Nontyphoidal salmonella, a foodborne pathogen, causes human gastroenteritis worldwide. Among >2,500 different Salmonella enterica serotypes, Salmonella Enteritidis and Salmonella Typhimurium are the most common serotypes responsible for human salmonellosis (1). In Europe, Salmonella Virchow has recently increased in prevalence, and a high proportion of isolated strains are resistant to multiple antimicrobial drugs (2–4).

Third-generation cephalosporins are widely used to treat major bacterial infections in humans and animals (5). However, the emergence and rapid spread of drug-resistant bacteria has become a serious public health concern. Extended-spectrum β-lactamases (ESBLs) are known to confer antimicrobial drug resistance by hydrolyzing most β-lactam antimicrobial drugs, including third-generation cephalosporins (5). Since the first report from Spain in 2000 of strains producing CTX-M-9 (6), which confers resistance to cefotaxime, various CTX-M–type ESBLs have been identified in Salmonella Virchow. In Spain, Belgium, and France, CTX-M-9 and CTX-M-2 producers spread clonally in humans and poultry (7,8). In addition, the bla CTX-M-15 gene was identified in porcine isolates in South Korea (9). These reports demonstrate that CTX-M–producing Salmonella Virchow clones can be easily transmitted to humans through food products of animal origin. In South Korea, the incidence of Salmonella Virchow infections in humans has increased over the years, necessitating a nationwide survey of antimicrobial drug resistance in Salmonella Virchow isolates.

The Study

Figure

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.

We used the broth microdilution method (10) to perform antimicrobial susceptibility testing of Salmonella Virchow; results showed that 54 (79.4%) of the 68 isolates were resistant to >1 of the 15 antimicrobial agents tested (Table; Technical Appendix Figure). The highest resistance rates were noted for nalidixic acid (77.9%), followed by ampicillin (44.1%), cefotaxime (44.1%), tetracycline (42.6%), and gentamicin (23.5%). Only 2 (2.9%) isolates were resistant to ciprofloxacin, but 50 (73.5%) had intermediate resistance. All of the isolates were susceptible to chloramphenicol, amikacin, or imipenem. Multidrug resistance, defined as resistance to at least 3 different classes of antimicrobial agents, was found in 30 (44.1%) isolates (Table).

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. 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% (Technical Appendix Figure), indicating the clonality of cefotaxime-resistant strains.