Antimicrobial susceptibility patterns of Campylobacter jejuni strains isolated from hospitalized children in Athens, Greece

S. Chatzipanagiotou1,*, E. Papavasileiou2, A. Lakumenta2, A. Makri2, C. Nicolaou1, K. Chantzis1, S. Manganas1 and N. I. Legakis3

1Department of Clinical Microbiology, Athens Medical School, Aeginition Hospital, Vass. Sophias av. 72–74, 115 28 Athens; 2Department of Microbiology, Penteli Children’s Hospital, Athens; 3Department of Microbiology, Athens Medical School, Athens, Greece

Received 27 March 2001; returned 6 December 2001; revised 15 January 2002; accepted 23 January 2002.


    Abstract
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The antimicrobial susceptibility of 129 Campylobacter jejuni strains, isolated from hospitalized children with gastroenteritis, to five antimicrobials, including nalidixic acid, ciprofloxacin, erythromycin, ampicillin and co-amoxiclav, was determined. Isolates belonged to two time periods: group A contained strains isolated in 1987–1988; and group B 1998–2000. Antimicrobial susceptibility patterns differed significantly between the two groups with respect to quinolones, with an increase in the percentage of resistant strains in group B (30.6% versus 0% in group A), whereas erythromycin, ampicillin and co-amoxiclav were effective drugs in both groups.


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Campylobacter jejuni, a microaerophilic Gram-negative bacillus, is one of the most common causes of bacterial gastroenteritis. This organism can colonize the intestinal tract of many animals, including poultry, cattle and swine, without causing illness, and is transmitted via contaminated food and water.1 In humans, ingestion of C. jejuni can cause diarrhoea associated with abdominal pain, fever and vomiting.2 The disease is self-limiting, lasting 3–5 days, but in some cases it can persist for up to 2 weeks or longer, whereas bacteraemia has occasionally been reported. Post-infection complications associated with a preceding C. jejuni infection are rare, and consist mainly of neurological immunopathological dis-orders, such as Guillain–Barré syndrome.3

Because of the low mortality and the self-limiting nature of C. jejuni infection, treatment is not justified unless the patient is immunocompromised.4 When indicated, erythromycin is the antimicrobial of choice. Ampicillin, co-amoxiclav and fluoroquinolones are recommended as alternative therapies. However, in the last 5 years there have been several reports of increasing resistance of C. jejuni to quinolones.5,6 In Greece the first reported ciprofloxacin-resistant C. jejuni strains were isolated from infected children in 1993.7

The aim of the present study was to determine and compare the in vitro antimicrobial susceptibility patterns to five antimicrobials of C. jejuni isolated from hospitalized children with gastroenteritis, and collected during two time periods: 1987–1988 (group A) and 1998–2000 (group B).


    Patients and methods
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients and strains

All patients were children up to 14 years with gastroenteritis, presenting in the out-patient department of the Children’s Hospital of Penteli in Athens. Stool specimens were collected and sent for bacteriological examination on the day of hospitalization. None of the patients had received any kind of antibiotic treatment before bacteriological examination. A total of 129 C. jejuni strains were collected during two periods: 31 during 1987–1988 (group A) and 98 during 1998–2000 (group B). All strains were kept frozen in liquid nitrogen until the day of examination. MIC determination was performed simultaneously in both groups.

Stool culture for Campylobacter

For the isolation of Campylobacter, stools were plated on Skirrow’s selective medium and incubated for 48 h at 42°C in a microaerophilic atmosphere (Genbox-microaer; bio-Mérieux, Marcy l’Étoile, France). C. jejuni was identified by typical growth, microscopic examination, hippurate hydrolysis, and positive catalase and oxidase reactions. Strains were suspended in stock culture medium containing trypticase soy broth with 20% glycerol and stored at –80°C.

MIC determination

Determination of MICs of erythromycin, nalidixic acid, ciprofloxacin, ampicillin and co-amoxiclav was performed by the standard agar dilution method.8 Antimicrobials were obtained as pure powders (Sigma Diagnostics, St Louis, MO, USA). Serial dilutions of antimicrobial stock solutions were prepared and incorporated in Mueller–Hinton agar containing 5% lysed horse blood. Bacterial suspensions in Mueller–Hinton broth were inoculated using a multipoint inoculator (MAST Laboratories, Bootle, UK) at a density of 105 cfu/mL and a volume of 10 µL per spot. Plates were incubated for 24 h at 42°C in a microaerophilic atmosphere. The MIC was defined as the lowest concentration of the antimicrobial that completely prevented visible growth. Results were interpreted according to the criteria of the NCCLS for Enterobacteriaceae (breakpoints are given in Table 1).


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Table 1.  Antimicrobial resistance of C. jejuni strains isolated from children with gastroenteritis during the periods 1987–1988 (group A) and 1998–2000 (group B)a
 

    Results
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 Abstract
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 Patients and methods
 Results
 Discussion
 References
 
There were significant differences in the antimicrobial susceptibility patterns between groups A and B. In group A, all strains were susceptible to ciprofloxacin, nalidixic acid, ampicillin and co-amoxiclav, and 12.9% were resistant to erythromycin (Table 1). In group B, resistance patterns were as follows: ciprofloxacin 30.6%, nalidixic acid 30.6%, erythromycin 0%, ampicillin 5.1% and co-amoxiclav 1% (Table 1).

Determination of MIC50s and MIC90s are shown in Table 2. Particularly noteworthy is the net shift of MIC90 to the side of resistance from group A to group B for nalidixic acid and ciprofloxacin, and the reverse shift (to the side of susceptibility) for erythromycin (Table 2). The MIC90s of ampicillin and co-amoxiclav remained unchanged from one time period to the other, although there was a significant decrease in MIC50s of these antimicrobials in group B compared with those of group A (Table 2)


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Table 2.  Determination of MIC50s and MIC90s (mg/L) of nalidixic acid, ciprofloxacin, erythromycin, ampicillin and co-amoxiclav for C. jejuni strains isolated from infected children during the periods 1987–1988 (group A) and 1998–2000 (group B)a
 

    Discussion
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
The antimicrobial susceptibility patterns of C. jejuni isolates differed between the two groups of isolates. The increase in quinolone resistance during recent years (1998–2000) in comparison with the incidence of quinolone resistance in the period 1987–1988 (Table 1) is a phenomenon reported worldwide, and was first observed in Greece in 1993.7,911 C. jejuni gastroenteritis is usually self-limiting, lasting only for a few days and does not require treatment unless the patient is immunocompromised or the disease becomes generalized. On the other hand, quinolones are frequently prescribed empirically for diarrhoeal illness because of their effectiveness against a range of enteropathogenic bacteria.12 Resistance to quinolones is chromosomally acquired (mutation in GyrA or GyrB gene) and the appearance of resistant strains probably reflects a wide use of these antimicrobials in both human infections and veterinary medicine (especially in poultry).9 In the present study all subjects were paediatric patients, a group for whom quinolone use is not recommended. Unfortunately, there is no official information about the use of quinolones in food animals in Greece. The only information we have, obtained through personal communication, is that oxolinic acid, a quinolone, has been widely used in poultry since 1990. Regarding our results, erythromycin, ampicillin and co-amoxiclav, antimicrobial agents with a therapeutic use restricted to humans, appear to remain effect-ive against C. jejuni (Table 1).

The antimicrobial treatment for Campylobacter is often initiated before the results of antimicrobial susceptibility testing are available; therefore, a general knowledge of the expected susceptibility of Campylobacter is a prerequisite to start treatment with an appropriate antimicrobial agent. The establishment of a simple surveillance system for recording and reporting data would be of great assistance and would improve the implementation of correct treatment.


    Acknowledgements
 
We thank the Research Account of the University of Athens for supporting a part of the present study.


    Footnotes
 
* Corresponding author. Tel: +30-1-7289192; Fax: +30-1-6004608; E-mail: chatlouk{at}hotmail.com Back


    References
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 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
1 . Centers for Disease Control. (1988). Campylobacter isolates in the United States, 1982–1986. Morbidity and Mortality Weekly Report SS-2, 1.

2 . Cover, T. L. & Blaser, M. J. (1989). The pathobiology of Campylo-bacter infections in humans. Annual Review of Medicine 40, 269–85.[ISI][Medline]

3 . Speed, B., Kaldor, J. & Cavanogh, P. (1984). Guillain–Barré syndrome associated with Campylobacter jejuni enteritis. Journal of Infection 8, 85–6.[ISI][Medline]

4 . Mishu, B. & Blaser, M. J. (1975). Campylobacter jejuni and the expanding spectrum of related infections. Clinical Infectious Diseases 20, 1091–8.

5 . Reina, J., Ros, M. J. & Serra, A. (1994). Susceptibilities to 10 antimicrobial agents of 1220 Campylobacter strains isolated from 1987 to 1993 from feces of pediatric patients. Antimicrobial Agents and Chemotherapy 38, 2917–20.[Abstract]

6 . Sanchez, R., Fernandez-Baca, V., Diaz, M. D., Miñoz, P., Rodriguez, M. & Bouza, E. (1994). Evolution of susceptibility of Campylobacter species to quinolones and macrolides. Antimicrobial Agents and Chemotherapy 38, 1879–82.[Abstract]

7 . Chatzipanagiotou, S., Papavassiliou, E. & Malamou-Lada, E. (1993). Isolation of Campylobacter jejuni strains resistant to nalidixic acid and fluoroquinolones from children with diarrhea in Athens, Greece. European Journal of Clinical Microbiology and Infectious Diseases 12, 566–8.[ISI][Medline]

8 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A4. NCCLS, Wayne, PA, USA.

9 . Gaunt, P. N. & Piddock, L. J. V. (1996). Ciprofloxacin resistant Campylobacter spp. in humans: an epidemiological and laboratory study. Journal of Antimicrobial Chemotherapy 37, 747–57.[Abstract]

10 . Husmann, M., Feddersen, A., Steitz, A., Freytag, C. & Bhakdi, S. (1997). Simultaneous identification of campylobacters and prediction of quinolone resistance by comparative sequence analysis. Journal of Clinical Microbiology 35, 2398–400.[Abstract]

11 . Ruiz, J., Goni, P., Marco, F., Gallardo, F., Mirelis, B., Jimenez De Anta, T. & Vila, J. (1998). Increased resistance to quinolones in Campylobacter jejuni: a genetic analysis of gyrA gene mutations in quinolone-resistant clinical isolates. Microbiology and Immunology 42, 223–6.[ISI][Medline]

12 . Hooper, D. C. & Wolfson, J. S. (1991). Fluoroquinolone antimicrobial agents. New England Journal of Medicine 324, 384–94.[ISI][Medline]