Fluoroquinolone-resistant Salmonella Paratyphi A.

To the Editor: Fluoroquinolones have been the drug of choice for treating typhoid and paratyphoid fever since the beginning of the 1990s. Multidrug-resistant strains began to prevail in disease-endemic areas, and former first-line antimicrobial drugs, such as chloramphenicol, were sometimes ineffective (1). In recent years, however, strains with decreased susceptibility to quinolones have emerged, and clinical treatment failure is a serious concern (2-5).

An 87-year-old woman was referred from a local clinic to Yokohama Municipal Citizen's Hospital in July 2002 because Salmonella enterica serovar Paratyphi A was detected in her urine. She had no subjective symptoms such as pain on urination or urinary urgency, and her temperature was normal. She had never had paratyphoid fever, and she had not traveled abroad. No other person in the community had paratyphoid. Before being admitted to the hospital, she had experienced frequent episodes of urinary tract infection and had been empirically treated each time with oral antimicrobial drugs, including ciprofloxacin. She had been given a dose of 600 mg/day for 7 days, 25 times in the last 4 years.

The patient did not display any abnormal findings on physical examination. S. Paratyphi A was not detected in the urine but was confirmed in the stool; therefore, the previous report of bacteriuria could have been due to contamination of a urine sample with feces. An ultrasound showed a polyp and multiple stones in her gallbladder. A carrier state was suspected. Bile was obtained by duodenal aspiration and was positive for S. Paratyphi A. The patient was considered to be an asymptomatic cholecystic carrier of S. Paratyphi A.

On disk diffusion susceptibility testing, the isolate was resistant to nalidixic acid (NA) and to ofloxacin. The MIC of ofloxacin was as high as 256 [micro]g/mL, and the MIC of ciprofloxacin was 128 [micro]g/mL (Table). An open cholecystectomy was performed for treatment of the polyp, the stones, and the highly quinolone-resistant bacteria. A routine perioperative intravenous antimicrobial agent, cefmetazole, was administered as surgical prophylaxis. The polyp was malignant, and the operation was curative. The patient resumed normal activities, and had no further fecal excretion of S. Paratyphi A.

Polymerase chain reaction (PCR) amplification and DNA sequencing were conducted to detect mutations responsible for the fluoroquinolone resistance. Nucleotide sequences of gyrA, gyrB, parC, and parE genes were investigated. The primers used for PCR amplification and DNA sequencing have been previously described (6,7). An ABI Prism dye terminator cycle sequencing kit (Perkin-Elmer, Applied Biosystems, Foster City, CA) and an automated sequencer (311A; Perkin-Elmer, Applied Biosystems) were used. The isolated strain possessed triple point mutations. The first 2 mutations were in the gyrA gene, which encodes DNA gyrase, at codon 83 (TCC to TTC), which substitutes phenylalanine for serine, and at codon 87 (GAC to AAC), which substitutes asparagine for aspartic acid. The third mutation was in the parC gene, which encodes DNA topoisomerase IV, at codon 84 (GAA to AAA), which substitutes lysine for glutamic acid. No mutations were found in gyrB and par E. S. Typhi and Paratyphi A with decreased susceptibility to fluoroquinolones emerged on the Indian subcontinent, Southeast Asia, and Central Asia in the mid-1990s (2-5). On disk diffusion testing, these strains were NA-resistant, and susceptible to ofloxacin or ciprofloxacin; however, the MICs of ciprofloxacin increased to 0.25-4 [micro]g/mL, 10- to 100-fold higher than the usual NA-susceptible strains (5,8,9). NA-resistant strains of S. Typhi have 1 point mutation at the target site of quinolones, DNA gyrase, in the quinolone resistance-determining region of gyrA, either at codon 83 or codon 87 (2,3). Several epidemics of typhoid and paratyphoid fever caused by NA-resistant strains with clinical failure of quinolone treatment have been reported (4,5).

An experimental attempt had been previously made to induce the production of strains with high-level fluoroquinolone resistance by culturing strains of S. Typhi and Paratyphi A in ciprofloxacin-supplemented medium (7). One of these in vitro-induced, high-level resistant strains of S. Paratyphi A had triple mutations in the gyrA gene at codons 83 and 87 and in the parC gene at codon 84, which are the same triple mutations as those seen in the current in vivo case.

Full fluoroquinolone resistance has already emerged in the community in nontyphoid Salmonella species. In a clinical isolate of S. enterica serovar Typhimurium, the MIC of ciprofloxacin was 32 [micro]g/mL, and mutations in both gyrA and gyrB were noted (10).

Our findings strongly suggest that high-level quinolone resistance was induced through the long-term carrier state of S. Paratyphi A under selective pressure of frequent quinolone administration. The resistance is associated with the 2 mutation sites in gyrA and 1 site in parC, and multiple point mutations are likely related to the acquisition of full resistance. Physicians should be aware of the emergence of S. Typhi and Paratyphi A, as well as nontyphoid Salmonella species, which are highly quinolone-resistant.

Table. MICs of antimicrobial agents for the isolate of Salmonella
enterica serovar Paratyphi A *

                                     MIC ([micro]g/mL)

Antimicrobial agent             Etest   Broth microdilution

Ampicillin                        4             ND
Chloramphenicol                  16             ND
Gentamicin                      0.06            ND
Kanamycin                         1             ND
Streptomycin                    0.75            ND
Sulfamethoxazole/trimethoprim    0.5            ND
Tetracycline                      8             ND
Cefoperazon                      1.5            ND
Cefotaxime                      0.38            ND
Ceftriaxone                     0.19            ND
Imipenem                        0.19            ND
Aztreonam                       0.125           ND
Fosfomycin                       64             ND
Nalidixic acid                  >256            ND
Norfloxacin                     >256           1024
Ofloxacin                        >32            256
Sparfloxacin                     >32            256
Ciprofloxacin                    >32            128
Levofloxacin                     ND             128
Tosufloxacin                     ND             128

* ND, no data.

Takuya Adachi, * Hiroko Sagara, * Kenji Hirose, ([dagger]) and Haruo Watanabe ([dagger])

* Yokohama Municipal Citizen's Hospital, Yokohama, Japan; and ([dagger]) National Institute of Infectious Diseases, Tokyo, Japan

References

(1.) Rowe B, Threlfall EJ, Ward LR. Spread of multiresistant Salmonella typhi. Lancet. 1990:336:1065.

(2.) Brown JC, Shanahan PM, Jesudason MV, Thomson CJ, Amyes SG Mutations responsible for reduced susceptibility to 4-quinolones in clinical isolates of multi-resistant Sahnonella typhi in India. J Antimicrob Chemother. 1996;37:891-900.

(3.) Wain J, Hoa NTT, Chinh NT, Vinh H, Everett M J, Diep TS, et al. Quinolone-resistant Salmonella typhi in Viet Nam: molecular basis of resistance and clinical response to treatment. Clin Infect Dis. 1997;25:1404-10.

(4.) Murdoch DA, Banatvala NA, Bone A, Shiosmamlloev BI, Ward LR, Threlfall EJ. Epidemic ciprofloxacin-resistant Salmonella typhi in Tajikistan. Lancet. 1998;351:339.

(5.) Chandel DS, Chaudhry R, Dhawan B, Pandey A, Dey AB. Drug-resistant Salmonella enterica serotype Paratyphi A in India. Emerg Infect Dis. 2000;6:420-1.

(6.) Giraud E, Brisabois A. Martel JL, Chaslus-Dancla E. Comparative studies of mutations in animal isolates and experimental in vitro- and in vivo- selected mutants of Salmonella spp. suggest a counterselection of highly fluororquinolone-resistant strains in the field. Antimicrob Agents Chemother. 1999;43:2131-7.

(7.) Hirose K, Hashimoto A, Tamura K, Kawamura Y, Ezaki T, Sagara H, et al. DNA sequence analysis of DNA gyrase and DNA topoisomerase IV quinolonr resistance-determining regions of Salmonella enterica serovar Typhi and serovar Paratyphi A. Antimicrob Agents Chemother. 2002;46:3249-52.

(8.) Atkins BL, Gottlieb T. Emerging drug resistance and vaccination for typhoid fever [letter]. JAMA. 1998;279:579-80.

(9.) Threlfall EJ, Skinner JA, Ward LR. Detection of decreased in vitro susceptibility to ciprofloxacin in Salmonella enterica serotypes Typhi and Paratyphi A. J Antimicrob Chemother. 2001;48:740-1.

(10.) Heisig P. High-level fluoroquinolone resistance in a Salmonella typhimurium isolate due to alterations in both gvrA and gyrB genes. J Antimicrob Chemother. 1993;32:367-77.

Address for correspondence: Takuya Adachi, 56 Okazawacho. Hodogaya-Ku, Yokohama 240-8555, Japan; fax: [phone]; email: [e-mail]