Differential Single Nucleotide Polymorphism-Based Analysis of an Outbreak Caused by Salmonella enterica Serovar Manhattan Reveals Epidemiological Details Missed by Standard Pulsed-Field Gel Electrophoresis

Abstract

We retrospectively analyzed a rare Salmonella enterica serovar Manhattan outbreak that occurred in Italy in 2009 to evaluate the potential of new genomic tools based on differential single nucleotide polymorphism (SNP) analysis in comparison with the gold standard genotyping method, pulsed-field gel electrophoresis. A total of 39 isolates were analyzed from patients (n ‫؍‬ 15) and food, feed, animal, and environmental sources (n ‫؍‬ 24), resulting in five different pulsed-field gel electrophoresis (PFGE) profiles. Isolates epidemiologically related to the outbreak clustered within the same pulsotype, SXB_BS.0003, without any further differentiation. Thirty-three isolates were considered for genomic analysis based on different sets of SNPs, core, synonymous, nonsynonymous, as well as SNPs in different codon positions, by Bayesian and maximum likelihood algorithms. Trees generated from core and nonsynonymous SNPs, as well as SNPs at the second and first plus second codon positions detailed four distinct groups of isolates within the outbreak pulsotype, discriminating outbreak-related isolates of human and food origins. Conversely, the trees derived from synonymous and third-codon-position SNPs clustered food and human isolates together, indicating that all outbreak-related isolates constituted a single clone, which was in line with the epidemiological evidence. Further experiments are in place to extend this approach within our regional enteropathogen surveillance system. Salmonellosis is a major food-borne disease worldwide, with an estimated 93.8 million cases occurring each year, resulting in 155,000 deaths (1). The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks (2) indicated that nontyphoid salmonellosis was the second most reported food-borne zoonosis in Europe in 2012, trailing only behind Campylobacter jejuni infection. The 2012 overall notification rate for human salmonellosis in the European Union (EU) was 22.2 episodes per 100,000 population, for a total of 91,034 confirmed cases, with hospitalization and mortality rates of 45.1% and 0.14%, respectively. The highest proportions of Salmonella-positive foodstuff samples were reported for fresh turkey, poultry, and pork at 4.4%, 4.1%, and 0.7%, respectively (2). In order to manage this food-borne infection and to limit its health and economic burdens, surveillance programs have developed and implemented DNA-based subtyping methods to identify outbreaks in a timely manner and to trace infections back to their food sources. Over the past decades, the two most intensively used protocols for Salmonella subtyping have been pulsed-field gel electrophoresis (PFGE) and multilocus variable-number tandemrepeat analysis (MLVA) (3). Unfortunately, these methods rely on just few features of the entire bacterial genome (rare restriction sites for PFGE or few polymorphic loci for MLVA) to assess the relatedness of different isolates. During epidemiological investigations of food-borne outbreaks, this limitation mi...

Whole genome sequencing analysis of multiple Salmonella serovars provides insights into phylogenetic relatedness, antimicrobial resistance, and virulence markers across humans, food animals and agriculture environmental sources

“…1 ). A number of studies have reported the use of SNP-based analysis as a potential molecular subtyping tool for outbreak investigation in multiple Salmonella serovars including Dublin [ 29 ], Enteritidis [ 22 , 30 ], Heidelberg [ 31 ], Manhattan [ 21 ], Montevideo [ 32 , 33 ], and Typhimurium [ 34 – 36 ].…”

“…The phylogenetic analysis based on WGS-derived SNPs has been shown to provide greater cluster resolution than the gold standard subtyping method, pulsed-field gel electrophoresis (PFGE), resulting in discrimination of outbreak-related human clinical isolates and food or environmental origins [ 21 – 23 ]. ParSNP was run for all the 200 Salmonella genomes and separately for each of the serovars.…”

“…A number of studies have used the classical molecular typing methods such as pulsed-field gel electrophoresis (PFGE), multilocus sequence-based typing (MLST), and multilocus variable-number tandem repeat analysis (MLVA) to assess the relatedness and the subsequent transmission of antimicrobial resistant (AMR) Salmonella in human, animals, and environment [ 19 – 21 ]. However, the limitation of these methods lies in insufficient discriminatory power to separate closely related Salmonella isolates in outbreak investigations and to differentiate between the intra-serovar isolates from different hosts [ 20 – 22 ].…”

Investigation of Outbreaks ofSalmonella entericaSerovar Typhimurium and Its Monophasic Variants Using Whole-Genome Sequencing, Denmark

“…The nature of the outbreak must be taken into account when defining a single outbreak, given that many parameters are possibly affecting the results (e.g., the genetic makeup of the serovar, routes of infection, source types, and time). The SNP analysis and other WGS methods for typing provide a higher discrimination of isolates in comparison with other conventional typing methods ( 12 – 19 ) and gives the advantage of many additional analyses. However, new typing approaches also lead to many new questions on how to interpret data and, in a surveillance context, how to define outbreak cases and sources.…”

“…The vast amount of data provided by this method not only enables high-resolution typing for surveillance but also provides valuable additional data regarding further characterization of emerging clones based on genetic differences and evolutionary studies. Several studies have proven WGS-based typing to have an enhanced discriminatory power in comparison to current molecular typing methods used for Salmonella ( 12 – 19 ), although few studies have evaluated WGS analysis in real-time surveillance. A retrospective study of Salmonella Enteritidis showed that single-nucleotide polymorphism (SNP)–based WGS analysis was suitable for surveillance purposes ( 15 ).…”