Recent reports suggest high-level fluoroquinolone (FQ) resistance is emerging in Salmonella enterica serovar Typhimurium, S. enterica serovar Choleraesuis, and S. enterica serovar Schwarzengrund in different parts of the world (1-9).
In this study we analyzed high-level FQ resistance mechanisms in four S. enterica serovar Schwarzengrund strains which showed resistance levels to ciprofloxacin (Cip) MICs of 16 or 64 μg/ml and were isolated from stools or blood from patients in Taiwan (Table 1). Three of them were also multidrug resistant (Table 1).
TABLE 1.
Characteristics of the S. enterica serovar Schwarzengrund strains studied
| Serovar | Strain | Origina | Year of isolation | Antibiotic resistance profileb | MICs quinolones (μg/ml)b
|
Substitutions in QRDRc
|
||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| NAL | FLU | ENR | MAR | CIP | GyrA | ParC | ParE | |||||
| Schwarzengrund | CGST2 | T | 2000 | Ap Cm Sm Sp Su Tc Tm | >1,024 [512] | >1,024 [64] | 256 [4] | 32 [4] | 64 [16] | Ser83Phe; Asp87Gly | Thr57Ser, Ser80Arg | Ser458Pro |
| Schwarzengrund | CGST3 | T | 2000 | Ap Cm Sm Sp Su Tc Tm | >1,024 [512] | 1,024 [64] | 64 [4] | 16 [4] | 16 [8] | Ser83Phe; Asp87Asn | Thr57Ser; Glu84Lys | |
| Schwarzengrund | CGST4 | T | 2001 | Ap Cm Sm Sp Su Tc Tm | >1,024 [512] | >1,024 [64] | 256 [4] | 32 [4] | 64 [16] | Ser83Phe; Asp87Gly | Thr57Ser; Ser80Arg | Ser458Pro |
| Schwarzengrund | CGST5 | T | 2002 | >1,024 [512] | >1,024 [128] | 64 [4] | 16 [2] | 16 [4] | Ser83Phe; Asp87Gly | Thr57Ser; Ser80Arg | ||
| Controls Schwarzengrund | 383SA99 | B | 1999 | 4 | 1 | 0.060 | 0.030 | 0.030 | Thr57Ser | |||
| Hadar | 13SA02 | B | 2002 | 4 | 1 | 0.125 | 0.060 | 0.030 | Thr57Ser | |||
| Typhimurium | S/921495 | S | 1992 | 4 | 0.5 | 0.125 | 0.060 | 0.030 | ||||
T, Taiwan; B, Belgium; S, Scotland.
Antibiotics are ampicillin (Ap), chloramphenicol (Cm), streptomycin (Sm), spectinomycin (Sp), sulfonamide (Su), tetracycline (Tc), trimethoprim (Tm), nalidixic acid (NAL), flumequine (FLU), enrofloxacin (ENR), marbofloxacin (MAR), and ciprofloxacin (CIP). Numbers in brackets are the MICs in the presence of PABN at 80 μg/ml.
No GyrB substitutions were detected.
Strains CGST2, CGST3, CGST4, and CGST5 carried up to five mutations in the quinolone target genes relative to those from S. enterica serovar Typhimurium, i.e., all carried a double mutation in the quinolone resistance-determining region (QRDR) of gyrA, leading to amino acid changes Ser83Phe and Asp87Asn or Asp87Gly, and a double mutation in the QRDR of parC, leading to amino acid changes Thr57Ser and Ser80Arg or Glu84Lys. Two strains displaying the highest levels of resistance to Cip carried an additional single mutation in the QRDR of parE, leading to amino acid change Ser458Pro. The ParC Thr57Ser amino acid change is likely not involved in quinolone resistance, because it was also identified in quinolone-susceptible S. enterica serovar Schwarzengrund control strain 383SA99 (Table 1). This amino acid change was, however, recently reported to be involved in decreased FQ susceptibility of S. enterica strains isolated in Hong Kong (7). Because we also found this amino acid change in a quinolone-susceptible S. enterica serovar Hadar strain (Table 1), it is probably the result of genetic divergence between the parC genes of S. enterica serovar Typhimurium, serovar Schwarzengrund, and serovar Hadar rather than being related to quinolone resistance. The ParC Ser80Arg and Glu84Lys amino acid changes found in the FQ-resistant S. enterica serovar Schwarzengrund strains have also been recently reported in FQ-resistant serovar Typhimurium isolates from patients in France, Hong Kong, Japan, and Taiwan (3, 6, 7, 8). The ParE Ser458Pro amino acid change found in strains CGST2 and CGST4 was also recently reported for FQ-resistant S. enterica serovar Typhimurium isolates from patients in Hong Kong (7). According to our data, this amino acid change, which is located at a same position as that found in the homologous GyrB protein (amino acid change Ser464Phe in FQ-resistant S. enterica serovar Typhimurium DT204 [2]), could account for a two- to fourfold increase in resistance levels to FQs (Table 1). The use of the efflux pump inhibitor Phe-Arg-β-naphthylamide (PAβN) decreased the FQ resistance levels 2- to 64-fold depending on FQ and the strain, suggesting participation of efflux in high-level FQ resistance (Table 1). The combination PAβN-enrofloxacin was the most effective in decreasing the resistance levels, as previously demonstrated (2).
In conclusion, high-level FQ resistance in S. enterica serovar Schwarzengrund strains appears to be linked to multiple target gene mutations at codon positions 83 and 87 for gyrA, codon positions 80 and 84 for parC, and codon position 458 for parE, as well as being linked to active efflux.
Acknowledgments
We thank C. Mouline and V. Verbeeren for expert technical assistance.
This study was funded by INRA, projet Transversalité.
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