Molecular Epidemiology of Salmonella enterica Serovar Typhimurium Isolates from Cattle in Hokkaido, Japan: Evidence of Clonal Replacement and Characterization of the Disseminated Clone

Abstract

The molecular epidemiology of 545 Salmonella enterica serovar Typhimurium isolates collected between 1977 and 2009 from cattle in Hokkaido, Japan, was investigated using pulsed-field gel electrophoresis (PFGE). Nine main clusters were identified from 116 PFGE patterns. Cluster I comprised 248 isolates, 243 of which possessed a sequence specific to definitive phage type 104 (DT104) or U302. The cluster I isolates were dominant in 1993 to 2003, but their numbers declined beginning in 2004. Beginning in 2002, an increase was observed in the number of cluster VII isolates, consisting of 21 PFGE patterns comprising 165 isolates. A total of 116 isolates representative of the 116 PFGE profiles were analyzed by multilocus variable-number tandem-repeat analysis (MLVA). Other than two drug-sensitive isolates, 19 isolates within cluster VII were classified in the same cluster by MLVA. Among the cluster VII isolates, an antibiotic resistance type showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, kanamycin, cefazolin, and sulfamethoxazole-trimethoprim and a resistance type showing resistance to ampicillin, streptomycin, sulfonamides, tetracycline, and kanamycin were found in 23 and 125 isolates, respectively. In the 19 isolates representative of cluster VII, the bla TEM-1 gene was found on a Salmonella serotype Typhimurium virulence plasmid, which was transferred to Escherichia coli by electroporation along with resistance to two to four other antimicrobials. Genomic analysis by subtractive hybridization and plasmid analysis suggested that the bla TEM-1 -carrying virulence plasmid has a mosaic structure composed of elements of different origin. These results indicate an emerging multidrug-resistant S. Typhimurium clone carrying a virulence-resistance plasmid among cattle in Hokkaido, Japan.Salmonella enterica serovar Typhimurium is a common cause of salmonellosis in humans and animals. Detailed characterization of this bacterium is necessary for studying the epidemiology of outbreaks and determining the source of contamination to avoid recurrence. Phage typing is a commonly used method for epidemiological surveillance of S. Typhimurium infection (2), but it requires special reagents and a specialized laboratory and fails to reflect the evolutionary relationships of bacterial strains. Over the last decade, new techniques in molecular biology have been developed, and new approaches have become available. Pulsed-field gel electrophoresis (PFGE) is now the gold standard for discriminating among strains at the DNA level (32). However, multiple-locus variable-number tandem-repeat analysis (MLVA), based on amplification of a variable number of tandem repeat areas, is considered to have greater discriminatory power than PFGE and has been proposed as an alternative for genotyping highly clonal groups of bacteria (18,19). Molecular subtyping of S. Typhimurium isolates by standard procedures and development of a DNA fingerprinting database of these isolates would assist in identifying Salmonel...

Complete Nucleotide Sequences of Virulence-Resistance Plasmids Carried by Emerging Multidrug-Resistant Salmonella enterica Serovar Typhimurium Isolated from Cattle in Hokkaido, Japan

“…Typhimurium DT104 increased, particularly in adult cattle in Hokkaido, Japan [8]. In 2002, we identified a non-DT104 multidrug-resistant clone with a pulsed-field gel electrophoresis (PFGE) pattern designated PFGE cluster VII and no recognized phage-type disseminating among cattle in Hokkaido [8]. These isolates carry the bla TEM-1 gene encoding TEM-1 β-lactamase on S .…”

Change in Antimicrobial Resistance Pattern in Salmonella enterica serovar Typhimurium Isolates Detected in a Beef Cattle Farm

“…The replacement of penta-resistant S. Typhimurium DT104 by a novel group of genetically related multidrug-resistant S. Typhimurium showing pulsed-field gel electrophoresis (PFGE) pattern cluster VII (ST-VII) has been reported in Hokkaido, Japan [10]. The ST-VII isolates were detected for the first time in 2000 and have been dominant since 2002 among bovine isolates in this area.…”

“…Serovar identification was performed by microtitre and slide agglutination methods according to the latest version of the Kaufmann and White scheme [8] using antisera (Denka Seiken, Tokyo, Japan). Macrorestriction analysis using XbaI endonuclease (Takara Bio, Shiga, Japan) was performed by PFGE according to the PulseNet protocol and interpreted as described previously [10].…”

“…These polymorphisms can be explained by one or two genetic differences, suggesting a close relationship among the isolates [11]. These isolates were classified in cluster VII based on the macrorestriction patterns (data not shown) [10].…”

Antimicrobial Resistance in Salmonella Strains Clinically Isolated in Hyogo, Japan (2009^|^ndash;2012)

“…1B), and there was no difference in subtypes between the antimicrobial-sensitive and -resistant strains. In Hokkaido, Japan, the number of S. Typhimurium isolates of bovine-origin that showed multidrug resistance in the same PFGE cluster was increased (10). Therefore, we need to carefully monitor the spread of this multidrug resistant S. Enteritidis with the CTX-M-2 type ESBL and gyrA and parC mutations.…”