program known as apoptosis, which likely explains how nanobacteria stymie scientists trying to grow mammalian cells.

 The investigators have been able to develop antibodies that bind to surface proteins of nanobacteria and have also isolated some of their DNA. In particular, they've sequenced the gene encoding a component of ribosomes, the protein-making machinery in all cells. By comparing that gene's sequence to those of the corresponding genes of other organisms, the Finnish scientists conclude that nanobacteria are closely related to Brucella and Bartonella bacteria. Some species of these bacteria are also known to infect the blood of animals and people.

 Kajander and his colleagues have found that about 5 percent of Finnish people are, or have been, infected with nanobacteria. The scientists have detected nanobacterial proteins in human blood and also have grown the organisms from blood samples. Yet, blood may not be the primary habitat of these microbes. When injected into animals, the bacteria seem to move quickly to the kidneys and end up in the urine, says Kajander.

 At least one other microbiologist has confirmed the existence of the nanobacteria. James W. Coulton of McGill University in Montreal, Quebec, who specializes in the surface proteins found on bacteria, has also isolated slow-growing microbes that build calcified shells. Coulton says his organisms are the same as those observed by the Finnish group and that they are indeed bacteria.

"We've isolated proteins from these organisms which are quite like the proteins of other bacteria," Coulton says. These proteins, called porins, are usually employed by bacteria to form channels that allow nutrients to enter through the cell wall.

 As Kajander and his colleagues learned more about nanobacteria, they grew increasingly curious as to whether the microbes were responsible for any human diseases, particularly ones not normally attributed to infectious agents. Such connections are not unprecedented. Take ulcers, for example. Though many physicians resisted the idea for years, most now agree that the bacterium Helicobacter pylori causes the majority of ulcers.

 Although the evidence is not yet nearly as compelling, scientists have also built cases that illnesses as diverse as heart disease, diabetes, arthritis, and multiple sclerosis might in some cases stem from bacterial or viral infections.

 An unusual feature of nanobacteria immediately suggested that the Finnish researchers look into kidney stones. Under certain growth conditions, including the acidity and mineral concentrations found in urine, the microbes somehow induce precipitation of dissolved calcium, phosphates, and other minerals into carbonate apatite, a major component of many kidney stones.

 Through this still unexplained process, the nanobacteria build a mineralized shell around themselves, a structure large enough to be visible even with low-powered light microscopes. These "castles," as Kajander calls them, may be made by the microbes as a protective measure. Or they may simply result from the interaction of proteins on the nanobacterial surface with minerals dissolved in the surrounding fluid.

 Whatever the explanation for the apatite-encrusted bacteria, the researchers began to wonder if such microbes provided the foundation for kidney stones, as grains of sand or other irritants provide the nucleus for pearls.

 Kidney stones strike an estimated 10 percent of people in the United States at least once. Although scientists know that the painful stones form when minerals precipitate out of urine, they're unable to explain why this process begins.

"We still don't know what the nucleus is that starts the precipitation. We've never really been able to determine that," says Nyberg. "We've plateaued in stone research for almost the last decade. We really need something to open it up again."

 Nanobacteria may offer that opening. Nyberg notes that some bacteria have already been linked to kidney stone formation, although the mechanism behind that connection is dramatically different. For the 5 to 15 percent of kidney stones formed largely of the mineral struvite, bacteria that produce an enzyme called urease are to blame. Urease makes urine more acidic, a condition under which mineral precipitation occurs more readily, and so indirectly encourages kidney stone formation.

 Kajander and Ciftcioglu have linked nanobacteria more directly to kidney stones. In PNAS, they describe how they broke apart human kidney stones and then looked for evidence of the microbes.

 In all 30 stones tested, antibodies that bind specifically to nanobacteria proteins attached to material derived from the stone. Electron microscope images also showed nanobacteria in samples of dissolved stones. Finally, the researchers were able to grow nanobacteria from some of the dissolved stones.

 Kajander is confident enough of his group's result to suggest that physicians should begin testing whether antibiotics can help people with recurrent kidney stones. Nanobacteria are resistant to many antibiotics, perhaps because of their mineral shells, but tetracycline has proven effective against them in test-tube experiments, he notes. Because tetracycline accumulates on apatite, it may concentrate near nanobacteria, says Kajander.

 The association with kidney stones should draw much more attention to nanobacteria, predict researchers. Coulton suggests that microbiologists may have so far shunned these microbes because they prefer to study bacteria, such as Escherichia coli or Bacillus subtilis, that grow quickly and can be genetically manipulated with ease. 

"But because of the disease link, I think people are going to be a lot more open to the idea that [nanobacteria] warrant further study," he says.

 The medical significance of nanobacteria may eventually extend beyond kidney stones. In a variety of human disorders, including dementias, atherosclerosis, cancers, and arthritis, unexplained calcium precipitation occurs in various tissues of the body, notes Carson in his commentary. And people who get frequent blood transfusions as part of dialysis treatment often develop dangerous calcium deposits, he adds.

 Adding to the intrigue, Kajander and Ciftcioglu have conducted a preliminary study of Turkish people who are undergoing dialysis and found that 80 percent displayed evidence of nanobacteria in their blood.

"There is ample cause to investigate thoroughly the part that nanobacteria play not only in renal stone formation but also in the many perplexing diseases associated with pathological extraskeletal calcification," concludes Carson.