Amikacin

Background & objectives: Multi drug resistant Shigellae pose a major threat in control of shigellosis with. Shigella dysenteriae being the most prevalent species at our centre before 1994. A gradual decrease in S. dysenteriae occurred over the years with a corresponding increase in S. flexneri which became the predominant serotype. From May to November 2003, an increase in number of patients admitted with clinical diagnosis of dysentery was noted in comparison to previous years, with a corresponding increase in the isolation of multi drug resistant S. dysenteriae. We report here the re-emergence of multi drug resistant S. dysenteriae at our tertiary care centre in north India after a gap of about 10 yr. Plasmid analysis of S. dysenteriae was also performed to study the origin and clonality of the isolates.

Methods: Stool samples were collected in Cary-Blair medium and processed by standard methods. Shigellae were confirmed by serotyping. Minimum inhibitory concentration was done by agar dilution method and E-test. Plasmid profiling of 18 isolates (16 S. dysenteriae 1 and 2 S. dysenteriae 2) was performed by modified alkali lysis method. Clinical details of patients were noted.

Results: A total of 64 patients with dysentery were admitted during the study period. Patients presented with unusually severe symptoms and six developed complications. Treatment failure with ciprofloxacin occurred in six patients who received cefotaxime and amikacin. There were 38 children below 5 yr of age. S. dysenteriae (18 isolates of 5. dysenteriae 1 and 2 isolates of S. dysenteriae 2) were isolated from 20 of the 64 (31.2%) stool samples. S. dysenteriae re-emerged as the commonest isolate after a gap of nearly 10 yr. Fourteen of the 20 S. dysenteriae isolates were multi drug resistant; 12 were resistant to ciprofloxacin with MIC of 8-32 µg/ml. Plasmid profile analysis revealed that 6 of 11 ciprofloxacin resistant S. dysenteriae 1 had similar profiles.

Interpretation & conclusion: Emergence of a clone of ciprofloxacin resistant S. dysenteriae 1 in north India is disturbing as treatment options in our geographic area are limited in view of already existing high drug resistance to nalidixic acid, co-trimoxazole and amoxycillin. A close monitoring of shifts in serogroup distribution and antibiotic resistance is required to guide clinicians for treatment of shigellosis.

Key words Ciprofloxacin - multi drug resistant Shigella dysenteriae - plasmid profile - re-emergence

Shigellosis is one of the major causes of morbidity and mortality among children less than 5 yr of age in developing countries'. The number of annual episodes of Shigella throughout the world has been estimated to be 164.7 millions, of which 163.2 millions were in developing countries (including 1.1 million deaths). A total of 69 per cent of all episodes and 61 per cent of all deaths attributable to shigellosis involved children below 5 yr of age1. Of all serotypes, Shigella dysenteriae 1 causes the most serious disease and has epidemic potential2"7. Increasing incidence of multi drug resistance among Shigellae poses a major threat in the control of shigellosis89. Moreover, shifts in the prevalent serogroups in a geographic area make it further difficult to choose an agent for empiric treatment. At our tertiary care centre in north India, Shigellae were found to be the second commonest bacterial agents causing diarrhoea after Escherichia coli, isolated from 2.6 per cent of stool samples10. 5. dysenteriae used to be the most prevalent species at our centre before 1994 accounting for 57 per cent of all Shigellae". A gradual decrease in S. dysenteriae occurred over the years to 12.7 per cent in 2002 with a corresponding increase in S.flexnerifrom 35.8 percent in 1993 to 65.5 percent in 200210.

Changing trends in epidemiology of shigellosis and antimicrobial resistance pattern of Shigella isolates have been noticed in many parts of the world over the last two decades12 B. Similar trends have been seen in Kolkata, India where multi drug resistant 5. dysenteriae serotype 1 re-emerged after a gap of 9 yr14. In this communication, we report re-emergence of multi drug resistant S. dysenteriae in 2003 after a gap of nearly 10 yr, with added resistance to ciprofloxacin.

Plasmid profile analysis is a useful epidemiological tool for investigating outbreaks of enteric diseases15,16. Shigellae are known to harbour a number of plasmids. Few studies have documented the usefulness of plasmid profiles for investigation of outbreaks caused by Shigellail17,18. Therefore, in the present study plasmid analysis of 5. dysenteriae isolates was also done to provide insight into the origin of these isolates and to study the relatedness amongst them.

Material & Methods

The study was performed at Post Graduate Institute of Medical Education & Research, Chandigarh, a 1268bedded tertiary care referral centre in northern India during May to November 2003. An increase was noticed during this period in the number of patients admitted with dysentery. Only complicated and referred cases were admitted. All available clinico-demographic details of patients were noted. Faecal samples were collected in Cary-Blair medium and cultured on Mac Conkey agar, Xylose-lysine-deoxycholate (XLD) agar, ampicillin blood agar, and enrichment was done in selenite broth (Hi-Media Laboratories, Mumbai, India). Bacterial identification was done using standard biochemical tests19. Shigellae were confirmed and serotyped using antisera from Denka-Seiken, Japan. The isolates were stored at room temperature in nutrient agar slopes, and at -20"C in brain heart infusion broth with 15 per cent glycerol. Antimicrobial susceptibility was performed by Kirby-Bauer method2". The antibiotics (Hi-media Laboratories. Mumbai, India) tested ^g/disc) were amoxycillin (30), nalidixic acid (30), co-trimoxazole (25). furazolidone (300). amikacin (10), cefotaxime (30), ciprofloxacin (5), norfloxacin (10), ofloxacin (5), chloramphenicol (30) and gentamicin (10). Minimum inhibitory concentration (MIC) to ciprofloxacin was done by agar dilution method21 and E test (AB Biodisk Solna, Sweden). Escherichia coli ATCC 25922 (gifted by Dr M.K. Lalitha, Christian Medical College, Vellore) was used as control strain.

Plasmid DNA from 18(165. dysenteriae 1 and two S. dysenteriae 2) isolates was extracted by the modified rapid alkaline lysis method of Birnboim & DoIy (1979)22 and Ish Horowicz & Burke (1981)2\ Two isolates of S. dysenteriae 1 could not be revived for plasmid analysis. The size of the plasmid DNA was determined with two super coiled DNA ladders ranging from 0.3 to 8.0 kb (0.2 to 5.6 MDa) and 2 to 23.1 kb (1.3 to 15.2 MDa) (Roche Diagnostics GmbH, Mannheim, Germany).

Results

A total of 64 patients (43 males, 21 females) were admitted with clinical diagnosis of acute dysentery during the study period: 54 children and 10 adults were affected. Twenty one children were below the age of 2 yr, 17 were between 2 to 5 yr and 16 were >5 to 14 yr. All had fever, blood and mucus in stool and abdominal cramps. Average number of stools per patient was 12-15 stools/day (range 8-65). Three patients developed severe dehydration. One patient developed haemolytic uraemic syndrome. Two patients presented with typhoid like illness with encephalopathy. Treatment failure with ciprofloxacin occurred in six patients, they were treated with cefotaxime and amikacin. All patients survived. S. dysenteriae was isolated as the sole pathogen from 20 of 64 (31.25%) stool samples. Of the 18 isolates serotyped, 16 were 5. dysenteriae 1 and two were typed as S. dysenteriae 2. Twelve isolates (115. dysenteriae serotype 1, one S. dysenteriae serotype 2) were resistant to ciprofloxacin with MIC of 8 to 32 pg/ml. Fourteen of the 20 S. dysenteriae isolates were multi drug resistant (resistant to 3 or more antibiotics, each antibiotic belonging to different groups) (Table I).

Number of plasmids ranged from 3 to 6 varying in size from approximately 1.1 to 20.9 MDa. No plasmid could be isolated from one isolate. Most common plasmids detected were of about 1.1 MDa (14 of 17 isolates), -3.7 (10 isolates), -2.3 (9 isolates), -5.5 (8 of 17), -9.8 (7 of 17), -15 (7 of 17), -6.3 (6 of 17) and -1.7 MDa (4 of 17). Three isolates had large plasmids of size varying from 18.8 to 20.9 MDa (Table I). Six out of 11 ciprofloxacin resistant S. dysenteriae 1 had similar plasmid profiles (P^sub 1^ to P^sub 1D^, Table I). Three isolates of S. dysenteriae 1 (nos. 7, 9 and 10) carried identical plasmids, of these, one (isolate no. 7) was sensitive to fluoroquinolones. Two drug sensitive isolates (isolate numbers 4 and 15) shared common plasmids of 15, 3.5 and 1.7 MDa (P^sub 2A^ & P^sub 2B^, Table I). The two isolates of S. dysenteriae 2 (nos. 2 and 6) shared 2 common plasmids of 4 and 1.7 MDa (P^sub 7^ and P^sub 8^, Table I and Figs. 1, 2).

Discussion

5. dysenteriae re-emerged as the commonest serogroup after a gap of nearly 10 yr. It used to be the most prevalent serogroup before 1994 accounting for 57 per cent of all Shigellae[[. A gradual decrease in isolation of 5. dysenteriae occurred over the years from 56.6 per cent in 1993 to 13.8 per cent in 2002 (Table II) with a corresponding increase in S. flexneri from 35.8 per cent 199323 to 65.5 per cent in 2002 (Table II). At Kolkata S. dysenteriae 1which was the most prevalent serotype of 1990s, was totally replaced with S.flexneri during 1995-96. Fluroquinolone resistant S. dysenteriae 1 emerged in 2002 at Kolkata14.

Being a tertiary care referral centre, only severe and complicated cases of dysentery are admitted. During the preceding 3 yr from January 2000 to December 2002, only 12 cases of dysentery were admitted out of which Shigellae were isolated from 8 cases (2 isolates of 5. dysenteriae, 3 of S. flexneri, 2 of 5. sonnei and 1 of 5. boydii) (unpublished data). In 2003, 64 cases of dysentery occurring in a period of 6 months indicated a probable outbreak. Majority of patients were children, 38 out of 64 (59%) being below 5 yr of age. In developing countries shigellosis is more common in this age group and is one of the major causes of morbidity and mortality1. In the present study there was no mortality inspite of the severity of illness and drug resistance to ciprofloxacin, as those not responding to ciprofloxacin were treated with cefotaxime and amikacin.

Epidemic dysentery caused by multi drug resistant S. dysenteriae type 1 has been a recurrent challenge in many parts of the developing world. This organism caused an extensive epidemic of shigellosis in eastern India in 19845. After a lapse of about 18 yr, an outbreak of bacillary dysentery with high morbidity and mortality was reported in April 2002 among the labourers of tea gardens in the same region24. The organism isolated was resistant to ampicillin, cotrimoxazole, nalidixic acid and norfloxacin. Emergence of multi drug resistant 5. dysenteriae 1, in patients hospitalized with diarrhoea in Kolkata, India has been reported recently25. Pulse-field gel electrophoresis (PFGE) performed on these isolates revealed that these were clonally related and were spreading from outbreak affected areas in eastern India25. It will be interesting to see the PFGE analysis of S. dysenteriae 1 isolated at our centre in northern India, which is quite far from eastern India. Though we could not perform PFGE, we analysed plasmid profile of our isolates which has long been used as an epidemiological tool for strain discrimination in both epidemic and endemic shigellosis926"28. Shigellae are known to harbour a number of plasmids of which the smaller ones (

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Neelam Taneja, Valarie Lyngdoh, Archa Vermani, Balwinder Mohan, Pooja Rao, Malkit Singh Ashok Dogra, Mini P. Singh & Meera Sharma

Department of Medical Microbiology, Postgraduate Institute of Medical Education & Research Chandigarh, India

Received July 5, 2004

Reprint requests: Dr Neelam Taneja, Assistant Professor, Department of Medical Microbiology, Postgraduate Institute of Medical Education & Research, Chandigarh 160012, India

e-mail: drneelampgi@yahoo.com

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