Introduction
Human beings have a rich and unique microecology in the channels that connect them to the outside world, such as the skin, digestive tract, respiratory tract, oral cavity, and genitourinary tract [
1]. Among them, the intestinal microbiota is the most representative microbial community [
1]. Previous studies [
2,
3] on intestinal microecology have mainly focused on intestinal bacteria or selected adults as subjects while neglecting the role of fungi in the gut of children due to the much lower abundance of intestinal fungi than bacteria, limitations of techniques for detecting fungi, the difficulty of obtaining samples, and the absence of large-scale reference databases. In particular, people still know little about the dynamic influence of fungi on the formation of intestinal microecological populations in neonates.
VLBWI, defined as newborns with birth weight <1500 g, are a special category of newborns characterized by low immunity and susceptibility to infectious diseases such as late-onset sepsis (LOS) and necrotizing enterocolitis (NEC) [
4]. It has been reported that the development of complications such as feeding intolerance (FI), NEC, and LOS in VLBWI may be related to their unique gut microflora composition [
5,
6].
A limited number of studies has been found that symbiont fungi may be able to calibrate host immunological responses [
7,
8], promote development of peripheral lymphoid organs [
9], promote T cell responses [
10], and even may be associated with the development of certain diseases, such as inflammatory bowel disease (IBD), NEC, and allergic diseases [
11‐
15].
There still are few studies on the intestinal fungi of very low birth weight infants. Based on the above-mentioned, first of all, we collected stool samples from 62 preterm infants at different time points, including 34 VLBWI and 28 preterm infants with birth weight >1500 g, then analyzed their intestinal fungi by ITS sequencing. At last, we explored the characteristics of intestinal fungi in VLBWI.
Method
Trial participants
After obtaining informed consent from parents, preterm infants who were admitted to the NICU directly after birth with a birth weight of less than 1500 g were eligible to participate in the experimental group, and preterm infants who had a birth weight of greater than 1500 g were eligible to participate in the control group. Exclusion criteria were preterm infants with severe asphyxia at birth or had congenital malformations, genetic metabolic disorders, or the presence of shock or multi-organ failure. The trial was in line with the Declaration of Helsinki and approved by the Ethics Committee of Ningbo Women’s and Children’s Hospital, Zhejiang Province, China.
Sampling
Medical personnel collected 2 g of stool samples on day 1, day 3, day 7, day 14, day 21, day 28, and day 60 (experimental group only) after the admission of study subjects who met the criteria with a special specimen box for stool cleaning, and stored them immediately in a −80 °C refrigerator. Collection was stopped for study subjects who were discharged or died in the middle of the study.
DNA extraction and sequencing of the ITS genes
Microbic DNA was extracted by a QIAamp DNA Stool Mini Kit (Qiagen) according to the manufacturer’s instructions. Then, DNA was quantified by Nanodrop, and the quality of DNA extraction was assessed by 1.2% agarose gel electrophoresis. The fungal-specific gene fragment sequence was amplified by the universal fungal primers. The purification of polymerase chain reaction PCR products was performed using Vazyme VAHTSTM DNA Clean Beads, and Illumina bridge PCR-compatible primers were introduced. The amplified products were detected by Quant-iT PicoGreen dsDNA Assay Kit fluorescent reagent and Microplate reader (BioTek, FLx800) quantification instrument. Sequencing libraries were prepared using Illumina’s TruSeq Nano DNA LT Library Prep Kit, and finally, the resulting samples were subjected to ITS high-throughput sequencing.
The raw data obtained from the Illumina platform turned into effective Amplicon Sequence Variants (ASVs) after removing the primer sequence, trimming, merging, and filtering. The alpha diversity refers to the fungal diversity within each sample, and it was calculated by using Simpson’s reciprocal index, which describes how many ASVs prevail in each sample. The beta diversity expresses the difference between the samples in terms of the number and abundance of ASVs within an age group, and it was calculated with the Bray-Curtis dissimilarity index, predicting the metabolic function of a sample’s fungus, identifying differential pathways, and obtaining the species composition of specific pathways. Functional prediction of ITS gene sequences in the MetaCyc database using PICRUSt2 was followed by functional unit PCoA analysis, i.e., using Bray-Curtis distance matrix combined with principal coordinates analysis to expand sample functional differences in low dimensions. After obtaining the abundance data of metabolic pathways, we used Student’s t-test to try to identify metabolic pathways with significant differences between groups.
Statistical analyses
Student’s t-test and analysis of variance were performed for the measurement data, and chi-square test analysis was performed for the count data by SPSS 23.0. Graphpad Prism v.8.0.2. was used for plotting.
Discussion
Previous studies have mainly investigated the composition and role of intestinal bacterial communities, but little is known about fungal communities, especially about the long-term effects of fungi on the formation of early intestinal microecological populations [
16]. However, intestinal fungi are a non-negligible part of the intestinal microbiota [
17]. It has been found that colonization of neonates by intestinal fungi may not only be associated with the development of invasive fungal disease, but may also be involved in the establishment and maturation of the human immune system [
12,
18‐
20], which may even be associated with the development of certain future diseases.
In this study, 34 VLBWI and 28 preterm infants with a birth weight of more than 1500 g who were hospitalized in Ningbo Women’s and Children’s Hospital from January 2021 to April 2022 were enlisted. The two groups differed in terms of gestational age, birth weight, preterm rupture of membranes, asphyxia, amniotic fluid contamination, mechanical ventilation, and central veins. Disease severity tends to be negatively correlated with gestational age, so gestational age can be associated with many covariates and potential factors of disease associated with preterm birth [
21], which lead to these differences in participant’s characteristics between the two groups. By collecting stool samples from these participants at 7 time points in the experimental group and 6 time points in the control group for sequencing, trends in their intestinal fungal composition and dynamics were described. The result of quality control indicated that the sample size of this study was sufficient for data analysis, and the sequencing depth met the requirements.
Willis et al. [
2] were able to detect Microeukaryotes (mainly of fungal origin) in the first meconium sample after birth, suggesting that the presence of intestinal fungi may retrace to the fetal period. In the present study, the fungal detectable rate was 50.82% on the first day for all stool samples and up to 64.71% for the VLBWI, which corroborate the above findings.
Experimental results shown that the fungal detectable rate was significantly higher in the experimental group (
χ2 = 13.59,
P = < 0.001). Microbial infestation in the uterine cavity may cause preterm labor [
22], such as intrauterine inflammation. And as a fungal causative agent, Candida intrauterine inflammation caused by
Candida is a rare but recognized cause of preterm birth [
22]. Therefore, we hypothesized that intrauterine fungal infection was one of the reasons for the higher detection rate of fungal infection in VLBWI.
The total fungal detectable rate in the experimental group increased from the first day (64.71%) to the third day (85.19%), followed by a smoothly decreasing trend, while in the control group, in contrast, gradually increased from a lower postnatal detectable rate (33.33%) to 62.5% on day 28. Candida detection rate showed a fluctuating trend within 2 months after birth, which peaked on the third day and troughs on the 21st day. Since there are few known studies using high-throughput sequencing technology for the analysis of neonatal intestinal fungi, therefore the literature available for comparison is insufficient. However, in the study [
23] by Nahid Kondori et al. on the fungal cultures of Swiss infants, it was found that the Candida detectable rate from 3 days of age maintains an increase to 18 months of age; this difference with our experimental results may be caused by the different detection method [
3], resulting in bias in the final results. More studies of the same experimental method are needed to compare with this experiment.
A comparison of alpha diversity between the two groups revealed a greater abundance of fungus in the VLBWI group. We speculate that the fact that VLBWI face more exposure to the environment, healthcare professionals, and the occurrence of more invasive operations (mechanical ventilation, central venous line placement, etc.) [
16,
24,
25] after birth may contribute to their having a more abundance of fungus. There were differences in alpha diversity among VLBWI groups at time point. At day 60, alpha diversity was significantly higher than ever before, suggesting a significant increase in fungal diversity in the stool at day 60. In the prospective study of parent-offspring fungal colonization by Kasper Schei et al. [
26], it was found that fungal alpha diversity in infants reached a minimum in 10-day samples and thereafter start increasing steadily from birth to 2 years of age. It was also found that species diversity gradually increased with infant age and diet [
25]. In addition, a study [
27] found that fungi were also present on the surface of the NICU environment and in breast milk. All of the above suggest that the differences in alpha diversity at different times may be related to the environment, the age itself, and the dietary intake in which the VLBWI was born.
In VLBWI, the combined abundance of Ascomycota (50.3%) and Basidiomycota (48.8%) was 99.1%, suggesting that the majority of intestinal fungus in the samples belonged to the above two phyla. This is generally consistent with the results of previous studies [
2,
25,
26,
28]. At the genus level, most of the fungi in the VLBWI group consisted of a few fungal genus, with the most dominant fungal genera being
Malassezia (21.2%) and
Candida (10.8%). In contrast to the second-highest abundance of
Candida found in this study, previous studies found that
Candida ranked first in abundance among the detected intestinal fungi [
2,
23,
25,
26].
Malassezia and
Candida were present at different samples at different points in time, so we consider they more likely to be intestinal colonizing fungus rather than transient population. A study [
2] found that the main composition of the fungal population shifted from
Malassezia to
Candida could be observed at 1 to 5 months, but the abundance of
Malassezia was consistently higher than
Candida during the period observed (postnatal to 2 months of age) in our study. A longer follow-up may be needed to verify previous observations.
In our study, in addition to the two fungal genera mentioned above, other dominant fungal genera are
Leucosporidium (8.45%),
Cystofilobasidium (8.11%),
Pseudogymnoascus (6.17%),
Mycosphaerella (3.92%),
Alternaria (3.74%),
Cutaneotrichosporon (3.60%),
Cladosporium (3.54%), and
Aspergillus (3.37%). This is not exactly the same as the previous studies by James et al. [
25]. Previous studies found that the composition and abundance of intestinal fungi varied greatly between individuals [
13,
25,
29], and symbiotic fungal populations are more variable than bacterial populations [
30]. Some of the fungal genera found in these studies on neonates were similar to adults [
3,
31,
32]. In the Human Microbiome Project, ITS2 and 18S rRNA sequencing results indicated that
Saccharomyces,
Malassezia,
Candida,
Cyberlindnera,
Penicillium,
Cladosporium,
Aspergillus,
Debaryomyces,
Pichia,
Clavispora, and
Galactomyces are the most common fungal genera in the human gut [
29]. Based on the above observation, we speculate that the fungal community in the gut during the earliest stages of life has an influence on the composition of the gut fungi in adults.
The most basic condition for fungal colonization in humans is the ability to survive at a temperature of 37 °C. Some of the detected genera of fungi, such as
Cladosporium, are not able to grow at human temperatures, and its ability to produce spores suggests that it may be caused by the inhalation of spores present in the environment by the host [
25]. Many fungi are usually present in soil and air and bound to plants as pathogens or saprophytes and enter the digestive tract by ingestion (as foodborne contaminants) or inhalation [
25]. In our study, some fungi such as Sonoraphlyctis, Anthracocystis, and Thecaphora occur only a few times in very low abundance, so we consider that they are of environmental origin and are not able to colonize the intestine.
The VLBWI group showed enhanced D-myo-inositol (1,4,5)-trisphosphate biosynthesis. This pathway mediates the biological response of a large number of hormones and neurotransmitters in target cells by regulating calcium release from intracellular stores and make roles in controlling calcium homeostasis, transferring calcium between intracellular stores, and regulating calcium entry across the plasma membrane [
33], while the expression of all other metabolic functions was diminished. Rozlyn et al. [
13] reported that at 1 year of age, fungal communities in the infant gut demonstrate an increased capacity for functions related to energy metabolism and a decrease in degradation pathways relative to communities at 3 months of age, but no more experiments were performed to verify this finding.
In conclusion, this study describes the composition and dynamics of intestinal fungi over time during the first 2 months of life in VLBWI and provides information for future studies on the intestinal fungus of children. However, there are some limitations in this study: firstly, we can not determine whether the detected fungus is intestinal colonization or temporary foreign contamination, which requires more study subjects and a longer observation period to verify. Since fungi are ubiquitous in the environment, and fungal DNA represents a relatively low percentage of fecal DNA content (especially in meconium), rigorous extraction, purification, and amplification techniques are required [
2], and it is difficult to differentiate environmental fungi from colonizing fungi from the sample. A prominent reason why the study of fungi lagging behind that of the microbiome is the lack of standardization of fungal bioassay methods [
29]. Secondly, we did not include full-term newborns as a separate control group, so we cannot compare VLBWI with healthy infant for further analysis. Finally, the observation period of this study was only 2 months after the birth of the study subjects which is too short to identify and summarize characteristics. In the future, we will select full-term infants as a control group and extend the observation period to explore the correlation and influence of the establishment, change, and maturation of intestinal fungal community with some clinical factors, such as gestational age, sex, birth weight, invasive manipulation, and food intake. We also tried to further analyze the relationship between intestinal fungi and disease occurrence in the context of clinical diseases.
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