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Rapid Emergence and Clonal Dissemination of CTX-M-15–Producing Salmonella enterica Serotype Virchow, South Korea

The prevalence of cefotaxime-resistant Salmonella enterica serotype Virchow has dramatically increased in South Korea since the first isolation in 2011. Of 68 isolates collected over 10 years, 28 cefotaxime-resistant isolates harbored the blaCTX-M-15 extended-spectrum β-lactamase gene and were closely related genetically, demonstrating the clonal dissemination of CTX-M-15–producing Salmonella Virchow in South Korea.


The Study
During 2005-2014 in South Korea, local public health laboratories participating in the national surveillance network isolated 68 Salmonella Virchow strains from feces samples from patients with acute diarrhea. Until 2010, <5 Salmonella Virchow strains were isolated per year, but this number gradually increased to 17 in 2014 (Figure). Salmonella Virchow consistently ranked among the top 10 serotypes in prevalence during each study year in South Korea, accounting for ≈1.5%-2% of salmonellosis cases.
All multidrug-resistant isolates showed resistance to third-generation cephalosporins. In South Korea, cefotaxime-resistant strains were first isolated in 2011. Since then, 4, 9, and 14 isolates were collected in 2012, 2013, and 2014, respectively ( Figure). The rates of cefotaxime resistance in Salmonella Virchow have increased markedly, from 21.4% in 2011 to 82.3% in 2014. This annual trend of increasing cefotaxime resistance in South Korea is of interest because the rates were substantially higher than those reported in Spain during 2002-2006 (15%) (11) and Belgium during 2009-2013 (<10%) (12). Moreover, even in Israel and Switzerland, where the incidence of Salmonella Virchow was relatively higher than that in South Korea, antimicrobial drug resistance to third-generation cephalosporins was rare (3,4).
Among the 30 cefotaxime-resistant Salmonella Virchow isolates, 28 were confirmed to be ESBL-producers.

Rapid Emergence and Clonal Dissemination of CTX-M-15-Producing Salmonella enterica Serotype Virchow, South Korea
1 These co-first authors contributed equally to this article. PCR and sequencing of β-lactamase genes (13) confirmed that these 28 isolates harbored a bla CTX-M-15 gene, whereas the other 2 contained a bla CMY-2 gene. Cefotaxime resistance was transferred by conjugation from 9 Salmonella Virchow isolates to Escherichia coli J53 recipients, and the bla CTX-M-15 gene was identified in transconjugants. Southern blotting and PCR-based replicon typing (14) showed that all plasmids in transconjugants were ≈215 kb in size and possessed an IncHI2 plasmid, which was further assigned to sequence type (ST) 2 by plasmid double locus sequence typing (15). The analysis of the genetic environment surrounding the bla CTX-M-15 gene (13) in transconjugants showed that insertion sequences ISEcp1 and orf477 were detected 48 bp upstream and downstream of the bla CTX-M-15 gene, respectively. This ISEcp1-bla CTX-M-15 -open reading frame 477 transposable unit was also identified in other incompatibility groups of the plasmids in Enterobacteriaceae. Furthermore, it was identical to that of the ST2-IncHI2 plasmid of Salmonella Enteritidis isolated from humans and poultry meat in South Korea (J. Kim, unpub. data), suggesting that the bla CTX-M-15 gene in Salmonella Virchow might have originated from ISEcp1-mediated transposition followed by interspecies spread through the IncHI2-type plasmid studied here. All of the CTX-M-15-producing strains had reduced ciprofloxacin susceptibility (MICs of 0.25-0.5 µg/mL). All 10 randomly selected isolates harbored a single substitution within the quinolone resistance-determining region of GyrA at codon 83 (Ser→Phe), which is the major mutation described in Salmonella species (8). Because fluoroquinolones and third-generation cephalosporins are the drugs of choice for treating severe salmonella infections in humans, the reduced ciprofloxacin susceptibility in cefotaxime-resistant Salmonella Virchow is considered a critical risk factor for infections with these strains.
The genetic relationship of the 68 Salmonella Virchow isolates was determined by using multilocus sequence typing (http://mlst.warwick.ac.uk/mlst/dbs/Senterica) and pulsed-field gel electrophoresis (PFGE) according to a standardized protocol. Seven multilocus sequence typing loci displayed 4 different profiles, and most isolates belonged to sequence type (ST) 16 (n = 59), followed by ST197 (n = 6), ST359 (n = 2), and ST426 (n = 1). All of the 28 CTX-M-15-producing strains were typed as ST16, but the cefotaxime-susceptible isolates were also assigned to this type. The PFGE analysis yielded sufficient discriminatory power in typing Salmonella Virchow isolates; 22 XbaI and 21 BlnI PFGE patterns were generated. Although the isolates shared >70% similarity, CTX-M-15-producing strains clustered on the basis of a similarity value of 90% (online Technical Appendix Figure), indicating the clonality of cefotaxime-resistant strains.
For humans, the main route of Salmonella infection is the consumption of contaminated food of animal origin, and Salmonella Virchow is one of the most prevalent serotypes identified in poultry and poultry products. The use of cephalosporins in animal production has led to emergence of antimicrobial drug-resistant Salmonella Virchow clones among food animals (8), posing a threat to public health because of the possible transmission of these bacteria through the food chain. In fact, the XbaI PFGE pattern identified among human isolates in this study was identical to that in 2 cefotaxime-resistant Salmonella Virchow strains isolated in 2012 from poultry meat in our collection (online Technical Appendix Figure). Furthermore, this pattern appeared similar to the patterns of Salmonella Virchow harboring the bla CTX-M-15 gene on an ST2-IncHI2 plasmid that was isolated from organically raised pigs in South Korea (9). Resistant clinical strains were collected from 13 of 15 local public health laboratories in South Korea during 2011-2014; thus, although the mode of the spread of Salmonella Virchow in humans is not established, it has been postulated that CTX-M-15-producing Salmonella Virchow might have disseminated clonally through the nationwide distribution of contaminated food products rather than through independent emergence.

Conclusions
We analyzed the antimicrobial drug susceptibility and genetic relatedness of Salmonella Virchow isolates from patients with diarrhea in South Korea. Of 68 isolates obtained during 2005-2014, a total of 30 were resistant to third-generation cephalosporins. The prevalence rate of the resistant strains has dramatically increased since the isolation of cefotaxime-resistant strains in 2011. These findings suggest that the cefotaxime-resistant isolates are genetically closely related and harbor a plasmid carrying the bla CTX-M-15 gene of the same compatibility group (ST2-IncHI2), representing clonal dissemination of CTX-M-15-producing Salmonella Virchow in South Korea and posing an urgent threat to public health. Therefore, more comprehensive surveillance is required to prevent further spread of resistant clonal strains.