Macconkey agar mug

At each sampling time (T0, T1, and T2), vaginal discharge samples were collected, using swabs filled with the transport medium Transystem Amies Clear (Biolife, Milan, Italy), and were subjected to culture-dependent analysis according to Pino and co-workers (26 (link), 27 (link)). In detail, after dislodging bacterial cells in sterile phosphate-buffered saline (PBS), 10-fold dilutions were made and plated using the following agar media and conditions: Rogosa SL agar (Biolife, Milan, Italy) for Lactobacillus counts, incubated at 35–37 °C for 40–48 h; Streptococcus Selective Agar (Biolife, Milan, Italy) for the enumeration of streptococci, incubated at 32 °C for 24 h; Columbia Blood Agar base (Oxoid, Milan, Italy), supplemented with Gardnerella Vaginalis Selective Supplement (Oxoid, Milan, Italy), incubated at 37 °C for 40–48 h for Gadnerella spp. count; MacConkey Agar Mug (Biolife, Milan, Italy) incubated at 37 °C for 16–18 h for Escherichia coli; Mannitol Salt Agar (Oxoid, Milan, Italy) incubated at 32 °C for 48 h for the count of staphylococci; Slanetz Bartley Agar (Biolife, Milan, Italy) incubated at 37 °C for 48 h for enterococci; and Chromogenic Candida Agar (Biolife, Milan, Italy), incubated at 35–37 °C for 18–48 h, for the count of Candida spp.
Microbiological count was performed in triplicate and results were reported as mean log cfu/ml and standard deviation.

Vaginal discharge, collected using a sterile synthetic-swab tip Transystem Amies Medium Clear (Biolife Srl, Milan, Italy), was analyzed as follows. After dislodging the cells in sterile phosphate-buffered saline (PBS), serial dilutions were made and plated on the following agar media and conditions: Rogosa SL agar (Biolife) incubated at 35–37°C for 40–48 h for Lactobacillus counts; Streptococcus Selective Agar (Biolife) incubated at 32°C for 24 h for streptococci; Gardnerella vaginalis Selective Medium (Oxoid, Milan, Italy) incubated at 37°C for 40–48 h for G. vaginalis; MacConkey Agar Mug (Biolife) incubated at 37°C for 16–18 h for Escherichia coli; Mannitol Salt Agar (Oxoid) incubated at 32°C for 48 h for staphylococci; Slanetz Bartley Agar (Biolife) incubated at 37°C for 48 h for enterococci; and Chromogenic Candida Agar (Biolife) incubated at 35–37°C for 48 h for Candida albicans, C. tropicalis, and C. krusei. All analyses were performed in duplicate.