​ncbi.​nlm.​nih.​gov) and subsequently aligned to the sequence of the reference plasmid, pUTI89 [GenBank:CP000244]. Gap closure was performed using primer walking into the gaps with

the LongRange PCR Kit (Qiagen). ON-01910 research buy The complete sequence of the plasmid was annotated using Rapid Annotation using Angiogenesis inhibitor Subsystem Technology (RAST) [34]. Comparative genomics and phylogenetic analysis Comparative genomics of pRS218 with closely related IncFIB/FIIA plasmids of other E. coli was performed using Mauve 3.2.1 genome alignment web tool (http://​gel.​ahabs.​wisc.​edu/​mauve/​) [35]. An evolutionary relationship of 24 plasmids belonging to the IncFIB/FIIA group based on repA1 gene sequence was performed using the neighbor-joining method. A neighbor joining tree was constructed by using the MEGA4 web tool (http://​www.​megasoftware.​net/​mega4/​mega.​html) [36,37]. Analysis of plasmid profiles of NMEC strains Extraction of large plasmids from NMEC strains was performed using an alkaline lysis method described previously [33]. In brief, 1 ml of overnight culture of each E. coli strain was subjected to alkaline lysis using 10% sodium hydroxide followed by phenol-chloroform

extraction of plasmid DNA. Plasmid BMS202 profiles of NMEC strains

were evaluated by electrophoresis on a 0.7% agarose gel containing 0.5 μg/ml ethidium bromide. Evaluation of prevalence of selected pRS218 genes in other NMEC and fecal E. coli Specific polymerase chain reactions (-)-p-Bromotetramisole Oxalate (PCRs) were performed to determine the presence of selected gene coding regions (n = 59) of pRS218 in other NMEC and fecal E. coli strains. Primers were designed using the Primer 3.0 web tool (http://​bioinfo.​ut.​ee/​primer3-0.​4.​0/​) (Table 5). PCR amplifications were performed using crude DNA extracted by the rapid boiling method [38]. The PCR mixture contained 1 U of Taq polymerase (Qiagen), 1× Taq polymerase buffer, 3.5 mM MgCl2, 125 μM each deoxynucleotide triphosphate (dNTP) and150 nM each primer pair. PCR conditions were as follows: 1 cycle of 95°C for 1min, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1.5 min, and a final extension at 72°C for 10 min. Amplicons were visualized on a 1.5% agarose gel containing 0.5 μg/ml ethidium bromide. Table 5 Primers used for the screening of pRS218 genes among neonatal meningitis causing E. coli and fecal commensal E.