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Molecular typing of Salmonella enterica serovar Enteritidis isolates from food-producing animals in Japan by multilocus variable-number tandem repeat analysis: evidence of clonal dissemination and replacement
© Kobayashi et al.; licensee BioMed Central Ltd. 2014
Received: 21 October 2013
Accepted: 9 May 2014
Published: 13 May 2014
Salmonella enterica serovar Enteritidis is a zoonotic pathogen. Human infections are associated with contaminated eggs and egg products. In Japan, since 1989, the incidence of food-borne disease caused by S. Enteritidis has increased and a pandemic has occurred; however, little is known about changes that occurred before and after this pandemic event in the dominant lineage of isolates from food-producing animals. This study aimed to determine the S. Enteritidis lineages in Japan over the last few decades by using multilocus variable-number tandem repeat analysis (MLVA).
MLVA was used to analyse 79 S. Enteritidis isolates collected from chickens (n = 63), cattle (n = 12), pigs (n = 2), and goats (n = 2) during 1975–2009. The S. Enteritidis isolates showed 14 different MLVA allele combinations, which were classified into two major clusters (A and C) and a minor cluster (B). All the 62 isolates in cluster A were isolated after 1988, whereas 13 of the 17 isolates belonging to cluster B and C were isolated before 1989.
The MLVA results showed that cluster C was predominant before 1989, and isolates in cluster A disseminated since 1989 and replaced the previous dominant clone, suggesting that isolates of cluster A originated from imported S. Enteritidis infection.
Salmonella enterica serovar Enteritidis is a zoonotic pathogen that can be transmitted to humans via many different reservoir hosts. Most outbreaks of S. Enteritidis infections have been associated with contaminated eggs and egg products . Until the 1980s, S. Typhimurium was the serovar most commonly isolated from humans worldwide; however, during the 1980s and 1990s, S. Enteritidis emerged as a common cause of salmonellosis, first in European countries and then worldwide [2–4].
Similarly, in Japan, the serotype S. Enteritidis has been isolated most frequently, since 1989, from patients with food-borne illnesses . Moreover, S. Enteritidis phage type PT8 was the most prevalent human isolate reported until 1988, until PT34 became the predominant phage type; PT1 and PT4 have replaced PT34 as the predominant phage types since 1992 . However, little is known about changes that have occurred over the last few decades in the dominant lineage of isolates from food producing animals in Japan.
Phage typing is a commonly used method for epidemiological surveillance of S. Enteritidis infection ; however, it requires specialized reagents and laboratory equipment and does not always yield sufficient information for epidemiological purposes. Over the last decade, new techniques in molecular biology have been developed, such as pulsed-field gel electrophoresis (PFGE) [7, 8], 2-enzyme ribotyping (Pst I-Sph I) , and multilocus variable-number tandem repeat analysis (MLVA) [10, 11]. Of these methods, PFGE is now the gold standard for discriminating among strains at the DNA level . However, based on amplification of a variable number of tandem repeat areas, MLVA is considered to have greater discriminatory power than PFGE and has been proposed as an alternative for genotyping of S. Enteritidis [10, 11].
In this study, MLVA genotyping was performed to determine the lineages of S. Enteritidis before and after pandemics during the 1980s and 1990s. We have provided evidence that clonal dissemination and replacement have occurred among isolates from food-producing animals since 1989.
The 79 S. Enteritidis isolates included 14 different MLVA profiles (Figure 1). Nine distinct MLVA profiles were identified from chicken isolates, 7 were identified from cattle, 2 from goats, and 2 from pigs. A dendrogram was generated using the categorical coefficient and unweighted pair group method with arithmetic means by the BioNumerics software (version 6.0). MLVA profiles were classified into 3 clusters (A, B, and C) delineated with a 67.8% similarity cutoff value (Figure 1). Cluster A consisted of 7 profiles corresponding to 62 isolates, including 58 isolates from chickens, 2 isolates from goats, 1 isolate from cattle, and 1 isolate from pig. Cluster B included 2 isolates from cattle, and cluster C, which was composed of 6 MLVA profiles representing 15 isolates, included 9 isolates from cattle, 5 isolates from chickens, and 1 isolate from pigs.
Since MLVA profiles can be easily stored in a database, routine and long-term epidemiological surveillance with such a method may enable early recognition of potentially epidemic Salmonella clones.
This work was supported by a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan (research project ensuring food safety from farm to table LP-5201).
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