New insight into the analysis of amniotic fluid microflora using 16S rRNA gene sequencing

Introduction: Intra-uterine infection is a major cause of spontaneous pre-term birth before 30 weeks of gestation. In addition, intra-amniotic inflammation is present in approximately 80 % of patients with acute cervical insufficiency. The microbiome is known to cause intra-amniotic infection causing pre-term birth. Recently, surveys of the microbial communities in the vagina or in amniotic fluid using sequencing of the 16s rRNA gene have been carried out. Here, we reported the microbiome compositions in mid-trimester amniotic fluids in two pregnant women with cervical insufficiency identified using 16S rRNA sequencing. Case presentation: A 35-year-old primipara and a 37-year-old nullipara were admitted to Hallym University Medical Center, Korea. They were negative for nitrazine and Actim PROM (Medix Biochemica) tests, and their amniotic membranes had prolapsed beyond the external os. A physical examination indicated that cerclage using a uniconcave balloon should be carried out, and amnioreduction was performed. Thereafter, they delivered at very early pre-term birth of below 24 weeks and the birth outcomes were pre-natal death. Analysis of mid-trimester amniotic fluids in these two pregnant women with cervical insufficiency identified Sneathia sanguinegens and Fusobacterium nucleatum. These bacteria were not identified using vaginal or amniotic cultures. Conclusion: These results suggest that 16S rRNA gene sequencing can be used to identify the predominant microbiome causing pre-term birth in pregnant women with cervical insufficiency. Further studies are needed in large samples to improve our understanding of microbes causing pre-term birth and to prevent pre-term birth in pregnant women with cervical insufficiency.

section_content: Intra-uterine infection is a major cause of spontaneous preterm birth before 30 weeks of gestation (Gravett et al., 1986; Yoon et al., 2001). In addition, intra-amniotic inflammation is present in approximately 80 % of patients with acute cervical insufficiency (Lee et al., 2008). The microbiome is known to cause intra-amniotic infection causing pre-term birth. PCR analysis of amniotic fluid of pregnant women who delivered pre-term detected 11 % bacteria in culture-negative samples (Gardella et al., 2004). PCR also revealed 45 % bacteria in the culture-negative amniotic fluid of participants with pre-term premature rupture of membranes (DiGiulio et al., 2010). Recently, surveys of the microbial communities in the vagina have been carried out using sequencing of the 16S rRNA gene (MacIntyre et al., 2015; Baldwin, et al., 2015). Here, we report that Sneathia sanguinegens and Fusobacterium nucleatum were identified using 16S rRNA gene sequencing in midtrimester amniotic fluid from two women with cervical insufficiency who delivered before 24 weeks. weeks of gestation. Speculum examination revealed 5 cm cervical dilation and amniotic membranes prolapsed beyond the external os. The patient was afebrile and stable haemodynamically, and had no persistent uterine contractions or vaginal bleeding. A sample was taken from the posterior vaginal fornix. A nitrazine and an Actim PROM test (Medix Biochemica), performed to rule out pre-term rupture of the membranes, were negative. Ultrasonography was performed to examine fetal biometry and anomalies, the amount of amniotic fluid, the status of the cervix and the location of the placenta. The patient was observed for 6 h to exclude pre-term labour and chorioamnionitis before cerclage placement. The serum white blood cell count was 14 070 cells ml 21 and the C-reactive protein was 26. 1 mg l 21. Vaginal exudate cultures were positive for Ureaplasma sp. Physical examination indicated that cerclage using a uniconcave balloon should be performed. To reduce the amount of amniotic fluid and evaluate possible subclinical chorioamnionitis, transabdominal amniocentesis was performed under ultrasonographic guidance before the cerclage procedure. A portion of the amniotic fluid was submitted for Gram staining and culture for anaerobic and aerobic bacteria; the amniotic fluid cultures were negative. The patient was given cephalosporin intravenously in the operating room and for the next 3 days. For 3 days post-operatively, she was given 500 mg azithromycin orally and treated with bed rest and continued tocolysis with intravenous magnesium sulfate. She developed labour pains 6 days after surgery at 23 6/7 weeks of gestation and the cerclage suture was removed. She delivered a 510 g female baby vaginally; the baby died in the delivery room. The patient was treated conservatively and discharged 2 days after delivery (Table 1 ).
section_name: Introduction

section_content: A 37-year-old nullipara was admitted to Hallym University Medical Center for abdominal discomfort and vaginal discharge at 22 3/7 weeks gestation. Speculum examination revealed 2 cm cervical dilation and the amniotic membranes were visible at the external os. The patient was afebrile and haemodynamically stable, and had no persistent uterine contractions or vaginal bleeding. A nitrazine and an Actim PROM test were negative. The patient was observed for 6 h before cerclage placement. The serum white blood cell count was 13 920 cells ml 21 and the C-reactive protein was 49. 5 mg l 21. Vaginal exudate cultures were positive for Ureaplasma sp. Physical examination indicated that cerclage should be carried out using a uniconcave balloon, and amnioreduction was performed. The patient was given cephalosporin intravenously in the operating room. She developed a fever of 38. 1 uC and frequent uterine contractions 1 day after the cerclage. Clinical chorioamnionitis was suspected and the suture was removed. The next day, the patient delivered a 520 g male baby vaginally at 22 5/7 weeks gestation; the baby died in the delivery room. Three days later, amniotic fluid obtained from the patient was positive for Gardnerella vaginalis. The patient was treated with antibiotics (second-generation cephalosporin antibiotics) and discharged 2 days after delivery (Table 1 ).
section_name: Case 2

section_content: 16S RNA gene sequencing DNA in amniotic fluid was amplified in triplicate reactions, pooled, and purified using MO BIO 96-htp PCR clean-up kits. Briefly, genomic DNA was amplified using the 16S amplicon PCR forward primer 59-TCGTCGGCAGCGTCA-GATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-39 and 16S amplicon PCR reverse primer 59-GTCTCGT-GGGCTCGAGATGTGTATAAGAGACAGGACTACHVGG-GTATCTAATCC-39. The amplicons were sequenced on an Illumina MiSeq, and clusters for the cDNA libraries were generated on a MiSeq flow cell and sequenced for 300 bp paired-end reads (2|300) with a MiSeq Reagent kit v. 3 (Illumina). Raw image data were transformed by base calling into sequence data and stored in FASTQ format. Raw reads were generated by the MiSeq platform and initially filtered for quality Q30 w80 %. The resulting 10 000 highquality sequence reads were analysed using CL community. 

Fig. 1 shows the bacterial phyla and species found in the amniotic fluid of each case. Although fusobacteria comprised 96. 3 and 78. 3 % of the phyla in cases 1 and 2, respectively, the predominant bacterial species differed: in case 1, 75. 2 % was Sneathia sanguinegens, while in case 2, 69. 2 % was Fusobacterium nucleatum.
section_name: Investigations

section_content: Amniotic fluid infection occurs in 10-15 % of pregnancies complicated by pre-term labour (Hitti et al., 2001; Cao et al., 2014). Notably, intra-amniotic inflammation is present in approximately 80 % of patients with acute cervical insufficiency (Lee et al., 2008). Here, we report the micro- Young-Ah You and others biome compositions in mid-trimester amniotic fluid from two women who delivered pre-term. The bacterial cultures were negative, but S. sanguinegens and Fusobacterium spp. were identified using 16S rRNA gene sequencing. These infections caused very early pre-term birth (before 24 weeks) and resulted in pre-natal death.
section_name: Discussion

section_content: Fusobacteria in amniotic fluid are associated with preterm birth and stillbirth (Hassan et al., 2006; Thilesen et al., 2007). S. sanguinegens forms long, Gram-negative, non-motile rods that sometimes exhibit bulbous protrusions (Collins et al., 2001; Goto et al., 2007). The species was previously named Leptotrichia sanguinegens, but was reassigned to a separate genus. S. sanguinegens has been associated with infections of the female reproductive tract, as has Leptotrichia amnionii (Thilesen et al., 2007). Moreover, S. sanguinegens is part of the vaginal flora and causes ascending infections of the uterus, fetal membranes and fallopian tubes (Gauthier et al., 2011). Recently, Romero et al. (2014) reported that the abundance of S. sanguinegens in the vaginal microbiota did not differ between pre-term and term births However, their results included diverse phylotypes in low numbers, while in our case 1, 16S rRNA gene sequencing identified only a few species in the amniotic fluid, and 75. 2 % of organisms were S. sanguinegens, supporting the notion that overgrowth of one or a few strains is harmful, whereas a diverse community with low numbers of individuals of each species does not seem to interfere with pregnancy (Wassenaar and Panigrahi, 2014). In our cases, the amniotic fluid cultures were negative. 

F. nucleatum is a non-spore-forming, Gram-negative anaerobe (Bolstad et al., 1996) that is common in the human oral cavity (Moore and Moore, 1994; Coppenhagen-Glazer et al., 2015). Periodontal disease is a risk factor for pre-term labour (Han et al., 2009; Gauthier et al., 2011), and F. nucleatum has been associated with pre-term birth (Hill, 1998; Cahill et al., 2005), stillbirth (Han et al., 2010) and early-onset neonatal sepsis (Wang et al., 2013). 

In pregnant mice, F. nucleatum injected intravenously invaded the placenta, proliferated in peripheral tissues, spread to the amniotic fluid and resulted in pre-term birth (Han et al., 2004. ). In humans, some researchers have reported that F. nucleatum was part of the mother's or father's oral flora but was undetected in the mother's vaginal or rectal samples (Han et al., 2009; Gauthier et al., 2011). Gauthier et al. (2011) reported intra-amniotic F. nucleatum in pregnant women who delivered pre-term, and postulated that it could originate from the father's oral flora. In case 2, there were only a few species in the amniotic fluid, and F. nucleatum comprised 69. 2 % of the bacteria. Again, amniotic fluid cultures were negative. 

There have been several studies on the microbiome in the vagina or in amniotic fluid associated with pre-term birth (DiGiulio, 2012; Wang et al., 2013; Hyman et al., 2014). However, in patients with cervical insufficiency, to the best of our knowledge this is the first study using 16S rRNA gene sequencing to identify the predominant microbiome in amniotic fluid. S. sanguinegens and F. nucleatum detected in our study were also detected in previous studies on pre-term birth (DiGiulio, 2012; Wang et al., 2013; Hyman et al., 2014). S. sanguinegens and F. nucleatum appeared to be highly prevalent in patients with pre-term delivery, which was unexpected based on their near absence in culture-based studies (DiGiulio, 2012). This molecular microbiological approach enables the detection of uncultivated microbes, thereby improving our understanding of the diversity of microbes causing pre-term birth. 

In conclusion, our 16S rRNA gene sequencing results identified S. sanguinegens and F. nucleatum in amniotic fluid as causes of pre-term birth. These bacteria were not detected in amniotic fluid cultures, while Ureaplasma sp. was detected in both vaginal swabs and Gardnerella sp. was detected in the amniotic fluid culture of case 2. This suggests that 16S rRNA gene sequencing can be used to identify the predominant microbiome in pregnant women with cervical insufficiency. Further studies are needed with larger sample numbers to improve our understanding of microbes causing pre-term birth and to prevent pre-term birth in pregnant women with cervical insufficiency.
section_name: S. sanguinegens and other members of the phylum

section_name: Acknowledgements

section_content: This study was supported by the Ministry of Health & Welfare, Republic of Korea (grant no. HI14C0306 ) through the Korea Health Industry Development Institute (KHIDI). The authors declare no conflicts of interest.
section_name: Acknowledgements