Quinolone and macrolide resistance in Campylobacter jejuni and C. coli: resistance mechanisms and trends in human isolates.

The incidence of human Campylobacter jejuni and C. coli infections has increased markedly in many parts of the world in the last decade as has the number of quinolone-resistant and, to a lesser extent, macrolide-resistant Campylobacter strains causing infections. We review macrolide and quinolone resistance in Campylobacter and track resistance trends in human clinical isolates in relation to use of these agents in food animals. Susceptibility data suggest that erythromycin and other macrolides should remain the drugs of choice in most regions, with systematic surveillance and control measures maintained, but fluoroquinolones may now be of limited use in the empiric treatment of Campylobacter infections in many regions.


Synopses
Campylobacter jejuni subsp. jejuni (C. jejuni) and C. coli have been recognized since the late 1970s as important agents of gastrointestinal infections throughout the world; in the United States, these infections affect approximately 1% of the population each year (1). Contaminated food is the usual source of human infections; therefore, the presence of fluoroquinolone-and macrolide-resistant strains in the food chain has raised concerns that the treatment of human infections will be compromised. Most cases of Campylobacter enteritis do not require antimicrobial treatment, being brief, clinically mild, and self-limiting (2)(3)(4). However, a substantial proportion of these infections require treatment. These include severe and prolonged cases of enteritis, septicemia, and other extraintestinal infections. Erythromycin has been the most commonly used agent for treating Campylobacter enteritis (2,5).
In the 1980s, the introduction of fluoroquinolones, which are effective against most major pathogens causing bacterial enteritis, offered a new approach to antibiotic intervention (6). Fluoroquinolones initially had good in vitro activity for thermophilic Campylobacter species, as well as for members of the family of Enterobacteriaceae.
Early clinical trials of both communityacquired acute diarrhea and traveler's diarrhea caused by Campylobacter demonstrated that patients treated with a fluoroquinolone had good clinical response (6,7). It soon became apparent, however, that resistance in Campylobacter spp. could arise in vivo, sometimes after only one or two administrations of fluoroquinolones (8). Moreover, Endtz and colleagues (9) reported as early as 1991 that the emergence of quinoloneresistant C. jejuni and C. coli isolated from humans in the Netherlands coincided with the introduction of fluoroquinolones in veterinary medicine.
Fluoroquinolone resistance in Campylobacter from food animals is now recognized as an emerging public health problem. Smith et al. from Minnesota (10) found that patients infected with resistant C. jejuni had longer duration of The incidence of human Campylobacter jejuni and C. coli infections has increased markedly in many parts of the world in the last decade as has the number of quinoloneresistant and, to a lesser extent, macrolide-resistant Campylobacter strains causing infections. We review macrolide and quinolone resistance in Campylobacter and track resistance trends in human clinical isolates in relation to use of these agents in food animals. Susceptibility data suggest that erythromycin and other macrolides should remain the drugs of choice in most regions, with systematic surveillance and control measures maintained, but fluoroquinolones may now be of limited use in the empiric treatment of Campylobacter infections in many regions. > > > diarrhea than patients with fluoroquinolonesensitive isolates. As Campylobacter infections can be serious in immunocompromised patients, the identified treatment failure raises the concern that fluoroquinolone-resistant strains may increase Campylobacter-associated deaths in this group of patients.

Mechanism of Macrolide Resistance in Campylobacter
Erythromycin binds to the ribosome but, unlike larger macrolides, appears to cause dissociation of the peptidyl-tRNA, rather than blocking the peptidyltransferase activity (11).
In C. jejuni and C. coli, erythromycin resistance is chromosomally mediated and is due to alteration of the ribosome (12); the resistance mechanism is not consistent with presence of an rRNA methylase, modification of the antibiotic, or efflux (13). Whole ribosomes or 50S subunits were purified from erythromycin-resistant strains and shown to bind much less erythromycin than ribosomes from sensitive strains. In a closely related bacterium, Helicobacter pylori, resistance to clarithromycin is due to an alteration of one of two adenine residues in the 23S rRNA at the erythromycin-binding site (14). Sequencing of the 23S rRNA genes from erythromycinresistant Campylobacter spp. identified mutations at these same sites, which are most probably responsible for resistance ( Figure 1) (15).

Mechanism of Fluoroquinolone Resistance in Campylobacter
Fluoroquinolone resistance in C. jejuni appears to be due most often to mutations in the genes encoding subunits of DNA gyrase (gyrA) and only occasionally to topoisomerase IV (parC) ( Figure 1). DNA gyrase purified from quinoloneresistant mutants of C. jejuni was 100-fold less sensitive to inhibition by quinolones than the wildtype gyrase (19). Cloning and sequencing of the C. jejuni gyrA gene demonstrate that mutations in gyrA at positions Thr-86, Asp-90, and Ala-70 were responsible for resistance (16,17). Mutations at Thr-86 are associated with higher level resistance to nalidixic acid (MIC 64-128 µg/mL) and ciprofloxacin (MIC 16-64 µg/mL) than mutations at Asp-90 or Ala-70. C. jejuni isolates resistant to even higher levels of quinolones (ciprofloxacin MIC of 125 µg/mL) carry two mutations, one in gyrA Thr-86 and the other in the topoisomerase IV subunit parC at Arg-139 (18).
Evidence of efflux of fluoroquinolones in C. jejuni (20) also exists. Passage of the bacteria on pefloxacin-containing agar has led to the isolation of a fluoroquinolone-resistant strain. This strain was also resistant to tetracycline, erythromycin, chloramphenicol, and several βlactams. The pefloxacin-resistant strain carried a mutation at Thr-86 of gyrA, likely responsible, in part, for fluoroquinolone resistance. Broadspecificity efflux pumps in C. jejuni, which cause fluoroquinolone resistance, have not yet been shown to be clinically relevant.

Use of Macrolides and Quinolones in Food Animals
Antibiotics of the macrolide-lincosamide group have been used in treating food animals worldwide for several decades. The most commonly used agents have been lincomycin and tylosin for controlling dysentery and Mycoplasma infections in swine and spiramycin for treating mastitis in cattle. For the past 20 years, tylosin has also been the most commonly used agent for growth promotion in swine production worldwide, whereas spiramycin has been commonly used in poultry. The use of macrolides for growth promotion has been banned in all European Union countries since July 1999. Several fluoroquinolones are available for treating food animals, such as poultry, cattle, pigs, and fish, in many countries. While information on global use is limited, worldwide use in food animals was estimated at 120 tons in 1997; use in humans has been estimated at more than 800 tons (21). Data are available only for the year of veterinary licensing of fluoroquinolones by country (Table 1). Licensing for use does not necessarily mean that the drug is actually used, so even these data have to be considered with caution. However, quinolone treatment of Campylobacter-colonized broiler chickens has induced quinolone resistance under experimental conditions (23).

Macrolide and Quinolone Resistance in Isolates from Food Animals and Foods of Animal Origin
Campylobacter is carried in the intestinal tract of wild and domestic animals and, as result Synopses of fecal contact during processing, frequently contaminates foods derived from animals. C. jejuni is predominant in broilers and cattle but is infrequent in pigs (where C. coli predominates) (24). In food animals, the prevalence of resistance to erythromycin is generally higher in C. coli, in particular in C. coli isolates from pigs, than in C. jejuni (24)(25)(26). In a recent study from Spain (27), rates of erythromycin and quinolone resistance in C. coli from pigs were 81% and 100%, respectively. High erythromycin resistance in pigs may be related to extensive veterinary use of macrolides (5,28).
In food products of animal origin, the occurrence of Campylobacter is much higher in poultry than in other categories, e.g., pork or beef (29). Therefore, Campylobacter resistance data are primarily based on poultry products, especially broiler meat. For a number of countries, fluoroquinolone-resistance rates are similar in isolates from poultry products and humans (10,25,27,(30)(31)(32). In the United Kingdom, enrofloxacin (a derivative of ciprofloxacin) was first licensed in late 1993; before then, domestically bred chickens were less frequently infected with quinolone-resistant campylobacters than imported chicken products. Using a simple model, researchers were able to correlate the previously observed resistance percentage in domestically acquired cases with estimates of the amount of imported chicken consumed in the United Kingdom (32). In recent data from Spain and Taiwan, rates of erythromycin resistance were 17% and 17%, respectively, in C. jejuni isolated from foods, whereas for C. coli the figures were 50% and 83%, respectively (27,31).

Transmission of Resistant Campylobacter from Animals to Humans
Campylobacteriosis is primarily a zoonosis. Evidence to indicate that fresh raw meat, especially poultry, is a major source of infection is ample, even though other sources such as raw milk, water, and pets may contribute to human infection (1,5,(33)(34)(35)(36)(37)(38).
Studying the transmission of antimicrobial resistance from animals (especially poultry to humans) has been difficult because the chain of transmission is often complex. The number of macrolide-and fluoroquinolone-resistant isolates from humans is influenced by several factors including veterinary use of macrolides (approved for use as antimicrobial growth promoters or as therapeutic drugs) and fluoroquinolones (only approved as therapeutic drugs) at a given location (24,39); association with recent or current antimicrobial treatment of patients; the origin of isolates (children vs. adults; inpatients vs. outpatients); travel (10,(40)(41)(42)(43)(44)(45); and sampling strategy and susceptibility testing procedures (no consensus as to method, media, culture conditions, or breakpoints [43,46]). These factors stress the need for cautious interpretation and comparison of data from different centers. However, several studies have shown that food animals can be a substantial source of infection in humans and that the same serotypes and genotypes can be isolated from humans and food animals (29,36,37,(47)(48)(49). DNA profiling of Danish C. jejuni serotype O:2 strains using pulsed-field gel electrophoresis with four restriction enzymes identified common genotypes in humans, poultry, cattle and swine (SLW On, EM Nielsen, and J Engberg, unpub. data). Typing data on resistant isolates is sparse, but Smith and colleagues (10) found DNA fingerprints of quinolone-resistant C. jejuni from U.S.produced poultry identical to those of resistant C. jejuni from domestically acquired infections in humans. Therefore, the susceptibility of humans strains originating in animals to antibiotics can be related to the exposure of animal strains to antibiotic agents used in farming.

Is There a Link Between Macrolide and Fluoroquinolone Use in Humans and Resistant Campylobacter Infections?
Some investigators suggest that resistance in C. jejuni and C. coli is driven by use of antibiotics for treating human infections rather than by veterinary use. Induction of macrolide resistance during treatment has been reported infrequently (50). However, induction of macrolide resistance may play a role in areas with a large reservoir of asymptomatic Campylobacter carriers and frequent use of macrolides in humans.
reported within 24 hrs of treatment, but prolonged therapy, e.g., in immunosuppressed patients, is also a risk factor (52,54).
Smith et al. (10) showed that use of a quinolone before culture accounted for a maximum of 15% of resistant isolates during 1996 to 1998. In addition, an increasing number of reports claim that fluoroquinolone-resistant strains have been isolated from patients who had not received medical treatment, suggesting that strains were already fluoroquinolone resistant before causing the infection (7,31,32,(55)(56)(57).
Since human-to-human transmission of C. jejuni and C. coli is rare (9), patients infected with resistant Campylobacter are not an important source of resistant Campylobacter for other humans.
Before fluoroquinolones were introduced in veterinary medicine, they were widely used in human medicine in a number of countries, including the Netherlands and the United States (since 1985 and 1987, respectively), without emergence of quinolone resistance in Campylobacter in humans. In contrast, emerging quinolone resistance in humans often coincides with or follows the approval of fluoroquinolones in animal husbandry (Table 1, Figure 2). Thus, while human macrolide and fluoroquinolone use contributes to the increase in resistance in humans, their relative contribution to increase in resistance compared to the use of these agents in husbandry appears to be small.

Frequency of Macrolide Resistance in Human Isolates
Data on erythromycin and azithromycin resistance in C. jejuni, C. coli, and the two organisms combined, isolated from humans around the world since 1989, differ by country and species (Table 2). Almost all studies report a higher frequency of erythromycin resistance in C. coli than in C. jejuni (0% to 11% in C. jejuni vs. 0% to 68.4% in C. coli). Trends over time for erythromycin resistance show stable and low rates in Japan, Canada, and Finland, but recent development of resistance in Thailand and Sweden (45,73).

Trends over Time for Quinolone Resistance
Resistance to fluoroquinolones in Campylobacter has clearly increased over the past decade in many parts of the world (Figure 2). Before 1989, resistance was rare. With the introduction of enrofloxacin in veterinary medicine (Table 1) and (probably less important) fluoroquinolones in human medicine in mainland Europe (the Netherlands, Finland, France, and Spain), a rapid emergence of quinolone resistance in Campylobacter isolates from patients was noted (8,9,43,55,64,89,90).
Surveillance data on resistance rates in human isolates from Asia soon indicated an equal increase (84,91). Quinolones were approved for veterinary use in the United Kingdom and the United States in late 1993 and 1995, respectively; reports from these areas now show increasing quinolone-resistance profiles (10,39,88).
In the latest data from Taiwan, Thailand, and Spain, rates of fluoroquinolone resistance in C. jejuni, or C. jejuni and C. coli combined were 56.9%, 84%, and 75% to 88%, respectively (27,31,40,73). In these regions, where quinolone resistance is highly endemic and Campylobacter spp. predominate, fluoroquinolones cannot be recommended for community-acquired bacterial diarrhea. Although lower frequencies are reported from other regions, recent trends show a clear tendency of emerging quinolone resistance in many countries. Quinolone resistance in human isolates often coincides with or follows the approval of fluoroquinolones for use in animal husbandry (Table 1, Figure 2), although some differences in resistance rates between countries may be explained by differences in association with foreign travel, commerce, methods of testing, and surveillance activity.

Multidrug Resistance
Multidrug resistance to macrolides and fluoroquinolones must be considered highly undesirable in Campylobacter as these two classes are generally advocated as first-and second-line drugs for antimicrobial treatment of Campylobacter enteritis.
Additional resistance to other relevant therapeutic agents poses a risk when there is no effective antimicrobial regimen for Campylobacter infections. Recently, Hoge et al. (73) found 100% co-resistance between Thai isolates resistant to azithromycin and ciprofloxacin in the last 2 years Synopses of surveillance. In addition, the level of tetracycline and ampicillin resistance in Thailand is so high that these agents now have no role in the treatment of Campylobacter or noncholera diarrhea. Li et al. (31) reported that concomitant resistance rates among nalidixic acid-resistant C. jejuni isolates from their patients (exclusively children) were as follows: gentamicin 2%, erythromycin 12%, clindamycin 12%, tetracyline 97%, and ciprofloxacin 66%. All of these human erythromycin-resistant C. jejuni isolates and 90% of the C. coli isolates were concomitantly resistant to clindamycin.

Consequences of Resistance for the Clinical Decision-making Process
Distinguishing infections caused by different enteric pathogens is seldom possible, so antimicrobial-drug use in the clinical setting is not confined to the treatment of Campylobacter spp. but rather to empiric treatment of community-acquired diarrhea in general. Increased rates of resistance have also been reported from nontyphoidal salmonellae (25,92), and documented failures in the treatment of human Salmonella infections have been described (93). Therefore, having continuous information on the resistance patterns of the most common bacteria causing gastrointestinal infections is critical.

Control Measures
Surveillance of resistance in Campylobacter is of paramount importance when fluoroquinolones are used to treat human infections. Systematic surveillance and timely reporting of antibiotic resistance patterns in C. jejuni and C. coli and other enteric pathogens from different regions should become a high priority. The principal purpose of monitoring antimicrobial resistance trends in enteric pathogens is to provide clinicians with data that can be used to select appropriate treatment regimens. Surveillance should emphasize antibiotics that are being used routinely to treat diarrhea, as well as any alternatives, such as fluoroquinolones, macrolides, and gentamicin. Equally important is the accessibility of the data to those providing primary care. For quinolones, quantitative nalidixic acid susceptibility data are more sensitive than fluoroquinolone susceptibility data for detecting common first-step mutations causing reduced susceptibility.
To circumvent the development of resistance, we have two options: infection control (zoonoses control) and prudent use of antibiotics. Improved infection control strategies along the chain "stable to the table" and guidelines for prudent use of antimicrobial agents in food animal production should be developed (94,95). To prevent further development of resistance in Campylobacter, limiting the use of macrolides and fluoroquinolones for food animals as much as possible is recommended. In Denmark, fluoroquinolones are not essential for treatment of any type of infection in food animals, according to surveillance performed by the Danish Veterinary Laboratory, and their use is only recommended on the rare occasion when no other therapeutic option is available (22). Because of the selection for resistance, the use of macrolides for growth promotion has been banned in all European Union countries since July 1999. The effect on the occurrence of resistance in bacteria in food animals is still not known. However, preliminary results suggest that macrolide resistance in C. coli from pigs in Denmark has decreased along with the decreased use of tylosin (FM Aarestrup, unpub. data).

Conclusions
Review of in vitro macrolide and quinolone resistance prevalence and trends in Campylobacter isolated from humans showed a temporal relationship between use of quinolones in food animals and resistant Campylobacter isolates in humans. The public health effects of antibiotic use in agricultural practice, including resistance of C. jejuni and C. coli to macrolides and quinolones, should be estimated. Adequate actions for control are strongly needed in both veterinary and human medicine. The public health issue of resistance in Campylobacter has global dimensions because of ever-increasing international trade and travel.
This work was supported in part by the Natural Sciences and Engineering Research Council of Canada to D.E.T., a medical scientist with the Alberta Heritage Foundation for Medical Research.
Dr. Engberg is a physician at the Danish national reference laboratory for enteric pathogens at Statens Serum Institut. His research interests focus on the epidemiologic, antimicrobial susceptibility, and molecular typing aspects of Campylobacter.