Original Article ANTIBIOTIC RESISTANCE IN CAMPYLOBACTER JEJUNI IN RAWALPINDI AND ISLAMABAD A Preliminary Study Arif Maqsood Ali 1, Ayaz Hussain Qureshi 2, Shahid Rafi 3, Iqbal Ahmad Khan 4 & Shujaat Hussain 5 ABSTRACT: Objective: To determine the antimicrobial resistance in Campylobacter jejuni isolated from stools of children suffering from diarrhoea/dysentery in our setup against the antimicrobials commonly used as empirical therapy. Study: A prospective cross sectional descriptive study. Place and duration of study: Department of Microbiology, Army Medical College and Military Hospital, Rawalpindi from 29 August to 29 November 2002. Patients and methods: The study was carried out on eighteen isolates recovered from one hundred stool samples of children up to the age of twelve years admitted with diarrhoea/dysentery in Military hospital, Rawalpindi. The samples were collected in clean polypropylene containers containing Cary Blair medium. These were transported to the Microbiology Department, Army Medical College, Rawalpindi within 1-2 hours. The samples were inoculated on Modified Preston (Oxoid) and Karmali media (Oxoid) beside other routine stool culture media. The cultures were incubated at 42 o C under microaerophilic conditions. The growth after 48 hours was provisionally identified by colonial morphology, oxidase test, Gram staining and motility. The organisms were identified to species level by hippurate hydrolysis, urease test, nitrate reduction, catalase test, H 2 S production, resistance to cephalothin and sensitivity to nalidixic acid. Sensitivity testing was carried by Modified Kirby Bauer disc diffusion technique on lysed horse Blood Agar against ampicillin (10 ug), erythromycin (15ug), tetracycline (10ug), chloramphenicol (30ug), trimethoprim/ sulphamethoxazole (1.25ug/23.75ug), nalidixic acid (30ug) and ciprofloxacin (5ug). Results: One isolate (7.14%) was resistant to ciprofloxacin, three (16.66%) to chloramphenicol and four (22.22%) to nalidixic acid, five (27.77%) to erythromycin, seven (38.88%) to tetracycline, sixteen (88.88%) to trimethoprim/sulphamethoxazole and ampicillin respectively. Conclusion: The susceptibility pattern reflects variable susceptibility with maximum resistance to ampicillin and trimethoprim/sulphamethoxazole. Four isolates were resistant to nalidixic acid. KEY WORDS: Campylobacter jejuni, Diarrhoea/dysentery, Antimicrobial resistance, Children. Pak J Med Sci October-December 2003 Vol. 19 No. 4 272-276 www.pjms.com.pk 1. Major Arif Maqsood Ali Instructor class B and Trainee FCPS (Microbiology) 2. Brig. Ayaz Hussain Qureshi Head Department of Pathology 3. Lt. Col. Shahid Rafi Assistant Professor of Pathology 4. Lt. Col. Iqbal Ahmad Khan Professor of Community Medicine 5. Maj.Gen. Shujaat Hussain HI (M) Principal 1-5. Army Medical College, Rawalpindi, Pakistan Corresspondence: Major Arif Maqsood Ali E mail:drarifmaqsood@hotmail.com * Received for publication: May 24, 2003 Accepted: August 6, 2003 INTRODUCTION Diarrhoea is a common cause of increased morbidity and mortality in children in developing countries and Pakistan is no exception. According to WHO fact sheet diarrhoea occurs worldwide and causes 4% of all deaths and 5% of health loss to disability. It kills around 2.2 million people globally each year, mostly children in developing countries 1. Bacteria are important agents in the long list of the causes of diarrhoea and among them Campylobacter jejuni (C. jejuni) is now being recognized around the world as one of the principal causes of gastroenteritis 2. Pakistan being a developing 272 Pak J Med Sci 2003 Vol. 19 No. 4 www.pjms.com.pk
Antibiotic resistance in campylobacter jejuni country has a high incidence of diarrhoeal diseases especially in children and C. jejuni may be one of the principal causes of diarrhoea. Little information is available on the subject in our setup, as most of the laboratories are not carrying out cultures for C. jejuni routinely. This may be due to lack of awareness of the subject or due to financial constraints. A study was designed to assess the existing sensitivity pattern against routinely used antimicrobials using disc diffusion method as empirical therapy in children admitted with diarrhoea /dysentery in Military Hospital, Rawalpindi 3. PATIENTS AND METHODS The study was performed on 18 clinical isolates recovered from one hundred stool samples of children up to the age of twelve years admitted with diarrhoea/dysentery in Military hospital, Rawalpindi. The samples were collected in clean polypropylene containers with screw caps, containing Cary Blair medium for their transport to the Pathology Laboratories, Army Medical College, Rawalpindi 4,5. The samples so collected were inoculated on Modified Preston and Karmali media (Oxoid) in parallel with cultures on Deoxycholate Citrate Agar and Thiosulphate Citrate Bile Salt Agar (Oxoid) 6. The cultures were incubated at 42 o C in Anaerobic Jar (Oxoid USA, Columbia, MD) under microaerophilic conditions using gasgenerating kit CN 035 (Oxoid). The growth was identified after 48 hours by colony morphology, oxidase test, Gram staining and motility 7. The organisms were identified to species level by a positive catalase test, a negative urease test, failure to produce H 2 S, non-fermentation of sugars, resistance to cephalothin, nitrate reduction and hippurate hydrolysis 8,9. Sensitivity testing was carried out on lysed horse Blood Agar using Modified Kirby Bauer technique against ampicillin (10ug), erythromycin (15ug), tetracycline (10ug), chloramphenicol (30ug), trimethoprim/ sulphamethoxazole (1.25ug/23.75ug), nalidixic acid (30ug) and ciprofloxacin (5ug). The sensitivity plates were incubated at 37 o C under microaerophilic conditions generated in a similar manner as for primary isolation. The results were read after 24 hrs. and interpreted in accordance with NCCLS criteria 3,10. Inclusion Criteria: Stool samples of either sex children up to the age of 12 years suffering from diarrhoea/dysentery were included in the study. The samples containing mucus, pus and /or blood were preferred. Exclusion Criteria: Children on antibiotics, three days prior to sample collection were excluded. RESULTS Out of 100 stool samples studied eighteen samples (18%) yielded the growth of C. jejuni (Table-I). Sixteen out of 18 isolates were resistant to trimethoprim sulphamethoxazole (88.88%), four were resistant to nalidixic acid (22.22%) and one was resistant to ciprofloxacin (5.55%) (Table-II). Table-I: Frequency of Faecal Isolates (n = 100) Organism No. of Isolates Campylobacter jejuni 18 (18%) Vibrio cholerae biotype El Tor 2 (2%) serotype Ogawa Shigella flexneri 1 (1%) Aeromonas hydrophila 1 (1%) Table-II: Antibiotic Resistance of Campylobacter Jejuni Isolates (n = 18) Antibiotic Sensitive Resistant Tetracycline 11 (61.11%) 7 (38.89%) Ampicillin 2 (11.11%) 16 (88.89%) Sulphamethoxazole- 2 (11.11%) 16 (88.89%) Trimethoprim Chloramphenicol 15 (83.33%) 3 (16.67%) Erythromycin 13 (72.22%) 5 (27.78%) Nalidixic acid 14 (77.78%) 4 (22.22%) Ciprofloxacin 17 (94.45%) 1 (5.55%) Pak J Med Sci 2003 Vol. 19 No. 4 www.pjms.com.pk 273
Arif Maqsood Ali et al. DISCUSSION Campylobacter jejuni (C.jejuni) enteritis is usually self-limiting. However, antibiotics have a role in reducing the symptoms, shortening the span of illness and controlling the transmission in the community. Erythromycin, tetracycline and quinolones have all been recommended in different clinical settings to treat campylobacter gastroenteritis 11-16. Fluoroquinolones are frequently prescribed empirically for diarrhoeal illness including travelers diarrhoea because of their effectiveness against a wide range of enteric bacteria 14-18. Fluoroquinolones also shorten the diarrhoea and severity of symptoms caused by C. jejuni 19,20. Since the late 1980s resistance in campylobacter isolates to fluoroquinolones has been increasing especially in Europe 13,21. A causal relationship has been found between the use of fluoroquinolones in animals and an increase in fluoroquinolone resistant campylobacter infection in humans 21,22. In our study one isolate (5.55%) was resistant to ciprofloxacin whereas four isolates (22.22%) were resistant to nalidixic acid. Ciprofloxacin resistant isolate was also resistant to nalidixic acid. Ciprofloxacin and nalidixic acid resistance reported from Tokyo, Japan was 7.3% and 10.3 % respectively 23. In Montreal, Canada these were 12.7% and 13.9% respectively 24. In a study from Mallorca, Spain 27.8% was resistant to ciprofloxacin and 24.2% were resistant to nalidixic acid 25. In Chennai, India while all the isolates (100%) were resistant to nalidixic acid, none was resistant to ciprofloxacin 26. Nalidixic acid resistant C.jejuni isolates recovered from diarrhoeal stools were 15% and 40% in Germany and in Egypt respectively 27,28. The highest reported resistance to quinolones is from Villarroel, Spain where nalidixic acid and ciprofloxacin resistance has almost doubled from 47.5% in 1990 to 88% in 1994 29. Quinolone resistance reported from Lugo, Spain and Norway were 34% and 23.3% respectively 30,31. However, other studies report minimal resistance to ciprofloxacin. In a study from Lukhnow, India only 2.7% were resistant to ciprofloxacin 32. In a study carried out in Sweden from 1978 to 1988 no general increase in vitro resistance to antibiotics commonly used for human gastroenteritis caused by C. jejuni was observed. The number of fluoroquinolone resistant strains from 1988 to 1989 was 0.7% and 1.4% respectively 33. The resistance to ciprofloxacin in our study is higher than that reported from Lukhnow, India but is lower than the resistance reported from Tokyo, Japan, Chennai, India, Spain, Germany and Egypt. However, the resistance to nalidixic acid in our isolates was similar to that reported from Spain. It was higher than that reported from Japan, Canada, Germany and Lukhnow, India but was lower than Chennai, India. Twenty seven percent of C. jejuni isolates in our study were resistant to erythromycin. In studies reported from Harare, Zimbabwe and Wroclow, Poland 15% of C.jejuni isolates were resistant to erythromycin while in Brazil 18.2% were resistant to erythromycin 34,35. A very high degree of resistance to erythromycin (65%) in C.jejuni was observed in a study from Thailand where the antibiotic was found to have no effect on the duration of diarrhoea 36. Similarly, a high level resistance to erythromycin was seen in a study from Singapore General hospital 37. However, most of the other studies had shown low level of resistance to erythromycin 23,29,30. In Lukhnow, India resistance in C.jejuni to erythromycin was 1.3%. It was 1% in London, UK 4%, in Kuala Lumpur, Malaysia while in Saudi Arabia 7.3% 31,38,36,39. In a study carried out in Mallorca, Spain the rate of erythromycin resistance remained low (0.9% - 03.5%) during the period 1987-1991 25. The high rate of erythromycin resistance in our study may be due to erythromycin being frequently prescribed in our set up. The resistance of C. jejuni to trimethoprim/ sulphamethoxazole and ampicillin was very high among our isolates (88.89%), as compared to the resistance reported from Brazil where 56.8% out of 22 isolates of C. jejuni were resistant to both the drugs 35. All the strains were resistant to trimethoprim/sulphamethoxazole 274 Pak J Med Sci 2003 Vol. 19 No. 4 www.pjms.com.pk
Antibiotic resistance in campylobacter jejuni in the study carried out in Mallorca, Spain during the period 1987-1991 25. Ampicillin and trimethoprim/sulphamethoxazole are frequently prescribed to treat diarrhoea/dysentery and are readily available in our clinical setting. This clinical practice in our setting might be a contributing factor in increased resistance to trimethoprim/sulphamethoxazole and ampicillin in our study. In our study 16.67% isolates were resistant to chloramphenicol. Resistance to chloramphenicol was common in studies from Vietnam and Thailand 40. All isolates in studies reported from Brazil and Zimbabwe were susceptible to chloramphenicol 35,41. Only 2.6% resistance was seen in isolates in Mallorca, Spain 25. Fifty percent of the isolates were found resistant in a study carried out in Egypt 33. Our figures remain less than those reported from Egypt but are higher than those reported from Spain, Brazil and Zimbabwe. Tetracycline resistance (38.89%) in our setup is similar to the studies from Quebec, Canada, Lugo, Spain, Tokyo, Japan and Saudi Arabia where it was 40.7 %, 43.1%, 43.2% and 32.7% respectively 42,29,23,39. Our results are higher than those reported from Lukhnow, India and Wroclow, Poland where 10% and 9.3% were found resistant to tetracycline respectively 31,34. The C. jejuni revealed significant resistance to commonly used antimicrobials like ampicillin, trimethoprim/sulphamethoxazole, tetracycline, erythromycin and nalidixic acid in our study. In contrast chloramphenicol and fluoroquinolones had adequate activity against most of the C.jejuni. However, because of the adverse effects of chloramphenicol on bone marrow and fluoroquinolones on cartilage both the drugs may have to be reserved as a last option for C.jejuni. ACKNOWLEGEMENT The author (s) are grateful to Pak-US Laboratory for Sero-epidemiology (PULSE) Army Medical College, Rawalpindi for providing funding, lab facilities and technical assistance without which this project could not be completed. REFERENCES 1. World Health Organization. World Water Day 2001, Disease fact sheet: Diarrhoea. Geneva: WHO, 2000 May; 1-2. 2. Lopez Ortiz W, Sullivan RA, Campylobacter jejuni among patients with gastroenteritis: incidence at a reference microbiology laboratory in San Juan, Puerto Rico, and PR Health Sci J 1999; 18(33): 273-6. 3. Huysmans MB, Turnidge JD. Disc susceptibility testing for thermophilic Campylobacter. Pathology 1997; 29(2): 209-16. 4. Wasfy M, Oyofo B, Elgindy A, Churilla A, Comparison of Preservation media for storage of stool samples, J Clin Microbiol 1995; 33(8): 2176-8. 5. Nachamkin I. Campylobacter and arcobacter. In: Murray PR eds. Manual of Clinical Microbiology, 6 th Edition, Washington: ASM; 1995: 483-86. 6. Merino FJ, Agulla A, Villasante PA, Diaz A, Saz JV, Velasco AC, Comparative efficacy of seven selective media for isolating Campylobacter jejuni. J Clin Microbiol 1986; 24(3): 451-2. 7. Old DC. Vibrio, aeromonas, plesiomonas, campylobacter, arcobacter, helicobacter, wolinella. In: Collee JG, Marmion BP, Fraser AG, Simmons A, eds. Mackie & McCartney Practical medical microbiology. New York: Churchill Livingstone, 1996: 437. 8. Elmer W, Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Win WC Jr, eds. Color atlas and textbook of diagnostic microbiology, 4 th ed. Pennsylvania: J.B. Lippincott, 1992: 244-51. 9. Harvey SM. Hippurate hydrolysis by Campylobacter fetus.j Clin Microbiol. 1980; 11:435-437. 10. National Committee for Clinical Laboratory Standards: Performance Standards for Antimicrobial Disk Susceptibility Tests (Approved Standard, M2- A4).Villanova,PA,National Committee for Clinical Laboratory Standards,1984. 11. Vanhoof R, Vanderlinden MP, Dierickr R, Lawers S, Yurasowsky E, Butzler JP. Susceptibility of Campylobacter fetus subsp. jejuni to twenty nine antimicrobial agents. Antimicrob Agents Chemother. 1978; 14:153-156. 12. Engberg J, Aarestrup FM, Taylor DE, Garner Smidt P, Nachamkin I. Quinolone and macrolide resistance in Campylobacter jejuni and C. coli: resistance mechanisms and trends in human isolates. Emerg Infect Dis 2001; 7(1): 24-34. 13. Smith KE, Besser JM, et al. Quinolone resistant Campylobacter jejuni infections in Minnesota, 1992-1998. N Eng J Med, 1999; 340:1525-1532. 14. Allos BM, Blaser MJ. Campylobacter jejuni and the expanding spectrum of related infections. Clin Infect Dis 1995; 20: 1092-1099. Pak J Med Sci 2003 Vol. 19 No. 4 www.pjms.com.pk 275
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