Indian J Med Res 123, February 2006, pp 145-150 Isolation & antimicrobial susceptibility of Shigella from patients with acute gastroenteritis in western Nepal Godwin Wilson, Joshy M. Easow, Chiranjoy Mukhopadhyay & P.G. Shivananda Department of Microbiology, Manipal College of Medical Sciences, Pokhara, Nepal Received January 13, 2005 Background & objectives: Shigellae play an important role as a causative organism of acute gastroenteritis, which is a global health problem with significant morbidity and mortality in especially in developing countries. This study was carried out to determine the isolation and pattern of antimicrobial resistance of Shigella in patients with acute gastroenteritis in western Nepal. Methods: The study included all patients with acute gestroenteritis who visited a tertiary care hospital at Pokhara, Nepal during a 2-year period (2002-2004). The isolates was confirmed as Shigella by biochemical reaction and slide agglutination test using specific antisera. Antibiotic sensitivity test was determined by agar diffusion method and minimum inhibitory concentration (MIC) of the drugs was detected. Results: Of the 770 stool samples, 83 (10.8%) yielded Shigella. Shigella flexneri caused 56 (67.4%) of the total cases of shigellosis followed by S. dysenteriae 12 (14.5%), S. sonnei 10 (12%) and S. boydii 5 (6%). Of the 83 isolates, 67 (80.7%) showed resistance to various drugs and 62 (74.7%) were resistant to two or more drugs. Resistance to cotrimoxazole was 80.7 per cent followed by tetracycline 74.7 per cent, ampicillin 53.0 per cent, nalidixic acid 31.3 per cent and ciprofloxacin 2.4 per cent. The MIC 50 and MIC 90 values of those drugs were also very high. All isolates were sensitive to cefotaxime and ceftriaxone. Interpretation & conclusion: The findings of our study suggested that Shigellae was an important etiological agent for acute gastroenteritis, with a high rate of drug resistance and requires constant monitoring in this region. Key words Agar dilution - antibiogram - MIC - seasonal variation - Shigella Diarrhoeal diseases and enteric infections are major causes of morbidity and mortality in the developing world 1. Epidemiology reports show that about 140 million people suffer from shigellosis with estimated 600,000 deaths per year worldwide 2. In Nigeria 3 as well as in Bangladesh 4, both children and young adults are at a higher risk. In Vellore, shigellosis was found to be one of the common causes 145
146 INDIAN J MED RES, FEBRUARY 2006 of gastroenteritis 5. Over a decade ago, the active surveillance studies showed that in most endemic countries especially in Asia and sub-saharan Africa 6,7, there was an emergence of multidrug resistance to different antibiotics including ampicillin, trimethoprim-sulphamethoxazole and nalidixic acid. Drugs like fluoroquinolones, azithromycin and pivamdinocillin have been found to be efficacious for the treatment of shigellosis in children and adults 8,9. Every year a large number of patients suffer from acute gastroenteritis in the western region of Pokhara, Nepal and isolation of Shigella from these patients is not uncommon; no studies were conducted so far from this region to evaluate the prevalence of Shigella in patients with acute gastroenteritis and the status of drug resistance in shigellosis. The present study was therefore carried out in a tertiary care hospital in Pokhara, Nepal to isolate Shigella from children and adults with suspected acute gastroenteritis, and to study its seasonal distribution and antimicrobial susceptibility pattern. Material & Methods Patients and sample collection: During October 2002 to September 2004, 770 stool samples were collected from all pre-school children (<5 yr), school-going children (6-15 yr) and adults (>15 yr) with acute gastroenteritis attending out patients department in a tertiary care hospital in Pokhara, Nepal, in clean, open-mouth disposable containers. All the samples were cultured within 2 h of collection and analyzed according to standard methods 10. Though most of the patients had suffered from dysentery, some patients had only mild diarrhoea and never developed dysenteric symptoms. Dysentery was characterized by frequent passage (usually 10 to 13 times/day) of small volume stools consisting of blood, mucus, and pus; often accompanied by abdominal cramps and tenesmus. Diarrhoea was defined as the passage of 3 or more liquid stools without blood and mucus in a 24 h period. Bacteriological analysis: The samples were primarily cultured on deoxycholate citrate agar (DCA) and MacConkey agar media (Himedia Laboratories Pvt. Ltd.). All plates were incubated aerobically at 37 º C overnight. The non-lactose-fermenting (NLF) colonies from both DCA and MacConkey agar were identified on urea hydrolysis, triple sugar iron (TSI) medium, sulphide-indole and motility medium (SIM), and Simmon s citrate test 11. They were further identified at group level by slide agglutination test 12 with specific antisera (DIFCO Laboratories, Detroit, Michigan, USA). Antimicrobial susceptibility testing: Resistance patterns of the Shigella isolates to various antibiotics were determined by the agar diffusion technique 13,14. Every inoculum was prepared by inoculating 5 ml of Mueller-Hinton broth with five colonies of an 18 h old pure Shigella culture followed by incubation in ambient air and at 37 º C for 16 h. The resulting turbid culture was standardized to a turbidity of 0.5 McFarland using 0.85 per cent NaCl as a diluent. A sterile cotton swab was dipped into the standardized suspension, drained, and used for inoculating 25 ml of Mueller-Hinton agar (MHA) in a 90 mm plate. The inoculating plates were air dried and antibiotic disks included ampicillin (10 µg), tetracycline (30 µg), cotrimoxazole i.e., trimethoprim/ sulphamethoxazole (1.25/23.75 µg), cefotaxime (30 µg), ceftriaxone (30 µg), ciprofloxacin (5 µg), nalidixic acid (30 µg), chloramphenicol (30 µg) and gentamicin (10 µg) [Oxoid (UK) and Hi-Media (Mumbai, India) were mounted on them. The plates were inverted and incubated in ambient air at 37 º C for 18 h. Zones of inhibition were recorded in millimeters and were compared with those of Escherichia coli ATCC25922 from (ICDDR, B, Dhaka, Bangladesh) which served as control strain. Determination of minimum inhibitory concentration (MIC): Minimum concentration of each antibiotic inhibitory to the growth of 50 per cent (MIC 50 ) and 90 per cent (MIC 90 ) of the isolates was determined on MHA in a 90 mm plate. The agar contained concentration ranges of the antibiotics prepared by two-fold serial dilution according to the National Committee for Clinical Laboratory Standards (NCCLS) 15. Manual inoculation with micropipette for dispensing 20 µl of standardized inoculum (10 7 cfu/ ml) of each isolate onto the surface of the antibiotic plate was done to obtain a final inoculum size of
WILSON et al: ANTIMICROBIAL SUSCEPTIBILITY OF SHIGELLA IN WESTERN NEPAL 147 10 4-10 5 cfu/spot. Antibiotic-free plates were inoculated at the end and were used as negative controls. The positive controls were the plates (one plate per antibiotic tested) inoculated with the reference strain E. coli ATCC25922. MIC 50 and MIC 90 of each antimicrobial agent against Shigella isolates were evaluated after incubating the plates, containing completely absorbed inocula, in ambient air at 37 º C for 18 h. Results Shigella was isolated from 83 of 770 (10.8%) stool samples. Of these, S. flexneri 56 (67.5%) was the most common isolate in all age groups, followed by S. dysenteriae 12 (14.5%), S. sonnei 10 (12%) and S. boydii 5 (6.0%). Isolation rate of S. flexneri was observed to be more among children less than 5 yr (71.4%, 15/21) and schoolgoing children (70.6%,12/17) compared to adults (64.4%, 29/45). Detection rates was highest in summer-monsoon i.e., June-September (54.2%, 45), and moderate in spring (March-May 22.9%, 19), and autumn (October-November 12.0%, 10), and lowest in winter (December-February 10.8%, 9). All 83 isolates were sensitive to cefotaxime and ceftriaxone and 67 showed variable resistance against the remaining seven antibiotics (Table I). Resistance to co-trimoxazole was highest (80.7%), followed by tetracycline (74.7%), gentamicin (55.4%), ampicillin (53%). chloromphenicol (39.7%) and nalidixic acid (31.3%). Ciprofloxacin (2.4%) had the least resistance. In 67 isolates, 7 patterns of antibiotic resistance were found (Table II), which on further analysis revealed that nearly 62 (74.7%) isolates were resistant to 2 or more drugs. Table I. Number of resistant isolates Shigella and per cent resistance among serogroups Antimicrobial drugs Resistant isolates Serogroups no. (%) S. flexneri S. dysenteriae S. sonnei n=51 (%) n=08 (%) n=07 (%) Ampicillin 44 (53) 33 (64.7) 06 (75) 05 (71.4) Cefotaxime 00 (00) 00 (00) 00 (00) 00 (00) Ceftriaxone 00 (00) 00 (00) 00 (00) 00 (00) Co-trimoxazole 67 (80.7) 51 (100) 08 (100) 07 (100) Tetracycline 62 (74.7) 49 (96) 07 (87.5) 06 (85.7) Chloramphenicol 33 (39.7) 23 (45.1) 05 (62.5) 05 (71.4) Gentamicin 46 (55.4) 34 (66.7) 06 (75) 06 (85.7) Nalidixic acid 26 (31.3) 16 (31.4) 05 (62.5) 05 (71.4) Ciprofloxacin 02 (2.4) 01 (2.0) 01 (12.5) 00 (00) Single isolate of S. boydii has shown resistance only against co-trimoxazole Table II. Patterns of antimicrobial resistance in Shigella isolates Antibiotic resistance pattern No. of resistant Shigella isolates (n=67) S. flexneri S. dysenteriae S. sonnei S. boydii (n=51) (n=08) (n=07) (n=01) Cot Tet Gen Amp Chl Nal Cif 01 01 00 00 Cot Tet Gen Amp Chl Nal 15 04 05 00 Cot Tet Gen Amp Chl 07 00 00 00 Cot Tet Gen Amp 10 01 00 00 Cot Tet Gen 01 00 01 00 Cot Tet 15 01 00 00 Cot 02 01 01 01 Cot, cotrimoxazole; Tet, tetracycline; Gen, gentamicin; Amp, ampicillin; Chl, chloramphenicol; Nal, nalidixic acid; Cif, ciprofloxacin
148 INDIAN J MED RES, FEBRUARY 2006 Table III. Minimum inhibitory concentrations (MICs) of antimicrobial agents for Shigella isolates Antimicrobial drugs Range MIC 50 MIC 90 (µg/ml) (µg/ml) (µg/ml) Ampicillin 8-32 16 64 Cefotaxime 8-64 16 32 Ceftriaxone 8-64 16 32 Co-trimoxazole (trimethoprime/sulphamethoxazole) 2/38-4/76 4/76 8/152 Tetracycline 4-16 16 64 Chloramphenicol 8-32 16 64 Gentamicin 4-16 8 32 Nalidixic acid 16-32 16 64 Ciprofloxacin 1-4 4 8 At 16 and 32 µg/ml, cefotaxime and ceftriaxone inhibited the growth of 50 and 90 per cent of the isolates respectively. The MIC 50 and MIC 90 values of co-trimoxazole, tetracycline, gentamicin, ampicillin, and nalidixic acids were very high and those of ciprofloxacin were within range (Table III). Discussion Shigellosis is a public health problem in western Nepal as the community is ravaged by poverty, poor sanitation, lack of personal hygiene and use of contaminated water supplies. It is a mountainous region and has a population of around 1,26,000 (2001 Census). The temperature ranges from 40º F (4º C) in January to approximately 100º F (38º C) in June, just before the monsoon. The summer-monsoon causes the climatic variation since rainwater is a major cause of rapid deterioration in the surface water quality in this area. The high isolation rate of Shigella (10.78%) from the stool samples of the acute gastroenteritis patients in the present study is also a reflection of poor hygiene and inadequate supply of clean drinking water. The finding of S. flexneri as the predominant and most active serogroup in western Nepal was similar to the study from Lagos, Nigeria where the predominance of S. flexneri did not change since last decade 3. However, this was unlike the situation in the islands of Bengal 16 where S. flexneri and S. dysenteriae alternated as most active agents of shigellosis or in endemic communities of Israel 17 and Pakistan 18 where S. sonnei was the predominant etiological agent. Children (both pre-school and school-going) as those from Bangladesh,were at a higher risk of getting affected by the disease, which might be a reflection of secondary infection from the adults as well as poor personal hygiene 8. Antimicrobial therapy is the cornerstone of treatment of shigellosis, as mortality especially with S. dysenteriae type 1 infection is appreciable (10%) in the young and elderly, and it prevents the more serious complications of the infection 19. The guiding principle for the choice of antimicrobials in developing countries includes cost, availability of the drug and pattern of resistance in the community. When compared with the study from Nigeria 3, we found decreased resistance for ampicillin, tetracycline, chloramphenicol and increased resistance for nalidixic acid, gentamicin and ciprofloxacin. The resistance against co-trimoxazole was similar in both the studies. All isolates were sensitive to cefotaxime and ceftriaxone. A study from north-western part of India documented nearly 100 per cent sensitivity of Shigella against drugs like chloramphenicol, nalidixic acid, co-trimoxazole, gentamicin and norfloxacin 20. The alarming rise in resistance in Shigella in this part against these drugs might be related to the indiscriminate use of drugs during the last few years and failure of prevention of spread of multidrug resistant strains.
WILSON et al: ANTIMICROBIAL SUSCEPTIBILITY OF SHIGELLA IN WESTERN NEPAL 149 Our findings showed that third generation cephalosporins should be kept in reserve, only for the treatment of drug-resistant non-responsive cases of acute gastroenteritis. Also co-trimoxazole, tetracycline, and ampicillin had no reasonable role in the empirical treatment of gastroenteritis in this part of Nepal and should be replaced with quinolones. We have found that ciprofloxacin had the least resistance among oral antibiotics with MIC 50 and MIC 90 values within susceptibility range. Nalidixic acid was introduced to cure shigellosis caused by ampicillin and co-trimoxazole resistant strains 21. Nalidixic acid still remains the drug of choice for shigellosis in our institution. However, the high resistance against this drug comparative to other study 22 has made it imperative that this drug should be used only in patients, especially in children, whose etiological agents are susceptible to nalidixic acid in vitro and not as an empirical therapy in all cases of suspected gastroenteritis. Although the safety of fluoroquinolones in young children is controversial, several reports about the safe use in childhood have been published 23. As resistance to nalidixic acid due to first step resistance mutations is generally thought to precede resistance to fluoroquinolones 24, closed continued monitoring to these drugs is warranted. It is concluded that Shigellae are predominant organisms for acute gastroenteritis, especially in children in western Nepal. The emergence of multiple drug resistance demands continuous monitoring of the susceptibility pattern of Shigella isolates. Simple measures like hand washing coupled with strict public health strategies like clean water supply, good sewage management and a clean environment should help reduce morbidity and mortality due to diarrhoeal diseases. References 1. Jousilahti P, Madkur SM, Lambrechts T, Sherwin E. Diarrhoeal disease morbidity and home treatment practices in Egypt. Public Health 1997; 111 : 5-10. 2. World Health Organization. Diarrhoeal disease due to Shigella disease. In: Vaccines, immunization and biologicals. Geneva: World Health Organization; 1998 p. 1-5. 3. Iwalokun BA, Gbenle GO, Smith SI, Ogunledun A, Akinsinde KA, Omonigbehin EA. Epidemiology of shigellosis in Lagos, Nigeria: Trends in antimicrobial resistance. J Health Popul Nutr 2001; 19 : 183-90. 4. Khan M, Curlin GT, Huq I. Epidemiology of Shigella dysenteriae type 1 infections, in Dacca urban area. Trop Geogr Med 1979; 31 : 213-23. 5. Jesudason MV. Shigella isolation in Vellore, south India (1997-2001). Indian J Med Res 2002; 115 : 11-3. 6. Bennish ML, Salam MA, Hossain MA, Myaux J, Khan EH, Chakraborty J. Antimicrobial resistance to Shigella isolates in Bangladesh1983-1990: increasing frequency of strains multiply resistant to ampicillin, trimethoprimsulfamethoxazole, and nalidixic acid. Clin Infect Dis 1992; 14 : 1055-60. 7. Bogaerts J, Verhaegen J, Munyabikali JP, Mukantabana B, Lemmens P, Vandeven J. Antimicrobial resistance and serotypes of Shigella isolates in Kigali, Rwanda (1983 to 1993): increasing frequency of multiple resistance. Diagn Microbiol Infect Dis 1997; 28 : 165-71. 8. Khan WA, Seas C, Dhar U, Salam MA, Bennish ML. Treatment of shigellosis: V. Comparison of azithromycin and ciprofloxacin. A double-blind randomized, controlled trial. Ann Intern Med 1997; 126 : 697-703. 9. Kabir I, Rahaman MM, Ahmed SM, Akhter SQ, Butler T. Comparative efficacies of pivmecillinam and ampicillin in acute shigellosis. Antimicrob Agent Chemother 1984; 25 : 643-5. 10. World Health Organization. Programme for Control of Diarrhoeal Diseases. Manual for laboratory investigations of acute enteric infections. Geneva: World Health Organisation; 1987 p. 1-113. (CDD/83.3 rev 1) 11. Duguid JP. Shigella. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Practical medical microbiology, Mackie & McCartney, 14th ed. New York: Churchill Livingstone; 1996 p. 405-11. 12. Ahmed K, Shakoori FR, Shakoori AR. Aetiology of Shigellosis in Northern Pakistan. J Health Popul Nutr 2003; 21 : 32-9. 13. Agarwal KC. Antibiotic sensitivity test by disc diffusion method: Standardization and interpretation. Indian J Pathol Bacteriol 1974; 17 : 149-59. 14. National Committee of Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing: ninth international supplement. Wayne, Pernnsyslvania: National Committee for Clinical Laboratory Standards, 1999. (NCCLS document no. M100-S9).
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