GBS serotypes and culture conditions
A total of 30 clinical GBS isolates collected previously  were used in this study. All these strains were inoculated in Todd Hewitt broth medium (THB; Himedia Laboratories, India) and were stored in 25% glycerol at -80oC for further use. Briefly, cultures were streaked from frozen stock on 0.5 % sheep blood agar plate (BAP) with the help of sterile loop and incubated at 37oC for 24-30 hrs.
Antibiotic susceptibility test
To determine the antibiotic resistance profile, all the 30 GBS clinical isolates were screened against six antibiotics according to the Clinical and Laboratory Standards Institute Guidelines . The Kirby-Bauer disk-diffusion method was used to test penicillin (0.06µg), clindamycin (2µg), erythromycin (15µg), gentamicin (10µg), ofloxacin (2µg) and azithromycin (15µg) resistance. Briefly, fresh sheep blood agar plates were inoculated with homogeneous GBS suspension of 0.5 McFarland standards, prepared from fresh bacterial cultures according to the CLSI . 0.5 McFarland standards were used as reference with an optical density comparable to 1.5 x 108 bacterial colony forming units (CFU/ml).
Pure cultures were streaked on Todd Hewitt broth (THB) agar plates with 1 % glucose and incubated at 37°C for overnight. Single purified colony was picked and mixed into phosphate buffer saline (1X), and used to form a bacterial lawn on fresh 5 % sheep blood agar plate (BAP) (Life Science Media, Delhi India). Specific antibiotic discs were placed onto the plate using sterile forceps and were incubated overnight at 37°C. The zone of inhibition was measured using mathematical scale manually and the results were interpreted as susceptible, intermediate, and resistant according to the Clinical and Laboratory Standard Institute Guidelines (CLSI) .
Biofilm formation assay
Biofilm formation of GBS clinical isolates was assessed by Congo Red Agar (CRA) and Crystal Violet Assay (CVA) as described . CRA is a qualitative assay whereas, CVA is a quantitative method for biofilm detection. In brief, THB medium supplemented with 1% glucose and 0.08% CRA plates was used to assess the biofilm formation phenotype. Briefly, CRA plates were inoculated with GBS cell suspension and incubated at 37°C for 24 hours. After overnight incubation, isolates were interpreted according to their colony phenotypes and change in color. The formation of black colored colonies with slime production was used as an indicator for biofilm formation .
In case of CVA, 5ml THB broth with 1% glucose was inoculated with single isolated colony from the fresh blood agar plate and incubated at 37°C until their OD600 reaches between 0.4-0.6. At desired OD600 (~ 0.5), 100μL culture was dispensed in 96-well microtiter plate along with 100μL fresh THB with 1% glucose and incubated at 37°C for 48 hours under static condition. After incubation plates were gently washed 3 times with 1X PBS (pH 7.4) followed by heat fixation at 60°C for 1 hour and was stained with 100μL of 0.5% crystal violet (CV) for 5 minutes. After CV staining, plates were washed three times with 1X PBS and the remaining CV was solubilized by addition of 200μL of 95% ethanol, and plates were incubated at room temperature for 10 minutes. The optical density of each well was measured by the ELISA plate reader at 595nm (BioTek Synergy™ H1 hybrid multi-mode microplate reader, USA). OD values less than 2 was considered as non-biofilm producers, 2-4 as weak biofilm producers, 4-8 as moderate biofilm producers and more than 8 as strong biofilm producers . Pseudomonas aeruginosa (MTCC 2297) and THB broth +1% glucose (only medium) was used as positive and negative controls, respectively . All experiments were performed in triplicate with three independent experiments.
Detection of virulence genes by PCR
Six virulence genes (gbs67, cylE, cfb, scpB, lmb, and pavA) were targeted their expression and role in biofilm formation using semi-quantitative PCR using gene specific primer (Supplementary Table 1). For this, genomic DNA was isolated by CTAB (Cetyltrimethylammonium bromide) method . In brief, 50 ml of log phase GBS culture (OD600 0.5) in THB was centrifuged at 6000 rpm for 5 min at room temperature. Pellet, obtained was washed twice with 5 ml of 0.1M tris buffer (pH 8.0). To this, 50 µl of lysozyme (100mg/ml), 200 µl of 10% SDS and 60 µl of proteinase K (60mg/ml) were added and this mixture was incubated at 65oC for 2 h. Further, 500 µl of 5M NaCl and 800 µl of 10% CTAB was added and mixed gently and incubated for 30 min at 65oC. After incubation, 15ml of chloroform: isoamylalcohol (24:1) was added and vortexed for 10 sec and centrifuged at 12,000 rpm for 10 min. Upper aqueous phase was collected and an equal volume of phenol: chloroform (1:1) was added to it. After vortexing for 5 sec, and centrifugation at 12,000 rpm for 5 min, upper aqueous phase was collected and 2 µl of RNAse (10mg/ml) was added followed by incubation at 37oC. After 30 min, equal volume of chilled absolute ethanol was added and incubated for 2 hrs at -20oC and centrifuged at 12,000 rpm for 10 min. Supernatant was discarded and the pellet obtained was washed with chilled 70% ethanol (1 ml) and centrifuged for 15 min at 12,000 rpm at 4oC. After centrifugation DNA pellet was dissolved in nuclease free water. Quantification and purity of extracted genomic DNA was checked by measuring the absorbance at 260nm and 280nm on UV spectrophotometer. The PCR reaction contains 2mM MgCl2, 5pmole of each forward and reverse primer,0.5mM dNTPs mix, 100ng genomic DNA template, 2 units high-fidelity DNA Polymerase. The PCR conditions used were as follows: initial denaturation at 95◦C for 5 min, 30 cycles of 95◦C for 45 s, 40–57◦C (depending on primer melting temperature) for 45 s, and extension at 72◦C for 1 min, followed by a ﬁnal extension at 72◦C for 5 min. The PCR amplified DNA was resolved on 1.2% TAE agarose gel and visualized under UV light. The gyrase subunit A(gyrA)gene was used as an internal standard .