Male Circumcision Reduces the Quantity of Genital Bacteria
Contraction of HIV, human papilloma virus (HPV) and herpes simplex virus 2 (HSV-2) in males is significantly reduced by circumcision. These viruses are also less likely to be transmitted to women by circumcised men. The reason for this is that the number of cells on the penis that HIV can target are lowered, thereby reducing the risk of infection. Circumcision also alters the microbiome located on the penis, removing the moist conditions and thereby affecting the bacteria that are propagating.
These bacteria, such as the species that cause inflammation, are noted for increasing the likelihood of contracting diseases such as HIV. They cause immune responses which bring about the aforementioned inflammation and thereby expose cells targeted by the virus. Therefore, removing the habitat of the bacteria reduces the frequency of immune responses and is thereby likely to reduce the occurrence of infection.
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In this study, Liu et al. (2013) used absolute abundance in order to measure the changes in the microbiome brought about by circumcision. The coronal sulcus microbiota, in particular, was examined in a number of people. 77 control and 79 experimental men were required. The hypothesis going into this study was that bacterial abundance would be greatly lowered as a result. The microbiota in people that underwent circumcision was expected to be affected, while the microbiota in men that remained uncircumcised should have been unchanged.
Methods and Materials
A sample of men unaffected by HIV were used for this study. The control used for this study consisted of men who were only circumcised after 24 months. The intervention group (experimental group for this experiment) were circumcised immediately. The circumcisions were all carried out in the same location, with the same procedure (the "sleeve method") being used for each participant. These men were then monitored over an extensive period of time to track incidence of sexually transmitted infections. Information was gathered every 6 months in the process for a total of 24 months. Several men from both the control and intervention groups underwent treatment when they contracted sexually transmitted diseases over the experimental period.
Samples from the coronal sulcus of each participant were gathered at various points throughout the 24 month period. Sterile cotton swabs were rubbed over the region in order to collect the samples and then incubated at -80°C to prevent spoilage of the samples during transport. 77 control and 79 intervention samples from HIV-negative participants were then selected for testing purposes. These men were specifically kept HIV-negative during the experimental period.
The samples were then processed together at the same time in order to prevent any variation occurring during the procedure. 100 µl of transport medium from each sample underwent mechanical and chemical lysis using an enzyme-free process. The product of the lysing process was then purified. Finally, the samples underwent elution in order to extract the DNA, to be used for further purposes.
Next, a PCR assay was conducted in order to determine the quantity of bacteria present in the samples. This was measured by the amount of bacterial 16S rRNA that was in each µl of eluent from the swab samples. PCR primers were then used to make a set of amplicons for the purpose of being sequenced afterwards. The amplicons were also checked for quality and for the occurrence of chimeras.
After the bacterial quantity was measured, it was necessary to map it to the overall time taken during the experiment. This was done by the calculation of a log ratio, using the formula ln[(bacterial quantity after 12 months/initial bacterial quantity)] in order to discover how much bacteria had been formed since the start of the experiment. These ratios were then plotted onto a graph in order to see trends that the data showed, and also to make comparisons between different readings. A t test was also conducted in order to determine statistical differences between the mean values of the different groups of readings.
Following this, the unique microbial composition of the microbiota of the coronal sulcus samples was documented. This was done by analysing each taxonomic level to discover which groups of bacteria were present at each level (operational taxonomic unit). This was able to give an overall view of what microbes were contained within this section of the penis, an area particularly significant in contracting sexually transmitted infections. The data obtained was used to determine the abundance of bacteria at each taxonomic level.
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It was then important to carry out various other statistical tests in order to further analyze the data obtained. The abundances of various bacterial genera from the beginning of the timeframe were compared using chi squared tests, as well as the t test, as mentioned previously. The types of microbes contained within the microbiota changed during the experiment, and this was also statistically analyzed. This was done by constructing nonmetric multidimensional scaling plots (nMDS), as well as using permutational multivariate analysis of variance calculations (PerMANOVA). Therefore, the changes in the microbial composition over the time period of the experiment could be made apparent.
The participants in the experiment were similar to each other in terms of the sexually transmitted diseases they exhibited, the types of sexual activities they engaged in and the symptoms they displayed as a result of their diseases. This was necessary to ensure fair and accurate results that were easily comparable. It also makes sure that results are statistically significant and therefore relevant in terms of applying the findings to the overall population.
It was found that the species of bacteria and their prevalence in the penises of uncircumcised men were largely similar. These findings were observed after examining the coronal sulcus and documenting each taxonomic level, as described in the previous section. A few species of bacteria were particularly common, such as the Clostridiales family, the Prevotellaceae family and the Corynebacteriaceae family.
The relative abundances of bacterial species in the coronal sulcus were overall found to be low. The most abundant bacteria were those of the Prevotella family. In addition, Corynebacterium and unclassified Clostridiales were also rather abundant, exhibiting proportional abundances of over 10% each. Asides from these particular bacterial families, however, all other families were observed to have mostly insignificant abundances, ranging from 5% to as low as 0.1%. These families are not expected to have much of an effect on sexual transmission of diseases or related factors.
Elaborating further, the study found that the quantity of bacteria contained in the coronal sulcus was reduced as a result of male circumcision. At the start of the experiment, both the control and intervention groups displayed similar numbers of bacteria, as measured by the 16s rRNA gene counts. This gene was found to be present in 1.4 x 105 copies in the control arm, and 2.0 x 105 copies in the arm of intervention.
However, after the first year, it was observed that the bacterial count had dropped by a sizeable amount in both control and intervention groups. Uncircumcised men displayed 5.7 x 104 copies of the 16s rRNA gene, while men that had been circumcised had 3.8 x 104 copies of the gene. This can be seen in Figure 1 below:
Figure 1: Figure showing the changes in the quantity of bacteria in the control groups as plotted against the intervention groups. Control groups are represented by red dots while the intervention readings are shown as orange dots. Figure adapted from Liu et al., 2013.
It can be seen that the number of copies was much lower in circumcised men when compared to men that are uncircumcised. Therefore the circumcision was directly related to the reduction in bacterial count.
In addition, circumcision also altered the prevalences of various bacteria occurring in the coronal sulcus. It was observed that mostly anaerobic bacteria decreased in quantity, such as Prevotella spp. and Porphyromonas spp. Although this was a general trend in the anaerobic bacteria, there were species that were not affected by circumcision, such as the Atopobium species.
In contrast, there were several species of bacteria that actually rose in prevalence in circumcised men. Examples of these included the Facklamia spp. and the aerobic Kocuria spp.. However, it was noted that most of these species also increased in prevalence in the uncircumcised men. It is therefore hypothesised that the reason for this increase in prevalence is not related to the circumcision procedure. The increase in prevalence may have instead been caused by other factors such as time and behaviour during the experimental period.
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Male circumcision also caused the composition of the microbiota in the coronal sulcus to be significantly altered. As can be seen in Figure 2A, the microbiota did not change very much in uncircumcised men after the period of 1 year as compared with the original microbiota in place. However in contrast, as seen in Figure 2B, the microbiota composition was affected quite largely in circumcised males after the 1 year period.
Figure 2: Figure showing two nonmetric multidimensional scaling plots (nMDS). Blue points represent the microbiota that were present at the beginning of the trial, compared to the orange points representing the state of the microbiota after the 12 month period. Figure adapted from Liu et al., 2013.
There was a large shift that could be observed on the nMDS plot. Therefore it is evident that circumcision definitely has an effect on what species of microbes are present in the coronal sulcus.
From the results obtained in this study, it could be observed that male circumcision played a major role in the reduction of abundance in bacterial quantity in the coronal sulcus. After the one year period, bacterial species were much less abundant in circumcised males in comparison to uncircumcised men, after the two started off with similar abundances at the experiment's initiation. Any decreases in abundance of bacterial species in uncircumcised men were attributed to alternate factors such as behavioural abnormalities, and were therefore unrelated to circumcision. Clearly, circumcision causes prevalence to decrease as well as causing significant changes in the composition of the microbiota.
It is likely that the bacterial species found on the foreskin make HIV target cells more easily infected by the virus. Langerhans cells on the foreskin are usually able to break down any HIV that comes into contact with them. If inflammation occurs on the foreskin as a result of the bacteria occurring there, it becomes increasingly more likely that the Langerhans cells will no longer break down the HIV, and instead present it to CD4+ T cells, which will thereby cause it to infect the person in question.
In addition, male circumcision is correlated with a decrease in bacterial vaginosis in female partners. Certain bacterial species become less prevalent in the female microbiota after the male partner has been circumcised. This leads onto the idea that transmission of sexually transmitted diseases to females is likely to be decreased as a result of the lower bacterial count.
It can therefore be concluded that male circumcision has a very notable significant effect on the microbiota of the coronal sulcus. Circumcision was a major factor contributing to the lowered abundance of various species of bacteria on the penis. It would be useful to have more concrete evidence on the link between bacteria and foreskin inflammation, so that it can be more solidly confirmed that lowering bacterial counts reduces the risk of contracting HIV.
Liu, C. M., Hungate, B. A., Tobian, A. A. R., Serwadda, D., Ravel, J., Lester, R., Kigozi, G., Aziz, M., Galiwango, R. M., Nalugoda, F., Contente-Cuomo, T. L., Wawer, M. J., Keim, P., Gray, R. H., and Price, L. B. (2013) Male Circumcision Significantly Reduces Prevalence and Load of Genital Anaerobic Bacteria. mBio. 4(2): 00076-13.