Penile microbiota dysbiosis associates with subclinical HPV infection in HIV-negative men

Abstract

Background

Human papillomavirus (HPV) is the most frequent sexually transmitted virus, with high importance due its oncogenic risk. Previous studies have reported an association between genital dysbiosis and HPV infection in women and also in men co-infected with HPV and HIV. However, it remains to be determined whether penile skin dysbiosis is associated with HPV infection in men who are HIV-negative. This study characterizes the penile skin microbiota (PSM) of HPV-positive and HPV-negative men, hypothesizing that HPV infection is linked to dysbiotic anaerobic-dominated communities.

Methods

A cross-sectional study was conducted on 103 sexually active HIV-negative men (70 HPV-negative, 33 HPV-positive). Those who tested HPV-positive were genotyped. The PSM of all samples was analyzed using 16S rRNA sequencing of the V4 region. Alpha and beta diversity were compared. Community State Types (CSTs) were identified using hierarchical clustering. Associations between CSTs and HPV status were tested adjusting for sexual preference.

Results

HPV-positive men exhibited significantly higher microbial richness than HPV-negative men (Chao1 p = .02), particularly those with high-risk genotypes (Chao1 p = .03). Five CSTs were identified, with CST-5 (dominated by Finegoldia and other anaerobes) showing a three-fold higher likelihood of HPV positivity (OR = 3.11, 95% CI: 1.22–8.22) compared to other CSTs. CST-5 also displayed reduced abundance of commensals like Corynebacterium and Staphylococcus.

Conclusions

Subclinical HPV infection in HIV-negative men was associated with a dysbiotic PSM, characterized by an increased abundance of anaerobic bacteria alongside with a reduced proportion of facultative anaerobic genera. These findings suggest that PSM composition may influence HPV susceptibility or persistence. Longitudinal studies are needed to explore causality.

Keywords

Genital microbiota high-risk genotype human papillomavirus penile microbiota sexual health sexually transmitted infection skin microbiome

Introduction

Human papillomavirus (HPV) is one of the most common sexually transmitted viral infections and is of great concern because of its oncogenic risk.¹ In men, HPV-related cancers are primarily in the anogenital region, with 90% of anal cancers and 40% of penile cancers attributed to persistent HPV infection.^2,3 Although most subclinical HPV infections in men are cleared by the immune system within 2 years, 10% progress to persistent infection.⁴ While certain factors influencing viral susceptibility or persistence are well established -such as a high number of sexual partners, tobacco use, and unprotected sexual activity- others, including the role of dysbiotic microbiota, remain poorly understood, particularly in HIV-negative men.

Accumulating evidence highlights the role of the local bacterial microbiota as a critical modulator of viral infection dynamics. In women, the pathogenesis of cervical HPV is closely associated with dysbiosis of the vaginal microbiota. A shift in the Lactobacillus-dominant community and an increase of anaerobic genera promotes a pro-inflammatory environment that facilitates viral persistence and neoplastic progression.⁵ In contrast, on the skin surface, resident bacteria such as Corynebacterium and Staphylococcus stabilize the epithelial barrier and enhance antiviral defenses through metabolites and immune crosstalk.^6,7 For example, Staphylococcus epidermidis activates Toll-like receptors to enhance interferon production,⁷ a key pathway for viral clearance. However, the penile skin microbiota (PSM), a community dominated by Corynebacterium, Staphylococcus, and some other anaerobic genera, remains understudied in the context of HPV pathogenesis. This gap persists despite evidence that PSM composition correlates with HIV susceptibility,⁸ suggesting broader implications for genital tract health, particularly in HIV-negative men. To date, the only published study on this topic analyzed samples from circumcised men, some of whom were co-infected with HPV and HIV.⁹ In that study, HIV-negative men who exhibited a higher abundance of anaerobes and a lower abundance of beneficial commensals were significantly more likely to have HPV or HR-HPV infections. Therefore, to achieve a more comprehensive understanding of the association between penile skin microbiota and HPV infection in HIV-negative men, further studies are required.

Recent studies have revealed that the penile skin microbiota is distinct from that of other skin sites and relatively stable.¹⁰ The most commonly detected genera include Anaerococcus, Corynebacterium, Finegoldia, Peptoniphilus, Prevotella, and Staphylococcus, most of which are facultative anaerobes. Anaerobic bacteria, often overlooked, are also resident commensals found in healthy individuals. Circumcision has been shown to alter the microbial composition of the coronal sulcus by reducing the abundance of anaerobes and increasing that of aerobes in the coronal sulcus.¹¹ However, despite growing interest, the microbiota of the healthy male genitalia remains poorly characterized.

The cross-sectional study presented here addresses these gaps by characterizing the PSM of young HPV-positive and HPV-negative men, including sexual preference as a variable. We hypothesize that subclinical HPV infection is associated with a dysbiotic PSM characterized by an increase in anaerobes and a decrease in commensal taxa, a microbial signature theorized to compromise local antiviral defenses through immune-barrier dysfunction. This study lays the groundwork for future mechanistic investigations of microbiota-driven immune modulation.

Methods

Setting, participants and sample collection

The project was approved by the ethics committee of the “Centro de Investigaciones Regionales Dr Hideyo Noguchi” of the “Universidad Autónoma de Yucatán” (identification number: CEI- 01-2020). This study was designed to be a cross-sectional. A recruitment campaign was conducted via social networks (Facebook™) to invite sexually active men aged 18-45 living in the city of Merida, Mexico. Exclusion criteria were: presence of genital warts, known to be living with HIV, antibiotic use within the 4 weeks prior to sample collection, and the presence of genital lesions. Enrolled participants were instructed to abstain from sexual intercourse and refrain from genital hygiene for 24 hours prior to sample collection. HPV vaccination status was not documented; however, public vaccination policy in Mexico covers only females, and private vaccination is rarely delivered in men.

Sample collection

Trained project staff obtained two samples by swabbing the penile skin of each subject with a flocked dacron swab moistened with saline solution. One sample was preserved in 50% ethanol, while the other sample was preserved in DNA/RNA shield solution (Zymo Research).

Detection of HPV DNA and genotyping

DNA from samples preserved in ethanol was extracted for HPV identification using a commercial kit (DNeasy Blood & Tissue, Qiagen). The presence of HPV was determined using classical PCR with two pairs of universal primers targeting the genes L1, MY09/MY11 and L1C1.¹² Negative controls included PCR reactions without DNA, while the positive control comprised the HPV-16 genome cloned in the pBR322 vector.

HPV genotyping was performed using a proprietary real-time PCR assay (Biomédicos de Mérida S.A. de C.V., a certified diagnostic laboratory under ISO 15189:2012). The assay detects high-risk HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and low-risk genotypes (6, 11, 13, 40, 42, 43, 44, 54, 61, 70, 72, 81, 84, 89, and 91). Men who had at least one high risk genotype were considered as HR-HPV positive, and only those who had only low risk genotypes were considered low risk.

DNA extraction and sequencing

Metagenomic DNA was extracted from each DNA/RNA shield-preserved sample using a ZymoBIOMICS™ DNA Miniprep Kit (Zymo Research) following the manufacturer’s protocol. The extracted DNA was then used to generate amplicon libraries targeting the V4 region of the rRNA gene, which were subsequently sequenced on the Illumina MiSeq 2x300 PE platform (Illumina, San Diego, CA) at the Research and Testing Laboratory (Lubbock, TX, USA).

16S rRNA amplicon sequence analyses

Sequence analyses were performed using R 4.2.2 (Team, 2013). Specific parameters are in Supplemental methods. Raw pair-end sequences were processed in DADA2 1.18¹³ for denoising, merging, and chimera removal. Classification was performed against the Silva database 138.1.¹⁴ Samples were rarefied to the depth of the sample with the lowest number of reads (12,223). To capture general patterns of the resident microbiota and minimize individual-specific variation, ASVs were filtered out if they were present in less than 10% of the samples.

Alpha diversity metrics (Observed species, Chao1 and Shannon diversity) were calculated using the vegan package.¹⁵ Beta diversity was visualized using a Non-metric Multidimensional Scaling (NMDS) based on Jensen-Shannon divergence distance constructed using the vegan package.

Community state types (CSTs) were established following methods described elsewhere,¹⁶ with the number of clusters validated through bootstrapping. The heatmap shows only the 10 most abundant ASVs (by mean relative abundance).

Statistical analyses

The statistical analyses were performed in R with the rstatix package.¹⁷ Since some characteristics are known to have a predictive effect for HPV, comparisons between positive and negative were made using Fisher’s exact test to control for confounding effects (e.g., smoking, sexual preference, number of sexual partners). Linear regression models were constructed to assess the relationships between alpha diversity metrics and predictor variables, including HPV status, HPV genotype (non-genotyped samples were not taken into account). For beta diversity comparisons, the Adonis nonparametric test was used. Associations between CSTs and HPV were initially assessed using Fisher’s exact test. CST-5 was subsequently compared with all other CSTs combined, and the strength of the association was quantified using odds ratios (OR) and 95% confidence intervals (CIs). Taxa that were differentially abundant in CST-5 relative to the other CSTs were grouped and compared using the Wilcoxon rank-sum test. A p-value of <0.05 was used as the level of significance.

Results

General characteristics of the study population

A total of 103 men were included in the study. Their basic characteristics are shown in Table 1. They were uncircumcised. Their mean age was 30.2 years (+/− 6.7) for HPV-negative and 31.2 years (+/− 6.6) for HPV-positive. Their median age was 29 years. HPV was detected in 33 (32.03%) men, with 15 high-risk, 10 low-risk, and 8 non-genotyped infections (Supplemental Table 1).

Table 1.

General characteristics of the male population studied.

General characteristic	# HPV-negative (%)	# HPV-positive (%)	p. value
Sampled men	70 (68%)	33 (32%)
Age			0.88
20–24	13 (13%)	6 (6%)
25–29	27 (26%)	11 (11%)
30–49	30 (29%)	16 (16%)
Sex of sexual partners			0.01
Men who have sex with men (MSM)	28 (27%)	5 (5%)
Men who have sex with women (MSW)	42 (41%)	28 (27%)
Number of sexual partners^a			0.57
0–4	9 (9%)	1 (1%)
5–9	18 (17%)	10 (10%)
10–19	22 (21%)	11 (11%)
<20	20 (19%)	11 (11%)
Unanswered	1 (1%)	0 (0%)
Tobacco use	3 (3%)		0.65
Smoker	23 (22%)	9 (9%)
Non-smoker	47 (46%)	24 (23%)

^aSum of previous and current sexual partners. p values are calculated using Fisher’s exact test.

Sexual preference was the only factor significantly linked to HPV status (Fisher’s test, p = .013). Men who have sex with women (MSW) were 3.69 times (95% CI: 1.21–13.73) more likely to be HPV positive than men who have sex with men (MSM). Consequently, subsequent multiple linear regression models were adjusted to control for the effect of sexual preference.

Alpha diversity

Multiple linear regression analyses revealed that HPV-positive men exhibited significantly higher observed and predicted richness (S.obs: +9.34 ASVs, p = .003; Chao1: +8.33 ASVs, p = .02) compared to HPV-negative individuals (Supplemental Table 2). However, no significant differences were observed in Shannon diversity (p = .23) or Pielou’s evenness (p = .68) between the two groups.

When stratified by HPV genotype present (Supplemental Table 3), men with high-risk (HR) genotypes showed significantly higher observed richness (S.obs: +11.79 ASVs, p = .003; Chao1: +10.06 ASVs, p = .03) compared to HPV-negative individuals, while no significant differences were observed for low-risk (LR) genotypes (S.obs: p = .16; Chao1: p = .26).

Beta diversity

NMDS ordination revealed overlapping in microbial community composition between HPV-positive and HPV-negative individuals (Figure 1). However, the PERMANOVA analysis revealed a modest but statistically significant effect of infection status on microbial composition (adonis2: R² = 0.0298, p = .0130). The PERMANOVA was not significant when evaluating according to genotype risk (adonis2 p = .1289).

Figure 1.

Community structure based on NMDS computed by Jensen-Shannon distance of the penile skin microbiota. Ellipses represent 95% confidence intervals. HPV-positive samples are closer together compared to HPV-negative samples (adonis2 R² = 0.0298, p = .0130).

Community state types of the penile skin microbiota and HPV status

The bacterial community was characterized and grouped into five distinct Community State Types (CSTs) (Figure 2, Table 2). CST-1 was dominated by Staphylococcus (n = 11), CST-2 by Corynebacterium (n = 20), CST-3 by Fenollaria and Prevotella (n = 14), CST-4 by Mannheimia (n = 12), and CST-5 by Finegoldia (n = 46).

Figure 2.

Community State Types of the penile skin microbiota. Heatmap representing the relative abundance of the most abundant ASV’s in the penile skin microbiota of subclinical HPV-positive and HPV-negative samples. The dendrogram was constructed using the Ward’s linkage clustering based on the Jensen-Shannon Divergence distance.

Table 2.

Community State Types in the penile skin microbiota of HPV-infected men.

CST	Dominant taxa (relative abundance)	Number of samples	# HPV-positive (%)
CST-5	Finegoldia (45%)	46	21 (84%)
CST-2	Corynebacterium (41%)	20	5 (33.3%)
CST-3	Fenollaria (11%)	14	4 (40%)
CST-4	Mannheimia (41%)	12	1 (9.1%)
CST-1	Staphylococcus (31%)	11	2 (22.2%)

Summary of the five identified Community State Types (CSTs) in the penile skin microbiota of HPV- infected individuals. The table shows the dominant taxa for each CST, the number of individuals belonging to each CST, and the prevalence of each CST.

All CSTs included at least one HPV-positive sample (Table 2). CST-5 had the highest proportion of HPV-positive individuals (45.7%), while CST-1 had the lowest (12.5%). Although Fisher’s exact test showed no overall association between CSTs and HPV status (p = .085), CST-5 exhibited a trend towards encompassing most HPV-positive samples. Comparing CST-5 to all other CSTs combined (CST-1 to CST-4) revealed a significant association (Fisher’s exact test: p = .01; OR = 3.11, CI 95%: 1.22–8.22), indicating a three-fold increased likelihood of HPV positivity in CST-5.

CST-5 was dominated by Finegoldia (45.13% relative abundance). To investigate other taxonomic drivers of this CST, we compared the mean abundance of taxa between CST-5 and the other CSTs combined. This revealed significant differences in several taxa (Supplemental Table 4). ASVs belonging to the genera Peptoniphilus, Negativicoccus, Anaerococcus, and Prevotella_7 showed higher abundance in CST-5, whereas common commensals such as Corynebacterium and Staphylococcus were significantly less represented.

Discussion

In this cross-sectional study, we analyzed the penile skin microbiota (PSM) of 103 men (70 HPV-negative, 33 HPV-positive) to explore its potential association with subclinical HPV infections. Our findings reveal two key insights: (i) HPV-positive men exhibit greater microbial richness compared to HPV-negative individuals, particularly those with high-risk genotypes, and (ii) an anaerobic-rich community state type (CST-5) is significantly associated with HPV positivity. These results support our hypothesis that a dysbiotic PSM, characterized by an increase in anaerobes and a reduction in facultative anaerobes commensals, is linked to HPV infection in HIV-negative uncircumcised men. To our knowledge, this is the first study to address this association while controlling for sexual preference.

With respect to alpha diversity, no differences were observed in the Shannon H′ index between HPV-negative and HPV-positive samples. However, HPV-positive men exhibited significantly higher richness than HPV-negative men. This finding aligns with previous research on the association between PSM and microbiota.⁹ One possible explanation is that certain anaerobic genera are usually present in very low abundances, often falling below detection thresholds, and that their increased abundance -although associated with HPV infection- does not substantially affect PSM alpha diversity.

Although beta diversity appeared broadly similar, statistical analyses revealed subtle but significant differences in bacterial community composition between HPV-negative and HPV-positive individuals.

In this study, we found that there are differences in the richness of ASV’s of those positive for HPV, specifically, and importantly, those with high-risk genotypes infections. Interestingly, there were no differences in evenness. This pattern of increased richness without differences in evenness has also been previously observed in the context of vaginal microbiota and HPV infection.¹⁸ These findings suggest that HPV infection may not drastically alter the overall microbial community structure, but rather favors the proliferation of opportunistic anaerobic bacteria that are in low proportion.

CST analysis simplifies complex bacterial community data by grouping similar samples together, facilitating the correlation of microbial profiles with factors such as infection status. This approach has been used to associate microbial profiles with HPV status in women.¹⁹ Changes in the dominant Lactobacillus community, which are accompanied by an increase in pH, allow the proliferation of anaerobic and opportunistic bacteria, which modify the microenvironment, making it more susceptible to HPV persistence²⁰ and progression to cancer lesions.²¹ In our study, this pattern is observed by CST-5, dominated by Finegoldia and rich in anaerobic bacteria genera such as Peptoniphilus, Negativicoccus, and Anaerococcus, while showing reduced proportions of commensals like Corynebacterium and Staphylococcus. Staphylococcus commensals have been shown to interact with the immune system to facilitate antiviral responses by stimulating Type I interferons.^7,22 These molecules are crucial for immune recognition and activation processes against HPV.²³ Their low proportion in CST-5 may compromise local immune-barrier function, creating a microenvironment permissive to HPV infection susceptibility or persistence.

The strong association between CST-5 and HPV positivity raises important questions about the role of anaerobic bacteria in viral pathogenesis. Finegoldia has been associated with an increased risk of HIV acquisition in a cohort of uncircumcised Ugandan men.⁸ While our study cannot establish causality, it is plausible that a preexisting dysbiotic microbiota, rich in anaerobes, may facilitate HPV infection by impairing the local immune response. Alternatively, HPV infection itself could induce changes in the penile microenvironment, promoting the growth of anaerobic bacteria. Longitudinal studies are needed to shed light into these possibilities and determine whether dysbiosis precedes or results from HPV infection.

The only previous study on this topic focused on Black South African men, many of whom (94.3%) were circumcised and some (26.5%) co-infected with both HPV and HIV, thereby limiting the extent to which meaningful comparisons can be drawn with our study population.⁹ Nonetheless, their results are in accordance with those of this study. They found a microbial signature of dysbiosis (higher proportion of anaerobes) associated with prevalent HR- HPV infections. However, circumcision is known to reduce HPV infection risk²⁴ and anaerobic load,¹¹ which may explain the lower prevalence and different microbial profiles observed in their circumcised cohort compared to our uncircumcised population. This suggests that the modification of the microbiome induced by this procedure may be an additional protective factor.

With regard to the penile microbiota of healthy men, our findings are consistent with previous studies of the penile microbiota, which frequently report the presence of the genera Anaerococcus, Corynebacterium, Finegoldia, Peptoniphilus, Prevotella, and Staphylococcus in the penis of healthy individuals (Gonçalves et al., 2022). All of these genera were detected in our cohort, albeit with varying relative abundances. For instance, whereas Corynebacterium and Staphylococcus are often predominant in studies such as Onywera et al. (2020), Finegoldia was the most abundant genus in our cohort, in agreement with the findings of Liu et al. (2017) in uncircumcised men. CST-5 was enriched in Anaerococcus, Negativicoccus, Peptoniphilus, and Prevotella, all of which have previously been reported as common members of the penile microbiota.

Our study had several strengths, and we had a large number of participants to be able to conduct robust analyses. Our population considered both MSW and MSM, taking into account that MSM have a higher oncogenic risk anogenital caused by high-risk HPV infection.^25,26 We also had a non-circumcised population, allowing a basis for comparison with circumcised populations in future studies. This study had also certain limitations; as a cross-sectional analysis, we cannot declare causative relationships between HPV and the observed microbiome differences. Longitudinal and mechanistic studies are needed to determine whether dysbiosis of the penile microbiota is a cause or a consequence of HPV infection.

Research in the vaginal microbiome has advanced significantly toward translational medicine, by the usage of probiotics.²⁷ In contrast, the study of the penile microbiome in men is still in early stages. Our findings suggest that dysbiosis of the penile microbiome, characterized by an increase in anaerobes such as Finegoldia, is associated with HPV infection. This opens new lines of research to explore whether manipulation of the microbiome, through targeted antimicrobial therapies, could reduce the risk of HPV progression to neoplastic lesions.

Conclusions

This study demonstrates an association between the penile skin microbiota and subclinical HPV infection in HIV-negative men, suggesting that dysbiosis (anaerobic predominance), may facilitate HPV susceptibility or persistence. Longitudinal studies are needed to determine if dysbiosis precedes the infection, or it occurs after.

Supplemental Material

Supplemental Material - Penile microbiota dysbiosis associates with subclinical HPV infection in HIV-negative men

Supplemental Material for Penile microbiota dysbiosis associates with subclinical HPV infection in HIV-negative men by Diego A Garza-González, Jesús G Gómez-Carballo, Stephany D Burgos-Vela, Laura Conde-Ferráez, Ian M Carroll, Aileen O'Connor-Sánchez and María del Refugio González-Losa in International Journal of STD & AIDS.

Footnotes

The authors thank Diego I. Sosa-Hernández and Mirbella del Rosario Cáceres-Farfán for their technical assistance. José Reyes Canché Pech on behalf of Biomédicos de Mérida S.A. de C.V. for providing the genotyping for this study. E.M. De Villiers for donating the pBR322 vector.

Ethical considerations

The study was conducted in accordance with the Declaration of Helsinki and was approved by the ethics committee of the “Centro de Investigaciones Regionales Dr Hideyo Noguchi” of the “Universidad Autónoma de Yucatán” (identification number: CEI- 01–2020).

Consent to participate

Informed consent was obtained from all eligible participants.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The research leading to this manuscript was funded by the CONAHCYT, “Ciencia de Frontera 2019” grant number: 87172 and Grant INFR2016 01-269833. Garza-González was awarded the CONAHCYT doctoral scholarship 800614.

ORCID iDs

Diego A Garza-González

Jesús G Gómez-Carballo

Stephany D Burgos-Vela

Laura Conde-Ferráez

Ian M Carroll

Aileen O'Connor-Sánchez

María del Refugio González-Losa

Data Availability Statement

Data available at NCBI: PRJNA1244442.

Supplemental Material

Supplemental material for this article is available online.

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Supplementary Material

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