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
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 MIC50 and MIC90 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 world1. Epidemiology reports show that
about 140 million people suffer from shigellosis with
estimated 600,000 deaths per year worldwide2. In
Nigeria3 as well as in Bangladesh4, both children and
young adults are at a higher risk. In Vellore,
shigellosis was found to be one of the common causes
Indian J Med Res 123, February 2006, pp 145-150
of gastroenteritis5. Over a decade ago, the active
surveillance studies showed that in most endemic
countries especially in Asia and sub-Saharan Africa6,7,
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
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
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 methods10. 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 37ºC
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 test11. They were further
identified at group level by slide agglutination test12
with specific antisera (DIFCO Laboratories, Detroit,
Antimicrobial susceptibility testing: Resistance
patterns of the Shigella isolates to various antibiotics
were determined by the agar diffusion technique13,14.
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 37ºC 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 37ºC
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 (MIC50) and
90 per cent (MIC90) 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 (107 cfu/
ml) of each isolate onto the surface of the antibiotic
plate was done to obtain a final inoculum size of
146 INDIAN J MED RES, FEBRUARY 2006
Table I. Number of resistant isolates Shigella and per cent resistance among serogroups
Antimicrobial drugs Resistant isolates
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)
Cot Tet Gen Amp Chl Nal Cif
Cot Tet Gen Amp Chl Nal
Cot Tet Gen Amp Chl
Cot Tet Gen Amp
Cot Tet Gen
Cot, cotrimoxazole; Tet, tetracycline; Gen, gentamicin; Amp, ampicillin; Chl, chloramphenicol; Nal, nalidixic acid; Cif, ciprofloxacin
WILSON et al: ANTIMICROBIAL SUSCEPTIBILITY OF SHIGELLA IN WESTERN NEPAL147
104-105 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. MIC50 and MIC90
of each antimicrobial agent against Shigella isolates
were evaluated after incubating the plates, containing
completely absorbed inocula, in ambient air at 37º C
for 18 h.
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 school-
going 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%),
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.
At 16 and 32 µg/ml, cefotaxime and ceftriaxone
inhibited the growth of 50 and 90 per cent of the
isolates respectively. The MIC50 and MIC90 values of
co-trimoxazole, tetracycline, gentamicin, ampicillin,
and nalidixic acids were very high and those of
ciprofloxacin were within range (Table III).
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
decade3. However, this was unlike the situation in
the islands of Bengal16 where S. flexneri and
S. dysenteriae alternated as most active agents of
shigellosis or in endemic communities of Israel17 and
Pakistan18 where S. sonnei was the predominant
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
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 infection19. 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 Nigeria3, 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 norfloxacin20. 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.
Table III. Minimum inhibitory concentrations (MICs) of antimicrobial agents for Shigella isolates
Ampicillin 8 - 32 1664
Cefotaxime 8 - 64 1632
Ceftriaxone8 - 641632
Co-trimoxazole (trimethoprime/sulphamethoxazole) 2/38 - 4/764/768/152
Tetracycline 4 - 1616 64
Chloramphenicol8 - 32 16 64
Gentamicin4 - 168 32
Nalidixic acid16 - 3216 64
Ciprofloxacin1 - 448
148 INDIAN J MED RES, FEBRUARY 2006
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 MIC50 and
MIC90 values within susceptibility range. Nalidixic
acid was introduced to cure shigellosis caused by
ampicillin and co-trimoxazole resistant strains21.
Nalidixic acid still remains the drug of choice for
shigellosis in our institution. However, the high
resistance against this drug comparative to other
study22 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 published23. As resistance to nalidixic acid due
to first step resistance mutations is generally thought
to precede resistance to fluoroquinolones24, 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
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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