Cephalosporin and ciprofloxacin resistance in Salmonella, Taiwan

Emerging Infectious Diseases, June, 2005 by Jing-Jou Yan, Chien-Shun Chiou, Tsai-Ling Yang Lauderdale, Shu-Huei Tsai, Jiunn-Jong Wu

We report the prevalence and characteristics of Salmonella strains resistant to ciprofloxacin and extended-spectrum cephalosporins in Taiwan from January to May 2004. All isolates resistant to extended-spectrum cephalosporins carried [bla.sub.CMY-2], and all ciprofloxacin-resistant Salmonella enterica serotype Choleraesuis isolates related.

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Resistance to extended-spectrum cephalosporins (ESCs) or fluoroquinolones in Salmonella enterica has become a global concern (1). ESC resistance in Salmonella strains is usually due to the production of plasmid-mediated extended-spectrum [beta]-lactamases (ESBLs) or AmpC [beta]-lactamases, and among these [beta]-lactamases, the CMY-2 AmpC enzyme has been reported most often (1-3). Resistance to fluoroquinolones in Salmonella strains is usually due to the accumulation of mutations in the quinolone resistance--determining regions (QRDRs) of DNA gyrase genes (1,4,5). Resistance to both ESCs and fluoroquinolones remains extremely rare in salmonellae.

In Taiwan, increasing resistance to fluoroquinolones and the emergence of CMY-2--producing ESC-resistant strains in salmonellae have been noted (3-6). The emergence of Salmonella strains resistant to both ceftriaxone and ciprofloxacin was reported more recently in Taiwan and may pose a serious therapeutic problem (7,8). We conducted the present study to investigate the prevalence and characteristics of Salmonella strains resistant to ciprofloxacin and ESCs in Taiwan.

The Study

From January to May 2004, a total of 600 Salmonella isolates from 585 patients were obtained from 5 medical centers and 14 district hospitals throughout Taiwan; these isolates were serotyped with commercial antisera (Difco, Detroit, MI, USA). The 4 most common serotypes of Salmonella enterica (Enteritidis, Typhimurium, Stanley, and Choleraesuis) accounted for 66.8% of all isolates. Two isolates were untypeable, and the remainder were typed into 42 serotypes (data not shown), which were each represented by 1 to 23 isolates.

MICs of antimicrobial agents were determined by the agar dilution method (9). Resistance to ciprofloxacin (MIC [greater than or equal to] 4 [micro]g/mL) was seen in 50 (8.3%) isolates (Table 1); 20 (3.3%) were resistant (MICs ranging from 8 to >64 [micro]g/mL) to ceftazidime, ceftriaxone, cefotaxime, or aztreonam (Table 2); 6 isolates showed decreased susceptibilities to 1 or 2 of the 4 ESCs (MICs 0.5-2 [micro]g/mL); 10 (1.7%) isolates were resistant to both ciprofloxacin and ESCs. S. Choleraesuis had high rates of resistance to ciprofloxacin (84.4%), ESCs (17.8%), and both (17.8%). None of the 26 Salmonella isolates with resistance or decreased susceptibility to ESCs produced ESBL, according to the double-disk synergy method (10). Among the 20 ESC-resistant isolates, 10 isolates were ciprofloxacin-resistant, 4 isolates showed decreased susceptibility to ciprofloxacin (MIC 0.25-1 [micro]g/mL) and resistance to nalidixic acid, and 6 isolates were susceptible to ciprofloxacin and nalidixic acid (Table 2). All 20 ESC-resistant isolates were susceptible to cefepime (MIC <0.03 [micro]g/mL) and imipenem (MIC <1 [micro]g/mL), and 17 isolates were resistant to >1 non-[beta]-lactam agent.

All 20 ESC-resistant isolates expressed a [beta]-lactamase of pI 9.0 by isoelectric focusing (3,11); 11 of these isolates expressed an additional pI 5.4 [beta]-lactamase (Table 2). [bla.sub.CMY-2] was detected in all ESC-resistant isolates, [bla.sub.TEM-1] was detected in the 11 isolates with the pI 5.4-[beta]-lactamase by polymerase chain reaction (PCR) and sequence analyses with the primers for the entire [bla.sub.TEM]-related and [bla.sub.CMY-2]-related structural genes (2,3).

The QRDR sequences of gyrA, gyrB, parC, and parE of the 20 ESC-resistant Salmonella isolates were determined by PCR and sequence analyses (5). All 10 ciprofloxacin-resistant isolates showed 2 mutations at the Ser-83 and Asp-87 codons in gyrA and a single mutation at the Ser-80 codon in parC (Table 2). Four isolates with decreased susceptibility to ciprofloxacin had a single mutation at either the Ser-83 or the Asp-87 codon in gyrA. All 20 ESC-resistant isolates showed no mutations in the QRDRs of gyrB and parE.

ESC resistance was transferred from 18 of the 20 ESC-resistant Salmonella isolates to Escherichia coli C600 in the liquid mating-out assay (3,12). All transconjugants showed decreased susceptibilities to the 4 ESCs tested (MICs 16-64 [micro]g/mL) and cefoxitin (MIC 64-128 [micro]g/mL) and were susceptible to all non-[beta]-lactam agents tested. A pI 9.0 vz [beta]-lactamase and [bla.sub.CMY-2] were detected by isoelectric focusing and PCR assays, respectively, in all transconjugants. Restricted by the endonuclease EcoRI, the 18 transferred plasmids produced 9 major restriction patterns (Figure 1 and Table 2). Patterns E and I were further divided into 4 and 2 subtypes, respectively, [bla.sub.CMY-2] on the transferred plasmids was demonstrated by Southern hybridization with the [bla.sub.CMY-2] probe.

[FIGURE 1 OMITTED]

The 38 ciprofloxacin-resistant S. Choleraesuis isolates were genotyped by pulsed-field gel electrophoresis on a CHEF Mapper apparatus (Bio-Rad Laboratories, Hercules, CA, USA) according to the PulseNet protocol (13). Banding patterns generated by XbaI restriction were compared with BioNumerics software (Applied Maths, Kortrijk, Belgium). The 38 isolates showed a close relationship (Dice correlation coefficient of 90%) and had only 1 pulsotype, based on Tenover criteria (Figure 2) (14). The pulsotype was divided into 7 pulsosubtypes, among which were 1-4 band differences. Five ESC-resistant isolates displayed the same pulsosubtypes (IA or IC) as ESC-susceptible isolates (Table 1 and Figure 2).