MST (Molecular Serotyping Tool): a Program for Computer-Assisted Molecular Identification of Escherichia coli and Shigella O Antigens

Abstract

Escherichia coli and Shigella O antigens can be inferred using the rfb-restriction fragment length polymorphism (RFLP) molecular test. We present herein a dynamic programming algorithm-based software to compare the rfb-RFLP patterns of clinical isolates with those in a database containing the 171 previously published patterns corresponding to all known E. coli/Shigella O antigens.Classical Escherichia coli/Shigella O serogrouping is expensive, labor-intensive, and susceptible to errors due to cross-reactivity between adsorbed O-antigen rabbit antisera, as reviewed in reference 2. Furthermore, corrupted expression of genes involved in O-antigen synthesis renders some strains nontypeable ("rough"). Importantly, classical serotyping does not detect new O antigens. These drawbacks are surmounted by the rfb-restriction fragment length polymorphism (RFLP) test (1, 2). Briefly, the rfb locus containing most of the genes involved in O-antigen synthesis is amplified by PCR and digested with MboII, and products are resolved by electrophoresis. A database with 171 rfb-RFLP patterns representing each known Shigella and E. coli O antigen has been published and used to type reference and clinical strains, including an isolate that had become rough in the lab, with 100% specificity and sensitivity (1, 2). We present herein a web-based software program to compare the rfb-RFLP patterns of clinical isolates with those of known E. coli and Shigella O antigens.For the purpose of this work, the concepts of similarity and alignment between two rfb-RFLP patterns were adopted from Needleman and Wunsch's dynamic programming algorithm (7), which detects insertions, deletions, and substitutions as changes in the strings that represent nucleic acid or protein sequences. By analogy, restriction patterns represented by ordered fragment sizes can be aligned and their similarity can be calculated as the sum of penalties for the edit operations that transform one pattern into the other. These edit operations are deletions (missing bands) and transformations (errors on fragment sizing). A scoring function assigns a penalty to each transformation, and a dynamic programming algorithm computes the best editing scores between two patterns, producing an editing matrix. For two patterns, a ϭ a 1 a 2 ⅐ ⅐ ⅐ a m and b ϭ b 1 b 2 ⅐ ⅐ ⅐ b n , where a i is the fragment size at position i of test pattern a and b j is the fragment size at position j of reference pattern b, the editing matrix has (m ϩ 1) ϫ (n ϩ 1) positions, where n is the number of fragments in pattern a and m is the number of fragments in pattern b. The matrix is initialized, filling the first column with each fragment of pattern a and the first row with each fragment of pattern b. The aim is to move across the matrix from the first column and first row to the last column and last row. At each position, it is possible to move only one step either in the diagonal to align two fragments and advance one position in both patterns or in the horizontal (or vertical) to acknowledge a missing fragmen...

Comparative Genomic Analysis of a Clinical Isolate of Klebsiella quasipneumoniae subsp. similipneumoniae, a KPC-2 and OKP-B-6 Beta-Lactamases Producer Harboring Two Drug-Resistance Plasmids from Southeast Brazil

“…Seven clusters were disclosed and the isolate named 142, which displayed the most divergent DNA pattern by RAPD cluster analysis selected for sequencing analysis (Figure S1 ). Fragment sizes in RAPD fingerprints were estimated using GelAnalyzer (GelAnalyzer.com) and a distance matrix was calculated with MST (Coimbra et al, 2010 ). Distance tree was built with NEIGHBOR program within the PHYLIP package (Felsenstein, 1989 ) and drawn with MEGA 6.0 (Tamura et al, 2013 ).…”

Recent Advances in Understanding Enteric Pathogenic Escherichia coli

“…Alternative approaches to antiserum-based serotyping have also been developed. Coimbra et al designed a software-supported and restriction fragment length polymorphism-based technique which is able to distinctly identify most but not all Shigella serotypes (540,541). In another study, molecular serotyping of S. flexneri was achieved by a multiplex PCR assay targeting several specific O-antigen synthesis and modification genes (542).…”

Revisitingmolecular serotyping of Streptococcus pneumoniae

“…This in silico analytical pipeline was applied also to the endonuclease HinfI, which had already been proposed for molecular serotyping of a subset of pneumococcal serotypes [ 19 ]. The most discriminant enzyme was chosen using a standalone version of Molecular Serotyping Tool (MST), our previously published software for computer-assisted molecular serotyping [ 12 ]. Briefly, pairwise alignments of all cps -RFLP patterns were performed and the pairwise distances were calculated as the sum of the penalties for the edit operations required to transform one pattern to the other.…”

“…The cps -RFLP patterns obtained in silico were uploaded to the database of the web-based MST software [ 12 ], which is freely accessible at http://www.cebio.org/mst . In addition, the database was complemented with the in vitro patterns that did not match any of the in silico ones.…”

“…The method named rfb -RFLP relies on restriction fragment length polymorphisms (RFLP) of the rfb loci, responsible for the synthesis of the somatic antigen in E. coli and Shigella spp. A database with rfb -RFLP patterns of all known serogroups/serotypes of this genospecies has been published, and a web-based software has been developed to compare the rfb -RFLP patterns of clinical isolates with those in the database [ 12 ]. This technique has been successfully used for more than a decade and allowed the discovery of new putative serotypes [ 13 - 15 ].…”