In Vivo Development of Quinolone Resistance in Salmonella enterica Serotype Typhimurium DT104

Abstract

Salmonella enterica serotype Typhimurium definitive phage type 104 was isolated several times from the same patient over a period of 2 years. The strain developed reduced sensitivity to fluoroquinolones, and a mutation in the gyrA gene that is associated with reduced sensitivity to quinolones was identified.

Salmonella enterica serotype Typhimurium definitive phage type 104 (DT104) has been reported with increasing frequency worldwide throughout the last decade in both humans and farm animals. The majority of Danish Salmonella serotype Typhimurium DT104 isolates are pentaresistant, and the genes encoding the resistance marker have been characterized and sequenced (2, 11). The five resistance genes are located on the chromosome in a large gene cluster that was sequenced by Briggs and Fratamico in 1999 (2) and have been shown to be mobile (14). In contrast to that finding, the resistance mechanism for the quinolones is single-point mutations in the gyrase genes resulting in decreased affinity of DNA gyrase for the quinolone (3, 6).

This study describes the course of illness of a 51-year-old woman over a period of 2 years. Prior to the study, she had undergone sigmoid resection due to perisigmoiditis complicated by a ureter lesion and leakage of the sigmoid anastomosis, both of which had been corrected surgically, and a colostomy had just recently been corrected. An abdominal ultrasound had shown cholecystolithiasis. She was admitted to the medical ward on 15 May 1999 due to diarrhea and dehydration. Within 24 h, the patient's temperature spiked to 40°C and her blood pressure dropped to 65 mm Hg, necessitating her transfer to the intensive care unit, where she received IVs containing metronidazole (500 mg every 8 h [q8h]), ceftriaxon (2g twice a day), and dopamine (10 μg/kg of body weight/min). On 26 May, blood cultures yielded Salmonella serotype Typhimurium DT104 organisms that were fully susceptible to fluoroquinolones. The antibiotic treatment was changed to oral ciprofloxacin (250 mg q12h), which was continued until 4 June, after which the patient was deemed to be free of the Salmonella serotype Typhimurium DT104 infection and was discharged from the hospital.

The patient was readmitted 9 August 2000 due to hypotension, fever, and pain in her left buttocks. An abscess in the gluteal region due to a fistula emanating from the sigmoid anastomosis was identified. The gluteal fascia was split, pus was cultured, and the patient was administered IVs of ampicillin (1g q6h) and metronidazole (500 mg q12h) until 1 September and gentamicin from 9 August to 12 August. Gentamicin was replaced by intravenous ciprofloxacin (200 mg q12h) on 12 August, and ciprofloxacin therapy was continued until 1 September due to culture yielding Streptococcus, ampicillin-resistant Escherichia coli, and Bacteroides. The patient gradually recovered and was deemed free of Salmonella serotype Typhimurium DT104. The patient was readmitted 11 December 2000 due to retraction of the colostomy. Her colon was rejoined, and an ileostomy was performed. Shortly after surgery, she fell ill and produced thin green stool in her stoma. A stool specimen was cultivated, and there was growth of Salmonella serotype Typhimurium DT104 once again. The patient was left untreated and recovered spontaneously. Feces specimens were cultivated again in mid-January 2001 and 5 September 2001, and both yielded Salmonella serotype Typhimurium DT104. In the interim, an attempt had been made to eradicate the Salmonella with a 4-week course of oral ciprofloxacin (250 mg q12h).

Colonies of Salmonella subsp. were serially isolated and serotyped as Salmonella enterica serotype Typhimurium DT104 (Table 1), described by Callow (4) and Anderson et al. (1). A susceptibility test was conducted in accordance with the method and materials described by Threlfall et al. (15), and the MIC of ciprofloxacin was determined by the E-test (AB Biodisk, Solna, Sweden). The extraction and preparation of DNA were performed as described by Gautom (5), and XbaI and BlnI were used for digestion (Roche Holding Ltd., Basel, Switzerland). Isolation of plasmid DNA from bacterial strains and subsequent profiling were performed as described by Kado and Liu (7). DNA preparation and PCR were performed according to the methods of Sandvang et al. (13) and Sandvang (12). The gyrA gene (GenBank accession no. X06744) (16) was amplified by using the primers gyrA-F (base pairs 186 to 213) and gyrA-B (base pairs 811 to 784) and resulted in a PCR product of 626 bp. The PCR products were purified and sequenced according to the methods of Sandvang (12).

TABLE 1.

Comparison of Salmonella serovar Typhimurium DT104 strains

Strain	Date of isolation	Antibiotics to which strain shows resistancea	Ciprofloxacin MIC (μg/ml)	Plasmid content (kb)	Nucleotide at 21329b	Amino acid in codon 83
99-K3404	8 May 1999	AMP, CHL, SPT, STR, SUL, TET	0.008	95, 2.2, 1.9	C	Serine
00-H10725	28 December 2000	AMP, CHL, CIP, NAL, SPT, STR, SUL, TET	0.190	95, 2.2, 1.9	T	Phenylalanine
01-W4281	5 September 2001	AMP, CHL, CIP, NAL, SPT, STR, SUL, TET	0.190	95, 2.2, 1.9	T	Phenylalanine
01W10929	19 September 2001	AMP, CHL, CIP, NAL, SPT, STR, SUL, TET	0.190	95, 2.2, 1.9	T	Phenylalanine

^aAMP, ampicillin; CHL, chloromphinicol; CIP, ciprofloxacin; NAL, nalidixic acid; STR, streptomycin; SPT, spectinomycin; SUL, sulfonamides; TET, tetracycline.

^bAccording to GenBank accession number AE008801 (8). C, cytosine; T, thymine.

The pulsed-field gel electrophoresis (PFGE) pattern of the DNA digests yielded approximately 15 bands in the area between 50 and 1,000 kb (Fig. 1). Visual comparison showed that the PFGE patterns of the four Salmonella strains were indistinguishable. Plasmid profiling revealed that the four strains all had two small plasmids of approximate 2.2- and 2.6-kb lengths as well as the large 95-kb serotype-associated plasmid (virulence plasmid). All the Salmonella serotype Typhimurium DT104 strains contained the 626-bp gyrase PCR amplicons (16). The three nalidixic acid-resistant Salmonella serotype Typhimurium DT104 strains showed one-point mutations in the gyrA gene. A point mutation from C to T at nt 431 in the gyrA gene (16) was found. The location of this substitution corresponds to the codon 83 (numbered according to Yoshida et al.) (16). A point mutation from TCC to TTC (with the change marked in bold) resulted in an amino acid substitution from serine to phenylalanine. This substitution is well known to result in decreased susceptibility to quinolones (9, 10).

FIG. 1.

PFGE illustrating the DNA pattern from the four Salmonella enterica serotype Typhimurium DT104 strains from the same patient, digested with restriction enzyme BlnI (left) or XbaI (right).

In the above case, the patient developed Salmonella serotype Typhimurium DT104 septicemia secondary to a gastrointestinal infection. The Salmonella serotype Typhimurium DT104 primarily found in the patient's blood before antibiotic treatment showed full susceptibility to fluoroquinolones. The patient was treated with low doses of ciprofloxacin (250 mg q12h) for 9 days, and three succeeding cultivations of feces over the next year still yielded Salmonella serotype Typhimurium DT104 organisms. Although the indistinguishable PFGE patterns indicate complete identity among all four serial isolates, the second isolate of this series had reduced susceptibility to ciprofloxacin, (MIC, 0.190 μg/ml) compared with that of the first ciprofloxacin-susceptible isolate (MIC, 0.008 μg/ml). Resistance to ciprofloxacin remained for the next isolates and correlated with a mutation present in the gyrA gene. Although epidemiological studies suggest a relationship between the use of quinolones and the development of quinolone resistance, to our knowledge this is the first time that the evolution of ciprofloxacin resistance in Salmonella serotype Typhimurium DT104 has been directly linked to quinolone therapy in a patient.