Methods to rapidly identify serotypes of Salmonella enterica subspecies I are of vital importance for protecting the safety of food. To supplement the serotyping method dkgB-linked intergenic sequence ribotyping (ISR), single-nucleotide polymorphisms were characterized within adenylate cyclase (cyaA). The National Center for Biotechnology Information (NCBI) database had 378 cyaA sequences from S. enterica subspecies I, which included 42 unique DNA sequences and 19 different amino acid sequences. Five representative isolates, namely serotypes Typhimurium, Kentucky, Enteritidis phage type PT4, and two variants of Enteritidis phage type PT13a, were differentiated within a microsphere-based fluidics system in cyaA by allele-specific primer extension. Validation against 25 poultry-related environmental Salmonella isolates representing 11 serotypes yielded a ˜89% success rate at identifying the serotype of the isolate, and a different region could be targeted to achieve 100%. When coupled with ISR, all serotypes were differentiated. Phage lineages of serotype Enteritidis 13a and 4 were identified, and a biofilm-forming strain of PT13a was differentiated from a smooth phenotype within phage type. Comparative ranking of mutation indices to genes such as the tRNA transferases, the diguanylate cyclases, and genes used for multilocus sequence typing indicated that cyaA is an appropriate gene for assessing epidemiological trends of Salmonella because of its relative stability in nucleotide composition.
Highlights
Alcaine SD, Soyer Y, Warnick LD, et al. Multilocus sequence typing supports the hypothesis that cow- and human-associated
Salmonella isolates represent distinct and overlapping populations. Appl Environ Microbiol 2006;72:7575–7585.
Aravind L, Koonin EV. DNA polymerase beta-like nucleotifyltransferase superfamily: Identification of three new families, classification and evolutionary history. Nucleic Acids
Res 1999;27:1609–1618.
Baker DA, Kelly JM. Structure, function and evolution of microbial adenylyl and guanylyl cyclases. Mol Microbiol 2004;
52:1229–1242.
Bell RL, Gonzalez-Escalona N, Stones R, Brown EW. Phylogenetic evaluation of the ‘Typhimurium’ complex of Salmonella strains using a seven-gene multi-locus sequence analysis. Infect Genet Evol 2011;11:83–91.
Benson DA, Cavanaugh M, Clark K, et al. GenBank. Nucleic
Acids Res 2013;41(Database issue):D36–D42.
[CDC-NCEZID] The Centers for Disease Control and Prevention–
National Center for Emerging and Infectious Disease. An Atlas of Samonella in the United States, 1968–2011: LaboratoryBased Enteric Disease Surveillance. Atlanta, GA: U.S. Department of Health and Human Services, 2013, p 246.
Chan KH, Li T, Wong CO, Wong KB. Structural basis for GTPdependent dimerization of hydrogenase maturation factor
HypB. PLoS One 2012;7:e30547.
Csonka LN, Ikeda TP, Fletcher SA, Kustu S. The accumulation of glutamate is necessary for optimal growth of Salmonella typhimurium in media of high osmolality but not induction of the proU operon. J Bacteriol 1994;176:6324–6333.
Curtiss III R, Hassan JO. Nonrecombinant and recombinant avirulent Salmonella vaccines for poultry. Vet Immunol Immunopathol 1996;54:365–372.
Ducey TF, Page B, Usgaard T, Borucki MK, Pupedis K, Ward
TJ. A single-nucleotide-polymorphism-based multilocus genotyping assay for subtyping lineage I isolates of Listeria monocytogenes. Appl Environ Microbiol 2007;73:133–147.
Dunbar SA, Jacobson JW. Quantitative, multiplexed detection of Salmonella and other pathogens by Luminex xMAP suspension array. Methods Mol Biol 2007;394:1–19.
Dunbar SA, Ritchie VB, Hoffmeyer MR, Rana GS, Zhang H.
Luminex((R)) multiplex bead suspension arrays for the detection and serotyping of Salmonella spp. Methods Mol Biol
2015;1225:1–27.
Fabre L, Zhang J, Guigon G, et al. CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections. PLoS One 2012;7:e36995.
Fakhr MK, Nolan LK, Logue CM. Multilocus sequence typing lacks the discriminatory ability of pulsed-field gel electrophoresis for typing Salmonella enterica serovar Typhimurium. J Clin Microbiol 2005;43:2215–2219.
Feng Y, Johnston RN, Liu GR, Liu SL. Genomic comparison between Salmonella Gallinarum and Pullorum: Differential pseudogene formation under common host restriction. PLoS
One 2013;8:e59427.
Fresno M, Barreto M, Gutierrez S, Dougnac C, Abalos P, Retamal P. Serotype-associated polymorphisms in a partial rpoB gene sequence of Salmonella enterica. Can J Microbiol
2014;60:177–181.
Galitski T, Roth JR. Pathways for homologous recombination between chromosomal direct repeats in Salmonella typhimurium. Genetics 1997;146:751–767.
Gilberthorpe NJ, Poole RK. Nitric oxide homeostasis in Salmonella typhimurium: Roles of respiratory nitrate reductase and flavohemoglobin. J Biol Chem 2008;283:11146–
11154.
Guard J, Morales CA, Fedorka-Cray P, Gast RK. Single nucleotide polymorphisms that differentiate two subpopulations of Salmonella enteritidis within phage type. BMC Res Notes
2011;4:369.
Guard J, Sanchez-Ingunza R, Morales C, et al. Comparison of dkgB-linked intergenic sequence ribotyping to DNA microarray hybridization for assigning serotype to Salmonella enterica. FEMS Microbiol Lett 2012;337:61–72.
Han H, Zhou HJ, Cui ZG, Du PC, Kan B. [Multilocus sequence typing and pulsed-field gel electrophoresis analysis of Salmonella Paratyphi A isolates from 2000 to 2008, China]. Zhonghua Yu Fang Yi Xue Za Zhi 2010;44:810–814. (in Chinese.)
Hensel M, Hinsley AP, Nikolaus T, Sawers G, Berks BC. The genetic basis of tetrathionate respiration in Salmonella typhimurium. Mol Microbiol 1999;32:275–287.
Jean-Gilles Beaubrun J, Ewing L, Jarvis K, et al. Comparison of a
PCR serotyping assay, Check&Trace assay for Salmonella, and
Luminex Salmonella serotyping assay for the characterization of Salmonella enterica identified from fresh and naturally contaminated cilantro. Food Microbiol 2014;42:181–187.
Jenal U, Malone J. Mechanisms of cyclic-di-GMP signaling in bacteria. Annu Rev Genet 2006;40:385–407.
Ji R, Li YJ, Wang YP, Cui SH, Jiang T. [Comparison of multilocus sequence typing system and pulsed-field gel electrophoresis in typing of Salmonella enteritidis]. Zhonghua Liu
Xing Bing Xue Za Zhi 2006;27:1065–1068. (in Chinese.)
Kennedy MJ, Yancey Jr. RJ, Sanchez MS, Rzepkowski RA,
Kelly SM, Curtiss III R. Attenuation and immunogenecity of
Dcya Dcrp derivatives of Salmonella choleraesuis in pigs.
Infect Immun 1996;67:4628–4636.
Le Hello S, Harrois D, Bouchrif B, et al. Highly drug-resistant
Salmonella enterica serotype Kentucky ST198-X1: A microbiological study. Lancet Infect Dis 2013;13:672–679.
Lettini AA, Saccardin C, Ramon E, et al. Characterization of an unusual Salmonella phage type DT7a and report of a foodborne outbreak of salmonellosis. Int J Food Microbiol 2014;
189:11–17.
Liebana E, Clouting C, Garcia-Migura L, et al. Multiple genetic typing of Salmonella Enteritidis phage-types 4, 6, 7, 8 and
13a isolates from animals and humans in the UK. Vet Microbiol 2004;100:189–195.
Liebana E, Garcia-Migura L, Clouting C, et al. Investigation of the genetic diversity among isolates of Salmonella enterica serovar Dublin from animals and humans from England,
Wales and Ireland. J Appl Microbiol 2002;93:732–744.
Lindstedt B-A, Vardund T, Aas L, Kapperud G. Multiple-locus variable-number tandem-repeats analysis of Salmonella enterica subsp. enterica serovar Typhimurium using PCR multiplexing and multicolor capillary electrophoresis. J Microbiol Methods 2004;59:163–172.
Liu WB, Liu B, Zhu XN, Yu SJ, Shi XM. Diversity of Salmonella isolates using serotyping and multilocus sequence typing. Food Microbiol 2011;28:1182–1189.
Lory S, Wolfgang M, Lee V, Smith R. The multi-talented bacterial adenylate cyclases. Int J Med Microbiol 2004;
293:479–482.
Malcova M, Karasova D, Rychlik I. aroA and aroD mutations influence biofilm formation in Salmonella Enteritidis. FEMS
Microbiol Lett 2009;291:44–49.
McClelland M, Sanderson K, Spieth J, et al. Complete genome sequence of Salmonella enterica serovar Typhimurium LT2.
Nature 2001;413:852–856.
McQuiston J, Waters R, Dinsmore B, Mikoleit M, Fields P.
Molecular determination of H antigens of Salmonella by use of a microsphere-based liquid array. J Clin Microbiol 2011;
49:565–573.
Morales CA, Musgrove M, Humphrey TJ, Cates C, Gast R, GuardBouldin J. Pathotyping of Salmonella enterica by analysis of single-nucleotide polymorphisms in cyaA and flanking 23S ribosomal sequences. Environ Microbiol 2007;9:1047–1059.
Ogle JM, Ramakrishnan V. Structural insights into translational fidelity. Annu Rev Biochem 2005;74:129–177.
Olson AB, Andrysiak AK, Tracz DM, et al. Limited genetic diversity in Salmonella enterica serovar Enteritidis PT13.
BMC Microbiol 2007;7:87.
Rehman MA, Ziebell K, Nash JH, et al. High-quality draft wholegenome sequences of 162 Salmonella enterica subsp. enterica
Serovar Enteritidis strains isolated from diverse sources in Canada. Genome announcements 2014;2: pii: e00348-14.
Romling U. Small molecules with big effects: Cyclic di-GMPmediated stimulation of cellulose production by the amino acid L-arginine. Sci Signal 2015;8:fs12.
Seong WJ, Kwon HJ, Kim TE, Lee DY, Park MS, Kim JH.
Molecular serotyping of Salmonella enterica by complete rpoB gene sequencing. J Microbiol 2012;50:962–969.
Song Y, Roumagnac P, Weill FX, et al. A multiplex single nucleotide polymorphism typing assay for detecting mutations that result in decreased fluoroquinolone susceptibility in
Salmonella enterica serovars Typhi and Paratyphi A. J Antimicrob Chemother 2010;65:1631–1641.
Stepan RM, Sherwood JS, Petermann SR, Logue CM. Molecular and comparative analysis of Salmonella enterica Senftenberg from humans and animals using PFGE, MLST and
NARMS. BMC Microbiol 2011;11:153.
Sukhnanand S, Alcaine S, Warnick LD, et al. DNA sequencebased subtyping and evolutionary analysis of selected Salmonella enterica serotypes. J Clin Microbiol 2005;43:3688–3698.
Swaminathan B, Barrett TJ, Hunter SB, Tauxe RV. PulseNet: The molecular subtyping network for foodborne bacterial disease surveillance, United States. Emerg Infect Dis 2001;7:382–389.
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S.
MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and meximum parsimony methods. Mol Biol Evol 2011;28:2731–2739.
Tankouo-Sandjong B, Sessitsch A, Liebana E, et al. MLST-v, multilocus sequence typing based on virulence genes, for molecular typing of Salmonella enterica subsp. enterica serovars. J Microbiol Methods 2007;69:23–36.
Tedin K, Norel F. Comparison of DeltarelA strains of Escherichia coli and Salmonella enterica serovar Typhimurium suggests a role for ppGpp in attenuation regulation of branched-chain amino acid biosynthesis. J Bacteriol 2001;183:6184–6196.
Thomson N, Clayton D, Windhorst D, et al. Comparative genome analysis of Salmonella Enteritidis PT4 and Salmonella
Gallinarum 287/91 provides insights into evolutionary and host adaptation pathways. Genome Res 2008;18:1624–1637.
Threlfall EJ, Chart H, Ward LR, de Sa JD, Rowe B. Interrelationships between strains of Salmonella enteritidis belonging to phage types 4, 7, 7a, 8, 13, 13a, 23, 24 and 30.
J Appl Bacteriol 1993;75:43–48.
Van Stelten A, Simpson JM, Ward TJ, Nightingale KK. Revelation by single-nucleotide polymorphism genotyping that mutations leading to a premature stop codon in inlA are common among Listeria monocytogenes isolates from readyto-eat foods but not human listeriosis cases. Appl Environ
Microbiol 2010;76:2783–2790.Versalovic J, Lupski JR. Molecular detection and genotyping of pathogens: More accurate and rapid answers. Trends Microbiol 2002;10:s15–s21.
Ward TJ, Ducey TF, Usgaard T, Dunn KA, Bielawski JP. Multilocus genotyping assays for single nucleotide polymorphismbased subtyping of Listeria monocytogenes isolates. Appl
Environ Microbiol 2008;74:7629–7642.
Wolfgang MC, Lee VT, Gilmore ME, Lory S. Coordinate regulation of bacterial virulence genes by a novel adenylate cyclasedependent signaling pathway. Dev Cell 2003;4:253–263.
Zhang X, Kelly SM, Bollen W, Curtiss III R. Protection and immune responses induced by attenuated Salmonella typhimurium UK-1 strains. Microb Pathog 1996;26:121–130.
Zheng J, Pettengill J, Strain E, et al. Genetic diversity and evolution of Salmonella enterica serovar Enteritidis strains with different phage types. J Clin Microbiol 2014;52:1490–
1500.