Comparison of typing methods with a new procedure based on sequence characterization for Salmonella serovar prediction

Abstract

As the development of molecular serotyping approaches is critical for Salmonella spp., which include >2,600 serovars, we performed an initial evaluation of the ability to identify Salmonella serovars using (i) different molecular subtyping methods and (ii) a newly implemented combined PCR- and sequencing-based approach that directly targets O- and H-antigen-encoding genes. Initial testing was performed using 46 isolates that represent the top 40 Salmonella serovars isolated from human and nonhuman sources, as reported by the U.S. Centers for Disease Control and Prevention and the World Health Organization. Multilocus sequence typing (MLST) was able to accurately predict the serovars for 42/46 isolates and showed the best ability to predict serovars among the subtyping methods tested. Pulsed-field gel electrophoresis (PFGE), ribotyping, and repetitive extragenic palindromic sequence-based PCR (rep-PCR) were able to accurately predict the serovars for 35/46, 34/46, and 30/46 isolates, respectively. Among the methods, S. enterica subsp. enterica serovars 4,5,12:i:-, Typhimurium, and Typhimurium var. 5- were frequently not classified correctly, which is consistent with their close phylogenetic relationship. To develop a PCR- and sequence-based serotyping approach, we integrated available data sources to implement a combination PCR-based O-antigen screening and sequencing of internal fliC and fljB fragments. This approach correctly identified the serovars for 42/46 isolates in the initial set representing the most common Salmonella serovars, as well as for 54/63 isolates representing less common Salmonella serovars. Our study not only indicates that different molecular approaches show the potential to allow for rapid serovar classification of Salmonella isolates, but it also provides data that can help with the selection of molecular serotyping methods to be used by different laboratories.

Fig 1

Midpoint-rooted maximum-likelihood phylogenetic tree of partial fliC amino acid sequences from 116 Salmonella isolates representing 46 common, 63 uncommon, and 7 untypeable serovars. The scale represents the estimated number of amino acid substitutions per site. Numerical values represent the percentage of bootstrap replications that support the respective node. Bootstrap values of >60 are shown for major clades. Each label shows the H1 antigen followed by Food Safety Laboratory (FSL) number; e.g., b_S5-410 indicates H1 antigen b, isolate FSL S5-410.

Fig 2

Midpoint-rooted maximum-likelihood phylogenetic tree of 90 partial fljB amino acid sequences from Salmonella isolates representing 32 common, 54 uncommon, and 4 untypeable serovars. The scale represents the estimated number of amino acid substitutions per site. Numerical values represent the percentage of bootstrap replications that support the respective node. Bootstrap values of >60 are shown for major clades. Each label shows the H2 antigen followed by the Food Safety Laboratory (FSL) number; e.g., 1,2_R8-457 indicates H2 antigen 1,2, isolate FSL R8-457.