Abstract
The mutations that are responsible for fluoroquinolone resistance in the gyrA, gyrB, parC, and parE genes of Salmonella enterica serovar Typhi and serovar Paratyphi A were investigated. The sequences of the quinolone resistance-determining region of the gyrA gene in clinical isolates which showed decreased susceptibilities to fluoroquinolones had a single mutation at either the Ser-83 or the Asp-87 codon, and no mutations were found in the gyrB, parC, and parE genes.
Fluoroquinolones have become the first-line drugs for the treatment of typhoid fever (3, 12, 18, 23). However, some Salmonella enterica serovar Typhi strains that exhibit decreased susceptibilities to fluoroquinolones have been already reported (2, 7, 13, 21). Furthermore, several clinical treatment failures after the administration of ciprofloxacin and other fluoroquinolones to patients with typhoid fever due to strains with decreased susceptibilities to fluoroquinolones have also been reported (17, 21). The emergence and spread of these organisms have been reported in developing countries. There is evidence that the incidence of strains that are resistant to nalidixic acid and that exhibit decreased susceptibilities to the most recent fluoroquinolones used for the treatment of typhoid fever is increasing. In most strains, the acquired fluoroquinolone resistance was attributed to mutations in the genes encoding DNA gyrase (GyrA, GyrB) (10, 24-26) or DNA topoisomerase IV (ParC, ParE) (8, 9). The purpose of this study was to investigate the association of quinolone resistance with mutations in the genes coding for gyrase and topoisomerase IV of S. enterica serovar Typhi and serovar Paratyphi A, which are especially clinically important serotypes of Salmonella spp.
The bacterial strains used in this study were collected from regional public health offices in Japan between 1995 and 2001, and all isolates were obtained from a culture of either blood or stool from individual patients and identified by biochemical tests and serological tests on the basis of standard criteria. S. enterica serovar Typhi Ty2 and ATCC 19430 and S. enterica serovar Paratyphi A NCTC13, NCTC5702, and RIMD 1015 were used as reference and control strains (Tables 1 and 2). The MICs of several fluoroquinolones, including norfloxacin, levofloxacin, ofloxacin, sparfloxa cin, and ciprofloxacin, and nalidixic acid were determined by the Etest (AB Biodisk, Solna, Sweden), according to the instructions of the manufacturer. The quality control strains Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were included in each test. The criterion for ciprofloxacin resistance was an MIC of ≥4 μg/ml, according to the NCCLS breakpoint criteria for members of the family Enterobacteriaceae. The criterion for decreased susceptibility to ciprofloxacin that we used in this study was an MIC between ≥0.25 and <4 μg/ml, and that for ciprofloxacin susceptibility was an MIC <0.25 μg/ml (19, 20). An attempt to increase the level of fluoroquinolone resistance was done by culturing the fluoroquinolone-susceptible strains and the strains with decreased susceptibilities to fluoroquinolones in medium supplemented with ciprofloxacin, as described by Giraud et al. (4). The sequences of the primers used for PCR amplification and determination of the nucleotide sequences of the gyrA, gyrB, parC, and parE genes were previously described by Giraud et al. (4). The PCR products were directly used as templates for sequencing. DNA sequencing was performed with an ABI Prism dye terminator cycle sequencing kit (Perkin-Elmer, Applied Biosystems, Foster City, Calif.) with an automated sequencer (311A; Perkin-Elmer, Applied Biosystems). The amino acid sequences between positions 54 and 171 of GyrA, positions 397 and 520 of GyrB, positions 12 and 130 of ParC, and positions 421 and 524 of ParE, which contain the quinolone resistance-determining regions (QRDRs), were determined from the DNA sequences.
TABLE 1.
MICs for strains and nucleotide changes in the gyrA gene of Salmonella serovar Typhi
| Classificationa and strain no.b | MIC (μg/ml)c
|
Nucleotide change at GyrA position:
|
||||||
|---|---|---|---|---|---|---|---|---|
| NOR | LVX | OFX | SPX | CIP | NAL | 83 (TCC [Ser]) | 87 (GAC [Asp]) | |
| A | ||||||||
| NIHP3-4 (990102) | 64 | >32 | >32 | >32 | >32 | >256 | TTC (Phe) | TAC (Tyr) |
| NIHP3-43 (010063) | 16 | >32 | >32 | >32 | 16 | >256 | TTC (Phe) | TAC (Tyr) |
| NIHP3-39 (010053) | 16 | 16 | >32 | >32 | 8 | >256 | TTC (Phe) | TAC (Tyr) |
| NIHP3-9 (990113) | 16 | 4 | 8 | 2 | 4 | >256 | TTC (Phe) | None |
| B | ||||||||
| 990018 | 1 | 0.25 | 4 | 0.25 | 0.25 | >256 | TTC (Phe) | Noned |
| 000006 | 2 | 0.5 | 0.5 | 0.25 | 0.5 | >256 | TAC (Tyr) | None |
| 000007 | 2 | 0.5 | 0.5 | 0.25 | 0.5 | >256 | TAC (Tyr) | None |
| 000008 | 2 | 0.5 | 0.5 | 0.25 | 0.5 | >256 | TAC (Tyr) | None |
| 000009 | 2 | 0.25 | 0.5 | 0.25 | 0.5 | >256 | TAC (Tyr) | None |
| 000012 | 2 | 0.5 | 0.5 | 0.25 | 0.5 | >256 | TAC (Tyr) | None |
| 000015 | 1 | 0.5 | 0.5 | 0.25 | 0.5 | >256 | None | GGC (Gly) |
| 000016 | 2 | 0.5 | 0.5 | 0.25 | 0.5 | >256 | TAC (Tyr) | None |
| 000019 | 2 | 0.5 | 0.5 | 0.25 | 0.5 | >256 | TAC (Tyr) | None |
| 990069 | 2 | 0.5 | 0.5 | 0.25 | 0.5 | >256 | TAC (Tyr) | None |
| 990026 | 2 | 0.25 | 1 | 0.25 | 0.5 | >256 | TAC (Tyr) | None |
| 980083 | 2 | 0.5 | 1 | 0.25 | 0.5 | >256 | TAC (Tyr) | None |
| 990106 | 2 | 0.5 | 1 | 0.25 | 0.5 | >256 | TTC (Phe) | None |
| 990097 | 2 | 0.5 | 0.5 | 0.25 | 0.5 | >256 | TAC (Tyr) | None |
| 990120 | 1 | 0.25 | 0.5 | 0.25 | 0.25 | >256 | TTC (Phe) | None |
| 990102 | 1 | 0.25 | 1 | 0.25 | 0.25 | >256 | TTC (Phe) | None |
| 000022 | 2 | 0.5 | 0.5 | 0.25 | 0.5 | >256 | TAC (Tyr) | None |
| 000023 | 2 | 0.5 | 0.5 | 0.25 | 0.5 | >256 | TAC (Tyr) | None |
| 000025 | 2 | 0.5 | 0.5 | 0.25 | 0.5 | >256 | TAC (Tyr) | None |
| 970004 | 1 | 0.25 | 0.5 | 0.25 | 0.25 | >256 | TTC (Phe) | None |
| 990020 | 1 | 0.25 | 0.5 | 0.25 | 0.25 | >256 | None | GGC (Gly) |
| 990104 | 2 | 0.25 | 0.25 | 0.25 | 0.25 | 64 | TTC (Phe) | None |
| 000015 | 1 | 0.25 | 0.25 | 0.125 | 0.25 | 64 | None | TAC (Tyr) |
| 000027 | 1 | 0.25 | 0.5 | 0.125 | 0.25 | 64 | None | TAC (Tyr) |
| 000037 | 1 | 0.25 | 0.25 | 0.125 | 0.25 | 64 | None | TAC (Tyr) |
| C | ||||||||
| 990053 | 0.064 | 0.032 | 0.064 | 0.016 | 0.032 | 4 | None | None |
| 980118 | 0.064 | 0.016 | 0.064 | 0.016 | 0.032 | 4 | None | None |
| 990113 | 0.032 | 0.032 | 0.064 | 0.016 | 0.016 | 2 | None | None |
| 990100 | 0.25 | 0.032 | 0.064 | 0.064 | 0.032 | 2 | None | None |
| 010053 | 0.25 | 0.032 | 0.125 | 0.008 | 0.016 | 2 | None | None |
| 010063 | 0.25 | 0.032 | 0.125 | 0.008 | 0.016 | 2 | None | None |
| Reference strains Ty2 (ATCC 19430) | 0.064 | 0.032 | 0.032 | 0.008 | 0.006 | 2 | None | None |
| 0.125 | 0.032 | 0.064 | 0.008 | 0.032 | 2 | None | None | |
A, ciprofloxacin-resistant strains (in vitro-selected strains); B, ciprofloxacin-strains with decreased susceptibilities to fluoroquinolones (clinical isolates); C, ciprofloxacin-susceptible strains (clinical isolates).
The strains with the NIHP3 designation are ciprofloxacin-resistant mutants obtained by in vitro selection. The designations in parentheses are those for the original laboratory mutants.
NOR, norfloxacin; LVX, lerofloxacin; OFX, ofloxacin; SPX, sparfloxacin, CIP, ciprofloxacin; NAL, nalidixic acid.
None, no alterations were found in the genes.
TABLE 2.
MICs for strains and nucleotide changes in the gyrA gene of Salmonella serovar Parayphi A
| Classificationa and strain no.b | MIC (μg/ml)c
|
Nucleotide change at GyrA position:
|
||||||
|---|---|---|---|---|---|---|---|---|
| NOR | LVX | OFX | SPX | CIP | NA | 83 (TCC [Ser]) | 87 (GAC [Asp]) | |
| A | ||||||||
| NIHP3-1 (950040) | >256 | >32 | >32 | >32 | >32 | >256 | TTC (Phe) | AAC (Asn) |
| NIHP3-44 (990118) | 16 | >32 | >32 | >32 | 8 | >256 | TTC (Phe) | TAC (Tyr) |
| NIHP3-3 (990021) | 16 | 24 | >32 | >32 | 8 | >256 | TTC (Phe) | TAC (Tyr) |
| NIHP3-41 (010044) | 16 | 12 | >32 | 24 | 8 | >256 | TTC (Phe) | TAC (Tyr) |
| NIHP3-6 (980043) | 32 | 8 | >32 | 12 | 8 | >256 | TTC (Phe) | Noned |
| NIHP3-10 (990110) | 32 | 8 | >32 | 4 | 8 | >256 | TTC (Phe) | None |
| NIHP3-16 (970087) | 16 | 4 | >32 | 4 | 4 | >256 | TTC (Phe) | None |
| NIHP3-40 (000066) | 16 | 8 | 16 | 8 | 4 | >256 | TTC (Phe) | None |
| NIHP3-45 (000056) | 16 | 8 | 8 | 4 | 4 | >256 | TTC (Phe) | None |
| B | ||||||||
| 950040 | 8 | 4 | 16 | 4 | 2 | >256 | TTC (Phe) | None |
| 990112 | 2 | 1 | 2 | 0.5 | 0.5 | >256 | TTC (Phe) | None |
| 990110 | 2 | 1 | 2 | 1 | 0.5 | >256 | TTC (Phe) | None |
| 000040 | 2 | 1 | 2 | 1 | 0.5 | >256 | TTC (Phe) | None |
| 000055 | 2 | 1 | 2 | 1 | 0.5 | >256 | TTC (Phe) | None |
| 990021 | 2 | 1 | 2 | 1 | 0.5 | >256 | TTC (Phe) | None |
| 980043 | 2 | 1 | 2 | 0.5 | 0.5 | >256 | TTC (Phe) | None |
| C | ||||||||
| 970087 | 0.25 | 0.125 | 0.25 | 0.064 | 0.064 | 4 | None | None |
| 970131 | 0.25 | 0.125 | 0.125 | 0.064 | 0.032 | 4 | None | None |
| 000056 | 0.25 | 0.125 | 0.25 | 0.064 | 0.064 | 4 | None | None |
| 000066 | 0.125 | 0.064 | 0.125 | 0.032 | 0.016 | 4 | None | None |
| 990118 | 0.25 | 0.064 | 0.125 | 0.032 | 0.064 | 2 | None | None |
| 010044 | 0.25 | 0.064 | 0.125 | 0.064 | 0.032 | 1 | None | None |
| Reference strains | ||||||||
| NCTCI3 | 0.032 | 0.032 | 0.064 | 0.032 | 0.016 | 2 | None | None |
| NCTC5702 | 0.064 | 0.032 | 0.064 | 0.032 | 0.016 | 2 | None | None |
| RIMD1015 | 0.064 | 0.064 | 0.125 | 0.032 | 0.032 | 2 | None | None |
A, ciprofloxacin-resistant strains (in vitro-selected strains); B, strains with decreased susceptibilities to fluoroquinolones (clinical isolates); C, ciprofloxacin-susceptible strains (clinical isolates).
The strains with the NIHP3 designation are ciprofloxacin-resistant mutants obtained by in vitro selection. The designation in parentheses are those for the original laboratory mutants.
NOR, norfloxacin; LVX, lerofloxacin; OFX, ofloxacin; SPX, sparfloxacin; CIP, ciprofloxacin; NA, nalidixic acid.
None, no alterations were found in the genes.
A total of 31 clinical isolates of S. enterica serovar Typhi and 13 clinical isolates of S. enterica serovar Paratyphi A were examined in this study. The data are shown in Tables 1 and 2. Twenty-five and six strains of serovar Typhi had decreased susceptibility to ciprofloxacin and were ciprofloxacin susceptible, respectively; and seven and six strains of serovar Paratyphi A had decreased susceptibility to ciprofloxacin and were ciprofloxacin susceptible, respectively. Typical resistant strains were never found among the strains tested. The serovar Typhi and serovar Paratyphi A clinical isolates with decreased susceptibility to fluoroquinolone had only a single mutation in the gyrA gene, at either position 83 or 87 of GyrA. Strains with high-level resistance to fluoroquinolones induced by in vitro selection with ciprofloxacin had double mutations in the gyrA gene at both position 83 and position 87 of GyrA (Tables 1 and 2). Only one serovar Paratyphi A strain (strain NIHP3-1, Table 2) had a mutation in the parC gene, at Glu-84 of ParC, in addition to double mutations in the gyrA gene. For the parC gene, the mutation was a change of GAA (Glu) to AAA (Lys) at codon 84. Alterations in the QRDRs of the gyrB and parE genes were not found in any of the strains tested.
In this study, three groups could be distinguished among the clinical isolates and the strains in which resistance was experimentally selected in vitro on the basis of the ciprofloxacin MICs and gyrA mutations (Tables 1 and 2). The first group consisted of strains which were susceptible to fluoroquinolones and which had no mutations in the QRDR of the gyrA gene (group C in Tables 1 and 2). The second group consisted of strains which exhibited slightly reduced susceptibilities to fluoroquinolones and which had only a single mutation in the QRDR of the gyrA gene (group B in Tables 1 and 2). The third and last group consisted of strains which were highly resistant to fluoroquinolones and which had a single mutation or double mutations in the QRDR of the gyrA gene (group A in Tables 1 and 2); all strains in the third group were experimentally selected. These findings indicate that gyrA mutations are of principal importance for the fluoroquinolone resistance of serovars Typhi and Paratyphi A. Alterations at position 83 or 87 of the GyrA amino acid sequence have been described previously for Salmonella strains (1, 4, 6, 14, 16, 22). Double mutations at positions 83 and 87 of the GyrA amino acid sequence were also reported in clinical isolates of serovar Schwarzengrund, which caused nosocomial infections in the United States and which exhibited ciprofloxacin resistance (11). Although strains with high-level fluoroquinolone resistance due to double mutations at codons 83 and 87 in the GyrA amino acid sequence have not been found in clinical isolates of serovars Typhi and Paratyphi A, several cases of the failure of treatment for typhoid fever due to strains with decreased susceptibilities to fluoroquinolones have been reported (21). Since we obtained isolates with double mutations in the gyrA gene by in vitro selection and a mutation in parC caused by a novel substitution in Lys-84, such mutations in clinical isolates of serovars Typhi and Paratyphi A may appear in the future. Establishment of a surveillance system for the detection of gyrA mutations will be important for the detection of fluoroquinolone resistance in S. enterica serovars Typhi and Paratyphi A.
We do not rule out the possibility that mutations which may contribute to fluoroquinolone resistance may be present outside of the regions sequenced. Although both NIHP3-4 and NIHP3-39 strains have double mutations in the gyrA gene, the MICs of ciprofloxacin for these two strains were very different. These results suggest that other mechanisms may also be responsible for high-level fluoroquinolone resistance in S. enterica serovars Typhi and Paratyphi A. Although high-level-resistant strains are commonly reported among clinical isolates of E. coli, resistant clinical isolates of S. enterica serovars Typhi and Paratyphi A were not found. The difference in fluoroquinolone resistance between two closely related species may be explained by differences in outer membrane permeabilities for fluoroquinolones and differences in active efflux activities (5, 15).
In conclusion, surveillance for antimicrobial resistance among clinical isolates of S. enterica serovars Typhi and Paratyphi A should be continued, particularly to monitor the emergence of strains with double mutations in the gyrA genes.
Nucleotide sequence accession numbers.
The partial DNA sequences of the gyrA, gyrB, parC, and parE genes of S. enterica serovar Typhi Ty2 and S. enterica serovar Paratyphi A NCTC13 reported here were registered in the DDBJ, EMBL, and GenBank nucleotide sequence databases under the following accession numbers: S. enterica serovar Typhi gyrA, accession no. AB071870; S. enterica serovar Typhi gyrB, accession no. AB072396; S. enterica serovar Typhi parC, accession no. AB071987; S. enterica serovar Typhi parE, accession no. AB072701; S. enterica serovar Paratyphi A gyrA, accession no. AB071871; S. enterica serovar Paratyphi A gyrB, accession no. AB072393; S. enterica serovar Paratyphi A parC, accession no. AB072700; and S. enterica serovar Paratyphi A parE, accession no. AB072702.
Acknowledgments
This work was partially supported by a grant-in-aid for International Health Cooperation Research (grant 12A-1) from the Ministry of Health, Labor and Welfare (to K.H.).
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