Background. Preterm Premature Rupture of Membranes (PPROM) is a major leading cause of preterm births.While the cause for PPROM remains unidentified, it is anticipated to be due to subclinical infection, since a large proportion of PPROM patients display signs of chorioamnionitis. Since subclinical infections can be facilitated by dysbiosis, our goal was to characterize the vaginal microbiome and amniotic fluid discharge upon PPROM, through latency antibiotic treatment, and until delivery, to detect the presence of pathogens, microbiota alteration, and microbial response to treatment. Methods. Enrolled subjects (15) underwent routine institutional antenatal care for PPROM, including the administration of latency antibiotics. Serial vaginal swabs were obtained fromdiagnosis of PPROMthrough delivery and the sequencing of the V3-V5 region of the 16S rRNA gene was performed for all collected samples. Results. The results show that Lactobacilli species were markedly decreased when compared to vaginal swabs collected from uncomplicated pregnancy subjects with a matched gestational time. Prevotella and Peptoniphilus were the most prevalent taxa in PPROM subjects at presentation. The vaginal microbiome of the PPROM subjects varied substantially intra- and inter-subjects. Several taxa were found to be significantly reduced during and after the antibiotic treatment: Weeksella, Lachnospira, Achromobacter, and Pediococcus. In contrast, Peptostreptococcus and Tissierellaceae ph2 displayed a significant increase after the antibiotic treatment. However, the relative abundance of Lactobacillus, Prevotella, and Peptoniphilus was not substantially impacted during the hospitalization of the PPROM subjects. The deficiency of Lactobacillus, and constancy of known pathogenic species, such as Prevotella and Peptoniphilus during and after antibiotics, highlights the persistent dysbiosis and warrants further investigation into mitigating approaches. Discussion. PPROM is responsible for one third of all preterm births. It is thought that subclinical infection is a crucial factor in the pathophysiology of PPROM because 25-40% of patients present signs of chorioamnionitis on amniocentesis. Here we sought to directly assess the bacterial content of the vagina and leaking amniotic fluid of subjects at presentation, throughout treatment and up until delivery, in order to search for common pathogens, microbiota changes, and microbial response to latency antibiotic treatment.We have found that the vaginal microbiome of PPROM subjects is highly variable and displays significant changes to treatment. However, the unchanging deficiency of Lactobacillus, and persistence of known pathogenic species, such as Prevotella and Peptoniphilus frompresentation, through antibiotic treatment and up until delivery, highlights the persistent dysbiosis and warrants further investigation into mitigating approaches.