Structure of Vaginal Microbiome Community After Perineal Disinfection and Its Effects on Neonatal Oral Microbiome

Background: Vaginal microbiota is not only an important source of bacterial colonization for neonates, but also plays a key role in maternal and neonatal health. In China, povidone iodine is used to disinfect vaginal during delivery. To date there has been no comprehensive study to investigate the vaginal microbiome composition after disinfection. Results: In this study, 27 women were recruited from Bao an Maternal and Child Health Hospital (Shenzhen, China). Vaginal samples before and after delivery were collected. Neonatal oral samples were also collected right after birth. Bacterial compositions of these study subjects were investigated using 16S rRNA sequencing of V3-V4 hyper-regions based on Hiseq 2500 platform. The results showed that vaginal microbiome during pregnancy were dominated by Lactobacillus spp. The identied microbiomes were separated into three community state types (CSTs), and a new CST (dominated by L. helveticus) was observed in this study. After disinfection, the relative abundance of Lactobacillus decreased and alpha diversity increased signicantly. Moreover, most CST III and CST VI during pregnancy, both them dominated by Lactobacillus, shifted to CST IV in vaginal samples after disinfection. Additionally, the similar change pattern was observed in neonatal oral microbiome, and they overlapped with vaginal samples after disinfection in NMDS analysis. Conclusions: Perineal disinfection resulted in the decrease of genus Lactobacillus and increase of alpha diversity both in maternal vaginal microbiome and neonatal oral microbiome. In further, it is vital to understand the inuence on maternal and neonatal health of vaginal microbiome community structure change after disinfection.

reduces richness and diversity of vaginal microbiome. Compared to more attention focused on the vaginal microbiome during pregnancy, only a few studies investigated postpartum vaginal microbiome (Freitas et al., 2017;DiGiulio et al., 2015). They showed that vaginal communities with less Lactobacillus species and characterized by more rich and diverse during the post-partum period. However, all the vaginal samples included in the postpartum period were a few weeks after delivery, a comprehensive characterization of vaginal microbiome signature right after delivery has not yet been undertaken.
In China, almost all hospitals in China use povidone iodine to disinfect the vulva before transvaginal examination during maternal delivery according to the latest edition of the medical education book "Obstetrics and Gynecology" (Xie and Guo, 2013). Yet relatively little is known about the community structure of vaginal microbiome after perineal disinfection and its affection on maternal and neonatal health. In this study, we aimed to characterize the composition of the vaginal microbiome after disinfection and compared the microbial pro les to those of pregnant period.
The neonate has been exposed to the maternal vaginal microbial ecosystem during the delivery process. . As an important source of pioneer bacteria for neonatal oral microbiome, it is important to discern the relative potential contribution of the maternal vaginal community to the neonate oral microbiome. Our previous study also demonstrated that relative abundance of genus Lactobacillus decreased in neonatal oral microbiome after maternal perineal disinfection (Li et al., 2019). It is therefore crucial to understand how the vaginal microbial community restructured after perineal disinfection and its association with neonatal oral microbiome.

Maternal and neonatal clinical data
Demographic and clinical characteristics of the women and newborns were provided in Table 1 The average birth weight of 27 newborns was 3205.9 g (range 2600-3850 g), including 15 boys and 12 girls.
Community structure of vaginal and oral microbiome To compare the overall vaginal community structures before disinfection and after disinfection, nonmetric multidimensional scaling (NMDS) analysis was implemented on the bacterial abundances. Figure 1A revealed that vaginal samples before disinfection (group BD) separated well from the subjects after disinfection (group AD, p < 0.01, PERMANOVA analysis), whereas vaginal samples after disinfection overlapped with neonatal oral samples (group NO).
Moreover, the vaginal microbiota after disinfection and neonatal oral microbiota was associated with a higher observed species index, evenness index and alpha diversity ( Figure 1B) Figure 1B (a)). Accompanied with signi cantly increased mean pielou index value of group AD (0.55 ± 0.16, p < 0.01) and NO (0.56 ± 0.24, p < 0.01) versus 0.25 ± 0.22 of group BD ( Figure 1B (b)). Notably, the mean Shannon index value was 0.92 ± 1.06 of BD group, signi cantly lower than that of group AD (4.22 ± 1.50, p < 0.01) and NO (4.19 ± 2.22, p < 0.01) ( Figure 1B (c)). Consistent with these observations, indices of alpha-diversity and richness were the smallest in samples obtained during pregnancy, with a signi cant increase in diversity detected in vaginal samples after disinfection and in neonatal oral samples. Further, the weighted UniFrac value of group BD was 0.35 ± 0.29, signi cant lower than group AD (0.71 ± 0.29, p < 0.01) and NO (0.69 ± 0.27, p < 0.01), which indicated that vaginal microbial communities were more similar within each other in group BD than AD ( Figure 1B (d)).
Vaginal and oral microbiome pro ling Microbiome of study participants was characterized using high-throughput sequencing of the 16S rRNA high-variable regions. A total of 3,788,402 reads were included in the analysis. The average sequence read count was 46,770 per sample, with a median of 46,158 (range 13,434-71,182), and the mean and median read lengths were 420 and 423 bp, respectively. The relative abundance of each participant at phylum and genus levels was showed in Figure 2. It showed that the top 10 phyla were Acidobacteria, Actinobacteria, Bacteroidetes, Chloro exi, Cyanobacteria, Firmicutes, OD1, Planctomycetes, Proteobacteria and Tenericutes ( Figure 2A). Meanwhile, the top 10 genera were Atopobium, Enterococcus, Gardnerella, Lactobacillus, Prevotella, Pseudomonas, Ralstonia, Staphylococcus, Streptococcus and Thiobacillus ( Figure 2B).

Different genus between vaginal and oral microbiome
Next, the STAMP tool was used to analyze bacterial communities in vaginal samples and to detect potential signi cant differences in relative abundances of genus. Figure 4A included a list of genera that signi cantly different between vaginal samples during pregnancy and neonatal oral samples. Among them, genera Acinetobacter, Burkholderia, Delftia, Mesorhizobium, Paracoccus, Ralstonia, Reyranella, Salinispora and Shewanella increased signi cantly in the neonatal microbiome, whereas genus Lactobacillus decreased signi cantly. In addition, the greatest differences in genus between vaginal samples before disinfection and after disinfection were presented in Figure 4B. Compared to vaginal samples during pregnancy, the relative abundance of genera Acinetobacter, Bradyrhizobium, Burkholderia, Comamonas, Delftia, Mesorhizobium, Mycobacterium, Ralstonia, Reyranella and Salinispora were higher, while genus Lactobacillus was dramatically lower in vaginal microbiome after disinfection. Notably, there was no difference between vaginal samples after disinfection and neonatal oral samples. Collectively, these observations suggested that the microbial composition of the vaginal samples signi cantly differed between pregnancy and after disinfection according to the relative abundance of sequences.

Community state types analysis and its changes after delivery
Categorizing microbiome pro les based on the taxon with the largest proportion of reads, and hierarchical clustering analysis of bacterial species from the pregnant vaginal microbiome pro les revealed 3 major community state types (CSTs), which showed in Figure 5A. Among all samples, 8 samples were dominated by species L. iners (CST III). Six samples were assigned to CST IV, which were dominated by genus Gardnerella, and also typi ed by higher proportions of Aerococcus, Atopobium, Bi dobacterium, Corynebacterium, Dialister, Finegoldia, Megasphaera, Mobiluncus, Peptoniphilus, Prevotella, Ralstonia, Staphylococcus, Streptococcus and Sneathia than other CSTs. The rest 13 samples were dominated by species L. helveticus or L. delbrueckii, which were clustered into a new observed community type and names as CST VI in this study.

Discussion
In this study, the composition of the vaginal microbiota of women after disinfection was determined and compared to the pro les before disinfection. Increased microbial richness and diversity was observed in the postpartum samples, which was consistent with previous studies (Huang et al., 2015;MacIntyre et al., 2015). In addition, differences in the microbiota between the two phases regarding bacterial abundance and prevalence were also identi ed. The results indicated that the composition of the vaginal microbiome was dynamically restructured after disinfection, where phyla Firmicutes decreased and Bacteroidetes, Proteobacteria increased signi cantly. Additionally, in concordance with previous observations ( , genus Lactobacillus was the predominate bacterium in most of the samples during pregnancy, with Atopobium, Gardnerella, Prevotella, and Streptococcus presented in a low proportion. Whereas after disinfection, as the most common genus in phylum Firmicutes, Lactobacillus decreased signi cantly, with genera Acinetobacter, Bradyrhizobium, Burkholderia, Comamonas, Delftia, Mesorhizobium, Mycobacterium, Ralstonia, Reyranella and Salinispora increased signi cantly. It had been reported that Lactobacillus was the most prevalent and dominant bacterium in female vaginal, which appeared to ensure normal vaginal microbiota and Dominance of CST IV was observed in neonatal sample and postpartum vaginal samples (n=20 and n=17 in group NO and AD respectively) ( Figure 5B). Most subjects of group BD belonged to CST III and CST VI (15/21) switched to CST IV of group NO. Similarly, they (11/21) shifted towards CST IV in vaginal microbiome after disinfection. An interesting change pattern was observed in sample S6, it changed from CST IV to CST VI in the neonatal oral microbiome.  In this study, analysis of the microbiota community using hierarchical cluster analysis showed that the vaginal microbiome clustered into 3 major groups. CST III and CST IV were previously described in North American ( In the further study, large cohort studies should be taken into account to con rm this observation. Beyond this, V3-V4 region of 16S rRNA was used to pro le the vaginal microbiome community in this study, which showed differences with a previous study based on cpn60 gene. To cull out the discrepancy caused by the ampli ed region, lower taxonomic levels should be investigated with whole genome sequencing in the next study. Additionly, the results showed that most samples during pregnancy belonged to CST III and CST VI, dominated with Lactobacillus spp. While after disinfection, the majority of CST III and CST VI during pregnancy switched to CST IV, in concordance with previous studies ( . The mechanism of passage through the vaginal canal to neonatal might be largely impacted by the disinfecting conduction. It would be helpful to understand the process that establishes the rst microbial colonizers of newborns, which was essential for gaining a comprehensive understanding of neonatal development.
The absence of longitudinal data set of maternal and neonatal health revealed some insights that need to be determined in the further study. First, during pregnancy, some vaginal microbiota pro les belonged to CST III, some belonged to CST IV, and some were clustered into CST VI, their longitude impact on maternal and neonatal health should be followed in the next study. Second, after disinfection, some vaginal microbiome communities were still dominated by Lactobacillus, where others with a small proportion of Lactobacillus, and similar phenomena was observed in neonatal oral microbiome, but its effects on maternal and neonatal health were not clear. Third, some pregnant women might need not to be disinfected at labor, and some neonates might need to be supplemented with probiotics during growth and developmental process, the clinical operations should be changed according to the result of large cohort study.

Conslusions
In summary, it was observed in this study that perineal disinfection restructured the vaginal microbiome and affected the colonization of neonatal oral microbiome. With the relative abundance of Lactobacillus decreased and some opportunistic bacteria increased signi cantly. These observations will be helpful to uncover the impact of perineal disinfection on maternal and neonatal growth and developmental health.

Study Subjects
Twenty-seven healthy and reproductive-ages women with gestational age (GA) > 37 weeks were recruited in this study. They were asymptomatic and showed no clinical signs of vaginal disease upon examination by obstetrician with evidence of vaginal discharge and amine or shy odor. And they also were with an uncomplicated singleton pregnancy, and without medical problems or adverse outcomes during pregnancy, without known fetal anomalies or complications, without antibiotics or other antimicrobial therapy during pregnancy. The study received ethics approval from Bao an Maternal and Child Health Hospital (Shenzhen, China). Written informed consents were obtained from all participants, and parents/guardians of the recruited newborns. Women who were incapable of understanding the informed consent or assent forms, or incarcerated were not recruited in this study. After obtaining written informed consent, demographic and clinical characteristics were collected from all participants via interview and by reviewing medical charts, including gestational age, height, weight, blood pressure, body mass index, ethnicity, age, and so on. Relevant clinical information was also obtained from neonates at birth.
Vaginal samples before delivery were collected at the rst examination of hospital admissions of all participants (group BD, before disinfection). And vaginal samples after disinfection were collected before mothers left delivery room with incision stitched (group AD, after disinfection). Where sterile swabs were placed carefully on the vaginal sidewall about halfway between the introitus and the cervix, follow the instructions reported previously , pressed rmly into the sidewall to a depth of roughly the diameter of the swab, rolled dorsally-ventrally back and forth four times to coat the swab. Neonatal oral samples were taken as soon as the newborns delivered and before feeding by carefully swabbing the oral mucosa (group NO, neonatal oral), which reported in the previous study (Li et al., 2019).
Three sterile swabs were obtained for every sample by trained nurses to avoid insu cient DNA concentration. All samples were stored at 4°C and transferred to -80°C storage within 30 minutes after collection until DNA extraction. Sequencing reads were assigned to each sample, and ltered reads containing ambiguous bases or mismatches in the primer regions using custom perl scripts. Then the 16S rRNA gene sequences generated were analyzed using the bioinformatics software package QIIME2 (version 2019.4) (Bolyen et al., 2019). Paired-end reads were rstly denoised via DADA2 (Callahan et al., 2016) offered by QIIME2 with command "qiime dada2 denoise-paired", to merge paired-end reads, quality ltering and to exclude chimeric and phiX sequences. Further sequences were classi ed against Greengenes (13_8 revision) database (DeSantis et al., 2006) at the species level using command "qiime feature-classi er classifysklearn". Meanwhile, an array of alpha-and beta-diversity measures was generated using command "qiime phylogeny align-to-tree-mafft-fasttree" and "qiime diversity core-metrics-phylogenetic" at a sampling depth of 1000. Where alpha diversity was calculated by Shannon's diversity index, observed OTUs (operational taxonomic units) numbers, Pielou's measure of species evenness. And beta diversity was calculated by weighted UniFrac distance.
Moreover, sequences were separately analyzed using Mothur software against Greegenes (13_8 revision) database as described previously (Schloss et al., 2009;Li et al., 2019). The sequences were clustered into OTUs based on the similarity threshold of 0.97.

Statistical analysis
The continuous demographic variables were presented as the mean ± standard deviation (SD), including alpha (Shannon diversity and observed OTUs number estimated species richness) and beta diversity (weighted UniFrac matrices). And the categorical characteristics were reported as numbers (percentages, %). All comparisons of this study were performed in R software at 0.05 level of signi cance using chisquare and t-tests for categorical and continuous variables, respectively. Diversity of microbiome pro les were also analyzed based on a nonmetric multidimensional scaling (NMDS) analysis, which was performed on R software using the metaMDS function in the vegan package, and accompanied with permuational multivariate analysis of variance (PERMANOVA, 999 permutations).
To determine statistical differences between the vaginal microbiome throughout pregnancy and after disinfection, the Statistical Analysis of Metagenomic Pro les (STAMP) software package (Parks and Beiko, 2010) was used. P values were calculated using Welch's t-test with Bonferroni correction. A corrected p-value < 0.05 was considered signi cant.
Assignments of vaginal microbial pro les to community state types (CSTs) were according to community compositions, and the clustering of communities based on vaginal microbial pro les was performed using mcquitty linkage hierarchical clustering analysis with the R package, essentially as previously reported (Ravel et

Declarations
Ethics approval and consent to participate This study was approved by the Ethics Committee of Bao an Maternal and Child Health Hospital (Shenzhen, No. QKTLL-2017-05-04) and complied with the Helsinki Declaration. Informed consents were obtained from all participants included in this study. All experiments were performed in accordance with the approved guidelines.

Consent for publication
Not applicable.

Availability of data and materials
The 16S rRNA sequence data generated and analysed during the current study is in the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA BioProject ID PRJNA596821).

Competing interests
The authors declare that they have no competing interests.