Increasing Ceftriaxone Resistance in Salmonellae, Taiwan

In Taiwan, despite a substantial decline of Salmonella enterica serotype Choleraesuis infections, strains resistant to ciprofloxacin and ceftriaxone persist. A self-transferable blaCMY-2-harboring IncI1 plasmid was identified in S. enterica serotypes Choleraesuis, Typhimurium, Agona, and Enteritidis and contributed to the overall increase of ceftriaxone resistance in salmonellae.


The Study
Since 1999, computerized records of bacterial culture results have been stored at Chang Gung Memorial Hospital, a 3,500-bed medical center in northern Taiwan. Periodic review of these records indicated a reverse trend in the prevalence of serogroups D (increase) and B (decrease) isolates during the past decade ( Figure 1, panel A). A signifi cant decrease in the prevalence of S. enterica ser. Choleraesuis was also evident (Figure 1, panel A). Nevertheless, in recent years, ceftriaxone resistance has increased from <5% to >10% in S. enterica ser. Choleraesuis and in serogroup B salmonellae (Figure 1, panel B).
Since isolation of SC-B67 in 2002 (4), 10 CIP r /CRO r S. enterica ser. Choleraesuis isolates have been recovered. All CIP r /CRO r isolates were resistant to nalidixic acid and ciprofl oxacin, but SC-B134 remained susceptible to ciprofl oxacin (Table 1). PCR and sequencing with specifi c primers (online Technical Appendix Table 1, www.cdc.gov/ EID/content/17/6/1086-Techapp.pdf) revealed 3 identical amino acid changes in GyrA and ParC among all CIP r /CRO r isolates except SC-B134 (Table 1). Reduced fl uoroquinolone susceptibility of SC-B134 could be explained by the single amino acid change at codon 87 of GyrA. Amino acid changes were not found in GyrB or ParE.
In terms of clinical features (online Technical Appendix Table 2), most patients with these infections were adults who had a wide spectrum of underlying diseases. Antimicrobial agents were prescribed for all patients, and extended-spectrum cephalosporins were used most frequently. Two patients died. Seven blood isolates from the 10 patients with CIP r /CRO r S. enterica ser. Choleraesuis infections, together with the ceftriaxone-resistant isolates noted below, were investigated further. SC-B67 was used as a reference.
During the fi rst 6 months of 2010, a total of 6 cases of ceftriaxone-resistant Salmonella infection were found: serogroup B in 5 patients (S. enterica ser. Agona, n = 1; S.  (Table 1). All isolates were derived from fecal specimens of patients <3 years of age, except S. enterica ser. Typhimurium SB-28, which was isolated from the urine of a 77-year-old patient. In contrast to S. enterica ser. Choleraesuis, these ceftriaxone-resistant isolates generally remained susceptible to fl uoroquinolones (Table 1).
Ceftriaxone resistance was investigated by using PCR and sequencing as described (6). The specifi c bla CMY-2carrying Tn6092 was present in all isolates tested (Table  1). Tn6092 was located within a fi nQ gene at a position identical to that in SC-B67. The only difference was in SC-B134; a 1,338-bp insertion sequence, IS10, was inserted 262 bp upstream of the bla CMY-2 . No CTX-M and SHV genes were found in these isolates.
Using an alkaline lysis method (8), we found various numbers of plasmids among the isolates studied (Table  1). DNA-DNA hybridization indicated that Tn6092 was located on large (85-kb to 150-kb) plasmids (Table 1) (9). An identical Rep_3 replicon was found in all CIP r / CRO r S. enterica ser. Choleraesuis isolates studied (5). Similar to SC-B67, the Rep_3 replicon was located on the Tn6092-harboring resistance plasmid in the 5 resistant isolates recovered before 2004 (Table 1). However, in SC-B134, the Rep_3 replicon was found on the smaller 40-kb plasmid, and in SC-B136, the Rep_3 replicon was on the 115-kb large virulence plasmid that contained the spvC gene (Table 1; Figure 2, panels B, C). Replicons FIIA and FIB were simultaneously present in all the spvCcontaining virulence plasmids among the S. enterica ser. Choleraesuis isolates studied (Table 1). Virulence plasmids in the 2 recent isolates, SC-B134 and SC-B136, appeared larger than those in earlier isolates, including SC-B67 (Table 1; Figure 2, panels B, C). The 2 bands †Antimicrobial drug susceptibility to chloramphenicol, trimethoprim/sulfamethoxazole, and quinolones. Results were the same for the 2 quinolones (nalidixic acid and ciprofloxacin) tested, except that SC-B134 was resistant to nalidixic acid and susceptible to ciprofloxacin. All isolates were resistant to ampicillin and ceftriaxone.
‡Pulsed-field gel electrophoresis; pulsotypes are expressed as serogroup-major type-subtype. demonstrated by the spvC probe in SC-B134 were from the same single virulence plasmid, as proven by hybridization experiments on BamHI-digested plasmid DNA of SC-B134 (data not shown).
Replicon typing through a published multiplex PCR system revealed a replicon I1 from the Tn6092harboring resistance plasmids in SC-B134 and SC-B136 (Table 1; Figure 2, panel C) (10). Similarly, Tn6092carrying plasmids among the other ceftriaxone-resistant salmonellae isolates all belonged to the IncI1 group (Table 1). Conjugation experiments using a fi lter mating method showed that all IncI1 resistant plasmids were selftransferrable (11). With azide-resistant Escherichia coli J53 and S. enterica ser. Typhimurium LBNP4417 as the recipients, the IncI1-resistant plasmids were confi rmed to be self-transferrable.
Subtyping of the 8 conjugated IncI1 plasmids was achieved by using a recently described plasmid multilocus sequence typing (pMLST) method specifi cally set up for IncI1 plasmids (12). Six combinations of allele variants were obtained (Table 2). Because these pMLST patterns differed from those reported elsewhere, 6 new sequence types (STs) were designated ( Table 2). Two major groups were further derived: ST54 (pSB28, pSB193) and ST52 (pSC-B136) that differed only in trbA, and ST56 (pSD166, PSB105) and ST53 (pSB5) that only differed in pilL ( Table  2). pMLST patterns of representative bla CMY-2 -carrying IncI1 plasmids published in recent years (Table 2) were derived from E. coli or various Salmonella serotypes in Europe or North America (12)(13)(14). Nine STs and 2 major clonal complexes, CC-2 and CC-12, were observed. pMLST patterns found in the present study differed from these STs by at least 3 alleles (Table 2).

Conclusions
Resistance to ciprofl oxacin and ceftriaxone remains high, indicating persistence of antimicrobial drug-resistant traits in S. enterica ser. Choleraesuis. The conserved genotypes found in the clinical isolates suggest a mode of clonal dissemination. However, plasmid analysis indicates that the location of the Tn6092-containing resistance element had shifted from the nonconjugative Rep_3 plasmids in early isolates to the self-transferable IncI1 plasmids in recent isolates. The emergence of such self-transferable resistance plasmids seems to provide an effi cient way for S. enterica ser. Choleraesuis to spread its ceftriaxone resistance trait.
Because infections with nontyphoid salmonellae are rampant in Asia, emergence of a conjugative IncI1 resistance plasmid in ceftriaxone-resistant salmonellae from an Asian country is of public health concern. Presence of bla CMY-2carrying IncI1 plasmids in a variety of Salmonella serotypes has been reported, but to our knowledge, not in S. enterica serotypes Enteritidis or Choleraesuis (12)(13)(14). IncI1 plasmids of the same or similar STs have been found in isolates of different bacterial species; with different resistance genes; or from different countries or sources, including human, animals, and the environment (12)(13)(14)(15). Emergence of the IncI1 plasmid in Taiwan represents a need for continuous efforts to monitor and control its further spread.
This work was supported by the grants CMRPG350023, CMRPG381591, and CMRPG460102 from Chang Gung Memorial Hospital, Taoyuan, Taiwan.
Ms Su is an infection control practitioner and clinical microbiologist at the Chang Gung Memorial Hospital and an associate professor at the Chang Gung University College of Medicine. Her research interests include antimicrobial drug resistance mechanisms, molecular epidemiology of infectious diseases, and microbial pathogenesis.