Cryptococcal meningitis (CM) is a global invasive fungal disease associated with high morbidity and mortality (Iyer et al., 2021; Rajasingham et al., 2022). Cryptococcus neoformans (C. neoformans) and C. gattii are the main species responsible for life-threatening CM (Hagen et al., 2015; Kwon-Chung et al., 2017); immunocompromised individuals are the most vulnerable, but there are also reports of cryptococcal infections in immunocompetent hosts (Perfect et al., 2010; Kwon-Chung et al., 2014). It is estimated that 223,100 cases of CM occur globally each year, leading to 181,100 deaths (Rajasingham et al., 2017). Most CM-related deaths occur in resource-limited settings in which access to drugs is limited and effective treatments are expensive, signifying the need to develop affordable therapeutics against these deadly fungal pathogens (Loyse et al., 2019; Vilas-Bôas et al., 2020; Driemeyer et al., 2022; Rajasingham et al., 2022).

Recent increases in our understanding and analysis of the gut microbiota have shed light on the impact of alterations to human health. The gut microbiota is a dynamic and highly diverse microbial ecosystem that affects many aspects of the host’s physiology (Kamada et al., 2013; Erny et al., 2021), and is involved in the host immune response, protection against pathogen overgrowth, biosynthesis, and metabolism. For example, the microbiota was found to be important for the host response against C. gattii, with germ-free mice more susceptible to infection, and showing lower survival, higher fungal burden in the lungs and brain, reduced levels of interferon-γ, interleukin (IL)-1β, and IL-17, and lower nuclear factor κB p65 phosphorylation than wild-type mice (Costa et al., 2016). Additionally, bloodborne Candida albicans infection was reported to decrease the diversity of the gut microbiota (Hu et al., 2021), while the gut bacterial microbiota affected generation of the pulmonary IL-17 response to Aspergillus fumigatus infection in mice (McAleer et al., 2016).

These findings highlight the commensal microbiota modules immune responses in infectious diseases and show that altering the composition could be a therapeutic approach for disease. However, it is unclear whether and how Cryptoccocus infection induces changes in the gut microbiota in human. In particular, the effect of anti-fungal treatments on the gut microbiota is unknown. Previous studies primarily focused on anti-bacterial and anti-viral treatment (Villanueva-Millán et al., 2017; Wipperman et al., 2017; Ponziani et al., 2018), but little is known about the effects of anti-fungal treatment on the intestinal microbiome. Additionally, most earlier studies on the gut microbiota concentrated on the bacterial community, while research into gut fungal communities only started more recently. Thus, more comprehensive research into the role of intestinal bacteria and fungi in the pathobiology of fungal diseases is needed.

To this end, we performed a cross-sectional study of CM patients and healthy controls (HCs) to characterize the bacterial and fungal gut microbiota changes in response to Cryptococcus infection and anti-fungal therapy in individuals from China.

Since the importance of microbiota was established, numerous studies have revealed associations between the gut microbiota and human disease (Qian et al., 2021; Trebicka et al., 2021; Wu et al., 2021). Dysbiosis of the gut microbiota during Cryptococcus infection or anti-fungal treatment may play an important role in the pathophysiology of CM. To investigate this, we examined the gut microbiota of CM patients with and without anti-fungal treatment and healthy subjects using shotgun metagenomic sequencing. Our results revealed that CM patients have distinct bacterial and fungal compositions compared with controls, but that anti-fungal treatment only led to minor changes in the gut microbiota. Moreover, both positive and negative correlations existed between fungi, bacteria, and clinical indicators in CM. Our study contributes valuable information to the field about the gut microbiota in CM patients, implying the existence of links between gut microbiota changes and CM.

We showed that the gut microbiota composition of CM patients differed significantly from that of healthy individuals with respect to beta diversity, but not alpha diversity. This contrasts with previous findings which revealed the alpha diversities of gut microbiota to be reduced in patients with infectious diseases compared with controls (Wang et al., 2017; Hu et al., 2019; Tuddenham et al., 2020).

Enterococcus is a genus of Gram-positive lactic acid bacteria of the Firmicutes phylum. Some enterococcal strains cause antibiotic-induced biological disorders, play antitumor or anticancer roles, and modulate the immune system. For example, cultured E. faecium from the human intestinal epithelium demonstrated bactericidal effects against enteroaggregative Escherichia coli, as well as membrane damage, and cell lysis (Tarasova et al., 2010; Fusco et al., 2017), while in another study it increased the expression of proinflammatory cytokines without appearing as a pathogen (Seishima et al., 2019). The present study observed significant increases in the abundance of six Enterococcus species in CM patients compared with controls, including E. dispar, E. avium, and E. faecalis. Considering the beneficial actions of Enterococcus bacteria on the host immune system, it seems that Cryptococcus infection-induced increases in the abundance of bacteria reflect a compensatory response in the host.

We also found that CM patients were significantly enriched in Blautia marasmi. The Blautia genus of the Lachnospiraceae family in the Firmicutes phylum was initially described in 2008 (Liu et al., 2008). Blautia species function in the degradation of indigestible carbohydrates (Sheridan et al., 2016), and some such as B. coccoides produce short-chain fatty acids as metabolic mediators between the microbiota and host (Liu et al., 2021). Considering its relatively high abundance in CM patients, B. marasmi may play an important role in Cryptococcus infection.

We observed a significant decrease in the abundance of Prevotella spp. in the CM group compared with controls. Prevotella is a diverse genus of Gram-negative anaerobes, which is highly abundant in different parts of the body and plays a key role in the balance between health and disease (Shah and Collins, 1990; Larsen, 2017; Posteraro et al., 2019). Conflicting findings have been reported about whether Prevotella spp. are beneficial or detrimental to gut health, especially with regard to glucose homeostasis (Ley, 2016; Cani, 2018; Claus, 2019). The emergence of Prevotella-rich microbiota is associated with inflammatory disorders such as rheumatoid arthritis, periodontitis, metabolic disease, and inflammation in HIV patients (Ibrahim et al., 2017; Armstrong et al., 2018; Wells et al., 2020). Several studies have further demonstrated the potential pro-inflammatory role of Prevotella spp., including enhanced T cell activation and T cell recruitment (Neff et al., 2018; Rolhion et al., 2019; Wells et al., 2020). Considering the important role of T cell immunity in preventing C. neoformans infection, the observed decrease in Prevotella abundance in CM patients of the present study may represent one of the mechanisms of C. neoformans against host immunity.

KEGG is a widely used annotated database (Kanehisa et al., 2004). Genes can be projected into the KEGG PATHWAY database uncover interactions with other genes that may influence the health of the host (Altermann and Klaenhammer, 2005). our data showed that gluconeogenesis, glycolysis and lipoic acid biosynthesis much more abundant in CM groups. Some studies have found that gluconeogenesis, glycogenolysis, and glycolytic pathways produce glucose-6-phosphate and further releases abundant nicotinamide adenine dinucleotide phosphate through the pentose phosphate pathway (Agius et al., 2002; Gomis et al., 2003). High levels of glutathione promote inflammatory macrophages to mediate inflammatory responses. Additionally, glycogen metabolism not only upregulates STAT1 expression by activating RARβ but also promotes STAT1 phosphorylation by downregulating phosphatase TC45 in macrophage, thereby regulating inflammatory responses (Ma et al., 2020). Therefore, alterations in the function of the gut microbiota may play an important role in the pathogenesis of CM. In our study, lipoic acid biosynthesis much more abundant in CM groups, lipoic acid is an antioxidant that has been suggested to have beneficial immunomodulatory effects on the innate and adaptive immune systems in autoimmune diseases (Liu et al., 2019). Further investigations are needed to better understand the relationship between different features of the gut microbiota in CM.

Our results showed that anti-fungal treatment induced only a minor alteration in the gut microbiota, with the clustering driven by CM, CM-2, CM-4 groups not reaching significance based on PCoA analysis. The effect of anti-infection treatments on the gut microbiota is unclear. Antibiotics, especially broad-spectrum antibiotics, can destroy the gut microbiota during anti-tuberculosis (TB) treatment in mice (Namasivayam et al., 2017). However, a human study by Wipperman et al. (2017) reported no significant difference in the overall microbiota diversity of active TB patients after using first-line drugs compared with uninfected or latent TB patients, as estimated by the Shannon diversity index. Moreover, anti-infection treatments, especially anti-viral treatment for non-intestinal infections, may help restore the intestinal flora. For example, it was reported that a combination of nucleoside reverse transcriptase inhibitors reduced the fecal bacterial diversity caused by HIV infection (Villanueva-Millán et al., 2017). However, a similar restoration of the intestinal flora was not seen in our study.

Fungi and bacteria share microhabitats and participate in complex communications within the microbial community. The correlation network of fungi and bacteria observed in our study shows these co-occurrence patterns in CM patients after treatment. The bacterium S. constellatus showed a negative correlation with the fungus B. bassiana, Streptococcus is the most common genus of bacteria present in patients with bacterial brain abscesses and is often isolated from mixed infections. S. stellate typically resides in the oral cavity, appendix, and female reproductive tract, and tends to form abscesses (Lanks et al., 2019). Our study found negative correlation between S. constellatus and B. bassiana, and this negative relationship need confirmed in larger cohorts.

There are several limitations of this study, including its relatively small sample size, which may have resulted in a lack of power for some comparisons, especially in post-treatment patients. Future studies should therefore expand the sample size. Additionally, the lack of follow-up prevented a longitudinal analysis. Moreover, the mechanisms underlying increases in the abundance of bacteria in the gut of mice with Cryptococcus infections should be investigated.

In conclusion, this study shows that Cryptococcus infection induces significant changes in the gut microbiota, with CM patients showing distinct bacterial and fungal compositions compared with controls. Both positive and negative correlations were found to exist between fungal and bacterial species after treatment. These data serve as a basis for further investigations into the role of the gut microbiome in CM patients.

The Metataxonomic raw data have been deposited in the NCBI online repository under the accession number PRJNA935276.

The studies involving human participants were reviewed and approved by the Institute Ethics Committee of Jiangxi Chest Hospital. The patients/participants provided their written informed consent to participate in this study.

HL and LZ performed the experiments and wrote the manuscript. KZ, YH, XDL, and YL contributed to the methodology and helped with data analysis. WL, XGL, WP, and QZ contributed to the study design and modified the manuscript for final submission. All authors contributed to the article and approved the submitted version.