Increasing incidence of quinolone resistance in human non-typhoid Salmonella enterica isolates in Korea and mechanisms involved in quinolone resistance

Sang-Ho Choi1–3,, Jun Hee Woo1,2, Jung Eun Lee1,2, Su Jin Park1,2, Eun Ju Choo1,2, Yee Gyung Kwak1,2, Mi-Na Kim4, Myung-Sik Choi3, Nam Yong Lee5, Bok Kwon Lee6, Nam Joong Kim1,2, Jin-Yong Jeong1,2, Jiso Ryu1,2 and Yang Soo Kim1,2,*

1 Division of Infectious Diseases, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea; 2 Center for Antimicrobial Resistance and Microbial Genetics, University of Ulsan, Seoul, Republic of Korea; 3 Department of Microbiology and Immunology, Seoul National University College of Medicine, Seoul, Republic of Korea; 4 Department of Laboratory Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea; 5 Department of Laboratory Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea; 6 Department of Microbiology, Korea Center for Disease Control and Prevention, Seoul, Republic of Korea


* Correspondence address. Division of Infectious Diseases, Asan Medical Center, 388-1 Pungnap-dong, Songpa-gu, Seoul, 138-736, Korea. Tel: +82-2-3010-3303; Fax: +82-2-3010-6970; E-mail: yskim{at}amc.seoul.kr

Received 16 May 2005; returned 25 June 2005; revised 14 September 2005; accepted 15 September 2005


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Objectives: We investigated the trends of nalidixic acid resistance in human non-typhoid Salmonella enterica in a Korean population, and examined some possible mechanisms involved in this resistance.

Methods: A total of 261 clinical strains were tested. For all strains, the MICs of nalidixic acid were determined. Nalidixic acid-resistant strains underwent further analysis, including determination of MICs of other antibiotics, mutation analysis within the topoisomerase genes, organic solvent tolerance test, western blotting for AcrA, marOR mutation analysis, ciprofloxacin accumulation test, and PCR for the qnr gene. The clonal relationships of Salmonella strains were examined by random amplified polymorphic DNA analysis.

Results: The incidence of nalidixic acid resistance increased from 1.8% in 1995–96 to 21.8% in 2000–02. The resistance rate was higher in S. enterica serotype Enteritidis (21.6%) than in serotype Typhimurium (12.1%). The nalidixic acid resistance rates in Salmonella Enteritidis varied according to the phage type (PT) and Salmonella Enteritidis PT 1 was most commonly associated with resistance to nalidixic acid. Several cases of clonal spread, especially by Salmonella Enteritidis PT 1, were identified. Of the 46 nalidixic acid-resistant strains, 43 had single mutations in the gyrA gene. Four strains were organic solvent-tolerant and were associated with decreased ciprofloxacin accumulation; three of these showed increased expression of AcrA and had novel mutations in marOR (84L). The qnr gene was not identified.

Conclusions: Recently, the rate of nalidixic acid resistance in Korean clinical Salmonella strains markedly increased and it was partly due to the clonal spread of Salmonella Enteritidis, especially PT 1. The main mechanism of nalidixic acid resistance was a mutation in the gyrA region.

Keywords: S. enterica , nalidixic acid , drug resistance


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Salmonella species are a leading cause of human food-borne enteritis. Ciprofloxacin is often used for treatment of salmonellosis. However, the incidence of fluoroquinolone resistance or reduced susceptibility in Salmonella species has increased worldwide over the past decade and there have been reports of failed treatments with ciprofloxacin.1

Korea is one of the areas with the highest worldwide rates of antimicrobial resistance, including very high incidences of fluoroquinolone resistance for Enterobacteriaceae. However, few studies have investigated quinolone resistance in human non-typhoidal Salmonella isolates from Korea. Here, we sought to investigate the trends of nalidixic acid resistance in human non-typhoid Salmonella enterica in a Korean population, and examined some possible mechanisms involved in quinolone resistance.


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A total of 261 strains of non-typhoid Salmonella were prospectively collected from clinical specimens during the periods 1995–96 (55 strains) and 2000–02 (206 strains). The strains were serotyped and phage typed by standard procedures. Antimicrobial MICs were determined and interpreted according to the NCCLS guidelines.

The sequences of the primers for the PCR amplification of quinolone resistance-determining regions (QRDRS) of gyrA, gyrB, parC and parE were based on published sequences. The primers for marOR and acrR were designed from the relevant GenBank sequences. The recently described plasmid-mediated quinolone resistance-conferring gene, qnr, was PCR amplified using the qnr-specific primers previously reported for Klebsiella pneumoniae.2 PCR amplicons were sequenced using an ABI prism 3700 Automated DNA Analyzer (Perkin-Elmer Applied Biosystems).

Efflux pump activity was screened by an organic solvent tolerance assay.3 Strains AG 100 (wild-type Escherichia coli K-12 strain) and AG 100B (AG100 acrR::kan mutant, AcrA overexpressed) were used as the negative and positive controls, respectively. Accumulation of ciprofloxacin in samples treated with or without 100 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP) was determined as previously described.4

Western blotting for AcrA was done as previously described,3 with some modifications. The following controls were used: ATCC 13076 (S. enterica serotype Enteritidis having wild-type expression of AcrA), AG 100 (E. coli having wild-type expression of AcrA), and AG 100B (an AG100 acrR::kan mutant overexpressing AcrA).

Random amplified polymorphic DNA (RAPD) analysis was carried out using five primers: primers 23L, OPB-17, OPA-4, P1254 and OPB-15.5 The band patterns were compared both visually and with the Quantity One computer software (Bio-Rad Laboratories). We considered patterns to be non-matching when they differed by two or more bands.


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Serotypes and phage types of strains

We identified 38 serotypes, with the most frequently identified being serotype Enteritidis, accounting for 58.6% (153/261) of all tested strains. The other frequently identified serotypes were serotype Typhimurium (12.6%, 33 strains), serotype London (5.4%, 14 strains), serotype Sinstorf (3.1%, 8 strains) and serotype Hadar (2.3%, 6 strains). The most common phage types (PTs) in the 153 Salmonella Enteritidis strains were PT 21 (42.5%, 65/153), PT 4 (23.5%, 36/153) and PT 1 (20.3%, 31/153). In the 33 S. Typhimurium strains, definitive phage type 104-related complex (21.2%, 7/33) was the most common, followed by PT 190 (15.2%, 5/33) and PT 46b (15.2%, 5/33).

Resistance rates to nalidixic acid and ciprofloxacin

Overall, 17.6% (46/261) of the examined Salmonella strains were nalidixic acid-resistant. Of the 55 strains collected in 1995–96, only one strain (1.8%) was nalidixic acid-resistant, whereas 21.8% (45/206) of the more recently collected strains were nalidixic acid-resistant. According to the current NCCLS criteria, none of the nalidixic acid-resistant strains was resistant to ciprofloxacin. However, all of the nalidixic acid-resistant strains showed reduced susceptibility (MICs 0.125–0.50 mg/L) to ciprofloxacin. Several groups have reported clinical failures in ciprofloxacin treatment of susceptible strains with increased MICs of ciprofloxacin.1 Within this context, the increasing incidence of nalidixic acid resistance is of great concern. The resistance rate was higher among Salmonella Enteritidis strains (21.6%, 33/153) as compared with S. Typhimurium strains (12.1%, 4/33). This is in good agreement with previous data obtained in Spain and in an international survey in ten European Union countries,6 but contrasts with data obtained in Finland, where the nalidixic acid resistance was more frequent in S. Typhimurium.7 Among the 33 nalidixic acid-resistant Salmonella Enteritidis strains, PT 1 was the most commonly identified phage type (24 strains), followed by PT 21 (eight strains). The resistance rates to nalidixic acid for PTs 1, 21 and 4 were 67.7, 12.5 and 0%, respectively. Similar findings have been reported from Spain and the UK.8 Threlfall et al.8 suggested that the high rate of nalidixic acid-resistant PT 1 strains in the UK might be associated with travel to Spain. However, when we retrospectively reviewed the medical records of patients with nalidixic acid-resistant PT 1, we found little evidence of recent foreign travel in these patients.

Resistance rates to other antibiotics in nalidixic acid-resistant strains

The antibiotic resistance profiles of the 46 nalidixic acid-resistant strains are shown in Table 1. Overall, sulfadiazine was associated with the highest resistance rate (80.4%), followed by tetracycline (69.6%), streptomycin (45.7%), ampicillin (39.1%), chloramphenicol (23.9%), trimethoprim (15.2%) and ceftriaxone (0%). Multidrug-resistance, defined as resistance to three or more classes of antibiotics other than nalidixic acid, was observed in 24 strains (52.2%). Twelve (36.4%) of the 33 strains of Salmonella Enteritidis were multidrug-resistant. Historically, Salmonella Enteritidis has remained susceptible to most antibiotics. The emergence of resistance to both quinolones and traditional anti-Salmonella agents in this serotype, which is now the most common Salmonella serotype in Korea, would be a serious problem.


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Table 1. Antibiotic resistance profiles according to the serotypes and RAPD profiles in 46 nalidixic acid-resistant non-typhoid Salmonella strains

 
Mutations in the QRDRs of topoisomerase type II and IV

Forty-three of the 46 nalidixic acid-resistant strains (93.5%) were found to have single mutations in the QRDR of the gyrA gene at either codon 87 (39 strains) or codon 83 (4 strains). None of the 46 nalidixic acid-resistant strains showed mutations in the QRDR of the parC gene. The three strains that did not have mutations in the QRDR of gyrA were also negative for mutations in the QRDRs of the gyrB and parE genes. One possible resistance mechanism for these strains could be decreased permeability due to the loss of outer membrane proteins. Another possibility is the existence of as yet unknown additional resistance mechanisms. These possibilities warrant further investigation.

Organic solvent tolerance test, measurement of ciprofloxacin accumulation, mutational analysis of marOR and acrR, and western blotting for AcrA

Of the 46 nalidixic acid-resistant strains, four strains (8.7%) displayed increased organic solvent tolerance (Table 2). All four of these strains showed decreased ciprofloxacin accumulation and had single mutations in the QRDR of gyrA at codon 87. However, the MICs of ciprofloxacin in these organic solvent-tolerant strains did not differ significantly from those of the organic solvent-susceptible strains (MICs 0.25–0.5 mg/L). Baucheron et al.9 reported that the AcrAB-TolC efflux system plays a major role in high-level fluoroquinolone resistance in S. Typhimurium strains with double mutations in the gyrA gene. It might be possible that active efflux plays a minor role in the low-level resistance conferred by single gyrA mutations and a major role in high-level resistance in strains with double gyrA mutations. Three of the four organic solvent-tolerant strains showed increased expression of AcrA and had the same novel marOR (I84L) mutations. This mutation pattern was not detected in any of the 42 organic solvent-susceptible strains. None of the 46 nalidixic acid-resistant strains showed mutations in the acrR gene. Further studies will be necessary to fully assess the significance of our seemingly organic solvent tolerance-associated novel mutations in the marOR, and the relative contributions of active efflux and the number of gyrA mutations in the quinolone resistance phenotype.


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Table 2. Results of tests for ciprofloxacin accumulation, topoisomerase mutation, marOR mutation, acrR mutation and western blot for AcrA in four organic solvent-tolerant strains

 
PCR for the qnr gene

We did not detect the presence of the qnr gene in any of our strains. We recently found two E. coli strains with the qnr gene in clinical specimens from our hospital,10 indicating that this gene is present in the Korean population. Since the impact of plasmid-mediated quinolone resistance may be great, we will need to carefully monitor the qnr status of the relevant clinical strains.

RAPD characterization

The primers 23L and P1254 each revealed eight different patterns (identified by the letters A–H, respectively), whereas OPB-17, OPA-4 and OPB-15 each discriminated 12, 10 and 11 patterns, respectively (A–L, A–J, and A–K, respectively). A RAPD profile identification was assigned to each strain by use of a five-letter code identifying the patterns obtained with each primer (in the order of 23L, OPB-17, OPA-4, P1254 and OPB-15). Twenty-nine different RAPD profiles were identified in this manner. The RAPD profiles according to the antibiotic resistance profiles and serotypes/phage types are included in Table 1. In the Salmonella Enteritidis strains, RAPD analysis discriminated 14 and six different profiles among the PT 1 (24 strains) and PT 21 (8 strains) strains, respectively. Of 24 Salmonella Enteritidis PT 1 strains, 17 strains were clustered (four strains for AABAA, four for BACAA, three AAAAA, two for BABAA, two BCBAC and two for BABAC).

Collectively, our data stress that continued surveillance is essential for researchers to detect the emergence and spread of quinolone-resistant strains, and suggest that it may be necessary to routinely test for nalidixic acid susceptibility as a marker of nascent quinolone resistance.


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No declarations were made by the authors of this paper.


    Acknowledgements
 
This study was supported by a grant (2005-389) from the Asan Institute of Life Sciences, Seoul, Korea.


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1. Aarestrup FM, Wiuff C, Molbak K et al. Is it time to change fluoroquinolone breakpoints for Salmonella spp.? Antimicrob Agents Chemother 2003; 47: 827–9.[Free Full Text]

2. Jacoby GA, Chow N, Waites KB. Prevalence of plasmid-mediated quinolone resistance. Antimicrob Agents Chemother 2003; 47: 559–62.[Abstract/Free Full Text]

3. Wang H, Dzink-Fox JL, Chen M et al. Genetic characterization of highly fluoroquinolone-resistant clinical Escherichia coli strains from China: role of acrR mutations. Antimicrob Agents Chemother 2001; 45: 1515–21.[Abstract/Free Full Text]

4. Mortimer PG, Piddock LJ. A comparison of methods used for measuring the accumulation of quinolones by Enterobacteriaceae, Pseudomonas aeruginosa and Staphylococcus aureus. J Antimicrob Chemother 1991; 28: 639–53.[Abstract]

5. Lin AW, Usera MA, Barrett TJ et al. Application of random amplified polymorphic DNA analysis to differentiate strains of Salmonella enteritidis. J Clin Microbiol 1996; 34: 870–6.[Abstract]

6. Threlfall EJ, Fisher IS, Berghold C et al. Antimicrobial drug resistance in isolates of Salmonella enterica from cases of salmonellosis in humans in Europe in 2000: results of international multi-centre surveillance. Euro Surveill 2003; 8: 41–5.[Medline]

7. Hakanen A, Siitonen A, Kotilainen P et al. Increasing fluoroquinolone resistance in salmonella serotypes in Finland during 1995–1997. J Antimicrob Chemother 1999; 43: 145–8.[Abstract/Free Full Text]

8. Threlfall EJ, Ward LR, Skinner JA et al. Antimicrobial drug resistance in non-typhoidal salmonellas from humans in England and Wales in 1999: decrease in multiple resistance in Salmonella enterica serotypes Typhimurium, Virchow, and Hadar. Microb Drug Resist 2000; 6: 319–25.[ISI][Medline]

9. Baucheron S, Tyler S, Boyd D et al. AcrAB-TolC directs efflux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium DT104. Antimicrob Agents Chemother 2004; 48: 3729–35.[Abstract/Free Full Text]

10. Jeong JY, Yoon HJ, Kim YS et al. Detection of qnr in clinical isolates of Escherichia coli from Korea. Antimicrob Agents Chemother 2005; 49: 2522–4.[Abstract/Free Full Text]





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