Comparison of Typing Methods with a New Procedure Based on Sequence Characterization for Salmonella Serovar Prediction

Abstract

bAs 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.

Distributions of Salmonella Subtypes Differ between Two U.S. Produce-Growing Regions

“…enterica isolates (n ϭ 754 tested) were incorrectly serotyped by traditional serotyping, and thus molecular serotyping may aid in identification (37). Serovar was confirmed by PCR detection of the Salmonella O serogroup genes by a multiplex PCR assay that simultaneously targets genes for five Salmonella O antigens (B, C1, C2-C3, D1, and E1), as described by Ranieri et al (35) and Herrera-Léon et al (38); in addition, PCR amplification and sequencing of the genes encoding the H1 and H2 antigens were performed. Primer sets, DNA amplification, and PCR conditions for fliC (encodes H1 antigen) and fljB (encodes H2 antigen) have been previously described by Imre et al (39) and Mortimer et al (40).…”

“…Primer sets, DNA amplification, and PCR conditions for fliC (encodes H1 antigen) and fljB (encodes H2 antigen) have been previously described by Imre et al (39) and Mortimer et al (40). DNA sequence data were compared to sequences in an internal database (CUFSL) of H1 and H2 sequences, as previously described (35). Molecular serotyping results were able to resolve serovar conflicts for the 12 isolates with different serovars identified by traditional serotyping and PFGE.…”

“…Fourdigit pattern codes were assigned by comparison of patterns to an internal reference database (CUFSL, Ithaca, NY) comprised of approximately 6,000 Salmonella isolates. Lastly, PFGE was used to predict the serovar of a given isolate by comparison to the CUFSL database using a similarity cluster analysis (as described above) (35).…”

Scientific Opinion on the evaluation of molecular typing methods for major food‐borne microbiological hazards and their use for attribution modelling, outbreak investigation and scanning surveillance: Part 1 (evaluation of methods and applications)

“…array technology , PCR (Paddock et al, 2012), real-time PCR (Anklam et al, 2012;Fratamico et al, 2011). For a more in depth study of molecular serotyping approaches for Salmonella see Ranieri et al, 2013; for E. coli see DebRoy et al, 2011; for L. monocytogenes see Doumith et al, 2005, Kérouanton et al, 2010and Vitullo et al, 2013; and for Campylobacter see Poly et al, 2011. For the four pathogens considered in this Opinion molecular serotyping is considered to provide a low to moderate discriminatory capability. This is normally similar or marginally higher than traditional serotyping as sub-types can often be recognised within serotypes.…”

Infectología en la era de la genómica

“…Sin embargo, el poder de discriminación del PFGE, es decir, su capacidad de diferenciar entre cepas, es limitada si se compara con la secuenciación de genomas completos. De hecho, se estima que sólo 1% del genoma es analizado cuando se observa el patrón de bandas generadas por el PFGE 10,11 . En varios estudios e investigaciones epidemiológicas ha quedado de manifiesto la limitación del PFGE, sobre todo en casos donde las cepas involucradas son de tipo monomórficas o clonales.…”