even after three randomized controlled trials in africa demonstrated reduction in hiv acquisition following circumcision,[13 ] the biologic mechanism of protection remains poorly understood . several hypotheses have been postulated that include differences in penile microbiota , rates of sexually transmitted infections , presence of langerhans cells , level of keratinization , and penile hygiene between circumcised and uncircumcised persons.[47 ] however , a clear understanding of the relationship between the foreskin and the penile microbiological environment is lacking . unlike considerable efforts to investigate vaginal characteristics that affect hiv transmission , such as particular microbiomes in bacterial vaginosis , little attention has been paid to the microbiota of the uncircumcised penis . a single study identified decreased numbers of anaerobic genera and increased numbers of facultative anaerobic genera including staphylococcus spp . after circumcision , which were examined through cultivation - independent methods . yet other known pathogens such as pseudomonas spp . were also prevalent in both circumcised and uncircumcised individuals . are predominantly found in and around the urethra ( ur ) , with circumcision increasing the presence of coagulase - negative staphylococcus spp . and decreasing the number of gram - negative pathogens and enterococci . after circumcision , relatively low levels of staphylococcus aureus and no changes in s. aureus prevalence are reported . however , these studies have focused on adolescents , populations with low levels of sexual activity and low hiv risk , those who have not evaluated resource - restricted regions , or those in which circumcision would be difficult to implement . between april and september 2009 , three groups of study participants were recruited from the andhra pradesh government chest hospital and an infertility clinic , both in hyderabad , andhra pradesh , india . the firs group included inpatients infected with hiv ( group 1 : hiv ) , the second group included inpatients infected with tuberculosis but not hiv ( group 2 : tb ) , and the third group included patients presenting at a male fertility clinic ( group 3 : control ) . exclusion criteria included use of short - term antibiotics ( within previous 4 weeks ) for an active bacterial or fungal infection other than tuberculosis . in group 1 , those who were on antibiotics other than trimethoprim - sulfamethoxazole were excluded , and in group 2 , those on a category ii , iii , or dots - plus regimen with agents other than rifampicin , ethambutol , isoniazid , and pyrazinamide were excluded . participants with unretractile foreskin , evidence of an active sexually transmitted infection ( sti ) , cellulitis , abscess , or a tight urethral meatus were excluded . data were collected through a face - to - face interview and chart abstraction , and all samples were collected by a single male physician trained by a local sti specialist . ethics review boards at share - india and the university of chicago approved the protocol and procedures . genital examination and specimen collection consisted of a penile swab of the cs and a second swab 1 cm into the proximal ur . samples from uncircumcised men were taken after foreskin retraction , and no external cleansing was performed before sampling . the specimen was inserted into a liquid thioglycollate medium and transported under ambient temperature to the laboratory within 1 h , where processing was completed upon arrival . aerobic culture was performed by inoculating swabs on 5% sheep blood agar ( cos , biomeriuex , france ) and on a chrome agar ( cps i d , biomeriuex , france ) . the culture plates were incubated at 37c for 24 h. anaerobic culture was conducted by inoculating the swabs on 5% sheep blood agar and incubating at 37c for 48 h under anaerobic conditions . the isolation of fungi was attempted by inoculating the swabs on two tubes of saboraud 's dextrose agar and incubating each at 28 and 37c . the culture tubes were examined for yeast growth after 48 h. a single microbiologist reviewed all plates and isolates . all culture isolates were further identified by using the api or the vitek 2 system ( biomerieux , france ) by the department of microbiology , nizam 's institute of medical sciences , hyderabad , andhra pradesh , india . the chi square or fisher 's exact test for categorical variables and the mann  whitney or kruskal  wallis test for continuous variables were conducted for bivariate analyses , and multivariate logistic regression models were created for the primary analysis that included variables achieving p<0.10 from bivariate analyses  age , circumcision status , and participant group . multivariate logistic regression models for two sets of subgroup analyses were also developed by using variables that achieved p<0.10 from bivariate analyses . in the first subgroup analysis , groups 1 and 2 were evaluated to assess for the potential effect of clinical ( diabetes , history of antibiotic use in past 6 months , previous sti , and presence of warts or other lesions ) and behavioral ( time spent bathing , time since last genital cleaning , wearing undergarments , and number of sex partners ) characteristics , achieving p<0.10 from bivariate analyses associated with pathogen prevalence in the cs or the ur . uncircumcised participants were included in the second subgroup analysis to assess the potential effect of subpreputial wetness , difficulty retracting the foreskin , foreskin length , and the presence of smegma that might be associated with pathogen prevalence in the cs or the ur . the software spss version 16.0 ( spss inc . , between april and september 2009 , three groups of study participants were recruited from the andhra pradesh government chest hospital and an infertility clinic , both in hyderabad , andhra pradesh , india . the firs group included inpatients infected with hiv ( group 1 : hiv ) , the second group included inpatients infected with tuberculosis but not hiv ( group 2 : tb ) , and the third group included patients presenting at a male fertility clinic ( group 3 : control ) . exclusion criteria included use of short - term antibiotics ( within previous 4 weeks ) for an active bacterial or fungal infection other than tuberculosis . in group 1 , those who were on antibiotics other than trimethoprim - sulfamethoxazole were excluded , and in group 2 , those on a category ii , iii , or dots - plus regimen with agents other than rifampicin , ethambutol , isoniazid , and pyrazinamide were excluded . participants with unretractile foreskin , evidence of an active sexually transmitted infection ( sti ) , cellulitis , abscess , or a tight urethral meatus were excluded . data were collected through a face - to - face interview and chart abstraction , and all samples were collected by a single male physician trained by a local sti specialist . ethics review boards at share - india and the university of chicago approved the protocol and procedures . genital examination and specimen collection consisted of a penile swab of the cs and a second swab 1 cm into the proximal ur . samples from uncircumcised men were taken after foreskin retraction , and no external cleansing was performed before sampling . the specimen was inserted into a liquid thioglycollate medium and transported under ambient temperature to the laboratory within 1 h , where processing was completed upon arrival . aerobic culture was performed by inoculating swabs on 5% sheep blood agar ( cos , biomeriuex , france ) and on a chrome agar ( cps i d , biomeriuex , france ) . the culture plates were incubated at 37c for 24 h. anaerobic culture was conducted by inoculating the swabs on 5% sheep blood agar and incubating at 37c for 48 h under anaerobic conditions . the isolation of fungi was attempted by inoculating the swabs on two tubes of saboraud 's dextrose agar and incubating each at 28 and 37c . the culture tubes were examined for yeast growth after 48 h. a single microbiologist reviewed all plates and isolates . all culture isolates were further identified by using the api or the vitek 2 system ( biomerieux , france ) by the department of microbiology , nizam 's institute of medical sciences , hyderabad , andhra pradesh , india . the chi square or fisher 's exact test for categorical variables and the mann  whitney or kruskal  wallis test for continuous variables were conducted for bivariate analyses , and multivariate logistic regression models were created for the primary analysis that included variables achieving p<0.10 from bivariate analyses  age , circumcision status , and participant group . multivariate logistic regression models for two sets of subgroup analyses were also developed by using variables that achieved p<0.10 from bivariate analyses . in the first subgroup analysis , groups 1 and 2 were evaluated to assess for the potential effect of clinical ( diabetes , history of antibiotic use in past 6 months , previous sti , and presence of warts or other lesions ) and behavioral ( time spent bathing , time since last genital cleaning , wearing undergarments , and number of sex partners ) characteristics , achieving p<0.10 from bivariate analyses associated with pathogen prevalence in the cs or the ur . uncircumcised participants were included in the second subgroup analysis to assess the potential effect of subpreputial wetness , difficulty retracting the foreskin , foreskin length , and the presence of smegma that might be associated with pathogen prevalence in the cs or the ur . the software spss version 16.0 ( spss inc . , three hundred fifteen study participants were enrolled : 150 in group 1 , 115 in group 2 , and 50 in group 3 . overall , 37.1% of the participants were circumcised without variation across groups ( p=0.29 ) . smegma was observed in 18.7% of the uncircumcised participants with no cases observed in group 3 ( p<0.001 ) . among group 1 , cd4 counts of < 200 , 200 - 350 , and > 350 were 40 , 37.9 , and 22.1% , respectively , and 92.7% of the participants were on highly active antiretroviral therapy . the culture of organisms from the cs yielded 41.6% colonized with s. aureus , 3.5% with enterococcus spp . , 22.9% with a gram - negative bacterium ( e. coli , klebsiella spp . , or pseudomonas aeruginosa ) , 1.6% with an anaerobe ( clostridium ) , 0.3% with a yeast ( candida albicans ) , and 60.0% with any pathogen . a coagulase - negative staphylococcus sp . gram - negative pathogens were more prevalent among study participants in group 1 ( 22.7% ) and group 2 ( 30.4% ) as compared with those in group 3 ( 6.0% ) ( p=0.003 ) . the association of circumcision status with gram - positive pathogens , gram - negative pathogens , and all pathogens combined , controlling for age and participant group , are presented in table 1 . interaction terms between the participant group and the circumcision status were not found to be statistically significant and were not included in the final logistic regression models . presence of pathogens in the coronal sulcus ( cs ) and the urethra ( ur ) by circumcision status among a diverse group of circumcised and uncircumcised indian patients ( n=315 ) in 2009 multivariable regression analyses including groups 1 and 2 participants demonstrated the following significant associations : diabetes was positively associated with gram - positive pathogens in the cs and the ur ; increasing frequency of changing underwear was associated with gram - positive pathogens in the cs ; having one or more sex partners was associated with any pathogen of the cs and the ur ; and not having a genital wart , lesion , or sore was associated with the presence of any pathogen of the ur ( odd ratio ( ors ) 1.96.8 ; p=0.020.05 ) . not being circumcised remained a significant predictor of the pathogen presence in these analyses ( ors 1.93.1 ; all p<0.05 ) . in multivariate analyses of uncircumcised participants , the relationship between smegma , foreskin length , subpreputial wetness , and pathogens was not found to be statistically significant except for the positive association between smegma and gram - positive pathogens of the cs ( or 2.6 ; p=0.01 ) . we found that bacterial pathogens were independently associated with uncircumcised status in a diverse population after controlling for age and clinical population . findings of colonization in the uncircumcised participants were also robust , with consistent findings across two distinct penile anatomical sites . several additional clinical and hygiene factors such as diabetes , absence of abnormal genital findings , decreased frequency of changing underwear , and number of sex partners were associated with colonization of the cs and the ur . while increases in both pathogenic and nonpathogenic staphylococcus spp . were found in one study , ecological analyses of microbiota that suggested increases in staphylococcus spp . after circumcision did not differentiate between pathogenic organisms ( e.g. , s. aureus ) and typically nonpathogenic organisms . such analyses could potentially miss important shifts in species at the family or genus level . our finding of increased s. aureus in uncircumcised men was surprising and warrants further investigation and validation in other settings and contexts . the potential for misclassification of s. aureus for other species such as coagulase - negative staphylococcus spp . exists ; however , we would not have expected the misclassification to be differentially distributed to uncircumcised individuals . this is consistent with a previous work in which variations in colonization of gram - negative organisms in the prepuce and s. aureus in the nares are evident in adolescent males and the elderly , respectively . our finding that foreskin length and subpreputial wetness were not associated with the presence of pathogens suggests that if these two qualities are in fact associated with hiv infection , the mechanism may not be mediated by bacterial pathogens . the presence of smegma , however , was independently associated with gram - positive pathogens in this study . while the secretions related to smegma may have antimicrobial properties , smegma is generally a marker of poor hygiene and further research into the properties and effects of smegma on microbial communities is warranted . uncircumcised men in this population in south india are more likely to harbor bacterial pathogens in the cs than do their circumcised counterparts . the finding that both gram - positive and gram - negative pathogens are more likely to be found in uncircumcised participants differs from that of a previous work that suggests shifts in microbiota following circumcision with decreases in gram - negative pathogens and increases in gram - positive pathogens . an additional research to better understand host and microbiome interactions and their potential relationship with circumcision 's efficacy in reducing the risk of hiv and other sexually transmitted infections is required . in many settings or subpopulations , similar to considerable efforts examining important microbial vaginal interactions and the relation to hiv transmission , further study of these potential foreskin microbial interactions would be required to develop nonsurgical methods that could achieve at least similar risk reduction rates as circumcision .
background : the biological mechanism of circumcision as potentiating hiv prevention is poorly understood . 
 foreskin microbiota has been postulated as having a potential role ; however , little is known about the relationship between bacterial pathogens and circumcision in adults.materials and methods : we sampled the coronal sulcus of a diverse group of circumcised and uncircumcised men ( n=315 ) from a government chest hospital and fertility clinic in hyderabad , andhra pradesh , india . 
 genital examination was conducted on three groups of men : group 1  hiv infected ; group 2  tb infected ; group 3  control . 
 aerobic and anaerobic specimens were cultured according to standard clinical protocols , and results were analyzed following multivariate logistic regression models.results:three hundred fifteen study participants  47.6% of group 1 , 36.5% of group 2 , and 15.9% of group 3  were enrolled in the study and included in all analyses . 
 overall 37.1% of the participants were circumcised without variation across groups ( p=0.29 ) . 
 smegma was observed in 18.7% of the participants with no cases observed in group 3 ( p<0.001 ) . 
 gram - negative pathogens were more prevalent among study participants in group 1 ( 22.7% ) and group 2 ( 30.4% ) as compared with those in group 3 ( 6.0% ) ( p=0.003 ) . in multivariate regression analysis , 
 controlling for group , age , and presence of smegma , uncircumcised men were more likely to be colonized with gram positives [ adjusted odds ratio ( aor ) 1.9 ; p<0.05 ) ] , gram negatives ( aor 2.4 ; p<0.05 ) , or any pathogen ( aor 2.8 ; p<0.005).conclusions : uncircumcised men in this population in south india are more likely to harbor bacterial pathogens in the coronal sulcus than do their circumcised counterparts . 
 future studies should examine the relationship between foreskin microbiota and hiv transmission .