Specific gyrA Mutation at Codon 83 in Nalidixic Acid-Resistant Salmonella enterica Serovar Typhi Strains Isolated from Vietnamese Patients

Typhoid fever is commonly seen in the developing countries, and the acquired resistance of Salmonella enterica serovar Typhi to antibiotics is a serious problem for the treatment of this disease. Before 1993, no nalidixic acid (NAL)-resistant strain was found in Vietnam. Since then, however, NAL- as well as multidrug-resistant strains have appeared and rapidly spread in the country. The quinolone resistance-determining region (QRDR) of the gyrA gene (7) was sequenced in 15 S. enterica serovar Typhi strains that were isolated from the blood of patients with typhoid fever in Vietnam. All 15 strains and their details were kindly provided by the Vietnam Collection of Medical Microorganisms (VCMM, Hanoi, Vietnam). The history and antibiotics susceptibility of 15 strains are shown in Table ​Table1.1. All 15 strains were susceptible to a fluoroquinolone, ciprofloxacin (CIP), although three were highly resistant to NAL. TABLE 1. History, drug susceptibility, and mutational analysis of the gyrA gene in E. coli K12 and serovar Typhi (Ty2 and 15 VCMM strains) Bacterial DNA was extracted from three independent colonies for each strain, and PCR was performed by GeneAmp PCR System 9700 (Perkin-Elmer, Foster City, Calif.). Primers used were 5′-GGTACACCGTCGCGTACTTTA-3′ (position 17 to 37) and 5′-GAGACGGTGGATTTCGTGGAT-3′ (positions 309 to 329), based on the reported sequence of the gyrA gene of serovar Typhimurium (3) (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"X78977","term_id":"1845586","term_text":"X78977"}}X78977). The sequencing was carried out by the dideoxy chain termination method, using the automated DNA sequencer (Genetic Analyser ABI Prism 3100; Perkin-Elmer). The sequenced PCR products from each DNA sample showed the 313-bp length as expected. The sequences in QRDR of the gyrA gene of the 15 Vietnamese strains were compared to those of Escherichia coli K12 and of S. enterica serovar Typhi Ty2 (5) (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AB071870","term_id":"15721886","term_text":"AB071870"}}AB071870) (Table ​(Table1). 1). The QRDR of the gyrA gene in 12 NAL-susceptible strains showed an identical nucleotide sequence to each other and to S. enterica serovar Typhi Ty2. In all of three highly NAL-resistant strains (MIC >512 μg/ml), a mutation was found at the 2nd nucleotide position of the same codon (Table ​(Table1). 1). The mutated codon is considered to be equivalent to codon 83 (serine) of gyrase A protein in E. coli, since the deduced amino acid residue is nearly identical to that of E. coli. The mutations of the three NAL-resistant strains of Vietnamese origin were C-to-A transversions in VCMM 1138 and 1145 and a C-to-T transition in VCMM 1142 at the 2nd nucleotide of codon 83; the corresponding amino acid residue changed from Ser-83 to Tyr and to Phe, respectively (Table ​(Table1). 1). The mutation at Ser-83 to Tyr is the first report in S. enterica serovar Typhi, although Ser-83-to-Phe mutations and Asp-87-to-Gly or -Tyr mutations in NAL-resistant strains (MIC > 64 μg/ml) have been reported earlier (1, 6). No mutation was found at Asp-87 in 15 isolates of S. enterica serovar Typhi in the present study. In other Salmonella species, as in E. coli, frequent mutations at codon 83 (Ser to Phe, Tyr, or Ala) or codon 87 (Asp to Gly, Asn, or Tyr) have also been observed in NAL-resistant strains (2). Ser-83 in QRDR, one of the “mutational hot spots,” plays a major role in DNA gyrase A interaction with quinolone, and the change at this position is proposed to be associated with high-level resistance to quinolone. New quinolones remain the drug of choice for treatment of typhoid fever in Vietnam. In clinical trials, however, short-course therapy with a new quinolone (ofloxacin, 15 mg/kg of body weight/24 h) showed 2.0 days' more fever clearance time (FCT) in 98 NAL-resistant cases (7.4 ± 3.2 days, mean ± 95% clearance) than in 113 NAL-susceptible cases (5.4 ± 2.2, P < 0.001) (4). Consequently, a cephalosporin III (cefotaxime, 100 mg/kg/24 h) can be used to replace them in young patients and patients in pregnancy. Its FCT was 3 days longer than that for ofloxacin therapy, but there were no differences in FCT between NAL-resistant and NAL-susceptible cases (4).