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Gut dysbiosis of bacteria and fungi associated with human immunodeficiency virus infection

To the Editor: There have been growing concerns about the gut bacterial dysbiosis associated with HIV infection, addressing a critical role of gut bacteria in persistent chronic inflammation and severe intestinal dysfunction among the human immunodeficiency virus (HIV) infected population. However, little is known about the gut fungal dysbiosis caused by HIV-infection. Therefore, we launched a case-control study aiming to explore the gut dysbiosis of bacteria and fungi in HIV infection, which may reveal a more comprehensive feature of HIV infection-related gut dysbiosis of bacteria and fungi that possibly play a role in the chronic systemic immune activation observed in HIV infection.

To the Editor: There have been growing concerns about the gut bacterial dysbiosis associated with HIV infection, addressing a critical role of gut bacteria in persistent chronic inflammation and severe intestinal dysfunction among the human immunodeficiency virus (HIV) infected population. However, little is known about the gut fungal dysbiosis caused by HIV-infection. Therefore, we launched a case-control study aiming to explore the gut dysbiosis of bacteria and fungi in HIV infection, which may reveal a more comprehensive feature of HIV infection-related gut dysbiosis of bacteria and fungi that possibly play a role in the chronic systemic immune activation observed in HIV infection.
The ethics committee of West China Fourth Hospital approved the study protocol (No. KS2020310). A total of 130 participants were enrolled in Chengdu Public Health Clinic Center, including 75 HIV-infected patients (HIV+) and 55 HIV-uninfected participants (HIV-, matched with HIV+ for sex, age, and body mass index [BMI]). Fecal and peripheral blood samples were collected, and all participants reported without any antibiotic usage in the past one month at the time of fecal sample collection. The peripheral blood CD4+ and CD8+ cell counts and plasma HIV viral load were determined by standard methods for HIV+ participants (n = 75). [1] Ten immune biomarkers were quantitatively measured by quantitative enzymelinked immunosorbent assays (ELISA) (Jiangsu MEI-MIAN Industry Co., Ltd., Yancheng, Jiangsu, China), including 1) four biomarkers indicating the microbial translocation: lipopolysaccharide (LPS), lipopolysaccharide-binding protein (LBP), soluble form of the LPS coreceptor CD14 (sCD14) and mannose-binding protein/ lectin (MBP/MBL); 2) six inflammatory-related cytokines: interleukin (IL)-6, tumor necrosis factor (TNF)-a, IL-1b, IL-17, IL-10, and IL-22. DNA was extracted from fecal samples using an E.Z.N.A. ® Soil DNA Kit (Omega Biotek, Inc., Norcross, GA, USA) according to manufac-turer's protocols. The V3-V4 region of bacterial 16S ribosomal RNA (rRNA) gene and internal transcribed spacer (ITS) region were amplified using universal primers. Amplicons were sequenced on Ion Torrent S5 system (Life Technologies, Carlsbad, USA) and Illumina Hiseq 2500 platform (Illumina Inc., San Diego, CA, USA) for bacteria and fungi, respectively. Sequence data was analyzed by bioinformatics analysis using QIIME (v.  In the present study, the alpha-diversity of gut bacteria in HIV+ was significantly lower than HIV-according to the observed species (P < 0.001) and Shannon index (P = 0.033). The result of the beta-diversity analysis presented significantly different clusters in bacterial communities between HIV+ and HIV-(R = 0.041, P = 0.023). In HIV+, there was a difference in the composition of gut bacteria compared to HIV-. Firmicutes, Bacteroides, Proteobacteria, and Fusobacteria were the four predominant genera in phylum level for both HIV+ and HIV-. For example, expansion of Fusobacteria (P = 0.001) and decreased proportion of Firmicutes (P = 0.001) were observed in HIV+. Top 15 genera in relative abundance were observed, with increased proportions of Fusobacterium (P = 0.001), Prevotella 9 (P = 0.018), and decreased Faecalibacterium (P = 0.001), Alistipes (P = 0.004), Akkermansia (P = 0.042), and Our study demonstrated the gut fungal dysbiosis due to HIV infection with increased gut fungal alpha-diversity and altered fungal composition. According to the observed species (P < 0.0001), the alpha-diversity of fungi in HIV+ was marginally higher than that in HIV-. Furthermore, our findings on beta-diversity revealed distinct clusters of fungal community between HIV+ and HIVÀ participants (R = 0.195, P = 0.001), which could be attributed to the expansion of opportunistic fungal infection, which has also been observed in HIV+ with oral fungal dysbiosis. [2] In HIV+, the ratio of Basidiomycota-Ascomycota was 0.09, which was lower than in HIV-(0.15). The top fungal genus was Candida, followed by Cladosporium, Gibberella, Rhodotorula, and Saccharomyces. Bacteroidetes were presented, among which, Clostridia and Clostridiales were the top 2 taxa, both having scores greater than 4.0. The depletion of potentially beneficial bacteria like Bacteroidaceae and Ruminococcaceae in HIV+ people could be linked to the loss of CD4+ T cells, as both effector and regulatory CD4+ T cells were specifically targeted and attacked by HIV, resulting in beneficial bacteria being killed by innate immune cells. [3] For fungal LDA analysis, 4 taxa from Ascomycota and 16 taxa from Basidiomycota were found to be differentiated between HIV+ and HIV-. Nectriaceae, Hypocreales, and Sordariomycetes were the top 3 fungal taxa with scores greater than 4.0 in HIV+. While, Basidiomycota (LDA = 3.94), Phallaceae (LDA = 3.88), and Phallales (LDA = 3.82) were verified to be particularly enriched in HIV-. The gut fungal dysbiosis in HIV infection was characterized by altered fungal biodiversity and compositions, which might also play an important role in HIV pathogenesis.
Between HIV+ and HIV-, there were distinct bacteriafungi correlation patterns [Supplementary Figure 4A, http://links.lww.com/CM9/B85]. The top 15 taxa in relative abundance, as well as differential taxa at the genus level from each group, were used to analyze the relationship between bacteria (n = 25) and fungi (n = 15). Less bacteria-fungi associations with 15 pairs were identified in HIV+, compared to HIV-with 23 pairs. In HIV-, Candida was observed to be significantly related to 9 bacteria, such as positive associations with Escherichia Shigella and Ruminococcus gnavus group, and negatively correlated to Bacteroides and Alistipes. By contrast in HIV +, Candida was just associated with two bacteria: Bacteroides (negative) and Ruminococcaceae UCG-005 (positive). Gibberella was observed positively correlated with 7 bacteria such as Fournierella and Ruminococcaceae in HIV-, whereas no correlation between Gibberella and bacteria was found in HIV+. Rhodotorula exhibited significant correlations with 4 bacteria, including positive associations with Christensenellaceae R7 group, Ruminiclostridium 6 and Ruminococcaceae UCG-014, as well as negative association with Bacteroides. Furthermore, bacteria-fungi interactions were found to cause tissue damage by producing extracellular enzymes, which increased pro-inflammatory cytokine secretion, resulting in increased oxidative damage and apoptotic cell death. [4] Associations of immune biomarkers with gut bacteria and fungi in HIV+ and HIV-were presented [Supplementary Figure 4B, http://links.lww.com/CM9/B85]. Neither group had a significant association of biomarkers with fungi. For bacteria in HIV+, 1) Dialister exhibited a positive correlation with two microbial translocation markers (sCD14 and MBP/MBL) and five inflammatoryrelated cytokines (IL-6, IL-17, IL-1b, IL-22, and IL-10), which may suggest Dialister play a dual role of proinflammation and anti-inflammation in HIV infection; 2) Fournierella, was positively associated with three microbial translocation markers (LPS, LBP and MBP/MBL) and four inflammatory-related cytokines (IL-17, TNF-a, IL-22, and IL-10); 3) negative correlation was found between Fusobacterium and LPS, sCD14, IL-6, IL-1b, and MBP/ MBL. By contrast in HIV-, increased abundance of Ruminococcaceae UCG-014 was associated with elevated levels of LBP, sCD14, and MBP/MBL. It is widely recognized that the complexity of interactions between gut microbes and host and the proposed relationships between immune biomarkers and specific taxa remained to be verified by experimental evidence. [5] Our study should address several limitations, such as the small sample size and lack of experimental proof. Future research could include larger samples and the use of metagenomics and metabolomics tools, as well as experimental evidence, to confirm the key microorganisms and investigate the functions of these strains linked to gut dysbiosis and immune status in HIV infection.
In conclusion, our findings contribute to a better understanding of the alterations in the gut bacterial and fungal microbiome and their associations with chronic systemic immune activation observed in HIV infection by providing a more comprehensive picture of HIV infectionrelated dysbiosis of the gut microbiome.

Declaration of patient consent
The authors certify that participants' records were anonymized. Written informed consents were obtained from all participants prior to the study procedure.

Funding
The study was supported by grants from the Key Research and Development Project, Department of Science and Technology, Sichuan Province (Nos. 2018SZ0212 and 2018JY0383) and the Fundamental Research Funds for the Central Universities, Project of Sichuan Health and Family Planning Commission (No. 18PJ015).