Protein subcellular localizations and signal peptides were predicted using PSORTb 3.0 [41] with default parameters for Gram-negative bacteria. A score of 7.5 was considered to be the cutoff for identification of protein localization.
Transmembrane regions were analyzed using TMHMM [42]. Protein secondary structures were predicted using the PSIPRED web server [43], available at http://bioinf.cs.ucl.ac.uk/psipred. Prediction of promoters was performed using the in-house SABIA platform as well as the BPROM program (http://linux1.softberry.com), which searches for promoters under the control of the sigma factor 70. Ribosome binding sites search was performed using the RBS finder software PF-6463922 that is included in the SABIA platform. GS-9973 clinical trial EasyFig [44] was used to generate the structural comparison of cps Kp13 and other sequenced cps loci. In silico serotyping An in silico serotyping approach was applied using the Molecular Serotyping Tool (MST) [45]. MST is a program for computer-assisted molecular identification of restriction
fragment length polymorphisms (RFLP) patterns, in which the concepts of similarity and alignment between RFLP patterns were adapted from Needleman and Wunsch’s dynamic programming algorithm. By analogy, RFLP patterns represented by ordered fragment sizes can be aligned, and their similarity can be calculated as the sum of penalties for edit operations (insertions, deletions or substitutions) that transform one pattern
into another [45]. MST, available at http://www.cebio.org/mst, was originally designed for Nintedanib (BIBF 1120) the identification of RFLP patterns from Escherichia coli and the Shigella O-antigen gene clusters [46, 47]. At present, identification of K. pneumoniae serotypes can also be achieved because the RFLP patterns of the amplified capsular antigen gene clusters of all known Klebsiella serotypes were published by Brisse et al. [29].The RFLP of Kp13 was determined and compared to those already described. All scores were used to build a distance matrix in a PHYLIP compatible format [48]. The distance matrix was used to reconstruct a phylogeny by the UPGMA method with the NEIGHBOR program, available in the PHYLIP package. The tree generated by UPGMA was visualized with the graphical viewer FIGTREE (http://tree.bio.ed.ac.uk/software/figtree/). To improve the analysis of the UPGMA tree, the two-time-scales were applied. The MST distance cutoff that is able to distinguish between two serotypes is 1.5, and the scale-adjusted measure should be interpreted as 0.75. In vitro K-serotyping Isolate Kp13 was sent to the International Escherichia and Klebsiella Reference GSK2118436 purchase Center (WHO), Statens Serum Institut, Copenhagen, Denmark, for serotyping. Briefly, K-typing was done by counter-current immunoelectrophoresis (CCIE) against antiserum pools as previously described [49].