Multidrug resistance in Campylobacter jejuni strains collected from Finnish patients during 1995–2000

Antti J. Hakanen1,2,*, Mirva Lehtopolku1,2, Anja Siitonen3, Pentti Huovinen1 and Pirkko Kotilainen1,2

1 Antimicrobial Research Laboratory, National Public Health Institute, Turku; 2 Department of Medicine, Turku University Central Hospital, Turku; 3 Laboratory of Enteric Pathogens, National Public Health Institute, Helsinki, Finland

Received 16 July 2003; returned 15 August 2003; revised 25 September 2003; accepted 27 September 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: The resistance of Campylobacter jejuni to fluoroquinolones is increasing globally. This study was performed to delineate those antimicrobial agents that are effective in vitro against ciprofloxacin-resistant C. jejuni isolates and potentially suitable for the treatment of severe disease when fluoroquinolone resistance or multidrug resistance is known or suspected.

Methods: During 1995–2000 we collected 376 C. jejuni strains, of which 354 were of foreign origin from multiple countries and 22 were of domestic origin. The MICs of 12 antimicrobial agents against the isolates were determined.

Results: Of the 376 strains, 174 (46%) were resistant to ciprofloxacin. Among other antimicrobials, resistance was most common to tetracycline (46%) and ampicillin (17%). Of the ciprofloxacin-resistant strains, 68% and 25%, respectively, were resistant to tetracycline and ampicillin, and 3% were resistant to erythromycin, gentamicin or clindamycin. One (0.6%) ciprofloxacin-resistant isolate was resistant to co-amoxiclav and none was resistant to imipenem. Resistance to three or more antimicrobial groups was detected in 22% of the isolates. Multidrug resistance was significantly associated with ciprofloxacin resistance (33% versus 12%; P < 0.01). Eight (2%) strains were resistant to macrolides, of which 75% were also resistant to ciprofloxacin, but none was resistant to co-amoxiclav or imipenem.

Conclusions: Macrolides still appear to be the first-choice alternative for suspected C. jejuni enteritis, if antimicrobial treatment is needed. The in vitro susceptibilities suggest that clinical trials to treat enteritis caused by multidrug-resistant C. jejuni with co-amoxiclav, and life-threatening infections with a carbapenem, may be valuable.

Keywords: drug resistance, fluoroquinolones, macrolides, diarrhoea


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Campylobacter jejuni is a major causative agent of bacterial diarrhoea. Although campylobacters are naturally susceptible to fluoroquinolones, the resistance to these antimicrobials has increased rapidly in several countries during the 1990s.1 This increasing resistance has complicated the empirical treatment of bacterial diarrhoea, as well as of severe Campylobacter infections in countries where fluoroquinolone-resistant strains predominate.2 Although most C. jejuni infections do not require antimicrobial treatment, some may be fatal, especially if the patient is immunocompromised. The aim of this study was to determine the susceptibilities of 376 C. jejuni strains to 12 antimicrobial agents. These strains were recovered from Finnish patients during 1995–2000.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Campylobacter strains and susceptibility testing

A total of 376 clinical human faecal C. jejuni strains were isolated from Finnish patients (one isolate per patient). The strains were isolated in the laboratory of a large private hospital in Helsinki, Finland, over two distinct time periods between 1995 and 2000. Subjects were treated as outpatients and no data on antimicrobial usage prior to faecal sampling were available. Two hundred and sixteen consecutive strains were isolated between January 1995 and November 1997, and 160 between October 1998 and January 2000. The strains were identified by standard microbiological methods. The strains isolated from patients travelling abroad within 2 weeks preceding their symptoms were classified as foreign isolates, all other strains were classified as domestic isolates. The countries of origin for the foreign isolates have been described previously.1

The MICs of antibiotics for the isolates were determined by the agar plate dilution method. Mueller–Hinton II agar (BBL, Becton Dickinson and Company, Cockeysville, MD, USA) supplemented with 5% sheep blood was used as the culture medium. The plates were incubated at 35°C for 48 h in a microaerobic atmosphere (CampyPak; BBL). The antimicrobials evaluated were nalidixic acid, ciprofloxacin, erythromycin, azithromycin, clindamycin, ampicillin, co-amoxiclav, cefotaxime, imipenem, tetracycline, gentamicin and chloramphenicol. C. jejuni RH 3583 (a local control strain, originally isolated in Edinburgh, UK as C. jejuni 143483) was used as a control in susceptibility testing and also as a growth control strain.3 The MIC breakpoints used for resistance to ciprofloxacin, cefotaxime, imipenem, tetracycline, gentamicin and chloramphenicol were those recommended by the NCCLS for non-Enterobacteriaceae.4 For nalidixic acid, ampicillin and co-amoxiclav, which lack breakpoints for non-Enterobacteriaceae, we used those recommended by the NCCLS for Enterobacteriaceae.4 The resistance breakpoints for erythromycin, azithromycin and clindamycin were chosen on the basis of earlier publications and histogram analysis (Figure 1). They were >=16 mg/L for erythromycin,5 >=4 mg/L for azithromycin2 and >=8 mg/L for clindamycin.6 For the isolates exhibiting MICs >= 16 mg/L of erythromycin, we also determined MICs of clarithromycin and telithromycin. Multidrug resistance was defined as resistance to three or more antimicrobial groups. The antimicrobial groups were as follows: (i) quinolones (ciprofloxacin and nalidixic acid); (ii) macrolides (erythromycin and azithromycin) and clindamycin; (iii) tetracycline; (iv) ß-lactams; (v) gentamicin; and (vi) chloramphenicol.



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Figure 1. MIC histograms of 12 antimicrobials for 376 C. jejuni isolates collected from Finnish patients between January 1995 and January 2000. The vertical lines represent the breakpoint of resistance for erythromycin, azithromycin and clindamycin, and the breakpoints of resistance and susceptibility for other antimicrobials.

 
Data analysis

The susceptibility data were analysed using the WHONET5 computer program (available from www.who.int/emc/WHONET/WHONET.html).

Statistical analysis was made using the {chi}2-test and the Fisher’s exact test. The statistical data were analysed using the SAS (v. 8.2) program.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Of the 376 C. jejuni isolates, 354 were collected from travellers returning to Finland and 22 were from patients with no travel history within the preceding 3 weeks.

Of all 376 isolates, 174 (46%) were resistant to ciprofloxacin (Table 1). Among the other antimicrobial groups studied, resistance was most common to tetracycline (46%) and ampicillin (17%). Only 2% of the isolates were resistant to erythromycin, azithromycin or clindamycin. Similarly, 2% were resistant to cefotaxime, but the proportion of intermediately cefotaxime-resistant isolates was 40%. Resistance to chloramphenicol was 3% and to gentamicin 2%. There was no resistance to imipenem, and only one (0.3%) isolate was resistant to co-amoxiclav (Table 1). The histograms illustrating the MICs are presented in Figure 1.


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Table 1. MICs of 12 antimicrobials for 376 C. jejuni strains collected from Finnish patients between 1995 and 2000
 
Of the 354 isolates of foreign origin, 172 (49%) were resistant to ciprofloxacin, compared with only two (9%) of the 22 domestic isolates (P < 0.01). Of the 174 ciprofloxacin-resistant strains tested with 10 non-quinolone antimicrobial agents, 68% and 25%, respectively, were resistant to tetracycline and ampicillin, as compared with the resistance among the ciprofloxacin-susceptible strains (27% and 9%; P < 0.01). Of the ciprofloxacin-resistant isolates, 4% were resistant to chloramphenicol, and 3% to erythromycin, gentamicin or clindamycin. Resistance to cefotaxime was also 3%, but the proportion of intermediately cefotaxime-resistant isolates was 48%. One (0.6%) ciprofloxacin-resistant isolate was resistant to co-amoxiclav and none was resistant to imipenem.

Multidrug resistance was detected in 81 (22%) isolates. While 57 (33%) of the 174 ciprofloxacin-resistant isolates had three or more additional resistance properties, 24 (12%) of the 202 quinolone-susceptible isolates were resistant to three or more antimicrobial agents (P < 0.01). Cefotaxime was excluded from the multidrug-resistance profile analysis because of the high number of intermediately cefotaxime-resistant isolates.

All of the eight erythromycin-resistant C. jejuni isolates were multidrug resistant, six (75%) of them being resistant to ciprofloxacin. None was resistant to co-amoxiclav or imipenem. The five isolates for which the erythromycin MICs were between 16 and 32 mg/L had MICs of clarithromycin between 8 and 32 mg/L and of telithromycin between 4 and 32 mg/L. The three isolates for which the erythromycin MICs were >256 mg/L had MICs of clarithromycin >=128 mg/L and of telithromycin between 16 and >128 mg/L.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Campylobacter infections are often self-limiting, thus requiring no antimicrobial treatment. However, therapy may be needed, for example in severe and prolonged cases of diarrhoea, in septicaemia or when the patient is immunocompromised. At the present time, fluoroquinolones may be unsatisfactory in the empirical treatment of Campylobacter infections or severe community-acquired bacterial diarrhoea in countries where fluoroquinolone-resistant strains are prevailing, or for tourists returning from those areas. The high fluoroquinolone resistance rate (49%) among our foreign C. jejuni isolates suggests that most international holiday destinations popular among Finns now belong to such a category.

In this study, C. jejuni resistance to ciprofloxacin was found to be significantly associated with resistance to three or more antimicrobial groups. Multidrug resistance is problematic, but a number of drugs are still effective against these fluoroquinolone-resistant and multidrug-resistant C. jejuni strains. Macrolides are currently the first-choice antimicrobials for the empirical treatment of suspected C. jejuni enteritis in many countries.2,7,8 Fortunately, macrolide resistance has so far remained relatively uncommon, with only 2% of all our isolates and 3% of the ciprofloxacin-resistant isolates classified as macrolide resistant. Thus, also in Finland, macrolides still appear to be the best alternative in suspected C. jejuni enteritis, if antimicrobial therapy is needed.

Higher macrolide resistance rates have been reported from some other countries. According to a recent survey, the rate of erythromycin resistance among C. jejuni was 17% in both Spain and Taiwan.7,8 In another study, the azithromycin resistance rate in Thailand was only 6%, but it was alarming that all azithromycin-resistant isolates were also fluoroquinolone resistant.9 A similar finding has been made by another group.2 In the present work, as many as 75% of the erythromycin-resistant isolates were ciprofloxacin resistant, and all erythromycin-resistant isolates were multidrug resistant. Based on in vitro results, no apparent benefits are afforded by the use of newer macrolides or ketolides in the treatment of erythromycin-resistant C. jejuni infections, since all isolates with elevated erythromycin MICs exhibited elevated MICs of clarithromycin and telithromycin. Among our entire C. jejuni collection, however, only one isolate was resistant to co-amoxiclav, and none was resistant to imipenem. These in vitro susceptibilities suggest that co-amoxiclav might be a candidate for clinical trials in enteritis caused by multidrug-resistant C. jejuni, and if the situation is life-threatening, a carbapenem may be the drug of choice. Nevertheless, it must be kept in mind that very few data exist on the clinical efficacy of co-amoxiclav or carbapenems,10 or of the other ß-lactams,11 for the treatment of C. jejuni infections.

In Finland, ciprofloxacin-resistant strains that had not existed in 1980 composed 9% of the strains isolated in 1990 and tested by Rautelin et al.5 In that study, no efforts were made to determine the origin of all ciprofloxacin-resistant C. jejuni isolates identified. Yet, the authors regarded it as plausible that the majority, if not all, of their resistant strains were derived from abroad. This assumption is consistent with our finding that ciprofloxacin resistance is still significantly more common among the foreign isolates than among those domestic in Finland, with only 9% of the domestic isolates resistant to ciprofloxacin. Despite the low rate of ciprofloxacin resistance among our domestic C. jejuni isolates, fluoroquinolones are of limited usefulness in the treatment of campylobacteriosis in Finland, since at least 80% of the clinical strains in our country are acquired abroad.5 This may be the reason for the discrepancy between the numbers of the foreign and domestic isolates included in the present study: when consecutive C. jejuni isolates are collected in one hospital, domestic isolates inevitably remain in the minority in Finland.

In conclusion, multidrug resistance was found to be significantly associated with resistance to ciprofloxacin. Macrolides still appear to be the first-choice alternative for suspected C. jejuni enteritis. The in vitro susceptibilities found suggest that co-amoxiclav might be a candidate for clinical trials in enteritis caused by multidrug-resistant C. jejuni, and if the situation is life-threatening, a carbapenem may be the drug of choice. The widespread emergence of multidrug resistance among C. jejuni is of great concern.


    Acknowledgements
 
We are indebted to Jari Ahvenainen for statistical assistance, and to Liisa Immonen, Minna Lamppu, Tarja Laustola, Marja-Liisa Lindman, Erkki Nieminen, Saija Nylander and all the staff members at the laboratories of the study for expert technical assistance. This study was supported by grants from the Maud Kuistila Memorial Foundation, the Finnish Medical Foundation Duodecim, the Research Foundation of Orion Corporation and a special government grant (EVO grant) from Turku University Central Hospital (all to A.J.H.).


    Footnotes
 
* Correspondence address. Antimicrobial Research Laboratory, National Public Health Institute, PO Box 57, 20521 Turku, Finland. Tel: +358-2-3316600; Fax: +358-2-3316699; E-mail: antti.hakanen{at}utu.fi Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Hakanen, A., Jousimies-Somer, H., Siitonen, A. et al. (2003). Fluoroquinolone resistance in Campylobacter jejuni isolates in travelers returning to Finland: association of ciprofloxacin resistance to travel destination. Emerging Infectious Diseases 9, 267–70.[ISI][Medline]

2 . Kuschner, R. A., Trofa, A. F., Thomas, R. J. et al. (1995). Use of azithromycin for the treatment of Campylobacter enteritis in travelers to Thailand, an area where ciprofloxacin resistance is prevalent. Clinical Infectious Diseases 21, 536–41.[ISI][Medline]

3 . Hakanen, A., Huovinen, P., Kotilainen, P. et al. (2002). Quality control strains used in susceptibility testing of Campylobacter spp. Journal of Clinical Microbiology 40, 2705–6.[Free Full Text]

4 . National Committee for Clinical Laboratory Standards. (2001). Performance Standards for Antimicrobial Susceptibility Testing: Eleventh Informational Supplement M100-S11. NCCLS, Wayne, PA, USA.

5 . Rautelin, H., Renkonen, O.-V. & Kosunen, T. U. (1991). Emergence of fluoroquinolone resistance in Campylobacter jejuni and Campylobacter coli in subjects from Finland. Antimicrobial Agents and Chemotherapy 35, 2065–9.[ISI][Medline]

6 . Sjögren, E., Kaijser, B. & Werner, M. (1992). Antimicrobial susceptibilities of Campylobacter jejuni and Campylobacter coli isolated in Sweden: a 10-year follow-up report. Antimicrobial Agents and Chemotherapy 36, 2847–9.[Abstract]

7 . Li, C. C., Chiu, C. H., Wu, J. L. et al. (1998). Antimicrobial susceptibilities of Campylobacter jejuni and coli by using E-test in Taiwan. Scandinavian Journal of Infectious Diseases 30, 39–42.[CrossRef][ISI][Medline]

8 . Sáenz, Y., Zarazaga, M., Lantero, M. et al. (2000). Antibiotic resistance in Campylobacter strains isolated from animals, foods, and humans in Spain in 1997–1998. Antimicrobial Agents and Chemotherapy 44, 267–71.[Abstract/Free Full Text]

9 . Isenbarger, D. W., Hoge, C. W., Srijan, A. et al. (2002). Comparative antibiotic resistance of diarrheal pathogens from Vietnam and Thailand, 1996–1999. Emerging Infectious Diseases 8, 175–80.[ISI][Medline]

10 . Kerstens, P. J., Endtz, H. P., Meis, J. F. et al. (1992). Erysipelas-like skin lesions associated with Campylobacter jejuni septicemia in patients with hypogammaglobulinemia. European Journal of Clinical Microbiology and Infectious Diseases 11, 842–7.[ISI][Medline]

11 . Leibovitz, E., Janco, J., Piglansky, L. et al. (2000). Oral ciprofloxacin vs. intramuscular ceftriaxone as empiric treatment of acute invasive diarrhea in children. Pediatric Infectious Disease Journal 19, 1060–7.[ISI][Medline]