INTRODUCTION

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Fluoroquinolones are antimicrobial agents related to the naphthyridine nalidixic acid. They are valuable for the treatment of infections caused by pathogenic bacteria in humans and animals because of their wide spectra and high levels of antimicrobial activity (38). They act by inhibiting DNA gyrase and topoisomerase IV in susceptible bacteria. The World Health Organization (WHO) estimated the production and usage of quinolones to be about 120 metric tons mainly in the United States, the European Union, Japan, and South Korea and 1,820 metric tons alone in China (38). During 1997, the usage of fluoroquinolones within the European Union was estimated to be 43 metric tons (3). In Germany the fluoroquinolone enrofloxacin was first licensed for use in veterinary medicine in May 1989. In The Netherlands, enrofloxacin had been approved 2 years earlier, and other European countries followed with the introduction of enrofloxacin in the early 1990s. In some instances the use of enrofloxacin was paralleled by a decrease in the susceptibility to quinolones of zoonotic bacteria isolated from food-producing animals and humans in Europe (8, 9, 19, 20). Quinolone and fluoroquinolone resistance in Campylobacter and Salmonella enterica has mainly been due to single point mutations in gyrA (12, 13, 26), which encodes the A subunit of DNA gyrase, and rarely in gyrB, which encodes the B subunit of DNA gyrase (10, 14). Other mechanisms have also been proposed, e.g., mutations in the parC gene (15, 35) and decreased uptake of antimicrobial agents (24, 37).

Salmonellae are known to cause severe disease in humans and animals and are the leading cause of food-borne infections in many countries. Serotyping is performed by the Kauffmann-White scheme (27), and in many countries S. enterica serotype Typhimurium (Salmonella Typhimurium) and serotype Enteritidis (Salmonella Enteritidis) predominate. These serotypes can be further differentiated by phage typing and molecular techniques (2, 23, 33, 36). The most commonly isolated salmonellae are often clonally distributed, for example, the recently predominating clones of Salmonella Typhimurium definitive phage type (DT) 104 (5, 33) or Salmonella Enteritidis phage type 4 (16, 30).

In humans most infections caused by nontyphoidal salmonellae are self-limiting and antibiotic therapy is not indicated. However, in life-threatening situations treatment with fluoroquinolones is recommended, and this is particularly applicable for infections caused by multidrug-resistant salmonellae (1). Quinolone resistance in veterinary salmonella isolates from Germany was first observed in 1988. Strains of multidrug-resistant Salmonella Typhimurium DT 204c highly resistant to fluoroquinolones were isolated from cattle in a defined area near the Dutch border with Germany (11, 17). Such resistance was caused by mutations in the gyrA and gyrB genes (14). Hof et al. (18) reported on a nonfatal case of salmonellosis in an 11-year-old girl who was infected with such a strain presumably by ingestion of contaminated meat. In 1990 the number of isolates of Salmonella Typhimurium DT 204c decreased in Germany. However, in parallel, the number of pentadrug-resistant isolates (resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracyclines) of Salmonella Typhimurium DT 104 increased, and the strains soon became the most prevalent salmonella serotype and phage type among veterinary isolates (28). In England and Wales a decrease in susceptibility to fluoroquinolones in isolates of Salmonella Typhimurium DT 104 was observed following the licensing of enrofloxacin for veterinary use in 1993 (31, 34). Recently, in Denmark, a fatal case of quinolone-resistant Salmonella Typhimurium DT 104 was reported in an outbreak associated with the consumption of contaminated pork (4). The outbreak highlighted the transfer of fluoroquinolone-resistant strains from animals to humans and the potential problems associated with the treatment of patients infected with such clones.

This paper analyzes the incidence in Germany of quinolone resistance among isolates of S. enterica from animal sources during the last 13 years, with particular reference to the susceptibility to enrofloxacin of veterinary isolates of Salmonella Typhimurium DT 104 obtained in 1997 and 1998.

	MATERIALS AND METHODS

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Bacterial strains. In a 13-year period (1986 to 1998), 29,563 nonhuman salmonella isolates from different locations in Germany were received at the National Salmonella Reference Laboratory (NRL-Salm) in Berlin. The majority of strains were from food-producing animals, domestic animals, and other animals, for example, horses, rats, and reptiles. The remaining strains were from food, animal feed, or environmental samples. The isolates were distributed into four categories: bovine, poultry, porcine, and other. The categories bovine, poultry, and porcine refer to isolates from the respective animals or the corresponding food items; the category other contained the remaining isolates and other isolates for which the source was not well indicated. The typing of isolates at NRL-Salm follows a hierarchical protocol, including serotyping, phage typing, and molecular typing. Serotyping was performed by the Kauffmann-White scheme (27), and phage typing was performed by using the scheme of Anderson et al. (2) for Salmonella Typhimurium and the system of Ward et al. (36), as applied in the Central Public Health Laboratory, London, United Kingdom, for Salmonella Enteritidis.

Agar diffusion test. Susceptibility to the quinolone nalidixic acid was tested by the agar diffusion test in accordance with the guidelines of the German Institute for Standards (DIN 58940, part 3) (6). Briefly, about 10⁶ CFU of salmonella cells was inoculated onto Mueller-Hinton agar plates (diameter, 9 cm), and antibiotic-containing discs (Oxoid Ltd., London, England) were applied. The plates were incubated at 37°C for 20 h, resulting in a semiconfluent growth. The discs contained 30 µg of nalidixic acid. Strains are described as resistant if the diameter of the inhibition zone was 13 mm. Control strain Escherichia coli ATCC 25922 was included on each set of plates and exhibited an inhibition zone of 22 to 28 mm (22).

MIC determination. The MICs of nalidixic acid and enrofloxacin were determined by a broth microdilution method in accordance with the German standard guideline DIN 58940, part 8 (7). Each well contained a final bacterial inoculum of about 10⁵ CFU/ml in Mueller-Hinton broth. For nalidixic acid (Sigma, Deisenhofen, Germany) the range of antibiotic concentrations tested was 0.25 to 256 µg/ml, and for enrofloxacin (Bayer AG, Leverkusen, Germany) the range was 0.0075 to 8 µg/ml. For enrofloxacin susceptibility, strains which grew at a concentration of 8 µg/ml were further tested with between 2 and 256 µg/ml for determination of MICs. All plates were incubated at 37°C for 20 h. The MIC was defined as the lowest concentration that produced no visible growth. Control strain E. coli ATCC 25922 was included on each set of plates. The MICs for the control strain were 2 µg/ml for nalidixic acid and 0.015 µg/ml for enrofloxacin (21). The breakpoints for the interpretation of resistance and susceptibility were those recommended by the German standard guideline DIN 58940. For nalidixic acid-susceptible strains MICs were 8 µg/ml, and MICs were 16 µg/ml for resistant strains. MICs for enrofloxacin-susceptible strains were 0.5 µg/ml, MICs for intermediate strains were 1 µg/ml, and MICs for resistant strains were 2 µg/ml.

	RESULTS

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Quinolone resistance in isolates from various sources. A total of 24,591 salmonella strains isolated between 1986 and 1998 were investigated by the agar diffusion test for their nalidixic acid susceptibility. Of these, 1,198 (4.9%) were resistant to nalidixic acid. Resistant strains isolated between 1991 and 1998 were investigated in more detail. With one exception, the nalidixic acid MIC for all strains was in excess of 256 µg/ml; the single exceptional strain required only 128 µg/ml for growth inhibition. The MICs of enrofloxacin ranged from 0.5 to 128 µg/ml. The prevalence of isolates resistant to enrofloxacin (MIC, 2 µg/ml) in relation to the total number of strains investigated varied between 3.4% in 1992 and 0.5% in 1998 (Fig. 1A). The incidence of intermediate strains (MIC, 1 µg/ml) ranged from 1.1% in 1991 to 5.6% in 1994, and the incidence of susceptible strains (MIC, 0.5 µg/ml) ranged from 0.2% in 1992 to 1% in 1994. In contrast, the proportion of isolates resistant to nalidixic acid ranged from 0.2% in 1986 to 14.8% in 1990 (Fig. 1A). The percentage of resistant strains varied considerably among strains from pigs, cattle, or poultry, but in all animals the proportion of isolates resistant to nalidixic acid increased in those years which followed the first use of enrofloxacin (Fig. 1A to C).

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Percent resistance to nalidixic acid (NAL; inhibition zone, 13 mm) and relative MICs of enrofloxacin (ENR; susceptible, intermediate, resistant) for nalidixic acid-resistant, nonhuman salmonella strains isolated in Germany between 1986 and 1998 (received between January and October 1998). (A) Total rate for all isolates. (B) Isolates from cattle. (C) Isolates from poultry. (D) Isolates from pigs. The arrows indicate the dates of licensing of enrofloxacin for veterinary use in Germany (for pigs and cattle, May 1989; for poultry, January 1990). N is the number of isolates investigated.

Susceptibilities of selected serotypes to quinolones. Higher prevalences of resistance to nalidixic acid were mainly detected in specific serotypes, for example, Salmonella Hadar, Paratyphi B (D-tartrate positive), and Saintpaul (Table 1). These serotypes are primarily found in poultry. In the last 5 years the prevalence of nalidixic acid-resistant poultry isolates was 57% for Salmonella Hadar, 69% for Salmonella Paratyphi B (D-tartrate positive), and 67% for Salmonella Saintpaul.

Susceptibility of Salmonella Typhimurium phage type DT 104 isolates to enrofloxacin. DT 104 has become the predominant Salmonella Typhimurium phage type among German veterinary isolates since 1994 (28). Of all Salmonella Typhimurium strains studied, 8.7% in 1996, 14.4% in 1997, and 12.8% in the first 9 months of 1998 were of phage type DT 104. These strains originated most frequently from cattle or pigs (Table 2). All DT 104 strains isolated in 1997 and 1998 were investigated for their susceptibility to enrofloxacin by measuring the MICs for the strains (Table 2). The range of enrofloxacin MICs was 0.015 to 2 µg/ml in 1997 and 0.03 to 1 µg/ml in 1998. In 1997 and 1998 the prevalences of DT 104 isolates for which the enrofloxacin MIC was 0.06 µg/ml were 76 and 83%, respectively. In 1997, the MIC was 0.25 µg/ml for 10% of strains; in contrast, in 1998 only one strain for which the MIC was 0.25 µg/ml was observed. The high prevalence in 1997 of strains for which MICs were >0.25 µg/ml was caused by an outbreak of DT 104 in cattle in Berlin, in which enrofloxacin was frequently used for therapy. In contrast, in 1998 the seven DT 104 isolates for which MICs were 1 µg/ml were isolated in different regions of Germany. These data show that, according to the recommended breakpoint for enrofloxacin (2 µg/ml), only one strain isolated in 1997 can be regarded as resistant.

	DISCUSSION

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In the present study the prevalence of resistance to quinolones among 24,591 German veterinary salmonella isolates obtained between 1986 and 1998 was examined. The results show that in three food-producing animal species, cattle, poultry, and pigs, the incidence of quinolone-resistant strains increased substantially in the years following the licensing of quinolones. A high prevalence of resistant strains was especially detected in isolates that originated from cattle, and such isolates were mainly to Salmonella Typhimurium DT 204c (nalidixic acid MIC range, 128 to >256 µg/ml). The MICs of enrofloxacin for these strains ranged from 0.25 to 128 µg/ml.

Among all isolates, the incidence of nalidixic acid-resistant strains varied between 0.2 and 14.8%. However, isolates from different animals showed considerable differences in the prevalence of nalidixic acid resistance per year. While in 1990 nearly 50% of isolates from cattle were highly resistant to nalidixic acid and enrofloxacin, for isolates from poultry an increase in the incidence of resistance has only occurred since 1994. Among isolates from pigs the incidence of resistance was, in general, low.

The data presented here have shown that the use of quinolones in food-producing animals leads, sometimes even before official approval of use of the drugs, to an initial increase in the prevalence of resistance. In subsequent years the levels of resistance have decreased, and in some cases they have reached low levels. Why this decrease has happened is speculative. One explanation could be that increasing levels of resistance lead to changes in prescription habits, resulting in a decline in the rate of use of such drugs. In order to show that this is the case, the incidence of resistance needs to be correlated to the consumption of the compounds in the individual animal species. Unfortunately, such data are not available for Germany.

Direct comparison of the data presented here with those from other reports is difficult because of differences in the breakpoints, fluoroquinolones, and methods used for the determination of resistance. However, reports from many countries have indicated that zoonotic bacteria including Salmonella spp. have shown decreased susceptibility to fluoroquinolones (9, 13, 20, 25, 39). This study shows that in Germany only specific serovars are responsible for the increasing prevalence of resistance and decreased susceptibility to fluoroquinolones. The selection of Salmonella strains resistant to fluoroquinolones has occurred in a stepwise fashion over several years. An increasing prevalence of quinolone-resistant Salmonella Hadar in animals and humans has been documented (29, 32), possibly indicating the epidemic spread of a particular clone which might have a greater potential for the development of resistance.

This seems to be true for Salmonella Typhimurium DT 104. Until 1995 no nalidixic acid-resistant isolates were identified. In 1996, 2.3% of isolates were nalidixic acid resistant. This number dropped to 1% in 1997 but increased to 2.5% in 1998. The range of enrofloxacin MICs for these strains was 0.5 to 4 µg/ml. It is possible, that in the coming years the relative proportion of quinolone-resistant DT 104 will increase if the selection pressure persists and the enrofloxacin MIC distribution will be shifted toward higher values, as has been observed in England and Wales (31, 32).

On the basis of the data presented above, it is important that the prudent use of fluoroquinolones in the veterinary field be encouraged and, in particular, that their application maximize the therapeutic effect and minimize the emergence of resistance (38). Global resistance-monitoring programs should be established and the consumption of fluoroquinolones should be registered. If resistance increases to dramatic levels, the veterinary use of fluoroquinolones must be curtailed in order to lower the selective pressure.