Genital bacterial community profiles differ with racial background. Dominant Lactobacillus in the vaginal flora of pregnant women is more prevalent amongst Caucasian and Asian women, and anaerobic bacterial communities are more common amongst African American and Hispanic women (Ma et al., 2012; Romero et al., 2014; MacIntyre et al., 2015; Freitas et al., 2017; Fettweis et al., 2019). Studies of the vaginal microbiome also have found racial differences in the context of preterm birth (Callahan et al., 2017; Elovitz et al., 2019). The associations of intrauterine microbiota with racial background have been studied for placental samples of African, Caucasian and Chinese women (Zheng et al., 2015; Collado et al., 2016; Doyle et al., 2017; Gomez-Arango et al., 2017; Parnell et al., 2017; Zhou et al., 2018), but there are little data on the influence of racial background on other intra-amniotic bacterial populations. There have not been investigations of the genital microbiota of pregnant Australian Aboriginal women; this study aims at addressing this gap by providing data on their vaginal and placental microbiomes and comparing them to the genital microbiota of Non-aboriginal women from the same Pilbara region in Western Australia. Knowledge of bacterial taxa residing in the vagina and placenta in normal pregnancies provides a foundation for healthcare professionals to identify abnormal flora, enabling the possibility of targeted prevention of genital infections.

The genital microbiomes have an important role in maternal and neonatal health. At the beginning of pregnancy, microbial richness and diversity of bacterial populations are reduced. At the same time, in healthy pregnancies the prevalence of potential pathogens is reduced too (Goltsman et al., 2018). The taxonomic composition of the microbial community of the vagina remains stable during pregnancy with an increase of the microbial diversity before birth of a healthy infant at term (DiGiulio et al., 2015). The historical view that the amniotic cavity constitutes a sterile environment has been challenged by findings that the healthy maternofetal unit is colonized with microbes, and that this is a prerequisite for immune maturation as well as metabolic and hormonal homeostasis (Staude et al., 2018). However, a review of recent findings concluded that the evidence supporting the hypothesis of a uterine microbiome is extremely weak (Perez-Muñoz et al., 2017). In addition, a recent study with placental samples concluded that the human placenta does not have a microbiome (De Goffau et al., 2019). Nonetheless, there is evidence that the placenta, the amnion, and the fetus share large proportions of a common microbiome, and that the maternal microbiome drives the development of the fetal immune system (Collado et al., 2016; De Aguero et al., 2016) These data lead to the conclusion that the composition of the vaginal microbiota is tightly regulated during pregnancy and that the switch to the non-pregnant situation precedes and maybe even triggers birth (Romero et al., 2014; DiGiulio et al., 2015; Freitas et al., 2017).

Owing to the polymicrobial nature of the genital microbiota the definition of what is normal or abnormal genital tract microflora is very difficult; normal microflora is assumed to be present in the absence of disease (Lamont, 2015). Spontaneous preterm labor leading to preterm birth (PTB) is recognized as a syndrome caused by a number of pathological processes leading to activation of the common terminal pathway of parturition. Abundant evidence supports the view that local or systemic infection or inflammation is a major cause of early PTB (Lamont, 2015). Bacterial infections threaten pregnant women and the fetus by gaining access to gestational tissues, such as the decidua, placenta, and fetal membranes (Vinturache et al., 2016). There are correlations between the risk of PTB and genital bacterial populations in both the vaginal (Wen et al., 2014) and intrauterine (Mendz et al., 2013) microbiota. These correlations differ between racial groups; for example, a study observed significant associations of vaginal bacteria and PTB in African and Hispanic American women, but Caucasian Americans did not show significant associations between vaginal microbiota and birth outcome (Wen et al., 2014).

It is estimated that 25%-40% of PTB are attributable to intrauterine infection (Goldenberg et al., 2008), and the relationship between the presence of pathogens and spontaneous preterm birth is complex. These findings underline the need to consider women's race when evaluating the links of specific genital bacteria with preterm birth. Similarly, early-onset neonatal sepsis (EOS) is associated with pathogenic microorganism acquired from the mother during pregnancy or at birth (Schrag et al., 2016). Firm connections have been established between several taxa such as E. coli and Streptococcus B and EOS, but the association of other taxa such as Enterococcus and Haemophilus with this type of sepsis appears to depend on human populations (Simonsen et al., 2014; Singh et al., 2019).

The rate of preterm birth (PTB) of Aboriginal Australian mothers is 14% and of Non-aboriginal mothers is 8% (Australian Institute of Health and Welfare, 2016), the former is higher than those of many underdeveloped countries. Spontaneous preterm birth was the most commonly identified cause of perinatal death in infants of Aboriginal and/or Torres Strait Islander (ATSI) mothers: 26% compared with 19% for Non-indigenous mothers (Australian Institute of Health and Welfare, 2018). Historically, infants of ATSI mothers have been over-represented in EOS data, although recent studies suggest that this rate may be decreasing (Braye et al., 2019). The current study investigated the presence of bacterial taxa associated with clinical signs of infection and PTB or EOS in the participant Australian Aboriginal women with the aim of providing data that would serve to devise strategies to reduce adverse pregnancy outcomes in this population.

Investigations of the genital microbiomes of pregnant Aboriginal Australians were needed to understand their characteristics and identify taxa whose presence were associated with infection in this population. The study compared the genital microbiomes at the time of delivery of pregnant Aboriginal and Non-aboriginal women living in the Pilbara region employing high throughput 16S-rRNA sequencing. Some differences in demographic and clinical characteristics between both racial groups were statistically significant; however, the small size of each group in the cohort and the values of these differences made them in most cases not relevant to understanding the microbiological data. Nonetheless, the differences in infection and PTB rates between both groups of women helped to interpret the abundances of some taxa (Table 1).

The relative abundances of various Phyla showed differences between vaginal and placental microbiomes and between the microbiomes of Aboriginal and Non-aboriginal women (Figure 1), opening up the possibility for significant differences at lower taxonomic levels. The predominance of Firmicutes in the vagina was in agreement with the results of previous studies including Black, Asian, Caucasian and Hispanic American women (Aagaard et al., 2012; MacIntyre et al., 2015). The predominance of Proteobacteria in the placental microbiome agreed with results for Caucasian European (Collado et al., 2016) and Caucasian and Black American women (Parnell et al., 2017), but not with those of Chinese women in whom the predominant placental Phylum was Firmicutes (Zheng et al., 2015), indicating a dependence of placental bacterial populations on racial background.

The overall composition of the microbiomes of women in both racial groups showed significant differences in distLM calculations. However, comparison of the microbiomes of Aboriginal and Non-aboriginal women yielded similar OTU composition, species richness, evenness and Shannon diversity index for the vaginal and placental microbiomes (Table 2). A prospective study by Hyman et al. (2014), found that the vaginal microbiome species diversity was greatest among African American (n = 8) followed by Hispanic (n = 13) women compared with that of Asian (n = 16) and Caucasian (n = 40) women; in addition, statistically significant differences were found between Asian and Caucasian women relative to African American or Hispanic women. This greater diversity in the vaginal microbiome of women of African descent was confirmed in an investigation comprising 300 pregnant women (Serrano et al., 2019). A study of the vaginal microbiome in the third trimester by Fettweis et al. (2019) that included women of African ancestry (71 delivered at term; 35 preterm) and European ancestry (13 delivered at term; 6 preterm) observed greater species diversity in women who delivered preterm. In contrast, Stout et al. (2017) reported that the vaginal microbiome of 24 non-African American women had lower species diversity than that of 53 African American women. It remains to be determined whether the absence of significant differences observed in the present work between Aboriginal and Non-aboriginal women will persist in analyses of larger numbers of samples. The presence of infection resulted in significant changes in evenness and diversity (Table 2) in agreement with the results of a study that observed greater species diversity in women who delivered preterm and included women of African ancestry (n = 106) and European ancestry (n = 19) (Fettweis et al., 2019).

Comparison of the vaginal and placental microbiomes indicated that they clustered separately in nMDS plots (Figure 2), and distLM calculations showed a significant difference between both genital locations in agreement with the results of previous studies (Aagaard et al., 2014). In healthy pregnant women, midpoint vaginal microbiomes are stable in time and are less rich and diverse than in healthy non-pregnant women (Aagaard et al., 2012; Walther-António et al., 2014). The numbers of OTU, richness and evenness, but not the Shannon diversity, were different between vaginal and placental samples (Table 2). Analyses by location and racial background showed nMDS clustering of the placental microbiomes in these populations (Figure 2); but no significant differences were found in PERMDISP calculations. Nonetheless, the differences in OTU composition, richness and evenness remained after stratification by racial background (Table 2). The results of this work suggested that the microbiome in the normal placenta has significant lower abundance, richness and diversity than in the vagina in agreement with previous investigations (Aagaard et al., 2014; Taddei et al., 2018; Seferovic et al., 2019).

Differences in relative abundances of specific taxa with frequencies > 15% were identified between both genital microbiomes (Table 4). More taxa were enriched in the vagina than in the placenta reflecting the greater diversity and abundances found in the former genital location; exceptions were Enterococcus and Flavobacterium. The higher relative abundance of Enterococcus found in placental samples agreed with the results of a study comparing placental microbiomes of Chinese women who gave birth to normal or low weight infants (Zheng et al., 2015); also, in a study of 1,832 placental samples from Chinese women with healthy pregnancies, Enterococcus was recovered by cultivation in 24% of the placental samples second in abundance only to E. coli. (Zhou et al., 2018). Viable bacteria have been detected in 15 normal placentas from pregnancies delivered at term by Caesarean section with Enterobacter and Escherichia/Shigella as the most abundant taxa and smaller abundances of Lactobacillus, Staphylococcus and Streptococus taxa (Collado et al., 2016). In situ hybridization of the placentas of 52 women in term and preterm births without evidence of infection and independent of delivery mode yielded relative abundances greater than 5% for Lactobacillus, Prevotella, Staphylococcus, and Streptococcus taxa. (Seferovic et al., 2019).

Flavobacterium has been found as a common contaminant in reagents, but after applying the quality control with the reagents employed in the sequencing and in the analyses described in the Methods section, this taxon appeared at significant relative abundances in a number of placental microbiomes of both Aboriginal and Non-aboriginal women, and at over twice the abundance in the latter group of women. This higher relative abundance identified in the participating women, concurred with the results of a study of the endometrial microbiome of women undergoing assisted reproductive technology treatments (Franasiak et al., 2016).

The microbiomes of Aboriginal and Non-aboriginal women showed differences in relative abundances of taxa with frequencies > 15% (Table 5). The results were consistent with those of genital microbiome studies of healthy women from various racial backgrounds (Romero et al., 2014; MacIntyre et al., 2015). An unusually high relative abundance of Pseudomonas was present in genital microbiomes of Aboriginal women, three times more abundant than in the microbiomes of Non-aboriginal women. Pseudomonas has been detected in placental samples of overweight and obese Caucasian Australian women (Gomez-Arango et al., 2017); also, it has been identified at high relative abundance in samples from the endometrium of the healthy control group of a study of Chinese women with endometrial polyps (Fang et al., 2016), endometrial and Fallopian tube samples of a large cohort of reproductive age Chinese women operated for conditions not known to involve infection (Chen et al., 2017), and endometrial samples of women with chronic endometritis (Moreno and Franasiak, 2017). Considering that the distLM logistic regression confirmed that significant differences in microbiomes were due to racial background and genital location (Table 3), these results suggested that colonization with Pseudomonas is characteristic of the Aboriginal population of this study.

Comparison of the vaginal microbiomes of Aboriginal and Non-aboriginal women yielded significant differences in the relative abundances of a number of taxa (Table 6). In the vagina of Non-aboriginal women, Corynebacterium was found only in negligible or very small concentrations; also, these women had negligible or very low Lactobacillus spp. abundance. The highest relative abundances of Corynebacterium found in Aboriginal women with no clinical signs of infection suggested that the taxa present were non-pathogenic species. These species of corynebacteria produce organic acids and stimulate the production of anti-inflammatory cytokines that enhance the capacity of the vaginal mucosa to protect against opportunistic and pathogenic microorganisms (Gladysheva and Cherkasov, 2018); Corynebacterium may play a similar role in the vaginal microbiome of Aboriginal women.

Vaginal microbiomes abundant in Atopobium and low in Lactobacillus can reach up to 40% amongst Black and Hispanic women (Ma et al., 2012), and Atopobium and Megasphaera have been associated with vaginal infections (Hocevar et al., 2019). In the vaginal microbiota of Aboriginal women Atopobium was commonly found in microbiomes with low Lactobacillus abundance but it was not associated with clinical signs of disease. Megaspahaera also was not found in women with clinical signs of infection.

Differences in the community structures (CST) have been identified in the vaginal microbiomes of non-pregnant (Ravel et al., 2011) and of pregnant (Freitas et al., 2018) healthy women from various racial backgrounds. CST III is found at a greater frequency in women with Asian, Black and Hispanic racial backgrounds, and CST I is found in Asian and Caucasian women Ravel et al., 2011; Gajer et al., 2012; Freitas et al., 2018). In terms of population structures, the predominant species in the microbiomes of Aboriginal women was L. iners which is associated with vaginal community state type CST III, and in Non-aboriginal women the most abundant taxon was identified as L. crispatus which is associated with CST I. The study by Hyman et al. (2014) concluded that L crispatus had a higher relative abundance in the vaginal microbiome of Caucasian participants, and L iners in African-American and Hispanic participants, and both species had similar relative abundances in Asian women. An investigation of the vaginal microbiome in the third trimester found higher relative abundances of L. iners in women of African descent, and of L. crispatus in women of European descent (Fettweis et al., 2019). Serrano et al. (2019) studied a matched cohort of women of African ancestry (n = 49) and European ancestry (n = 41) who delivered at term and found in the third trimester a predominance of L. iners in the former group and of L. crispatus in the latter. In contrast, Stout et al. (2017) determined that L. iners was significantly more abundant than L. crispatus in a study comprising both African Americans and non-African Americans. Gardnerella and Atopobium are dominant genera in the vaginal microbiome of CST IV, and present at lower abundances in the other CST (Freitas et al., 2018). In the study of women with predominantly African ancestry by Fettweis et al. (2019), the next most abundant taxa after Lactobacillus were Gardnerella, BVAB1, Atopobium and Prevotella cluster 2. No significant differences between the abundance of Atopobium and Prevotella were observed for women of African or European ancestry, but Gardenerella was more abundant in women of African ancestry. The work of Serrano et al. (2019) showed a linkage between Gardnerella clade 3 and women of African ancestry, and no Gardnerella showed a significant association with women European ancestry. On the other hand, Stout et al. (2017) found no difference in Gardnerella abundance between were African Americans and non-African Americans. In Aboriginal women the most abundant vaginal taxon was L. iners followed by L. crispatus, Gardnerella, Pseudomonas, and Enterococcus. Considering that the genital microbiomes of these women were dominated by L. iners, and the abundance in healthy Aboriginal women of other taxa such as Corynebacterium, Atopobium and Megaspahera suggested in the context of the reduced diversity of the vaginal microbiomes of pregnant women, that there were some similarities with the vaginal microbiome of women of African descent, but overall the Aboriginal pregnant women showed an uncommon vaginal community structure.

Significant differences between the placental microbiomes of Aboriginal and Non-aboriginal women were the result of LEfSe analyses (Table 7). In addition to L. crispatus. The placental microbiomes of Non-aboriginal women were enriched with Streptococcus and Veillonella. The former taxon has been found commonly in placental samples of women of various racial backgrounds: Chinese (Zheng et al., 2015; Zhou et al., 2018), Caucasian European (Collado et al., 2016), Caucasian and African American (Parnell et al., 2017), Caucasian Australian (Gomez-Arango et al., 2017), and African Malawian women (Doyle et al., 2017), suggesting the colonization of a broad range of racial backgrounds. Streptococcus has been identified at high relative abundance also at the endometrium: in samples of Chinese women with endometrium polyps (Fang et al., 2016), of predominantly Caucasian American (Tao et al., 2017) and Caucasian Italian (Moreno and Simon, 2018) women undergoing IVF cycles. Similarly, Veillonella has been identified in the placenta of women from various racial backgrounds (Aagaard et al., 2014; Zheng et al., 2015; Doyle et al., 2017; Gomez-Arango et al., 2017; Moreno and Simon, 2019).

In Aboriginal women in addition to L. iners, the placental microbiomes were enriched with Paracoccus and Pseudomonas (Table 7). The former taxon has been identified in the placenta of women from only few racial backgrounds and at low relative abundances (Zheng et al., 2015; Theis et al., 2019), and has been considered a contaminant by others (Perez-Muñoz et al., 2017; Leiby et al., 2018). In the present study after elimination of OTU with an abundance <1% in the negative controls, Paracoccus was present at relative abundances > 1% and with a frequency higher than 15%, as were all the other taxa presented in tables.

Comparisons of the effects of infection on the genital microbiomes of Aboriginal women did not yield any overall significant differences; likewise, for the vaginal microbiomes of Non-aboriginal women. However, at the taxon level, LEfSe analyses showed important differences in the presence of infection (Table 8). Potential effects of colonization by Atopobium, Corynebacterium, Gardnerella, and Megasphaera and have been discussed. The presence of Acinetobacter has been associated with PTB and neonatal infections in American women (He et al., 2013), spontaneous preterm and chorioamnionitis in an Australian Caucasian woman (Quinlivan et al., 2014), and vaginal infection in Indian women (Gopalan et al., 2017). Genital infections of Acinetobacter during pregnancy are associated with adverse outcomes in various racial backgrounds.

Post-delivery increases in the abundance of Anaerococcus and Prevotella with disturbance in the vaginal microbiota unrelated to gestational age at delivery were observed in a population of mostly Caucasian American women (DiGiulio et al., 2015). In contrast, significant Anaerococus vaginal colonization was detected in studies with mostly African American with normal pregnancies (Romero et al., 2014) and of Chinese women with uncomplicated pregnancies (Chen et al., 2019). Anaerococcus appeared to colonize the female genital tract of women of various racial backgrounds albeit with different pregnancy outcomes. In a study with mostly African American women, Prevotella was found with higher abundance in the vagina of women who delivered preterm (Subramaniam et al., 2016). Several Prevotella spp. were associated with both term and preterm in predominantly Caucasian American women. Prevotella amnii and P. tannerae had greater prevalence in the term cohort, whereas P. timonensis, P. bivia, P. corporis, and P. bucalis were more prevalent in the preterm group (Freitas et al., 2018). Prevoltella was strongly associated with vaginal infection in a study of mostly Caucasian Brazilian women (Dobbler et al., 2019). These results suggest an overall association of Prevotella with vaginal infection in different racial backgrounds.

Amongst the 23 Aboriginal women in the study, 12 presented clinical signs of infection. The relative abundance of Pseudomonas, E. coli/Shigella and the prevalence of GBS in Aboriginal women with sign of infection is given in Table 9; included in the table are the metadata for PTB and sepsis. In the placental microbiome of 3 of these women the dominant taxon was Pseudomonas with relative abundances > 93%; this taxon was dominant also in the vaginal microbiome (99% relative abundance) of one of these women. Pseudomonas was identified at low relative abundance in another Aboriginal woman. This taxon has been found in the placenta of women from different racial backgrounds, e.g., Chinese women who delivered of low birth weight infants (Zheng et al., 2015), Chinese women with gestational diabetes mellitus (Zheng et al., 2017), overweight and obese Caucasian Australian women (Gomez-Arango et al., 2017), and African American women who delivered at term (Theis et al., 2019). Commonly, intrauterine presence of Pseudomonas has been associated with infection but not in all instances.

Many taxa have been associated with infection during pregnancy (DiGiulio et al., 2015; Freitas et al., 2018). Table 10 shows the relative abundance of 12 of these taxa in eight of the Aboriginal women who had adverse pregnancy outcomes, either PTB and/or neonatal sepsis. In women A6005, A6007, and A6009 there is a correlation between these outcomes and the presence of Pseudomonas at high relative abundance in the absence of significant abundance of other potential pathogens. The presence of several potential pathogens at low and intermediate abundances in the genital microbiomes of woman A6003 would account for her infection. The Group B Streptococcus positive status of women A6004, A6006, and A6010 may explain the presence of infection in these participants. In addition, the low abundance of Pseudomonas, Gardnerella and other potentially pathogenic taxa in woman A6006, and the intermediate abundance of E. coli and Paracoccus in woman A6010 may have contributed to their respective infections. Participant A6021 did not show abundance of E. coli, GBS or other taxa that would explain satisfactorily that she delivered preterm a child with sepsis. However, Acinetobacter has been associated with preterm birth and was detected at relatively high abundance in the placenta of this woman.

Early-onset neonatal sepsis almost always is associated with pathogenic microorganisms acquired from the mother during pregnancy or at birth (Klinger et al., 2009); they are commonly GBS and E. coli (Schrag et al., 2016), but a significant proportion of EOS is due to other infections. Pseudomonas has been associated with PTB and late-onset sepsis (Harnaen et al., 2015), but in Australia and New Zealand also with EOS (Braye et al., 2019; Singh et al., 2019).

The abundance of Pseudomonas and E. coli, and the GBS status in the genital microbiomes of Aboriginal and Non-aboriginal women in this study are given in Supplementary Table 2, together with clinical data on PTB and sepsis. These data suggested the association of Pseudomonas with PTB and/or sepsis, particularly in Aboriginal women, also in the absence of other potential infections.

The study investigated the genital microbiomes at delivery in Aboriginal women of the Pilbara region of Western Australia by comparing them to Non-aboriginal women in the same region. The study is the first report on the genital microbiomes of pregnant Australian Aboriginal women using culture independent techniques. The results support previous findings that the most abundant vaginal Phyla during pregnancy are Firmicutes, Proteobacteria, and Actinobacteria, and that the vaginal microbiome of normal pregnancy commonly consolidates into communities dominated by Lactobacillus. At a global level distLM analyses yielded differences between the genital microbiomes of both groups of women, and at the taxon level significant differences were found between the vaginal or placental microbiomes of Aboriginal and Non-aboriginal women. Pseudomonas appeared to be associated with infection and adverse pregnancy outcomes, particularly in the group of Aboriginal women.

The study focused on a rural population with a high rate of spontaneous preterm birth and demonstrated differences in the microbiomes at the taxon level in women with infection. The findings served to enhance the current understanding of microbiota associated with health and disease in Aboriginal and Non-Aboriginal women, and will help the development of targeted prevention of chorioamnionitis. Further studies with larger cohorts will serve to verify the results of this work and the routes of invasion of pathogenic bacteria into the intra-uterine environment that would enable earlier detection of subclinical infection, and allow for further reduction of premature deliveries for both Aboriginal and Non-aboriginal women.

The datasets generated for this study can be found in the European Nucleotide Archive under the accession number PRJEB39698.

The studies involving human participants were reviewed and approved by WA Country Health Service Human Research Ethics Committee (HREC #2015/40) and Western Australian Aboriginal Health Ethics Committee (HREC #686). The patients/participants provided their written informed consent to participate in this study.

ND, JQ, and GM contributed to the design of the study. ND and JQ acquired the data. ND, NC-R, and GM helped with the analyses and interpretation of the data. ND, JQ, NC-R, and GM wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.