Molecular Typing and Epidemiological Study of Salmonella enterica Serotype Typhimurium Isolates from Cattle by Fluorescent Amplified-Fragment Length Polymorphism Fingerprinting and Pulsed-Field Gel Electrophoresis

Abstract

One hundred twenty Salmonella enterica serotype Typhimurium strains, including 103 isolates from cattle gathered between 1977 and 1999 in the prefecture located on the northern-most island of Japan, were analyzed by using fluorescent amplified-fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis (PFGE) to examine the genotypic basis of the epidemic. Among these strains, there were 17 FAFLP profiles that formed four distinct clusters (A, B, C, and D). Isolates that belonged to cluster A have become increasingly common since 1992 with the increase of bovine salmonellosis caused by serotype Typhimurium. PFGE resolved 25 banding patterns that formed three distinct clusters (I, II, and III). All the isolates that belonged to FAFLP cluster A, in which all the strains of definitive phage type 104 examined were included, were grouped into PFGE cluster I. Taken together, these results indicate that clonal exchange of serotype Typhimurium has taken place since 1992, and they show a remarkable degree of homogeneity at a molecular level among contemporary isolates from cattle in this region. Moreover, we have sequenced two kinds of FAFLP markers, 142-bp and 132-bp fragments, which were identified as a polymorphic marker of strains that belonged to clusters A and C, respectively. The sequence of the 142-bp fragment shows homology with a segment of P22 phage, and that of the 132-bp fragment shows homology with a segment of traG, which is an F plasmid conjugation gene. FAFLP is apparently as well suited for epidemiological typing of serotype Typhimurium as is PFGE, and FAFLP can provide a source of molecular markers useful for studies of genetic variation in natural populations of serotype Typhimurium.Salmonella infections in livestock have been a concern for both animal and human health. In particular, a common serotype causing salmonellosis in humans is Salmonella enterica serotype Typhimurium, a globally distributed zoonotic serotype that is common in both cattle and poultry. In order to study the epidemiology of its outbreaks and determine the source of contamination so that a recurrence can be avoided, detailed characterization is necessary. Although the majority of outbreaks in livestock are caused by a select number of serotypes, serotyping is not an adequate method for determination of the source of contamination during an outbreak. One subtyping method for epidemiological investigations of human and animal salmonellosis outbreaks is phage typing (3), which discriminates phenotypically at the intraserotype level. However, phage typing requires access to special reagents and a specialized laboratory and fails to reflect evolutionary relationships of bacterial strains. In the last decade, with the development of new techniques in molecular biology techniques, new approaches have become available. Widely used are plasmid analysis (29, 39), chromosomal fingerprinting by Southern hybridization (12,16,31,36,37), and macrorestriction analysis of chromosomal DNA by pulsed-field gel electrophoresis (PFGE) (...

Caracterización por electroforesis de campo pulsado de aislamientos de Salmonella Typhimurium recuperados en el programa de vigilancia de enfermedad diarreica aguda en Colombia, 1997-2004

“…El alto poder discriminatorio de la PFGE aplicada a los 468 aislamientos de S. Typhimurium estudiados no constituyó una sorpresa, ya que la PFGE ha sido exitosa en la subtipificación de Salmonella spp., según se informa en varios estudios (10,13,(24)(25)(26). Nuestro estudio confirma que, en las regiones donde S. Typhimurium es altamente endémico, la PFGE es de gran utilidad epidemiológica para la caracterización y comparación de aislamientos de este serotipo, a diferencia de los métodos de vigilancia convencionales que carecen de la sensibilidad para detectar aislamientos relacionados con brotes de S.Typhimurium, especialmente cuando involucran un número reducido de casos con cercanía en el tiempo.…”

“…Las técnicas de subtipificación molecular como la electroforesis en gel de campo pulsado (PFGE), considerada la prueba de oro para la diferenciación y el agrupamiento de aislamientos de Salmonella spp. de un serotipo específico, la cual es en la actualidad el método más ampliamente utilizado en las redes de vigilancia de enfermedades transmitidas por alimentos (12)(13)(14)(15)(16).…”

Use of AFLP and PFGE to discriminate between Salmonella enterica serovar Typhimurium DT126 isolates from separate food-related outbreaks in Australia

“…AFLP has been used for a variety of analyses of Salmonella, from separation of serotypes and serovar phage types [30] to the determination of genetic variability of multidrug-resistant isolates [29] and separation of serovar Typhimurium phage types and detection of potential markers for phage typing by molecular methods [3,9]. AFLP offers a level of discrimination at least equal to that of PFGE (or any other typing methods) and often provides a greater level of separation of individual isolates (although there are some exceptions to this rule).…”

“…Typhimurium is routinely subtyped by phage typing where resistance or sensitivity to a panel of typing phage determines phage type [8]. Phage typing provides a first level of isolate discrimination ; however, it requires a specialist laboratory and can be highly subjective [9]. The predominance of one particular phage type within a serovar in a geographical and/or temporal region often reduces the effectiveness of this method in discriminating between outbreak strains and epidemiologically unrelated isolates.…”

“…More recently amplified fragment length polymorphism (AFLP) has been developed to separate bacterial isolates including Salmonella at the genetic level [3,7,9,[15][16][17][18]. In this procedure, PCR primers target specific sequences ligated to either end of restriction endonuclease-digested genomic DNA rather than genomic DNA itself, generating a high number of fragments for analysis.…”