In the human body, bacterial cells outnumber our own cells by ten to one. Up to 50 percent of these bacterial species remain poorly characterized, as they don’t thrive under standard laboratory growth conditions. While scientists have been able to identify many of these bacteria by detecting their unique DNA sequences, new methods to grow them in the lab would help researchers understand much more about their positive and negative effects on human health. To further this goal, the National Institutes of Health started the Human Microbiome Project, a 5-year initiative to improve understanding of the bacteria that live in our mouth, gut, skin, vagina, and other organs.
VIDD member Dr. David Fredricks has just received $2 million from this project for a 3-year study to develop novel methods for cultivating the many bacterial species found in the human vagina, including those that cause the common female disease bacterial vaginosis (BV). BV happens when the normal vaginal bacteria, primarily Lactobacillus species, are replaced by a complex mixture of other bacterial species. It affects up to 29 percent of U.S. reproductive age women, and can increase risk for preterm labor, HIV infection and transmission, and other infections, including pelvic inflammatory disease.
Many BV-associated bacteria have proven difficult to cultivate in the laboratory, Fredricks said. He thinks this may be in part because certain bacterial species rely on other species present in the mix to produce essential nutrients or growth factors, so fail to grow when isolated from their brethren. Alternatively, there might be something about the environment of the human vagina that is impossible to recreate in the lab setting.
“It’s obviously difficult to assess the properties of these bacteria when we can’t grow them in the laboratory,” Fredricks said. “That’s why we set out on this path of trying to propagate them.” Normally, researchers grow bacteria in liquid broth or on jelly-like agar poured into petri dishes, supplemented with certain nutrients, but these standard cocktails haven’t worked for many BV-associated bacteria. In the planned study, Fredricks’ group will use three novel cultivation approaches to try to make these tricky bacteria feel more at home in the lab, using vaginal samples from study participants with BV to isolate the bacteria. First, they will use conventional agar plates or broth supplemented with filtered, sterilized vaginal fluid. “There may just be something in vaginal fluid that we haven’t identified that is important,” Fredricks said. Second, they will use special dishes to grow a given bacterial species in the presence of other “feeder” bacteria, BV-associated bacteria that can be cultured in the lab. This method may work if the feeder bacteria secrete a needed growth factor or other nutrient that the bacteria of interest needs to survive.
Finally, they will use a novel device called an in situ isolation chamber, originally developed to grow hard-to-culture marine bacteria. The adapted version of the device is about one-quarter the size of a penny, and contains many tiny holes filled with miniature agar plugs. These holes are seeded with BV-associated bacteria from vaginal samples so that each plug gets a single bacterial cell, and a permeable membrane then seals the chip to allow fluids to wash over the plugs but keep the bacteria in. The chip can then be incubated either in vaginal fluid in the lab, or even inside volunteers’ vaginas. After this “domestication,” getting the bacteria used to growing and dividing on agar in their natural environment, the plugs are then transferred to standard laboratory conditions, a process shown to work for certain marine microbes.
Ultimately, even though antibiotics exist to treat BV, the disease is still poorly understood so cultivating these bacteria is a necessary step, Fredricks said. For one, recurrence of BV is very common even after antibiotic treatment, but researchers don’t know why. “Understanding the capabilities of these bacteria and their interactions may lead to new ways to prevent or treat BV,” he said.