Indian J Med Res ***, February ****, pp ***-***

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 1 2 (14.5%), S . sonnei 1 0 (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 MIC50 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

estimated 600,000 deaths per year worldwide 2. In

Diarrhoeal diseases and enteric infections are

Nigeria3 as well as in Bangladesh 4, both children and

major causes of morbidity and mortality in the

developing world1. Epidemiology reports show that young adults are at a higher risk. In Vellore,

about 140 million people suffer from shigellosis with shigellosis was found to be one of the common causes

145

146 INDIAN J MED RES, FEBRUARY 2006

o f gastroenteritis 5. Over a decade ago, the active Ltd.). All plates were incubated aerobically at 37 C

surveillance studies showed that in most endemic overnight. The non-lactose-fermenting (NLF)

countries especially in Asia and sub-Saharan Africa6,7, colonies from both DCA and MacConkey agar were

there was an emergence of multidrug resistance to identified on urea hydrolysis, triple sugar iron (TSI)

different antibiotics including ampicillin, medium, sulphide-indole and motility medium (SIM),

and Simmon s citrate test 11 . They were further

trimethoprim-sulphamethoxazole and nalidixic acid.

identified at group level by slide agglutination test12

Drugs like fluoroquinolones, azithromycin and

pivamdinocillin have been found to be efficacious with specific antisera (DIFCO Laboratories, Detroit,

for the treatment of shigellosis in children and Michigan, USA).

adults 8,9.

Antimicrobial susceptibility testing : Resistance

Every year a large number of patients suffer from patterns of the Shigella isolates to various antibiotics

were determined by the agar diffusion technique13,14.

acute gastroenteritis in the western region of Pokhara,

Nepal and isolation of Shigella from these patients Every inoculum was prepared by inoculating 5 ml of

is not uncommon; no studies were conducted so far Mueller-Hinton broth with five colonies of an 18 h

from this region to evaluate the prevalence of Shigella old pure Shigella culture followed by incubation in

ambient air and at 37 C for 16 h. The resulting turbid

in patients with acute gastroenteritis and the status

of drug resistance in shigellosis. The present study culture was standardized to a turbidity of 0.5

was therefore carried out in a tertiary care hospital McFarland using 0.85 per cent NaCl as a diluent. A

in Pokhara, Nepal to isolate Shigella from children sterile cotton swab was dipped into the standardized

and adults with suspected acute gastroenteritis, and suspension, drained, and used for inoculating 25 ml

to study its seasonal distribution and antimicrobial of Mueller-Hinton agar (MHA) in a 90 mm plate.

susceptibility pattern. The inoculating plates were air dried and antibiotic

disks included ampicillin (10 g), tetracycline

Material & Methods (30 g), cotrimoxazole i .e., trimethoprim/

sulphamethoxazole (1.25/23.75 g), cefotaxime

Patients and sample collection: During October 2002 (30 g), ceftriaxone (30 g), ciprofloxacin (5 g),

to September 2004, 770 stool samples were collected nalidixic acid (30 g), chloramphenicol (30 g) and

from all pre-school children (

children (6-15 yr) and adults (>15 yr) with acute (Mumbai, India) were mounted on them. The plates

were inverted and incubated in ambient air at 37 C

gastroenteritis attending out patients department in

a tertiary care hospital in Pokhara, Nepal, in clean, for 18 h. Zones of inhibition were recorded in

open-mouth disposable containers. All the samples millimeters and were compared with those of

were cultured within 2 h of collection and analyzed Escherichia coli A TCC25922 from (ICDDR, B,

according to standard methods10. Though most of the Dhaka, Bangladesh) which served as control strain.

patients had suffered from dysentery, some patients

had only mild diarrhoea and never developed Determination of minimum inhibitory concentration

dysenteric symptoms. Dysentery was characterized (MIC) : Minimum concentration of each antibiotic

by frequent passage (usually 10 to 13 times/day) of inhibitory to the growth of 50 per cent (MIC50) and

small volume stools consisting of blood, mucus, and 90 per cent (MIC90) of the isolates was determined

pus; often accompanied by abdominal cramps and on MHA in a 90 mm plate. The agar contained

tenesmus. Diarrhoea was defined as the passage of 3 concentration ranges of the antibiotics prepared by

or more liquid stools without blood and mucus in a two-fold serial dilution according to the National

24 h period. Committee for Clinical Laboratory Standards

(NCCLS)15. Manual inoculation with micropipette for

dispensing 20 l of standardized inoculum (107 cfu/

Bacteriological analysis: The samples were primarily

cultured on deoxycholate citrate agar (DCA) and ml) of each isolate onto the surface of the antibiotic

MacConkey agar media (Himedia Laboratories Pvt. 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 c fu/spot. Antibiotic-free plates were going children (70.6%,12/17) compared to adults

inoculated at the end and were used as negative (64.4%, 29/45). Detection rates was highest in

controls. The positive controls were the plates (one summer-monsoon i .e., June-September (54.2%,

plate per antibiotic tested) inoculated with the 45), and moderate in spring (March-May 22.9%,

reference strain E. coli ATCC25922. MIC50 and MIC90 19), and autumn (October-November 12.0%, 10),

of each antimicrobial agent against Shigella isolates and lowest in winter (December-February 10.8%,

were evaluated after incubating the plates, containing 9). All 83 isolates were sensitive to cefotaxime

completely absorbed inocula, in ambient air at 37 C and ceftriaxone and 67 showed variable resistance

for 18 h. against the remaining seven antibiotics (Table I).

Resistance to co-trimoxazole was highest

Results (8 0 . 7 %), fo l l o wed b y t et racy cl i n e (7 4 . 7 % ),

gentamicin (55.4%), ampicillin (53%).

Shigella w as isolated from 83 of 770 (10.8%) chloromphenicol (39.7%) and nalidixic acid

stool samples. Of these, S . flexneri 5 6 (67.5%) (31.3%). Ciprofloxacin (2.4%) had the least

was the most common isolate in all age groups, resistance. In 67 isolates, 7 patterns of antibiotic

followed by S . dysenteriae 1 2 (14.5%), S . sonnei resistance were found (Table II), which on further

10 (12%) and S . boydii 5 ( 6.0%). Isolation rate of analysis revealed that nearly 62 (74.7%) isolates

S. flexneri w as observed to be more among were resistant to 2 or more drugs.

children less than 5 yr (71.4%, 15/21) and school-

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

( g/ml) ( g/ml) ( g/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

shigellosis or in endemic communities of Israel 17 and

At 16 and 32 g/ml, cefotaxime and ceftriaxone

Pakistan 18 w here S . sonnei w as the predominant

inhibited the growth of 50 and 90 per cent of the

isolates respectively. The MIC 50 and MIC90 values of etiological agent.

co-trimoxazole, tetracycline, gentamicin, ampicillin,

Children (both pre-school and school-going) as

and nalidixic acids were very high and those of

those from Bangladesh,were at a higher risk of getting

ciprofloxacin were within range (Table III).

affected by the disease, which might be a reflection

of secondary infection from the adults as well as poor

Discussion

personal hygiene8.

Shigellosis is a public health problem in western

Antimicrobial therapy is the cornerstone of

Nepal as the community is ravaged by poverty, poor

treatment of shigellosis, as mortality especially with

sanitation, lack of personal hygiene and use of

S. dysenteriae type 1 infection is appreciable (10%)

contaminated water supplies. It is a mountainous

in the young and elderly, and it prevents the more

region and has a population of around 1,26,000 (2001

serious complications of the infection19. The guiding

Census). The temperature ranges from 40 F (4 C)

principle for the choice of antimicrobials in

in January to approximately 100 F (38 C) in June,

developing countries includes cost, availability of the

just before the monsoon. The summer-monsoon

drug and pattern of resistance in the community.

causes the climatic variation since rainwater is a

When compared with the study from Nigeria3, we

major cause of rapid deterioration in the surface water

found decreased resistance for ampicillin,

quality in this area. The high isolation rate of Shigella

tetracycline, chloramphenicol and increased

(10.78%) from the stool samples of the acute

resistance for nalidixic acid, gentamicin and

gastroenteritis patients in the present study is also a ciprofloxacin. The resistance against co-trimoxazole

reflection of poor hygiene and inadequate supply of was similar in both the studies. All isolates were

clean drinking water. sensitive to cefotaxime and ceftriaxone. A study from

north-western part of India documented nearly 100

The finding of S. flexneri as the predominant and per cent sensitivity of Shigella against drugs like

most active serogroup in western Nepal was similar chloramphenicol, nalidixic acid, co-trimoxazole,

to the study from Lagos, Nigeria where the gentamicin and norfloxacin 20. The alarming rise in

predominance of S. flexneri did not change since last resistance in Shigella in this part against these drugs

decade 3. However, this was unlike the situation in might be related to the indiscriminate use of drugs

the islands of Bengal 16 w here S . flexneri a nd during the last few years and failure of prevention of

S. dysenteriae alternated as most active agents of spread of multidrug resistant strains.

WILSON et al : ANTIMICROBIAL SUSCEPTIBILITY OF SHIGELLA IN WESTERN NEPAL 149

3. Iwalokun BA, Gbenle GO, Smith SI, Ogunledun A,

O ur findings showed that third generation

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4. Khan M, Curlin GT, Huq I. Epidemiology of S higella

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in the empirical treatment of gastroenteritis in this

Geogr Med 1979; 31 : 213-23.

part of Nepal and should be replaced with quinolones.

We have found that ciprofloxacin had the least 5. Jesudason MV. Shigella isolation in Vellore, south India

(1997-2001). Indian J Med Res 2002; 115 : 11-3.

resistance among oral antibiotics with MIC 50 a nd

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13 (Suppl 4) : S245-S51.

Reprint requests: Dr P.G. Shivananda, Professor & Head, Department of Microbiology, Manipal College of Medical Sciences

P.O. Box no.155, Deep Heights - 16, Pokhara, Nepal

e-mail: abqbxk@r.postjobfree.com

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