Bacterial culture on GAM-agar plates was performed for 24 meconium samples (5 corresponding negative field controls) and 24 amnion samples (4 negative field controls). The frozen samples were first transferred to an anaerobic workstation (Ruskinn Concept Plus), mixed thoroughly and plated, using an aseptic technique, at +37°C. After this, the samples were transferred to a laminar flow cabinet and plated in aerobic conditions at +37°C. The culture plates were checked for growth daily for 14 days. All visible bacterial colonies were subcultured until pure isolates were obtained. Fresh cultures were then identified using MALDI-TOF (Bruker Microflex LT) at the Central laboratory of the Faculty of Veterinary Medicine (Helsinki, Finland).
Samples were prepared using MALDI Biotyper MSP Identification Standard Method v 1.1. Mass spectra were analyzed in a mass/charge range from 2,000 to 20,000 Da with MBT Compass v4.1 on flexControl v3.4 (Bruker Daltonik GmbH) using BDAL-7311 as the reference library. The Bruker Bacterial Test Standard (RUO) (Bruker Daltonik GmbH) was used for instrument calibration. If the identification confidence score was < 2.00, further identification was done with 16S rRNA gene amplicon Sanger sequencing at the Institute of Biotechnology (University of Helsinki, Finland).
Samples were prepared using MALDI Biotyper MSP Identification Standard Method v 1.1. Mass spectra were analyzed in a mass/charge range from 2,000 to 20,000 Da with MBT Compass v4.1 on flexControl v3.4 (Bruker Daltonik GmbH) using BDAL-7311 as the reference library. The Bruker Bacterial Test Standard (RUO) (Bruker Daltonik GmbH) was used for instrument calibration. If the identification confidence score was < 2.00, further identification was done with 16S rRNA gene amplicon Sanger sequencing at the Institute of Biotechnology (University of Helsinki, Finland).