Antimicrobial susceptibility profiles of thermophilic campylobacters isolated from patients in the town of Niš Profil osetljivosti termofilnih kampilobaktera izolovanih kod obolelih u Nišu

Background/Aim. In some clinical forms of human Campylobacter infections, such as prolonged diarrhea or associated with postinfections sequels, antibacterial treatment is necessary. The aim of the present study was to evaluate the antimicrobial susceptibility of thermophilic Campylobacter strains isolated from patients with diarrhea, as well as from patients with diarrhea followed by postinfections sequels, to drugs used in the therapy of enterocolitis, and to nalidixic acid used in laboratory identification and differentiation of thermophilic Campylobacter spp. Methods. We studied the antimicrobial susceptibility profiles of 131 Campylobacter strains isolated from patients with diarrhea (122 strains), diarrhea associated with rheumatic disorders (8 strains), and one strain isolated from a patient with Guillain-Barré Syndrome following Campylobacter enterocolitis. Susceptibility testing to erythromycin, gentamicin, tetracycline, chloramphenicol, ciprofloxacin and nalidixic acid was performed by the agar dilution method. Results. In the strains we investigated, resistance to gentamicin and chloramphenicol was not recorded, whereas a low rate of strains resistant to erythromycin (2.4%), a higher prevalence of strains resistant to tetracycline (9.9%), and a high level of resistance to ciprofloxacin (29.8%) and nalidixic acid (33.3%) were registered. All strains resistant to nalidixic acid were also resistant to ciprofloxacin. In addition, there was no difference in the occurrence of resistance between strains isolated from patients with diarrhea as compared to those isolated from patients with diarrhea followed by postinfection disorders. Conclusion. The fact that the most of Campylobacter strains were sensitive to erythromycin and all to gentamicin, makes erythromycin an antibiotic of choice in the treatment of Campylobacter diarrhea and gentamicin when parenteral therapy should be administered. Resistance to tetracycline and, especially, ciprofloxacin, necessitates antibiotic susceptibility testing.


Introduction
Although human Campylobacter enterocolitis is often a self-limiting disease, treatment is necessary in illness with severe symptoms, prolonged disease, in immunocompromised patients and in patients with chronic sequels, such as Guillain-Barré syndrome (GBS) 1 .In the therapy of Campylobacter enterocolitis, macrolides and quinolones are very effective 2,3 .However, reports on resistance to erythromycin and also increasing Campylobacter resistance to quinolones may pose a threat to efficient therapy 4,5 .In addition, the rate of sensitivity to drugs recommended for therapy differs between different geographic regions 6 .
The aim of the present study was to evaluate the antimicrobial susceptibility of thermophilic Campylobacter strains isolated from patients with diarrhea, as well as from patients with diarrhea followed by postinfections sequels, against drugs used in the therapy of enterocolitis, and to nalidixic acid used in laboratory identification and differentiation of thermophilic Campylobacter spp.

Methods
We investigated antimicrobial susceptibility of thermophilic Campylobacter strains isolated at the Institute for Public Health, the town of Niš, Serbia, in 2002 and 2003 from the stool of patients with diarrhea (n = 122) and diarrhea followed by rheumatic disorder (n = 8) in clinic and outclinic patients in Niš.We also included a strain of Campylobacter jejuni associated with GBS isolated at the Republic Institute for Public Health, Belgrade.A total of 131 strains was thus included in the study.
Strains were isolated on Columbia agar base supplemented with 5% sheep blood and antibiotics (cefoperazone 1.5 g/L, colistin 10 6 U, vancomycin 1 g/L, amphotericin B 0.2 g/L), (bioMérieux, Marcy l'Etoile, France), following incubation in a jar under microaerobic conditions (Gas generating system "Torlak", Belgrade, Serbia), at 42º C, 48 hours.Identification to the level of genus was made using colony morphology, Gram staining ("gull wings", S-or spiralshaped bacteria), oxidase and catalase tests.Strains were stored at -20º C in a glucose broth supplemented with 5% horse serum until susceptibility testing was performed.
Strains grown after 48 hours of incubation at 37º C on Columbia agar base (bioMérieux, Marcy l'Etoile, France) with 5% defibrinated horse blood under microaerophilic conditions described above were resuspended in sterile saline to obtain a density of 0.5 on a McFarland scale.Susceptibility testing was performed using the agar dilution method to erythromycin, gentamicin, tetracycline, chloramphenicol, ciprofloxacin and nalidixic acid.Pure substances of antibiotics were purchased from the manufacturer ("Galenika", Belgrade).Erythromycin and chloramphenicol were suspended in 95% ethanol, gentamicin in phosphate-buffered saline (PBS) (pH 8), tetracycline in distilled H 2 O, ciprofloxacin in PBS (pH6) and nalidixic acid in 1N NaOH for stock dilutions.They were prepared as serial dilutions, and added to agar base at 50º C in 90 mm agar plates.
A minimal inhibitory concentration was defined as the lowest concentration producing no visible growth.
As no official recommendations for breakpoints exist, we used from the literature data for erythromycin 4 mg/L 7 , and for gentamicin and tetracycline, 8 mg/L, cholaramphenicol 16 mg/L, ciprofloxacin 4 mg/L, nalidixic acid 32 mg/L.We used MIC interpretative standards for Enterobacteriaceae 8 .
Campylobacter jejuni NCCLS 11951 and Staphylococcus aureus ATCC 29213 were used as control for growth.
A multiresistant strain was defined as a strain resistant to three or more antibiotics.
In order to determine the difference in frequency of resistant strains occurring in the two groups of patients, Fisher's exact test was performed.Statistical calculation was performed using a standard statistical program (EpiInfo ver 6.04).

Results
By using the agar dilution method, we detected antimicrobial resistance in 47 strains: to one antibiotic in 32 strains, to two in 13 strains and to three in two strains.The results of the susceptibility testing, along with the values of MIC 50 and MIC 90 , are presented in Table 1.
When strains associated with postinfections sequels were selected, MIC 50 and MIC 90 (mg/L) for erythromycin were 0.25 and 0.5, for gentamicin 1 (both values), for tetracycline 0.5 and 4, for chloramphenicol 2 and 4, and for ciprofloxacin 0.25 and 8, respectively.
In strains isolated from patients with diarrhea only, MIC 50 and MIC 90 were not changed as compared with values obtained for all investigated strains.Minimal inhibitory concentrations (mg/L) for the strain isolated from the patient with GBS were 0.12 for erythromycin, 2 for chloramphenicol, 0.5 for ciprofloxacin, and 1 for tetracycline and gentamicin.
A closer investigation of strains isolated from diarrhea associated with postinfections sequels (patients with rheumatic disorders and GBS) showed resistance to two antibiotics: one strain was resistant to tetracycline (11%) and three strains to ciprofloxacin (33%).Resistance to erythromycin, chloramphenicol and gentamicin was not recorded.The strain isolated from the patient with GBS was susceptible to all antibiotics tested.
When the frequencies of detected resistance to antibiotics in the group of strains isolated from patients with diarrhea and from patients with diarrhea complicated with rheumatic or neurological disorders were compared, no differences were found (Fisher' exact test p = 1.00) for erythromycin, tetracycline and ciprofloxacin.Since there was no recorded resistance to gentamicin and chloramphenicol in both investigated groups, statistical analysis was not performed for those antibiotics.

Discussion
Depending on the geographic localization, the success of treating Campylobacter spp.infection with drugs recommended for the therapy may differ considerably.The present susceptibility testing of strains isolated in the town of Niš, Serbia, revealed occurrence of antimicrobial resistance to erythromycin, ciprofloxacin, tetracycline and nalidixic acid.All strains were sensitive to gentamicin and chloramphenicol.Higher percentage of resistant strains was proved in the study conducted in north Indian rural community -antibiotic resistance of Campylobacter species was as follows: cipro-floxacin 71.4%, tetracycline 26.5%, furazolidine 14.3%, gentamicin 10.2% and erythromycin 6.1%; 30.6% of strains were multidrug resistant 9 .In the study conducted in Poland, the highest resistance was observed for ciprofloxacin (more than 40%), followed by ampicillin, and tetracycline, with significant resistance increase to tetracycline between 2003 and 2005 10 .
The growth of 50 and 90% of our isolates was inhibited by erythromycin concentrations of 0.5 and 1 mg/L, respectively.In a Finnish study on domestic and foreign strains of thermophilic Campylobacter strains, MIC 50 and MIC 90 values were 1 and 2 μg/ml for domestic strains, whereas the values for foreign strains were 1 and 4 μg/ml, respectively 11 .At the breakpoint of MIC ≥ 0.4 mg/L, we detected strains resistant to erythromycin in 2.4% of isolates.That fact underlines the possibility of an increasing prevalence of strains resistant to erythromycin in the future.
In a comprehensive study published in Spain in 1994, resistance to erythromycin was detected in only 3.2% of strains, with MIC of ≥ 4 μg/ml, while later studies reported an increase of strains resistant to erythromycin 7,12 .In the study conducted in the Netherlands, resistance to erythromycin increased from 1.9% (in their wide 2001) to 2.7% (in 2004) 13 .In Crete, 14.9% of thermophilic Campylobacter spp.strains were resistant to erythromycin 14 .In some reports, an increasing resistance to macrolides (50%) seems to be a real threat; however, other studies report on quite low or absent resistance rates to erythromycin 15 .
Values of MIC 50 and MIC 90 for gentamicin in our strains were 0.5 and 1 mg/L, respectively.Our strains did not exhibit resistance to gentamicin (MIC ranged from ≤ 0.25 to 4 mg/L).In strains studied in Germany MIC 50 and MIC 90 were 2, without detection of resistant strains at the breakpoint of MIC ≥ 16 mg/L 16 .In the Spanish study referred above, 1% of strains investigated were resistant to gentamicin 7 .In Crete, resistance was detected in 2.3% of Campylobacter spp.isolates 14 .
In this study, MIC 50 and MIC 90 (mg/L) of tetracycline were ≤ 0.5 and 8, respectively, and resistance to tetracycline was seen in 10% of the strains, at the breakpoint of 8 mg/L.For the strains isolated in Germany, MIC 50 and MIC 90 (mg/L) were 0.06 and 16, respectively, with resistant strains occurring in 13.5% of isolates at the same breakpoint 16 .Resistance to tetracycline was recorded in the Spanish study in 21.2% of strains 8 .
In this study, MIC 50 and MIC 90 (mg/L) of ciprofloxacin were ≤ 0.25 and 8.0, respectively.In addition, 29.8% of strains investigated were resistant to ciprofloxacin.One of the first reports of ciprofloxacin resistance (9%) was in 1991, in Finland 17 .Since then, the prevalence of strains resistant to ciprofloxacin has increased several times 9 .In a new Finnish study, MIC 50 and MIC 90 for domestically acquired strains were 0.25 and 0.5 μg/mL, respectively and for imported strains 1 and 64 μg/mL 11 .Those findings suggest a progressively reduced therapeutic value of ciprofloxacin.A resistance rate of 39% was found in human isolates in a study recently conducted in Austria 18 .In another recent study conducted in Thailand, 90% of strains were resistant to ciprofloxacin 15 .In Crete, 42.5% of Campylobacter spp.strains were resistant to ciprofloxacin 14 .
Resistance to quinolones in Campylobacter spp.from human infections may be related to clinical use, or use of fluoroquinolones in animal husbandry, or both 19 .A more thorough investigation of this problem is necessary to prevent its increase.A study conducted in England and Wales 20 recommended that both veterinary and clinical use should be reconsidered and that fluoroquinolone antibiotics should be used only to treat serious infections requiring hospital admission.Also, using antibiotics in a month before the is the risk factor for acquering a ciprofloxacin-resistant strain of Campylobacter 21 .Resistance rates increased with increasing urbanisation, too 13 .Increased resistance to macrolide and quinolone antibiotics poses major risks for treatment failure 22 .
We detected a relatively high proportion of resistance to nalidixic acid.Resistance to nalidixic acid in both Campilobacter jejuni and Campilobacter coli strains was observed during preliminary identification.All of the strains, which were resistant to nalidixic acid, were simultaneously resistant to ciprofloxacin.
This study did not detect strains resistant to chloramphenicol.In the study conducted in Spain in 1994 resistance to chloramphenicol occurred in 2.6 % of isolates 7 .In strains isolated in Crete, Greece, 7.9% of investigated strains were resistant to that antibiotic 14 .In England and Wales, resistance to chloramphenicol was recorded in 5.4 % of investigated strains at the breakpoint of 8 mg/L 20 .
Since we have detected two strains that were simultaneously resistant to quinolones (ciprofloxacin and nalidixic acid) and tetracycline, we can not discuss the presence of multiple resistance in our strains.Multiple resistance in Campylobacter can occur, but is usually seen in animal isolates 23 .A relatively high rate of multiple resistant strains (14.8%) was described in Harare, Zimbabwe 24 .In human isolates, multidrug resistance may include antibiotics important for infection treatment, such as erythromycin, tetracycline, and gentamicin or ciprofloxacin, tetracycline, and erythromycin 24,25 .The appearance of resistant strains may be due to less prudent use of antibiotics in veterinary and/or human practice 26 .

Conclusion
Strains isolated from patients with enterocolitis and enterocolitis associated with postinfections sequels expressed a similar pattern of sensitivity.Low levels of resistance to erythromycin makes it as the antibiotic of choice in the treatment of diarrhea or in diarrhea complicated with post infections sequels.When parenteral therapy should be included, gentamicin is also a drug of choice.Resistance to tetracycline and fluoroquinolones, ciprofloxacin, necessitates sensitivity testing.Resistance to nalidixic acid diminished its value in preliminary identification, but in our strains, it was a marker of resistance to ciprofloxacin.Further investigation should be considered in Serbia in the future.