The World Health Organization declared three years ago that circumcision should be part of any strategy to prevent HIV infection in men. The organization based its recommendation on three randomized clinical trials in Africa that found the incidence of HIV was 60 percent lower in men who were circumcised. Although this "research evidence is compelling," wrote the WHO panel assigned to the topic, there was little evidence explaining how circumcision might reduce a man's risk of acquiring HIV.



Now comes an answer in a new study, published in the January 6 issue of PLoS ONE, which found that there are gross changes in the penis's microbiome following circumcision, suggesting that shifts in the bacterial environment could account, in part, for the differences in HIV infection. Families of anaerobic bacteria, which are unable to grow in the presence of oxygen, are abundant before circumcision but nearly disappear after the procedure. The researchers suspect that in uncircumcised men, these bacteria may provoke inflammation in the genitalia, thereby improving the chances that immune cells will be in the vicinity for HIV viruses to infect.



"We never knew that there were that many anaerobic bacteria on the uncircumcised penis before [this study]," says Ronald Gray, a reproductive epidemiologist at Johns Hopkins Bloomberg School of Public Health and one of the lead authors on the current study. According to a 2006 survey, 56.1 percent of boys in the U.S. are circumcised. In its recommendation, the WHO panel stated that circumcision efforts would be most beneficial in parts of the world where less than 20 percent of boys are circumcised.



Gray, who is also working with one of the three randomized clinical trials on which WHO based its recommendation, adds that, "If we can show that these anaerobic bacteria are associated with HIV, then one could develop microbicides—antiseptics or targeted antibiotics —that might provide protection."



In the current study Gray and his colleagues compared the microbiota of 12 HIV-negative Ugandan men ages 15 to 49 before and after they were circumcised. It was important to limit the study to HIV-negative participants because infection itself can throw off the bacterial environment of the penis, says Lance Price, a research director at the Translational Genomics Research Institute in Flagstaff, Ariz., and co-author on the study. The team collected swabs from an area between the head and shaft of the men's penises before and one year after circumcision. Then the researchers performed polymerase chain reaction analysis of a gene that is shared by, although not identical in, numerous bacterial families. The analysis allowed for identification of different bacterial families as well as abundance counts.



At 12 months after circumcision, the microbiome's predominant bacterial population had shifted fromanaerobic to aerobic, which require oxygen to grow. Whereas the researchers detected similar number of bacteria belonging to aerobic families in circumcised and uncircumcised samples, they found that the abundance of anaerobic family members plummeted after circumcision. As the authors wrote, this decrease makes sense because there is an oxygen-deprived area under the foreskin that is lost after circumcision.







Whereas the current analysis could only detect gene sequences that were specific to bacterial families, the researchers are now working to identify the specific species that presumably cannot survive on circumcised penises. Price says that the researchers should begin this PCR-based analysis in the next six months, as soon as they finish improving the databases of bacterial sequences and developing new PCR tools.



Once the researchers identify the anaerobic species, the plan is to determine which can create an inflammatory environment that favors HIV infection, Gray says. Certain species of bacteria, both anaerobic and aerobic, can cause the release of inflammatory cytokines (immunoregulatory proteins) from cells present in the skin and body surfaces, such as the foreskin and vagina. These cytokines then activate a type of immune cell, called Langerhans cells. Scientists think that HIV transmission requires that the virus first infect activated Langerhans cells, which then pass the virus to T cells.



Even if the researchers demonstrate that specific species of anaerobic bacteria on the uncircumcised penis are potent activators of Langerhans cells, the microbiome is probably only one aspect of the environment that affects HIV infection. As Gray points out, anatomical changes following circumcision also "almost certainly" account for part of the protection against HIV infection. Specifically, the authors wrote, after the foreskin is removed the penis head develops extra layers of skin, which reduces the abundance of Langerhans cells.



In any case, changes that occur to the penis microbiome following circumcision could hamper the transmission of other sexually transmitted diseases. Similar to HIV reduction, Gray's clinical trial in Uganda found that the incidence of genital herpes and human papillomavirus were about 27 and 35 percent lower, respectively, in circumcised men.



Moreover, the current study found that two of the most abundant anaerobic organisms present on uncircumcised penises, Clostridiales and Prevotellaceae, have been associated with bacterial vaginosis, an uncomfortable condition in which the vagina's bacterial balance is upset. This finding could help explain why there are reduced rates of bacterial vaginosis in the wives of circumcised men, Gray says. He adds that the microbiome study will help doctors understand the extent of the potential health benefits of circumcision.