Eswab

All patient samples were collected as part of routine care for ASB screening prior to performing an examination. A midstream voided urine sample (described hereafter as “urine” samples) was collected in a sterile blue cap collection cup (BD #364956; Becton, Dickinson and Company). A portion of this sample was placed in a BD Vacutainer Plus C&S Preservative Tube (Becton, Dickinson and Company), as is standard practice for clinical microbiology samples. At the time of the routine pelvic examination prior to performing a sterile digital examination, a vaginal sample (described hereafter as “vaginal” sample types) was collected with a sterile speculum from the posterior fornix of the vagina using the BD ESwab (BD#220245; Becton, Dickinson and Company, Sparks, Maryland), which contains a swab resting in 1 mL of the bacterial preservative. The microbiomes of all collected samples were determined using two methods: EQUC and 16S rRNA gene amplicon sequencing (described hereafter as the “amplicon” method).

To mark the area where the child defecated on the floor (concrete or stone) or a material laid down on the floor, we followed a similar method using toothpicks as noted above. We collected samples from the place of defecation and a location approximately 30 cm away from the place of defecation, but used sterile nylon-flocked swabs stored in liquid Amies elution solution (BD ESwabTM, Franklin Lakes, NJ) for sampling the floor surface. Swab sample collection and analysis followed methods modified from Hedin et al. (2010) (link). Swabs were removed from the elution solution, pressed against the side of the tube to remove excess solution from the swab, and then the tip was used to swab an approximately 10 cm by 10 cm square of the floor, thoroughly wiping in the horizontal direction, and then the vertical direction of the square, constantly rotating the swab head while swabbing the surface. The swab was returned to the tube and stored in the elution solution until processing.

All sample self-collection methods were approved by the Johns Hopkins University Institutional Review Board under IRB study IRB00099798, and written informed consent was obtained from each participant. The approved protocol granted access to medical history, which was used to confirm delivery dates, numbers of previous pregnancies, and history of sexually transmitted infections. Participants provided additional information about their medical history and behaviors on a written questionnaire (Supplementary Figure 1). Participants (n = 92) were between 18-45 years old and provided a total of n = 432 samples over the course of their pregnancies. In general, samples were collected approximately every 4 weeks during pregnancy. A post-partum sample was collected up to 8 weeks post-delivery. CVM samples were self-collected, as previously described (Boskey et al., 2003 (link); Hoang et al., 2020 (link)). Participants were instructed to insert an Instead Softcup^® (Evofem) into their vagina and twist while pulling out the cup (< 30 s total) to collect undiluted CVM. They placed the Softcup^® into a 50 mL conical tube, which was stored on ice until processing within 12 h of collection. Additionally, participants self-collected a vaginal swab (BD Eswab) sample to be used for sequencing and a urine specimen to confirm pregnancy status with hCG urine test strips.

Rectal E. coli isolates were collected from healthy adult volunteers in St. Louis, Missouri, USA, from 2014 to 2015. Exclusion criteria included age < 18 years old, pregnancy, current urinary tract infection, previous urogenital surgery, ongoing treatment for urogenital cancer, the use of systemic antibiotics within 30 days of the study visit, or the use of a urinary catheter within 30 days of the study visit. Each study participant used a previously published protocol (70 (link)) to procure a self-collected rectal swab (BD Eswab) and submitted it with a study survey. Swabs were processed by the clinical microbiology lab at Barnes-Jewish Hospital to identify a dominant E. coli isolate and assess its antibiotic susceptibilities. Fifty-seven individuals were consented, 48 individuals submitted study materials, 41 E. coli isolates had matching demographic data, and 13 of those E. coli isolates were randomly selected for the current study.